FIELD

The invention relates to a protease-deficient Bacillus anthracis and its use as a production microorganism to produce stable (i.e., intact) proteins, including recombinant secreted proteins.

BACKGROUND

Species of Bacillus, such as Bacillus anthracis, Bacillus cereus, and Bacillus subtilis, are attractive microorganisms for recombinant protein production in view of their fast growth rate, high yield, and ability to secrete produced products directly into the medium. Bacillus anthracis is also attractive in view of its ability to produce anthrax toxin and ability to fold proteins correctly. However, the attractiveness of Bacillus is reduced in view of the large quantities of extracellular proteases that the microorganisms secrete into the medium, leading to protein degradation, and the fact that they form spores.

The Gram-positive bacterial pathogen Bacillus anthracis (also referred to herein as

B. anthracis) secretes high levels of the three proteins that are collectively termed anthrax toxin: protective antigen (PA), edema factor (EF), and lethal factor (LF), when grown under conditions thought to mimic those in an infected animal host. PA is a receptor- binding component which acts to deliver LF and EF to the cytosol of eukaryotic cells; EF is a calmodulin-dependent adenylate cyclase, and LF is a zinc metalloprotease that cleaves most members of the mitogen-activated protein kinase kinase family; see, for example, Leppla SH et al, 2000, in Anthrax toxin, bacterial protein toxins, 445-472, Aktories K et al, (Eds.), Springer, Berlin; Moayeri M et al., 2011, Anthrax toxins, Bacillus anthracis and Anthrax, 121-156, Bergman (Ed.), John Wiley & Sons, Inc., Hoboken, NJ; Moayeri M et al, 2009, Mol. Aspects Med. 30, 439-455; Young JA et al, 2007, Annu. Rev. Biochem. 76, 243-265. PA, EF, and LF are encoded on virulence plasmid pXOl by pag, cya, and lef, respectively. Virulence plasmid pX02 encodes proteins required for capsule formation and depolymerization. B. anthracis that lack one or both virulence plasmids are typically attenuated in most animal hosts. Single PA, EF, and LF components are non-toxic; a combination of one PA and at least two EFs, at least two LFs, or mixtures of EF and LF are required for toxicity.

Because anthrax pathogenesis is highly dependent on the actions of the anthrax toxin proteins, vaccine and therapeutic development efforts have focused on countering toxin action, typically by generating antibodies to PA. The anthrax vaccine currently licensed in the USA, and developed almost 50 years ago (see, e.g., Puziss M et al, 1963, J. Bacteriol. 85, 230-236), consists of a partially purified culture supernatant of a protease-deficient B. anthracis strain (V770-NP1-R). PA is the most abundant protein and the key immunogen in this vaccine. Efforts to produce a recombinant PA vaccine from B. anthracis by scale-up of an established process (see, e.g., Farchaus JW et al, 1998, Appl. Environ. Microbiol. 64, 982-991) appear to have been hampered by instability of the final product.

While the toxin components can be purified as recombinant proteins from B. anthracis culture supernatants (see, e.g., Farchaus JW et al, ibid.; Varughese M et al, 1999, Infect. Immun. 67, 1860-1865; Singh Y et al, 1991, J. Biol. Chem. 266, 15493- 15497; Park S et al, 2000, Protein Expr. Purif. 18, 293-302), the integrity and yields are limited by the B. anthracis proteolytic enzymes that are co-secreted.

Two extracellular proteases are reported to be abundant in the B. anthracis secretome: NprB (GBAA 0599), neutral protease B, a thermolysin-like enzyme highly homologous to bacillolysins from other Bacillus species; and InhAl (GBAA 1295), immune inhibitor Al, a homo log of the immune inhibitors A from other members of the Bacillus cereus group (see, e.g., Antelmann H et al, 2005, Proteomics 5, 3684-3695; Chitlaru T et al, 2006, J. Bacteriol 188, 3551-3571; Chung MC et al, 2006, J. Biol. Chem. 281, 31408-31418. These two proteases contain zinc-binding motifs typical for the zincin tribe of metallopeptidases (His-Glu-Xxx-Xxx-His (SEQ ID NO:31)) and belong, respectively, to the M4 and M6 families of metalloproteases according to the MEROPS database, Wellcome Trust Sanger Institute (see e.g., website of Wellcome Trust Sanger Institute).

A third metalloprotease, camelysin (GBAA 1290), belonging to the M73 family is found in the secretome of several B. anthracis strains. This protease is similar to the camelysin of B. cereus, a novel surface metalloprotease; see, e.g., Grass G et al, 2004, Infect. Immun. 219-228.

B. anthracis also contains a gene encoding InhA2 metalloprotease (GBAA 0672, M6 family), although it is not known whether this protease is expressed and secreted. This gene is an ortholog of the InhAl described above (68% amino acid identity). Similarly, the genome of B. anthracis also contains genes encoding TasA (GBAA 1288, M73 superfamily), which is an ortholog of camelysin (60% amino acid identity), and MmpZ (GBAA 3159, ZnMc superfamily), which is a putative extracellular zinc- dependent matrix metalloprotease, a member of the metzincin clan of metallopeptidases. This clan is characterized by an extended zinc-binding motif (His-Glu-Xxx-Xxx-His- Xxx-Xxx-Gly/Asn-Xxx-Xxx-His/Asp (SEQ ID NO:32)) (see, e.g., Gomis-Ruth, FX, 2009, J. Biol. Chem. 284, 15353-15357).

Bacillus subtilis strains having more than one protease inactivated have been produced and analyzed. For example, Wu XC et al, 1991, J. Bacteriol. 173, 4952-4958 produced a B. subtilis strain deficient in six extracellular proteases (WB600), namely neutral protease A, subtilisin, extracellular protease, metalloprotease, bacillopeptidase F, and neutral protease B. WB600 showed only 0.32% of the extracellular protease activity of wild-type B. subtilis strains. Kurashima K et al, 2002, J. Bacteriol. 184, 76-81, expressed apparently intact Clostridium cellulovorans EngB cellulase in a B. subtilis strain deficient in eight proteases (WB800). WB800 was derived from WB600 through inactivation of VpR protease and cell wall protease WprA. WB700 was derived from WB600 through inactivation of VpR (see, e.g., Wu et al, 2002, Appl. Environ. Microbiol. 68, 3261-3269).

There have been reports of inactivation of certain individual B. anthracis proteases:

Inactivation of B. anthracis NprB led to reduced proteolysis of casein (see, e.g., Pomerantsev AP et al., 2006, Infect. Immun. 74, 682-693. Inactivation of InhAl indicated that coagulation of human blood by B. anthracis required InhAl for proteolytic activation of prothrombin and factor X (see, e.g., Kastrup CJ et al., 2008, Nat. Chem. Biol. 4, 742-750. However, production of anthrax toxin proteins in both of these strains led to protein degradation over time, albeit at a later time than production in B. anthracis A35.

There remains a need for a B. anthracis that can produce large amounts of stable (i.e., intact) proteins, such as anthrax toxin proteins PA, EF, and LF.

SUMMARY

The invention relates to a B. anthracis in which more than one secreted protease is inactivated by genetic modification. Such a protease-deficient B. anthracis has an improved ability to produce recombinant secreted proteins compared to other bacteria, particularly other Bacillus. Improvements include production of intact (i.e., mature full- length) proteins, often at high yield.

The disclosure provides a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, and a genetic modification that inactivates a protease of the M73 family of metalloproteases. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, and a genetic modification that inactivates a B. anthracis protease of the ZnMc superfamily of zinc-dependent metalloproteases. An example of a M4 protease is NprB. Examples of M6 proteases include InhAl and InhA2. Examples of M73 proteases are camelysin and TasA. An example of a ZnMc protease is MmpZ.

One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease and a genetic modification that inactivates InhAl protease. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhAl protease. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhAl protease. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhAl protease. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhAl protease. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhAl protease, and a genetic modification that inactivates MmpZ protease.

The disclosure provides that any of these protease-deficient B. anthracis can also comprise a genetic modification selected from the group consisting of a genetic modification that inactivates BA1995 protease, a genetic modification that inactivates VpR protease, a genetic modification that inactivates BA5414 protease, and combinations thereof.

The disclosure also provides that any of these protease-deficient B. anthracis can lack SinR and Sinl regulatory proteins. The disclosure also provides that any of these protease-deficient B. anthracis can be sporulation deficient. The disclosure also provides that any of these protease-deficient B. anthracis can lack one or more virulence plasmids. Genetic modification of any of these protease-deficient B. anthracis can be selected from the group consisting of deletion, insertion, inversion, substitution, derivatization, and combinations thereof, wherein such genetic modification affects one or more nucleotides in a gene encoding the protein. In one embodiment, genetic modification comprises deletion of one or more nucleotides in a gene encoding a protein, insertion of one or more nucleotides in a gene encoding a protein, or a combination thereof.

The disclosure provides a B. anthracis comprising: a genetic modification that inactivates NprB protease, wherein the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; a genetic modification that inactivates InhA2 protease, wherein the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA 0672 of B. anthracis; a genetic modification that inactivates TasA protease, wherein the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA 1288 of the B. anthracis; a genetic modification that inactivates camelysin, wherein the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA 1290 of the B. anthracis; a genetic modification that inactivates InhAl protease, wherein the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis; a genetic modification that inactivates MmpZ protease, wherein the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA 3159 of the B. anthracis; and a genetic modification that inactivates SpoOA protein, wherein the inactivated SpoOA protein is encoded by a genetically modified spoOA gene at locus GBAA_4394 of the B. anthracis, wherein the B. anthracis lacks virulence plasmids pXOl and pX02. One embodiment is a B. anthracis having the identifying characteristics of BH460, deposited under ATCC Accession No. PTA-12024. One embodiment is B. anthracis BH460, deposited under ATCC Accession No. PTA- 12024. The disclosure provides that any of these protease-deficient B. anthracis can also comprise a genetic modification that inactivates a protease selected from the group consisting of a protease of the transglutaminase-like superfamily, a protease of peptidase family S8, and a combination thereof. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a B. anthracis protease of the ZnMc superfamily of zinc-dependent metalloproteases, a genetic modification that inactivates a B. anthracis protease of the transglutaminase-like superfamily, and a genetic modification that inactivates a B. anthracis protease of peptidase family S8. One example of a transglutaminase-like superfamily protease is CysPl protease. One example of a peptidase family S8 protease is VpR protease. In one embodiment, the protease of the transglutaminase-like superfamily is CysPl protease. In one embodiment, the protease of peptidase family S8 is VpR protease. In one embodiment, the protease of the transglutaminase-like superfamily is CysPl protease, and the protease of peptidase family S8 is VpR protease. As used herein, CysPl protease is also referred to as BA1995 protease.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhAl protease, a genetic modification that inactivates MmpZ protease, a genetic modification that inactivates CysPl protease, and a genetic modification that inactivates VpR protease. In one embodiment, the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA 0672 of B. anthracis; the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA 1290 of the B. anthracis; the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis; the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA 3159 of the B. anthracis; the inactivated CysPl protease is encoded by a genetically modified cysPl gene at locus GBAA 1995 of the B. anthracis; and the inactivated VpR protease is encoded by a genetically modified vpR gene at locus GBAA_4584 of the B. anthracis.

The disclosure provides that any of the protease-deficient B. anthracis can also comprise a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof. As used herein, SprA protease is also referred to as BA5414 protease.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhAl protease, a genetic modification that inactivates MmpZ protease, a genetic modification that inactivates CysPl protease, a genetic modification that inactivates VpR protease, and a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof.

The disclosure also provides that any of these protease-deficient B. anthracis can lack SinR and Sinl regulatory proteins. The disclosure also provides that any of these protease- deficient B. anthracis can be sporulation deficient. The disclosure also provides that any of these protease-deficient B. anthracis can lack one or more virulence plasmids. Genetic modification of any of these protease-deficient B. anthracis can be selected from the group consisting of deletion, insertion, inversion, substitution, derivatization, and combinations thereof, wherein such genetic modification affects one or more nucleotides in a gene encoding the protein.

The disclosure provides a B. anthracis comprising: a genetic modification that inactivates NprB protease, wherein the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; a genetic modification that inactivates InhA2 protease, wherein the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA 0672 of B. anthracis; a genetic modification that inactivates TasA protease, wherein the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA 1288 of the B. anthracis; a genetic modification that inactivates camelysin, wherein the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA 1290 of the B. anthracis; a genetic modification that inactivates InhAl protease, wherein the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis; a genetic modification that inactivates MmpZ protease, wherein the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA 3159 of the B. anthracis; a genetic modification that inactivates CysPl protease, wherein the inactivated CysPl protease is encoded by a genetically modified cysPl gene at locus GBAA 1995 of the B. anthracis; a genetic modification that inactivates VpR protease, wherein the inactivated VpR protease is encoded by a genetically modified vpR gene at locus GBAA 4584 of the B. anthracis; and a genetic modification that inactivates SpoOA protein, wherein the inactivated SpoOA protein is encoded by a genetically modified spoOA gene at locus GBAA 4394 of the B. anthracis, wherein the B. anthracis lacks virulence plasmids pXOl and pX02. One embodiment is a B. anthracis having the identifying characteristics of BH480, deposited under ATCC Accession No.

. One embodiment is B. anthracis BH480, deposited under ATCC

Accession No. .

The disclosure provides a method to produce a B. anthracis comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases and to inactivate a protease of the M6 family of metalloproteases. The disclosure provides a method to produce a B. anthracis comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, and to inactivate a protease of the M73 family of metalloproteases. The disclosure provides a method to produce a B. anthracis comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, to inactivate a protease of the M73 family of metalloproteases, and to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases. The disclosure provides a method to produce a B. anthracis comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, to inactivate a protease of the M73 family of metalloproteases, to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases, to inactivate a protease of the transglutaminase-like superfamily, and to inactivate a protease of peptidase family S8. The disclosure provides a method to produce any B. anthracis of the embodiments. Such a method comprises culturing the B. anthracis in a medium; and recovering the B. anthracis.

The disclosure provides a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. The disclosure provides a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, and a genetic modification that inactivates a protease of the M73 family of metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. The disclosure provides a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, and a genetic modification that inactivates a protease of the ZnMc superfamily of zinc-dependent metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. The disclosure provides a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a protease of the ZnMc superfamily of zinc-dependent metalloproteases, a genetic modification that inactivates a B. anthracis protease of the transglutaminase-like superfamily, and a genetic modification that inactivates a B. anthracis protease of peptidase family S8; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. One embodiment of a product is a product selected from the group consisting of a protein, an amino acid, a nucleic acid molecule, a compound produced via a recombinant biosynthetic pathway, a small molecule, a drug, a vitamin, a drug conjugate, and a peptide nucleic acid conjugate. In one embodiment, such a product is a toxin. Examples of toxins include an anthrax toxin, a cholera toxin, a diphtheria toxin, a hemolysin, and a ricin. Additional examples of toxins include a Pseudomonas toxin, a Haemophilus ducreyi toxin, an Escherichia coli toxin, and a ribosome-inactivating protein (RIP) toxin.

The disclosure provides a recombinant molecule that comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. Examples of recombinant molecules include a recombinant molecule comprising a nucleic acid sequence selected from the group consisting of a recombinant molecule comprising nucleic acid sequence SEQ ID NO:62, a recombinant molecule comprising nucleic acid sequence SEQ ID NO: 65, a recombinant molecule comprising nucleic acid sequence SEQ ID NO:67, and a recombinant molecule comprising nucleic acid sequence SEQ ID NO:69.

One embodiment is a recombinant molecule comprising a pSJl 15 plasmid that encodes a protein comprising amino acid sequence SEQ ID NO:91. One embodiment is a recombinant molecule comprising a pYS5 plasmid that encodes a protein comprising amino acid sequence SEQ ID NO:92. One embodiment is a recombinant molecule selected from the group consisting of recombinant molecule pSJ136EF-His, recombinant molecule pSJ136EF-Cys, and recombinant molecule pSJ136EF-NEHY. The disclosure also provides a protein comprising an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO:93, amino acid sequence SEQ ID NO:94, and amino acid sequence SEQ ID NO: 95.

The disclosure provides a method to produce a product comprising: culturing a modified B. anthracis of the embodiments in a medium to produce the product; and recovering the product.

BRIEF DESCRIPTION OF THE FIGURES

Figure 1 provides sequences of truncated and inactivated B. anthracis proteases. Figure 1 A provides sequences of truncated and inactivated B. anthracis NprB and InhAl proteases. Figure IB provides sequences of truncated and inactivated B. anthracis InhA2, TasA, camelysin, and MmpZ proteases, as well as sequences used to produce the deletion of a B. anthracis genome spanning a portion of tasA through a portion of inhAl, as described in the Examples. Protease genes were inactivated using a procedure that results in the insertion of the 34-bp pair loxP sequence (underlined in the nucleotide sequences) flanked by several endonuclease restriction sites (positions not indicated). The large region between the TasA and InhAl genes was replaced by a loxP sequence using the same procedure. For each protease, the amino acid and nucleotide sequences shown in bold in the upper section identify the final three amino acids retained from the original protease. The amino acid sequences following this trimer are nonsense and/or out of frame translations encoded by the restriction sites and loxP sequences; translation terminates at the codons indicated by asterisks. The lower section of each of InhA2, TasA, camelysin, and MmpZ protease shows the entire amino acid sequence (including signal sequence) for each respective protease. The frame shift that occurs following the three amino acids shown in each upper section causes only the underlined portion of the respective original amino acid sequence to be translated. The zinc-binding active site sequences for NprB and InhAl are shown in bold in Figure 1A. Identical zinc-binding active site sequences are shown in bold in the InhA2 and MmpZ amino acid sequences in Figure IB. For the large TasA-InhAl deletion, truncation of the TasA protein occurs at the same site as for the single TasA deletion. The deleted DNA sequence extends through the corresponding sequence encoding the first 402 amino acids of InhAl (not indicated). The remaining DNA begins with the sequence that would encode the last 393 amino acids of InhAl, which begins with the sequence IMSGGSWAGKIAGTTPTSFS (SEQ ID NO:33) (dashed underlining). Figure 1C provides sequences of truncated and inactivated B. anthracis CysPl and VpR proteases. These protease genes were inactivated using a procedure that results in replacement of at least a portion of the genes with a 48-bp FRT- site (underlined in the nucleotide sequences) using Flp recombinase.

Figure 2 provides growth curves of certain B. anthracis strains and production analyses of edema factor (EF), anthrolysin O (ALO), protective antigen (PA), lethal factor (LF), and camelysin by these strains. Growth curves in LB medium are shown for ten B. anthracis strains over 24 h. Western blot analyses of EF, ALO, PA, LF, and camelysin at various time points are shown for each strain. The most slowly migrating band in each set of blots appears to correspond to the respective intact, or nearly intact, protein.

Figure 3 provides production analyses of LF and ALO by certain genetically modified strains, some of which had a deficient protease function complemented by a plasmid encoding active protease. Western blot analyses are shown of (A) LF production by A35AMmpZ compared to A35AMmpZC and (B) ALO production by A35TM compared to A35TMC. (C at the end of the strain name indicates a complemented strain.) Numbers on the top of each lane indicate time in hours at which samples were taken.

Figure 4 provides a comparison of EF proteins purified from E. coli BL21(DE3), B. anthracis BH450 (with a genetic modification in nprB and in spoOA), and B. anthracis BH460. A) SDS-PAGE analysis of the EF proteins. M - molecular mass markers (Page Ruler Unstained Protein Ladder, Fermentas). B) Electron Spray Ionization - Mass Spectra (ESI-MS) of the EF samples shown in A). The Y-axis represents relative abundance and the X-axis represents mass/charge ratio (m/z). C) Comparison of the experimental molecular masses of the EF samples extrapolated from ESI-MS data to the molecular masses calculated from the amino acid sequences. Relative abundances of the resulting components from the ESI-MS data are shown in parentheses. The final line shows differences between experimental and calculated masses.

Figure 5 provides EF activity analyses. A) cAMP production by different EF preparations was measured following treatment of RAW264.7 cells for 1 h with a range of EF concentrations and a set PA concentration (250 ng/ml). B) Potency of EF prepared from BH460 was compared to EF prepared from BH450 or from E. coli BL21(DE3) in Balb/cJ mice challenged with either 25 μg EF + 25 μg PA (for EF made from BH450, BH460, or E. coli, respectively) or 50 μg EF+ 50 μg PA (for EF made from BH450 only).

Figure 6 provides a schematic map of recombinant molecule pSJ136EFOS (SEQ ID NO:62), which comprises a nucleic acid molecule encoding mature EF with a PA signal sequence (SEQ ID NO:63) operatively linked to a pag promoter. The pag promoter spans approximately nucleotides 3496-3658 of SEQ ID NO:62. The PA signal sequence is encoded by nucleotides beginning at nucleotide 3659 of SEQ ID NO:62. The EF mature protein (SEQ ID NO:64) is encoded by nucleotides beginning at nucleotide 3746 of SEQ ID NO: 62. The E. coli ORI (not shown) spans approximately nucleotides 6286-6755 of SEQ ID NO:62. Figure 7 provides a schematic map of recombinant molecule pSW4-HBL LI His (SEQ ID NO:65), which comprises a nucleic acid molecule encoding HBL LI His operatively linked to a pag promoter. The HBL LI His protein (SEQ ID NO: 66) is encoded by nucleotides beginning at nucleotide 6611 of SEQ ID NO:65.

Figure 8 provides a schematic map of recombinant molecule pSW4-HBL L2 His

(SEQ ID NO: 67), which comprises a nucleic acid molecule encoding HBL L2 His operatively linked to a pag promoter. The HBL L2 His protein (SEQ ID NO:68) is encoded by nucleotides beginning at nucleotide 3660 of SEQ ID NO: 67.

Figure 9 provides a schematic map of recombinant molecule pSW4-HBL B His (SEQ ID NO:69), which comprises a nucleic acid molecule encoding HBL B His operatively linked to a pag promoter. The HBL B His protein (SEQ ID NO:70) is encoded by nucleotides beginning at nucleotide 2 of SEQ ID NO: 69.

Figure 10 provides native Phast gel (native 8-25% acrylamide gradient) analysis of production of EF proteins EFOS (lane 4), EF-His (lane 6), EF-Cys (lane 7), and EF- NEHY (lane 8) by B. anthracis BH480 transformed with recombinant molecule ^• pSJ136EFOS, B. anthracis BH480 transformed with recombinant molecule pSJ136EF- His, B. anthracis BH480 transformed with recombinant molecule pSJ136EF-Cys, or B. anthracis BH480 transformed with recombinant molecule pSil 36F.F~NEFiY, respectively. Lanes 1 and 2 show production of PA-U2f and PA-U7f by modified B. anthracis BH480. Lane 3 shows production of mature lethal factor (LF-OS) by B. anthracis BH480 transformed with pSJl 15-LF-OS. Lane 5 shows production of Hfql-FLAG by B. anthracis BH480 transformed with pSJ136 Hfql-FLAG.

Figure 11 provides SDS Phast gel analysis of production of LFnBlaY (SEQ ID NO:91) and PA-SNKE-deltaFF-E308D (SEQ ID NO:92) proteins by B. anthracis BH480 transformed with recombinant molecules encoding either LFnBlaY or PA-SNKE- deltaFF-E308D, respectively. Lane 1 : SDS molecular weight marker mix. Lane 2: PA- SNKE-deltaFF-E308D produced by B. anthracis BH480 transformed with a pYS5 plasmid encoding PA-SNKE-deltaFF-E308D; Lane 3: LF-BLA, recombinantly produced in E. coli; Lanes 4 and 5: LFnBlaY produced by B. anthracis BH480 transformed with a pSJl 15 plasmid encoding LFnBlaY.

DETAILED DESCRIPTION

The present disclosure describes the novel finding that genetic modifications of Bacillus anthracis (B. anthracis) that inactivate more than one secreted protease provide a protease-deficient B. anthracis with an improved ability to produce recombinant secreted proteins compared to other bacteria, e.g., other i?. anthracis strains, including those with a single inactivated secreted protease. Improvements include production of intact (i.e., mature full-length) proteins, often at high yield. For example, one embodiment is B. anthracis BH460. BH460 has genetic modifications that inactivate six proteases, is sporulation-deficient, and is free of the virulence plasmids. This strain provides an improved host for production of recombinant proteins. As an example, EF produced from BH460 is highly active, whereas previous B. anthracis host strains produced truncated EF proteins having low potency. The ability to produce an intact EF allows EF to be a component of a recombinant anthrax vaccine. As another example, one embodiment is B. anthracis BH480. BH480 has genetic modifications that inactivate eight proteases, is sporulation-deficient, and is free of the virulence plasmids. This strain also provides an improved host for production of recombinant proteins as shown herein.

Before the present invention is further described, it is to be understood that this invention is not limited to particular embodiments described, as such may, of course, vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the claims.

It must be noted that as used herein and in the appended claims, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. It is further noted that the claims may be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as "solely," "only" and the like in connection with the recitation of claim elements, or use of a "negative" limitation.

It should be understood that as used herein, the term "a" entity or "an" entity refers to one or more of that entity. For example, a genetic modification refers to one or more genetic modifications. As such, the terms "a", "an", "one or more" and "at least one" can be used interchangeably. Similarly the terms "comprising", "including" and "having" can be used interchangeably.

The publications discussed herein are provided solely for their disclosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, the preferred methods and materials are now described. All publications mentioned herein are incorporated herein by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

It is appreciated that certain features of the invention, which are, for clarity, described in the context of separate embodiments, may also be provided in combination in a single embodiment. Conversely, various features of the invention, which are, for brevity, described in the context of a single embodiment, may also be provided separately or in any suitable sub-combination. All combinations of the embodiments are specifically embraced by the present invention and are disclosed herein just as if each and every combination was individually and explicitly disclosed. In addition, all sub-combinations are also specifically embraced by the present invention and are disclosed herein just as if each and every such sub-combination was individually and explicitly disclosed herein.

The disclosure provides a B. anthracis comprising genetic modifications that inactivate more than one protease. In one embodiment, such inactivated proteases are inactivated secreted proteases. As used herein the phrase "a B. anthracis comprising a genetic modification that inactivates a protease" refers to a B. anthracis strain (also referred to as a B. anthracis organism or a B. anthracis microorganism) in which at least one of its proteases has been inactivated by at least one genetic modification. It is to be noted that the terms inactivated, inactive, defective, and deficient can be used interchangeably.

As used herein, a protease (also referred to as a proteinase or a peptidase) is any enzyme that conducts proteolysis, i.e., an enzyme that initiates protein catabolism by hydrolyzing the peptide bonds that link amino acids together in a chain to form a protein. A protein is any compound that has two or more amino acids linked together by a peptide bond between the carboxyl and amino groups of adjacent amino acids; as such, the term protein includes polypeptides and peptides. A secreted protease is a protease that is secreted from the cell (e.g., B. anthracis) that produces it. An inactivated protease is a protease that no longer functions to hydro lyze peptide bonds. An inactivated protease can have amino acids that have been deleted (e.g., to form a truncated protein), inserted, inverted, substituted, derivatized (e.g., by glycosylation, phosphorylation, acetylation, myristylation, prenylation, palmitation, amidation and/or addition of glycosylphosphatidyl inositol), or subjected to any other change known to those skilled in the art in order to deactivate the protease. An inactivated protease can be produced by effecting deletion, insertion, inversion, substitution, derivatization, and/or or any other change of amino acids at the amino acid level in order to deactivate the protease. Alternatively, inactivation can occur at the nucleic acid level by genetic modification (also referred to herein as mutation). Genetic modification includes deletion, insertion, inversion, substitution, derivatization, and/or any other change known to those skilled in the art of one or more nucleotides in a gene encoding the protease such that the genetically modified nucleic acid does not encode an active protease. In some embodiments, no protease is produced at all. In some embodiments, the encoded protease is truncated. In one embodiment, genetic modification comprises deletion of one or more nucleotides in a gene encoding a protease. In one embodiment, such a deletion comprises Cre-loxP gene knockout, a technique further described in the Examples herein. One embodiment is a conditionally inactivated protease, which can be produced, for example, using inducible antisense or anti-parallel loxP sites bracketing the structural gene encoding such a protease so that the Cre recombinase flips the gene between active and inactive forms. In one embodiment, a protease gene is inactivated via a Saccharomyces cerevisiae Flp- i?7 recombinase system.

One embodiment is a B. anthracis comprising genetic modification that inactivates at least two secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least three B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least four B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least five B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least six B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least seven B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least eight B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least nine B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least ten B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least eleven B. anthracis secreted proteases. One embodiment is a B. anthracis comprising genetic modification that inactivates at least twelve B. anthracis secreted proteases

The disclosure provides a B. anthracis comprising a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases. The M4 and M6 families of metalloproteases are as defined in the MEROPS database, Wellcome Trust Sanger Institute, ibid. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family and an inactivated protease of the M6 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family and at least two inactivated proteases of the M6 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family and two inactivated proteases of the M6 family.

A non-limiting example of a protease of the M4 family of metalloproteases is B. anthracis NprB protease, also referred to herein as NprB. In one embodiment a genetic modification that inactivates a protease of the M4 family of metalloproteases is a genetic modification that inactivates NprB. Non-limiting examples of a protease of the M6 family of metalloproteases are B. anthracis InhAl protease and B. anthracis InhA2 protease (also referred to herein as InhAl and InhA2, respectively). In one embodiment a genetic modification that inactivates a protease of the M6 family of metalloproteases is a genetic modification that inactivates a protease of the M6 family selected from the group consisting of InhAl, InhA2, and combinations thereof. As such, genetic modification can inactivate InhAl, genetic modification can inactivate InhA2, or genetic modification can inactivate both InhAl and InhA2. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB and InhAl . One embodiment is a B. anthracis that has genetic modifications that inactivate NprB and InhA2. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, and InhA2.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, and a genetic modification that inactivates a protease of the M73 family of metalloproteases. The M73 family of metalloproteases is as defined in the MEROPS database, Wellcome Trust Sanger Institute, ibid. Surprisingly, a B. anthracis comprising such genetic modifications produced significantly more intact secreted protein than did strains having inactivated proteases of the M4 and/or M6 families of metalloproteases but an active protease of the M73 family. Such improved production is exemplified in the Examples.

One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, and an inactivated protease of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, and at least two inactivated proteases of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, at least two inactivated proteases of the M6 family, and an inactivated protease of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, at least two inactivated proteases of the M6 family, and at least two inactivated proteases of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, and two inactivated proteases of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, two inactivated proteases of the M6 family, and an inactivated protease of the M73 family. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, two inactivated proteases of the M6 family, and two inactivated proteases of the M73 family.

Non-limiting examples of a protease of the M73 family of metalloproteases are B. anthracis camelysin protease and B. anthracis TasA protease (also referred to herein as camelysin and TasA, respectively.) In one embodiment a genetic modification that inactivates a protease of the M73 family of metalloproteases is a genetic modification that inactivates a protease of the M73 family selected from the group consisting of camelysin, TasA, and combinations thereof. As such, genetic modification can inactivate camelysin, genetic modification can inactivate TasA, or genetic modification can inactivate both camelysin and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, and camelysin. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, and camelysin. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, InhA2, and camelysin. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, InhA2, and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, camelysin, and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, camelysin, and TasA. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, InhA2, camelysin, and TasA.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, and a genetic modification that inactivates a protease of the ZnMc superfamily of zinc-dependent metalloproteases. The ZnMc superfamily of zinc-dependent metalloproteases is as defined in the MEROPS database, Wellcome Trust Sanger Institute, ibid. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, an inactivated protease of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, at least two inactivated proteases of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, at least two inactivated proteases of the M6 family, an inactivated protease of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, at least two inactivated proteases of the M6 family, at least two inactivated proteases of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, two inactivated proteases of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, two inactivated proteases of the M6 family, an inactivated protease of the M73 family, and an inactivated protease of the ZnMc superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, two inactivated proteases of the M6 family, two inactivated proteases of the M73 family, and an inactivated protease of the ZnMc superfamily.

A non-limiting example of a protease of the ZnMc superfamily of metalloproteases is B. anthracis MmpZ protease, also referred to herein as MmpZ. In one embodiment a genetic modification that inactivates a protease of the ZnMc superfamily of metalloproteases is a genetic modification that inactivates MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, camelysin, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, camelysin, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, InhA2, camelysin, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, TasA, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, TasA, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, InhA2, TasA, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, camelysin, TasA, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, camelysin, TasA, and MmpZ. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhAl, InhA2, camelysin, TasA, and MmpZ.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates at least two proteases, wherein the proteases are selected from the group consisting of a protease of the M4 family of metalloproteases, a protease of the M6 family of metalloproteases, a protease of the M73 family of proteases, and a protease of the ZnMc superfamily of zinc-dependent metalloproteases. The disclosure encompasses any combination of two or more such inactivated proteases. One or more genetic modifications can lead to the inactivation of such two or more proteases. For example, a deletion spanning at least a portion of two genes that encode such proteases can effect at least two inactivated proteases. One embodiment is a B. anthracis comprising a genetic modification that inactivates at least two proteases, wherein the proteases are selected from the group consisting of NprB, InhA2, TasA, camelysin, InhAl, and MmpZ.

The disclosure provides a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhAl protease, and a genetic modification that inactivates MmpZ protease. It is to be appreciated that a genetic modification that inactivates one protease can be the same genetic modification that inactivates one or more additional proteases. For example, a genetic modification that deletes a region of the B. anthracis genome that spans from at least a portion of the tasA gene through at least a portion of the inhAl gene is an example of a genetic modification that inactivates a TasA protease, a genetic modification that inactivates a camelysin protease, and a genetic modification that inactivates an InhAl protease. In one embodiment, such a deletion spans from the codon that encodes amino acid residue 63 of SEQ ID NO:9 through the codon that encodes amino acid 402 of SEQ ID NO: 17. SEQ ID NO:9 and SEQ ID NO: 17 encode wild-type TasA and InhAl, respectively. Such a deletion also deletes the genes encoding regulatory proteins SinR and Sinl.

One embodiment is a B. anthracis that comprises a genetic modification that inactivates NprB protease and a genetic modification that inactivates InhAl protease. One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB protease and InhAl proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; and (b) the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis "Ames ancestor" strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment is B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhAl protease. One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB protease, camelysin, and InhAl proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; (b) the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA 1290 of the B. anthracis; and (c) the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis "Ames ancestor" strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment is a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhAl protease. One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB protease, TasA, camelysin, and InhAl proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; (b) the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA 1288 of the B. anthracis; (c) the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA 1290 of the B. anthracis; and (d) the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis "Ames ancestor" strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment is a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, and a genetic modification that inactivates InhAl protease. One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB protease, InhA2, TasA, camelysin, and InhAl proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; (b) the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA 0672 of B. anthracis; (c) the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA 1288 of the B. anthracis; (d) the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA 1290 of the B. anthracis; and (e) the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis "Ames ancestor" strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment is a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhAl protease, and a genetic modification that inactivates MmpZ protease.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl and MmpZ proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; (b) the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA_0672 of B. anthracis; (c) the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA_1288 of the B. anthracis; (d) the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA 1290 of the B. anthracis; (e) the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis; and (f) the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA 3159 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis "Ames ancestor" strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl and MmpZ proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a nprB gene that encodes an inactivated NprB comprising amino acid sequence SEQ ID NO:2; (b) the inactivated InhA2 protease is encoded by an inhA2 gene that encodes an inactivated InhA2 comprising amino acid sequence SEQ ID NO:6; (c) the inactivated TasA protease is encoded by a tasA gene that encodes an inactivated TasA comprising amino acid sequence SEQ ID NO: 10; (d) the inactivated camelysin protease is encoded by a calY gene that encodes an inactivated camelysin comprising amino acid sequence SEQ ID NO: 14; (e) the inactivated InhAl protease is encoded by an inhAl gene that encodes an inactivated InhAl comprising amino acid sequence SEQ ID NO: 18; and (f) the inactivated MmpZ protease is encoded by a mmpZ gene that encodes an inactivated MmpZ comprising amino acid sequence SEQ ID NO:22. In one embodiment, the amino acid sequence of the encoded inactivated NprB protease is SEQ ID NO:2. In one embodiment, the amino acid sequence of the encoded inactivated InhA2 protease is SEQ ID NO:6. In one embodiment, the amino acid sequence of the encoded inactivated TasA protease is SEQ ID NO: 10. In one embodiment, the amino acid sequence of the encoded inactivated camelysin protease is SEQ ID NO: 14. In one embodiment, the amino acid sequence of the encoded inactivated InhAl protease is SEQ ID NO: 18. In one embodiment, the amino acid sequence of the encoded inactivated MmpZ protease is SEQ ID NO:22.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl and MmpZ proteases is a B. anthracis in which: (a) the genetic modification that inactivates NprB protease is a nprB gene that encodes an inactivated NprB comprising amino acid sequence SEQ ID NO:2; (b) the genetic modification that inactivates InhA2 protease is an inhA2 gene that encodes an inactivated InhA2 comprising amino acid sequence SEQ ID NO:6; (c) the genetic modification that inactivates TasA protease, camelysin protease, and InhAl protease is a deletion that spans from the codon that encodes amino acid residue 63 of SEQ ID NO: 9 through the codon that encodes amino acid 402 of SEQ ID NO: 17 (such a deletion also deletes genes encoding SinR and Sinl); and (d) the genetic modification that inactivates MmpZ protease is a mmpZ gene that encodes an inactivated MmpZ comprising amino acid sequence SEQ ID NO:22.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, and MmpZ proteases is a B. anthracis in which: (a) the genetic modification that inactivates NprB protease is a nprB gene that encodes an inactivated NprB consisting of amino acid sequence SEQ ID NO:2; (b) the genetic modification that inactivates InhA2 protease is an inhA2 gene that encodes an inactivated InhA2 consisting of amino acid sequence SEQ ID NO:6; (c) the genetic modification that inactivates TasA protease, camelysin protease, and InhAl protease is a deletion that spans from the codon that encodes amino acid residue 63 of SEQ ID NO: 9 through the codon that encodes amino acid 402 of SEQ ID NO: 17 (such a deletion also deletes genes encoding SinR and Sinl); (d) the genetic modification that inactivates MmpZ protease is a mmpZ gene that encodes an inactivated MmpZ consisting of amino acid sequence SEQ ID NO:22.

In some embodiments, B. anthracis of the disclosure can also include genetic modifications that inactivate regulatory protein SinR, regulatory protein Sinl, or both SinR and Sinl. SinR and Sinl are encoded by genes located at GBAA 1292 and GBAA_1293, respectively, of the B. anthracis genome.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a protease of the ZnMc superfamily of metalloproteases and a genetic modification selected from the group consisting of a genetic modification that inactivates BA1995 protease, a genetic modification that inactivates VpR protease, a genetic modification that inactivates BA5414 protease, and combinations thereof. BA1995, VpR and BA5414 proteases have been identified as secreted proteases present in the supernatant of B. anthracis in which NprB, InhA2, TasA, camelysin, InhAl, and MmpZ proteases have been inactivated. BA1995 is encoded by a gene located at locus GBAA 1995 of B. anthracis (see, e.g., Sastalla I et al, 2010, Microbiology 156, 2982- 2993). VpR, or BA4584, is a minor extracellular protease that is encoded by the vpR gene located at locus GBAA_4584 of B. anthracis. BA 5414,is a serine protease that is encoded by a gene located at locus GBAA 5414 of B. anthracis. The disclosure provides a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhAl protease, a genetic modification that inactivates MmpZ protease, and a genetic modification selected from the group consisting of a genetic modification that inactivates BA1995 protease, a genetic modification that inactivates VpR protease, a genetic modification that inactivates BA5414 protease, and combinations thereof.

Additional secreted proteases, the inactivation of which would further improve protein production by B. anthracis of the embodiments, can be identified using techniques known to those skilled in the art. For example, a protease-deficient B. anthracis of the embodiments can be cultured in a medium, and the resulting medium, or supernatant produced from the medium, can be tested for protease activity against desired proteins being produced by such B. anthracis. Protease(s) responsible for such activity can be isolated, sequenced, and the sequence(s) compared to the genomic map of B. anthracis in order to identify the gene(s) that encode it (them). Such gene(s) can then be inactivated using techniques described herein.

The disclosure provides that any of the B. anthracis of the embodiments can be sporulation-deficient. That is, any of the B. anthracis described herein can also comprise a genetic modification that prevents sporulation, such as a genetic modification that inactivates the SpoOA protein. The SpoOA protein, a key regulator of sporulation in Bacillus (see, e.g., Molle V et al, 2003, Molec. Microbiol. 50, 1683-1701), is encoded by the spoOA gene, located at locus GBAA 4394 of B. anthracis. B. anthracis that are unable to sporulate are advantageous as production organisms.

The disclosure also provides that any of the B. anthracis of the embodiments can be deficient in one or more virulence plasmids. One embodiment is a B. anthracis of the embodiments that is pXOl-, i.e., the B. anthracis lacks virulence plasmid pXOl . One embodiment is a B. anthracis of the embodiments that is pXOl-, pX02-; i.e., the B. anthracis lacks virulence plasmids pXOl and pX02. One embodiment is a B. anthracis of the embodiments that is pXOl-, pX02+.

The disclosure provides a B. anthracis comprising: a genetic modification that inactivates NprB protease, wherein the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; a genetic modification that inactivates InhA2 protease, wherein the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA 0672 of B. anthracis; a genetic modification that inactivates TasA protease, wherein the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA 1288 of the B. anthracis; a genetic modification that inactivates camelysin, wherein the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA 1290 of the B. anthracis; a genetic modification that inactivates InhAl protease, wherein the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis; a genetic modification that inactivates MmpZ protease, wherein the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA 3159 of the B. anthracis; and a genetic modification that inactivates SpoOA protein, wherein the inactivated SpoOA protein is encoded by a genetically modified spoOA gene at locus GBAA_4394 of the B. anthracis, wherein the B. anthracis lacks virulence plasmids pXOl and pX02.

The disclosure provides a Bacillus anthracis having the identifying characteristics of BH460, deposited under ATCC Accession No. PTA-12024. A deposit of Bacillus anthracis BH460 has been made on August 9, 2011 at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209, USA, under ATCC accession number PTA-12024. The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure. Identifying characteristics of BH460 include (a) inactivated NprB, InhA2, TasA, camelysin, InhAl, and MmpZ proteases, (b) sporulation-deficiency, and (c) lack of virulence plasmids pXOl and pX02. One embodiment of the disclosure is Bacillus anthracis BH460.

The disclosure provides that any of these protease-deficient B. anthracis can also comprise a genetic modification that inactivates a protease selected from the group consisting of a protease of the transglutaminase-like superfamily, a protease of peptidase family S8, and a combination thereof. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a B. anthracis protease of the ZnMc superfamily of zinc-dependent metalloproteases, and a genetic modification that inactivates a B. anthracis protease of the transglutaminase-like superfamily. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a B. anthracis protease of the ZnMc superfamily of zinc-dependent metalloproteases, and a genetic modification that inactivates a B. anthracis protease of peptidase family S8. One embodiment is a B. anthracis that comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a B. anthracis protease of the ZnMc superfamily of zinc-dependent metalloproteases, a genetic modification that inactivates a B. anthracis protease of the transglutaminase-like superfamily, and a genetic modification that inactivates a B. anthracis protease of peptidase family S8. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, an inactivated protease of the M73 family, an inactivated protease of the ZnMc superfamily, and an inactivated protease of the transglutaminase-like superfamily. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, an inactivated protease of the M73 family, an inactivated protease of the ZnMc superfamily, and an inactivated protease of peptidase family S8. One embodiment is a B. anthracis comprising an inactivated protease of the M4 family, an inactivated protease of the M6 family, an inactivated protease of the M73 family, an inactivated protease of the ZnMc superfamily, an inactivated protease of the transglutaminase-like superfamily, and an inactivated protease of peptidase family S8.

A non-limiting example of a protease of the transglutaminase-like superfamily is B. anthracis CysPl protease, also referred to herein as CysPl . In one embodiment, a genetic modification that inactivates a protease of the transglutaminase-like superfamily is a genetic modification that inactivates CysPl . A non- limiting example of a protease of peptidase family S8 is B. anthracis VpR protease (also referred to herein as VpR). In one embodiment, a genetic modification that inactivates a protease of peptidase family S8 is a genetic modification that inactivates VpR. As such, genetic modification can inactivate CysPl, genetic modification can inactivate VpR, or genetic modification can inactivate both CysPl and VpR. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, MmpZ, and CysPl . One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, MmpZ, and VpR. One embodiment is a B. anthracis that has genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, MmpZ, CysPl, and VpR.

The disclosure provides a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhAl protease, a genetic modification that inactivates MmpZ protease, a genetic modification that inactivates CysPl protease, and a genetic modification that inactivates VpR protease. It is to be appreciated that a genetic modification that inactivates one protease can be the same genetic modification that inactivates one or more additional proteases. For example, as disclosed above, a genetic modification that deletes a region of the B. anthracis genome that spans from at least a portion of the tasA gene through at least a portion of the inhAl gene is an example of a genetic modification that inactivates a TasA protease, a genetic modification that inactivates a camelysin protease, and a genetic modification that inactivates an InhAl protease. In one embodiment, such a deletion spans from the codon that encodes amino acid residue 63 of SEQ ID NO: 9 through the codon that encodes amino acid 402 of SEQ ID NO: 17. SEQ ID NO:9 and SEQ ID NO: 17 encode wild-type TasA and InhAl, respectively. Such a deletion also deletes the genes encoding regulatory proteins SinR and Sinl.

One embodiment is a B. anthracis that comprises a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhAl protease, a genetic modification that inactivates MmpZ protease, a genetic modification that inactivates CysPl protease, and a genetic modification that inactivates VpR protease.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, MmpZ, CysPl, and VpR proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; (b) the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA 0672 of B. anthracis; (c) the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA 1288 of the B. anthracis; (d) the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA 1290 of the B. anthracis; (e) the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis; (f) the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA 3159 of the B. anthracis; (g) the inactivated CysPl protease is encoded by a genetically modified cysPl gene at locus GBAA 1995 of the B. anthracis; and (h) the inactivated VpR protease is encoded by a genetically modified vpR gene at locus GBAA 34584 of the B. anthracis. Each of these gene loci corresponds to an equivalently named locus of B. anthracis "Ames ancestor" strain chromosome (GenBank Accession No. NC 003997), as described in the Examples.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, MmpZ, CysPl, and VpR proteases is a B. anthracis in which: (a) the inactivated NprB protease is encoded by a nprB gene that encodes an inactivated NprB comprising amino acid sequence SEQ ID NO:2; (b) the inactivated InhA2 protease is encoded by an inhA2 gene that encodes an inactivated InhA2 comprising amino acid sequence SEQ ID NO:6; (c) the inactivated TasA protease is encoded by a tasA gene that encodes an inactivated TasA comprising amino acid sequence SEQ ID NO: 10; (d) the inactivated camelysin protease is encoded by a calY gene that encodes an inactivated camelysin comprising amino acid sequence SEQ ID NO: 14; (e) the inactivated InhAl protease is encoded by an inhAl gene that encodes an inactivated InhAl comprising amino acid sequence SEQ ID NO: 18; (f) the inactivated MmpZ protease is encoded by a mmpZ gene that encodes an inactivated MmpZ comprising amino acid sequence SEQ ID NO:22; (g) the inactivated CysPl protease is encoded by a cysPl gene that encodes an inactivated CysPl comprising amino acid sequence SEQ ID NO:72; and (h) the inactivated VpR protease is encoded by a vpR gene that encodes an inactivated VpR comprising amino acid sequence SEQ ID NO: 76. In one embodiment, the amino acid sequence of the encoded inactivated NprB protease is SEQ ID NO:2. In one embodiment, the amino acid sequence of the encoded inactivated InhA2 protease is SEQ ID NO: 6. In one embodiment, the amino acid sequence of the encoded inactivated TasA protease is SEQ ID NO: 10. In one embodiment, the amino acid sequence of the encoded inactivated camelysin protease is SEQ ID NO: 14. In one embodiment, the amino acid sequence of the encoded inactivated InhAl protease is SEQ ID NO: 18. In one embodiment, the amino acid sequence of the encoded inactivated MmpZ protease is SEQ ID NO:22. In one embodiment, the amino acid sequence of the encoded inactivated CysPl protease is SEQ ID NO: 72. In one embodiment, the amino acid sequence of the encoded inactivated VpR protease is SEQ ID NO: 76. One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, MmpZ, CysPl, and VpR proteases is a B. anthracis in which: (a) the genetic modification that inactivates NprB protease is a nprB gene that encodes an inactivated NprB comprising amino acid sequence SEQ ID NO:2; (b) the genetic modification that inactivates InhA2 protease is an inhA2 gene that encodes an inactivated InhA2 comprising amino acid sequence SEQ ID NO:6; (c) the genetic modification that inactivates TasA protease, camelysin protease, and InhAl protease is a deletion that spans from the codon that encodes amino acid residue 63 of SEQ ID NO:9 through the codon that encodes amino acid 402 of SEQ ID NO: 17 (such a deletion also deletes genes encoding SinR and Sinl); (d) the genetic modification that inactivates MmpZ protease is a mmpZ gene that encodes an inactivated MmpZ comprising amino acid sequence SEQ ID NO:22; (e) the inactivated CysPl protease is encoded by a cysPl gene that encodes an inactivated CysPl comprising amino acid sequence SEQ ID NO:72; and (f) the inactivated VpR protease is encoded by a vpR gene that encodes an inactivated VpR comprising amino acid sequence SEQ ID NO: 76.

One embodiment of a B. anthracis that comprises genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, MmpZ, CysPl, and VpR proteases is a B. anthracis in which: (a) the genetic modification that inactivates NprB protease is a nprB gene that encodes an inactivated NprB consisting of amino acid sequence SEQ ID NO:2; (b) the genetic modification that inactivates InhA2 protease is an inhA2 gene that encodes an inactivated InhA2 consisting of amino acid sequence SEQ ID NO:6; (c) the genetic modification that inactivates TasA protease, camelysin protease, and InhAl protease is a deletion that spans from the codon that encodes amino acid residue 63 of SEQ ID NO:9 through the codon that encodes amino acid 402 of SEQ ID NO: 17 (such a deletion also deletes genes encoding SinR and Sinl); (d) the genetic modification that inactivates MmpZ protease is a mmpZ gene that encodes an inactivated MmpZ consisting of amino acid sequence SEQ ID NO:22; (e) the inactivated CysPl protease is encoded by a cysPl gene that encodes an inactivated CysPl consisting of amino acid sequence SEQ ID NO:72; and (f) the inactivated VpR protease is encoded by a vpR gene that encodes an inactivated VpR consisting of amino acid sequence SEQ ID NO: 76.

In some embodiments, a B. anthracis of the disclosure also includes a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof. B. anthracis NprC is a neutral metalloprotease encoded by nprC, located at gene locus GBAA 2183 of B. anthracis "Ames ancestor" strain chromosome (GenBank Accession No. NC 003997). B. anthracis SprA is a serine protease encoded by sprA, located at gene locus GBAA 5414 of B. anthracis "Ames ancestor" strain chromosome. B. anthracis HtrA is a serine protease encoded by htrA, located at gene locus GBAA 3660 of B. anthracis "Ames ancestor" strain chromosome. HsIV protease is ATP-dependent protease HsIV that has been found in BH480 secrotome and is encoded at gene locus GBAA 3968 of B. anthracis "Ames ancestor" strain chromosome. Protease HsIV and the ATPase/chaperone HslU are part of an ATP- dependent proteolytic system that is the prokaryotic homo log of the proteasome. HsIV is a dimer of hexamers (a dodecamer) that forms a central proteolytic chamber with active sites on the interior walls of the cavity. HsIV shares significant sequence and structural similarity with the proteasomal beta-subunit and both are members of the Ntn-family of hydrolases. HsIV has a nucleophilic threonine residue at its N-terminus that is exposed after processing of the propeptide and is directly involved in active site catalysis.

The disclosure provides a B. anthracis comprising a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a protease of the ZnMc superfamily of metalloproteases, a genetic modification that inactivates a protease of the transglutaminase-like superfamily, a genetic modification that inactivates a protease of peptidase family S8, and a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof.

The disclosure provides a B. anthracis comprising genetic modification that inactivates NprB, InhA2, TasA, camelysin, InhAl, MmpZ, CysPl, and VpR proteases that also includes a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof. One embodiment is a B. anthracis comprising a genetic modification that inactivates NprB protease, a genetic modification that inactivates InhA2 protease, a genetic modification that inactivates TasA protease, a genetic modification that inactivates camelysin protease, a genetic modification that inactivates InhAl protease, a genetic modification that inactivates MmpZ protease, a genetic modification that inactivates CysPl protease, a genetic modification that inactivates VpR protease, and a genetic modification selected from the group consisting of a genetic modification that inactivates NprC protease, a genetic modification that inactivates SprA protease, a genetic modification that inactivates HtrA protease, a genetic modification that inactivates HsIV protease, and combinations thereof.

In some embodiments, B. anthracis of the disclosure that comprise genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, MmpZ, CysPl, and VpR proteases also include genetic modifications that inactivate regulatory protein SinR, regulatory protein Sinl, or both SinR and Sinl. SinR and Sinl are encoded by genes located at GBAA 1292 and GBAA 1293, respectively, of the B. anthracis genome.

Additional secreted proteases, the inactivation of which would further improve protein production by B. anthracis of the embodiments, can be identified using techniques known to those skilled in the art and as described herein.

In some embodiments, B. anthracis of the disclosure that comprise genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, MmpZ, CysPl, and VpR proteases are sporulation-deficient. That is, any of the B. anthracis described herein can also comprise a genetic modification that prevents sporulation, such as a genetic modification that inactivates the SpoOA protein. The SpoOA protein, a key regulator of sporulation in Bacillus (see, e.g., Molle V et al, 2003, Molec. Microbiol. 50, 1683-1701), is encoded by the spoOA gene, located at locus GBAA 4394 of B. anthracis. B. anthracis that are unable to sporulate are advantageous as production organisms.

In some embodiments, B. anthracis of the disclosure that comprise genetic modifications that inactivate NprB, InhA2, TasA, camelysin, InhAl, MmpZ, CysPl, and VpR proteases are deficient in one or more virulence plasmids. One embodiment is a B. anthracis of the embodiments that is pXOl-, i.e., the B. anthracis lacks virulence plasmid pXOl . One embodiment is a B. anthracis of the embodiments that is pXOl-, pX02-; i.e., the B. anthracis lacks virulence plasmids pXOl and pX02. One embodiment is a B. anthracis of the embodiments that is pXOl-, pX02+.

The disclosure provides a B. anthracis comprising: a genetic modification that inactivates NprB protease, wherein the inactivated NprB protease is encoded by a genetically modified nprB gene at locus GBAA 0599 of B. anthracis; a genetic modification that inactivates InhA2 protease, wherein the inactivated InhA2 protease is encoded by a genetically modified inhA2 gene at locus GBAA 0672 of B. anthracis; a genetic modification that inactivates TasA protease, wherein the inactivated TasA protease is encoded by a genetically modified tasA gene at locus GBAA 1288 of the B. anthracis; a genetic modification that inactivates camelysin, wherein the inactivated camelysin is encoded by a genetically modified calY gene at locus GBAA 1290 of the B. anthracis; a genetic modification that inactivates InhAl protease, wherein the inactivated InhAl protease is encoded by a genetically modified inhAl gene at locus GBAA 1295 of the B. anthracis; a genetic modification that inactivates MmpZ protease, wherein the inactivated MmpZ protease is encoded by a genetically modified mmpZ gene at locus GBAA 3159 of the B. anthracis; a genetic modification that inactivates CysPl protease, wherein the inactivated CysPl protease is encoded by a genetically modified cysPl gene at locus GBAA 1995 of the B. anthracis; a genetic modification that inactivates VpR protease, wherein the inactivated VpR protease is encoded by a genetically modified vpR gene at locus GBAA 34584 of the B. anthracis, and a genetic modification that inactivates SpoOA protein, wherein the inactivated SpoOA protein is encoded by a genetically modified spoOA gene at locus GBAA 4394 of the B. anthracis, wherein the B. anthracis lacks virulence plasmids pXOl and pX02.

The disclosure provides a Bacillus anthracis having the identifying characteristics of B. anthracis BH480, deposited under ATCC Accession No. . A deposit of Bacillus anthracis BH480 has been made on July 31, 2012 at the American Type Culture Collection (ATCC), 10801 University Boulevard, Manassas, VA 20110-2209,

USA, under ATCC accession number . The ATCC deposit was made pursuant to the terms of the Budapest Treaty on the International Recognition of the Deposit of Microorganisms for Purposes of Patent Procedure. Identifying characteristics of BH480 include (a) inactivated NprB, InhA2, TasA, camelysin, InhAl, MmpZ, CysPl, and VpR proteases, (b) sporulation-deficiency, and (c) lack of virulence plasmids pXOl and pX02. One embodiment of the disclosure is Bacillus anthracis BH480.

B. anthracis lacking more than one secreted protease can be produced using the methods disclosed herein. The disclosure provides a method to produce a B. anthracis of the embodiments comprising: effecting a genetic modification to inactivate a protease of the M4 family of metalloproteases; and effecting a genetic modification to inactivate a protease of the M6 family of metalloproteases. The disclosure also provides a method to produce a B. anthracis of the embodiments comprising: effecting a genetic modification to inactivate a protease of the M4 family of metalloproteases; effecting a genetic modification to inactivate a protease of the M6 family of metalloproteases; and effecting a genetic modification to inactivate a protease of the M73 family of metalloproteases. The disclosure also provides a method to produce a B. anthracis of the embodiments comprising: effecting a genetic modification to inactivate a protease of the M4 family of metalloproteases; effecting a genetic modification to inactivate a protease of the M6 family of metalloproteases; effecting a genetic modification to inactivate a protease of the M73 family of metalloproteases; and effecting a genetic modification to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases. The disclosure also provides a method to produce a B. anthracis of the embodiments comprising: effecting a genetic modification to inactivate a protease of the M4 family of metalloproteases; effecting a genetic modification to inactivate a protease of the M6 family of metalloproteases; effecting a genetic modification to inactivate a protease of the M73 family of metalloproteases; effecting a genetic modification to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases; effecting a genetic modification to inactivate a protease of the transglutaminase-like superfamily; and effecting a genetic modification to inactivate a protease of peptidase family S8. One embodiment is a method to produce a B. anthracis of the embodiments comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases and to inactivate a protease of the M6 family of metalloproteases. One embodiment is a method to produce a B. anthracis of the embodiments comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, and to inactivate a protease of the M73 family of metalloproteases. One embodiment is a method to produce a B. anthracis of the embodiments comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, to inactivate a protease of the M73 family of metalloproteases, and to inactivate a protease of the ZnMc superfamily of zinc- dependent metalloproteases. One embodiment is a method to produce a B. anthracis of the embodiments comprising genetically modifying a B. anthracis to inactivate a protease of the M4 family of metalloproteases, to inactivate a protease of the M6 family of metalloproteases, to inactivate a protease of the M73 family of metalloproteases, to inactivate a protease of the ZnMc superfamily of zinc-dependent metalloproteases, to inactivate a protease of the transglutaminase-like superfamily, and to inactivate a protease of peptidase family S8. Genetic modifications taught herein as well as those known to those skilled in the art can be used to inactivate proteases specified herein. Sporulation- deficient B. anthracis can be produced using techniques as taught herein or other techniques known to those skilled in the art. Those skilled in the art can also produce B. anthracis free of one or both virulence plasmids.

The disclosure provides a method to produce B. anthracis of the embodiments comprising: culturing the B. anthracis in a medium; and recovering the B. anthracis. Any suitable medium and culture conditions known to those skilled in the art can be used to produce such B. anthracis. Non-limiting examples of media include LB and FA, the compositions of which are specified in the Examples. Methods to recover the bacteria are also known to those skilled in the art.

The disclosure also provides a method to produce an endogenous B. anthracis protein, such as an endogenous secreted protein. Such a method comprises culturing a B. anthracis of the embodiments in a medium; and recovering the protein. In one embodiment, the protein is an endogenous secreted protein of B. anthracis. Culturing methods, recovery methods, and media to use are known to those skilled in the art.

The disclosure provides a modified Bacillus anthracis (B. anthracis) transformed with a recombinant molecule encoding a product, wherein the B. anthracis comprises any of the genetically modified B. anthracis disclosed herein (i.e., any B. anthracis of the embodiments), and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. One embodiment is a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases and a genetic modification that inactivates a protease of the M6 family of metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. One embodiment is a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, and a genetic modification that inactivates a protease of the M73 family of metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. One embodiment is a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, and a genetic modification that inactivates a protease of the ZnMc superfamily of zinc- dependent metalloproteases; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector. One embodiment is a modified B. anthracis transformed with a recombinant molecule encoding a product, wherein: the B. anthracis comprises a genetic modification that inactivates a protease of the M4 family of metalloproteases, a genetic modification that inactivates a protease of the M6 family of metalloproteases, a genetic modification that inactivates a protease of the M73 family of metalloproteases, a genetic modification that inactivates a protease of the ZnMc superfamily of zinc-dependent metalloproteases, a genetic modification that inactivates a protease of the transglutaminase-like superfamily, and a genetic modification that inactivates a protease of peptidase family S8; and the recombinant molecule comprises a nucleic acid molecule encoding the product operatively linked to an expression vector.

As used herein, a recombinant molecule comprises an expression vector operatively linked to a nucleic acid molecule encoding a desired product. A modified B. anthracis can comprise one or more recombinant molecules. A recombinant molecule can comprise one or more nucleic acid molecules encoding one or more desired products. As used herein, the phrase operatively linked means that the nucleic acid molecule encoding a desired product is joined to the vector in such a way that the product is produced. An expression vector is any vector that can transform B. anthracis (i.e., deliver a nucleic acid molecule encoding a product into B. anthracis) and that comprises expression control sequences that can effect expression in B. anthracis of the nucleic acid molecule encoding a desired product. Non-limiting examples of expression vectors are plasmid expression vectors, viral expression vectors, and other vectors known to those skilled in the art. Such a vector can be DNA, RNA, or a derivative of DNA or RNA. Examples of expression control sequences include, but are not limited to, a promoter, an enhancer, a repressor, a ribosome binding site, an RNA splice site, a polyadenylation site, a transcriptional terminator sequence, and a microRNA binding site. Examples of promoters include, but are not limited to, a promoter that controls expression of the pag gene that encodes B. anthracis protective antigen (PA), referred to herein as a pag promoter or PA promoter, and other promoters that function in B. anthracis. In one embodiment, the promoter is a pag promoter. A recombinant molecule can also comprise replication control sequences that can effect vector replication, such as an origin of replication. Selection of replication and expression control sequences to include can be accomplished by one skilled in the art. One embodiment is a recombinant molecule that is heterologous to B. anthracis; i.e., at least a portion of the recombinant molecule is not a natural B. anthracis plasmid. One embodiment is a recombinant molecule comprising a B. anthracis plasmid that is attenuated, for example, a virulence plasmid from which at least one component that would lead to virulence has been removed.

One embodiment is recombinant molecule pSJ136EFOS. One embodiment is a recombinant molecule comprising nucleic acid sequence SEQ ID NO:62.

One embodiment is a recombinant molecule selected from the group consisting of a recombinant molecule comprising nucleic acid sequence SEQ ID NO: 65, a recombinant molecule comprising nucleic acid sequence SEQ ID NO:67, and a recombinant molecule comprising nucleic acid sequence SEQ ID NO: 69. One embodiment is a recombinant molecule selected from the group consisting of pSW4-HBL LI His, pSW4-HBL L2 His, and pSW4-HBL B His. One embodiment is recombinant molecule pSW4-HBL LI His. One embodiment is recombinant molecule pSW4-HBL L2 His. One embodiment is recombinant molecule pSW4-HBL B His.

One embodiment is a recombinant molecule comprising a pSJl 15 plasmid that encodes a protein comprising amino acid sequence SEQ ID NO:91. One embodiment is a recombinant molecule comprising a pYS5 plasmid that encodes a protein comprising amino acid sequence SEQ ID NO:92. One embodiment is a recombinant molecule selected from the group consisting of recombinant molecule pSJ136EF-His, recombinant molecule pSJ136EF-Cys, and recombinant molecule pSJ136EF-NEHY. One embodiment is recombinant molecule pSJ136EF-His. One embodiment is recombinant molecule pSJ136EF-Cys. One embodiment is recombinant molecule pSJ136EF-NEHY.

The disclosure also provides a protein comprising an amino acid sequence selected from the group consisting of amino acid sequence SEQ ID NO: 93, amino acid sequence SEQ ID NO:94, and amino acid sequence SEQ ID NO:95. One embodiment is a protein comprising amino acid sequence SEQ ID NO: 93. One embodiment is a protein comprising amino acid sequence SEQ ID NO: 94. One embodiment is a protein comprising amino acid sequence SEQ ID NO:95.

Methods to transform B. anthracis of the embodiments and to select and produce expression vectors and recombinant molecules with which to transform B. anthracis are described in the Examples, and can also be found, for example, in Sambrook J et al., 2001, Molecular Cloning: a Laboratory Manual, 3 ^rd edition, Cold Spring Harbor Laboratory Press, and Ausubel F et al, 1994, Current Protocols in Molecular Biology, John Wiley & Sons.

A nucleic acid molecule encoding a desired product of the embodiments can encode a product that B. anthracis produces naturally (e.g., an anthrax toxin protein) or a product heterologous to B. anthracis (e.g., a toxin from a different organism). A heterologous product can be a combination of an endogenous and non-endogenous product (e.g., a tumor-targeting anthrax toxin). Examples of products include, but are not limited to, a protein, an amino acid, a nucleic acid molecule, a compound produced via a recombinant biosynthetic pathway, a small molecule, a drug, a vitamin, a drug conjugate, and a peptide nucleic acid conjugate.

One embodiment is a product comprising a toxin. Examples of toxins include, but are not limited to, an anthrax toxin, a cholera toxin, a diphtheria toxin, a hemolysin, and a ricin. Additional examples include, but are not limited to a Pseudomonas toxin, a Haemophilus ducreyi toxin, an Escherichia coli toxin, and a ribosome-inactivating protein (RIP) toxin. One embodiment is a product selected from the group consisting of an anthrax edema factor (EF), an anthrax lethal factor (LF), an anthrax protective antigen (PA), and combinations thereof. One embodiment is a product comprising an anthrax edema factor. One embodiment is a product comprising intact anthrax edema factor. One embodiment is a product comprising an anthrax lethal factor. One embodiment is a product comprising intact anthrax lethal factor. One embodiment is a product comprising an anthrax protective antigen. One embodiment is a product comprising intact anthrax protective antigen. One embodiment is a product comprising an anthrolysin. One embodiment is a product comprising intact anthrolysin. One embodiment is a product comprising a hemolysin. One embodiment is a product comprising a Bacillus cereus hemolysin HBL. One embodiment is a product selected from the group consisting of a B. cereus hemolysin HBL LI, a B. cereus hemolysin HBL L2, a B. cereus hemolysin HBL B, and combinations thereof. One embodiment is a product comprising a diphtheria toxin. One embodiment is a product comprising a cholera toxin. One embodiment is a product comprising a ricin. One embodiment is a product comprising a Pseudomonas toxin. One embodiment is a product comprising a Haemophilus ducreyi toxin. One embodiment is a product comprising an Escherichia coli toxin. One embodiment is a product comprising a ribosome-inactivating protein (RIP) toxin. One embodiment is a product comprising a toxin fusion protein. One embodiment is a product comprising a toxin conjugated to a tumor target. Examples of tumor targets are known to those skilled in the art. Examples of conjugates include, but are not limited to, an anthrax toxin conjugated to a tumor target, a cholera toxin conjugated to a tumor target, a diphtheria toxin conjugated to a tumor target, a hemolysin conjugated to a tumor target, and a ricin conjugated to a tumor target. One embodiment is an anthrax toxin conjugated to a tumor target. One embodiment is a product comprising a fusion protein comprising a PA binding moiety located in the amino terminal region of EF or LF (e.g., the amino terminal about 250 to about 260 amino acids of the mature protein) joined to a protein of interest that can be delivered to a cell via the protective antigen (PA) cellular translocation mechanism known to those skilled in the art.

A nucleic acid molecule of the embodiments can encode a natural product or any variant thereof that retains the activity of the natural product. Nucleic acid molecules of the embodiments can be produced using a number of methods known to those skilled in the art; see, for example, Sambrook J et al, ibid, and Ausubel F et al, ibid. For example, nucleic acid molecules can be modified using a variety of techniques including, but not limited to, classic mutagenesis techniques and recombinant DNA techniques, such as site- directed mutagenesis, chemical treatment of a nucleic acid molecule to induce mutations, restriction enzyme cleavage of a nucleic acid fragment, ligation of nucleic acid fragments, polymerase chain reaction (PCR) amplification and/or mutagenesis of selected regions of a nucleic acid sequence, synthesis of oligonucleotide mixtures and ligation of mixture groups to "build" a mixture of nucleic acid molecules and combinations thereof. Nucleic acid molecules of the embodiments can be selected from a mixture of modified nucleic acids by screening for the function of the protein encoded by the nucleic acid (e.g., EF expressed from a nucleic acid molecule that encodes an EF protein can be tested for its activity in a potency assay, such as that described in the Examples).

Products of the embodiments can be produced by culturing modified B. anthracis transformed with a recombinant molecule encoding a product of the embodiments. The disclosure provides a method to produce a product comprising: culturing a modified B. anthracis transformed with a recombinant molecule encoding a product of the embodiments; and recovering the product. Methods to effect such production, including culturing such modified B. anthracis and recovering such products are described in the Examples and are known to those skilled in the art, see for example Sambrook J et al., ibid, and Ausubel, F et al, ibid. The disclosure provides antibodies against products produced by B. anthracis of the embodiments. Examples of antibodies include polyclonal antibodies, monoclonal antibodies, and functional equivalents such as antibody fragments and genetically- engineered antibodies (including, but not limited to, single chain antibodies and chimeric antibodies that can bind to more than one epitope) that retain antibody binding activity. One embodiment is an antibody against an anthrax edema factor produced by a modified B. anthracis of the embodiments.

The disclosure provides methods to use products of the embodiments. The disclosure provides that antibodies produced against products produced by modified B. anthracis of the embodiments can be used to detect such products or, as appropriate, organisms or toxins corresponding to such products. One embodiment is a method to determine if a sample comprises anthrax toxin, the method comprising: contacting a sample with an antibody against an anthrax edema factor produced by a modified B. anthracis of the embodiments; and determining whether the antibody forms a complex, wherein detection of a complex indicates that the sample comprises anthrax. One embodiment is a method to determine if a sample comprises anthrax toxin, the method comprising: contacting a sample with an antibody against intact anthrax edema factor produced by a B. anthracis of the embodiments; and determining whether the antibody forms a complex, wherein detection of a complex indicates that the sample comprises anthrax. Methods to accomplish such detection are known to those skilled in the art.

The disclosure provides that products produced by modified B. anthracis of the embodiments can be used for their intended purpose. For example, products of agents that cause disease can be used to protect (e.g., prevent or treat) an animal from such disease. One embodiment is a method to protect an animal from anthrax, the method comprising administering an anthrax edema factor produced by a modified B. anthracis of the embodiments to the animal. One embodiment is a method to protect an animal from anthrax, the method comprising administering intact anthrax edema factor produced by a modified B. anthracis of the embodiments to the animal. Such an anthrax edema factor can be administered alone or in combination with one or more other agents, such as with an anthrax lethal factor and/or protective antigen. One embodiment is a method to protect an animal from anthrax, the method comprising administering an antibody against intact anthrax edema factor produced by a modified B. anthracis of the embodiments to the animal. Such an anti-anthrax edema factor can be administered alone or in combination with one or more other agents, such as with an antibody against an anthrax lethal factor and/or protective antigen. Methods to accomplish such administration are known to those skilled in the art.

The following is a listing of the SEQ ID NOs disclosed in the application. It is to be appreciated that since nucleic acid sequencing technology is not entirely error-free, the nucleic acid sequences and amino acid sequences presented herein represent, respectively, apparent nucleic acid sequences of nucleic acid molecules of the embodiments and apparent amino acid sequences of proteins of the embodiments.

SEQ ID Species Description

NO:

34 Synthetic Primer 0672LL: GCTCGAGCGGATGTACATCTGTAATGAGT

35 Synthetic Primer 0672LR: GGATATCTTGAACGATGTGACCAAATG

36 Synthetic Primer 0672RL: GCCCGGGCCTGTCGAAGCTTGGTCATT

37 Synthetic Primer 0672RR: GCCGCGGTTTGCATACCTGTGTTACCG

38 Synthetic Primer 0672seqF: GGTCAAGAAGCTGGTGGAGGTA

39 Synthetic Primer 0672seqR: TCTGTTCCTGCAATTTTCCC

40 Synthetic Primer 1288LL: GCTCGAGTAATTTGGAAGGTGATTAGC

41 Synthetic Primer 1288LR: GCCCGGGTTCACATCTTCTACATTGTAAT

42 Synthetic Primer 1288RL: GACTAGTAACAATCGTAAAAGAAACAGCG

43 Synthetic Primer 1288RR: GGAGCTCTATCGATCGCCTGTAAATTC

44 Synthetic Primer 1288seqF: GGGGATATGGACATGACTTT

45 Synthetic Primer 1288seqR: CAGTAAGTGTGTCACCCTTC

46 Synthetic Primer 1290L: GAGAAGATAGCTGCTGAGAG

47 Synthetic Primer 1290R: TAGAGGGAGTTTAATGGGGA

48 Synthetic Primer 1290seqF: GAAATTGCGCAAAAAGAT

49 Synthetic Primer 1290seqR: AGAGCCATTCCAGAACGC

50 Synthetic Primer 3159LL: GCTCGAGGGGTAATAACTTTCAATTAATAC

51 Synthetic Primer 3159LR: GGATATCGAAAAAACAAACACAGTACC

52 Synthetic Primer 3159RL: GCCCGGGGGTTGGCAAGCTGCCGATTC

53 Synthetic Primer 3159RR: GCCGCGGCGAATGGTTCAATTGCTCCG

54 Synthetic Primer 3159seqF: GGTACTGTGTTTGTTTTTTC

55 Synthetic Primer 3159seqR: GAATCGGCAGCTTGCCAACC

56 Synthetic Primer 3159CF: CCTCGAGTTTCATTTTTGAAGTCTTCTTC

57 Synthetic Primer 3159CR: CACTAGTCAGCGAAACGATGATTGATTTT

58 Synthetic Primer deltaF: TCCGATTAGGAAGTTGACAA

59 Synthetic Primer deltaR: CAGTTACCACCAATTGTTTT

60 Synthetic Primer deltaCF: CCTCGAGTTTTCTTATTGCATTTCTAATGTG

61 Synthetic Primer deltaCR: CCCGCGGTTAGCGATATAAGCGAACAG

62 Synthetic pSJ136EFOS

63 Synthetic Translation of EF from SEQ ID NO:62

64 Synthetic Translation of Mature EF from SEQ ID NO: 62

65 Synthetic PSW4-HBL LI His

66 Synthetic Translation of HBL LI from SEQ ID NO:65

67 Synthetic PSW4-HBL L2 His

68 Synthetic Translation of HBL L2 from SEQ ID NO:67

69 Synthetic PSW4-HBL B His

70 Synthetic Translation of HBL B His from SEQ ID NO:69

71 B. anthracis Wild-type CysPl protein (full-length encoded protein); note that the mature

CysPl protease begins at amino acid 31 of SEQ ID NO:71

72 Synthetic Inactivated CysPl protease (full-length encoded protein)

73 Synthetic Portion of cysPl nucleic acid comprising FRT insert

74 Synthetic Translation of SEQ ID NO: 73

75 B. anthracis Wild-type VpR protein (full-length encoded protein); note that the mature VpR protease begins at amino acid 26 of SEQ ID NO: 75

76 Synthetic Inactivated VpR protease (full-length encoded protein)

77 Synthetic Portion of vpR nucleic acid comprising FRT insert

78 Synthetic Translation of SEQ ID NO: 77

79 Synthetic Primer 1995LL: ACTGCTCGAGTGGGCTGACACATTTAAAAG SEQ ID Species Description

NO:

80 Synthetic Primer 1995LR: ACTGACTAGTAGTTGAACAAAGTGCGGCAG

81 Synthetic Primer 1995RL: ACTGCTCGAGAATGAAATAAACTGGCCAAAAGGTG

82 Synthetic Primer 1995RR: ACTGACTAGTCGGGAAAAACTTCAAATCCA

83 Synthetic Primer 1995 seqF: TTGCCAGAGCTTTTCATTGA

84 Synthetic Primer 1995 seqR: CGCTAATGAATAATCTGCCA

85 Synthetic Primer 4584LL: ACTGCTCGAGAAGCTGTCGGTACTGCTAAA

86 Synthetic Primer 4584LR: ACTGACTAGTCGAGTGCCATACTTAAAAGTATAGA

87 Synthetic Primer 4584RL: ACTGCTCGAGATCCTTGGGAGAAAAATTACGGCATT

88 Synthetic Primer 4584RR: ACTGACTAGTCGCCAAACATTCATTCATTTCTTCT

89 Synthetic Primer 4584 seqF: TGAGTGAAACGGCGTAACTT

90 Synthetic Primer 4584 seqR: TATTCCTTCAAAGCCGATAT

91 Synthetic LFn-BlaY protein

92 Synthetic PA SNKE dFF E308D protein

93 Synthetic EF-His protein

94 Synthetic EF-Cys protein

95 Synthetic EF-NEHY protein

EXAMPLES

The following examples are put forth so as to provide those of ordinary skill in the art with a complete disclosure and description of how to make and use the embodiments, and are not intended to limit the scope of what the inventors regard as their invention nor are they intended to represent that the experiments below are all or the only experiments performed. Efforts have been made to ensure accuracy with respect to numbers used (e.g. amounts, temperature, etc.) but some experimental errors and deviations should be accounted for. Unless indicated otherwise, parts are parts by weight, molecular weight is weight average molecular weight, and temperature is in degrees Celsius. Standard abbreviations are used.

Example 1. Materials and Methods

Materials

Proteases and genes encoding such proteases that were used and/or analyzed herein are listed in Table 1.

Table 1. B. anthracis Ames ancestor strain genes and proteins inactivated and/or analyzed herein.

Protein Gene Function/Name Locus Tag

Sinl sinl regulatory protein GBAA 1293

InhAl inhAl Metallopeptidase GBAA 1295

MmpZ mmpZ Metallopeptidase GBAA 3159

CysPl cysPl putative cysteine protease GBAA 1995

VpR vpR minor extracellular protease GBAA 4584

SprA sprA serine protease GBAA 5414

NprC nprC neutral metalloprotease GBAA 2183

HtrA htrA serine protease GBAA 3660

Prokaryotic homolog of

HsIV hsIV GBAA 3968

proteasome HsIV

thiol-activated cytolysin

ALO alo GBAA 3355

(anthrolysin)

SpoOA spoOA sporulation regulator GBAA 4394

EF cya edema factor GBAA_pXOl_0142

PA pag protective antigen GBAA_pXOl_0164

LF lef lethal factor GBAA_pXOl_0172

Oligonucleotide primers produced and/or used herein are listed in Table 2.

Table 2. Primers produced and/or used herein.

Primer Sequence " (5'-3') (location) Relevant property Site

1290R TAGAGGGAGTTTAATGGGGA (SEQ ID

NO:47)

1290seqF GAAATTGCGCAAAAAGAT (SEQ ID NO:48) Primer pair to verify calY gene

disruption

1290seqR AGAGCCATTCCAGAACGC (SEQ ID NO:49)

3159LL GCTCGAGGGGTAATAACTTTCAATTAATAC Primer pair to amplify left Xhol

(SEQ ID NO:50) fragment of mmpZ gene to clone

it into pSC

3159LR GGATATCGAAAAAACAAACACAGTACC EcoRV

(SEQ ID NO:51)

3159RL GCCCGGGGGTTGGCAAGCTGCCGATTC Primer pair to amplify right Smal

(SEQ ID NO:52) fragment of mmpZ gene to clone

it into pSC

3159RR GCCGCGGCGAATGGTTCAATTGCTCCG SacII

(SEQ ID NO:53)

3159seqF GGTACTGTGTTTGTTTTTTC (SEQ ID NO: 54) Primer pair to verify mmpZ gene

disruption

3159seqR GAATCGGCAGCTTGCCAACC (SEQ ID

NO:55)

CCTCGAGTTTCATTTTTGAAGTCTTCTTC

3159CF Xhol

(SEQ ID NO:56)

Primer pair to complement mmpZ gene disruption

CACTAGTCAGCGAAACGATGATTGATTTT

3159CR Spel

(SEQ ID NO:57)

ACTGCTCGAGTGGGCTGACACATTTAAAAG

1995LL Xhol

(SEQ ID NO:79)

Primer pair to amplify left

ACTGACTAGTAGTTGAACAAAGTGCGGCAG fragment of cysPl gene to

1995LR clone it into pSCF Spel

(SEQ ID NO:80)

ACTGCTCGAGAATGAAATAAACTGGCCAAA

1995RL Xhol

AGGTG (SEQ ID NO:81)

Primer pair to amplify right

ACTGACTAGTCGGGAAAAACTTCAAATCCA fragment of cysPl gene to

1995RR

(SEQ ID NO:82) clone it into pSCF Spel

1995

TTGCCAGAGCTTTTCATTGA (SEQ ID NO:83)

seqF

Primer pair to verify cysPl gene disruption

1995

CGCTAATGAATAATCTGCCA (SEQ ID NO: 84)

seqR

ACTGCTCGAGAAGCTGTCGGTACTGCTAAA

4584LL Xhol

(SEQ ID NO:85) Primer pair to amplify left fragment of vpR gene to clone

ACTGACTAGTCGAGTGCCATACTTAAAAGT it into pSCF

4584LR Spel

ATAGA (SEQ ID NO: 86)

ACTGCTCGAGATCCTTGGGAGAAAAATTAC

4584RL Xhol

GGCATT (SEQ ID NO: 87) Primer pair to amplify right fragment of vpR gene to clone

ACTGACTAGTCGCCAAACATTCATTCATTTC it into pSCF

4584RR Spel

TTCT (SEQ ID NO:88)

4584 TGAGTGAAACGGCGTAACTT (SEQ ID Primer pair to verify vpR gene seqF NO:89) disruption Primer Sequence " (5'-3') (location) Relevant property Site

4584

TATTCCTTCAAAGCCGATAT (SEQ ID NO: 90)

seqR

TCCGATTAGGAAGTTGACAA (SEQ ID

deltaF

NO:58) Primer pair to verify deletion of

tasA-inhAl region

deltaR CAGTTACCACCAATTGTTTT (SEQ ID NO:59)

CCTCGAGTTTTCTTATTGCATTTCTAATGTG

deltaCF Xhol

TTCG (SEQ ID NO:60)

Primer pair to complement tasA- inhAl region deletion

CCCGCGGTTAGCGATATAAGCGAACAG

deltaCR SacII

(SEQ ID NO:61)

Restriction site is underlined

Plasmids produced, used, and/or analyzed herein are listed in Table 3.

Table 3. Plasmids produced and/or used herein.

Plasmid Relevant characteristic(s) pCysPlLI pSCF containing cysPl fragment amplified with primer pair

1995LL/1995LR

pCysPlRI pSCF containing cysPl fragment amplified with primer pair

1995RL/I995RR

pVpRLI pSCF containing vpR fragment amplified with primer pair 4584LL/4584LR pVpRRI pSCF containing vpR fragment amplified with primer pair 4584RL/4584RR pCrePA Contains ere gene and strongly temperature-sensitive replicon for both E.

coli and gram-positive bacteria; Em ^R in both E. coli and B. anthracis pCrePAS Contains ere gene and strongly temperature-sensitive replicon for both E.

coli and gram-positive bacteria; Sp ^R in both E. coli and B. anthracis pFPAS Contains flp gene and strongly temperature-sensitive replicon for both E.

coli and gram-positive bacteria; Sp ^R in both E. coli and B. anthracis pMmpZC pSC with 3159F/3159R PGR fragment containing entire mmpZ gene pATasA- pSC with deltaCF/dcitaC fragment containing entire tasA-inhAl DNA InhAlC region

pSDL304 pDC with ΙοχΡ-Ω-sp-loxP flanked 3' and 5' by spoOA sequences pSJ136EFOS Contains B. anthracis cya gene instead of the lef gene in pSJl 15 (Park S et al., 2000, Protein Expr. Purif. 18, 293-302.

pSW4-HBL LI Encodes B. cereus HBL LI His

His

pSW4-HBL L2 Encodes B. cereus HBL L2 His

His

pSW4-HBL B Encodes B. cereus HBL B His

His

Bacterial strains produced, used, and/or analyzed herein are listed in Table 4. ble 4. Bacterial strains produced and/or used herein.

Strain Relevant characteristic(s)

A33 B. anthracis Ames 33 strain (ρΧΟΓ pX02 ^" )

A35 B. anthracis Ames 35 strain (pX01 ⁺ pX02 ^" )

A35ASpoOA SpoOA knockout containing one loxP site, previous name of this strain is

SDL35

A35ANprB nprB knockout containing one loxP site

A35AInhA2 inhA2 knockout containing one loxP site

A35ATasA tasA knockout containing one loxP site

A35ACam calY knockout containing one loxP site

A35AInhAl inhAl knockout containing one loxP site

Α35ΔΜηιρΖ mmpZ knockout containing one loxP site

A35AmmpZC A35AMmpZ complemented in situ by insertion of native mmpZ gene

A35DM Ames 35 double mutant with nprB and inhAl knockouts; each gene Strain Relevant characteristic(s)

contains one loxP site

A35TM Ames 35 tetra-protease mutant; has nprB knockout containing one loxP site and deleted DNA region including tasA, calY, sinl, sinR, and inhAl genes

A35TMC A35TM with tasA-inhAl deletion restored in situ by insertion of native

DNA region including tasA, calY, sinl, sinR, and inhAl genes

A35PM Ames 35 penta-protease mutant; 35TM with inhA2 knockout containing one loxP site; total of 3 loxP sites in chromosome

A35HM Ames 35 hexa-protease mutant: A35PM with mmpZ knockout containing one loxP site; total of 4 loxP sites in chromosome

A35HMS A35HM with spoOA knockout containing one loxP site; total of 5 loxP sites in chromosome

BH460 A35HMS cured ofpXOl

BH480 BH460 with both cysPl and vpR knockout containing FRT site in each knockout gene; total of 5 loxP and two FRT sites in chromosome

BH450 A33; nprB and spoOA knockouts, residual loxP sites in both nprB and

spoOA genes; strain was previously named MSLL33

Bacterial growth conditions and phenotypie characterization

E. coli strains were grown in Luria-Bertani (LB) broth and used as hosts for cloning, LB agar was used for selection of transformants (Sarabrook i et aL, 2001 , Molecular Cloning: A Laboratoiy Manual, Third Edition, Cold Spring Harbor Laboratoiy Press, Cold Spring Harbor, New York). B. anthracis strains were also grown in LB or FA medium (Singh Y et aL 1989, J. Biol. Chem. 264, 19103- 19107). FA medium contains, per liter, 33 g of tryptone, 20 g of yeast extract, 7.4 g of NaCl, 8 g of Na ₂ HP0 ₄ , and 4 g of KH ₂ PO ₄ , pH 7.5. Antibiotics (Sigraa-Aldrich, St. Louis, MO) were added to the medium when appropriate to give the following final concentrations: ampicillin (Ap), 100 ,ug/'ml (only for E. coli)', erythromycin (Em), 400 p.g/ml for E. coli and 10 fig/ml for B. anthracis: spectinomycin (Sp), 150 .ag/mi for both E. coli and 5. anthracis; kanamycin (Km), 20 ag/ml (only for B. anthracis). SOC medium (Quality Biologicals, Inc., Gaithersburg, MD) was used for outgrowth of transformation mixtures prior to plating on selective medium. B, anthracis spores were prepared from strains capable of sporuiating as previously described (Thome C, 1993, in Bacillus subtil is and other gram-positive bacteria: biochemistry, physiology, and molecular genetics, 113-124, Sonenshein AB et aL, (Eds.), American Society for Microbiology, Washington, D.C.) after growth on NBY mi imal agar (nutrie t broth, 8 g liter; yeast extract, 3 g/liter: MnSC^'ELO, 25 mg/liter; agar, 15 g/liter) at 30 °C for 5 days. Spores and vegetative cells were visualized with a Nikon Eclipse E600W light microscope (Nikon Instrument Inc., New York). DNA isolation and manipulation

Preparation of plasmid DNA from E. coli, transformation of E. coli, and recombinant DN A techniq ues were carried out by standard procedures (Sambrook J et ah, ibid,). E. coli SCSI 10 competent ceils were purchased from Stratagene (La Jolia, CA), and E. coli TOP 10 competent cells were purchased from Iiivitrogen (Carlsbad, CA). Recombinant plasmid construction was carried out in E. coli TOP 10. Plasmid DN A from B. anthraeis was isolated according to the protocol for the piuification of plasmid DNA from B. snhtilis (Qiagen, Valencia, CA). Chromosomal DNA from B. anthraeis was isolated with the Wizard genomic purification kit (Promega, Madison, Wi). B. anthraeis was eiectroporated with unmethylated plasmid DNA. isolated from E. coli SCSI .10 (dam ^" dcm ^" ). Electroporation-competent B. anthraeis cells were prepared and transformed as previously described (Pomerantsev AP et al, 2009, J, Bacteriol. 191, 5134-5136). Restriction enzymes, T4 [igase, and Antarctic phosphatase were purchased from New England Biolabs (Ipswich, MA). Ta polymerase. Platinum PCR SuperMix High Fidelity kit and the TOPO TA cloning kit. were from Invitrogen. The pGEM~T Easy Vector system was from Promega. Ready-To-Go PCR Beads were from GE Healthcare Biosciences Corp. (Piscataway, NJ). For routine PCR analysis, a single colony was suspended in 200 μί of TE buffer (pH 8.0), heated to 95 °C for 45 s, and then cooled to room temperature (Sambrook J et al., ibid.). Cellular debris was removed by centrifugation at 15,000 x g for 10 min. Two microliters of the lysate contained sufficient template to support PCR. The GeneRuler DNA Ladder Mix from MBI Fermentas (Glen Burnie, MD) was used to assess DNA fragment, length. All constructs were verified by DNA sequencing and/or restriction enzyme digestion.

B. anthraeis Ames 35 (pXOK- ρ.Χ02-) (A35) was used for genetic manipulations. The GenBank database (GenBank Accession No. for the Ames strain is NC 003997) was analyzed for the ident fication of target genes and for she corresponding primer design. The Cre/Lox genetic modification method was adapted to introduce precise genetic knockouts into B. anthraeis genes encoding putative proteases. The general schemes for producing B. anthraeis mutants using CxQ-hxP system were described previously (Pomerantsev AP et al., 2006, ibid.; Pomerantsev AP et al., 2009, ibid.). The system employs vectors designated generic-ally as pDC, for double-crossover plasmid or pSC, for single-crossover plasmid. These plasmids are derived from the highly temperature- sensitive plasmid p!-IY304 iPriiz!aff et al., 200 ! . o! . Microbiol. 39, 236-247), which has permissive and restrictive temperatures of 30 °C and 37 °C, respectively. The pDC plasmid (Pomerantsev AP et al ,, 2006, ibid. ) was used to inactivate the spoQA (GBAA_4394), nprB (GBAA_0599) (Pomerantsev AP et al., 2006, ibid.), inhAi (GBAA 1295) ( astrup CJ et al., ibid.), inhA2 (GBAA 0672), and calY (GBAA 1290) genes. The pSC pias id (Pomerantsev AP et al,, 2009, ibid.) was used to inactivate the tasA (GBAA 1288) and mmpZ (GBAA 3159) genes. Both plasmids were used in the production of a genomic deletion of the region from tasA (GBAA 1288) to inhA i (GBAAJ295).

The nprB gene was inactivated as described in Pomerantsev et al., 2006, ibid. The inhA i gene was inactivated as described in Kastrup CJ et al., ibid. To inactivate the inhAi ge e, left and right fragments were amplified with primer pairs 0672LL/0672LR and 0672R.L/0672 RR, respectively, (Table 2) and inserted into pDC to produce the pInhA2I plasmid (I at the end of the proteases gene number means inactivation). To inactivate the tasA gene, left and right fragments were amplified wit primer pairs 1288LL/ 1288LR and 1288RL/1288R , respectively, and inserted into pSC to produce the pTasALI and pTasARI plasmids. To inactivate the camelysin gene calY, D .A fragment overlapping the protease gene was amplified with primer pair I290L/I290R and inserted into the EcoRJ-site of pHY304. The internal Tig/ll-frag ent of the calY gene was replaced with a ΙοχΡ-Ω-sp-loxP cassette flanked by two Bglll sites (Pomerantsev AP et al, 2006, ibid.) to create the pCaml plasmid for the gene inactivation. To inactivate the mmpZ gene, left and right fragments were amplified with primer pairs 31.59LL/3159LR and 3159RL/3159RR, respectively, and inserted into pSC to produce pMmpZLI and pMmpZRI plasmids. To delete the tasA-inkA l gene cluster, the double protease mutant A35 DM strain (having a LoxP site in the inhAi gene) was transformed with pTasALI, and the plasmid was integrated into the genome as described previously (Pomerantsev AP et al., 2009, ibid.). Subsequent transformation of the recombinant strain with the pCrePAS plasmid (Pomerantsev AP et al., 2009, ibid.) eliminated the complete tasA- inhAi gene cluster and produced the tetra-protease mutant strain A35TM.

The Cre recombinase-expressing plasmids pCrePAS (Pomera tsev AP et al., 2009, ibid.) and pCrePA (Pomerantse AP et al, 2006, ibid.) both have permissive and restrictive ternperamres of 30 °C and 37 °C, respectively, a d differ only in the selectable marker. The pCrePA was used for elimination of DMA regions containing a spectinomycin resistance cassette located between two similarly oriented loxP sites (Pomerantsev AP et aL 2006, ibid.), while pCrePAS was used in a similar way when the recipient strain did not contain a spectinomycin marker. In that case, the region to be deleted generally contained an erythromycin resistance gene along with backbone plasmid pSC (Pomerantsev AP et al., 2009, ibid.), in both cases, a single loxP site replaced the DNA region targeted for deletion.

To complement the mutation in the mmpZ gene (A35AMmpZ strain) and to restore the deleted taksA-inhAl region i the A35TM strain, the 3159CF/3159CR PGR fragment (amplified using primer pair 3 I59CF/3159CR) and the deltaCF/ ^' deltaCR PGR fragment (amplified using primer pair delta CF/delta CR) were inserted into the pSC plasmid. The resulting pMmpZC and pATasA-InbAI C piasmids containing, respectively the intact mmpZ gene and the whole tasA-inhAl region (C at the end of the proteases gene means complementation), were used for complementation. Each plasmid was inserted separately into the corresponding mutant by a single crossover event. During the crossover, both the mmpZ gene a d whole tasA-inhAl region were inserted into the genomes of the corresponding mutants. Subsequent elimination of the plasmid sequences by Cre recombinase left an intact functional copy of the originally mutated gene along with an inactive duplicate copy of a fragment of the gene. Prcparauo-; oP p:X)tc;;sc-dcndc: !i </;?. ;;·. _' /; ·;¾ ^·

B. anthracis strains A33 (Pomerantsev AP et al., 2003, infect immun 71, 6591- 6606), A35, A35ASpoOA, A35A prB, BH450 (the latter four ail described in Pomerantsev AP et al,, 2006, ibid,), and A35AinhAl (Kastrup Ci et al., ibid.) and their relevant characteristics are listed in Table 4. Also listed in Table 4 are protease-deficient B. anthracis strains produced as described herein. B. anthracis that was genetically modified to inactivate NprB, I hA2, TasA, camelysin, InhAl, or MmpZ, respectively, was constructed in the A35 strain by the replacement of the respective coding sequences with the loxP element as described herein.

The double NprB, InhA l muta t (A35D ), a B. anthracis comprising a. genetic modification to inactivate NprB and a genetic modification to inactivate InhA l, was created starting from the A35ANprB strain. The tetra -protease mutant, A35TM (a B. anthracis comprising a genetic modification to inactivate NprB, a genetic modification to inactivate TasA, a genetic modification to inactivate camelysin, and a genetic modification to inactivate InhAl ) was created by deletion of the tasA-inhAl gene region in the A35DM strain. The A35TM was then used for inactivation of the inhA2 and mmpZ genes with piasmids plniiA21 and pMmpZLI/pMmpZRI. The resulting penia- and hexa- protease mutants were designated A35 PM (a B. anthracis comprising a genetic modification to inactivate NprB, a genetic modification to inactivate TasA, a genetic modification to inactivate ca elysin, a genetic modification to inactivate InhAl, and a genetic modification to inactivate IiiliA2) and A35HM (a B. anthracis comprising a genetic modification to inactivate NprB, a genetic modification to inactivate TasA, a genetic modification to inactivate camelysin, a genetic modification to inactivate InhAl, a genetic modification to inactivate inhA2, and a genetic modification, to inactivate MmpZ), respectively.

The mutant strains were checked at each, step to ensure they had retained the ability to sporulate (Sastalla et al, 2010, Ap l. Environ. Microbiol. 76, 6318-632 1 ). To intentionally produce a sporulation-deficient hexa-protease mutant of A35HM, A35HMS was produced by inactivating the spoOA (GBAA_4394) gene in A35HM using the plasmid pSQL304 (Pomerantsev et al., 2006). The final protease-deficient spoOA- negative mutant lacking pXOl was obtained by repeated passage of the A35HMS utant at elevated temperatures to cure pXOl as described previously (Pomerantsev et al, 2003, ibid.). The final strain was designated. B. anthracis BH460 (Table 4). P( ^' R and sCi.UjCi ^' KJO q ^j' K- i ysis orchroinosomai modi c^iion-.

PCR fragments containing loxP sites within mutated (i.e., inactivated) genes were amplified and sequenced using primers listed in Table 2 (0672seqF/0672seqR, 1288seqF/1288seqR, 1290seqF/1290seqR, 3159seqF/3159seqR). All primers for PCR and sequencing were synthesized by Operon Biotechnologies, Inc. (Huntsville, AL) or the FDA core facility (Bethesda, MD). Sequences were determined from both, sides of the PCR fragments (Macrogen, Rockvil le, MD). For verification of the genomic deletion in the tasA-inhAl gene area, the region encompassing the start of tasA gene and the end of inhA! gene was amplified in the mutant strains by PCR using the primer pair deltaF/deitaR.. The location of the loxP site inside the PCR. fragments was determined by sequencing the fragments. csjcrn Not analysis

The A35 strain and genetically modified B. anthracis mutants were grown at 37' ^' C in LB in air to analyze B. anthracis toxin production by Western blot Overnight cultures were diluted into fresh LB to give A6oc ⁼ 0.002, and growth was measured at I , 2, 3, 4, 5, 6, 7, 8, 9, 10, 17 and 24 h. Supernatant samples (6 ml) from each time point w e e filtered (0.22 μιτϊ Miliex syringe-driven filter units, illipore, Cork, Ireland) and concentrated 10-fold using Ami con U!tra~4 membranes (Millipore). Samples of 5 μΐ were mixed with 5 μΐ of 2X Tris-glycine SDS sample loading buffer (126 niM Tris-HCI, pH 6.8, 4% SDS, 20% glycerol, 0.005% bromophenol blue) (Invitrogen), heated (96 °C, 10 min) and separated on 4-12% Bis-Tris NuPAGE gels) using NuPAGE MOPS SDS running buffer (Invitrogen). Precision Plus Protein Standard (All Blue, Bio-Rad. Hercules, CA) was used as a molecular weight marker. Proteins were transferred to MagnaCharge 0.45 μιη nylon membranes (Osmonics inc., Minnetonka, MN) using transfer buffer (25 mM Tris, 190 mM glycine, and 20% methanol), blocked in PBS + 3% skim milk (Difeo, Lawrence, KS) for 1 h at room temperature, followed by three 10 min washes in PBST (PBS + 0.05% Tween20). Membranes were then incubated with primary antibody diluted in PBS + 1% skim milk overnight at 4 °C. Mouse monoclonal antibody PA-05-A-G1 Lot#071100-02, 5.9 mg/ml (Naval Medical Research Center, Biological Defense Research Directorate), for detection of PA, and mouse monoclonal anti body LF-03-A-G1 Lot# 150900-01 , 7.4 mg ml (NMRC, BD D), for detection of LF, were both used at 1 :2000. Rabbit antisera for development of blots included anti-EF serum #5900 (used at 1 :8000; produced in inventors' laboratory), anti-camelysin serum (used at 1 :2000; Molecular Biology Institute of Barcelona, Spain), and anti-recombinant A LO serum (used at 1 :2000; R. Rest, Drexel University College of Medicine, Philadelphia, PA). Appropriate HRP-eonjugated secondary IgGs ( PL, Gaithersburg, MD) were used at 1 : 10000 fol lowed by development with TMB (3,3 5,5 ^; -tetramethylbenzidine) (KPL).

EF (B. anthracis edema factor) protein with an -terminal six-histidine tag was expressed from plasmid pProEx-H6-EF (provided by Wei-Jen Tang) in E. coli BL21(DE3) (Promega) as previously described (Soelaiman S et ah, 2003, J. Biol. Chem, 278, 25990-25997). pProEx-H6-EF comprises a nucleic acid molecule encoding EF with an N-terminal six-histidine tag operatively linked to pPROEX HTa (Addgene, Cambridge, MA).

EF was expressed in B. anthracis host strains from plasmid pSJ136EFOS (SEQ ID NO:62 and Figure 6, which contains the EF structural gene in plasmid pYS5 under the control of (i.e., operatively linked to) the PA (B. anthracis protective antigen) promoter and signal sequence (Singh Y et al, 1989, J. Biol. Chem. 264, 19103-19107). Host strains BH450 and BH460 containing pSJ136EFOS were grown m FA medium containing 15 μg/ml of kanamycin at 37 °C for 14 h, essentially following procedures previously used for production of LF (Park S et al, 2000, Protein Expr. Purif. 18, 293- 302). The cultures were cooled, supplemented with 2 μg/ml of AEBSF [4-(2- Aminoethyl)-benzenesulfonylfluoride ^' HCl] (US Biological, Swampscott, MA) and centrifuged at 4550 x g for 30 min. All subsequent steps were performed at 4 °C.

The supernatants were filter sterilized and supplemented with 5 mM EDTA. Solid ammonium sulfate was added to the supernatants to obtain 40% saturation. Phenyl- Sepharose Fast Flow (low substitution, GE Healthcare Biosciences Corp.) was added and supernatants gently mixed in the cold for 1.5 h. The resins were collected on porous plastic funnels (BelArt Plastics, Pequannock, NJ) and washed with buffer containing 1.5 M ammonium sulfate, 10 mM Tris-HCl, and 1 mM EDTA (pH 8.0). The EF proteins were eluted with 0.3 M ammonium sulfate, 10 mM Tris-HCl, and 1 mM EDTA (pH 8.0), precipitated by adding an additional 30 g ammonium sulfate per 100 ml eluate, and centrifuged at 18,370 x g for 20 min. The proteins were dissolved and dialyzed against 5 mM HEPES, 0.5 mM EDTA (pH 7.5). The dialyzed samples were applied to a Q- Sepharose Fast Flow column (GE Healthcare Biosciences Corp.) and eluted with a 0-0.5 M NaCl gradient in 20 mM Tris-HCl, 0.5 mM EDTA (pH 8.0). The EF-containing fractions identified by SDS-PhastGel analysis were purified on a column of ceramic hydroxyapatite (Bio-Rad Laboratories, Hercules, CA) with a gradient of 0.02-1.0 M potassium phosphate containing 0.1 M NaCl (pH 7.0). The fractions containing EF were dialyzed overnight against 5 mM HEPES and 0.5 mM EDTA, pH 7.5, concentrated as necessary, frozen, and stored at -80 °C. The molecular mass of purified EF was estimated by liquid chromatogram-electrospray mass spectrometry using an HP/ Agilent 1100 MSD instrument (Hewlett Packard, Palo Alto, CA) at the NIDDK core facility, Bethesda, MD).

Analysis of EF activity in vitro and in vivo

EF activity was measured by analysis of cyclic AMP (cAMP) production in the RAW264.7 macrophage cell line (ATCC TIB-71). Cells were grown in Dulbecco's Modified Eagle Medium (DMEM) with 10% fetal bovine serum, 2 mM Glutamax, 2 mM HEPES and 50 μg/ml gentamicin (all from Invitrogen) at 37 °C in 5% C0 ₂ . Cells were seeded in 96-well plates 24 h prior to assays. EF preparations were serially diluted in a constant PA (B. anthracis protective antigen) concentration (250 ng/ml) prior to addition to cells and incubation for 1 h at 37 °C. Total cAMP levels were assessed using the BioTRAK cAMP enzyme immunoassay kit (GE Healthcare Biosciences Corp.) according to the manufacturer's protocol. For analysis of EF potency, groups of five 8-week old female Balb/cJ mice (Jackson Laboratories, Bar Harbor, ME) were injected via tail vein with EF preparations combined with an equal dose of PA. Toxin was prepared in sterile PBS. Mice were monitored for survival for 168 h. All experiments involving animals were performed under protocols approved by the Animal Care and Use Committee of the National Institute of Allergy and Infectious Diseases, National Institutes of Health. Example 2. Genetic modification of B. anthracis protease genes

This Example demonstrates that a genetic modification of B. anthracis in which loxP is inserted into genes encoding B. anthracis proteases results in truncation of corresponding proteins.

A number of protease genes on the B. anthracis chromosome as well as the spoOA gene were targeted for inactivation in this study. They are identified here with the gene numbers assigned by The Institute for Genomic Research, now the J Craig Ventner Institute (Rockville, Maryland), for the "Ames ancestor" strain chromosome (GenBank Accession No. NC 007530, gene designation GBAA gene number) (Ravel J et al, 2009, J. Bacteriol. 191, 445-446). The gene numbers are coincident with the previous "Ames" strain chromosome (GenBank Accession No. NC 003997, gene designation BA gene number) (Read TD et al, 2003, Nature 423, 81-86). All genes and proteins inactivated or analyzed in this study are listed in Table 1 together with corresponding locus tags.

The inactivation of the nprB and inhAl genes has been previously described (Pomerantsev AP et al, 2006, ibid., Kastrup CJ, ibid.). The inhA2, tasA, calY, and mmpZ genes were inactivated as described herein. The B. anthracis InhA2 protein is 96% identical in sequence to the InhA2 protease of Bacillus thuringiensis, which is an essential virulence factor in that insect pathogen (Fedhila S et al., 2003, J. Bacteriol. 185, 2820- 2825). The tasA gene (GBAA_1288) located downstream of the putative signal protease gene sipW (GBAA _. 1287) is only 5 genes upstream of the InhAl gene (GBAA 1295). The intervening genes include the calY protease gene, and the two regulatory genes sinl and sinR (Table 4). The Sinl and SinR proteins play important roles in B. subtilis biofilm formation, acting through protease-dependent processes (Chai Y, 2010, Mol. Microbiol. 78, 218-229), while in B. anthracis these proteins regulate secreted proteases (Pflughoeft KJ et al, 2010, J. Bacteriol. 193, 631-639). It is interesting that the genes corresponding to calY and inhAl are not found in the sinl, sinR region of the B. subtilis genome. Only the gene for the TasA protease, tasA, is located downstream of sipW and upstream of sinR and sinl (Pflughoeft et al., ibid.). Both TasA and camelysin are similar to the B. subtilis TasA protease (36% and 34% sequence identities, respectively). The final gene selected for inactivation, mmpZ, is reported to form an operon with the downstream gene (GBAA 3160) (Passalacqua KD et al, 2009, J. Bacteriol. 191, 3203-3211). The latter gene encodes a hypothetical secreted protein that is overproduced in B. anthracis (Antelmann H et al., ibid.). The absence of the MmpZ protease in the B. anthracis secretome indicates that this protease could be a target for proteolytic degradation by other proteases during the stationary phase of growth Antelmann H et al., ibid.).

The protease genes were inactivated as described in Example 1, followed by sequencing to locate the loxP insertions and infer the corresponding amino acid changes in the mutated proteins. These are shown in Figure 1. Typically, the 34-bp loxP sequence will generate a frameshift and early downstream occurrence of a stop codon in an alternative reading frame either within the loxP sequence or soon thereafter. Thus, all four inactivated protease genes encoded greatly shortened proteins.

Example 3. Comparison of protein degradation by genetically modified B. anthracis and A35 strain

This Example compares protein degradation by several genetically modified B. anthracis, including some B. anthracis of the embodiments, and the Ames 35 strain.

The levels of the three B. anthracis toxin components, ALO, and camelysin produced by the B. anthracis mutants were compared to those of the parental A35 strain by Western blot (Figure 2). The ten strains were grown in LB at 37 °C over a 24-h period. All strains grew similarly except the six -protease mutant BH460, which appeared to have a slight lag before reaching exponential growth phase.

Expression of PA by Ames 35 was detectable starting at 5 h of growth. However, intact PA (83 kDa) disappeared by the 9th hour of growth due to proteolytic degradation. Inactivation of the InhA2 protease did not influence production of PA while inactivation of SpoOA or camelysin actually reduced the half-life of PA by 1-2 h. Inactivation of NprB, TasA, or MmpZ resulted in increased PA stability up to 10 h. However, PA produced by all these strains was completely degraded after 17 h. The most stable production of PA among the single knockout mutants was found in the InhAl strain. PA was present even at 24 h of growth with this strain, although some degradation occurred. Surprisingly, the A35DM double mutant did not demonstrate enhanced PA production. PA degradation generally began at 6-7 h of growth and continued through 24 h. Strikingly, the A35HMS strain with six inactivated proteases produced PA with minimal to no degradation during the full 24 h of growth. Similar analyses were performed to follow production of the other B. anthracis toxin components, EF and LF. Intact EF (89 kDa) was found to be more vulnerable to degradation than PA, while LF (90 kDa) was quite stable when produced from most mutant strains. The levels of intact EF and LF, and the timing of their production, paralleled what was seen for PA for each mutant strain.

The enhanced breakdown of PA and EF found in the A35DM strain may be explained by increased production of camelysin in A35DM compared with A35 or the two corresponding single protease knockouts. Although both the NprB and InhAl knockout strains had increased levels of camelysin, the double mutation produced what seems to be a greater than additive effect. The camelysin levels produced by the A35DM strain were similar to those by the SpoOA mutant strain, A35ASpoOA. Both strains produced camelysin (19 kDa) that remained stable over the 24-h period. These observations on post-translational regulation of camelysin production by several proteases support and expand recently published data demonstrating that the concentration of InhAl in culture supernatants is inversely proportional to the concentration of camelysin (Pflughoeft et al, ibid . Another interesting finding of these studies was that the global transcriptional regulator SpoOA inhibited camelysin production. Pflughoeft et al.. ibid., recently demonstrated that B. anthracis sinR (which was deleted in those mutants containing the tasA-ihhAl deletion) also negatively regulates transcription of camelysin and InhAl, both of which have been suggested to be associated with virulence (Chung MC et al, ibid., Liu YT et al, 2008, Protein Expr. Purif. 57, 72-80).

Knocking out TasA also increased production of camelysin but to a lesser extent than elimination of NprB and InhAl . The InhA2 knockout did not result in any change in camelysin degradation or production when compared to A35, while MmpZ elimination was actually detrimental to camelysin production. These studies indicate that inactivation of six proteases in B. anthracis leads to increased production of intact toxin proteins in culture supernatants relative to A35, single, or double protease mutants.

Very low levels of ALO were produced from the A35 strain compared to all the protease knockouts, with the exception of the InhA2 mutant. The ALO produced by the A35 strain completely disappeared after 9 hours of growth. Every protease knockout strain showed increased levels of production or greater stability of ALO (53-kDa band). It is interesting that ALO breakdown started in A35DM (less in A35AInhAl) only after 17 h of growth. A similar effect was not seen in the A35HMS strain, which allowed accumulation of intact ALO throughout the 24 h of growth. The ALO gene is under control of a PlcR-dependent promoter, so that the truncation of the PlcR protein in B. anthracis is expected to greatly limit ALO synthesis, along with all the other PlcR- dependent proteins (Sastalla, 2010, Microbiology 156, 2892-2993). The fact that ALO can be observed in several of the protease-deficient mutants implies that previous reports of low ALO production may be attributed, at least in part, to its degradation rather than to low expression. Camelysin overexpression did not influence ALO production, indicating that this toxin is not a target for camelysin. Taken together, the results demonstrate the involvement of multiple proteases in controlling the accumulation of extracellular proteins in B. anthracis.

Example 4. Complementation of genetic modifications to inactivate a B. anthracis protease

This Example demonstrates that complementation of genetic modifications to inactivate a B. anthracis protease restores proteolytic activity.

To verify that the changes observed above were due to the intended gene knockouts rather than to unrecognized second-site mutations, studies to assess complementation of several mutants were conducted. To complement the mmpZ mutation, the Α35ΔΜητρΖ strain was transformed with pMrapZC. This piasmid undergoes a single crossover to insert the full length, wild-type mmpZ gene next to the mutated one. The pSC vector was eliminated by Cre-recombi ase treatment as described previously (Pomerantsev AP et al., 2009, ibid. ). The presence of the intact mmpZ gene in A35MmpZC was confirmed by PGR and sequencing. Western blot analysis of LF production from A35AMmpZC (Figure 3A) over 24 li indicated that proteolytic activity was restored to levels similar to that of A35 (Figure 2). To restore the large tasA- i« j,4ideletion in the genome of the A.35TM strain, the strain was transformed with the piasmid pATasA-InliAlC (containing the entire tasA-inhAI region) and complementation was assessed in a manner similar to that described above. Analysis of ALO production from the complemented strain verified restoration of proteolytic activity (Figure 3B). ComplcTnentation of she A35/VlnhA2 strain, and restoration of the lnhA2 protease in the same manner, however, did not result in any difference in secreted proteins (data not shown).

All the studies described above were done in derivatives of Ames 35, where the toxin proteins are encoded on. the large virulence plasmk! pXOl . Production of the toxin proteins and secreted proteases in these strams is highly dependent on the growth medium. In particular, the production of the three toxin proteins and certain proteases is greatly enhanced by the addition of bicarbonate (Chitlaru T et a!., ibid., Passa!acqua D et al., ibid., Bartkus JM, 1989, Infect. Immun. 57, 2295-2300. Thus, it is likely that growth in certain media could produce higher concentrations of both proteases and substrates (e.g., PA, LF, EF, etc.), and this could lead to even greater degradation than observed here.

Bacillus host strains are widely used in biotechnol.ogi.cal processes, and aviruient B. anthracis strains have been used for a number of years to produce PA and LF (see, for example, Varughese M et al, ibid.. Park S et al, ibid., Gupta P et aL 2008, PLoS. ONE 3, e313(). However, these strains have not been useful as generic hosts for recombinant protein production due to the secreted proteases demonstrated by the work presented above. The successful elimination of many of the most abundant proteases described herein suggested that the resulting strains could have value as protein expression hosts. To create an optimal host, the A35HMS strain was further modified by curing it of plasmid pXO l . The resulting strain, designated BH460, is non-toxigenic, and can be considered innocuous since it lacks the major virulence factors of B. anthracis. The permanent deletion of the spoOA gene assures that the strain dies rapidly at the end of exponential growth, eliminating concerns regarding laboratory contamination.

Example 5. Production of intact EF protein by B. anthracis BH460

This Example demonstrates production of intact EF by a modified B. anthracis of the embodiments, namely B. anthracis BH460 transformed with recombinant molecule pSJ136EFOS.

Production of EF from B. anthracis hosts has previously been difficult because tills protein is more susceptible to proteolytic degradation than are PA and LF. The plasmid pSJ136EFOS (the nucleic acid sequence of which is SEQ ID NO:62) encodes the mature EF protem with its native N-terminus (thus, the "OS" for original sequence) fused to the PA signal sequence and under the control of the pag promoter. This plasmid is otherwise similar to the plasmids pYS5 and pSJl 15 that are routinely used by the inventors to produce PA and LF, respectively; see, e.g., Park S et al, ibid. Protein purified from ihe transformant BH460 (pSJ 136EFOS) was compared to a preparation made in a similar way from the single protease mutant host BH450, and to a His6~tagged EF protein purified from E. coli (Soelaiman S et al., ibid.), the latter being the type of material used in previous toxicity analyses reported in Firoved AM et al., 2005, Am. J. Pathol. 167, 1306-1320. SDS-PAGE profiles of the recombinant EF proteins are shown in Figure 4A. The EF produced from BH460 appeared to be slightly less degraded than that isolated from BH450. This finding was confirmed by mass-spectrometry analyses (Figure 4B). The molecular mass of the recombinant protein isolated from BH460 (88,820 Da) compared well with the theoretical molecular weight for EF (88,822 Da), differing by 2 Da, which is within the instrumental error. A second species was found that had a lower mass (88,687 Da) consistent with loss of the N-terminal methionine. These two protein species were present in about equal amounts (47% for the larger, 53% for the smaller). Mass spectra of the EF produced from BH450 showed degradation as indicated by losses of 1495.7 and 2455.7 Da, resulting in proteins of 87,326 and 86,366 Da, found with similar abundances of 54% and 46%, respectively (Figure 4C). Apparent protease cleavage sites in mature EF produced by BH450 were mapped to amino acid residues Arg-12 and Lys-20. EF purified from E. coli was monomorphic, with a mass of 89,995 Da, differing by only 6 Da from the theoretical molecular weight (89,989 Da, Figure 4C). These results clearly demonstrate production of intact EF from BH460 compared to the truncated proteins made by BH450.

Example 6. Activity of EF produced by genetically modified protease-deficient B. anthracis

This Example demonstrates the EF produced by a modified B. anthracis of the embodiments, namely B. anthracis BH460 transformed with recombinant molecule pSJ136EFOS, is active in a potency assay.

As noted above, recombinant EF has previously been difficult to produce from B. anthracis host strains transduced with plasmids such as pSJ136. Furthermore, the EF that was obtained either from B. anthracis Sterne strain culture supernatant (Leppla SH, 1991, Methods Enzymol. 195, 153-168) or E. coli (Soelaiman S et al, ibid.) consistently had higher potency in inducing cAMP production in cultured cells or lethality to mice than EF produced from B. anthracis (data not shown). In fact, no previous recombinant EF preparations from B. anthracis have been lethal to mice even when injected in doses as high as 100 μg (combined with equimolar PA) (data not shown). The ES-MS analyses shown in Figure 4 suggest that the low potency of previous B. anthracis-derived EF preparations could be due to degradation. Consistent with prior results, the BH450- derived EF displayed an extremely low level of specific activity and was not lethal for mice (Figure 5A and Figure 5B). However, the recombinant EF purified from the BH460 culture supernatant had a specific activity exceeding that of highly active E. coli BL21(DE3)-derived EF (Figure 5 A). Similarly, when injected with equal doses of PA, recombinant EF prepared from BH460 was lethal to animals at the 25 μg dose, whereas this dose of E. co/z-derived EF had minimal effect (Figure 5B). EF purified from BH450 was not lethal at 50 μg (Figure 5B) and even at doses up to 100 μg (data not shown). Thus, the BH460 strain, which produces 5-7 mg of EF per liter of culture, allows for the first time the purification of substantial amounts of highly active EF from B. anthracis. Because other proteins purified in the same way from B. anthracis have consistently been free of endotoxin, use of these EF preparations also eliminates concerns regarding endotoxin-mediated cAMP co-signaling associated with E. co/z ^' -derived preparations. The BH460 strain has also proven very useful for expression of a variety of other proteins, typically yielding in excess of 10 mg of final pure protein per liter of culture, an Example of which is provided in Example 7.

Example 7. Production of intact B. cereus hemolysin HBL toxins by B. anthracis BH460

This Example demonstrates that modified a B. anthracis of the embodiments, namely B. anthracis BH460 transformed with recombinant molecule pSW4-HBL LI His, pSW4-HBL L2 His, or pSW4-HBL B His, produces large quantities of intact HBL LI, HBL L2, or HBL B, respectively.

B. cereus can cause infections in immune-compromised patients, such as sepsis, meningitis, pneumonia, and wound infections. There are also reports on impetigo-like lesions in non-compromised patients. HBL toxins have been shown to contribute to B. cereus caused diarrheal food poisoning, and their presence is used as an indicator for B. cereus food contaminations.

B. cereus 569 has two HBL operons. The first operon is located on the chromosome, and the second on a plasmid. The chromosomal operon appears to comprise three genes that encode the following proteins: L2 (1320 bp, 439 amino acids, 49.3 kDa with signal sequence); LI (1221 bp, 406 amino acids, 43.8 kDa with signal sequence); and B (1146 bp, 381 amino acids, 42.5 kDa with signal sequence). These proteins do not share high homology on the protein level: LI vs. L2 has 24% identity; LI vs B has 26% identity; and L2 vs B also has 26% identity. All of these HBL proteins have been shown to be secreted via the Sec pathway and contain a Gram-positive signal sequence (Fagerlund A et al, 2010, BMC Microbiol 10, 304).

Single HBL toxin components are non-toxic; all three components (i.e., LI, L2, and B) are needed to effect cell lysis. The exact mechanism by which the toxin components function in concert is still not well understood.

HBL has been shown to affect a variety of cell types and tissues such as retinal tissue (Beecher DJ et al, 1995, Infect. Immun. 63, 4423-4428), rabbit skin, ileum, CHO cells (Beecher DJ et al, 1997, J. Biol. Chem. 272, 233-239), and red blood cells from guinea pig, swine, bovine, sheep, rabbit, goat, and human (activity in descending order).

Studies performed by Beecher et al, 1997, ibid, showed that the B (binding) moiety can prime erythrocytes to lyse when followed by incubation with LI and L2 components.

Cells can also be primed with either L component, indicating that all three components can bind to erythrocyte membranes. In addition, toxin action can be inhibited by addition of antibodies specific to the binding component, as well as by addition of excess LI component, indicating that LI binds either L2 or the binding component (Beecher et al.,

1997, ibid.).

Recombinant molecule pSW4-HBL LI His (depicted in Figure 7) was produced by operatively linking a nucleic acid molecule encoding HBL LI (including its natural signal sequence) and a C-terminal 6xHis tag to expression vector pSW4, such that expression of HBL LI His was under the control of the B. anthracis pag promoter. B. anthracis BH460 was transformed with recombinant molecule pSW4-HBL LI His, and the modified B. anthracis, when grown in 2 liters FA broth overnight (-16 h), produced 28 mg of pure HBL LI His protein.

Recombinant molecule pSW4-HBL L2 His (depicted in Figure 8) was produced by operatively linking a nucleic acid molecule encoding HBL L2 (including its natural signal sequence) and a C-terminal 6xHis tag to expression vector pSW4, such that expression of HBL L2 His was under the control of the B. anthracis pag promoter. B. anthracis BH460 was transformed with recombinant molecule pSW4-HBL L2 His, and the modified B. anthracis, when grown in 2 liters FA broth overnight (-16 h), produced 151 mg of pure HBL L2 His protein.

Recombinant molecule pSW4-HBL B His (depicted in Figure 9) was produced by operatively linking a nucleic acid molecule encoding HBL B (including its natural signal sequence) and a C-terminal 6xHis tag to expression vector pSW4, such that expression of HBL B His was under the control of the B. anthracis pag promoter. B. anthracis BH460 was transformed with recombinant molecule pSW4-HBL B His, and the modified B. anthracis, when grown in 2 liters of FA broth overnight (-16 h), produced 75 mg of pure HBL B His protein.

Purification of the respective proteins was accomplished by the following steps: adsorbed protein to phenyl-Sepharose at 2 M ammonium sulfate; eluted protein from phenyl-Sepharose with 0.3 M ammonium sulfate; precipitated protein, resuspended, and dialyzed against 5 mM HEPES, 0.5 mM EDTA, pH 7.5 prior to loading onto a Q- Sepharose column (GE Healthcare); eluted protein from Q-Sepharose using a NaCl gradient (buffer A was 20 mM Tris-HCl, 0.5 mM EDTA, pH 8.0; buffer B was 20 mM Tris-HCl, 0.5 mM EDTA, pH 8.0, plus 0.5 M NaCl); combined protein-containing fractions and dialyzed against 5 mM HEPES, 0.5 mM EDTA, pH 7.5 prior to loading onto a hydroxyapatite (BioRad ceramic type) column; eluted protein from hydroxyapatite using a phosphate gradient (buffer A was 0.02 mM potassium phosphate, pH 7.0, 0.10 M NaCl; buffer B was 1 M potassium phosphate, pH 7.0, 0.10 M NaCl); collected protein peak; and dialyzed pure protein prior to concentration on an ultrafiltration unit.

Example 8. Genetic modification of B. anthracis protease genes

This Example demonstrates that genetic modification of a gene encoding a protease protein in B. anthracis using a Flp-FRT recombinase system results in truncation of the corresponding protein and production of a B. anthracis strain that no longer expresses such active protease.

Culture supernatants of strain BH460 were analyzed by mass spec to determine which proteases continued to be produced that might degrade valuable endogenous proteins (e.g., anthrax toxin proteins that are vaccine candidates) or heterologous proteins. Two additional proteases were identified (GBAA 1995 cysteine protease CysPl and GBAA 4584 minor extracellular protease VpR). At least a portion of genes encoding these proteases were genetically deleted from B. anthracis BH460, leading to B. anthracis BH480, which lacks eight active proteases, due to genetic modification of the following genes leading to inactivation of the respectively encoded proteins: nprB, inhA2, tasA, calY, inhAl, mmpZ, cysPl, and vpR. Mutations engineered into B. anthracis cysPl and vpR are indicated in Figure 1C. Specifically, a Saccharomyces cerevisiae Flp- i?r recombinase system (Park, YN, et al, 2011, Yeast 28, 673-681) was adapted to use for B. anthracis cysPl and vpR gene deletions by introducing S. cerevisiae Flp recombinase and S. cerevisiae FRT sites into the genome of B. anthracis; both FRT sites and Flp-recombinase genes were inserted into vectors suitable for replication, expression, and recombination in B. anthracis. The system was used in a manner similar to the Cre-loxP recombinase system described previously (Pomerantsev AP et a!., 2009, ibid.) and herein. The Flp-FRT method allowed the replacement of both cysPl and vpR genes with a 48-bp FRT-site using Flp recombinase. Both FRT-site and ^Zp-genes were cloned into plasmids pSCF and pFPAS, respectively, which are described in Table 3. Additional plasmids used in the genetic modifications of the cysPl and vpR genes included plasmid pCysPlLI, pCysP! RI, pVpRLI, and pVpRRI, which are described in Table 3. Primer pair 1995 seqF and 1995 seqR was used to verify cysPl gene disruption; and primer pair 4584 seqF and 4584 seqR was used to verify vpR gene disruption. Table 5 provides mass spec (MS) analysis that confirms the absence of both CysPl and VpR proteases in the secretome of the B. anthracis BH480 strain.

Table 5. MS-peptide hits for CysPl and VpR proteases in supernatants of B. anthracis BH460 and B. anthracis BH480.

Table 10 demonstrates the absence of both CysPl and VpR proteases in the supernatant of B. anthracis BH480 in comparison to that of B. anthracis BH460.

Supernatants of BH480 have been examined as described above, and several additional proteases have been identified: (GBAA 2183 neutral metalloprotease NprC, GBAA 5414 serine protease SprA, GBAA 3660 serine protease HtrA and GBAA 3968 prokaryotic homo log of proteasome HslV).

Example 9. Production of intact EF, LF, and uPA proteins by B. anthracis BH480 This Example demonstrates production of EF proteins by modified B. anthracis of the embodiments, namely B. anthracis BH480 transformed with recombinant molecules encoding a variety of EF proteins. Also demonstrated is production of intact LF and uPA proteins.

B. anthracis BH480 was transformed with recombinant molecule pSJl 36EFOS in a manner similar to transformation of B. anthracis BH46Q with recombinant molecule pSJ136EFOS as described herein. B. anthracis BH480 was also transformed with recombinant molecule pSJ136EF-His, recombinant pSJ136EF-Cys, or recombinant molecule pSJ 136EF-NEHY, which are identical to pSJl 36EFOS except that they encode mature EF proteins having small modifications in the -terminal sequence as indicated in Table 6.

Table 6. Recombinant molecules and proteins encoded by such recombinant molecules

B, anthracis strains transformed with pSJ136EFOS, pSJ136EF-His, pSJ136EF-

C y-;. or pSil 36EF-NEHY were inoculated into 10 ml FA media that included 10 micrograms per ml (iig/ml) kanamycin. Protein production for each sample was demonstrated by native Phast gel (native 8-25% acrylamide gradient) analysis, the proteins being stained by Coomassie blue. Results are shown in Figure 10. The results indicate production of intact EF proteins by B. anthracis BH480 transformed with pSJ136EFOS, pSJ136EF-eis, pSJ136EF-Cys, or pSJ] 36EF-NFHY, as shown in lanes 4, 6, 7, and 8 of Figure 10, respectively.

Figure 10 also shows production of intact urokinase plasminogen activator (uPA) variants PA-U2f (lane 1) and PA-U7f (lane 2) by B. anthracis BH480 transformed with pYS5 plasmids encoding PA-U2f and PA-U7f, respectively, and cultured as described herein; PA-U2, PA-U7, and recombinant molecules comprising nucleic acid molecules encoding such proteins are described in International Publication No. WO 01/21656, published 29 March 2001; PA-U2f and PA-U7f lack a C-terminal serine present in PA- U2 and PA-U7. Figure 10, lane 3 shows production of intact mature lethal factor (LF-OS) by B. anthracis BH480 transformed with pSJ115-LF-OS and cultured as described herein. Lane 5 shows that B. anthracis BH480 transformed with pSJ136 Hfql-FLAG and cultured as described herein did not produce detectable amounts of protein. Hfql-FLAG is a small RNA chaperone Hfql with a C-terminal FLAG tag.

Example 10. Production of intact recombinant proteins by B. anthracis BH480

This Example demonstrates production of recombinant proteins by modified B. anthracis of the embodiments.

Intact fusion protein LFnBlaY was produced as follows: B. anthracis BH480 was transformed with a recombinant molecule comprising a pSJl 15 plasmid encoding LFnBlaY. LFnBlaY is a fusion protein of B. anthracis lethal factor LFn to B. cereus beta-lactamase (BlaY) that has an amino acid sequence represented by SEQ ID NO:91. A similar fusion protein, LF-BLA, produced in E. coli, is described in Hobson JP et al, 2006, Nature Methods 3, 259-261. The modified B. anthracis was cultured and LFnBlaY purified as described herein. A 2-liter preparation yielded 184 mg of intact LFnBlaY, the purity of which is demonstrated in Figure 11, lanes 4 and 5, and compared to the purity of LF-BLA from E. coli (lane 3).

Intact protective antigen (PA) variant PA-SNKE-deltaFF-E308D was produced as follows: B. anthracis BH480 was transformed with a recombinant molecule comprising a pYS5 plasmid encoding PA-SNKE-deltaFF-E308D. PA-SNKE-deltaFF-E308D is described in Ramirez DM et al., 2002, J Industrial Microbiology & Biotechnology 28, 232-238. The PA variant's amino acid sequence is represented by amino acid sequence SEQ ID NO:92. The modified B. anthracis was cultured and PA-SNKE-deltaFF-E308D purified using techniques similar to those described in US 2004/0076638 Al, published April 22, 2004. A 2-liter preparation yielded 122 mg of intact PA-SNKE-deltaFF-E308D, the purity of which is shown in Figure 5, lane 2.

This Example along with other examples herein demonstrate the abilities of B. anthracis BH460 and B. anthracis BH480 to make a variety of intact recombinant proteins.

Example 11. Summary and Conclusions

These Examples describe the adaptation of an improved Cre-loxP system for sequentially deleting additional protease-encoding genes of B. anthracis. They also describe a role of each protease in degradation of B. anthracis toxin components and another potential virulence factor, anthrolysin O (ALO) (Shannon JG et al., 2003, Infect. Immun. 71, 3183-318).

Bacillus anthracis produces a number of extracellular proteases that impact the integrity and yield of other proteins in the B. anthracis secretome. This study shows that anthrolysin O (ALO) and the three anthrax toxin proteins, protective antigen (PA), lethal factor (LF), and edema factor (EF), produced from the B. anthracis Ames 35 strain (pX01 ⁺ , pX02 ^~ ), are completely degraded at the onset of stationary phase due to the action of proteases. An improved Cre-loxP gene knockout system was used to sequentially delete the genes encoding six proteases (NprB, InhA2, TasA, camelysin, InhAl, and MmpZ). The role of each protease in degradation of the B. anthracis toxin components and ALO was demonstrated. Levels of the anthrax toxin components and ALO in the supernatant of the sporulation defective, pX01 ⁺ A35HMS mutant strain deleted for the six proteases were significantly increased and remained stable over 24 h. A pXOl-free variant of this six-protease mutant strain, designated BH460, provides an improved host strain for the preparation of recombinant proteins. As an example, BH460 was used to produce recombinant EF, which previously has been difficult to obtain from B. anthracis. The EF protein produced from BH460 had the highest in vivo potency of any EF previously purified from B. anthracis or E. coli hosts. BH460 is recommended as an effective host strain for recombinant protein production, typically yielding greater than 10 mg pure protein per liter of culture.

These Examples also describe the successful adaptation of a modified Saccharomyces cerevisiae Flp- i?r recombinase system for deleting additional pro tease- encoding genes of B. anthracis. This system was used to inactivate CysPl and VpR proteases in BH460, thereby creating B. anthracis BH480. BH480 is also recommended as an effective host strain for recombinant protein production, typically yielding greater than 10 mg pure intact protein per liter of culture. In some embodiments, the yield is greater than 50 mg pure intact protein per liter of culture. In some embodiments, the yield is greater than 90 mg pure intact protein per liter of culture.

While the present invention has been described with reference to the specific

embodiments thereof, it should be understood by those skilled in the art that various changes may be made and equivalents may be substituted without departing from the true spirit and scope of the invention. In addition, many modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, to the objective, spirit and scope of the present invention. All such modifications are intended to be within the scope of the claims.