Actinobacillus pleuropneumoniae hlyX gene homology with the fnr gene of Escherichia coli.

The hlyX gene from Actinobacillus pleuropneumoniae, which confers a hemolytic phenotype on Escherichia coli, was sequenced, and its role in regulation of gene expression was investigated. No similarity was found between the hlyX sequence and sequences of known hemolysin or cytotoxin genes. However, the hlyX sequence was very similar to that of the fnr gene of Escherichia coli which encodes the global regulatory protein, FNR. Comparison of the deduced amino acid sequence of the hlyX gene product (HlyX) with that of FNR revealed a high degree of well-aligned sequence correlation throughout the polypeptide chain. For example, 23 of 24 amino acids in the DNA-binding region of FNR are identical in the corresponding region of HlyX. Four cysteine residues in the amino-terminal region are also conserved. The promoter region of hlyX is very similar to that of fnr. It has a putative -10 sequence which closely resembles the E. coli -10 consensus sequence. This sequence is overlapped by a potential operator which is very similar to the FNR-binding-site consensus sequence. Functional homology between HlyX and FNR was also demonstrated. Plasmids carrying hlyX complemented the nutritional lesion of an fnr deletion strain of E. coli. These data suggest that HlyX may regulate, rather than mediate, hemolytic activity in E. coli, but the possibility that HlyX is both a regulator of gene expression and a hemolysin cannot be excluded.     

Deciphering global gene expression and regulation strategy in Escherichia coli during carbon limitation

Despite decades of studies meant to analyse the bacterial response to carbon limitation, we still miss a high-resolution overview of the situation. All gene expression changes observed in such conditions cannot solely be accounted for by the global regulator Crp either free or bound to its effector, cyclic AMP. Here, for the first time, we evaluated the response of both CDS (protein-coding sequence) and ncRNA (non-coding RNA) genes to carbon limitation, revealed cellular functions of differentially expressed genes systematically, quantified the contribution of Crp-cAMP and other factors to regulation and deciphered regulation strategies at a genomewide scale. Approximately one-third of the differentially expressed genes we identified responded to Crp-cAMP via its direct or indirect control, while the remaining genes were subject to growth rate-dependent control or were controlled by other regulators, especially RpoS. Importantly, gene regulation mechanisms can be established by expression pattern studies. Here, we propose a comprehensive picture of how cells respond to carbon scarcity. The global regulation strategies thus exposed illustrate that the response of cell to carbon scarcity is not limited to maintaining sufficient carbon metabolism via cAMP signalling while the main response is to adjust metabolism to cope with a slow growth rate.     

Metabolic limitation of DNA-polymerase I synthesis by Escherichia coli strain CM5199

The work was aimed at studying DNA polymerase I synthesis after induction of the vector prophage lambda polA (NM 964) in lysogenic Escherichia coli CM 5199 in the course of batch cultivation in different media and at various growth phases as well as upon "nutrient shifts" caused by adding organic compounds to the minimal medium. The enzyme activity was highest when the phage was induced at the exponential phase of growth and in media richer in their composition. The enzyme synthesis, the dynamics of protein and RNA content were studied after induction of the phage and enrichment of the medium; the studies have shown that synthesis of DNA polymerase I is influenced by limitation at two levels: (1) biosynthesis of amino acids and (2) biosynthesis of components of the protein-synthesizing apparatus. There is a direct correlation between DNA polymerase I biosynthesis under the control of vector DNA and the growth rate of the culture.     

Increased a-lytic protease production from recombinant Escherichia coli grown with oxygen limitation

Production of a mutant of the enzyme a-lytic protease from recombinant E. coli JM109 increased from 50 units/l to 750 units/l as the dissolved oxygen was decreased from above 30% saturation to near zero during batch growth on a complex medium. This represents a 60-fold increase in specific protease production (units/g cell dry wt.). Limiting the oxygen supply decreased the maximum growth rate from 0.55 h -1 to 0.066 h -1 which may in turn regulate the synthesis of recombinant protease in this system so that increased levels of product are obtained.     

Temperature-dependent regulation of the Escherichia coli lpxT gene

The Lipid A moiety of the lipopolysaccharide can be covalently modified during its transport to the outer membrane by different enzymes, among which the LpxT inner membrane protein. LpxT transfers a phosphate group from the undecaprenyl pyrophosphate to the Lipid A, a modification affecting the stability of the outer membrane and its recognition by the host immune system in Enterobacteria. We previously found that the expression of the Pseudomonas aeruginosa lpxT gene, encoding LpxT, is induced in response to a temperature upshift and we proposed that an RNA thermometer was responsible for such regulation. Here we show that the Escherichia coli lpxT orthologous gene is down-regulated upon a temperature upshift and investigated the mechanism of this regulation. We found that the LpxT protein stability is not affected by the temperature change. Conversely, the lpxT mRNA levels strongly decrease upon a shift from 28 to 42 °C. The lack of MicA sRNA, which was previously implicated in lpxT regulation, does not affect lpxT thermal regulation. We identified the lpxTp promoter and demonstrated that lpxTp has temperature-sensitive activity depending on its peculiar −10 region. Moreover, we found that RNase E-dependent degradation of the lpxT mRNA is also modulated by temperature causing a strong destabilization of the lpxT mRNA at 42 °C. In vitro data argue against the involvement of factors differentially expressed at 28 and 42 °C in the temperature–dependent modulation of lpxT mRNA stability.     

Growth-phase regulation of the Escherichia coli thioredoxin gene

The two promoters of Escherichia coli trxA gene were separately cloned into pKO100 as well as pJEL170. Galactokinase expression in cells containing the pKO100 derivatives was found to be negatively correlated with growth rate and was 6- to 20-fold higher in stationary cultures than in exponential cultures. The expression of trxA-galK was induced by amino acid starvation in a RelA(+) strain but not in an isogenic Rel(-) strain indicating that the control involves guanosine 3',5'-bispyrophosphate (ppGpp). RpoS, which appears to be essential for expression of most stationary phase expressed genes, is not required for trxA expression. Increased expression of relA, which increases ppGpp concentration, increases trxA expression.     

Effect of a single-gene knockout on the metabolic regulation in Escherichia coli for D-lactate production under microaerobic condition

The effects of several single-gene knockout mutants (pykF, ppc, pflA, pta, and adhE mutants) on the metabolic flux distribution in Escherichia coli were investigated under microaerobic condition. The intracellular metabolite concentrations and enzyme activities were measured, and the metabolic flux distribution was computed to study the metabolic regulation in the cell. The pflA, pta and ppc mutants produced large amount of lactate when using glucose as a carbon source under microaerobic condition. Comparing the flux distribution and the enzyme activities in the mutants, it was shown that the lactate production was promoted by the inactivation of pyruvate formate lyase and the resulting overexpression of lactate dehydrogenase. The flux through Pta-Ack pathways and the ethanol production were limited by the available acetyl coenzyme A. It was shown that the glycolysis was activated in pykF mutant in microaerobic culture. The glycolytic flux was related with Pyk activity except for pykF mutant. The cell growth rate was shown to be affected by the flux through phosphoenolpyruvate carboxylase. The quantitative regulation analysis was made based on the deviation indexes.     

Effect of oxygen limitation and starvation on the benzalkonium chloride susceptibility of Escherichia coli

Aims: To investigate the effect of oxygen limitation, glucose-starvation and temperature on the susceptibility of Escherichia coli towards the quaternary ammonium biocide benzalkonium chloride (BAC). Methods and Results: The effect of BAC on planktonic and sessile cells were investigated using the gfp-tagged E. coli K-12 strain MG1655[pOX38Km]. Increasing temperature from 10°C to 30°C increased the bactericidal effect of BAC for both starved and nonstarved E. coli under aerobic and anaerobic conditions. The lowest minimum bactericidal concentration was observed for cells in anaerobic media at 30°C (30 mg l⁻¹ BAC). Decreasing cell densities increased the decay rate for BAC-exposed cells for both starved and nonstarved E. coli. Biofilms of E. coli exposed to BAC in anaerobic medium showed a greater percentage of membrane-compromised cells than biofilms grown in aerobic medium. Image analyses of BAC-exposed biofilms showed that membrane-compromised cells were occasionally located in the interior structure of the biofilm microcolonies. Conclusions: Increasing temperatures and the absence of oxygen, and energy substrates increased the antimicrobial effect of BAC towards E. coli. Significance and Impact of the Study: The results are relevant for understanding the disinfection efficacy of quaternary ammonium compounds towards planktonic and sessile bacteria.     

Effect of oxygen limitation and medium composition on Escherichia coli fermentation in shake-flask cultures

Shake-flask cultures are widely used for screening of high producing strains. To select suitable strains for production scale, cultivation parameters should be applied that provide optimal growth conditions. A novel method of measuring respiratory activity in shake-flask cultures was employed to analyze Escherichia coli fermentation under laboratory conditions. Our results suggest that the length of fermentation, choice of medium, and aeration do not normally satisfy the requirements for unlimited growth in shake flasks. Using glycerol rather than glucose as a carbon source greatly reduced the accumulation of overflow and fermentative metabolites when oxygen supply was unlimited. A rich buffered medium, Terrific Broth (TB), yielded 5 times more biomass compared to LB medium but also caused oxygen limitation in standard shake-flask cultures at shaking frequencies below 400 rpm. These results were used to optimize the production of benzoylformate decarboxylase from Pseudomonas putida in E. coli SG13009, resulting in a 10-fold increase in volumetric enzyme production. This example demonstrates how variation of medium composition and oxygen supply can be evaluated by the measurement of the respiratory activity. This can help to efficiently optimize screening conditions for E. coli.     

Metabolic regulation of the histidine operon in Escherichia coli and Salmonella typhimurium

Expression of the histidine operon in Escherichia coli cells in contrast to the one in Salmonella typhimurium is changed proportionally to cells growth rate on the different carbon sources. The specific activity of histidinol-dehydrogenase is repressed by addition of 19 amino acids both in Escherichia coli and Salmonella typhimurium independent of the growth medium used. Using of Escherichia coli and Salmonella typhimurium strains containing the heterologous histidine operons made possible to demonstrate the dependence of the histidine operon metabolic regulation to be determined by the operon itself but not by the specificity of the recipient cells. ppGpp was shown to be a positive regulator of the histidine operon expression in Escherichia coli.     

Use of gene fusions to study the expression of fnr, the regulatory gene of anaerobic electron transfer in Escherichia coli

Abstract Fusions between fnr , the regulatory gene for anaerobic electron transfer, and lacZ were obtained by insertion of Mu d II1734 bacteriophage in the fnr gene cloned in a plasmid. After transfer onto the chromosome, study of the fusion showed that the expression of fnr is independent of anaerobiosis, negatively regulated by its own product and partly positively controlled by cyclic AMP.