Control of CydB and GltA1 expression by the SenX3 RegX3 two component regulatory system of Mycobacterium tuberculosis

Two component regulatory systems are used widely by bacteria to coordinate changes in global gene expression profiles in response to environmental signals. The SenX3-RegX3 two component system of Mycobacterium tuberculosis has previously been shown to play a role in virulence and phosphate-responsive control of gene expression. We demonstrate that expression of SenX3-RegX3 is controlled in response to growth conditions, although the absolute changes are small. Global gene expression profiling of a RegX3 deletion strain and wild-type strain in different culture conditions (static, microaerobic, anaerobic), as well as in an over-expressing strain identified a number of genes with changed expression patterns. Among those were genes previously identified as differentially regulated in aerobic culture, including ald (encoding alanine dehydrogenase) cyd,encoding a subunit of the cytochrome D ubiquinol oxidase, and gltA1, encoding a citrate synthase. Promoter activity in the upstream regions of both cydB and gltA1 was altered in the RegX3 deletion strain. DNA-binding assays confirmed that RegX3 binds to the promoter regions of ald, cydB and gltA1 in a phosphorylation-dependent manner. Taken together these data suggest a direct role for the SenX-RegX3 system in modulating expression of aerobic respiration, in addition to its role during phosphate limitation.     

Effect of aeration and sodium on the metabolism of citrate by Klebsiella aerogenes.

Anaerobic growth of Klebsiella aerogenes NCDO 711 (NCTC 418) on citrate was dependent on the presence of Na+ in the medium, and fermentation of citrate was mediated via the fermentation pathway enzymes, citrate lyase and a Na+-dependent oxalacetate decarboxylase. This confirms the previous findings on strain NCTC 418. Growth under aerobic conditions was independent of Na+. The mean generation time for cells grown aerobically on either Na+ or K+ citrate medium was about 60 min, with a molar growth yield of about 40 g (dry weight) of cells per mol of citrate utilized. Citrate was apparently metabolized aerobically in both the Na+ and K+ citrate cells via the citric acid cycle, since cell extracts contained alpha-ketoglutarate dehydrogenase but not the citrate fermentation enzymes. The presence of theother enzymes of the citric acid cycle in K. aerogenes was shown in earlier studies. Under aerated conditions (no detectable oxygen tension in the culture), growth was faster on the Na+ citrate medium (mean generation time, 85 min) than on the K+ citrate medium (mean generation time, 120 min). Both cultures grew slower than under aerobic conditions, presumably because of oxygen limitation. Despite the faster growth rate, the molar growth yield of the aerated Na+ citrate culture was one-half that observed for the aerated K+ citrate culture. Citrate was metabolized via the citric acid cycle in cells grown in the K+ citrate medium under aerated conditions since alpha-ketoglutarate dehydrogenase, but not the fermentation enzymes, was detected in extracts prepared from these cells. Metabolism of citrate in the Na+ citrate medium under aerated conditions occurred via both the fermentation pathway (approximately 75 percent) and the citric acid cycle (about 25 percent), as evidenced by (i) the presence of the fermentation enzymes and alpha-ketoglutarate dehydrogenase in extracts of cells grown under these conditions, (ii) a molar growth yield which was intermediate between that obtained for anaerobic and aerated K+ citrate cultures, and (iii) the excretion of acetate, which also occurred in anaerobic cultures but not in aerated K+ citrate or aerobic cultures.     

过量表达苹果酸脱氢酶对大肠杆菌NZN111产丁二酸的影响

大肠杆菌NZN111是敲除了乳酸脱氢酶的编码基因 (ldhA) 和丙酮酸-甲酸裂解酶的编码基因 (pflB) 的工程菌,厌氧条件下由于辅酶NAD(H) 的不平衡导致其丧失了代谢葡萄糖的能力。构建了苹果酸脱氢酶的重组菌大肠杆菌NZN111/pTrc99a-mdh,在厌氧摇瓶发酵过程中通过0.3 mmol/L的IPTG诱导后重组菌的苹果酸脱氢酶 (Malate dehydrogenase,MDH) 酶活较出发菌株提高了14.8倍,NADH/NAD+的比例从0.64下降到0.26,同时NAD+和NADH浓度分别     

Effects of oxygen on invertase expression in continuous culture of recombinant Saccharomyces cerevisiae containing the SUC2 gene

The yeast SUC2 gene, cloned on a multicopy plasmid pRB58, was used to study the effect of oxygen on the invertase expression of the recombinant Saccharomyces cerevisiae. Glucose repression was not the only factor affecting the invertase expression. The results obtained from the single-stage continuous cultures under microaerobic conditions showed that invertase expression was also strongly dependent on oxygen availability, and moving from anaerobic to aerobic conditions led to a five-fold increase in specific invertase activity. However, the cell yields under anaerobic conditions were quite low compared to those under aerobic conditions. These opposite effects of oxygen on cell growth and gene expression offer a strategy for maximizing invertase productivity by a two-stage continuous culture. The first stage was operated at a low level of glucose, around 100 mg/l, under aerobic conditions in order to obtain a high yield of yeast biomass, and the second stage maintained anaerobic conditions with residual glucose levels of 50 mg/l to derepress and fully induce invertase expression. The two-stage continuous culture resulted in a 2.5-fold increase in invertase productivity over that of a single-stage continuous culture. Received: 28 July 1998 / Received revision: 22 September 1998 / Accepted: 7 November 1998     

Identification of the mcrC gene product in Methanococcus vannielii

Abstract The polypeptide encoded by the mcr C gene has been identified in Methanococcus vannielii by immunoblotting using rabbit antibodies raised against the product of a lac Z- mcr C gene fusion synthesized and purified from Escherichia coli . The mcr C gene product (gp mcr C) was located in both supernatant and pellet fractions after centrifugation of Mc. vannielii cell extracts for 2 h at 100 000 × g . When anaerobic reducing conditions were maintained during purification, gp mcr C co-sedimented through sucrose gradients to the same position as molecules of the methyl coenzyme M reductase holoenzyme (approx. 300 kDa). This co-sedimentation was lost under aerobic, nonreducing conditions.     

Cytochrome o (cyoABCDE) and d (cydAB) oxidase gene expression in Escherichia coli is regulated by oxygen, pH, and the fnr gene product.

The aerobic respiratory chain of Escherichia coli contains two terminal oxidases that catalyze the oxidation of ubiquinol-8 and the reduction of oxygen to water. They are the cytochrome o oxidase complex encoded by cyoABCDE and the cytochrome d oxidase complex encoded by cydAB. To determine how these genes are regulated in response to a variety of environmental stimuli, including oxygen, we examined their expression by using lacZ protein fusions in wild-type and fnr mutant strains of E. coli. Anaerobic growth resulted in a 140-fold repression of cyoA'-'lacZ expression relative to aerobic growth and a 3-fold increase in cydA'-'lacZ expression. Anaerobic repression of both fusions was mediated in part by the fnr gene product, as evidenced by a 30-fold derepression of cyoA'-'lacZ expression and a 4-fold derepression of cydA'-'lacZ expression in an fnr deletion strain. Supplying wild-type fnr in trans restored wild-type repression for both fusions. Fnr thus functions as an anaerobic repressor of both cyoABCDE and cydAB expression. Reduced-minus-oxidized difference spectrum analyses of cell membranes confirmed the effect of the fnr gene product on the production of cytochrome d oxidase in the cell. Based on the pattern of anaerobic cydAB expression observed, we propose the existence of a second, as yet unidentified, regulatory element that must function either to activate cydAB expression as oxygen becomes limiting or to repress cydAB expression aerobically. Whereas cytochrome o oxidase encoded by cyoABCDE appears to be produced only under oxygen-rich growth conditions, in keeping with its biochemical properties, cytochrome d oxidase is expressed moderately aerobically and is elevated yet further when oxygen becomes limiting so that the organism can cope better under oxygen starvation conditions. We also examined cyoABCDE and cydAB expression in response to growth on alternative carbon compounds and to changes in the culture medium pH and osmolarity.     

Metabolic regulation analysis of wild-type and arcA mutant Escherichia coli under nitrate conditions using different levels of omics data

It is of practical interest to investigate the effect of nitrates on bacterial metabolic regulation of both fermentation and energy generation, as compared to aerobic and anaerobic growth without nitrates. Although gene level regulation has previously been studied for nitrate assimilation, it is important to understand this metabolic regulation in terms of global regulators. In the present study, therefore, we measured gene expression using DNA microarrays, intracellular metabolite concentrations using CE-TOFMS, and metabolic fluxes using the (13)C-labeling technique for wild-type E. coli and the ΔarcA (a global regulatory gene for anoxic response control, ArcA) mutant to compare the metabolic state under nitrate conditions to that under aerobic and anaerobic conditions without nitrates in continuous culture conditions at a dilution rate of 0.2 h(-1). In wild-type, although the measured metabolite concentrations changed very little among the three culture conditions, the TCA cycle and the pentose phosphate pathway fluxes were significantly different under each condition. These results suggested that the ATP production rate was 29% higher under nitrate conditions than that under anaerobic conditions, whereas the ATP production rate was 10% lower than that under aerobic conditions. The flux changes in the TCA cycle were caused by changes in control at the gene expression level. In ΔarcA mutant, the TCA cycle flux was significantly increased (4.4 times higher than that of the wild type) under nitrate conditions. Similarly, the intracellular ATP/ADP ratio increased approximately two-fold compared to that of the wild-type strain.     

Involvement of ArcA and Fnr in expression of Escherichia coli thiol peroxidase gene

To explore the oxygen response regulators involved in thiol peroxidase gene (tpx) expression in Escherichia coli, we constructed a single-copy tpx-lacZ operon fusion and monitored tpx-lacZ expression in various genetic backgrounds. Expression of the tpx-lacZ fusion was increased 4-fold by aerobic growth. Anaerobic expression of tpx-lacZ in either (delta)arcA or delta(fnr) strains was 2.5-fold depressed compared with that of the wild-type strain. The results of immunoblotting experiments also demonstrated that ArcA and Fnr regulatory proteins repressed thiol peroxidase gene expression during anaerobic growth. Inspection of the tpx promoter region revealed putative binding sites for ArcA and Fnr. It thus appears that ArcA and Fnr function as repressors by blocking the binding of RNA polymerase to the tpx promoter in E. coli under anaerobic growth conditions.     

Use of a wild-type gene fusion to determine the influence of environmental conditions on expression of the S fimbrial adhesin in an Escherichia coli pathogen.

S fimbrial adhesins (Sfa) enable pathogenic Escherichia coli strains to bind to sialic acid-containing eucaryotic receptor molecules. In order to determine the influence of culture conditions on the expression of the sfa determinant in a wild-type strain, we fused the gene lacZ, coding for the enzyme beta-galactosidase, to the sfaA gene, responsible for the major protein subunit of S fimbriae. By using a plasmid which carries an R6K origin, the sfaA-lac hybrid construct was site-specifically integrated into the chromosome of the uropathogenic E. coli strain 536WT. The expression of lacZ, which was under the control of the sfa wild-type promoters, was now equivalent to the sfa expression of strain 536WT. With the help of this particular wild-type construct, it was demonstrated that the sfa determinant is better expressed on solid media than in liquid broth. The growth rate had a strong influence on Sfa expression under aerobic but not under anaerobic conditions. Production of Sfa was further regulated by catabolite repression, osmolarity, and temperature.     

Cloning and expression of Klebsiella pneumoniae genes coding for citrate transport and fermentation.

Three Escherichia coli clones (DH1/Cit1, DH1/Cit2 and DH1/Cit3) capable of utilizing citrate as a sole carbon source were isolated from a cosmid bank of Klebsiella pneumoniae wild-type DNA. Two of these clones (DH1/Cit1 and DH1/Cit2) only grew aerobically on citrate minimal medium, the third clone (DH1/Cit3) could also be cultured under fermentative conditions. The aerobic as well as the anaerobic generation times of the three clones were from 4.5 to 7 h. Whereas clone DH1/Cit3 showed a pronounced lag phase on citrate when the cells were pre-grown in medium without citrate, clone DH1/Cit1 immediately started growth, while with clone DH1/Cit2 a short lag phase could be observed upon transfer to citrate minimal medium. Restriction analyses of the three plasmids showed that no common fragments had been cloned. The length of the inserts were 13 and 6 kb for the aerobic Cit+ clones and 27 kb (10 kb) for the anaerobic one. Cultures of the anaerobic Cit+ clone were analyzed by immunoblotting techniques and shown to contain oxaloacetate decarboxylase, which confers citrate utilization under anaerobic conditions to K. pneumoniae. Enzyme assays demonstrated the active state of this biotin-containing membrane protein. The specific activity in vesicle preparations from the E. coli clone was 30% of the wild-type K. pneumoniae vesicles. Citrate acts as an inducer of enzyme protein synthesis in the E. coli clone as it does in K. pneumoniae.     

Molecular characterization of the Escherichia coli htrD gene: cloning, sequence, regulation, and involvement with cytochrome d oxidase.

The Escherichia coli htrD gene was originally isolated during a search for new genes required for growth at high temperature. Insertional inactivation of htrD leads to a pleiotropic phenotype characterized by temperature-sensitive growth in rich medium, H2O2 sensitivity, and sensitivity to cysteine. The htrD gene was cloned and sequenced, and an htrD::mini-Tn10 insertion mutation was mapped within this gene. The htrD gene was shown to encode a protein of approximately 17.5 kDa. Expression of the htrD gene was examined by using an phi (htrD-lacZ) operon fusion. It was found that htrD is not temperature regulated and therefore is not a heat shock gene. Further study revealed that htrD expression is increased under aerobic growth conditions. Conversely, under anaerobic growth conditions, htrD expression is decreased. In addition, a mutation within the nearby cydD gene was found to drastically reduce htrD expression under all conditions tested. These results indicate that htrD is somehow involved in aerobic respiration and that the cydD gene product is necessary for htrD gene expression. In agreement with this conclusion, htrD mutant bacteria are unable to oxidize the cytochrome d-specific electron donor N,N,N',N'-tetramethyl-p-phenylenediamine.     

Induction of citrate lyase in Enterobacter cloacae grown under aerated conditions and its effect on citrate metabolism.

Growth of Enterobacter cloacae on K+ citrate under aerated conditions (no detectable oxygen tension in the medium even though it was aerated) was slower (mean generation time, 130 min) than under aerobic conditions (mean generation time, 72 min), but with a faster utilization of citrate, resulting in a molar growth yield of 10.6 g (dry weight) of cells per mol of citrate utilized versus 40 g (dry weight) of cells per mol of citrate utilized for aerobic growth. The rapid utilization of citrate under aerated conditions was apparently due to the induction of citrate lyase and was supported by the finding that cells excreted acetate and a small amount of oxalacetate under aerated conditions, but not under aerobic conditions when the cells were devoid of citrate lyase activity. The activity of oxalacetate decarboxylase in aerated cells was slightly lower than in aerobic cells, indicating that little of the oxalacetate produced by the citrate lyase was metabolized by the decarboxylase. Oxalacetate was probably metabolized by malate dehydrogenase, previously shown to be present in anaerobic and aerobic cells. Thus, about 70% of the citrate was cleaved by the citrate lyase, resulting in little or no production of energy for growth. The remaining citrate was metabolized via the citric acid cycle under aerated conditions, since the cells contained alpha-ketoglutarate dehydrogenase at the same level as in aerobically grown cells. The presence of the other enzymes of the cycle was shown in earlier studies.     

Characterisation of a Pasteurella multocida esterase gene which confers a hemolytic phenotype in Escherichia coli under anaerobic conditions

Investigation of the hemolytic phenotype under anaerobic growth conditions of an avian Pasteurella multocida strain, PBA100, resulted in the identification and characterisation of a gene encoding an esterase enzyme, mesA, that conferred a hemolytic phenotype in Escherichia coli under anaerobic conditions. MesA appeared to be expressed and functional under anaerobic and aerobic conditions in both E. coli and P. multocida. A P. multocida mesA mutant was generated which resulted in the loss of acetyl esterase activity under anaerobic conditions. However, this mutation did not cause any attenuation of virulence for mice nor a detectable change to the anaerobic hemolytic phenotype of P. multocida. In E. coli MesA appeared to cause hemolysis indirectly by the induction of the latent E. coli K-12 cytolysin, sheA.     

Genome-wide expression analysis indicates that FNR of Escherichia coli K-12 regulates a large number of genes of unknown function

The major regulator controlling the physiological switch between aerobic and anaerobic growth conditions in Escherichia coli is the DNA binding protein FNR. To identify genes controlled by FNR, we used Affymetrix Antisense GeneChips to compare global gene expression profiles from isogenic MG1655 wild-type and Deltafnr strains grown in glucose minimal media under aerobic or anaerobic conditions. We found that 297 genes contained within 184 operons were regulated by FNR and/or by O2 levels. The expression of many genes known to be involved in anaerobic respiration and fermentation was increased under anaerobic growth conditions, while that of genes involved in aerobic respiration and the tricarboxylic acid cycle were repressed as expected. The expression of nine operons associated with acid resistance was also increased under anaerobic growth conditions, which may reflect the production of acidic fermentation products. Ninety-one genes with no presently defined function were also altered in expression, including seven of the most highly anaerobically induced genes, six of which we found to be directly regulated by FNR. Classification of the 297 genes into eight groups by k-means clustering analysis indicated that genes with common gene expression patterns also had a strong functional relationship, providing clues for studying the function of unknown genes in each group. Six of the eight groups showed regulation by FNR; while some expression groups represent genes that are simply activated or repressed by FNR, others, such as those encoding functions for chemotaxis and motility, showed a more complex pattern of regulation. A computer search for FNR DNA binding sites within predicted promoter regions identified 63 new sites for 54 genes. We suggest that E. coli MG1655 has a larger metabolic potential under anaerobic conditions than has been previously recognized.