Developing Gram-negative bacteria for the secretion of heterologous proteins

Gram-negative bacteria are attractive hosts for recombinant protein production because they are fast growing, easy to manipulate, and genetically stable in large cultures. However, the utility of these microbes would expand if they also could secrete the product at commercial scales. Secretion of biotechnologically relevant proteins into the extracellular medium increases product purity from cell culture, decreases downstream processing requirements, and reduces overall cost. Thus, researchers are devoting significant attention to engineering Gram-negative bacteria to secrete recombinant proteins to the extracellular medium. Secretion from these bacteria operates through highly specialized systems, which are able to translocate proteins from the cytosol to the extracellular medium in either one or two steps. Building on past successes, researchers continue to increase the secretion efficiency and titer through these systems in an effort to make them viable for industrial production. Efforts include modifying the secretion tags required for recombinant protein secretion, developing methods to screen or select rapidly for clones with higher titer or efficiency, and improving reliability and robustness of high titer secretion through genetic manipulations. An additional focus is the expression of secretion machineries from pathogenic bacteria in the “workhorse” of biotechnology, Escherichia coli, to reduce handling of pathogenic strains. This review will cover recent advances toward the development of high-expressing, high-secreting Gram-negative production strains.     

Culture medium for selective isolation and enumeration of Gram-negative bacteria from ground meats.

We developed a new medium, designated peptone bile amphotericin cycloheximide (PBAC) agar, which contains (per liter) 10 g of peptone, 300 mg of bile salts, 1 mg of amphotericin B, 1 g of cycloheximide, and 15 g of agar. When 21 samples of fresh ground beef were studied and plate count agar counts were used as references, we obtained a mean recovery of 28% of total counts with violet red bile agar overlay, whereas we obtained 48% recovery with PBAC agar. With 12 samples of frozen ground beef, recovery on violet red bile agar overlay was 29% of the recovery on plate count agar, whereas the corresponding value on PBAC agar was 45%. PBAC agar allowed the enumeration of 1.4 times as many gram-negative bacteria as violet red bile agar overlay. None of eight strains of gram-positive bacteria and none of eight strains of yeasts grew on PBAC agar. Of 158 colonies randomly selected from pour plates of eight fresh ground meat samples, 95% stained gram negative. In comparison, only 70% of 151 colonies selected from corresponding plate count agar plates were gram negative. The lack of background color, turbidity, and ease of use make PBAC agar easier to handle than other media used for gram-negative bacteria, such as violet red bile agar, violet red bile agar overlay, and crystal violet tetrazolium agar. In the preparation PBAC agar, all ingredients are autoclaved together except amphotericin B, which is filter sterilized and added before the plates are poured.     

Comparative polyacrylamide electrophoresis of periplasmic proteins released from gram-negative bacteria by polymyxin B

Summary Several cell-wall and membrane affecting agents were tested for causing release of periplasmic proteins of E. coli B as compared by gel electrophoresis. Osmotic shock and polymyxin-treatment yielded the best differentiated protein patterns. The periplasmic proteins derived from different E. coli strains and other gram-negative bacteria by polymyxin-treatment were compared. Whereas related strains showed similarities in the protein positions, unrelated gram-negative bacteria showed great differences of the protein bands. The polymyxin-induced liberation of periplasmic proteins was dependent upon the growth phase and growth media of the bacteria and was severely inhibited by 10^-2 M magnesium chloride.Abbreviations PX polymyxin B - CTABr cetyltrimethylammonium-bromide - tris trishydroxymethylaminomethane - EDTA ethylenediaminetetraacetate - LPS lipopolysaccharide     

Structural basis for the recognition of lysozyme by MliC, a periplasmic lysozyme inhibitor in Gram-negative bacteria

Lysozymes are an important component of the innate immune system of animals that hydrolyze peptidoglycan, the major bacterial cell wall constituent. Many bacteria have contrived various means of dealing with this bactericidal enzyme, one of which is to produce lysozyme inhibitors. Recently, a novel family of bacterial lysozyme inhibitors was identified in various Gram-negative bacteria, named MliC (membrane bound lysozyme inhibitor of C-type lysozyme). Here, we report the crystal structure of Pseudomonas aeruginosa MliC in complex with chicken egg white lysozyme. Combined with mutational study, the complex structure demonstrates that the invariant loop of MliC plays a crucial role in the inhibition of the lysozyme by its insertion to the active site cleft of the lysozyme, where the loop forms hydrogen and ionic bonds with the catalytic residues. Since MliC family members have been implicated as putative colonization or virulence factors, the structures and mechanism of action of MliC will be of relevance to the control of bacterial growth in animal hosts.     

Towards large scale methods for the selective release of periplasmic human cystatin C from E. coli

Summary Two promising methods, osmotic shock and guanidine treatment, for the selective release of human cystatin C from the periplasmic space of E. coli were developed at the laboratory scale. High release efficiencies were achievable by both approaches, however, the chemical membrane permeabilization proved to be much more selective. Both methods have excellent potential for scale up.     

Discriminative features of type I and type III secreted proteins from Gram-negative bacteria

The amino acid composition of sequences and structural attributes (α-helices, β-sheets) of C-and N-terminal fragments (50 amino acids) were compared to annotated (SWISS-PROT/ TrEMBL) type I (20 sequences) and type III (22 sequences) secreted proteins of Gram-negative bacteria. The discriminant analysis together with the stepwise forward and backward selection of variables revealed the frequencies of the residues Arg, Glu, Gly, Ile, Met, Pro, Ser, Tyr, Val as a set of strong (1-P < 0.001) predictor variables to discriminate between the sequences of type I and type III secreted proteins with a cross-validated accuracy of 98.6–100 %. The internal and external validity of discriminant analysis was confirmed by multiple (15 repeats) test-retest procedures using a randomly split original set of proteins; this validation method demonstrated an accuracy of 100 % for 191 non-selected (retest) sequences. The discriminant analysis was also applied using selected variables from the propensities for β-sheets and polarity of C-terminal fragments. This approach produced the next highest and comparable cross-validated classification accuracy for randomly selected and retest proteins (85.4–86.0 % and 82.4–84.5 %, respectively). The proposed sets of predictor variables could be used to assess the compatibility between secretion substrates and secretion pathways of Gram-negative bacteria by means of discriminant analysis.     

H-NS in Gram-negative bacteria: a family of multifaceted proteins

DNA-packaging and the control of gene expression constitute a major challenge for bacteria to survive and adapt to environmental changes. The use of multiple strategies to solve these problems could explain the presence of various nucleoid-associated proteins in bacteria. H-NS, one of these proteins, has been extensively studied in Escherichia coli, and a variety of phenotypes have been associated with a mutation in its structural gene. However, the role of H-NS in bacterial physiology and its mechanism of action are still a matter of debate. The expanding number of H-NS-related proteins identified in Gram-negative bacteria reveals interesting clues about their structure-function-evolution relationship.     

Structural analysis of lipopolysaccharides from Gram-negative bacteria

This review covers data on composition and structure of lipid A, core, and O-polysaccharide of the known lipopolysaccharides from Gram-negative bacteria. The relationship between the structure and biological activity of lipid A is discussed. The data on roles of core and O-polysaccharide in biological activities of lipopolysaccharides are presented. The structural homology of some oligosaccharide sequences of lipopolysaccharides to gangliosides of human cell membranes is considered.     

Novel mechanism of outer membrane targeting of proteins in Gram-negative bacteria

In Gram-negative bacteria, all the proteins destined for the outer membrane are synthesized with a signal sequence that is cleaved, either by the signal peptidase LepB for integral outer membrane proteins or by LspA for lipoproteins, when they cross the cytoplasmic membrane. The Dickeya dadantii protein PnlH does not possess a cleavable signal sequence but is anchored in the outer membrane by an N-terminal targeting signal. Addition of the 41 N-terminal amino acids of PnlH is sufficient for anchoring various hybrid proteins in the outer membrane. This targeting signal presents some of the characteristics of a Tat (twin arginine translocation) signal sequence but without an obvious cleavage site. We found that the Tat translocation pathway is required for the targeting process. This new mechanism of outer membrane protein targeting is probably widespread as PnlH was also addressed to the outer membrane when expressed in Escherichia coli . As PnlH was not detected as a substrate by Tat signal sequence prediction programmes, this would suggest that there may be many other unknown Tat-dependent outer membrane proteins.     

Gram-positive and Gram-negative bacteria synergize with oxidants to release CXCL8 from innate immune cells

We have recently demonstrated that oxidants can activate monocytes via an action on Toll-like receptor (TLR) 2; however, it is unclear what functional consequence this has on immune surveillance for Gram-negative and -positive bacteria. Gram-negative and -positive bacteria and their related pathogen-associated molecular patterns (PAMPs) are sensed by TLR4 and TLR2, respectively. In the current study, we used a human monocyte cell line to show that oxidants prime cells to subsequent challenge with Gram-negative or -positive bacteria as well as PAMPs specific for TLR4 (LPS), TLR2/1 (Pam(3)CSK4), TLR2/6 (FSL-1), Nod1 (FK565), and Nod2 (MDP Lys 18). Similarly, activation of TLR4 with LPS primed for subsequent activation of cells by agonists of the TLR2/6 or TLR2/1 complex. However, no synergy was noted when cells were costimulated with Pam(3)CSK4 and FSL-1. We then tested blood (and isolated monocytes) derived from healthy smokers, which is oxidant primed, making it more sensitive to bacterial or PAMP stimulation when compared with blood of nonsmokers. Thus an oxidant stimulation, possibly via an action on TLR2 or associated transduction pathways, provides a signal that initiates inflammatory responses and sensitizes cells to pathogenic insults.     

A potential role for periplasmic superoxide dismutase in blocking the penetration of external superoxide into the cytosol of Gram-negative bacteria

Superoxide is a key component of the antibacterial weaponry of phagocytes. Presumably, for this reason, strains of Salmonella typhimurium express a periplasmic superoxide dismutase (SOD) that is essential for full virulence. Because most anions cannot easily penetrate lipid membranes, it is thought that the phagosomal superoxide either damages an unknown target on the bacterial surface or reacts with nitric oxide to form peroxynitrite (HOONO), a toxic oxidant that can freely enter bacteria. However, in this study, we tested whether superoxide itself could penetrate membranes. Superoxide that was generated at high pH (>7.5) very slowly reduced cytochrome c that was encapsulated inside lipid vesicles. It did so much more quickly at lower pH (<7). Under the latter conditions, more superoxide was protonated and uncharged (HO2*), and the penetrance of superoxide was proportional to the concentration of this species. The permeability coefficient of HO2* was determined to be 9 x 10(-4) cm sec(-1), just slightly lower than that of water and far higher than the value of the anionic form (O2-, <10(-7) cm sec(-1). When Escherichia coli mutants that lack periplasmic SOD were exposed to super-oxide at pH 6.5, cytosolic fumarase B was damaged. Damage was minimal at higher pH or in strains that contained periplasmic SOD. Thus, in the acidic phagolysosome, superoxide may be able to penetrate and attack cytosolic targets of captive bacteria. This process may contribute to the potency of the oxidative burst. One role of periplasmic SOD may be to avert this damage. In contrast, periplasmic SOD was ineffective at lowering the extracellular super-oxide concentration and, therefore, may have little impact upon HOONO formation.     

The NodI and NodJ proteins from Rhizobium and Bradyrhizobium strains are similar to capsular polysaccharide secretion proteins from Gram-negative bacteria

The NodI and NodJ nodulation proteins have been described in different Rhizobium and Bradyrhizobium species. The NodIJ genes belong to the nod regulon. Other genes from this regulon are involved in the biosynthesis and modification of lipo-oligosaccharide molecule(s) which are morphogénic signals when acting on legume roots. It has been proposed that the NodI and NodJ proteins belong to a bacterial inner membrane transport system of small molecules. Nucleotide sequencing of MudII PR 13 insertions in the nodulation region of the symbiotic plasmid from a Rhizobium leguminosarum bv. phaseoli strain CE3 has revealed the presence of NodI and nodJ related sequences downstream of nodC. Computer nucleotide sequence analysis of the entire NodI and NodJ sequences from R. leguminosarum bv. viciae and Bradyrhizobium japonicum strains show that both proteins are similar to the KpsT and KpsM proteins from Escherichia coli Kl and K5 strains, to the BexB and BexA proteins from Haemophilis influenzae and to the CtrD and CtrC proteins from Neisseria meningitidis, respectively. Except for the NodI and NodJ proteins, all of them have been involved in the mechanism of secretion of polysaccharides in each of their harbouring species. On the basis of the similarity found, we propose that the NodI and the NodJ proteins could be involved in the export of a lipo-oligosaccharide.     

Bacterial degradation of glycol ethers

Assimilation of ethyleneglycol (EG) ethers by polyethyleneglycol-utilizing bacteria was examined. Ethyleneglycol ether-utilizing bacteria were also isolated from soil and activated sludge samples by enrichment-culture techniques. Three strains (4-5-3, EC 1-2-1 and MC 2-2-1) were selected and characterized as Pseudomonas sp. 4-5-3, Xanthobacter autotrophicus, and an unidentified gram-negative, non-spore-forming rod respectively. Their growth characteristics were examined: Pseudomonas sp. 4-5-3 assimilated EG (diethyleneglycol, DEG) monomethyl, monoethyl and monobutyl ethers, DEG, propanol and butanol. X. autotrophicus EC 1-2-1 grew well on EG monoethyl and monobutyl ethers, EG and primary alcohols (C₁-C₄), and slightly on EG monomethyl ether. The strain MC 2-2-1 grew on EG monomethyl ether, EG, primary alcohols (C₁-C₄), and 1,2-propyleneglycol (PG). The mixed culture of Pseudomonas sp. 4-5-3 and X. autotrophicus EC 1-2-1 showed better growth and improved degradation than respective single cultures towards EG monomethyl, monoethyl or monobutyl ethers. Intact cells of Pseudomonas sp. 4-5-3 degraded various kinds of monoalkyl ethers, which cannot be assimilated by the strain. Metabolic products were characterized from reaction supernatants of intact cells of Pseudomonas sp. 4-5-3 with EG or DEG monoethyl ethers: they were analyzed by thin-layer chromatography and GC-MS and found to be ethoxyacetic acid and ethoxyglycoxyacetic acid. Also, PG monoalkyl ethers (C₁-C₄), dipropyleneglycol monoethyl and monomethyl ethers and tripropyleneglycol monomethyl ether were assimilated by polypropyleneglycol-utilizing Corynebacterium sp. 7.     

Making a beta-barrel: assembly of outer membrane proteins in Gram-negative bacteria

The outer membrane (OM) of Gram-negative bacteria is an essential organelle that serves as a selective permeability barrier by keeping toxic compounds out of the cell while allowing vital nutrients in. How the OM and its constituent lipid and protein components are assembled remains an area of active research. In this review, we describe our current understanding of how outer membrane proteins (OMPs) are delivered to and then assembled in the OM of the model Gram-negative organism Escherichia coli.     

Amino-terminal amino acid sequences of electron transfer proteins from Gram-negative bacteria as indicators of their cellular localization: the sulfate-reducing bacteria

Abstract Data are presented that all known periplasmic redox proteins from the sulfate reducing bacteria included in the genus, Desulfovibrio have aminoterminal (N-terminal) amino-acid sequences commonly found in other Gram-negative bacteria and are indicative of recognition sites for signal peptides. In contrast, none of the cytoplasmic redox proteins exhibited these unique N-terminal amino-acid sequences. It is proposed that the N-terminal amino-acid residues of a given protein can be used as an indicator of its cellular localization within the bacterial cell.     

Simple, rapid, and quantitative release of periplasmic proteins by chloroform.

We introduce a method by which periplasmic proteins can be released rapidly, simply, and quantitatively by treating cells with chloroform. All the amino acid-binding proteins tested maintained their activity during chloroform treatment. This method makes practical the analysis of the periplasmic protein complement of a large number of strains.