AGONISTS OF TLR4/MD-2

CROSS REFERENCE

[0001] The present application is a non-provisional application claiming priority to U.S. Provisional Patent Application Serial Number 61/313,562, filed March 12, 2010, the disclosure of which is incorporated in its entirety herein by reference.

FIELD OF THE INVENTION

[0002] The present invention is directed to Gram-negative bacteria substantially lacking a iigand that acts as an agonist of TLR4/MD-2. The present invention is also directed to methods of generating such Gram-negative bacteria and uses thereof.

BACKGROUND OF THE INVENTION

[0003] Lipopolysaccharide (LPS) is the main outer membrane surface-associated component in Gram-negative bacteria and is associated with an array of pathological and physiological activities in mammalian host immune responses. LPS-mediated toxicity caused by these bacteria is generally due to lipid A, the hydrophobic moiety of LPS, which can function as an agonist of Toll-like receptor 4 (TLR4)/MD-2. Lipid A comprises two glucosamine residues with six acyi chains attached.

[0004] Kdo (3-deoxy-D-manno-octulosonate) is considered an essential component of LPS, and it is believed that the minimal LPS structure required for growth of E. coli is two Kdo residues attached to lipid A (Kd0 ₂ -lipid A). Biosynthesis of Kdo begins with API (D-arabinose 5-phosphate isomerase), which coverts D-ribuiose 5- phosphate (Ru5P) into ASP (D-arabinose 5-phosphate). In £. coll K-12, there are two API genes, kdsD and gutQ. Next, ASP is condensed with phosphoenolpyruvate (PEP) to form Kdo 8-phosphate (Kdo8P), which is then hydrolyzed forming Kdo. Kdo is subsequently activated as the sugar nucleotide CMP-Kdo, which is ultimately transferred to lipid IVA forming Kd0 ₂ -iipid IVA. The Kdo-dependent acyltransferases LpxL and Lpx transfer laurate and myristate, respectively, to Kd0 ₂ -lipid IV _A to form Kdo ₂ -Hpid A.

[0005] The strain E. coll K-12 TCM15, which has both API genes (kdsD and gutQ) deleted and thus lacks Kdo, is not viable unless supplied with exogenous A5P (e.g., see Meredith and Woodard, identification of GutQ from Escherichia coll as a D- arabinose 5-phosphate isomerase, J Bacteriol 187:6938, 2005). The present invention features viable Gram-negative bacteria that substantially lack a ligand that acts as an agonist of TLR4/MD-2. Additional information regarding TLR4/MD-2 may be found at, for example, Qureshi et al., J. Exp. Med., Vol. 189, No.4, February 15, 1999, Pages 615-625; Shimazu et al., J. Exp. Med., Volume 189, Number 1 1 , June 7, 1999, Pages 1777-1782; Poltorak et al., Science 282, 2085 (1998), the disclosures of which are incorporated in their entirety by reference herein to the extent that the disclosures are consistent with the present invention. The ligand, for example, may comprise lipid A or a 6-acyi lipid. The viable Gram-negative bacteria comprise suppressor mutations, which enable viability despite lacking otherwise essential Kdo. Mam at et al. have described suppressor mutations in the yhjD and msbA genes non-conditional of E coll TCM15 derivatives (see Moi Microbiol 67(3):633, 2008).

[0006] Any feature or combination of features described herein are included within the scope of the present invention provided that the features included in any such combination are not mutually inconsistent as will be apparent from the context, this specification, and the knowledge of one of ordinary skill in the art. Additional advantages and aspects of the present invention are apparent in the following detailed description and claims.

SUMMARY

[0ΘΘ7] The present invention features viable Gram-negative bacteria comprising an outer membrane that substantially lacks a ligand (e.g., lipid A, 6-acyl lipopoiysaccharide, etc.) that acts as an agonist of TLR4/MD2, especially that lacks an agonist of human TLR4/MD2. The ligand may activate a production of costimulatory immune response molecules in a mammal (e.g., inducing macrophages to synthesize mediators of inflammation). In some embodiments, the Gram-negative bacterium is an Escherichia coii strain (e.g., KPM22, KPM22 L1 , KPM22 L1 1 , KPM22 L13, KPM22 L14, KPM22 L15, KPM22 L18, KPM22 L20, KPM316, KPM318, KPM334 through KPM362) or a Pseudomonas strain.

[0008] The viable Gram-negative bacterium may further comprise reduced activity of functional arabinose-5-phosphate (ASP) isomerases KdsD and GutQ. In some embodiments, the remaining activity of the arabinose-5-phosphste isomerases with reduced activity is insufficient to provide substrate for the Kdo biosynthetic pathway (e.g., zero or inadequate substrate for the Kdo biosynthetic pathway as would be understood by one of ordinary skill). In some embodiments, this is achieved by complete deletion of both genes. In the present KPM318 and some earlier IpxL IpxM deletions allow full restoration of Kdo synthesis (by growing on A5P) while limiting the lipid form to lipid SVA, when grown in the presence of A5P as supplement.

[0009] In some embodiments, the viable Gram-negative bacterium further comprises one or more suppressor mutations, for example a suppressor mutation in a transporter (e.g., MsbA such as MsbA-P50S, sbA-Pf SS, MsbA-T283A, sbA- R310S, or MsbA~L48F) thereby increasing the transporter's capacity to transport lipid IVA, a suppressor mutation affecting membrane protein YhjD (e.g., YhjD- R134C), a suppressor mutation that enables growth at 42 degrees Celsius, etc.

[0010] in some embodiments, one or more genes (e.g., IpxL, IpxM, pagP, IpxP, and/or eptA) are substantially deleted and/or one or more enzymes (e.g., LpxL, LpxM, PagP, LpxP, and/or EptA) are substantially inactive. For example, the viable Gram-negative bacterium may comprise a site-specific recombinase scar sequence at a site of a partial or complete deletion of one or more of genes (e.g., IpxL, IpxM, pagP, IpxP, eptA), E. coli strains may include but are not limited to: KPM279, KP 280, KP 288, KP 290, KPM296, KP 300, KP 303, KPM310, KPM312, KPM314, KPM318, KP 317, KP 318, KP 334 through KP 362. In some embodiments, a gene encoding for a DNA restriction enzyme and/or a DNA methylation enzyme and/or a gene encoding for recA and/or endA is mutated or deleted.

[0011] The bacterium may exhibit various growth characteristics. For example, in some embodiments, the bacterium can grow exponentially at 42 degrees Celsius. For example, in some embodiments, the bacterium exhibits an exponential phase doubling time of less than about 35 minutes, less than about 40 minutes, or less than about 45 minutes at above 37 degrees Celsius. In some embodiments, the bacterium remains viable in a salt concentration between about 0.1 M and 0.9M.

[0012] In some embodiments, the bacterium comprises a means of conferring resistance to an antibiotic. In some embodiments, the bacterium comprises an F plasmid, an F' plasmid, or a gene encoding for F pilus production. In some embodiments, the bacterium can propagate bacteriophages fd, Ml 3, or bacteriophages related to fd or M13. In some embodiments, the bacterium is competent to take up extracellular DNA (e.g., electrocompetent).

[0013] The present invention also features outer membranes derived from such Gram-negative bacteria, the outer membranes substantially lacking a ligand that is an agonist of TLR4/MD2. For example, the outer membrane may be derived from the Gram-negative bacteria described herein. The present invention also features viable Gram-negative bacteria (e.g., Gram-negative bacteria described herein such as E. co!i and/or Pseudomonas) lacking an O-acyl transferase that can acylate a lipid A precursor iipidA or Lipid IVA. O-acyl transferases may include LpxL, LpxM, LpxP, and PagP.

[0014] In some embodiments, a Gram-negative bacterium as described herein is a donor bacterium. For example, the Gram-negative donor bacterium may comprise a DNA cassette comprising a gene (e.g., IpxL, IpxM, pagP, IpxP, eptA) having an open reading frame deletion and a means of conferring resistance to an antibiotic (e.g., kanamycin, penicillin, neomycin, tetracycline, chloramphenicol, or ampicillin), wherein the cassette is flanked by sites that are a target for a site-specific recombinase enzyme.

[0015] The present invention also features methods of selecting Gram-negative bacteria capable of exponential growth at a temperature above 40 degrees Celsius, wherein the Gram-negative bacterium substantially lacks a ligand that acts as an agonist of TLR4/MD2, In some embodiments, the method comprises growing a strain that substantially lacks the ligand that acts as an agonist of the TLR4/MD2 receptor and has a suppressor mutation that allows growth between about 30 to 40 degrees Celsius (e.g., a strain described herein); plating the strain on a suitable nutrient medium; and incubating the plated strain at 42 degrees Celsius until single colonies of bacteria appear.

[0016] The present invention also features methods of constructing a bacterium substantially lacking a gene such as IpxL, !pxM, pagP, IpxP, and eptA. The method comprises obtaining a viable Gram-negative bacterium comprising an outer membrane that substantially lacks a ligand that acts as an agonist of a TLR4/MD2 receptor (e.g., a strain described herein). In some embodiments, the viable Gram- negative bacterium comprises a DNA cassette comprising a modified gene (e.g., a modified antibiotic resistance gene), wherein the modified gene is a modified target gene having an open reading frame deletion. The DNA cassette may further comprise a means of conferring resistance to an antibiotic and/or may be flanked by appropriate target sites for a site-specific recombinase. The method further comprises subjecting the bacterium to P1 vir transduction and replacing the target gene with the modified gene (e.g., a modified antibiotic resistance gene) via homologous recombination. In some embodiments, the method further comprises deleting the antibiotic resistance gene by transient introduction of a site-specific recombinase capable of acting on sequences that flank the antibiotic resistance gene, thereby deleting the resistance gene and leaving a scar characteristic of the site-specific recombinase.

[0017] The present invention also features methods of producing a DNA sample and/or a protein sample substantially free of a ligand that acts as an agonist of TLR4/ D2. The method comprises obtaining a Gram-negative bacterium comprising an outer membrane that substantially lacks the ligand that acts as an agonist of the TLR4/ D2 receptor (e.g., a strain described herein), wherein the bacterium is competent to take up extracellular DNA. For producing the DNA sample, the method further comprises introducing a DNA vector to the bacterium, wherein the bacterium amplifies the DNA vector, and harvesting a DNA sample from the bacterium via a standard DNA isolation protocol. Such DNA isolation protocols are well known to one of ordinary skill in the art,

[0018] For producing the protein sample, the method may comprise introducing a DNA vector expression construct to the bacterium, wherein the vector comprises both a gene encoding a protein of interest expressed from a functional promoter and a selectable marker gene, growing the bacterium and allowing or inducing the bacterium to express the protein of interest, and harvesting the bacterium and treating the bacterium to release the protein of interest. In some embodiments, the bacterium comprises a mutation or deletion in one or more genes such as !olA, io!B and/or /pp. In some embodiments, for producing the protein sample, the method may comprise introducing a DNA vector expression construct to the bacterium, wherein the vector comprises both a gene encoding a protein of interest expressed from a functional promoter and a selectable marker gene, growing the bacterium and allowing or inducing the bacterium to express the protein of interest, and removing the bacterium from the culture medium to leave the protein of interest in the culture medium.

[0019] The present invention also features a library of bacteria substantially free of a ligand that acts as an agonist of the TLR4/MD2 receptor (e.g., a strain described herein), wherein the library of bacteria expresses a series of variants of a protein. The library of bacteria may be useful for screening the protein variants for activity in a mammalian ceil based assay. In some embodiments, the members of the library are generated by phage display or by mutagenesis of a piasmid vector expressing a prototype member of the library,

[0020] Preferably, the bacteria are able to grow at least as rapidly in rich media supplemented with arabinose-5-phosphate as they grow in the same rich media lacking arabinose-5-pbosphate, allowing a choice of outer membrane composition dependent upon the arabinose-5-phosphate without the supplement leading to a slower growth rate. Growth with supplement should be at least 95% of the unsupplemented growth rate and preferably a faster rate than the unsupplemented rate. [0021] The viable Gram-negative bacteria described herein, various components thereof, reagents, and/or materials, may be available in the form of a kit. For example, in some embodiments, the kit comprises a viable Gram-negative bacterium having an outer membrane substantially free of a iigand that acts as an agonist of TLR4/MD2 (e.g., a strain described herein), wherein the bacterium is competent to take up extracellular DNA.

DEFINITIONS

[0022] The term "viable non-toxic Gram-negative bacteria" refers to viable Gram- negative bacterial strains comprising an outer membrane substantially free of a Iigand that acts as an agonist of TLR4/MD2.

[0023] The terms "ceils" and "host cells" and "recombinant host cells", which are used interchangeably herein, refer to ceils that are capable of or have been transformed with a vector, typically an expression vector. The host cells used herein may be Gram-negative bacteria. It is understood that such terms refer not only to the particular subject ceil, but to the progeny or potential progeny of such a cell. Because certain modifications may occur in succeeding generations due to either mutation or environmental influences, such progeny may not, in fact, be identical to the parent cell, but are still included within the scope of the term as used herein.

[0024] The terms "media" and "culture medium" is recognized in the art, and refers generally to any substance or preparation used for the cultivation of living cells.

[0025] The term "derived from," as used, for example, in the context of deriving lipid IVA from a strain of Gram-negative bacteria, refers to lipid IVA that can be obtained from the bacteria or the protein, and is intended to include fragments or portions of proteins.

[0026] The term "defective", as used herein, with regard to a gene or gene expression, may mean that the gene is not a wild type gene and that the organism does not have a wild type genotype and/or a wild type phenotype. The defective gene, genotype or phenotype may be the consequence of a mutation in that gene, or of a gene thai regulates the expression of that gene (e.g., transcriptional or post- transcriptional), such that its normal expression is disrupted or extinguished. "Disrupted gene expression" is intended to include both complete inhibition and decreased gene expression (e.g., as in a leaky mutation), below wild type gene expression.

[0027] The term "Gram-negative bacteria" is recognized in the art, and refers generally to bacteria that do not retain Gram stain (e.g., the deposition of a colored complex between crystal violet and iodine). Sn an exemplary Gram stain, cells are first fixed to a slide by heat and stained with a basic dye (e.g., crystal violet), which is taken up by ail bacteria (i.e., both Gram-negative and Gram-positive). The slides are then treated with an iodine-K! mixture to fix the stain, washed with acetone or alcohol, and finally counterstained with a paler dye of different color (e.g., safranin). Gram-positive organisms retain the initial violet stain, while Gram-negative organisms are decolorized by the organic solvent and hence show the counterstain. Gram-negative bacteria and cell lines include, but are not limited to, Escherichia spp., Shigella spp., Salmonella spp., Campylobacter spp., Neisseria spp., Haemophilus spp., Aeromonas spp., Francisella spp., Yersinia spp., Klebsiella spp., Bordetelia spp., Legionella spp., Cifrobacter spp., Chlamydia spp., Brucella spp., Pseudomonas spp., Helicobacter spp. and Vibrio spp.

[0028] The term "mutant Gram-negative bacteria", "LPS mutant Gram-negative bacteria", "kdsD and gutQ mutant Gram-negative bacteria", "API mutant Gram- negative bacteria" or similar terms, as used herein, includes Gram-negative bacteria of the invention that have been mutated one or more times in, for example, one or more of the gutQ, kdsD, kdsA, kdsB, waaA, msbA, yhjD genes, of any other biosynthetic, processing, or trafficking gene thereby producing an outer membrane substantially lacking LPS or other iigand that acts as an agonist of TLR4/ D2.

[0029] An "immunogenic portion of a molecule" refers to a portion of the molecule that is capable of eliciting an immune reaction against the molecule in a subject.

[0030] The term "isolated" as applied to LPS or lipid !VA molecules, refers to LPS or lipid !VA which has been isolated (e.g., partial or complete isolation) from other bacterial components, in particular from the outer membrane.

[0031] As used herein, the term "portion" when used in reference to a sequence (e.g., an amino acid sequence of a protein, a nucleic acid sequence of a gene) represents any amount of the referenced sequence (e.g., 0.001 %, 0.1 %, 1 %, 10%, 30%, 50%, 75%, 80%, 85%, 90%, 95%, 98%, 99.999% of an amino acid sequence or nucleic acid sequence).

[0032] The term "modulation" as used herein refers to both upregulation (i.e., activation or stimulation, e.g., by agonizing or potentiating) and downregulation (i.e., inhibition or suppression, e.g., by antagonizing, decreasing or inhibiting). The term "inducible" refers in particular to gene expression which is not constitutive but which takes place in response to a stimulus (e.g., temperature, heavy metals or other medium additive).

[0033] The term "nucleic acid" refers to polynucleotides or oligonucleotides such as deoxyribonucleic acid (DNA), and, where appropriate, ribonucleic acid (RNA). The term should also be understood to include, as equivalents, analogs of either RNA or DNA made from nucleotide analogs and as applicable to the embodiment being described, single (sense or antisense) and double-stranded polynucleotides.

[0034] As used herein, the term "transfection" means the introduction of a nucleic acid (e.g., via an expression vector) into a recipient cell by nucleic acid-mediated gene transfer. "Transformation", as used herein, refers to a process in which a cell's genotype is changed as a result of the cellular uptake of exogenous DNA or RNA. In an illustrative embodiment, a transformed cell is one that expresses a mutant form of one or more of the kdsD and gutQ genes. A transformed cell can also be one that expresses a nucleic acid that interferes with the expression of a gutQ, kdsD, kdsA, kdsB, waaA, msbA, yhjD gene of any other biosynthetic, processing, or trafficking gene.

[0035] As used herein, the term "transgene" means a nucleic acid (e.g., a mutant kdsD, gutQ, kdsA, kdsB, waaA, msbA, yhjD gene of any other biosynthetic, processing, or trafficking gene, or an antisense transcript thereto) that has been introduced into a cell. A transgene could be partly or entirely heterologous, e.g., foreign, to the transgenic animal or cell into which it is introduced, or, can be homologous to an endogenous gene of the organism or- ceil into which it is introduced, but which is designed to be inserted, or is inserted, into the animal or cell's genome in such a way as to alter the genome of the cell into which it is inserted. A transgene can also be present in a cell in the form of an episome.

[0036] The term "treating" a subject for a condition or disease, as used herein, is intended to encompass curing, as well as ameliorating at least one symptom of the condition or disease.

[0037] The term "vector" refers to a nucleic acid molecule, which is capable of transporting another nucleic acid to which it has been linked. Vectors capable of directing the expression of genes to which they are operatively linked are referred to herein as "expression vectors."

[0038] The term "expression system" as used herein refers to an expression vector under conditions whereby an mRNA may be transcribed and/or an mRNA may be translated into protein, structural RNA, or other cellular component. The expression system may be an in vitro expression system, which is commercially available or readily made according to art known techniques, or may be an in vivo expression system, such as a eukaryotic or prokaryotic ceil containing the expression vector. In general, expression vectors of utility in recombinant DNA techniques are often in the form of "plasmids" which refer generally to circular double stranded DNA loops that, in their vector form, are not bound to the chromosome. In the present specification, "plasmid" and "vector" are used interchangeably as the piasmid is the most commonly used form of vector. However, the invention is intended to include such other forms of expression vectors which serve equivalent functions and are well known in the art or which become known in the art subsequently hereto (e.g., cosmid, phagemid and bacteriophage vectors).

[0039] The term "viable" means that the cells are able to grow and divide exponentially for multiple generations in a suitable nutrient medium, including rich media, and that the ceils can be stored under typical storage conditions for that cell type and subsequently re-grown,

BRIEF DESCRIPTION OF THE DRAWINGS

[0040] FIG. 1 shows growth curves of £. coli KPM22 and the KPM22-like strains KPM22 L1 , KPM22 L1 1 , KPM22 L13, KP 22 L14, KP 22 LI 5, KPM22 L18, and KPM22 L20. Generation times were determined in LB medium with shaking at 200 rpm at 37°C. Generation times (under these conditions) were calculated to be as follows: KPM22 40 min, KPM22 L1 37 min, KPM22L1 1 39 min, KP 22 L13 59 min, KPM22 L14 44 min, KPM22 L15 40 min, KPM22 L18 36 min, and KPM22 L20 55 min.

[0041] FIG, 2 shows growth curves of £. coli strain KPM288 (KPM22 L1 A/pxL::Km ⁺ ) and strain KPM290 (KPM22 L1 1 A/pxL::Km ⁺ ) in either Luria-Bertani (LB) media at 37°C or in LB media supplemented with 15 μΜ D-arabinose 5-phosphate (ASP) and 10 μΜ D-giucose8-phosphate (G6P) at 37°C. Generation times in LB media were calculated to be as follows: KPM288 45 min, KPM290 43 min. Results from growth in LB media supplemented with ASP and G6P were as follows: KP 288 ceased to grow exponentially after 2-3 generations, KPM290 46 min,

[0042] FIG. 3 shows growth curves of E. coil strain KPM303 (KPM22 L1 1 AlpxL AlpxM) at 37°C in either LB media, LB media supplemented with 15 μΜ ASP and 10 μΜ G6P, Super Broth (SB) media (containing 10 g/L NaCi), or SB media (containing 10 g/L NaCi) supplemented with 15 μΜ ASP and 10 μΜ G6P. SB media did not improve the growth rate of KPM303 as compared to LB media. However, in contrast to the growth in LB medium supplemented with ASP and G6P, KPM303 continued to grow exponentially after 2-3 generations when cultivated in SB supplemented with ASP and G6P.

[0043] FIG. 4 shows growth curves of £. coli strains KPM303 (KPM22 L1 1 AlpxL AlpxM), KPM312 (KP 22 L1 1 A!pxL AipxM ApagP), and KP 316 (KPM22 L1 1 Δ/pxL AlpxM ApagP AlpxP) at 37°C in either SB media or SB supplemented with 15 μΜ ASP and 10 μ G6P. Generation times in SB media were as follows: KPM303 54 min, KP 312 50 min, KP 318 46 min. Generation times in SB media supplemented with A5P and GBP were as follows: KP 303 39 min, KPM312 42 min, KPM316 42 min.

[0044] FIG. 5 shows growth curves of E coii strain KPM316 at 37°C in either SB medium supplemented with 10 mM MgSQ ₄ and 2 mM CaCi or SB medium supplemented with 10 mM MgS0 ₄ , 2 mM CaCI ₂ , 15 μ ASP, and 10 μ G6P. KPM316 had a generation time of 51 min in SB with 10 mM MgS0 ₄ and 2 mM CaCI ₂ . KPM316 had a generation time of 48 minutes in SB with 10 mM MgS0 , 2 mM CaCI _2i 15 μΜ ASP, and 10 μΜ G6P.

[0045] FSG. 6 shows growth curves of £. coii strains KPM318 and KPM318 grown in either SB or in SB supplemented with 15 μΜ ASP, and 10 μΜ GBP at 37 ^C C . The doubling times are very similar in the range 39-44 min.

[0046] FIG. 7 shows growth curves of £. coii strains KPM296-6 and KPM318 at 37°C in either SB medium supplemented with 10 mM MgS0 ₄ and 2 mM CaC or SB medium supplemented with 10 mM MgS0 ₄ , 2 mM CaC , 15 μΜ A5P, and 10 μΜ GBP. The growth rate of KPM296-6 in SB medium with 10 mM MgS0 ₄ and 2 mM CaCI ₂ was 48 min. In SB with 10 mM MgS0 ₄ , 2 mM CaCI ₂ , 15 μΜ ASP, and 10 μΜ GBP the growth rate of KPM296-8 was 42 min.

[0047] FIG. 8 Growth curves of a series of independent isolates selected from KPM296 at 42°C. Growth was in SB medium at 42°C. Individual isolates are numbered in the inset and keyed to the symbols used to represent their growth curve. Considerable variation in generation time is seen, but each shows exponential growth,

[0048] FIG. 9 Analysis of PGR products produced by primers flanking each gene to confirm deletions of G-acyi transferase genes and deletion of eptA gene in KPM318. The parental strain BW30270 is used as a control beside KPM318 for each gene analysed. Template DNAs and the gene for which the particular PGR primer pairs were designed are indicated. It can be seen in each case that the parental strain BW30270 gives rise to a larger PCR product than that seen for KPM318. Only the scar sequence remains in the place of the whole gene in KPM318.

[0049] FIG. 10A and 10B HEK-Blue™ Assays of strains BW3027G, KPM318 and KPM318. (A) Outer membrane preparations of strains, and (B) whole cells. Numbers of cells added to each well (as cfu - colony forming units) or mass of outer membrane preparation are indicated. Reference LPS from £. coli K-12 supplied with the assay kit was run in parallel with all experiments and is shown for comparison with the outer membrane data with which it is most directly comparable on a mass- to-mass basis.

[0050] FIG. 1 1 Charge deconvoluted electrospray-ionization Fourier-transformed ion cyclotron (ESI FT-ICR) mass spectra in negative ion mode of LPS molecules isolated from £. coli strains KPM316 (A) and KPM318 (B). The mass numbers given refer to the monoisotopic peaks of the neutral molecules. The peak at 1 178.68 u is most likely a triacylated degradation product of lipid IVA (1404.86 u) produced during LPS isolation as it is not consistent with a known pathway intermediate.

[0051] FIG. 12a-g The sequence for Escherichia coii KPM316 {msbA52 AkdsD AgutQ AlpxL A!pxM ApagP AlpxP). msbA52 replaces the wild type allele of msbA, wherein a C at 965895 is replaced by a T, resulting in a Serine instead of Proline at amino acid 18 in the MsbA protein. The following were deleted from the parental strain: AgutQ (FIG. 12b),. AkdsD (FIG. 12c), AipxL (FIG. 12d), AlpxM (FIG. 12e), ApagP (FIG. 12f), and AlpxP (FIG. 12g).

[0052] FIG. 13 Single nucleotide changes are present in KPM31 6 relative to the parental strain MG 655, in addition to the msbA52 mutation and the deletion/insertion to inactivate LPS synthetic genes. None are known or predicted to result in any distinctive phenotype.

[0053] FIG. 14a-h Escherichia coli KPM318 {msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA). msbA52 replaces the wild type allele of msbA, wherein a C at 965895 is replaced by a T, resulting in a Serine instead of Proline at amino acid 18 in the MsbA protein. The fo!iowing were deleted from the parentai strain: AgutQ (FIG. 14b), AkdsD (FIG, 14c), AipxL (FIG. 14d), AlpxM (FIG. 14e), ApagP (FIG. 14f), Δ/ρχΡ (FIG. 14g), and AeptA (FIG. 14f).

[0054] FIG. 15 The BVV30270 and KPM318 strains were sequenced at the Scripps Core DNA sequencing facility using lllumina sequencing. Ail the Single Nucleotide Polymorphisms (SNP) and Deletion/Insertion Polymorphisms (DIP) are indicated. These SNPs and DIPs are assigned based on the comparison of these strains to the Blattners IV1G1855 strain. Only those detected at 100% of reads are noted. The msbA-PI SS suppressor allele in the original KPM22 L1 1 strain was identified.

[0055] FIG. 16 The growth of the four KPM-318 temperature-resistant derivatives, KPM334, KPM335, KPIV1338, and KPM337 grown in SB medium at 30°C were assessed.

[0056] FIG. 17 The growth of the four KPM-318 temperature-resistant derivatives, KPM334, KPM335, KPM336, and KPM337 grown in SB medium supplemented with A5P/G6P at 30°C were assessed.

[0057] FIG. 18 The growth of the four KPM-318 temperature-resistant derivatives, KPM334, KPM335, KPM336, and KP 337 grown in SB medium at 37°C were assessed.

[0058] FIG. 19 The growth of the four KPM-318 temperature-resistant derivatives, KPM334, KPM335, KP 336, and KPM337 grown in SB medium supplemented with A5P/G6P at 37°C were assessed.

[0Θ59] FIG. 20 The growth of the four KPM-318 temperature-resistant derivatives, KPM334, KPM335, KPM336, and KPM337 grown in SB medium at 42°C were assessed.

[0060] FIG. 21 The growth of the four KPM-318 temperature-resistant derivatives, KPM334, KPM335, KPM336, and KPM337 grown in SB medium supplemented with A5P/G6P at 42°C were assessed.

[0061] FSG. 22a-d The four KP -318 temperature-resistant derivatives, KPM334, KPM335, KPM336, and KPM337 were sequenced at the Scripps Core DNA sequencing facility using liiumina sequencing. All the Single Nucleotide Polymorphisms (SNP) and Deletion/Insertion Polymorphisms (DIP) are indicated. These SNPs and D!Ps are assigned based on the comparison of these strains to the Blattner's MG1655 strain. Only those detected at 100% of reads are noted. The msM-P18S suppressor allele in the original KPM22 L1 1 strain was identified.

[0062] FIG. 23 ESI FT-!CR mass spectrum in negative ion mode of lipid IV, _\ (1404.88 u) isolated from £. co!i KPM337 grown at 42°C in LB medium. Mass numbers given refer to the monoisotopic masses of neutral molecules. The peak corresponding to triacylated lipid A (1 178.87 u) is likely an artefact produced during lipid IVA isolation and/or ionization as it is not consistent with a known pathway intermediate.

[0063] FIG. 24 ESI FT-ICR mass spectrum in negative ion mode of LPS isolated from £. coll KPM334 grown at 42°C in LB medium supplemented with A5P/G8P. Mass numbers given refer to the monoisotopic masses of neutral molecules.

[0064] FIG. 25 ESI FT-ICR mass spectrum in negative ion mode of LPS isolated from E coil KPM335 grown at 42°C in LB medium supplemented with A5P/G6P. Mass numbers given refer to the monoisotopic masses of neutral molecules.

[0065] FIG. 26 ESI FT-ICR mass spectrum in negative ion mode of LPS isolated from E coll KPM337 grown at 42 °C in LB medium supplemented with A5P/G6P. Mass numbers given refer to the monoisotopic masses of neutral molecules.

[0066] FIG. 27 ESI FT-ICR mass spectrum in negative ion mode of LPS isolated from E. coii KPM318 grown at 37°C in LB medium supplemented with A5P/G6P (control). Mass numbers given refer to the monoisotopic masses of neutral molecules. Peak assignments are listed in Table 1. [0067] FIG. 28 Detection of LPS in bacterial cell suspensions of KPM316 and KP 318 grown at 37°C in either LB medium or LB medium supplemented with A5P/G6P. The E.coii K-12 wild-type strain BW30270 was used as a control.

[0068] FIG. 29 Relative NF-κΒ induction in HEK-Blue ceils caused by LPS/lipid IV _A isolates from E. co// strains BW30270, KP 318, KPM318 and KPM334.

[0069] FSG. 30 Human TNF-aipha ELISA to determine the antagonistic activity of LPS/lipid IVA samples isolated from E. coli strains KP 316 and KPM318. For details, see the protocol in the text.

[0070] FIG. 31 TNF-aipba release in human macrophages caused by LPS/lipid IV _A samples from E. coli strains KPM318 and KPM318 in medium containing 4% AB- serum. The S-LPS of E. coli K-235 was used as a control.

[0071] FIG. 32 TNF-aipha release in human macrophages caused by LPS/lipid IVA samples from E. coii strains KPM316 and KPM318 in serum-free medium. The S- LPS of E. coli K-235 was used as a control.

[0Θ72] FIG. 33 IL-8 release in HEK293 hTLR4/MD2 #33 cells stimulated with LPS/lipid IVA samples from E. coli strains KPM316 and KPM318. The S-LPS of E. coii K-235 was used as a control.

[0073] FIG. 34 IL-8 release in HEK293 hTLR2 #2 cells stimulated with LPS/lipid !V _A samples from E. coli strains KPM316 and KPM318. The S-LPS of E. coli K-235 was used as a control.

[0074] FIG. 35 Maps of plasmids pMAL-c2 and pMAL-p2 for cytoplasmic (upper panel) and periplasmic Ma!E-LacZa expression (lower panel).

[0075] FSG. 36 SDS-PAGE analysis of protein extracts (10 μg each) and culture media (10 μΙ each). The protein extracts were prepared from uninduced cells and cells after an induction time of 3 nr. The culture media were obtained from cells grown under conditions of overnight IPTG induction. The samples were resolved using 10% polyacrylamide gels and stained with Coomassie blue. Molecular Mass protein markers (Broad Rnage - Bio-Rad) were run in lanes M.

[0076] FIG 37 SDS-PAGE analysis of protein extracts (10 μg each) and culture media (10 μΙ each). The protein extracts were prepared from uninduced cells and ceils after an induction time of 3 hr. The culture media were obtained from cells grown under conditions of overnight IPTG induction. The samples were resolved using 10% polyacrylamide gels and stained with Coomassie blue. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M.

[0077] FIG. 38 SDS-PAGE analysis (upper panel) of protein extracts (8 pg each) and culture media (10 μΙ each). The protein extracts were prepared from uninduced ceils and cells after overnight induction. The culture media were obtained from ceils grown under conditions of overnight IPTG induction. The samples were resolved using 10% polyacrylamide gels and stained with Coomassie blue. For the immunobiots (lower panel), 8 pg of each protein extract and 2.5 μΙ of each culture supernatant were subjected to SDS-PAGE. The blotted membranes were probed with anti-MalE and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibodies, and developed in the presence of nitroblue tetrazoiium (NBT) and 5- bromo-4-chloro-3-indoiylphosphate (BCIP) substrate. Molecular mass protein markers (Broad Range ^■■■■ Bio-Rad) were run in lanes M.

[0078] FIG. 39 SDS-PAGE analysis (upper panel) of protein extracts (8 pg each) and culture media (10 μΙ each). The protein extracts were prepared from uninduced ceils and cells after overnight induction. The culture media were obtained from cells grown under conditions of overnight !PTG induction. The samples were resolved using 10% polyacrylamide gels and stained with Coomassie blue. For the irnmunobiots (lower panel), 8 of each protein extract and 2.5 μ! of each culture supernatant were subjected to SDS-PAGE. The blotted membranes were probed with anti-Ma!E and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibodies, and developed in the presence of nitroblue tetrazoiium (NBT) and 5- bromo-4-chioro-3-indoly!phosphate (BCIP) substrate. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M.

[0079] FIG. 40 immunoblot analysis of protein extracts (8 pg each) and culture media (2.5 μ! each). The protein extracts were prepared from uninduced ceils and cells after an induction time of 3 hr. The culture media were from ceils grown under conditions of IPTG induction for 3 hr. The samples were resolved using 10% poiyacry!amide gels, followed by blotting and probing of the membranes with anti- Mai E and alkaline phosphatase-conjugated goat anti-mouse !gG (H+L) antibodies, and developed in the presence of NBT and BCSP substrate.

[0080] FIG. 41 8DS-PAGE analysis of protein extracts (6 g each) and culture media (8 μΙ each) of pMAL-p2 strains. The protein extracts were prepared from uninduced cells and ceils after induction times of 3 hr, 6 hr, 12 hr and 24 hr. The culture media were from cells after 3 hr, 6 hr, 12 hr and 24 hr of induction. The samples were resolved using 10% po!yacrylamide gels and stained with Coomassie blue. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M.

[0081] FIG 42 Immunoblot analysis of protein extracts (8 g each) and culture media (10 μ! each) of pMAL-p2 strains. The protein extracts were prepared from uninduced ceils and cells after induction times of 3 h, 6h, 12 h and 24 h. The culture media were from cells after 3 h, 8h, 12 h and 24 h of induction. The samples were resolved using 10% polyacrylamide gels, followed by blotting and probing of the membranes with anti-MalE and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibodies, and developed in the presence of NBT and BCIP substrate.

[0082] FIG. 43 Maps of plasmids pJexpress404:51149 and pJexpress404:51 150 for cytoplasmic (upper panel) and perip!asmic ApoAi expression (lower panel).

[0083] FSG. 44 SDS-PAGE analysis of protein extracts (6 pg each) prepared from strains BW3Q270/pJexpress404:51 149 and KPM318/pJexpress404:51 149. The protein extracts were obtained from uninduced ceils and cells after induction times of 3 hr, 8 hr, 12 hr and 24 hr. The samples were resolved using 12% polyacrylamide gels and stained with Coomassie blue. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M. The arrow indicates the ApoA1 protein,

[0084] FIG, 45 SDS-PAGE analysis of protein extracts (8 each) prepared from strains KPM334/pJexpress404:51 149 and KPM335/pJexpress404:51 149. The protein extracts were obtained from uninduced ceils and cells after induction times of 3 hr, 8 hr, 12 hr and 24 hr. The sampies were resolved using 12% poiyacrylamide gels and stained with Coomassie blue. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M. The arrow indicates the ApoA1 protein.

[0085] FIG, 48 SDS-PAGE analysis of protein extracts (8 each) prepared from strain KPM336/pJexpress404:51 149. The protein extracts were obtained from uninduced cells and ceils after induction times of 3 hr, 6 hr, 12 hr and 24 hr. The samples were resolved using 12% poiyacrylamide gels and stained with Coomassie blue. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M.

[0086] FIG. 47 SDS-PAGE analysis of protein extracts (8 each) prepared from strains BW30270/pJexpress404:51 150 and KPM318/pJexpress404:51 150. The protein extracts were obtained from uninduced ceils and cells after induction times of 3 hr, 6 hr, 12 hr and 24 hr. The sampies were resolved using 12% poiyacrylamide gels and stained with Coomassie blue. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M.

[0087] FIG. 48 SDS-PAGE analysis of protein extracts (8 each) prepared from strains KPM334/pJexpress404:51 150 and KPM335/pJexpress404:51 150. The protein extracts were obtained from uninduced ceils and cells after induction times of 3 hr, 8 hr, 12 hr and 24 hr. The sampies were resolved using 12% poiyacrylamide gels and stained with Coomassie blue. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M.

[0088] FIG. 49 SDS-PAGE analysis of protein extracts (8 each) prepared from strain KPM336/pJexpress404:51 150. The protein extracts were obtained from uninduced ceils and ceils after induction times of 3 hr, 8 hr, 12 hr and 24 hr. The samples were resolved using 12% po!yacrylamide gels and stained with Coomassie blue. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M.

[0089] FSG. 50 The optica! ceil density (ODeoo) vs. number of viable cells (cfu/ml) of strain KPM335/pJexpress404:51 150 was evaluated during the period of induced ApoA1 expression.

[0Θ90] FIG. 51 SDS-PAGE analysis of culture media from strains BW30270/pJexpress404:51 150 and KPM318/pJexpress404:51 150. The culture media were obtained from uninduced cells and ceils after induction times of 3 hr, 6hr, 12 hr and 24 hr. The samples were resolved using 12% poiyacrylamide gels and stained with Coomassie blue. For the amounts of samples loaded, see Tables 7 - 1 1 .

[0091] FIG. 52 SDS-PAGE analysis of culture media from strains KPM334/pJexpress404:51 150 and KPM335/pJexpress404:51 150. The culture media were obtained from uninduced cells and ceils after induction times of 3 hr, 8 hr, 12 hr and 24 hr. The samples were resolved using 12% poiyacrylamide gels and stained with Coomassie blue. For the amounts of samples loaded, see Tables 7 - 1 1 .

[0092] FIG. 53 SDS-PAGE analysis of culture media of KPM336/pJexpress404:51 150. The culture media were obtained from uninduced cells and cells after induction times of 3 hr, 8 hr, 12 hr and 24 hr. The samples were resolved using 12% poiyacrylamide gels and stained with Coomassie blue. For the amounts of samples loaded, see Tables 7 - 1 1. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M.

[0093] FIG. 54 !mmunobiot analysis of protein extracts (3 pg each) of strains BW30270/pJexpress404:51 149 and KP 318/pJexpress404:51 149. The protein extracts were prepared from uninduced cells and cells after induction times of 3 hr, 6 hr, 12 hr and 24 hr. The samples were resolved using 10% poiyacrylamide gels, followed by blotting and probing of the membranes with Penta-His antibody and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibodies, and developed in the presence of NBT and BCIP substrate. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M and labelled after blotting.

[0094] FIG. 55 immunob!ot analysis of protein extracts (3 g each) of strains KPM334/pJexpress404:51 149 and KPM335/pJexpress404:51 149. The protein extracts were prepared from uninduced cells and cells after induction times of 3 hr, 8 hr, 12 hr and 24 hr. The samples were resolved using 10% polyacrylamide gels, followed by blotting and probing of the membranes with Penta-His antibody and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibodies, and developed in the presence of NBT and BCIP substrate. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M and labelled after blotting.

[0095] FIG. 56 Immunoblot analysis of protein extracts (3 pg each) of KPM336/pJexpress404:51 149. The protein extracts were prepared from uninduced cells and cells after induction times of 3 hr, 6 hr, 12 hr and 24 hr. The samples were resolved using 10% polyacrylamide gels, followed by blotting and probing of the membranes with Penta-His antibody and alkaline phosphatase-conjugated goat anti- mouse IgG (H+L) antibodies, and developed in the presence of NBT and BCIP substrate. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M and labelled after blotting.

[0096] FSG. 57 Immunoblot analysis of protein extracts (3 each) of strains BW30270/pJexpress404:51 150 and KP 318/pJexpress404:51 150. The protein extracts were prepared from uninduced cells and cells after induction times of 3 hr, 6 hr, 12 hr and 24 hr. The samples were resolved using 12% polyacrylamide gels, followed by blotting and probing of the membranes with Penta-His antibody and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibodies, and developed in the presence of NBT and BCIP substrate. Molecular mass protein markers (Broad Range ^■■■■ Bio-Rad) were run in lanes M and labelled after blotting.

[0097] FIG. 58 Immunoblot analysis of protein extracts (3 pg each) of strains KPM334/pJexpress404:51 150 and KP 335/pJexpress404:51 150. The protein extracts were prepared from uninduced cells and cells after induction times of 3 hr, 6 hr, 12 hr and 24 hr. The samples were resolved using 12% polyacry!amide gels, followed by blotting and probing of the membranes with Penta-His antibody and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibodies, and developed in the presence of NBT and BC!P substrate. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M and labelled after blotting.

[0Θ98] FIG. 59 immunoblot analysis of protein extracts (3 pg each) of KPM336/pJexpress404:51 150. The protein extracts were prepared from uninduced cells and cells after induction times of 3 hr, 8 hr, 12 hr and 24 h. The samples were resolved using 12% poiyacrylamide gels, followed by blotting and probing of the membranes with Penta-His antibody and alkaline phosphatase-conjugated goat anti- mouse IgG (H+L) antibodies, and developed in the presence of NBT and BCIP substrate. Molecular mass protein markers (Broad Range - Bio-Rad) were run in lanes M and labelled after blotting.

[0099] FSG. 80 Immunoblot analysis of culture media from strains BW30270/pJexpress404:51 150 and KPM318/pJexpress404:51 150. The culture media were obtained from uninduced cells and cells after induction times of 3 hr, 6 hr, 12 hr and 24 hr. The samples were resolved using 12% poiyacrylamide gels, followed by blotting and probing of the membranes with Penta-His antibody and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibodies, and developed overnight in the presence of NBT and BCSP substrate. For the amounts of samples loaded, see Tables 7 - 1 1.

[00100] FIG. 61 Immunoblot analysis of culture media from strains KPM334/pJexpress404:51 150 and KPM335/pJexpress404:51 150. The culture media were obtained from uninduced cells and ceils after induction times of 3 hr, 6 hr, 12 hr and 24 hr. The samples were resolved using 12% poiyacrylamide gels, followed by blotting and probing of the membranes with Penta-His antibody and alkaline phosphatase-conjugated goat anti-mouse IgG (H+L) antibodies, and developed overnight in the presence of NBT and BCIP substrate. For the amounts of samples loaded, see Tables 7 - 1 1. [00101] FIG. 62 Immunoblot analysis of culture media of KP 338/pJexpress404:51 150. The culture media were obtained from uninduced cells and cells after induction times of 3 hr, 6 hr, 12 hr and 24 hr. The samples were resolved using 12% polyacrylamide gels, followed by blotting and probing of the membranes with Penta-His antibody and alkaline phosphatase-conjugated goat anti- mouse IgG (H+L) antibodies, and developed overnight in the presence of NBT and BCIP substrate. For the amounts of samples loaded, see Tables 7 - 1 1 .

[00102] FIG. 63 Agarose gel electrophoresis of pMAL-c2 plasmids isolated from four randomly selected transformants of E. coli strains BW30270/pMAL-c2, KPM318 (LB)/pMAL-c2, and KPM318 (LB A5P/G8P)/pMAL-c2, The samples were subjected to electrophoresis on a 0.8% agarose gel in TBE buffer. Lane 1, plV1AL-c2 control; lane 2, BW30270/p AL-c2 (1 ); lane 3, BW30270/p AL-c2 (2); lane 4, BW30270/p AL-c2 (3); lane 5, BW30270/p AL-c2 (4); lane 6, KPM318 (LB)/p AL- c2 (1 ); lane 7, KPM318 (LB)/pMAL-c2 (2); lane 8, KPM318 (LB)/pMAL-c2 (3); lane 9, KP 318 (LB)/p AL-c2 (4); lane 10, KPM318 (LB A5P/G8P)/pMAL-c2 (1 ); lane 11, KPM318 (LB A5P/G6P)/pMAL-c2 (2); lane 12, KPM318 (LB A5P/G6P)/p AL-c2 (3); lane 13, KPM318 (LB A5P/G6P)/pMAL-c2 (4); lane M, 1-kb Pius DNA Ladder (Invitrogen).

[00103] FIG. 64 Agarose gel electrophoresis of pMAL-c2 and pMAL-p2 plasmids isolated from two randomly selected transformants of £. coli strains KPM334/p AL-c2, KPM334/p AL-p2, KPM335/p AL-c2, and KP 335/pMAL-p2. Lane 1, pMAL-c2 control; lane 2, KP 334/p AL-c2 (1 ); lane 3, KP 334/p AL-c2 (2); lane 4, KPM335/p AL-c2 (1 ); lane 5, KPM335/pMAL-c2 (2); lane 6, pMAL-p2 control; lane 7, KPM334/plV1AL-p2 (1 ); lane 8, KP 334/p AL-p2 (2); lane 9, KPM335/p AL-p2 (1 ); lane 10, KPM335/p AL-p2 (2); lanes M, 1-kb Plus DNA Ladder (Invitrogen).

[00104] FIG. 65 Agarose gel electrophoresis of PGR products obtained by using the primer pair 5ECIpxPctrl/3ECipxPctri to distinguish between the presence of the IpxP wild-type gene and the Δ/ρχΡ knockout mutation in KPM strains. The biomasses of a potential BW30270 (F ^' 121 Tn fO) strain (lane 1) and eight tetracyciine-resistarit KPM318 (F ^' 121 Tn f O) transconjugants (lanes 2 - 9} were used as templates. The 1-kb Plus DNA Ladder (Snvitrogen) is shown in lane M.

[00105] FIG. 66 Map of the helper plasmid pMAK705 Krn ^R CATMUT.

[00106] FIG. 87 The E. coll strains TOP10 and XL1 -Blue were transformed with plasmid pMAK705 Km ^R CATMUT. The resulting strains TOP10/p AK705 Km ^R CATMUT and XL1 -Blue/p AK705 Km ^R CATMUT were streaked on both LB + 30 Mg/ml kanamycin and LB + 30 g/ml kanamycin +15 pg/ml chloramphenicol plates.

[00107] FIG. 68 shows TABLE 1 , which provides examples of £. coll strains constructed.

[00108] FIG. 89 shows TABLE 2, which lists the peak list of glycoforms detected in £. coll KPM318 using ESI FT-!CR MS (FIGURE 27).

[00109] FIG. 70 shows TABLE 3, which provides the optical density and ceil number of the bacterial suspensions immediately prior to induction with IPTG.

[00110] FIG. 71 shows TABLE 4, which provides the optical density and ceil number of the bacterial suspensions after 3 hr of !PTG induction.

[00111] FIG. 72 shows TABLE 5, which provides the optical density and ceil number of the bacterial suspensions after 6 hr of IPTG induction.

[00112] FIG. 73 shows TABLE 6, which provides the optical density and ceil number of the bacterial suspensions after 12 hr of IPTG induction.

[00113] FIG. 74 shows TABLE 7, which provides the optical density and cell number of the bacterial suspensions after 24 hr of IPTG induction.

[00114] FIG. 75 shows TABLE 8, which demonstrates the process for determining of the amount of culture media obtained from ceils immediately before the induction with IPTG.

[00115] FIG 76 shows TABLE 9, which demonstrates the process for determining the amount of culture media obtained from cells after 3 hr of induction.

[00116] FIG. 77 shows TABLE 10, which demonstrates the process for determining the amount of culture media obtained from ceils after 6 h of induction.

[00117] FIG. 78 shows TABLE 1 1 , which demonstrates the process for determining the amount of culture media obtained from ceils after 12 hr of induction.

[00118] FIG. 79 shows TABLE 12, which demonstrates the process for determining the amount of culture media obtained from ceils after 24 hr of induction.

[00119] FIG. 80 shows TABLE 13, which provides the number of competent ceils.

[00120] FIG. 81 shows TABLE 14, which provides the transformation efficiencies for E, coli strains BW30270, KPM318 (LB) and KPM318 (LB A5P/G6P).

[00121] FIG. 82 shows TABLE 15, which provides the transformation efficiencies for E. coli strains KPM334, KPM335, KPM336, and KPM337.

[00122] FIG. 83 shows TABLE 16, which provides the yields of p!asmids pMAL-c2 and pMAL-p2 isolated from £. coli strains BW30270, KPM318, KPM334, KPM335, KPM338, and KPM337.

[00123] FIG. 84 shows TABLE 17, which describes the calculation for the amount of the recipient (BW30270 and KPM318) to add to 500 μΙ of the donor (JC19022 F ^' 121 Tn f ).

[00124] FIG. 85 shows TABLE 18, which describes the calculation for the amount of the recipient (KPM334, KPM335, KPM336, and KPM337) to add to 500 μΙ of the donor (JC19022 F ^' 121 Tn fO).

[00125] FSG. 88 shows TABLE 19, which provides the optica! densities (OD500) of the strains immediately prior to the M13K07 infection.

[00126] FIG. 87 shows TABLE 20, which provides the number of kanamycin- resistant colonies following infection of potential F ^' 121 Tn f O transconjugants with 13K07 helper phage.

[00127] FSG. 88 shows TABLE 21 , which provides the sequences referred to in this application.

[00128] FSG. 89, 90 and 91 show the construction path for each E. Coii strain. [00129] FSG. 92A and 92B show a protocol for HEK-Blue assays.

DESCRIPTION OF PREFERRED EMBODIMENTS

[00130] Lipid A, a component of lipopoiysaccharide (LPS) which comprises two glucosamine residues to which six acyl chains are attached, functions as an agonist of Toil-like receptor 4 (TLR4/MD-2). TLR4/MD-2 is present on several immune system cells, for example macrophages, monocytes, and dendritic cells. Activation of TLR4/MD-2 via LPS/Lipid A can lead to activation of costimulatory immune response components and molecules, ultimately causing endotoxicity. For example, when LPS becomes bound to TLR4/MD-2 (in humans), cytokine production can be activated, complement can be activated, and coagulation can be activated. Cytokines may include interieukin IL-1 , SL-8, IL-8, tumor necrosis factor (TNF) (e.g., TNFct), and platelet activating factor. Such cytokines can stimulate production of mediators of inflammation and septic shock (e.g., prostaglandins, leukotrienes). Complement C3a and C5a can cause histamine release leading to vasodilation and/or affect neutrophil chemotaxis. Blood-clotting Factor XII can activate several responses resulting in thrombosis, plasmin activation (fibrinolysis, hemorrhaging), and the like.

[00131] Gram-negative bacteria normally comprise a majority of 8~acyi LPS in their outer membranes. The present invention features viable Gram-negative bacteria comprising an outer membrane that substantially lacks components (e.g., iigands) that act as agonists of Toll-like receptor 4 (TLR4)/MD-2. The components (e.g., Iigands) may comprise lipid A, a 6-acyi lipid (e.g., 8-acyi LPS), the like, or a combination thereof. In contrast to normal LPS (or lipid A, 6-acyl lipid), lipid !VA _, or 4- acyi LPS binds less tightly to human TLR4/MD-2 and acts as a mild antagonist to TLR-4/MD2 rather than as an agonist. In some embodiments, the iigand substantially lacking in the outer membrane comprises a Lipid !VA, or derivatives of lipid IV _A .

[00132] As used herein, the term "substantially lacks" means that the outer membrane has from about zero of the agonist Iigand up to a level of such Iigand that does not induce a response above about 25% of the maximal signal in the HEK-blue assay when 20 μ of outer membrane is added to a single assay well. In some embodiments, a HEK-Blue assay that may be used in accordance with the present invention may be obtained from InvivoGen, 3950 Sorrento Valley Blvd., Suite 100, San Diego, CA 92121 (USA), catalog # hkb-htir4, and the assay protocol is submitted in FIG. 92. For example, in some embodiments, outer membrane that substantially lacks a iigand that acts as an agonist of Toll-like receptor 4 (TLR4/MD- 2) means that the outer membrane does not have the Iigand at all. In some embodiments, the outer membrane may have a low amount of agonist for TLR4/MD- 2 present at a level that is below the detection limit in a HEK-blue cell-based assay when tested as a membrane extract at up to 20 ,ug per well. The human ceil line HEK-blue is engineered to be very sensitive to TLR4/MD-2 signaling, and this assay- will give a very strong signal when only 20 pg of LPS is present per well. In some embodiments, a very low signal (e.g., less than about 25% of the maximal value) is seen even at a level of 20 ug of outer membrane per well.

[00133] The Gram-negative bacteria of the present invention include but are not limited to Escherichia cols ^' . For example, distantly related Gram-negative species such as Pseudomonas species may also be engineered to grow while lacking TLR4/MD2 agonist as a component of their outer membrane. In some embodiments the Escherichia cols strain is K-12, W31 10, MG1855, B/r, BL21 , G157:h7, 042, 101 - 1,1180, 1357, 1412, 1520, 1827-70, 2362-75, 3431, 53838, 83972, 929-78, 98NK2, ABU 83972, B, B088, B171, B185, B354, B646, B7A, C, c7122, CFT073, DH1, DH5a, E110019, E128010, E74/68, E851/71, EAEC 042, EPECa11, EPECa12, EPECa14, ETEC, H10407, F11, F18+, FVEC1302, FVEC1412, GEMS_EPEC1, HB101, HT115, K011, LF82, LT-41, LT-62, LT-68, MS 107-1, MS 119-7, MS 124-1, MS 145-7, MS 79-2, MS 85-1, NCTC 86, Niss!e 1917, NT:H19, NT:H40, NU14, O103:H2, O103:HNM, O103:K+, O104:H12, O108:H25, O109:H9, 0111:H-, 0111:H19, 0111:H2, 0111:H21, 0111:NM, 0115:H-, 0115:HMN, 0115:K+, 0119:H6, 0119:UT, O124:H40, 0127a:H8, 0127:H6, 0128:H2, 0131:H25, 0136:H~ , 0139:H28 (strain E24377A / ETEC), 013:H11, 0142:H6, 0145:H-, 0153:H21, 0153:H7, 0154:H9, 0157:12, 0157:H-, 0157:H12, 0157:H43, 0157:H45, 0157:H7 EDL933, 0157:N , 015:NM, 0177:H11, > 017:K52:H18 (strain U N026 / ExPEC), O180:H-, 01:K1 / APEC, 026, 026:H~, 026:H11, O26:H11:K60, 026:NM, 041:H~, 045:K1 (strain S88 / ExPEC), 051 :H-, 055:H51, 055:H6, 055:H7, 05:H-, 06, 063:H6, 063:HN , 06:K15:H31 (strain 536 / UPEC), 07:K1 (strain IAI39 / ExPEC), 08 (strain IAI1), 081 (strain ED1a), 084:H-, 086a:H34, O86a:H40, O90:H8, 091:H21, 09:H4 (strain HS), 09:H51, ONT:H-, 0NT:H25, OP50, Orough:H12, Orough:H19, Orough:H34, Orough:H37, Orough:H9, 0UT:H12, OUT:H45, 0UT:H6, 0UT:H7, OUT:HNM, OUT:NM, RN587/1, RS218, 55989 / EAEC, B / BL21.B / BL21- DE3, SE11, SMS-3-5 / SECEC, UTI89 / UPEC, TA004, TA155, TX1999, or Vir68.

[00134] In some embodiments, the viable Gram-negative bacteria of the present invention have reduced activity of the functional D-arabinose-5-phosphate (ASP) isomerases (APIs) KdsD and GutQ, The reduced activity of functional APIs KdsD and GutQ may be a result of substantial inactivation of the APIs, for example a chemical agent functioning to substantially inactivate the APIs, a dominant negative protein functioning to substantially inactivate the APIs, an antibody of fragment thereof that binds and functions to substantially inactivate the APIs, or the like. In some embodiments, the reduced activity of functional APIs KdsD and GutQ may be a result of a partial or substantial deletion or a mutation in the genes encoding the APIs (e.g., kdsD, gutQ). The bacteria may include £. co!i K-12 TCM15, BL21, BL21(DE3), W3110. SUPPRESSOR MUTATIONS

[00135] The viable Gram-negative bacteria of the present invention comprise a suppressor mutation that enables growth despite lacking any TLR4 agonists in the membrane. For example, the viable gram-negative bacteria may be derived from E co!i K-12 TCM15, which lacks both API genes (thus lacks Kdo) and is not viable unless supplied with exogenous ASP. The viable Gram-negative bacteria may- comprise a suppressor mutation that allows the bacteria to be viable despite lacking both API genes (and Kdo). Suppressor mutations may include gain-of-function suppressor mutations as well as ioss-of-function suppressor mutations. In some embodiments, the suppressor mutation is a mutation in a gene encoding a transporter or a membrane protein, e.g., the transporter MsbA encoded by msbA or the membrane protein YhjD encoded by yhjD, The suppressor mutation may provide the transporter or membrane protein an increased capacity to transport lipid SVA. The suppressor mutation may include an amino acid substitution, for example a mutation at amino acid position 18, or at position 50, or at position 283, or at position 310, or at position 448 in sbA or a mutation at amino acid position 134 in YhjD. In some embodiments, the suppressor mutation results in up-regulated expression of a transporter (e.g., MsbA), increasing the bacterium's capability of transporting lipid IV _A . The suppressor mutation is not limited to the aforementioned examples. The bacteria may include E coli K-12 KPM22, KPM22 L1 , KPM22 L 1 1 . In some embodiments, the bacteria include E coli K-12 KPM22 L13, KP 22 LI 4, KP 22 L15, KPM22 L18, KPM22 L20. In some embodiments, the suppressor alleles of the E coli msbA gene may be used in distantly related Gram-negative bacterial species (such as Pseudomonas) even though the wild type E coli msbA gene itself cannot replace the native msbA gene.

NON-REVERTABLE STRA!NS

[00136] In wild-type Gram-negative bacteria, the O-acyi transferases LpxL and LpxM add Olinked lipids to Kdo2-Lipid IVA to form a 8-acyl lipid that functions as an agonist of TLR4/MD-2. In the absence of Kdo, the Oacyl transferases LpxL and LpxM fail to use Lipid IV _A as a substrate and do not add the O-linked lipids. However, it is possible that mutations in !pxL (encoding LpxL) or IpxM (encoding LpxM) may alter specificity of the enzymes, allowing the transferases to use lipid IVA as a substrate and thereby generating a 8-acyl lipid that functions as an agonist of TLR4/MD-2. Or, mutations may arise in promoters or regulators of IpxP or pagP, activating expression of these normally absent or very low abundance transferases. Or, mutations may arise in promoters or regulators of IpxL or !pxM, which may activate their overexpression.

[00137] To help avoid these situations, the bacteria of the present invention may further comprise a deletion (e.g. partial deletion, substantial deletion, etc.) in one or more of the following genes: IpxL, IpxM, pagP, and IpxP. Or, in some embodiments, one or more enzymes (e.g., LpxL, LpxM, PagP, LpxP) are substantially inactivated in the bacterium. In some embodiments, the gene eptA encoding for the lipid A phosphoethanolamine transferase is partially or substantially deleted. In some embodiments, the bacteria is E, coll K-12 KPM279 (KPM22 AipxL :Km*)„ KPM 280 (KPM22 A/px ::Km ⁺ ), KPM 288 (KPM22 L1 A/pxL::Km ⁺ ), KPM 290 (KPM22 L1 1 A/pxL::Km ⁺ ), KPM 296 (KPM22 L1 1 AlpxL), KPM 300 (KPM22 L1 1 MpxL Δ/ρχ ::Κίϊ , KPM 303 (KPM22 L1 1 AlpxL AlpxM), KPM310 (KPM22 L1 1 Δ/pxL AlpxM ApagP :Km ⁺ ), KPM312 (KPM22 L1 1 AlpxL A!pxM ApagP), KPM314 (KPM22 L1 1 AlpxL AlpxM ApagP Δ/ρχΡ: :Km ⁴ ), KPM316 (KPM22 L1 1 AlpxL AlpxM ApagP A!pxP), KPM317 (KPM22 L1 1 AlpxL AlpxM ApagP AlpxP AepiA:;Km ⁺ ), or KPM 318 (KPM22 L1 1 AlpxL AlpxM ApagP AlpxP AepiA) or further derivatives of these strains.

LABORATORY TOOLS

[00138] The present invention also features laboratory tools, reagents, strains (e.g., base strains, donor strains), and the like, derived from the viable Gram-negative bacteria as described. For example, in some embodiments, the bacteria of the present invention are competent to take up extracellular DNA. In some embodiments, the bacteria are electrocompetent. In some embodiments, the bacteria comprise a means of conferring resistance to an antibiotic (e.g., kanamycin, penicillin, neomycin, ampiciilin, etc.). In some embodiments, the bacteria comprise a means to express proteins for high-throughput screening on cells. In some embodiments, the bacteria comprise a means to express recombinant proteins for research and/or therapeutic and/or diagnostic use. [00139] The bacteria may comprise at least one additional suppressor mutation that enables growth above 37 degrees Celsius (e.g., at 42 degrees Celsius). The additional suppressor mutations may for example stabilize the outer membrane of the bacteria or enhance transport of a toxic precursor away from the inner membrane. In some embodiments, bacteria can grow exponentially above 37 degrees Celsius, for example at about 38 degrees Celsius, at about 39 degrees Celsius, at about 40 degrees Celsius, at about 41 degrees Celsius, at about 42 degrees Celsius.

[00140] In some embodiments, the bacteria exhibit an exponential phase doubling time of less than about 30 minutes at above 37 degrees Celsius. Or, the bacteria may exhibit an exponential phase doubling time of less than 35 minutes or less than 40 minutes or less than about 45 minutes at above 37 degrees Celsius. In some embodiments, the bacteria exhibit an exponential phase doubling time of less than about 30 minutes (or less than about 35 minutes or less than about 40 minutes or less than about 45 minutes) at above 40 degrees Celsius. In some embodiments, the bacteria exhibit an exponential phase doubling time of less than about 30 minutes (or less than about 35 minutes or less than about 40 minutes or less than about 45 minutes) at 42 degrees Celsius. In some embodiments, the bacterial strain is E. coil K-12 KP 296-6.

[00141] In some embodiments, additional suppressor mutations may alter (e.g., enhance) the bacteria's ability to grow in various salt concentrations. In some embodiments, the viable gram-negative bacteria are viable in a salt (NaCi) concentration between about 0.1 M and 0.9 .

[00142] Donor strains may provide the ability to rapidly construct new variant strains. The donor strains may comprise one or more of the aforementioned gene modifications, for example partial or substantial deletions in one of the following genes: ipxL, IpxM, !pxP, pagG, eptA, kdsD, gutQ, etc. Each donor deletion construct has a selectable marker gene cassette replacing the deleted DNA sequence, and the cassette is flanked by site-specific deletion sites in such an orientation and arrangement that they may be used subsequently to delete the selectable marker gene cassette when the corresponding recombinase is transiently or temporarily introduced into the strain.

[00143] In some embodiments, the bacteria further comprise an F piasmid, an F' p!asmid, or genes encoding for F piius production. In some embodiments, the bacteria can propagate bacteriophages fd, M13, or bacteriophages related to fd or M13. In some embodiments a gene encoding for a DNA restriction enzyme or a DNA methy!ation enzyme is mutated or deleted. This may allow for enhanced transformation and/or cloning of unmodified DNA or PCR-amplified DNA. In some embodiments a gene encoding for recA or endA is mutated, partially deleted, or substantially deleted. Such inactivation or deletion of recA results in a host that predominantly produces monomeric p!asmid DNAs following transformation with a plasmid. Inactivation of endA leads to increased yields of supercoiled DNA of a piasmid introduced into such a strain.

[00144] The present invention also features an outer membrane of a Gram-negative bacterium that substantially lacks a !igand that is an agonist of a TLR4/ D2 receptor.

[00145] Bacteria of the present invention may be used as donor strains. Sn some embodiments, the donor strains may comprise an outer membrane that substantially lacks a ligand that acts as an agonist of TLR4/MD2. In some embodiments, the donor strains comprise a DNA cassette comprising a gene having an open reading frame deletion. For example, the gene may be selected from the group consisting of IpxL, IpxM, pagP, IpxP, and eptA. The DNA cassette may further comprise a means of conferring resistance to an antibiotic and/or a recombinase component.

[00146] The bacteria of the present invention may retain "scar" DNA sequences at the site(s) of the engineered gene deletions, particularly in one or more of the genes selected from the group consisting of kdsD, gutQ, IpxL, IpxM, pagP, IpxP, and eptA. The "scar" is produced upon deletion of the selectable cassette by site-specific recombination. The recombinase may be one selected from the group consisting of FLP, Cre, Int Xis, Hin, Gin, or the like. The scar produced may be active for subsequent recombination by the same recombinase, or alternatively the scar may be inactive for further recombination events by the same recombinase. Deletion of the selectable cassette DNA results in a strain that may be used again as a recipient for subsequent transfer of a further gene deletion using a similar selectable cassette, thereby facilitating the construction of multiple gene deletions in a single strain.

METHODS

[00147] The present invention also features methods of selecting a Gram-negative bacterium substantially lacking a ligand that acts as an agonist of TLR4/MD2, wherein the bacterium is capable of exponential growth at a temperature above 40 degrees Celsius. In some embodiments, the method comprises growing the bacteria that substantially lack the ligand that acts as an agonist of the TLR4/MD2 receptor (e.g., lipid A, 8-acyi lipid, etc.) and has a suppressor mutation that allows growth between about 30 to 40 degrees Celsius. The bacteria can then be plated on a suitable nutrient medium and incubated at 42 degrees Celsius. Single colonies that appear under such conditions represent the bacterium substantially lacking the ligand that acts as an agonist of TLR4/MD2 and capable of exponential growth at 42 degrees Celsius.

[00148] The present invention also features methods of constructing bacteria substantially lacking a gene selected from the group consisting of !pxL, !pxM, pagP, ipxP, and eptA, The method comprises obtaining a viable Gram-negative bacterium comprising (i) an outer membrane that substantially lacks a ligand that acts as an agonist of TLR4/MD2 and (ii) a DNA cassette comprising a modified gene, the modified gene being a modified target gene having an open reading frame deletion, the DNA cassette further comprising a means of conferring resistance to an antibiotic and further being flanked by appropriate target sites for a site-specific recombinase. The method further comprises subjecting the bacterium to Pl v/r transduction and replacing the target gene with the modified resistance cassette gene via homologous recombination. Subsequently, a site-directed recombinase enzyme or DNA encoding the enzyme is introduced into the cells and this enzyme acts on the corresponding recognition sites flanking the cassette to catalyze recombination, leaving a scar sequence only in the chromosome (see Example 3 below). Alternatively, the gene deletion cassette described above may be introduced into the chromosome of the strain directly using a PCR-ampiified linear form of the cassette via the "red-gam" homologous recombination method (see BMC Molecular Biology 2008, 9:20). As above, the resistance gene is then subsequently deleted using a site-specific recombination step, again leaving a "scar" sequence. P1 v/r transduction, red-gam recombination using linear DNA, and site-directed recombination are all techniques well known to one of ordinary skill in the art.

[00149] Bacteria, for example Gram-negative bacteria such as E, coli, are commonly used as expression systems for amplifying DNA or expressing proteins of interest. In some cases, the harvested DNA or protein obtained from the bacteria may be contaminated with components of the bacteria, for example ligands that act as agonists of TLR4/ D-2 (e.g., lipid A, LPS). The contamination can have deleterious effects on further experiments and procedures, such as transient transfection of mammalian host cell lines, treatment of cultured mammalian ceils that serve as reporter lines in cell-based screening assay.

[00150] The present invention also features methods of producing DNA samples substantially free of a Iigand that acts as an agonist of TLR4/MD-2. The method may comprise obtaining gram-negative bacteria comprising an outer membrane that substantially lacks the iigand that acts as an agonist of TLR4/MD-2. The bacteria are generally competent to take up extracellular DNA. A DNA vector can be introduced to the bacteria via standard methods well known to one of ordinary skill in the art. The bacteria function to amplify the DNA vector, and the bacteria can be grown in appropriate media. The amplified DNA sample can be harvested from the bacteria via a standard DNA isolation protocol well known to one of ordinary skill in the art. Because the bacteria used for amplification of the DNA substantially lacks the iigand that acts as an agonist of TLR4/MD-2, the DNA sample also substantially lacks of the Iigand. DNA isolated from these bacteria is of particular use for transient transfection of mammalian cells for expression of proteins encoded by the plasmid. LPS from the normal E. coli employed as plasmid hosts can adversely affect the productivity and yield of protein from transient transfection procedures.

[00151] The present invention also features methods of producing protein samples substantially free of a ligand that acts as an agonist of TLR4/MD2, The method may comprise obtaining Gram-negative bacteria comprising an outer membrane that substantially lacks the ligand that acts as an agonist of TLR4/MD2 (the bacteria are competent to take up extracellular DNA) and introducing a DNA vector expression construct to the bacteria via standard protocols well known to one of ordinary skill in the art. The DNA vector expression construct comprises both a gene encoding a protein of interest expressed from a functional promoter and a selectable marker gene. In some embodiments, the bacteria comprise mutations in or deletions of at least one of the following genes: !oiA, lolB or /pp.

[00152] The bacteria transformed with the DNA vector expression construct can then be grown (e.g., via standard methods) so as to allow the bacteria to express the protein of interest. In some embodiments, the bacteria are induced to express the protein of interest. The protein of interest can then be harvested from the bacteria via standard methods. In some embodiments, the bacteria are removed from the culture medium to leave the protein of interest behind in the medium. Because the bacteria used for protein expression lack the ligand that acts as an agonist of TLR4/MD-2, the protein sample also substantially lacks of the ligand.

[00153] Libraries are extremely useful tools for screening various protein variants. The present invention also features a library of bacteria, the bacteria being substantially free of a ligand that acts as an agonist of TLR4/MD2. The library of bacteria may express a series of variants of a protein. In some embodiments, the library of bacteria is used to screen the protein variants for activity in a mammalian cell based assay, however the present invention is not limited to mammalian cell based assays. Members of the library may be generated via phage display, via mutagenesis of a plasmid vector expressing a prototype member of the library, or via other standard methods.

[00154] Various reagents, strains, and the like may be provided (or combined with other reagents, strains, and the like) in the form of a kit. For example, the kit may comprise a viable Gram-negative bacterium having an outer membrane substantially free of a ligand that acts as an agonist of TLR4/MD2, wherein the bacterium is competent to take up extracellular DNA, EXAMPLES

[00155] The examples provided below are merely examples to further clarify the present invention, and do not limit the scope of the invention in any way.

EXAMPLE 1: DETERMINATION OF GROWTH RATES

[00156] The following examples describe measurements of growth rates of various strains according to the present invention. The present invention is not limited to the following examples. Growth rates of strains can be determined by monitoring the cell densities at 600 nm of exponentially growing cultures. Fresh overnight cultures can be diluted 1 :50 in pre-warmed media to continue the growth of the bacteria to the mid-exponential growth phase (ODeoo of 0.5 to 0.6). Cultures can then be diluted again to an ODeoo of about 0.005 in pre-warmed media to keep the ceils growing exponentially for determination of generation times. Media may comprise Luria- Bertani (LB) media, and may further comprise 10 g/L NaCI. As shown in FIG. 1 , KPM22 LI , KP 22 L1 1 , KP 22 LI 3, KPM22 L18, and KPM22 L20 were grown at 37 degrees Celsius. KPM22 L1 , KPM22 L1 1 , and KPM22 L18 all had generation times between about 36 to 44 minutes.

[00157] As shown in FIG. 2, E coil strain KPM288 (KP 22 L1 A/pxL::KnV ^" ) and strain KPM290 (KPM22 L1 1 Δ/ρχί_::ΚΥη ⁺ ) were grown in either Luria-Bertani (LB) media at 37 degrees Celsius or in LB media supplemented with 15 μΜ D-arabinose 5- phosphate (ASP) and 10 μΜ D-giucose6-pbosphate (G6P) at 37 ^C C. Generation times in LB media were calculated to be as follows: KPM288 45 min, KPM290 43 min. Results from growth in LB media supplemented with ASP and G6P were as follows: KP 288 ceased to grow exponentially after 2-3 generations, KP 290 46 min.

[00158] As shown in FIG. 3, £. coli strain KPM303 (KPM22 L1 1 Δ/pxL AlpxM) was grown at 37°C in either LB media, LB media supplemented with 15 μΜ ASP and 10 μΜ G6P, Super Broth (SB) media (containing 10 g/L NaCI), or SB media (containing 10 g/L NaCI) supplemented with 15 μ ASP and 10 μΜ G6P. SB media did not improve the growth rate of KPM303 as compared to LB media. However, in contrast to the growth in LB medium supplemented with ASP and G6P, KPM303 continued to grow exponentially after 2-3 generations when cultivated in SB supplemented with ASP and G6P.

[00159] As shown in FIG, 4, £. coii strains KPM303 (KPM22 L1 1 A!pxL AlpxM)„ KPM312 (KP 22 L1 1 Δ/pxL A!pxM ApagP), and KP 316 (KP 22 L1 1 Δ/pxL A!pxM ApagP A!pxP) were grown at 37°C in either SB media or SB supplemented with 15 μΜ ASP and 10 μΜ G6P. Generation times in SB media were as follows: KPM303 54 min, KPM312 50 min, KPM318 46 min. Generation times in SB media supplemented with ASP and GBP were as follows: KP 303 39 min, KPM312 42 min, KPM316 42 min.

[00160] As shown in FIG. 5, E. coii strain KPM316 was grown at 37 ^C C in either SB medium supplemented with 10 mM MgS0 ₄ and 2 mM CaC or SB medium supplemented with 10 mM MgSG ₄ , 2 mM CaGI ₂ , 15 μΜ ASP, and 10 μΜ G6P. KP 318 had a generation time of 51 min in SB with 10 mM gS0 ₄ and 2 mM CaC . KPM316 had a generation time of 46 minutes in SB with 10 mM MgS0 ₄ , 2 mM CaC! _2l 15 μΜ ASP, and 10 μΜ G6P.

[00161] As shown in FIG. 6, E. coii strains KPM316 and KPM318 were grown at 37°C in either SB medium or SB medium supplemented with 15 μΜ A5P, and 10 μΜ GBP. KPM316 and KPM318 showed generation times of 39-44 min in these media.

[00162]

EXAMPLE 2: GENERATION OF TEMPERATURE-RESISTANT STRAINS

[00163] The following example describes generation of a temperature-resistant derivative of KPM296. The present invention is not limited to the following example. Fresh overnight cultures of KPM296 grown in LB medium at 37°C can be plated on LB agar plates and incubated at 42°C. Incubation at 42°C can be continued for several days, for example four to five days. Clones with colony-forming ability at 42°C may appear (representing 42°C-resistant derivatives of KPM296). The 42°C- resistant strains can be confirmed by overnight growth on LB agar plates at 42°C. KPM296-6 is a 42°C-resistant derivative of KPM296.

[00164] As shown in FSG. 7, E. co!i strains KPM298-6 and KPM318 (KPM22 L1 1 A!pxL ΔΙρχΜ ApagP Δ/ρχΡ) were grown at 37 ^C C in either SB with 10 mM gS0 ₄ and 2 mM CaCi ₂ , or in SB with 10 mM g80 ₄ , 2 mM CaQ ₂ , 15 μΜ ASP, and 10 μ G6P. The growth rate of KPM296-6 in SB medium with 10 mM MgS0 ₄ and 2 mM CaCi ₂ was 48 min. In SB with 10 mM MgSG ₄ , 2 mM GaCI ₂ , 15 μΜ ASP, and 10 μΜ G6P the growth rate of KPM296-8 was 42 min.

[00165] As shown in FIG 8, a series of £. coli strains isolated independently from parallel selections at 42°C from KPM298 were grown at in SB medium at 42°C. Virtually every isolate tested, including KPM296-6, exhibits exponential growth at 42°C, with a wide variation in doubling times. The parental strain, KPM298, was unable to grow at all in LB or in SB medium at 42°C.

[00166] KPM318 and KPM318 have also been successfully used to select a series of independent isolates able to grow at 42 ^C C by this method.

EXAMPLE 3: STRAIN CONSTRUCTION

[00167] The following example describes construction of various aforementioned strains. The present invention is not limited to these examples. Kanamycin resistance cassettes targeting the IpxL, !pxM, pagP, IpxP, and/or eptA genes can be generated in donor strains using the phage XRed recombinase procedure, and then transferred to a KPM strain by P1 v r transduction. Excision of the kanamycin resistance gene can be carried out in the presence of the FLP recombinase, and the temperature-sensitive helper piasmid pCP20 can be removed. P1 wr transduction and other such techniques are well known to one of ordinary skill in the art.

[00168] For example, the A!pxL: :Km ⁺ cassette can be generated in KPM22 to yield strain KPM279 (KPM22 Δ/p L: :Km ⁺ ). KPM279 can be used as a donor for transfer of the A/pxL::Km ^÷ cassette to KPM22 L1 via P1 wr transduction to yield strain KPM288 (KPM22 L1 Δ/pxL:: Km ⁺ ) or to KPM22 L1 1 via P1 vir transduction to yield strain KPM290 (KPM22 L1 A/pxL::Km ⁴ ). [00169] Further, strain KPM316 (KP 22 L1 1 MpxL pxM ApagP MpxP) can be obtained via successive deletions of the acyl transferases (in the foilowing order) using KPM280 (KPM22 MpxM: Km ⁺ ), BW30270 MpxP :Km and BW30270 ApagP::Km ^f as donor strains of the targeting cassettes, respectively: KP 290 (KPM22 L1 1 Δ/ρχί_::Κηι ⁺ ) > KPM 296 (KPM22 L1 1 AipxL) > KPM 300 (KPM22 L1 1 A!pxL AlpxM::K ⁺ ) > KPM 303 (KPM22 L1 1 AipxL MpxM) > KP 310 (KPM22 L1 1 MpxL MpxM ApagP::Km ⁺ ) > KPM312 (KPM22 L1 1 MpxL MpxM ApagP) > KPM314 (KPM22 L11 MpxL MpxM ApagP AipxP::KnY) > KPM316 (KPM22 L1 1 MpxL MpxM ApagP MpxP).

[00170] KP 318 was derived from KPM316 by P1 v r transduction of the AepiA::Km ⁺ created by the ,\Red recombinase procedure in BW30270Aep£4::Km ⁺ to give KPM317 (KP 316 AeptAvXm^) which was then cured of the Km cassette by transient expression of FLP recombinase. PGR amplification using primers that fiank each deletion were used to confirm the deletions against control DNA from the parental BW30270 (FIG 9). In each case the parental strain shows a larger PGR product with the primers that flank the gene deleted in KP 318.

EXAMPLE 4: OTHER REAGENTS AND METHODS

[00171] The following example describes other reagents and methods that may be used in accordance with the present invention. The present invention is not limited to these examples.

[00172] The presence of endotoxin (e.g., LPS) can be detected via standard assays. For example, HEK-Blue™ LPS Detection Kits (Invivogen, San Diego, CA) may be used. In this assay, HEK-Biue™-4 cells are extremely sensitive to LPS (lipid A), and can be used to detect concentrations of LPS (lipid A) as low as 0.3 ng/mL. The assay can be followed according to the manufacturer's protocol.

[00173] Whole cells and also outer membrane extracts of cells were tested in the HEK-Blue™ LPS Detection Kit. Control wild type cells and outer membranes were prepared from the parent BW30270, and these were tested with samples of cells and extracts from KPM318 and KPM318. The results, shown in FIG 10, demonstrate that the HEK-Blue™ LPS assay is extremely sensitive to LPS endotoxin, responding to as little as 2 pg of LPS. Signals in any of the samples prepared from KPM316 or KP 318 were at essentially background levels, even at 20 micrograms of outer membrane added to an assay, 9 orders of magnitude less potent for TLR4/MD2 signalling. Whole cells of KPM316 or KPM318 were also unable to elicit a significant signal at the highest levels tested, in contrast to the whole cells of BW30270, which gave significant response at levels 4 orders of magnitude lower.

[00174] Analysis of the ESI-Mass spectra of the outer membrane extracts of KPM318 showed the expected prominent masses for lipid IVA and lipid IVA- Ethano!amine (FIG 1 1 panel A). ESI-MS of KP 318 outer membranes showed only lipid IVA (FIG 10 panel A), as expected since the ethanolamine transferase has been deleted from this strain.

[00175] P1 ½r transduction is a standard method used to move genes/alleles from a donor strain to a recipient strain via P1 v r, a mutant bacteriophage that enters the lytic phase upon infection. P1 (P1 v r) can package approximately 90 kb of DNA, so it is generally used with a selectable marker. To perform such experiments, donor strains (and recipient strains) are cultured. P1 v r can then be added to the donor strain culture and monitored until the culture has completely lysed. Lysate is then harvested and added to the culture of recipient cells. Following incubation of the recipient cells with the lysate, the recipient ceils are plated on selective media to select for colonies with the selectable marker (and thus the gene of interest).

[00176] The complete sequence of the E. coli K-12 strain is provided in Blattner FR et al. (Science. 1997 Sep 5;277(5331 ):1453-62) and Riley M et al. (Nucleic Acids Res. 2008 Jan 5;34(1 ): 1-9. Print 2006). The generation of the KPM318 strain is outlined in Figure 12. mshA52 replaces the wild type allele of msbA, wherein a C at 965895 is replaced by a T, resulting in a Serine instead of Proline at amino acid 18 in the MsbA protein (Figure 12a). The following were deleted from the parental strain: AgutQ (Figure 12bJ, AkdsD (Figure 12c), AlpxL (Figure 12d), AlpxM (Figure 12e), pagP (Figure 12f), and A!pxP (Figure 12g), The KPM316 sequence was compared to that of the wild-type strain MG1855 (Figure 13). The functions of these mutations are unknown. [00177] The generation of the KPM318 strain is outlined in Figure 14. msbA52 replaces the wild type allele of msbA, wherein a C at 965895 is replaced by a T, resulting in a Serine instead of Proline at amino acid 18 in the MsbA protein (Figure 14a). The following were deleted from the parental strain: AgufQ (Figure 14b), AkdsD (Figure 14c), AlpxL (Figure 14d), AlpxM (Figure 14e), ApagP (Figure 14f), AipxP (Figure 14g), and AeptA (Figure 14f). Examples of generated strains are provided in Table 1.

[00178] The KPM318 and BW30270 strains were sequenced at the Scripps Core DNA sequencing facility using an il!umina DNA sequencer. The sequences were generated using paired end reads of 40 or 80 bases. DNA samples were prepared from each strain and used to generate end-tagged libraries. Up to six libraries were run per lane, along with a reference lane. The other lanes were used for additional unrelated DNA reads, which served as further internal controls. Single nucleotide polymorphisms (SNPs) refer to ail single base changes defined as being 100% mutant by the Scripps analysis programs when compared to the reference genome, the Blattners MG1655 strain. Deletion/ insertion polymorphisms (DiPs) refer to all the deletions or insertions (up to 8 bp) defined by the Scripps analysis programs, again using MG1655 as the reference. Only those detected at 100% of reads are provided. Whole gene deletions were independently confirmed by separate sequence alignments. The KPM318 sequence was compared to that of the wild-type strain BW30270 (Figure 15). Sequencing identified the msbA-P188 suppressor allele, which was previously established to exist in the original KPM strain (Figures 14 and 15). Ail known changes introduced by strain engineering were determined to be present at 100% frequency. This was true for all engineered deletions and point mutations.

[00179] Temperature-resistant derivatives of KPM318 were isolated in an attempt to identify more robust strains. Overnight cultures of KPM318 grown in LB medium at 37°C were plated onto LB-agar plates and incubated at 42°C. Only after continued incubation of the plates at 42°C for four to five days, a number of clones were obtained that regained their colony-forming ability at the elevated temperature. The 42°C~resistani phenotype of randomly selected clones was confirmed by overnight growth on LB-agar plates at 42 ^C C. A series of independent, nonclonal 42 ^c C-resistant derivatives of KP 318 were identified. Among a total of 24 temperature-resistant KPM318 derivatives, the strains KP 318-9, KPM318-10, KPM318-19 and KPM318- 23 displayed the best growth characteristics, being capable of exponentially growing at temperatures of 30°C, 37°C and 42°C in either SB medium (Figures 16, 18, and 20) or under conditions of restored LPS biosynthesis in SB medium supplemented with 15 μ D-arabinose 5~phosphate (ASP) and 10 μΜ D-glucose 6-phosphate (G6P) (Figures 17, 19, and 21 ). Therefore, these temperature-resistant strains exhibit robust growth characteristics over a range of temperatures.

[00180] The KPM318-9, KPM318-10, KPM31 S-19 and KPM318-23 strains were sequenced at the Scripps Core DNA sequencing facility using an Silumina DNA sequencer (Figure 22). Specific mutations were identified that may account for the viability of the strains at 42°C. Strains KP 318-9 and KPM318-10 express a frr- D61 Y mutation that alters a ribosomai recycling factor (Figures 22a and 22b). The KP 318-19 strain has a frameshift mutation in efp, which encodes elongation factor P (Figure 22c). The KPM318-23 strain has a deletion of P148 and L147 in dcd, which encodes the deoxycytidine deaminase gene (Figure 22d). The specific functions of these mutations are unknown.

[00181] The LPS/lipid IVA composition of the KPM318-9, KPM31 S-10, KPM318-19 and KPIV1318-23 strains was analyzed. The cultures (2.5 L each) were grown at 42°C to stationary phase in either LB medium or LB medium supplemented with A5P/G6P. The LPS/lipid IV _A was extracted from the dried biomass of each strain using either the original phenol-chloroform-light petroleum (PCP) procedure for those strains grown in LB medium with A5P/G6P or a modified PCP protocol for strains grown in LB-only medium. Then the extracts were subjected to ESI FT-ICR mass spectrometry. The KPM318-23 strain grown in LB medium predominantly expresses lipid SV _A (peak at 1404.86 u) at 42°C (Figure 23). When the 42°C-resistant KP 318 derivatives were grown in LB medium supplemented with A5P/G6P, biosynthesis of the E. coli K-12 core oligosaccharide was restored. There was expression of a similar mixture of different giycoforms in all strains, albeit attached to the tetraaeylated precursor lipid IV _A (Figures 24-26), LPS isolated from the parental strain KPM318 grown at 37°C in LB medium supplemented with A5P/G6P was used as a control for all these experiments (Figure 27). A number of the molecular masses of the different glycoforms expressed by KP 318 could be assigned and are provided in Table 2. Taken together, the overall LPS/lipid !VA composition of the 42°C-resistant derivatives of KP 318 did not show any peculiarities with respect to the typical heterogeneous oligosaccharide composition of the K-12 core. All outer membrane preparations of these strains showed no lipid A; they only contained lipid IVA-

[00182] The biological activity of both the bacterial celis and the LPS/lipid IVA isolated from various KPM strains grown in either LB/SB medium or LB/SB medium supplemented with A5P/G8P was examined using the HEK-Blue LPS detection assay (InvivoGen). For preparation of bacterial cells for this detection assay, 1 mi of an overnight culture of each strain was sedimented by centrifugation and washed in Dulbecco ^' s phosphate buffered saline (DPBS). To kill the bacterial cells, the pellet was subsequently resuspended in 1 ml of a 10 x cone, penicillin-streptomycin (Pen- Strep) in DPBS solution. The mixture was incubated at room temperature for 2 hr and stored at 4°C overnight. At the same time, serial dilutions of the overnight cultures were prepared for calculation of cell number (cfu/ml) of the overnight cultures/Pen-Strep killed ceils. The Pen-Strep suspensions of the strains were diluted to 10 ⁸ cfu/ml in 10 x cone. Pen-Strep in DPBS solution, and serial dilutions of the suspensions were then prepared in 10 x cone. Pen-Strep in DPBS solution to yield bacterial ceil suspensions of 10', 10 ⁶ , 10 ⁵ , 10 ⁴ , 10 ³ , 10 ² and 10 cfu/ml. A typical HEK-Blue LPS detection assay using serial dilutions of bacterial ceil suspensions from strains BW30270, KPM316 and KPM318 is shown in Figure 28. There is little to no LPS activity in the bacterial ceil suspensions from the KPM316 and KPM318 strains grown in LB medium, as compared to the control strain BW30270. There is a slight increase in activity for the KPM318 and KPM318 strains grown in media supplemented with A5P/G8P. There is little to no N FKB activation induced by serial dilutions of the LPS/lipid IV _A samples isolated from the KPM316 and KP 318 strains (Figure 29). [00183] The biological activity of the LPS/!ipid !V _A isolated from the temperature- resistant strain KPM31 S-9 grown at 42°C in LB-oniy medium and LB medium supplemented with A5P/G6P was compared with the ability of LPS/iipid IVA samples from BW30270, KPM318 and KPM318 to induce N FKB activation in H EK-Blue cells. Strain BW30270 was grown at 37°C in LB medium, whereas strains KPM316 and KPM318 were cultivated at 37°C in either LB medium or LB medium containing A5P/G6P. To examine the potential antagonistic activity of KPM31 8-9, constant amounts of the LPS/iipid IV _A isolated from KPM318-9 (256 pg) were mixed with decreasing amounts of BW30270-derived LPS. The LPS/iipid IV _A from LP 318-9 is a potent antagonist of LPS activity as demonstrated by the ability of BW30270 to compete with this antagonist when it is present at high concentrations (Figure 29, lanes 9-12).

[00184] A human TNF-alpha ELISA was performed with LPS/iipid IV _A samples isolated from the KPM316 and KPIV1318 strains grown at 37°C in either LB medium or LB medium containing A5P/G6P (Figures 30-32). The S-LPS isolated from £. co!i K-235 was used as a positive control. The blood was separated using Biocoll (density: 1 .077 g/mL). Mononuclear cells (MNCs) were isolated and washed twice in Hanks and once in VLE RPML The monocytes were differentiated into macrophages by incubating them in Teflon bags with M-CSF. The macrophages were harvested after 7 days. The number of ceils was determined. Macrophages were seeded into fiat-bottom 98 well plates at 1 x 10 ^s ceils/well in VLE RPMI + 100 U penicillin + 100Mg/ml streptomycin + 2 mM L-g!utamine + 4% AB-serum (and free of serum). The LPS/iipid IV _A samples were isolated from KPM318 and KPM318 strains and preincubated at 37°C and 5% CO ₂ for 30 min. S-LPS K-235 was added to the samples for 4 hr. Supernatants were removed and stored overnight at 4°C. An ELISA was performed to assess TNF-alpha activity (BD Biosciences #555212). Serial dilutions of the LPS samples in VLE RPMI without AB-serum were prepared. KPM318 LPS LB, KPM318 LPS LB, KPM 318 LPS LBAG, and KPM 316 LPS LBAG were prepared at_1 mg/ml in 20 mM HEPES, pH 7.0. Then serial dilutions from 100 ug/mL to 1 ng/mL were prepared. The plates were coated with capture antibody, which was diluted 1 :250 in coating buffer (PBS). The plate was agitated overnight and then washed three times with wash buffer. The plate was blocked with 80 μΙ assay di!uent/wel! and agitated at room temperature for 1 hr. The plate was washed three times with wash buffer,

[00185] Samples were diluted in assay diluent and serial dilutions of the standard were pr« jparec as follows:

1 :212 = = 500 pg/ml

1 :2 = - 250 pg/ml

1 :2 = = 125 pg/ml

1 :2 ^~ - - 62,5 pg/ml

1 :2 = - 31 ,25 pg/m

1 :2 - = 15,6 pg/ml

1 :2 - ^■ 7,8 pg/ml

1 :2 = = 3,9 pg/ml

The plates were agitated with standard, samples and blank at room temperature for 2 hr. The plate was washed three times with wash buffer. The detector solution and detection antibody (biotinyiated anti-human TNF-aipba) were added to the plate at 1 :1000. The enzyme reagent (streptavidin-horseradish peroxidase conjugate) was added at 1 :250. The plate was agitated at room temperature for 1 hr and then washed three times with wash buffer. The plate was agitated in the dark with TMB (1 :20) in substrate buffer. The plate was incubated for a minimum of 30 min until the wells turned blue. The reaction was stopped with 50 μΙ/weii H2SO4. The absorbance was measured at 450 nm. The LPS/iipid IVA isolated from the positive control K-235 potently increased TNF-aipha activity at all concentrations tested (Figure 30). The LPS/iipid !V, from the KPM316 and KPM318 strains was able to inhibit the induction of TNF-alpha by the K~235~derived LPS at ail concentrations tested (Figure 30); therefore, the LPS/iipid !VA isolated from the KPM316 and KPM318 strains is a potent antagonist of LPS activity. The LPS/iipid IV _A from the KPM318 and KP 318 strains grown in medium containing 4% AB-serum was able to inhibit the release of TNF-alpha from macrophages (Figure 31 ). The LPS/iipid IV _A from the KPM318 and KPM318 strains grown in serum free medium was able to inhibit the release of TNF- alpha from macrophages (Figure 32). Therefore, the LPS/iipid !V _A isolated from the KPM316 and KP 318 strains is a potent inhibitor of TNF-alpha activity. [00186] LPS is known to exert its inflammatory functions via activation of the TLR4 receptor in human cells. The biological activity of the LPS/iipid IV _A from the KPM316 and KP 318 strains was evaluated by assessing the release of IL-8 from HEK293 hTLR4/MD2 #33 (Figure 33) and HEK293 hTLR4 #2 cells (Figure 34). hTLR4/ D2 #33 cells respond to LPS binding, generating a signal, while HEK293 hTLR4 #2 cells respond to peptidogiycan, and not to LPS. Therefore, the latter cell line can be used to assess LPS specificity. S-LPS from the K-235 strain was used as a positive control in these assays. The HEK293 hTLR4/MD2 #33 cell medium was DME (Biochrom) supplemented with Pen/Strep/Glu and 10% FCS. There were 50,000 HEK293 hTLR4/ D2 #33 cells/well seeded in the wells. The cells were stimulated in the presence of the LPS/iipid IVA samples for a period of 24 hr, followed by measurement of human IL-8 release (BD Biosciences #555244). The LPS/iipid IV from the KPIV1316 and KPM318 strains was able to block the release of IL-8 from HEK293 hTLR4/MD2 #33 cells (Figure 33). The LPS/iipid IV _A extracts from the strains were crude extracts as they were able to promote the release of IL-8 from HEK293 hTLR2 #2 cells (Figure 34). TLR2 is a receptor for peptidogiycan, another bacterial component that can cause cytokine signalling. The abundance of free peptidogiycan is typically far lower than the levels of LPS, so it is usually a minor component. Here it is used to demonstrate that the extracts do indeed contain material derived from the outer membrane-periplasm of the cells, since this is where peptidogiycan is located. The HEK cells over expressing TLR4/MD2 (ceil line #33) responded to LPS binding, i.e., released IL-8 (Figure 33), while the HEK cells over expressing TLR2 (ceil line #2), which respond to peptidogiycan, do not respond to LPS (Figure 34). Therefore, this control confirms that something from the outer membrane extracts was indeed added to the assays in both cases.

[00187] The ability of the KPM318-9, KP 318-10, KPM318-19 and KP 318-23 strains to overexpress heterologous genes was assessed. The MalE-LacZa fusion protein was used as a model protein to investigate the capability of these strains to express cytoplasmic and periplasmic variants of the MalE protein in large quantities under standard growth and induction conditions. The BW30270, KPM318, KPM318- 9, KPM318-10, KPM318-19 and KPIV1318-23 strains were transformed each with plasmids p AL-c2 and pMAL-p2 for cytoplasmic (Figure 35, upper panel) and peripiasmic MalE-LacZa (Figure 35, lower pane!) expression, respectively. The following strains were obtained:

BW30270/p AL-c2 (control)

KPM318/p AL-c2

KPM318-9/pMAL-c2

KPM318-10/p AL-c2

KPM318-19/p AL-c2

KPM318-23/p AL-c2

BW30270/p AL-p2 (control)

KPM318/p AL-p2

KPM318-9/pMAL-p2

KPM318-10/p AL-p2

KPM318-19/p AL-p2

KPM318-23/p AL-p2

To induce expression of Ma!E-LacZau isopropy!-G-D-thioga!actoside (!PTG) was added to a final concentration of 0.4 mM when the cultures reached the mid- exponential growth phase at an GDeoo of approximately 0.8. For strains carrying the plasmid pMAL-c2, the cells were harvested prior to induction (uninduced), 3 hr after induction, as well as after overnight induction. For strains with plasmid pMAL-p2, samples were taken from uninduced cells and after induction times of 3 hr, 6 hr, 12 hr, and 24 hr. The ability to secrete the Ma!E-LacZa protein into the medium was additionally examined for strains carrying the pMAL-p2 plasmid, with pMAL-c2 strains used as controls. Protein extracts were prepared using the BugBuster reagent according to the manufacturers recommendations (Novagen). Western blot analyses using a monoclonal antibody against MalE (NEB) were performed to detect a!E-LacZa expression.

[00188] The expression levels of MalE-LacZa were highest at 3 hr post-induction in pMAL-c2 strains, and at 3 hr and 6 hr post-induction in pMAL-p2 strains (Figures 36- 42). Expression of MalE-LacZa was consistently highest in strains KPM318, KP 318-9 and KP 318-10, showing almost identical expression levels to the BW30270 wild-type controls (Figures 36-42). As expected, both Coomassie-b!ue stained polyacryiamide gels and immunobiots did not detect Ma!E-LacZa in the culture media of strains carrying the plasmid p AL-c2 for cytoplasmic expression of the protein (Figures 36-42). In contrast, the abundance of the MalE-LacZa protein in the culture media of pMAL-p2 strains apparently increased with increasing duration of IPTG induction. This did not only apply to all KPM mutants, but also to the BW30270 wild-type strain (Figures 36-42). It is unknown whether the presence of MaiE-LacZa in the culture medium is a result of secretion of the protein as a result of a compromised outer membrane in the KPM strains or simply cell lysis due to perip!asmic overexpression of the protein under the experimental conditions used in this study. MBP is known to be highly expressed in £. coli. The KPM18 temperature-resistant derivatives exhibited similar levels of protein expression to the wild-type control strain BW30270. These strains secrete or leak MBP more readily than the wild-type strain, which allows the collection of protein in the absence of ceil lysis.

[00189] The ability of KP 318 and its temperature-resistant derivatives to produce the human ApoA1 protein was investigated. The apoA1 gene optimized for codon usage of £. coli was synthesized and cloned under the control of the Tn5 promoter in pJexpress404 by the company DNA 2.0. For peripiasmic expression of ApoA1 , the gene was fused to the leader sequence of ompA for the major outer membrane protein A of £. coli. In addition, both the cytoplasmic and the peripiasmic version of the apoA 1 gene were C-terminaliy fused to a 8xHis:tag-coding sequence. The strains were transformed with pJexpress404:51 149 and pJexpress404:51 150 for cytoplasmic (Figure 43, upper panel) and peripiasmic ApoAi (Figure 43, lower panel) expression, respectively. Despite several attempts, the strain KP 318-23 could not be transformed with the pJexpress404:51 149 and pJexpress404:51 150 expression vectors. The following strains were obtained:

1. BW30270/pJexpress404:51 149 (control)

2. KP 318/pJexpress404:51 149

3. KP 318-9/pJexpress404:51 149

4. KP 318-10/pJexpress404:51 149

5. KP 318-19/pJexpress404:51 149

8. BW30270/pJexpress404:51 150 (control)

7. KPM318/pJexpress404:51 150 8, KPM31 S~9/pJexpress4Q4:51 150

9, KP 318-10/pJexpress404:51 150

10, KPM318-19/pJexpress404:51 150

The cultures were grown overnight at 37°C in 5 ml of SB medium containing 100 pg/m! ampicillin. The overnight cultures (1 :50) were diluted in 40 mi of pre-warmed SB medium containing 100 Mg/m! ampicillin. The cultures were grown at 37°C and agitated at 250 rpm to an ODeoo of approximately 0.8. The ceil number (cfu/ml) for each strain was determined (Table 3). The uninduced cultures were centnfuged for 10 min at 6,000 rpm (4°C). A 1 ml aliquot of each pJexpress404:51 150-cu!ture supernatant was obtained for further analysis. The 1 -mi culture supernatants and cell pellets were frozen and stored at -80°C. IPTG was added to the cultures (at a final concentration of 0.4 mM). The cells were incubated at 37°C and agitated at 250 rpm for 3 hr. The cell number (cfu/ml) for each strain (Table 4) was evaluated. After the 3 hr induction, 5 ml of each culture was removed:

1 . BW30270/pJexpress404:51 149 - induced (3 hr)

2. KPM318/pJexpress404:51 149 - induced (3 hr)

3. KPM318-42C 9/pJexpress404:51 149 - induced (3 hr)

4. KP 318-42C 10/pJexpress404:51 149 - induced (3 hr)

5. KPM318-42C 19/pJexpress404:51 149 - induced (3 hr)

6. BW30270/pJexpress404:51 150 - induced (3 hr)

7. KPM318/pJexpress404:51 150 - induced (3 hr)

8. KP 318-42C 9/pJexpress404:51 150 - induced (3 hr)

9. KP 318-42C 10/p Jexpress404:51 150 - induced (3 hr)

10. KPM318-42C 19/pJexpress4Q4:51 150 - induced (3 hr)

The induced cultures were centrifuged for 10 min at 6,000 rpm (4°C). A i m! aliquot of each pJexpress404:51 150-culture supernatant was obtained for further analysis. The 1 -ml culture supernatants and cell pellets were stored at -80°C. The induction was continued at 37°C and 250 rpm. The eel! number (cfu/ml) for each culture was determined strain after 6 hr of induction (Table 5). A 3 m! aliquot of each culture was removed after 6 hr of induction:

1 . BW30270/pJexpress404:51 149 - induced (6 hr)

2. KP 318/pJexpress404:51 149 - induced (6 hr)

3. KPM318-42C 9/pJexpress404:51 149 - induced (6 hr) 4, KPM318-42C 10/pJexpress404:51 149 - induced (8 hr)

5, KP 318-42C 19/pJexpress404:51 149 - induced (6 hr)

8. BW30270/pJexpress404:51 150 - induced (6 hr)

7. KP 318/pJexpress404:51 150 - induced (6 hr)

8. KPM318-42C 9/pJexpress404:51 150 - induced (6 hr)

9. KP 318-42C 10/pJexpress404:51 150 - induced (6 hr)

10. KP 318-42C 19/pJexpress404:51 150 - induced (6 hr)

The induced cultures were centrifuged for 10 min at 6,000 rpm (4°C)

There was a 1 ml aliquot of each pJexpress404:51 150 culture supernatant removed for further analysis. The 1-ml culture supernatanfs and cell pellets were frozen and stored at -SOX. The induction was continued at 37°C and 250 rpm. The cell number (cfu/ml) was determined for each strain after 12 hr of induction (Table 8). A 2 ml aliquot of each culture was removed after the 12 hr induction:

1. BW3027G/pJexpress404:51 149 - induced (12 hr)

2. KPM318/pJexpress404:51149 - induced (12 hr)

3. KPM318-42C 9/pJexpress404:51 149 - induced (12 hr)

4. KPM318-42C 1 Q/pJexpress4Q4:51 149 - induced (12 hr)

5. KP 318-42C 19/pJexpress404:51 149 - induced (12 hr)

8. BW30270/pJexpress404:51 150 - induced (12 hr)

7. KP 318/pJexpress404:51150 - induced (12 hr)

8. KPM318-42C 9/pJexpress404:51 150 - induced (12 hr)

9. KP 318-42C 10/pJexpress404:51 150 - induced (12 hr)

10. KP 318-42C 19/pJexpress404:51 150 - induced (12 hr)

The induced cultures were centrifuged for 10 min at 6,000 rpm (4°C). There was 1 ml of each pJexpress404:51 150-cuiture supernatant obtained for further analysis. The 1-mi culture supernatants and cell pellets were frozen and stored at -80°C. The induction was continued at 37°C and 250 rpm. The ceil number (cfu/ml) for each strain was determined after 24 hr of induction (Table 7). There was 1 ml of each culture removed after the 24 hr induction:

1. BW30270/pJexpress404:51 149 - induced (24 hr)

2. KPM318/pJexpress404:51149 - induced (24 hr)

3. KP 318-42C 9/pJexpress404:51 149 - induced (24 hr)

4. KPM318-42C 10/pJexpress404:51 149 - induced (24 hr) 5, KPM318-42C 19/pJexpress404:51 149 - induced (24 hr)

8, BW30270/pJexpress404:51 150 - induced (24 hr)

7. KPM318/pJexpress404:51 150 - induced (24 hr)

8. KP 318-42C 9/pJexpress404:51 150 - induced (24 hr)

9. KPM318-42C 10/pJexpress404:51 150 - induced (24 hr)

10. KP 318-42C 19/pJexpress404:51 150 - induced (24 hr)

The cultures were induced for 10 min at 6,000 rpm (4°C). The pJexpress404:51 150- culture supernatants were obtained for further analysis. The 1-mi culture supernatants and cell pellets were frozen and stored at -80°C. The uninduced and induced cells were thawed and all cell pellets were resuspended in 300 μΙ of 1 x BugBuster. There was 3 μ! of 20 mg/m! lysozyme solution added to the samples and 0.5 μΙ of Benzonase. The samples were incubated at room temperature for 20 min with shaking. The samples were centrifuged at 14,000 rpm for 20 min (4°C). The 1 x Bug Buster contains:

1 ml 10 x cone. BugBuster

200 μΙ 1 M Tris-HCI, pH 7.5 (20 m Tris-HCI, pH 7.5)

3.5 μΐ 2-mercaptoEtOH (5 mM 2-mercaptoEtOH)

[00190] Both the cytoplasmic and the peripiasmic version of the human ApoA1 protein, as analyzed by 12% SDS-PAGE, were expressed in E. coli strains BW30270, KPM318, KPM318-9 and KPM318-10, but not in KP 318-19 (Figures 44- 49). x e apparent relative molecular mass of the 29-kDa band was in accordance with the calculated mass of the protein (29.4 kDa) fused to the C- terminai histidine tag. Strikingly, periplasmicaliy expressed ApoA1 migrated as two bands with apparent relative molecular masses of 29 kDa and 31 kDa, the latter being consistent with the calculated mass of unprocessed ApoA1 of 31.4 kDa (Figures 47 and 48). It has been previously shown that ApoA1 is expressed in the cytoplasm of £. coli as a mixture of soiuble and insoluble protein, which suggests that the supposed unprocessed form of ApoA1 constitutes the insoluble fraction of the protein that is not accessible to transport to its final destination in the periplasm and processing. However, we currently cannot rule out the possibility that the appearance of the slower migrating band is a result of sample preparation at 95°C for 2 min prior to SDS-PAGE. In any case, the results indicate that the expression rate of ApoA1 in the KP strains was at least as high as in BW30270, showing a steady level for at least 6 hr.

[00191] In contrast to the experiments performed with MalE-LacZa, the volume of the culture media for SDS-PAGE analyses was adjusted in relation to the optical density (GDeoo) of pJexpress404:51 I SO-carrying cells so that the amount of the culture supernatant loaded onto the polyacrylamide gel came from one and the same Οϋβοο value (Tables 8 - 12). Remarkably enough and as presented for strain KPM318-10/pJexpress404:51 150 in Figure 50, the optical cell densities of all temperature-resistant strains increased while the number of viable cells decreased over the entire period of IPTG induction (Tables 3 - 7). As shown in Figures 51 - 53, the strains carrying the pJexpress404:51 150 plasmid were not capable of secreting ApoA1 into the culture medium, The weakly Coomassie-stained bands rather resembled the protein banding pattern of the ceil extracts and, thus, most likely originated from cell lysis. By using immunoblots with a Penta-His antibody (QIAGEN), we could corroborate ail findings of SDS-PAGE analyses (Figures 54 - 62). Taken together, our data suggests that the 42°C-resistant KP 318 derivatives KP 318-9 and KP 318-10 are suitable hosts for protein expression. Optimization of the conditions for bacterial growth, induction and sample preparation should further improve the expression rate and yield of the proteins of interest.

[00192] The DNA transformation ability of the cells was assessed. For preparation of eiectrocompetent ceils, the cultures were grown to mid-exponential growth phase (GDeoo = 0.5 - 0.7) at 37°C with vigorous shaking (220 rpm). The ceils were subsequently washed three times with ice-cold water and twice with ice-cold 10% glycerol. Finally, the cell sediments were resuspended in a small volume of ice-cold 10% glycerol, and 50-μΙ aliquots were prepared for storage at ~-8Q°C. First, we examined the influence of restored K-12 core oligosaccharide biosynthesis on the transformation of KPM318 with pMAL-c2. Strain BW30270 was grown in LB medium as a control, whereas KPM318 was cultivated in LB medium and LB medium containing A5P/G8P. The cells were harvested for preparation of eiectrocompetent ceils when strains BVV30270, KPM318 (LB) and KPM318 (LB A5P/G6P) reached an optical density (GDeoo) of 0.621 , 0.813, and 0.519, respectively. In order to use one and the same number of electrocompetent cells for transformation, we determined the colony forming units per milliliter of the competent cells of each strain (Table 13).

[00193] The electrocompetent ceils of KPM318 (LB) and KPM318 (LB A5P/G6P) were diluted to 7.3 x 10 ⁸ cfu/ml with ice-cold 10% glycerol (3.65 x 10' cfu/50 i) and transformed each with 25 ng of piasmid pMAL-c2 (Fig. 35). The transformation mixtures were serially diluted, plated onto LB agar plates containing 100 g/ml ampicil!in and incubated at 37°C. The results in Table 14 demonstrate that strains BW30270, KPM318 (LB) and KPM318 (LB A5P/G6P) were transformed with similar efficiencies. The data further shows that KPM318 predominantly expressing lipid IVA in the outer membrane when grown in LB~oniy medium (Figure 1 1 ) was not transformed with higher efficiency than BW30270 or KPM318 under conditions of restored core oligosaccharide biosynthesis.

[00194] The piasmid pMAL-c2 from four randomly selected transformants of each strain was isolated from 5-m! overnight cultures grown in LB medium containing 100 Mg/ml ampicillin at 37°C and 200 rpm. The plasmids were isolated using the Wizard Plus Minipreps DNA Purification System (Promega). Samples of 1 μΙ were subsequently run on a 0.8% agarose gel in TBE buffer (Figure 83). The electrophoresis results indicated that the yield of piasmid pMAL-c2 was apparently almost identical for £ coii strains BW30270/pMAL-c2, KPM318 (LB)/pMAL-c2, and KPM318 (LB A5P/G6P)/pMAL-c2.

[00195] As a next step, the 42°C-resistant KP 318 derivatives KP 318-9, KPM318- 10, KPM318-19 and KPM318-23 were transformed each with plasmids pMAL-c2 and pMAL-p2 (Figure 35). The ceils were grown in SB medium at 37°C and harvested for preparation of electrocompetent cells when KPM318-9, KPM318-10, KP 318-19 and KPM318-23 reached an optical density (OD ₆ oo) of 0.618, 0.598, 0.491 , and 0.702, respectively. The electrocompetent ceils were transformed each with 25 ng plV1AL-c2 and 25 ng pMAL-p2. Serial dilutions of the transformation mixtures were then plated onto LB agar plates with 100 pg/ml ampicillin and incubated at 37°C. The transformation efficiencies for the 42°C-resistant KPM318 strains are summarized in Table 15. While strains KPM318-9, KP 318-10 and KP 318-19 were transformed with high efficiency, the capability of KP 318-23 to uptake piasmids pMAL-c2 and pMAL-p2 was only marginal. In fact, the latter result was consistent with the failure to transform KPM318-23 with piasmids pJexpress404:51 149 and pJexpress4G4;51 150.

[00196] The piasmids pMAL-c2 and pMAL-p2 were isolated from 5-m! overnight cultures of two randomly selected transformants of KPM318-9/pMAL-c2, KPM318- 9/p AL-p2, KP 318-10/p AL-c2, and KPM318-10/p AL-p2, using the Wizard Plus inipreps DNA Purification System according to the manufacturer's recommendations (Prornega). The samples (1 μ! each) were run on a 0.8% agarose gel in TBE buffer (Figure 64). Finally, the yields of the isolated piasmids were determined for those strains that were subsequently used for the protein expression studies described above (Table 16). Taken together, both the high transformation efficiency and the acceptable yield of piasmid DNA obtained from a standard miniprep culture suggests that KPM318-9 and KP 318-10 are suitable "base strains" for the development of piasmid DNA production strains.

[00197] To develop a KP "base strain" dedicated to screening proteins derived from phage display libraries, the strain should have an F ^' piasmid to permit infection with M13 phage. VVe have used the E, coii JC19022 (F ^' 121 Tn fO) as a donor strain to transfer the F ^' 121 Tn fO piasmid to BW30270, KPM318, KP 318-9, KPM318-10, KPM318-19, and KPM318-23 by a tube mating protocol. The cultures were grown overnight in 5 ml of medium at 37°C with vigorous shaking (220 rpm). LB medium containing 10 g/m! tetracycline was used for JC19022 (F ^' 121 Tn f O), LB medium for BW30270, and LB medium supplemented with A5P/G6P was used for KP 318 and its temperature-resistant derivatives. The following dilutions of the overnight cultures were set-up using the same media as used for overnight growth:

Donor: 25 μΙ→ 5 mi

50 μΙ→ 5 mi

100 μΙ→ 5 ml

200 μΙ→ 5 ml

Recipients: 25 μΙ→ 5mi

50 μΙ→ 5m!

100 μΙ→5 ml 200 μΙ→ 5 ml

The samples were vigorously (220 rpm) shaken at 37°C for approximately 2 to 3 hr. The optical density (ODeoo) of the suspensions (1 ml) was recorded. The optimal ODeoo range is 0.08 to 0.2; the dilution with the ODeoo closest to 0.1 should be used if there are options available (this is true for the donor and recipient cultures). Sn order to remove the tetracycline from the medium of the donor strain, the culture should he centrifuged, and the cell pellet should be resuspended in 4 ml LB medium. A 1 :1 ratio of donor:recipient is used for the transfer. The dilution of the donor dilution is determined and multiplied by 500. This value is divided by the ODeoo of the recipient to obtain the amount of the recipient in μΙ to add to 500 μΙ of the donor. The donor culture (500 μ!) is incubated with the appropriate amounts of the recipient cuitures (Tables 17 and 18). The mating cultures are incubated at 37°C for 2 hr without agitation. The cuitures are then incubated for another 2 hr at 37°C and agitated at 220 rpm. The samples are vigorously vortexed to disrupt the mating pairs. Dilutions at 1 Q ^{_ i} , 10 ^"2 , 1 Q ^~3 , and 10 ^"4 in PBS (pre-warmed to room temperature) for each mating were prepared, and 100 μΙ of each undiluted and diluted mating culture was added to M9 agar plates containing 1 g/ml thiamine and 10 pg/mi tetracycline. The cuitures were incubated at 37°C. The potential transconjugants were streaked onto 9 agar plates containing 1 g/ml thiamine and 10 g/mi tetracycline and incubated at 37°C. The tube mating experiments yielded a number of potential transconjugants for all recipient strains. A subset of potential transconjugants was subsequently examined by PGR for the presence of the Δ/ρχΡ knockout mutation to distinguish between false positive clones and tetracycline-resistant KP strains. As shown in Figure 65, ail tested tetracycline-resistant KPM318 transconjugants contained the Δ/ρχΡ knockout mutation, which indicated the successful transfer of the F ^' 121 Tn fO piasmid to the KPM strain. By using the IpxP PGR, we also could identify several potential F ^' 121 Tn fO transconjugants of KP 318-9, KP 318-10, KPM318-19 and KPM318-23, which contained the required AipxP knockout mutation.

[00198] To provide evidence for the presence of the F ^' 121 Tn fO piasmid in KPM318 and its 42°C-resistant derivatives, as well as to show that the transconjugants are susceptible to M13 infection, the M13K07 helper phage was used to infect four randomly selected transconjugants of each strain. As 13K07 contains the origin of replication from P15A and the kanamycin resistance gene from Ίη903 both inserted within the M13 origin of replication, the development of kanamycin-resistant clones following infection is an indication for the susceptibility of a given strain to 13K07. Overnight cultures of E. coii JC19022 (F ^' 121 TnfO) and BW30270 (F ^' 121 TnfO) in LB medium containing 10 pg/m! tetracycline, and KPM318 (F ^' 121 TnfO) and 42° C- resistant KPM318 (F ^' 121 TnfO) derivatives in LB medium with 10 pg/rni tetracycline and A5P/G6P were set-up. The BW30270 and KPM318 strains were grown in LB medium and LB medium supplemented with A5P/G6P, respectively, as controls. The cultures were vigorously agitated (220 rpm) at 37°C. The following strains and potential transconjugants were grown;

1. BW30270 (control)

2. KPM318 (control)

3. JC19022 (F ^' 121 TnfO) (control)

4, BW30270 (F ^' 121 Tnf0)-1

5, BW30270 (F ^' 121 Tn 0)~2

8, BW30270 (F ^' 121 Tnf0)-3

7. BW30270 (F ^' 121 Tnf0)~4

8. KP 318 (F ^' 121 Tnf0)-4

9. KPM318 (F ^' 121 Tn 0)-6

10 KP 318 (F ^' 121 Tnf0)-7

11 KP 318 (F ^' 121 Tnf0)~8

12 KPM318-9 (F ^' 121 Tn 0)~1

13 KP 318-9 (F ^' 121 Tnf0)-2

14 KPM318-9 (F ^' 121 Tnf0)~3

15 KP 318-9 (F ^' 121 ^" fnf0)-4

16 KPM318-10 (F ^' 121 Tnf0)-1

17 KP 318-10 (F ^' 121 Tnf0)-3

18 KP 318-10 (F ^' 121 Tni0)-4

19 KP 318-10 (F ^' 121 Tnf0)-5

20 KP 318-19 (F ^' 121 Tnf0)-3

21 KPM318-19 (F ^' 121 Tnf0)-4

22 KP 318-19 (F ^' 121 Tnf0)-5

23 KPM318-19 (F ^' 121 Tnf0)-6 24. KPM318-23 (F ^' 121 Τη ί0)-1

25. KP 318-23 (F ^' 121 Τη ί0)-2

26. KP 318-23 (F ^' 121 Tn ?0)-3

27. KPM318-23 (F ^' 121 Tn f 0)-5

The overnight cultures of BW30270 and JC19022 (F ^' 121 Tn fO) were grown in 3 ml LB medium (1 : 100), and KPM318 and the potential transconjugants were cultured in 3 mi of LB medium supplemented with A5P/G8P (1 :50). The cultures were grown to an early exponential growth phase at 37°C with vigorous shaking (280 rpm). A i m! aliquot was removed for determination of the ODeoo o the suspension (Table 19)

[00199] The M13K07 infection experiments resulted in kanamycin resistant colonies for ail potential transconjugants of KPM318 and its temperature-resistant derivatives (Table 20). Although the number of kanamycin-resistant colonies significantly varied among the strains, the results suggest that: (i) the conjugative transfer of plasmid F ^' 121 Ύη 10 to the KPM strains was successful, and (ii) the strains became susceptible to M13 infection. This should provide a fundamental basis for the generation of phage display libraries in temperature-resistant KPM318 derivatives.

[00200] The supE44 gene from E. coii XL1 -Blue was transferred to BW30270, KPM318 and its 42°C-resistant derivatives. To facilitate selection for supE44 in BW30270, KP 318 and its derivatives, the helper plasmid pMAK705 KmR CAT UT was constructed (Figure 86). First, the kanamycin resistance gene of plasmid pKD4 was amplified using the primers SBamHIKmR and 3Hindl!!KmR, followed by digestion of the PGR product with BamH! and Hindi!! . Second, the BamHI/Hindlll digested PGR product was cloned into the BamHI/Hindlll sites of the temperature- sensitive plasmid pMAK705 to yield pMAK705 KmR. Finally, the codon ACC at position 10 was substituted for a TAG (amber) nonsense codon using primers CATamber and CATwtreverse in site-directed mutagenesis with the Change-IT Mutagenesis System (USB).

(SEQID1 ) SBamHIKmR: ATATGGATCCTTACATGGCGATAGCTAGACTGG

(SEQID2) 3HindillKmR: ATATAAGCTTGAAGAACTCCAGCATGAGATGC

(SEQID3) CATamber:

GAGAAAAAAATCACTGGATATACCTAGGTTGATATATCCCAATGGCA (SEQID4) CATwtreverse:

CAGTACTGTTGTAATTCATTAAGCATTCTGCCGACATGGAAGCCATC

To verify the introduction of the amber nonsense codon into the cat gene, the E. coii strains TOP10 and XL1 ~Blue were transformed with piasmid pMAK705 KmR CATMUT, The resulting strains TOP10/pMAK705 KmR CATMUT and XL1 - Biue/pMAK705 KmR CATMUT were streaked on both LB + 30 pg/ml kanamycin and LB + 30 Mg/m! kanamycin + 15 pg/ml chloramphenicol plates (Figure 87). In contrast to TOP10/p AK705 KmR CATMUT carrying the wi!d-type allele of ginV, strain XL1 - Blue/pMAK705 KmR CATMUT was capable of growing on LB + 30 Mg/m! kanamycin + 15 g/mi chloramphenicol plates, indicating the suppression of the nonsense mutation by supE44 (Figure 67).

EXAMPLE 5: Construction of the PM Derived AeradiA ArecA strains

The ArecA::KmR strains KPM348, KP 349, KPIV135Q, KPM351 and KPM352, were each derived from the corresponding AendA strains to KPM343, KPM344, KPM345, KPM346 and KPM347. Δ as used in this example means deletion. The ArecA::KmR targeting cassette was transferred to KPM343, KP 344, KPM345, KPM348 and KPM347 by P1 vir transduction essentially as described for construction of other knockout mutations:

1. The E.coii strain BW28547 carrying the ArecA::KmR mutation was used as a donor for transduction. The strain was obtained from the E.coii Genetic Stock Center.

2. Phage P1 vir was propagated on BW28547 grown at 37°C in LB medium with 30 g/mi kanamycin to obtain a ArecA::KmR transducing !ysate.

3. For transduction, the AendA recipient strains KPM343, KPM344, KPM345, KPM348 and KPM347 were grown at 37°C in LB medium supplemeted with

A5P/G6P. 4. Transduction was performed according to standard protocols, with selection of potential transductants at 37° on LB-agar plates containing 30 g/ml kanarnycin.

5. The distance between recA and gutQ is only about 6 kb (theoretical co~ transduction rate: about 82%), so potential kanarnycin-resistant transductants were tested by PCR for the presence of both the GrecA;:KmR cassette and the deleted gutQ gene in KPM strains.

gutQ control primers:

5'gutQctrt1 - GTCGATAAGCTGATTACCGACGC

3'gutQctri2 - GTGAAACTATTCGTCAGGCACTGG

recA control primers:

5'recActrl - CTACTGCGTATGCATTGCAG

3'recActrl - TCGTAATCTTCTGCCGTAGC

6. As a result strains KP 348, KP 349, KPM350, KPM351 and KP 352 containing ArecA::KmR and the original AgutQ mutation were obtained.

7. The kanarnycin resistance gene is removed using transient transformation by pCP20 to obtain strains KPM353, KP 354, KPM355, KP 356, and KPM357.

8. The above recA primers serve for identifying the elimination of the KanR insert in the ArecA final strains.

„E 6: E, coli strain s arne designations:

Strain Description

P 22 Lll [msbA52 AkdsD AgutQ )

KPM290 {msbA52 AkdsD AgutQ AlpxL: : πι ^÷ )

KPM296 AlpxL derivative of KPM22 Lll

(msbA52 AkdsD AgutQ AlpxL)

KPM3QQ (msbA52 AkdsD AgutQ AlpxL AlpxM: :i¾t*)

PM303 AlpxL AlpxM derivative of KPM22 Lll

{rnsbA52 AkdsD AgutQ AlpxL AlpxM)

KPM310 (i sbA52 AkdsD AgutQ AlpxL AlpxM ApagP: : Km ⁴ )

KPM312 AlpxL AlpxM. ApagP derivative of KP 22 Lll

(msbA52 AkdsD AgutQ AlpxL AlpxM ApagP)

PM31 {! sbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP: : Km ^' )

KPM316 AlpxL AlpxM ApagP AlpxP derivative of KPM22 Lll

(i sbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP) PM317 (msbA52 AkdsD AgutQ AlpxL AlpxM. ApagP AlpxP AeptA:: Km ^4" ) KPM318 AlpxL AlpxM ApagP AlpxP AeptA derivative of KPM22 Lll

{! sbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA)

Temperature-resistent KPM318 derivative KPM318-42C 9 (i sbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA. frrlSl)

Temperature-resistent KPM318 derivative KPM318-42C 1

KPM335

(insbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA fr

Temperature-resistent KPM318 derivative KPM318-42C 19

KPM336

imsbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA efp ^' )

Temperature-resistent KPM318 derivative KPM318-42C 23

KPM337

{rnsbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA dcd ^" )

Temperature-resistent KPM334 derivative

PM33S

{msbA52 AkdsD AgutQ AlpxL AlpxM. ApagP AlpxP AeptA.

frr181AendA : : Km ^÷ )

Tempera ture-resistent KPM335 derivati ve

KPM339

(msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA frrieiAendA :Km ⁺ )

Temperature-resistent KPM336 derivative

KPM340 {msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA efp ^"

AendA: : Km ⁺ ) emperature-re s istent KPM337 derivative

KPM341

(msbA52 AkdsD AgutQ IpxL AlpxM ApagP AlpxP Aep A dcd ^'

AendA : Km ^' )

KPM342 (rasbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA AendA: :Km ^f )

KPM343 Temperature-re3is ent AendA derivative of KPM334

{msb&52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA frrlSlAendA)

KPM344 Temperature-resistent AendA derivative of KP 335

(msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA frzlSlAendA)

KPM345 Tetnpera ~ ure-resistent AendA derivative of KPM336

< _R isbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA efp ^' AendA) PM346 Temperature-resistent AendA derivative of KPM337

( sbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA dcd ^' AendA) PM347 AendA derivative of P 318

{msb&52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA AendA ⁾

KPM348 Temperature-resistent derivative of KPM343

_[ msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA. frrlSlAendA

Ar cA: : Κ 3 )

KPM349 Temperacure- resistent derivative of KPM344

_{ msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA frrlSlAendA ArecA: : K"V )

PM350 Temperature-resistent derivative of KPM345

_{ m.sbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA ^, efp AendA Are A: : :v3)

KPM351 Temperature-resistent derivative of KP 346

(msbA52 AkdsD AgutQ AlpxL AlpxM. ApagP AlpxP AeptA dcd ^' AendA ArecA: : Km ^" )

KPM352 {msbA52 AkdsD AgacQ AlpxL AlpxM ApagP AlpxP AeptA AendA

ArecA: :KnT)

KPM353 Temperature- resistent AendA ArecA derivative of PM334

{msbA52 AkdsD AgutQ AlpxL AlpxM. ApagP AlpxP AeptA frrlSlAendA ArecA)

P 354 Temperature-resistent AendA ArecA derivative of KFM335

₍ msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP Aep A frrlSlAendA ArecA)

KPM355 Temperature-resistent AendA ArecA derivative of PM336

(msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA efp ^' AendA ArecA)

PM356 Temperature-resistent AendA ArecA derivative of KP 337 (msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA dcd ^' AendA ArecA)

KPM357 AendA ArecA derivative of KP 318

imsbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AepcA AendA ArecA)

P 358 Temperature-resistent KPM334 derivative carrying ;;he plasmid

F ^' 121 TnlO

\msbA52 AkdsD gatQ AlpxL AlpxM ApagP AlpxP AeptA frrlBl ( F ^' 121 TnlO) ]

KPM359 Temperauire-resistent KPM335 derivative carrying the plasmid

F ^" 121 TnlO

[msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA frrlBl (F ^' 12i TnlO) ]

KPM360 Temperature --resistent KPM336 derivative carrying the plasmid

F ^' 121 TnlO

[msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA efp ^' (F ^' 121 TnlO) ]

PM361 Temperature-resi stent KPM337 derivative carrying the plasmid

F'121 TnlO

[ sbA52 AkdsD AgutQ AlpxL AlpxM. ApagP AlpxP AeptA dcd ^' (F'121 TnlO) ]

KPM362 KP 318 derivative carrying the plasmid F ^' 121 TnlO

[msbA52 AkdsD AgutQ AlpxL AlpxM ApagP AlpxP AeptA (F ^' 121 TnlO) ]

[00201] The following disclosures of the following patents are incorporated in their entirety by reference herein: W!PO Application No. 2007/084633. References and publications cited herein are incorporated in their entirety by reference herein to the extent that these references are consistent with the present invention.

[00202] Various modifications of the invention, in addition to those described herein, will be apparent to those skilled in the art from the foregoing description. Such modifications are also intended to fall within the scope of the appended claims. Each reference cited in the present application is incorporated herein by reference in its entirety.

[00203] Although there has been shown and described the preferred embodiment of the present invention, it will be readily apparent to those skilled in the art that modifications may be made thereto which do not exceed the scope of the appended claims. Therefore, the scope of the invention is only to be limited by the following claims.