The invention relates to a microorganism comprising at least one heterologous genetic sequence from a donor microorganism. In particular it relates to a microorganism comprising a heterologous gene sequence encoding at least one gene involved in Sec- mediated protein translocation. The invention also relates to a method of producing a micro-organism by transforming at least one heterologous genetic sequence into the micro-organism. The invention also relates to a vector comprising the genetic sequence and method for secreting a protein from a micro-organism.

Bacteria are commonly used to produce industrial enzymes for the detergent, food, boifuels and beverage industries and heterologous proteins for pharmaceutical and other human therapeutic purposes, for example in vaccine, peptide hormone, antibody and antigen production.

Gram-positive bacteria such as members of the class Sac/7// ^' have the capacity to secrete enzymes across cell envelopes into a culture medium at high concentrations as they have an efficient secretory system and no outer cell membrane.

Using such secretory systems as industrial production tools or "cell factories" provides multiple benefits including increased production capacity, structural authenticity, and enhanced purity of the end product. Purification from a culture medium rather than from the cell cytoplasm also considerably reduces associated downstream processing and purification costs.

However, to date, there has been limited success in the use of such bacteria for industrial scale heterologous protein production.

This is due, at least in part, to the intrinsic properties of the secretion pathways in these bacteria. For example, gram-positive bacteria typically lack the specialised secretion pathways found in Gram-negative bacteria. They rely on a Sec dependent secretion pathway for secretion of unfolded proteins, including secretion of house-keeping proteins such as those involved in cell wall biosynthesis and cell division. Although other secretion pathways have been discovered in bacteria of the genus Bacillus, such as Bacillus subtilis, the majority of exported proteins are translocated and secreted via the Sec pathway.

The mechanism of protein secretion in such micro-organisms, is however not yet fully understood. In general, the Sec pathway or Sec translocase in B. subtilis consists of the membrane proteins SecY, SecE, and SecG, which together form a pore, and homologues of the E. coli proteins SecD, SecF, YajC and SecA (in B. subtilis SecDF, YrbF, SecA). SecA is a multifunctional membrane-associated ATPase component that acts as a pilot protein, chaperone, and biochemical motor for the translocation of proteins.

Although members of the Bacilli class of bacteria naturally produce and secrete high yields of proteins (grams per litre), yields of heterologous proteins secreted by these bacteria often drop to milligram or even microgram quantities per litre of culture.

One proposed solution to the problem is to generate strains in which genes encoding quality control proteases have been deleted (Construction and use of a Bacillus subtilis mutant deficient in multiple protease genes for the expression of eukaryotic genes. He XS, Shyu YT, Nathoo S, Wong SL, Doi RH. Ann N Y Acad Sci. 1991 vol 646:69-77). (Heterologous production of Clostridium cellulovorans engB, using protease-deficient Bacillus subtilis, and preparation of active recombinant cellulosomes. Murashima K, Chen CL, Kosugi A, Tamaru Y, Doi RH, Wong SL. J Bacteriol. 2002 vol 184:76-81.) Such strains have already improved the productivity of B. subtilis for the translocation of some substrates but not others.

Lin Fu et al Biotechnology Advances 25 (2007) suggest a number of approaches to optimise protein secretion in B. subtilis. Among their suggestions are the deletion of genes coding for side products and the removal and replacement of chromosomal segments to tailor foreign secretory proteins to meet the requirements of the expression system.

Bacillus subtilis is used extensively in various industries as a bioprocessing and bioproduction organism and as such its exploitation on an industrial scale is well established. The use of Bacillus subtilis to secrete pre-determined heterologous proteins by growing a strain of B. subtilis in which a plasmid containing a DNA sequence coding for a desired protein has been introduced is described in US4816396 (Cetus Corporation). The heterologous gene product is secreted by the host. The document describes methods of expressing human fibroblast interferon gene and the eukaryotic pre-insulin gene in B. subtilis, by using a plasmid, the transcription and translation of which is under the control of the gene of a DNA sequence indigenous to B. licheniformis.

Since β. subtilis is non-pathogenic, it would be a good organism to use for heterologous protein secretion, if it were not for the problems of low yield described above.

US5939317 (SKW Biosystems) relates to the use of a Sec-dependent secretion system from lactococci bacteria for secreting proteins, such as bacteriocins, normally secreted by a Sec-independent system in strains of industrial interest.

It is therefore desirable to improve efficiency of protein translocation in commercially useful bacteria.

The inventors have investigated protein secretion systems in bacteria. They have determined that components of the Sec accessory secretion system can surprising be used in heterologous micro-organisms to provide a secretion system in a host cell. That secretion system can be utilised to provide a means for secreting proteins of industrial interest from the host cell.

Hence a first aspect of the invention provides a micro-organism comprising at least one heterologous genetic sequence encoding at least one gene involved in Sec-mediated protein translocation, wherein the gene is secA1, secA2, sec Y1, secY2 and/or SecH or a fragment, variant or homologue thereof.

For the avoidance of doubt, as used herein the class Bacilli refers to bacterial species that are members of the Genus Bacillus and other related organisms. Such related organisms include Clostridia and Streptococci species. The taxonomic class Bacilli includes the orders of Bacillales and Lactobacillales bacteria. A well known bacterium within the genus Bacillus is Bacillus anthracis, the etiological agent of anthrax. It has two Sec-dependent protein secretion systems or translocases. Figure 1 shows a schematic representation of the Sec pathways in B. anthracis. B. anthracis encodes paralogues of components of the Sec pathway, including two copies of SecA - SecA1 and SecA2.

As shown in Figure 1 , translocation in β. anthracis can occur via a primary pathway involving the SecA1 dimer 1 or a secondary pathway, which involves the SecA2 dimer 2. The translocation pathway depends on the signal peptide 3 on a preprotein substrate 4. The SecY1/Y2EG protein complex 5 is located in the cell membrane 6 and defines a pore 7, through which protein substrates may be translocated from the cytoplasm 8 to the external milieu 9 of the cell.

Most proteins in B. anthracis are secreted via the primary pathway using SecA1. It has been discovered by the Applicant that certain substrates specifically use the SecA2 pathway in Bacillus anthracis and that the SecA2 pathway in B. anthracis is highly efficient for translocation of substrate proteins. Examples of substrates that specifically use the SecA2 pathway are surface associated protein (Sap) and E antigen protein 1 (EA1/Eag). Of all of the proteins secreted out of the cell by B. anthracis, those secreted in the highest concentration are surface associated protein (Sap) and Eantigen protein 1 (EA1).

Against this background the applicant decided to investigate whether components of the Sec-mediated pathway in Bacillus anthracis can be harnessed to provide an additional Sec-mediated translocase in microorganisms. They have surprisingly found that components of the Sec pathway can be transferred to other bacterial species and can be utilised for the secretion of proteins.

Specifically the inventors have found that secA1, secA2, secY1, secY2 and/or SecH genes from Bacillus anthracis can be used to provide a parallel or replacement Sec- mediated secretion pathway in heterologous bacterial species. Until the present application, this had previously not been disclosed or appreciated from ongoing research in this area. A key conclusion from this research is that SecA1 and SecA2 are functionally distinct. SecA2 interacts with specific substrates, Sap and EA1 , directing them to the translocase. While the elements on SecA2 and the substrates that are involved in the targeting are yet to be identified, the inventors believe on the basis of previous work that it involves the PPXD domain of SecA2 and the signal peptides of the substrates. It is their opinion that incorporation of SecA2, with or without SecY2, into a producer bacterium will increase the secretion proteins carrying the targeting signals of Sap/EA1 by reducing competition between essential housekeeping proteins and the target protein for SecA1.

Therefore the microorganism of the first aspect of the invention can be utilised as a host cell for the expression of proteins of interest, and as such can be used for industrial scale heterologous protein production.

By "micro-organism" we include that the first aspect of the invention relates to bacterial species.

It is well known that bacteria can be used for synthesising proteins of industrial interest. Such proteins are subsequently prepared from the cell cultures, subjected to various standard purification and preparation procedures, and routinely used for medical and/or industrial purposes. A wide range of bacterial species can be used for such purposes.

Bacterial species can generally be divided into "gram-positive" and "gram-negative" species, the difference being specifically attributable to the retention of particular dyes that depend on the structural differences of their bacterial cell walls.

Examples of "gram-negative" bacterial species include E. coli, Shigella, Salmonella, Yersinia or Klebsiella species, all of which can be used to prepare proteins of interest.

However it is preferred that the micro-organism of the first aspect of the invention is a "gram-positive" bacterial species, such as Aerococcus sp., Bacillus sp., Bifidobacterium sp., Clostridium sp., Corprococcus sp., Deinobacter sp., Deinococcus sp., Enterococcus sp., Gemella sp., Lactobacillus sp., Lactococcus sp., Leuconostoc sp., Marinococcus sp., Melissococcus sp., Micrococcus sp, Pediococcus sp., Peptococcus sp., Peptostreptococcus sp., Planococcus sp, Ruminococcus sp., Saccharococcus sp., Salinococcus sp., Carcina sp., Staphylococcus sp., Stomatococcus sp. Streptococcus sp., Trichococcus sp., and Vagococcus sp.

Preferably micro-organism is of the Glass Bacilli. Most preferably the micro-organism is selected from the group of Bacillus anthracis, Bacillus subtilis, Bacillus cereus, Bacillus cytotoxicus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus and Bacillus weihenstephanensis. Most preferably the micro-organism is Bacillus subtilis, B. licheniformis and/or β. amyloliquefaciens.

When preparing the microorganism of the first aspect of the invention, suitable samples of bacterial species can be readily obtained from a variety of different depositories. For example, the ATCC (American Tissue Collection Center; www.atcc.org) can supply bacterial cells that can be used to derive the micro-organism of the first aspect of the invention.

By "heterologous" we mean that the genetic sequence encoding at least one gene involved in Sec-mediated protein translocation is not native to that species, i.e. a nucleic acid of the sequence of that gene is not naturally found in the species.

Accordingly the first aspect of the invention requires the transfer of genetic material to a bacterial cell so as to derive the micro-organism of the first aspect of the invention. There are many standard laboratory techniques that can be adopted by the skilled person to introduce genetic material to host cells. Generally, not all of the host cells will be transformed by the genetic material and it will therefore be necessary to select for transformed host cells. One selection technique involves incorporating a DNA sequence marker that codes for a selectable trait in the transformed cell. These markers include dihydrofolate reductase, G418 or neomycin resistance for eukaryotic cell culture, and tetracycline, kanamycin or ampicillin resistance genes for culturing in bacteria. The selectable markers could also be those which complement auxotrophisms in the host. Alternatively, the gene for such a selectable trait can be on another vector, which is used to co-transform the desired host cell. By "gene involved in Sec-mediated protein translocation" we include that the microorganism of the first aspect of the invention can include genes encoding components of the Sec-mediated protein translocation pathway in bacteria.

As discussed above, the Sec pathway or Sec translocase consists of the membrane proteins SecY, SecE, and SecG, which together form a pore, and SecD, SecF, YajC and SecA SecA (in β. subtilis and other Gram-positive bacteria SecDF, YrbF and SecA). However the first aspect of the invention requires that the micro-organism includes heterologous genetic sequence of the secA1, secA2, sec Y1, secY2 and/or SecH genes. Examples of nucleic acid sequences corresponding to a gene involved in Sec-mediated protein translocation can be readily identified from, for example, GenBank (http://www.ncbi.nlm.nih.gov/qenbank/).

Bacillus anthracis encodes paralogues of components of the Sec pathway, namely two copies of SecA (SecA1 / SecA2), two copies of Sec Y (SecY1 / SecY2) and three copies of the extracytoplasmic chaperone PrsA (PrsAA, PrsAB and PrsAC). In addition, the inventors have identified that the nucleic acid sequence disclosed in Genbank Accession Number BA0881 is a further member of the Sec-mediated protein translocation pathway, herein termed "secH".

Of all of the proteins secreted out of the cell by B. anthracis, those secreted in the highest concentration are surface associated protein (Sap) and E antigen protein (EA1 ). β. anthracis genes having Genbank Accession Numbers BA5421 and BA0882 have been previously referred to as "secA-Z and ^u secA-T respectively (Read et al (2003) Nature 423: 81-86).

When comparing the secA homologues of B. anthracis and B. subtilis, it appears that there are some clear differences. B. subtilis secA shows 73% identity to B. anthracis "SecA-2", but only 48% identity to "SecA-1". "SecA-1° (788 residues) is also significantly shorter than both B. subtilis secA (841 residues) and B. anthracis ^u SecA-2' (835 residues). So called ^u SecA-2" is more similar to secA in B. subtilis and therefore in this document secA 1 is used to refer to the B. anthracis gene having Genbank Accession Number BA5421 and secA2 is used in this document to refer to the gene having Genbank Accession Number BA0882. This terminology is used to ensure that the terminology in B. anthracis is compatible with that used in other bacterial species.

Examples of a nucleic acid and polypeptide sequences of secA1, secA2, sec Y1, secY2 and SecH from B. anthracis are provided towards the end of the example section of the patent specification SEQ ID NO:1 provides a nucleic acid sequence of B. anthracis secA 1. SEQ ID NO:2 provides a polypeptide sequence of B. anthracis SecA1. SEQ ID NO:3 provides a nucleic acid sequence of B. anthracis secA2. SEQ ID NO:4 provides a polypeptide sequence of B. anthracis SecA2. SEQ ID NO:5 provides a nucleic acid sequence of B. anthracis secY1. SEQ ID NO:6 provides a polypeptide sequence of β. anthracis SecY1. SEQ ID NO:7 provides a polypeptide sequence of B. anthracis SecY2. SEQ ID NO:8 provides a nucleic acid sequence of B. anthracis secY2. SEQ ID NO:9 provides a nucleic acid sequence of B. anthracis secH. SEQ ID NO: 10 provides a polypeptide sequence of B. anthracis SecH.

By "fragment" we include where the micro-organism of the first aspect of the invention comprises at least a part of the heterologous genetic sequence encoding secA 1, secA2, sec Y1, secY2 and/or SecH. It is envisaged that such a fragment would retain biological activity, i.e. still able to function in Sec-mediated protein translocation.

The term "variant" as used herein used to describe a heterologous genetic sequence encoding secA1, secA2, sec Y1, secY2 and/or SecH which retains the biological function of that peptide, i.e. still able to function in Sec-mediated protein translocation. A skilled person would know that the sequence of any one of specific sequences provided herein can be altered without the loss of biological activity. In particular, single like for like changes with respect to the physio-chemical properties of the respective amino acid should not disturb the functionality, and moreover small deletions within non-functional regions of the secretion unit peptide can also be tolerated and hence are considered "variants" for the purpose of the present invention. The experimental procedures described below can be readily adopted by the skilled person to determine whether a 'variant' can still function as a secretion unit peptide, i.e. whether the variant is still able to function in Sec-mediated protein translocation. By "homologue" we include those genes in addition to the specific sequences mentioned above from further bacteria species. Such genes can be readily identified from genetic databases.

Preferably the secA1, secA2, sec Y1, secY2 and/or SecH gene is obtained from Bacillus anthracis or related bacteria (e.g. B. cereus and S. thurengiensis).

More preferably the micro-organism of the first aspect of the invention comprises heterologous genetic sequence encoding at least secA2 and secH genes. The secH gene is also known as BA0881.

As presented herein, the inventors have shown that SecH, a cytoplasmic protein only found in members of the B. cereus group that encode SecA2, interacts with SecA2 to improve the secretion of Sap and EA1. It is the inventor's view that this is a type of secretion specific chaperone. Until the present invention it was not known or suggested that the SecH gene played a role in the SecA2 pathway.

Preferably the micro-organism of the first aspect of the invention comprises the secA2 gene having the nucleic acid sequence provided in SEQ ID NO: 3 and the SecH gene having the nucleic acid sequence provided in SEQ ID NO: 9.

It can be readily appreciated that the micro-organism of the first aspect of the invention can provide an additional Sec-mediated translocase in the micro-organism, and indeed this is a prefered embodiment of the invention.

A futher embodiment of the first aspect of the invention is wherein a signal peptide sequence specifically directs proteins through the additional Sec-mediated translocase; for example the Sec-mediated translocase of Sap and/or Eag (EA1 ) surface layer proteins are directed through the additional Sec-mediated translocase.

As disclosed above, the inventors have determined that SecA1 and SecA2 are functionally distinct. SecA2 interacts with specific substrates, for example Sap and EA1 , directing them to the translocase. The SecA2 specific substrates are targeted to that Sec- mediated pathway by virtue of a signal peptide. Examples of amino acid sequences of signal peptides are provided in the accompanying examples, and listed below:

MAKTNSYKKVIAGTMTAAMVAGIVSP EA1 signal peptide (SEQ ID NO.11)

MAKTNSYKKVIAGTMTAAMVAGWSP SAP signal peptide (SEQ ID NO:12)

Signal peptides of Sap and EA1. The single conservative change is underlined.

It can be appreciated by the skilled person that the signal peptides provided above are examples of such amino acid sequences. The accompanying examples provide more information on signal peptide from additional proteins that can be used to specifically direct proteins to be secreted by the additional secA2 -mediated trans|ocase.

A further embodiment of the present invention is wherein the micro-organism further comprises a bacterial expression construct comprising nucleic acid sequence encoding a signal peptide sequence.

Nucleic acid sequences encoding a signal peptide can be readily identified by the skilled person from the accompanying examples. For example, additional information concerning the amino acid and nucleic acid sequence of EA1 from B. anthracis can be found in GenBank accession NP_843398.1 , while further information concerning Sap from B. anthracis can be found in GenBank accession ZP_02394181.1.

By "bacterial expression construct", we include expression constructs known in the art that can be used to direct the expression of recombinant polypeptides in bacterial host cells.

An "expression construct" is a term well known in the art. Expression construct are basic tools for biotechnology and the production of proteins. It generally includes a piasmid that is used to introduce a specific gene into a target cell, a "host cell". Once the expression construct is inside the cell, protein that is encoded by that gene is produced by the cellular-transcription and translation machinery ribosomal complexes. The piasmid also includes nucleic acid sequences required for maintenance and propagation of the vector. The goal of an expression vector is the production of large amounts of stable messenger RNA, and therefore proteins. Suitable expression constructs comprising nucleic acid for introduction into bacteria can be chosen or constructed, containing appropriate regulatory sequences, including promoter sequences, terminator fragments, enhancer sequences, marker genes and other sequences as appropriate. For further details see, for example, Molecular Cloning: a Laboratory Manual: 2nd edition, Sambrook et al., 1989, Cold Spring Harbor Laboratory Press.

The plasmid is frequently engineered to contain regulatory sequences that act as enhancer and promoter regions and lead to efficient transcription of the gene carried on the expression vector. Most parts of the regulatory unit are located upstream of coding sequence of the heterologous gene and are operably linked thereto. The expression cassette may also contain a downstream 3' untranslated region comprising a polyadenylation site. The regulatory sequences can direct constitutive or inducible expression of the heterologous coding sequence.

Expression constructs for use the micro-organism of the invention can be readily obtains. Many such constructs are commercially available.

A second aspect of the invention provides a vector comprising the at least one heterologous genetic sequence as defined in relation to the first aspect of the invention. Preferably the vector is a plasmid. A further embodiment is wherein the vector is an integration plasmid.

A third aspect of the invention provides a method of producing a micro-organism of the Class Bacilli comprising the steps of: (i) isolating at least one genetic sequence involved in Sec-mediated protein translocation in a donor, wherein the genetic sequence is secA1, secA2, sec Y1; secY2 and/or SecH or a fragment, variant or homologue thereof; and (ii) transforming the at least one genetic sequence into the micro-organism. Preferably the micro-organism is as defined in relation to the first aspect of the invention. A further embodiment is wherein the method employs a vector as defined in relation to the second aspect of the invention.

A fourth aspect of the invention provide a bacterial expression construct comprising a nucleic acid sequence encoding a signal peptide comprising SEQ ID NO:11 or SEQ ID NO: 12 or a variant thereof, wherein the variant is capable of mediating the secretion of a peptide via the SecA2 pathway. A preferred embodiment of this aspect of the invention is wherein the signal peptide is derivable from EA1 from B. anthracis and/or Sap from B. anthracis or a related bacterium.

The preparation of bacterial expression constructs of the present invention can be readily achieved using information in the art without any inventive requirement.

It can therefore be appreciated that commonly used laboratory techniques for manipulating recombinant nucleic acid molecules can be used to derive the claimed bacterial expression construct.

A variety of methods have been developed to operably link polynucleotides, especially DNA, to vectors for example via complementary cohesive termini. Suitable methods are described in Sambrook er al (1989) Molecular Cloning, A Laboratory Manual, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY.

A desirable way to modify the DNA encoding a polypeptide is to use the polymerase chain reaction. This method may be used for introducing the DNA into a suitable vector, for example by engineering in suitable restriction sites, or it may be used to modify the DNA in other useful ways as is known in the art.

An embodiment of the fourth aspect of the invention is wherein the construct further comprises a multiple cloning site located 3' to the nucleic acid sequence encoding the C-terminal amino acid of the signal peptide.

The term 'multiple cloning site' is well known in the art. Also called a 'polylinker', it is a short segment of DNA which contains many restriction sites hence facilitating the insertion of nucleic acid sequences in the expression construct using procedures involving molecular cloning or subcloning.

An embodiment of the fourth aspect of the invention is wherein the construct further comprises a second nucleic acid sequence encoding a protein of interest located at the multiple cloning site, the second nucleic acid arranged such that the protein of interest is operatively linked with the signal peptide. By "protein of interest", or other such terms like "recombinant protein", "heterologous protein", "heterologous coding sequence", "heterologous gene sequence", "heterologous gene", "recombinant gene" or "gene of interest", as can be used are interchangeably herein, these terms refer to a protein product that is sought to be expressed in the mammalian cell and harvested in high amount, or nucleic acid sequences that encode such a protein. The product of the gene can be a protein or polypeptide, but also a peptide.

The protein of interest may be any protein of interest, e.g. a therapeutic protein such as an interleukin or an enzyme or a subunit of a muitimeric protein such as an antibody or a fragment thereof, as can be appreciated by the skilled person.

For the avoidance of doubt, the bacterial expression construct of this aspect of the invention can comprise more than one nucleic acid encoding a protein of interest.

An embodiment of the fourth aspect of the invention is wherein the construct encodes a recombinant polypeptide having the following structure: (i) a signal peptide, operatively linked at the C-terminus with (ii) a protein of interest.

In such an arrangement, when a gene encoding a protein of interest is placed in the multiple cloning site such that the protein of interest is operatively linked with the signal peptide, upon introduction to a suitable host cell the bacterial expression construct will encode a heterologous polypeptide molecule having: (i) an N-terminal signal peptide; and (ii) a protein of interest.

Accordingly a fifth aspect of the invention provides a recombinant fusion protein comprising a signal peptide comprising SEQ ID NO:11 or SEQ ID NO:12 or a variant thereof, fused with a protein of interest.

A sixth aspect of the invention provides a method for secreting a protein from a microorganism comprising the steps of:

(i) obtaining a micro-organism as defined in the first aspect of the invention comprising a gene encoding a protein to be secreted and at least one heterologous genetic sequence from a donor encoding at least one gene involved in a Sec-mediated protein translocase; and,

(ii) cuituring the microorganism under conditions suitable for expression of the at least one gene involved in Sec-mediated protein translocation and secretion of the protein to be secreted.

Preferably the donor is of the Class Bacilli.

For the avoidance of doubt, by "a protein to be secreted" we mean the "protein of interest" as described above in relation to the other aspects of the invention.

As stated above, the micro-organism of the first aspect of the invention can be utilised as a host cell for the expression of proteins of interest, and as such can be used for industrial scale heterologous protein production.

Hence the sixth aspect of the invention provides a method secreting a protein from such a micro-organism.

The method of the sixth aspect of the invention comprises cuituring the micro-organism described above for a sufficient time and under appropriate conditions in a culture medium so as to obtain expression of the protein to be secreted. An optional step in the method of the invention comprises recovering the protein from the culture medium.

The protein to be secreted can be readily isolated from the culture medium using standard techniques known in the art including ammonium sulphate or ethanol precipitation, acid extraction, anion or cation exchange chromatography, phosphocellulose chromatography, hydrophobic interaction chromatography, affinity chromatography, hydroxylapatite chromatography and lectin chromatography.

A further embodiment of the sixth aspect of the invention comprises (i) preparing a micro-organism of the first aspect of the invention; (ii) preparing a bacterial expression construct of the fourth aspect of the invention, comprising a gene encoding a protein of interest; (ii) introducing the bacterial expression construct into the micro-organism; (iii) cuituring the micro-organism in conditions to promote the expression and secretion of the protein of interest into the culture medium; (iv) isolating the protein of interest from the culture medium.

A seventh aspect of the invention provides a kit of parts comprising: (i) the microorganism according to the first aspect of the invention; and/or a vector as defined in the second aspect of the invention; and optionally a bacterial expression construct according to the fourth aspect of the invention. The kit may also comprise a manual of operation.

The manual of operation can include information concerning, for example, the restriction enzyme map of the expression construct; the nucleic acid sequence of expression construct; how to introduce a gene encoding a protein of interest in to the expression construct; optional conditions for expression of the protein of interest in a suitable host cell, and other such information as appropriate.

An embodiment of this aspect of the invention is wherein the kit of parts further comprising one or more additional components selected from the group comprising: transformation competent host cells; restriction enzyme; DNA polymerase; control plasmid inserts; protein purification columns and resins.

The kit of parts can further comprise further components, for example, transformation competent host cells for expression of the expression construct; enzymes that can be used to prepare an expression construct harbouring a gene encoding a protein of interest, such as typical restriction enzymes; enzymes that can be used to amplify the copy number of a gene encoding a protein of interest, such as DNA polymerase, preferably Taq, Pfu, or further well-known thermostable DNA polymerases; 'test control' agents such as control plasmid inserts. The kit may also comprise reagents useful for the recovery of the protein of interest from the cell supernatant, such as protein purification columns or resins.

A further aspect of the invention provides a method of producing a micro-organism substantially as shown in and described with reference to the drawings.

A further aspect of the invention provides a micro-organism substantially as shown in and described with reference to the drawings. The invention is now described by reference to the following, non-limiting, figures and examples.

Further embodiments of the invention

In addition to the aspects of the invention provided above, the present application also provides aspects of the present invention as presented below.

A micro-organism of the Class Bacilli comprising at least one heterologous genetic sequence from a donor encoding at least one gene involved in Sec-mediated protein translocation.

Preferably, the at least one heterologous genetic sequence is selected from the genes secA 1, secA2, sec Y1, secY2 and BA0881 {secH) or a homologue thereof.

Preferably the at least one heterologous genetic sequence may comprise the Bacillus anthracis secA2 and secY2 genes.

Advantageously, the heterologous genetic sequence provides an additional Sec- mediated translocase in the micro-organism.

Advantageously, a signal peptide sequence specifically directs proteins through the additional Sec-mediated translocase.

Advantageously, the Sec-mediated translocase of Sap and/or Eag (EA1 ) surface layer proteins are directed through the additional Sec-mediated translocase.

Preferably, the micro-organism is selected from the group of Bacillus anthracis, Bacillus subtilis, Bacillus cereus, Bacillus cytotoxicus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus and Bacillus weihenstephanensis.

Preferably, the donor is selected from the group of Bacillus anthracis, Bacillus subtilis, Bacillus cereus, Bacillus cytotoxicus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus and Bacillus weihenstephanensis. A method of producing a micro-organism of the Class Bacilli comprising the steps of: (i) isolating at least one genetic sequence involved in Sec-mediated protein translocation in a donor (ii) transforming at least one genetic sequence into the micro-organism, wherein the genetic sequence is a heterologous sequence.

Preferably, the micro-organism is selected from the group of Bacillus anthracis, Bacillus subtilis, Bacillus cereus, Bacillus cytotoxicus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus and Bacillus weihenstephanensis.

Preferably, the donor is selected from the group of Bacillus anthracis, Bacillus subtilis, Bacillus cereus, Bacillus cytotoxicus, Bacillus thuringiensis, Bacillus mycoides, Bacillus pseudomycoides, Bacillus cytotoxicus and Bacillus weihenstephanensis.

The at least one heterologous genetic sequence may be selected from the genes secA1, secA2, sec Y1, secY2 or secH or homologue thereof.

The at least one heterologous genetic sequence may comprise the Bacillus anthracis secA2 and secY2 genes.

A vector comprising the at least one genetic sequence as described herein. A method for secreting a protein from a micro-organism comprising the steps of:

(i) Obtaining a micro-organism of the Class Bacilli comprising a gene encoding a protein to be secreted and at least one heterologous genetic sequence from a donor encoding at least one gene involved in a Sec-mediated protein translocase; and

(ii) Culturing the microorganism under conditions suitable for expression of the at least one gene involved in Sec-mediated protein translocation and secretion of the protein to be secreted.

Preferably, the donor is of the Class Bacilli. A method of producing a micro-organism substantially as shown in and described with reference to the drawings.

A micro-organism substantially as shown in and described with reference to the drawings

Brief Description of the Drawings

Figure 1 shows a schematic representation of secretion pathways in Bacillus anthracis.

Figure 2 shows a schematic representation of transformation of B. subtilis with B. anthracis secA1.

Figure 3 shows a schematic representation of transformation of B subtilis.

Figure 4 shows a schematic representation of transformation of B. subtilis with B. anthracis secA1.

Figure 5 shows a schematic representation of transformation of B. subtilis.

Figure 6 shows a schematic representation of transformation of β. subtilis with B. anthracis secA2 and secY2.

Figure 7 shows a schematic representation of transformation of B. subtilis with B. anthracis secA 1.

Figure 8 shows a schematic representation of transformation of B. subtilis with B. anthracis secA 1.

Figure 9 shows a schematic representation of transformation of B. subtilis with B. anthracis secA2.

Figure 10 shows a schematic representation of transformation of B. subtilis with B. anthracis secY1. Figure 11 shows a schematic representation of transformation of B. subtilis with B. anthracis secY2.

Figure 12 shows a schematic representation of transformation of B. subtilis with B. anthracis secA2 and secY1.

Figure 13 shows a schematic representation of transformation of B. subtilis with B. anthracis secA2.

Figure 14 shows a schematic representation of transformation of B. subtilis with B. anthracis secA2.

Figure 15 shows a schematic representation of transformation of B. subtilis with B. anthracis BA881.

Figure 16 shows a schematic representation of transformation of B. subtilis with B. anthracis BA881.

Figure 17. Complementation of B.anthracis AsecA2-secH with the complementing plasmids/proteins. PageRuler™ Prestained Protein Ladder (Fermentas) was used.

Figure 18. Secretion of EA1 (the product of the eag genes) by various knockout mutants. Knocking out secA2 prevents sap and eag expression and this is overcome by the complementation plasmid. Knocking out secH reduces but does not abolish EA1 secretion, but secA2 is essential.

Figure 19. Matrix of interactions of components of the translocation machinery and their substrates. The bacterial two-hybrid system showing interaction of translocations components fused to the T18 subunit versus T25 fusions of the adenylate cyclase are shown. SecH interacts with SecA2 and EA1. Blue spots show positive result, white spots show negative one.

Figure 20. Overlaid false colour images 2-dimensional polyacrylamide gels (2D-PAG) of the proteins secreted by the wild-type B. anthracis (green) and the secH-null mutant (red). The Sap and EA1 proteins are coloured red/orange indicating that their secretion by the mutant is significantly lower than that of the wild type.

Figure 21. The sequences of putative signal peptide binding domains and associated a-helices 1 , 2 and 3 are shown for various strains of Bacillus encoding either a single (SecA) or two (SecA1 and SecA2) SecA-like proteins. The locations of positively (light shading) and negatively (dark shading) charged residues are shown. The relevant region of E. coli SecA is included and the numbering relates to this protein to allow direct reference to the work of Musial-Siwek et al. (Musial-Siwek et al., 2007 J Mol Biol. 2007 Jan 19;365(3):637-48.). Eco, E. coli strain K12; Bsu, B. subtilis strain 168; Bee, B. cereus, strains ATCC 14579, ATCC10987 or E33L; Bth, B. thuringiensis serovars konkukian str. 97-27 or Al Hakam.

Example 1

Strains were grown and maintained in Luria-Bertani (LB) medium (per litre: 10g tryptone, 5g yeast extract, 10g NaCl) at 37°C. Antibiotics were used in the following concentrations: ampicillin 50μg/ml; chloramphenicol 5 μg/ml; erythromycin 1 μg/ml; kanamycin 10μg/ml.

Isopropyl β-D thiogalactopyranoside (IPTG) was added at 1 mM and xylose at 1% (w/v) to induce gene expression from the P _spac and P _xyl promoters, respectively.

A series of strains was constructed to analyse the ability of B. anthracis secA homologues to complement the essential activity of B. subtilis secA.

The integrative plasmid pMUTIN4 (Vagner et al 1998) was used to generate a strain of B. subtilis in which secA was under the control of the IPTG-inducible P _spac promoter.

The RBS and 5' terminus of secA was amplified with the following primer pair: BsuSecAF and BsuSecAR and the product was cloned into the Hind\\\ and BamH\ sites of pMUTIN4. The resulting plasmid pRCW103 was integrated into B. subtilis via a single crossover recombination to generate strain RCW203 in which secA was under the control of the Pspac promoter.

Multi-copy plasmid pMAP65 (Petit ef al. 1998) encoding an extra copy of the lactose repressor gene (lacl) was introduced into this strain to reduce the non-induced level of expression of secA, generating strain RCW204 whose growth was conditional on the addition of IPTG.

Subsequently, copies of β. anthracis homologues (i.e. secA1 and secA2) of the B. subtilis secA gene were integrated into the amyE locus of the B. subtilis chromosome via a single crossover combination, using pJPR1-based integration plasmids pRCW211 (secAl) and pRCW212 (secA2) and the following primer pairs: BanSecA1-fwd and BanSecA1-rev; BanSecA2fwd and BanSecA2-rev.

Expression of these secA homologues was under the control of the xylose-inducible Pxyl promoter.

These sequential integration events resulted in the creation of B. subtilis strains (secAl, RCW307; secA2 RCW308) in which the B subtilis secA gene and one or other of its B. anthracis homologues can be jointly or independently expressed by the addition of IPTG and/or xylose to the growth medium.

The secA2 (BA0882) and secY2 (BA2734) genes of B. anthracis were inactivated using plasmid pUTE583 (Chen et al 2004b). 5' and 3' fragments of secA2 or secY2 were amplified from the B. anthracis chromosome and cloned into pUTE583 flanking the OKm ^r kanamycin resistance cassette (Chen et al., 2004b). The primer pairs used to generate the secA2 and secY2 5' and 3' fragments were as follows: Ban5'SecA2Fwd and Ban5'SecA2Rev; Ban3 ^* SecA2Fwd and Ban3SecA2Rev; Ban5'SecY2Fwd and Ban5'SecY2Rev; Ban3SecY2Fwd and Ban3'SecY2Rev.

The resulting plasmids pSA102 (secA2) and pUM201 (secY2), were used to integrate the ΩΚηΥ cassette into the secA2 and secY2 genes via double crossover integration events. Example 2

Further examples are illustrated in Figures 2 to 17, which show various plasmids constructed in the experiments and used in transformation of B. anthracis genes into B. subtilis.

Figure 2 shows plasmid of pGEM-Aup-Km-A1-Adown was used to transform B. subtilis 168, selecting for Km (20μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for replacement of β. subtilis secA with B. anthracis secA1 by PCR.

Figure 3 shows plasmid DNA of pEag or pSap was. used to transform β. subtilis 168 selecting for Cm (10μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated eag or sap genes at the amyE locus by PCR.

Figure 4 shows gDNA of Bsu secA::secA1 was used to transform β. subtilis amyE::eag (or sap) selecting for Km (20μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for replacement of B. subtilis secA with B. anthracis secA1 by PCR.

Figure 5 shows plasmid DNA of pAB-Eag or pAB-Sap was used to transform B. subtilis 168 selecting for Cm(10μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated prsAB, eag or prsAB, sap genes at the amyE locus by PCR.

Figure 6 shows plasmid DNA of pAX01-secA2Y2 was used to transform 8 subtilis amyE::prsAB-eag (or sap) selecting for Cm(10μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated B. anthracis secA2 and secY2.

Figure 7 shows gDNA of Bsu secA::secA1 was used to transform B subtilis lacA::secA2Y2, amyE::prsAB-eag (or sap), colonies were selected by Km (20μg/ml). Chromosomal DNA from transformant colonies were checked for replacement of B. subtilis secA with B. anthracis secA1 by PCR. Figure 8 shows plasmid of pAX01-secA1 was used to transform B subtilis amyE::prsAB-eag (or sap) selecting for Em (1 μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated 6. anthracis secA1 gene.

Figure 9 shows plasmid of pAX01-secA2 was used to transform B. subtilis amyE::prsAB-eag (or sap) selecting for Em (1 μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated B. anthracis secA2 gene.

Figure 10 shows plasmid of pAX01-secY1 was used to transform B. subtilis amyE::prsAB-eag (or sap) selecting for Em (1μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated B. anthracis secY1 gene.

Figure 11 shows plasmid of pAX01 -secY2 was used to transform B subtilis amyE::prsAB-eag(or sap) selecting for Em (1 μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated B. anthracis secY2 gene.

Figure 12 shows plasmid of ρΑΧΟΊ -secA2Y1 was used to transform B subtilis amyE::prsAB-eag(or sap) selecting for Em (1 μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated B. anthracis secA2 and secY1 genes.

Figure 13 shows plasmid of pAX01-secA2-BA881 was used to transform B subtilis secA::secA1 , AmyE::prsAB-eag (or sap) selecting for Em (1 μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated B. anthracis secA2 and BA881 genes.

Figure 14 shows plasmid of pAX01-secA2 was used to transform B subtilis secA::secA1 , amyE::prsAB-eag (or sap)) selecting for Em (1 μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated B. anthracis secA2 gene. Figure 15 shows plasmid of pAX01-BA881 was used to transform B subtilis amyE::prsAB-eag (or sap) (or sap) ) selecting for Em (1 μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated B. anthracis BA881 gene.

Figure 16 shows plasmid of ρΑΧ01-ΒΑ881 was used to transform B subtilis secA::secA1 , amyE::prsAB-eag (or sap) ) selecting for Em (1 μg/ml) resistance. Chromosomal DNA from transformant colonies were checked for the incorporation of the xylose regulated B. anthracis BA881 gene.

Results were analysed using SDS-PAGE and showed that replacement of secA in B. subtilis with genes involved in the secondary pathway in B. anthracis (secA2, secY2) improved yield of proteins Eag (EA1 ) and Sap in the culture medium. Replacing Bacillus subtilis secA with B. anthracis secA 1 also increased the yield of protein Eag (EA1 ) and Sap in the culture medium.

Transforming both β. anthracis secA homologues secA1 and secA2 into B. subtilis increased protein yield but the highest protein yields were shown in transformants having secA2 and secY2. However, in the absence of secY2 resulted in lower yield, and demonstrating that secY2 is important for higher protein production via a secondary pathway in B. subtilis. This is in contrast to the situation in B. anthracis, in which knocking out secA2 leads to no detectable Sap or EA1 secretion while knocking out secY2 makes no difference to Sap or EA1 secretion. Therefore for optimum function of the secondary translocase in B. subtilis, both the secA2 and SecY2 genes were required, whereas in B. anthracis, only the secA2 gene is required.

Since other bacteria of the class Bacilli have homologues of the secA and sec V-like genes, elements of the secondary Sec pathway in B. anthracis can be successfully reconstructed in commercially important bacteria.

Other bacteria sharing some or all homologues of B. anthracis secA2, secY2 and BA0881 genes include, but not exclusively, strains of B. cereus, B. cytotoxicus, B. thuringiensis, and B. weihenstephanensis. The Bacillus cereus group of bacteria includes Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis and Bacillus weihenstephanensis and Bacillus cytotoxicus.

Not all representatives of the Bacillus cereus group have been sequenced. However, those that have appear to share two secY genes, while only the pathogenic members of the groups have two sec/* genes.

The invention provides a second (or additional) pathway that can be used to secrete commercially valuable proteins from Bacilli bacteria at much higher concentrations than can presently be achieved, and with improved strain viability.

This can be achieved by cloning in genes for a desired protein and directing that protein through the second pathway and/or by modifications to the existing pathway by replacing components of the pathway in one micro-organism of the class Bacilli with components of a pathway from another micro-organism of the class Bacilli.

For example, components of the B. subtilis primary pathway may be replaced with components from B. anthracis and/or other members of the class Bacilli, and in particular of the B. cereus group.

Presently, proteins are secreted for industrial purposes using Bacilli bacteria directing proteins through the existing primary translocase. However, the longer these bacteria are used, the more their integrity and viability declines, together with their productivity. This is likely to be due to the need for the overproduced industrial proteins having to competing with, and interfering with, the translocation of essential cell housekeeping proteins.

The advantage in using the secondary translocase from one species of Bacilli to channel targeted proteins across the membrane of other Bacilli bacteria is that this can be achieved without the need to disrupt the main or primary translocase pathway in those bacteria, which is required for the production/secretion of essential housekeeping proteins. The examples show the transfer of a heterologous genetic sequence from a B. anthracis donor to B. subtilis. However, similar transfers of genetic material between other bacteria of the class Bacilli are possible.

The invention is particularly applicable to members of the Bacillus cereus group, which includes Bacillus anthracis, Bacillus cereus, Bacillus mycoides, Bacillus pseudomycoides, Bacillus thuringiensis and Bacillus weihenstephanensis and Bacillus cytotoxicus.

Not all representatives of the group have been sequenced. However, those that have appear to share two secY genes, while only the pathogenic members of the groups have two secA genes.

Example 3: Further data for the present invention

Background

In bacteria, the vast majority of proteins are secreted by the Sec translocase, comprising proteins SecY, SecE, SecG, SecD, SecF (in various Gram-positive species SecD and SecF are fused as a single protein - SecDF). SecA (or SecA1 for the Gram- positive bacteria that have two SecA-like proteins) is a multifunctional component that identifies Sec substrates, pilots them to the translocase, and acts as the translocase motor to drive substrates through the pore of the translocase.

In the Gram-negative bacterium E. coli, the secretion of some Sec substrates is improved by SecB, an intracellular chaperone that is absent from all Gram-positive bacteria.

The present invention

SecA2.

Members of the Bacillus cereus group (including B, anthracis, B. cereus, thuringiensis) encode a homologue of SecA and therefore has two SecA proteins called SecA1 (BA5421 ) and SecA2 (BA0882). Alignment of the SecA/SecA1 homologues of β. subtiiis and B. anthracis reveals a high degree of identity (73%) throughout their lengths. In contrast, B. anthracis SecA2 (788 residues) is significantly shorter than both B. subtiiis SecA (841 residues) and B. anthracis SecAl (835 residues), and exhibits a lower degree of identify (48%), particularly in the C-terminal third of the proteins. All three proteins have two well-conserved Walker motifs (NBF1 and NBF2).

Complementation analysis was used to determine the functionality of the B. anthracis SecA-like proteins in . S. subtiiis. B. subtiiis strain RCW203 was generated in which secA, an essential gene, was placed under the control of an IPTG-inducible Pspac promoter via the pMUTIN4 integration plasmid. Although the Pspac promoter of pMUTIN4 is optimised to minimize non-induced levels of expression, the efficiency of plating (eop) of RCW203 was virtually the same irrespective of the presence of IPTG, indicating that Pspac regulation was not sufficiently stringent to prevent the synthesis of growth-sustaining levels of SecA. The required level of stringency was established (eop <0.01 ) by incorporating of pMAP65, encoding additional copies of the lactose repressor (Lacl), to generate strain RCW204.

A xylose-inducible B. anthracis secA l gene was integrated into the chromosome of RCW204 via integration plasmid pRCW21 1. The resulting strain (RCW307) grew in the presence of either IPTG or xylose, but not in their absence (eop. <0.01 ). This shows that B. anthracis SecAl complements B. subtiiis SecA. A similar construct (RCW308with pRCW212) with a xylose-inducible chromosomal copy of secA2 was only able to grow in the presence of IPTG, showing that SecA2 was not able to complement B. subtiiis SecA. These studies demonstrate that B. anthracis SecAl and SecA2 are distinguishable on the basis of their functional activity.

The roles of SecAl and SecA2 in B. anthracis strain UM23C1-2 were determined by attempting to generate secA l and secA2 knockout mutants using derivatives of plasmid pUTE583 with 5' and 3' fragments of secAl (pUM101 ) or secA2 (pSA102) separated by the OKrn element encoding kanamycin resistance. Despite extensive attempts, we failed to isolate a sec>A7-null mutant. This suggests that SecAl is essential and not able to be complemented by SecA2. As as implied by the B. subtiiis complementation studies, SecAl and SecA2 appear to have distinct functional activities. In contrast, the inventors did isolate a secA2-nu\\ mutant (SA102) and its growth characteristics that were indistinguishable from those of the parental. This indicates that SecA2 is not responsible for the secretion of proteins that are essential for growth in vivo.

The inventors next tested whether SecA2 was responsible for the secretion of specific substrates. The parental strain (UM23C1-2) and secA2-nu\\ mutant were grown in nutrient medium and proteins recovered from the culture medium analysed by 20- PAGE. The patterns of proteins secreted were similar excepting for two prominent, high molecular mass (~90kDa.) proteins (red-coloured spots, Fig. 20), identified by mass spectrometry as Sap (surface associated protein) and EA1 (E antigen 1 ). These surface (S-) layer proteins were the major extracellular proteins in the culture medium of the parental strain UM23C1 -2, while only traces were present in cultures of the secA2-null mutant.

A key conclusion from this research is that SecA1 and SecA2 are functionally distinct. SecA2 interacts with specific substrates, Sap and EA1 , directing them to the translocase. While the elements on SecA2 and the substrates that are involved in the targeting are yet to be identified, the inventors believe on the basis of previous work that it is the PPXD domain of SecA2 and the signal peptides of the substrates. It is their opinion that incorporation of SecA2, with or without SecY2, into a producer bacterium will increase the secretion proteins carrying the targeting signals of Sap/Ea1 by reducing competition between essential housekeeping proteins and the target protein for SecAI .

SecH

The gene disclosed in GenBank entry BA0881 , now called SecH, is encoded by the same operon as SecA2 and are expressed together. The inventors have make knockout mutants of secA2-H, secA2and secH. In the latter two mutants, they have confirmed that the other gene is expressed.

• The secA2-H mutant lacks both genes and fails to secreted Sap and EA1. If the mutant is complemented by a plasmid-based copy of secA2-H or just secA2, then secretion is restored, but not by secH alone. The data is presented in Figure 17. • The secA2 mutant only lacks SecA2, but produces SecH. This mutant fails to secrete Sap and EA1. The data is presented in Figure 18.

• The secH mutant only lacks SecH, but produces SecA2. This mutants secretes Sap and EA1 but at about 20% of the wild type (Fig. 20).

This data suggests that SecH, a cytoplasmic protein only found in members of the B. cereus group that encode SecA2 interacts with SecA2 to improve the secretion of Sap and EA1. It is the inventors view that this is a type of secretion specific chaperone. Bacterial two-hybrid analysis indicates that SecH interacts specifically with SecA2 and EA1. The data is presented in Figure 19.

SecH appears to interact with SecA2 and substrate to increase secretion. It is therefore a novel cytoplasmic secretion component.

Details of sequences of the invention

The inventors have identified components of the Sec-mediated protein translocation pathway relevant to the present invention.

Presented directly below is an alignment of the polypeptide sequence of B. subtilis SecA (Bsu_SecA), and B. anthracis SecA1 (Ban_SecA1 ) and SecA2 (Ban_SecA2).

Presented in Table 1 below are percentage identity and similarity between B. subtilis SecA and its B. anthracis homologues, SecA1 and SecA2, in the various identified structural/functional domains.

Table 1

Presented directly below is an alignment of the polypeptide sequence of Eco_secY, Bsu_SecY, Ban_secY and Ban_SecY2

Presented directly below is an alignment of the polypeptide sequence of Bsu_SecA, Ban_SecA, Eco_SecA and Ban_SecA2

NB THE SECA2 SEQUENCES FROM OTHER MEMBERS OF THE B. CEREUS GROUP ARE VIRTUALLY IDENTICAL.

Presented directly below is a table showing the presence or absence of SecA2 and SecY2 in Bacillus strains

Additional nucleic acid and polypeptide sequences relevant to the invention