Influence of oxygen availability on physiology, verocytotoxin expression and adherence of Escherichia coli O157

A strain of Escherichia coli serotype O157 was grown in steady state chemostat culture under aerobic, oxygen-limited and anaerobic conditions. The growth and metabolic efficiency of oxygen-limited and anaerobic cultures was impaired, with biomass yield and the molar growth yield for glucose, Yglucose, reduced markedly in comparison with aerobic cultures. Steady state cells were typically short rods 2-3 microns long, and were encapsulated by a layer of extracellular material. The majority of cells were non-flagellated and fimbriae were not observed. Chemostat-grown cells were significantly more adhesive for HEp-2 monolayers than cells grown in aerobic batch culture. Furthermore, oxygen-limited and anaerobic cultures were significantly more adhesive for Hep-2 cells when compared with cells grown in aerobic chemostat culture, possibly reflecting increased pathogenicity associated with the induction of novel adhesins. Type 1 pili were not responsible for increased adherence. Verocytotoxins, VT1 and VT2, were expressed constitutively and were not influenced by oxygen availability. This study demonstrates that E. coli O157 is a versatile micro-organism, which responds to environmental conditions likely to be encountered during infection by inducing a phenotype which is more adhesive for human epithelial cells.     

Engineering E. coli for improved microaerobic pDNA production

Escherichia coli strains W3110 and BL21 were engineered for the production of plasmid DNA (pDNA) under aerobic and transitions to microaerobic conditions. The gene coding for recombinase A (recA) was deleted in both strains. In addition, the Vitreoscilla hemoglobin (VHb) gene (vgb) was chromosomally inserted and constitutively expressed in each E. coli recA mutant and wild type. The recA inactivation increased the supercoiled pDNA fraction (SCF) in both strains, while VHb expression improved the pDNA production in W3110, but not in BL21. Therefore, a codon-optimized version of vgb was inserted in strain BL21recA⁻, which, together with W3110recA⁻vgb⁺, was tested in cultures with shifts from aerobic to oxygen-limited regimes. VHb expression lowered the accumulation of fermentative by-products in both strains. VHb-expressing cells displayed higher oxidative activity as indicated by the Redox Sensor Green fluorescence, which was more intense in BL21 than in W3110. Furthermore, VHb expression did not change pDNA production in W3110, but decreased it in BL21. These results are useful for understanding the physiological effects of VHb expression in two industrially relevant E. coli strains, and for the selection of a host for pDNA production.

     

代谢工程改造大肠杆菌合成D-1,2,4-丁三醇

【目的】D-1,2,4-丁三醇是一种四碳的多元醇,在军事和医药领域具有广泛的应用。为实现生物法一步转化生产D-1,2,4-丁三醇,对Escherichia coli W3100的木糖代谢途径进行改造。【方法】将来源于柄杆菌的D-木糖脱氢酶基因xylB和恶臭假单胞菌的苯甲酰甲酸脱羧酶基因mdlC克隆至E.coli W3100,得到重组菌E.coli(pEtac-mdlC-tac-xylB)。在此基础上对重组菌代谢木糖合成D-1,2,4-丁三醇的能力进行考察。【结果】在30°C下,以30 g/L D-木糖为底物,重组菌E.coli(pEtac-mdlC-tac-xylB)的D-1,2,4-丁三醇产量达到了0.9 g/L,摩尔转化率为4%。【结论】实现了D-1,2,4-丁三醇的一步法发酵生产,为国内开展相关研究奠定了坚实的基础。     

Characterization of a novel tetracycline resistance that functions only in aerobically grown Escherichia coli.

A tetracycline resistance (Tcr) gene that was found originally on two Bacteroides plasmids (pBF4 and pCP1) confers tetracycline resistance on Escherichia coli, but only when it is grown aerobically. Using maxicells, we have identified a 44-kilodalton protein which is encoded by the region that carries the Tcr gene and which may be the Tcr gene product. Localization experiments indicate that this 44-kilodalton protein is cytoplasmic. To determine whether the tetracycline resistance gene is expressed under anaerobic conditions, we have constructed a protein fusion between the Tcr gene and lacZ. In strains of E. coli carrying the fusion, beta-galactosidase activity was the same when the cells were grown under anaerobic conditions as when the cells were grown under aerobic conditions. This indicates that the tetracycline resistance gene product is made under anaerobic conditions but does not work. The failure of the Tcr protein to function under anaerobic conditions was not due to a requirement for function of the anaerobic electron transport system, because neither nitrate nor fumarate added to anaerobic media restored tetracycline resistance. Inhibition of the aerobic electron transport system with potassium cyanide did not prevent growth on tetracycline of cells containing the Tcr gene. A heme-deficient mutant, E. coli SHSP19, which carries the Tcr gene, was still resistant to tetracycline even when grown in heme-free medium. These results indicate that functioning of the Tcr gene product is not dependent on the aerobic electron transport system. Thus the requirement for aerobic conditions appears to reflect a requirement for oxygen. Spent medium from an E. coli strain carrying the Tcr gene, which was grown in medium containing tetracycline (50 micrograms/ml), did not inhibit growth of a tetracycline-susceptible strain of E. coli. Thus, the Tcr gene product may be detoxifying tetracycline.     

Significance of rpoS during maturation of Escherichia coli biofilms

Presence of starved, stationary phase-like zones in biofilms seems to be an important factor for biofilm formation. In this study, roles of rpoS gene in the formation of Escherichia coli biofilms were investigated. E. coli MG1655 wild type (WT) and rpoS mutant (DeltarpoS) strains were used to compare biofilm formation capacity and global gene expression. Even though the DeltarpoS strain could attach and form microcolonies on glass surfaces, it could not establish mature biofilms. DNA microarray analysis revealed that WT biofilms (WBF) showed similar pattern of gene expression with WT planktonic stationary phase, whereas DeltarpoS biofilms (MBF) showed similar pattern of gene expression with WT planktonic exponential phase. Genes involved in energy metabolism (atpIBEFHAG, atpC, cydAB) and flagella synthesis (flgB, flgC, flhD, fliA, fliC, fliY) showed increased expression in the MBF, but not in the WBF. Moreover, genes involved in stress responses (blc, cspG, dinD poxB, wcaF, wcaI, and yfcF) showed increased expression in the WBF compared to the MBF. These results suggested that the rpoS gene contributed in maturation of E. coli biofilms through regulation of global gene expression including energy metabolism, motility, and stress responses.     

Evaluation of Genetic Manipulation Strategies on d-Lactate Production by Escherichia coli

In order to rationally manipulate the cellular metabolism of Escherichia coli for D: -lactate production, single-gene and multiple-gene deletions with mutations in acetate kinase (ackA), phosphotransacetylase (pta), phosphoenolpyruvate synthase (pps), pyruvate formate lyase (pflB), FAD-binding D-lactate dehydrogenase (dld), pyruvate oxidase (poxB), alcohol dehydrogenase (adhE), and fumarate reductase (frdA) were tested for their effects in two-phase fermentations (aerobic growth and oxygen-limited production). Lactate yield and productivity could be improved by single-gene deletions of ackA, pta, pflB, dld, poxB, and frdA in the wild type E. coli strain but were unfavorably affected by deletions of pps and adhE. However, fermentation experiments with multiple-gene mutant strains showed that deletion of pps in addition to ackA-pta deletions had no effect on lactate production, whereas the additional deletion of adhE in E. coli B0013-050 (ackA-pta pps pflB dld poxB) increased lactate yield. Deletion of all eight genes in E. coli B0013 to produce B0013-070 (ackA-pta pps pflB dld poxB adhE frdA) increased lactate yield and productivity by twofold and reduced yields of acetate, succinate, formate, and ethanol by 95, 89, 100, and 93%, respectively. When tested in a bioreactor, E. coli B0013-070 produced 125 g/l D-lactate with an increased oxygen-limited lactate productivity of 0.61 g/g h (2.1-fold greater than E. coli B0013). These kinetic properties of D-lactate production are among the highest reported and the results have revealed which genetic manipulations improved D-lactate production by E. coli.     

Diversity of<Emphasis Type="Italic">fliC</Emphasis> gene in commensal<Emphasis Type="Italic">Escherichia coli</Emphasis> derived from various mammals

Relations between the diversity of the fliC gene conditioning flagellum protein in E. coli and the source of the strain origin are presented. The fliC genes have been identified and characterized in commensal E. coli derived from 10 healthy animal species living in Zoo Safari Park (Poland). The fliC gene was found in 150 strains by the PCR method. The amplifiedfliC products revealed single bands within the range 1.26-2.16 kbp. Forty restriction patterns (classed by restriction analysis with the use of RsaI (PCR-RFLP RsaI; R-types) were determined. The neighbor-joining method was employed to illustrate the distribution of the kinds of R-types. There are 3-8 various R-types of a diversified frequency of occurrence in strains. Application of PCR-RFLP RsaI permitted the identification of alleles of fliC genes characteristic for E. coli and the estimation of their diversity among the animal species. The transmission ways of E. coli fliC+ between organisms of different species were determined and confirmed the role of transmission and horizontal gene transfer in the generation of the allelic diversity of fliC gene in natural E. coli populations.     

Identification of Tn10 insertions in the rfaG, rfaP, and galU genes involved in lipopolysaccharide core biosynthesis that affect Escherichia coli adhesion

Escherichia coli was used as a model to study initial adhesion and early biofilm development to abiotic surface. Tn10 insertion mutants of Escherichia coli K-12 W3110 were selected for altered abilities to adhere to a polystyrene surface. Seven insertion mutants that showed a decrease in adhesion harbored insertions in genes involved in lipopolysaccharide (LPS) core biosynthesis. Two insertions were located in the rfaG gene, two in the rfaP gene, and three in the galU gene. These adhesion mutants were found to exhibit a deep-rough phenotype and to be reduced, at different levels, in type 1 fimbriae production and motility. The loss of adhesion exhibited by these mutants was associated with either the affected type 1 fimbriae production and/or the dysfunctional motility. Apart from the pleiotropic effect of the mutations affecting LPS on type 1 fimbriae and flagella biosynthesis, no evidence for an involvement of the LPS itself in adhesion to polystyrene surface could be observed. Received: 1 December 1998 / Accepted: 3 April 1999     

An oxygen dependent X-ray lesion in Escherichia coli strain B/r detected by penicillin.

Enhancement of lethal damage to E. coli B/r by penicillin was observed after X-irradiation under aerobic conditions but not after exposure to X-rays under anoxia or after U.V. (260 nm). No enhancement of damage occurred when incubation with penicillin was delayed for 2 hours after aerobic X-irradiation. This enhancing effect was only detected in this strain and not in the filamentous strain E. coli B. It is concluded that an X-ray induced lesion, sensitive to the presence of oxygen at the time of irradiation and probably located in the cell envelope, initiates filamentation in E. coli B/r, which results in lethal damage in this strain.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations.

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Comparison of growth, acetate production, and acetate inhibition of Escherichia coli strains in batch and fed-batch fermentations

The growth characteristics and acetate production of several Escherichia coli strains were compared by using shake flasks, batch fermentations, and glucose-feedback-controlled fed-batch fermentations to assess the potential of each strain to grow at high cell densities. Of the E. coli strains tested, including JM105, B, W3110, W3100, HB101, DH1, CSH50, MC1060, JRG1046, and JRG1061, strains JM105 and B were found to have the greatest relative biomass accumulation, strain MC1060 accumulated the highest concentrations of acetic acid, and strain B had the highest growth rates under the conditions tested. In glucose-feedback-controlled fed-batch fermentations, strains B and JM105 produced only 2 g of acetate.liter-1 while accumulating up to 30 g of biomass.liter-1. Under identical conditions, strains HB101 and MC1060 accumulated less than 10 g of biomass.liter-1 and strain MC1060 produced 8 g of acetate.liter-1. The addition of various concentrations of sodium acetate to the growth medium resulted in a logarithmic decrease, with respect to acetate concentration, in the growth rates of E. coli JM105, JM105(pOS4201), and JRG1061. These data indicated that the growth of the E. coli strains was likely to be inhibited by the acetate they produced when grown on media containing glucose. A model for the inhibition of growth of E. coli by acetate was derived from these experiments to explain the inhibition of acetate on E. coli strains at neutral pH.     

Aerobic sulfide production and cadmium precipitation by Escherichia coli expressing the Treponema denticola cysteine desulfhydrase gene

The cysteine desulfhydrase gene of Treponema denticola was over-expressed in Escherichia coli to produce sulfide under aerobic conditions and to precipitate metal sulfide complexes on the cell wall. When grown in a defined salts medium supplemented with cadmium and cysteine, E. coli producing cysteine desulfhydrase secreted sulfide and removed nearly all of the cadmium from solution after 48 h. A control strain produced significantly less sulfide and removed significantly less cadmium. Measurement of acid-labile sulfide and energy dispersive X-ray spectroscopy indicated that cadmium was precipitated as cadmium sulfide. Without supplemental cysteine, both the E. coli producing cysteine desulfhydrase and the control E. coli demonstrated minimal cadmium removal.     

Effect of glucose on the capacity of Escherichia coli to be infected by a virulent lamba bacteriophage

Howes, William V. (Massachusetts Institute of Technology, Cambridge). Effect of glucose on the capacity of Escherichia coli to be infected by a virulent lambda bacteriophage. J. Bacteriol. 90:1183-1193. 1965.-A substrate-dependent phenotypic resistance to phage lambda(vir) was observed among cells of Escherichia coli W3350 and C600 grown aerobically on glucose. Similar cells grown on glycerol were sensitive. When cells of W3350 grown on glycerol were transferred to glucose, the rate of appearance of the resistant fraction was proportional to the growth rate and became zero after 5.5 to 6.0 generations. Cells grown on glucose, upon transfer to glycerol, became sensitive within one generation. P(32) studies with W3350 indicated that the resistant cells did not adsorb phage. Furthermore, among the sensitive cells approximately the same number of particles adsorbed to each cell. Among five strains of E. coli K-12 investigated, three did not exhibit this aerobic phenotypic resistance. W3350 grown anaerobically on glucose was 100% resistant. Anaerobic growth had no further effect on C600. These inconsistent effects of anaerobiosis probably involved a mechanism different from that of aerobic growth.