Capacity of the bovine intestinal mucus and its components to support growth of Escherichia coli O157:H7

Colonization of the gastrointestinal tract of cattle by Shiga toxin-producing Escherichia coli increases the risk of contamination of food products at slaughter. Our study aimed to shed more light on the mechanisms used by E. coli O157:H7 to thrive and compete with other bacteria in the gastrointestinal tract of cattle. We evaluated, in vitro, bovine intestinal mucus and its constituents in terms of their capacity to support growth of E. coli O157:H7 in presence or absence of fecal inoculum, with and without various enzymes. Growth of E. coli O157:H7 and total anaerobic bacteria were proportionate to the amount of mucus added as substrate. Growth of E. coli O157:H7 was similar for small and large intestinal mucus as substrate, and was partially inhibited with addition of fecal inoculum to cultures, presumably due to competition from other organisms. Whole mucus stimulated growth to the greatest degree compared with other compounds evaluated, but the pathogen was capable of utilizing all substrates to some extent. Addition of enzymes to cultures failed to impact growth of E. coli O157:H7 except for neuraminidase, which resulted in greater growth of E. coli O157 when combined with sialic acid as substrate. In conclusion, E. coli O157 has capacity to utilize small or large intestinal mucus, and growth is greatest with whole mucus compared with individual mucus components. There are two possible explanations for these findings (i) multiple substrates are needed to optimize growth, or alternatively, (ii) a component of mucus not evaluated in this experiment is a key ingredient for optimal growth of E. coli O157:H7.     

The control of sulphate reduction in bacteria

1. An enzyme from Escherichia coli 9723 that reduces adenosine 3′-phosphate 5′-sulphatophosphate to inorganic sulphite is described. Extracts of E. coli K₁₂ and Bacillus subtilis 1379 contain a similar enzyme. 2. This reductase and sulphite reductase (EC 1.8.1.2) of E. coli 9723, E. coli K₁₂ and of B. subtilis are repressed by growth in the presence of l-cystine. Cysteine synthase (EC 4.2.1.22) is unaffected. 3. Growth of E. coli 9723 on inorganic sulphite represses the sulphate-activating enzymes (EC 2.7.7.4 and 2.7.1.25) almost completely but has little effect on sulphite reductase. Growth on 0·042–0·056mm-l-cystine gives a similar result. 4. Such differential repression by cyst(e)ine prevents E. coli, when growing on sulphite, from synthesizing unnecessary enzymes.     

Expression of a thermostable cellulase gene from a thermophilic anaerobe in Saccharomyces cerevisiae

A cellulase gene from a thermophilic anaerobe was recloned in the yeast Saccharomyces cerevisiae. The maximum level of the gene expression in the recombinant yeast was 4.4 times higher than that in the Escherichia coli transformant harboring the same plasmid. Cellulase activity was observed only within the yeast cells. To compare the enzymatic properties of cellulase produced by the yeast and E. coli transformants, cellulases were purified to homogeneous state by only three purification steps of heat treatment, and cellulose affinity and ion exchange chromatographies. The molecular weights of the enzymes produced by the yeast and E. coli were 3.8 × 10⁴ and 4.0 × 10⁴, respectively by SDS-polyacrylamide gel electrophoresis. Neither of the enzymes was glycosylated. Although the molecular weights were slightly different, enzymatic properties and thermostability were almost indistinguishable between the enzymes produced by the yeast and E. coli transformants.     

The Escherichia coli adherence factor plasmid of enteropathogenic Escherichia coli causes a global decrease in ubiquitylated host cell proteins by decreasing ubiquitin E1 enzyme expression through host aspartyl proteases

Ubiquitylation is a widespread post-translational global regulatory system that is essential for the proper functioning of various cellular events. Recent studies have shown that certain types of Escherichia coli can exploit specific aspects of the ubiquitylation system to influence downstream targets. Despite these findings, examination of the effects pathogenic E. coli have on the overall host ubiquitylation system remain unexplored. To study the impact that pathogenic E. coli have on the ubiquitylation levels of host proteins during infections, we analyzed the entire ubiquitylation system during enteropathogenic E. coli infections of cultured cells. We found that these microbes caused a dramatic decrease in ubiquitylated host proteins during these infections. This occurred with a concomitant reduction in the expression of essential E1 activating enzymes in the host, which are integral for the initiation of the ubiquitylation cascade. Control of host E1 enzyme levels was dependent on the E. coli adherence factor plasmid which acted on host aspartyl proteases within enteropathogenic E. coli. Hijacking of the ubiquitylation system did not require the plasmid-encoded regulator or bundle forming pilus expression, as enteropathogenic E. coli mutated in those factors did not revert the ubiquitylation of host proteins or the abundance of E1 enzyme proteins to uninfected levels. Our work shows that E. coli have developed strategies to usurp post-translational systems by targeting crucial enzymes. The ability of enteropathogenic E. coli to inactivate host protein ubiquitylation could enable more efficient effector protein functionality, providing increased bacterial control of host cells during enteropathogenic E. coli pathogenesis.     

Assimilation of E. coli cells by Daphnia magna on the whole organism level

Consumption of E. coli cells by Daphnia magna was studied. It was found that this organism not only ingested E. coli cells but digested them as demonstrated by the release of ¹⁴CO₂ originating from E. coli grown on ¹⁴C-glucose, and by the transfer of the radioactive label from parental Daphnia to their progenies. In addition the effect of antibiotics on the consumption of E. coli cells by Daphnia magna was studied. In long incubation times, antibiotics inhibited bacterial uptake by Daphnia. The microflora isolated from Daphnia was found to be capable of causing leakage of enzymes out of E. coli cells thus playing at least a partial role in the digestion of E. coli cells by Daphnia.     

tRNA methylases from HeLa cells: purification and properties of an adenine-1-methylase and a guanine-N2-methylase

Two enzymes (methylases) that catalyze the transfer of methyl groups from S-adenosyl-l-methionine to tRNA (prepared from Escherichia coli) have been partially purified from extracts of HeLa cells. One catalyzes the methylation of adenine residues of the tRNA to give 1-methyladenine units and the other is responsible for the conversion of guanine residues to N²-methylguanine and N²,N²-dimethylguanine (and may be a mixture of two enzymes). Activities of these relatively unstable enzymes could be maintained by storage at ?20 °C in the presence of 50% glycerol. Substrate specificity studies have revealed that bacterial tRNA (E. coli, Bacillus subtilis) can be used as substrate, whereas tRNA of animal origin (HeLa cells, rat liver) cannot be used. Of the specific tRNA's tested, E. coli tRNA^fMet was used as substrate by both enzymes. E. coli tRNA^Tyr was used by the adenine-1-methylase but not by the guanine-N²-methylase. The adenine-1-methylase catalyzed the transfer of approximately one methyl group per mole of either tRNA^fMet or tRNA^Tyr offered as substrate; in the presence of the guanine-N²-methylase 1 mole of E. coli tRNA^fMet accepted 1 mole of methyl. Studies with the use of both enzymes established that enzymic methylation of the guanine site of E. coli tRNA^fMet did not interfere with subsequent methylation of an adenine residue and neither did prior methylation of adenine interfere with the subsequent methylation of a guanine residue. In the presence of both enzymes, approximately 2 moles of methyl groups were accepted by 1 mole of the E. coli tRNA^fMet.     

Temperate Bacteriophage Which Causes the Production of a New Major Outer Membrane Protein by Escherichia coli

Under most conditions of growth, the most abundant protein in the outer membrane of most strains of Escherichia coli is a protein designated as “protein 1” or “matrix protein”. In E. coli B, this protein has been shown to be a single polypeptide with a molecular mass of 36,500 and it may account for more than 50% of the total outer membrane protein. E. coli K-12 contains a very similar, although probably not identical, species of protein 1. Some pathogenic E. coli strains contain very little protein 1 and, in its place, make a protein designated as protein 2 which migrates faster on alkaline polyacrylamide gels containing sodium dodecyl sulfate and which gives a different spectrum of CNBr peptides. An E. coli K-12 strain which had been mated with a pathogenic strain was found to produce protein 2, and a temperate bacteriophage was isolated from this K-12 strain after induction with UV light. This phage, designated as PA-2, is similar in morphology and several other properties to phage lambda. When strains of E. coli K-12 are lysogenized by phage PA-2, they produce protein 2 and very little protein 1. Adsorption to lysogenic strains grown under conditions where they produce little protein 1 and primarily protein 2 is greatly reduced as compared to non-lysogenic strains which produce only protein 1. However, when cultures are grown under conditions of catabolite repression, protein 2 is reduced and protein 1 is increased, and lysogenic and non-lysogenic cultures grown under these conditions exhibit the same rate of adsorption. Phage PA-2 does not adsorb to E. coli B, which appears to have a slightly different protein 1 from K-12. These results suggest that protein 1 is the receptor for PA-2, and that protein 2 is made to reduce the superinfection of lysogens.     

Microbial production of N-acetylneuraminic acid by genetically engineered Escherichia coli

Previously, we described the production of N-acetylneuraminic acid (NeuAc) from N-acetylglucosamine (GlcNAc) in a system combining recombinant Escherichia coli expressing GlcNAc 2-epimerase (slr1975), E. coli expressing NeuAc synthetase (neuB), and Corynebacterium ammoniagenes. However, this system was unsuitable for large-scale production because of its complexity and low productivity. To overcome these problems, we constructed a recombinant E. coli simultaneously overexpressing slr1975 and neuB. This recombinant E. coli produced 81 mM (25 g/L) NeuAc in 22 h without the addition of C. ammoniagenes cells. For manufacturing on an industrial scale, it is preferable to use unconcentrated culture broth as the source of enzymes, and therefore, a high-density cell culture is required. An acetate-resistant mutant strain of E. coli (HN0074) was selected as the host strain because of its ability to grow to a high cell density. The NeuAc aldolase gene of E. coli HN0074 was disrupted by homologous recombination yielding E. coli N18-14, which cannot degrade NeuAc. After a 22 h reaction with 540 mM (120 g/L) GlcNAc in a 5 L jar fermenter, the culture broth of E. coli N18-14 overexpressing slr1975 and neuB contained 172 mM (53 g/L) NeuAc.     

Production by Clostridium acetobutylicum ATCC 824 of CelG, a Cellulosomal Glycoside Hydrolase Belonging to Family 9

The genome sequence of Clostridium acetobutylicum ATCC 824, a noncellulolytic solvent-producing strain, predicts the production of various proteins with domains typical for cellulosomal subunits. Most of the genes coding for these proteins are grouped in a cluster similar to that found in cellulolytic clostridial species, such as Clostridium cellulovorans. CAC0916, one of the open reading frames present in the putative cellulosome gene cluster, codes for CelG, a putative endoglucanase belonging to family 9, and it was cloned and overexpressed in Escherichia coli. The overproduced CelG protein was purified by making use of its high affinity for cellulose and was characterized. The biochemical properties of the purified CelG were comparable to those of other known enzymes belonging to the same family. Expression of CelG by C. acetobutylicum grown on different substrates was studied by Western blotting by using antibodies raised against the purified E. coli-produced protein. Whereas the antibodies cross-reacted with CelG-like proteins secreted by cellobiose- or cellulose-grown C. cellulovorans cultures, CelG was not detectable in extracellular medium from C. acetobutylicum grown on cellobiose or glucose. However, notably, when lichenan-grown cultures were used, several bands corresponding to CelG or CelG-like proteins were present, and there was significantly increased extracellular endoglucanase activity.     

Osmoprotection of Escherichia coli by Peptone Is Mediated by the Uptake and Accumulation of Free Proline but Not of Proline-Containing Peptides

The effect of meat peptone type I (Sigma) on the growth of Escherichia coli cells under hyperosmotic stress has been investigated. Peptone is a complex mixture of peptides with a small content of free amino acids, which resembles nutrients found in natural environments. Our data showed that peptone enhances the growth of E. coli cells in high-osmolarity medium to levels higher than those achieved with the main compatible solute in bacteria, glycine betaine. The mechanism of osmoprotection by peptone comprises the uptake and accumulation of the compatible solute, proline. The main role of the peptides contained in peptone is the provision of nutrients rather than the intracellular accumulation of osmolytes. In contrast to Listeria monocytogenes (M. R. Amezaga, I. Davidson, D. McLaggan, A. Verheul, T. Abee, and I. R. Booth, Microbiology 141:41–49, 1995), E. coli does not accumulate exogenous peptides for osmoprotection and peptides containing proline do not lead to the accumulation of proline as a compatible solute. In late-logarithmic-phase cultures of E. coli growing at high osmolarity plus peptone, proline becomes the limiting factor for growth, and the intracellular pools of proline are not maintained. This is a consequence of the low concentration of free proline in peptone, the catabolism of proline by E. coli, and the inability of E. coli to utilize proline-containing peptides as a source of compatible solutes. Our data highlight the role that natural components in food such as peptides play in undermining food preservation regimes, such as high osmolarity, and also that the specific mechanisms of osmoprotection by these compounds differ according to the organism.     

Hydrogenase-3 Contributes to Anaerobic Acid Resistance of Escherichia coli

Background

Hydrogen production by fermenting bacteria such as Escherichia coli offers a potential source of hydrogen biofuel. Because H₂ production involves consumption of 2H⁺, hydrogenase expression is likely to involve pH response and regulation. Hydrogenase consumption of protons in E. coli has been implicated in acid resistance, the ability to survive exposure to acid levels (pH 2–2.5) that are three pH units lower than the pH limit of growth (pH 5–6). Enhanced survival in acid enables a larger infective inoculum to pass through the stomach and colonize the intestine. Most acid resistance mechanisms have been defined using aerobic cultures, but the use of anaerobic cultures will reveal novel acid resistance mechanisms.

Methods and Principal Findings

We analyzed the pH regulation of bacterial hydrogenases in live cultures of E. coli K-12 W3110. During anaerobic growth in the range of pH 5 to 6.5, E. coli expresses three hydrogenase isoenzymes that reversibly oxidize H₂ to 2H⁺. Anoxic conditions were used to determine which of the hydrogenase complexes contribute to acid resistance, measured as the survival of cultures grown at pH 5.5 without aeration and exposed for 2 hours at pH 2 or at pH 2.5. Survival of all strains in extreme acid was significantly lower in low oxygen than for aerated cultures. Deletion of hyc (Hyd-3) decreased anoxic acid survival 3-fold at pH 2.5, and 20-fold at pH 2, but had no effect on acid survival with aeration. Deletion of hyb (Hyd-2) did not significantly affect acid survival. The pH-dependence of H₂ production and consumption was tested using a H₂-specific Clark-type electrode. Hyd-3-dependent H₂ production was increased 70-fold from pH 6.5 to 5.5, whereas Hyd-2-dependent H₂ consumption was maximal at alkaline pH. H₂ production, was unaffected by a shift in external or internal pH. H₂ production was associated with hycE expression levels as a function of external pH.

Conclusions

Anaerobic growing cultures of E. coli generate H₂ via Hyd-3 at low external pH, and consume H₂ via Hyd-2 at high external pH. Hyd-3 proton conversion to H₂ is required for acid resistance in anaerobic cultures of E. coli.     

A Moonlighting Enzyme Links Escherichia coli Cell Size with Central Metabolism

Growth rate and nutrient availability are the primary determinants of size in single-celled organisms: rapidly growing Escherichia coli cells are more than twice as large as their slow growing counterparts. Here we report the identification of the glucosyltransferase OpgH as a nutrient-dependent regulator of E. coli cell size. During growth under nutrient-rich conditions, OpgH localizes to the nascent septal site, where it antagonizes assembly of the tubulin-like cell division protein FtsZ, delaying division and increasing cell size. Biochemical analysis is consistent with OpgH sequestering FtsZ from growing polymers. OpgH is functionally analogous to UgtP, a Bacillus subtilis glucosyltransferase that inhibits cell division in a growth rate-dependent fashion. In a striking example of convergent evolution, OpgH and UgtP share no homology, have distinct enzymatic activities, and appear to inhibit FtsZ assembly through different mechanisms. Comparative analysis of E. coli and B. subtilis reveals conserved aspects of growth rate regulation and cell size control that are likely to be broadly applicable. These include the conservation of uridine diphosphate glucose as a proxy for nutrient status and the use of moonlighting enzymes to couple growth rate-dependent phenomena to central metabolism.     

Development of a Continuous Bioconversion System Using a Thermophilic Whole-Cell Biocatalyst

The heat treatment of recombinant mesophilic cells having heterologous thermophilic enzymes results in the denaturation of indigenous mesophilic enzymes and the elimination of undesired side reactions; therefore, highly selective whole-cell catalysts comparable to purified enzymes can be readily prepared. However, the thermolysis of host cells leads to the heat-induced leakage of thermophilic enzymes, which are produced as soluble proteins, limiting the exploitation of their excellent stability in repeated and continuous reactions. In this study, Escherichia coli cells having the thermophilic fumarase from Thermus thermophilus (TtFTA) were treated with glutaraldehyde to prevent the heat-induced leakage of the enzyme, and the resulting cells were used as a whole-cell catalyst in repeated and continuous reactions. Interestingly, although electron microscopic observations revealed that the cellular structure of glutaraldehyde-treated E. coli was not apparently changed by the heat treatment, the membrane permeability of the heated cells to relatively small molecules (up to at least 3 kDa) was significantly improved. By applying the glutaraldehyde-treated E. coli having TtFTA to a continuous reactor equipped with a cell-separation membrane filter, the enzymatic hydration of fumarate to malate could be operated for more than 600 min with a molar conversion yield of 60% or higher.     

Characterization of Unexpected Growth of Escherichia coli O157:H7 by Modeling

Modeling of batch kinetics in minimal synthetic medium was used to characterize Escherichia coli O157:H7 growth, which appeared to be different from the exponential growth expected in minimal synthetic medium and observed for E. coli K-12. The turbidimetric kinetics of 14 of the 15 O157:H7 strains tested (93%) were nonexponential, whereas 25 of the 36 other E. coli strains tested (70%) exhibited exponential kinetics. Moreover, the anomaly was almost corrected when the minimal medium was supplemented with methionine. These observations were confirmed with two reference strains by using plate count monitoring. In mixed cultures, E. coli K-12 had a positive effect on E. coli O157:H7 and corrected its growth anomaly. This demonstrated that commensalism occurred, as the growth curve for E. coli K-12 was not affected. The interaction could be explained by an exchange of methionine, as the effect of E. coli K-12 on E. coli O157:H7 appeared to be similar to the effect of methionine.     

Biocontrol of Escherichia coli O157 with O157-Specific Bacteriophages

Escherichia coli O157 antigen-specific bacteriophages were isolated and tested to determine their ability to lyse laboratory cultures of Escherichia coli O157:H7. A total of 53 bovine or ovine fecal samples were enriched for phage, and 5 of these samples were found to contain lytic phages that grow on E. coli O157:H7. Three bacteriophages, designated KH1, KH4, and KH5, were evaluated. At 37 or 4°C, a mixture of these three O157-specific phages lysed all of the E. coli O157 cultures tested and none of the non-O157 E. coli or non-E. coli cultures tested. These results required culture aeration and a high multiplicity of infection. Without aeration, complete lysis of the bacterial cells occurred only after 5 days of incubation and only at 4°C. Phage infection and plaque formation were influenced by the nature of the host cell O157 lipopolysaccharide (LPS). Strains that did not express the O157 antigen or expressed a truncated LPS were not susceptible to plaque formation or lysis by phage. In addition, strains that expressed abundant mid-range-molecular-weight LPS did not support plaque formation but were lysed in liquid culture. Virulent O157 antigen-specific phages could play a role in biocontrol of E. coli O157:H7 in animals and fresh foods without compromising the viability of other normal flora or food quality.     

The UDP-glucose Dehydrogenase of Escherichia coli K-12 Displays Substrate Inhibition by NAD That Is Relieved by Nucleotide Triphosphates

UDP-glucose dehydrogenase (Ugd) generates UDP-glucuronic acid, an important precursor for the production of many hexuronic acid-containing bacterial surface glycostructures. In Escherichia coli K-12, Ugd is important for biosynthesis of the environmentally regulated exopolysaccharide known as colanic acid, whereas in other E. coli isolates, the same enzyme is required for production of the constitutive group 1 capsular polysaccharides, which act as virulence determinants. Recent studies have implicated tyrosine phosphorylation in the activation of Ugd from E. coli K-12, although it is not known if this is a feature shared by bacterial Ugd proteins. The activities of Ugd from E. coli K-12 and from the group 1 capsule prototype (serotype K30) were compared. Surprisingly, for both enzymes, site-directed Tyr → Phe mutants affecting the previously proposed phosphorylation site retained similar kinetic properties to the wild-type protein. Purified Ugd from E. coli K-12 had significant levels of NAD substrate inhibition, which could be alleviated by the addition of ATP and several other nucleotide triphosphates. Mutations in a previously identified UDP-glucuronic acid allosteric binding site decreased the binding affinity of the nucleotide triphosphate. Ugd from E. coli serotype K30 was not inhibited by NAD, but its activity still increased in the presence of ATP.     

Specific Secretion of Active Single-Chain Fv Antibodies into the Supernatants of Escherichia coli Cultures by Use of the Hemolysin System

A simple method for the nontoxic, specific, and efficient secretion of active single-chain Fv antibodies (scFvs) into the supernatants of Escherichia coli cultures is reported. The method is based on the well-characterized hemolysin transport system (Hly) of E. coli that specifically secretes the target protein from the bacterial cytoplasm into the extracellular medium without a periplasmic intermediate. The culture media that accumulate these Hly-secreted scFv's can be used in a variety of immunoassays without purification. In addition, these culture supernatants are stable over long periods of time and can be handled basically as immune sera.     

X-Ray and Ultraviolet Sensitivity of Synchronously Dividing Escherichia coli

A paper pile filtration technique was used to obtain synchronously dividing populations of E. coli strains B and B/r from cultures in the exponential growth phase. Three generations of highly phased cell division were obtained by rapid pressure filtration which selected approximately 1 per cent of the exponentially growing culture. The sensitivity of E. coli strain B to x-ray and UV inactivation as a function of the cell division cycle was determined on synchronous populations. E. coli strain B showed a sharp decrease in sensitivity to inactivation by both radiations in the middle of the division cycle, and a further decrease near the end of the cycle. The sensitivity of E. coli strain B/r to x-irradiation was also investigated. Only the mid-cycle decrease in sensitivity was found during the division cycle of this strain. It was concluded that the repetition of the observed sensitivity patterns in both strains through the first three cycles after synchronization indicates that the same basic sensitivity patterns are probably also present in the individual cells of an exponential phase culture.     

Polysaccharide hydrolysis with engineered Escherichia coli for the production of biocommodities

Escherichia coli can ferment a broad range of sugars, including pentoses, hexoses, uronic acids, and polyols. These features make E. coli a suitable microorganism for the development of biocatalysts to be used in the production of biocommodities and biofuels by metabolic engineering. E. coli cannot directly ferment polysaccharides because it does not produce and secrete the necessary saccharolytic enzymes; however, there are many genetic tools that can be used to confer this ability on this prokaryote. The construction of saccharolytic E. coli strains will reduce costs and simplify the production process because the saccharification and fermentation can be conducted in a single reactor with a reduced concentration or absence of additional external saccharolytic enzymes. Recent advances in metabolic engineering, surface display, and excretion of hydrolytic enzymes provide a framework for developing E. coli strains for the so-called consolidated bioprocessing. This review presents the different strategies toward the development of E. coli strains that have the ability to display and secrete saccharolytic enzymes to hydrolyze different sugar-polymeric substrates and reduce the loading of saccharolytic enzymes.     

Proteomic analysis of a multi-resistant clinical Escherichia coli isolate of unknown genomic background

Horizontal transfer of gene clusters occurs in Escherichia coli (E. coli), which could lead to evolution of new pathovars and improve survival fitness. However, this genetic event results in genomic plasticity which is a hindrance for proteomic characterization of strains with unknown genetic backgrounds. To characterize such isolate with many specific genetic variations we used the recently in-house designed MSMSpdbb software which merges protein databases from several sources of E. coli including type strains and other commensal and pathogenic isolates. We selected a multidrug resistant clinical isolate in order to check the capacity of our approach to identify selected protein markers. From the 1596 identified proteins, we found important virulence factors such as IutA, OmpA, TraT and selected enzymes conferring antibiotic resistance, such as CTX-M-15 (Extended-Spectrum Beta Lactamase - ESBL) and AAC(6′)-Ib-cr (to aminoglycoside + fluoroquinolone). In addition, we compared the protein identifications with E. coli gene annotation and found that 27% of the proteins identified in the present study corresponded to the pan-genome of E. coli species and are only present in a subset of strains. This demonstrates the ability of our approach to characterize the proteome of bacterial strains with complex genomic plasticity even without its genomic information.