Regulation of the pho regulon in Escherichia coli K-12. Genetic and physiological regulation of the positive regulatory gene phoB

phoB is a positive regulatory gene for phoA, which codes for alkaline phosphatase, as well as for other genes belonging to the phosphate (pho) regulon whose expression is inducible by phosphate limitation in Escherichia coli. A hybrid plasmid that contains a phoB-lacZ fused gene was constructed in vitro. This plasmid enabled us to study phoB gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the phosphate regulon, and phoB gene expression in these strains was studied under limited and excess phosphate conditions. It was found that the regulation of phoB expression was very similar to that of phoA expression. Expression of both genes was induced by phosphate starvation. Both genes were constitutively expressed in phoR, phoS, phoT and phoU mutants and were not expressed in a phoR-phoM double mutant. The implications of these findings for the regulatory mechanism of the pho regulon are discussed.     

Regulation of the phosphate regulon in Escherichia coli K-12: regulation of the negative regulatory gene phoU and identification of the gene product.

The phoU gene is one of the negative regulatory genes of the pho regulon of Escherichia coli. The DNA fragment carrying phoU has been cloned on pBR322 (Amemura et al., J. Bacteriol. 152:692-701, 1982). Further subcloning, Tn1000 insertion inactivation, and complementation tests localized the phoU gene within a 1.1-kilobase region on the cloned DNA fragment. The gene product of phoU was identified by the maxicell method as a protein with an approximate molecular weight of 27,000. A hybrid plasmid that contains a phoU'-lac'Z fused gene was constructed in vitro. This plasmid enabled us to study phoU gene expression by measuring the beta-galactosidase level in the cells. The plasmid was introduced into various regulatory mutants related to the pho regulon, and phoU gene expression in these strains was studied under limited and excess phosphate conditions. It was found that phoU is expressed at a higher level when the cells are cultured under the excess phosphate condition. The higher phoU expression was observed in a phoB mutant and a phoR-phoM double mutant. The implications of these findings for the regulation of pho genes are discussed.     

Identification and characterization of the sodA genes encoding manganese superoxide dismutases in Vibrio parahaemolyticus, Vibrio mimicus, and Vibrio vulnificus

Sequencing of Fur titration assay-positive clones obtained from genomic DNA libraries of Vibrio parahaemolyticus, V. mimicus and V. vulnificus revealed open reading frames encoding proteins of 202, 205 and 202 amino acid residues, respectively. Each open reading frame was preceded by a predicted Fur box which overlaps a likely promoter with similarity to the -10 and -35 consensus sequence of Escherichia coli. The deduced amino acid sequences shared considerable homology with bacterial Mn-containing superoxide dismutases (MnSODs). Consistent with this, these Vibrio strains produced proteins with SOD activity resistant to inhibition by H2O2 and KCN only when grown under iron-limiting conditions. Primer extension analysis of the total RNA from these vibrios revealed iron-repressible expression of the genes. Furthermore, when grown under iron-limiting conditions, E. coli carrying a plasmid with each cloned gene overexpressed protein with the same electrophoretic mobility and insensitivity of SOD activity to H2O2 and KCN. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis followed by N-terminal amino acid sequencing revealed that proteins (MnSODs) having N-terminal amino acid sequences consistent with those deduced from the corresponding genes were present in cell lysates of the vibrios grown under these iron-limited conditions. These results demonstrate that the genes cloned in this study are sodA homologs encoding MnSODs, whose expression is regulated by the iron status of the growth medium. PCR using a primer set based on the V. parahaemolyticus sodA sequence revealed the presence of homologous genes in certain other Vibrio species.     

Cloning and expression of the 5'-nucleotidase gene of Vibrio parahaemolyticus in Escherichia coli and overproduction of the enzyme

The gene encoding the membrane-bound 5'-nucleotidase of Vibrio parahaemolyticus was cloned and expressed in Escherichia coli. Cells of E. coli harboring a plasmid, pNUT5, which carries the 5'-nucleotidase gene were able to grow on ATP as the sole source of carbon, although the original cells were not. The 5'-nucleotidase activity was detected in whole cells of E. coli harboring pNUT5 and in membrane vesicles prepared from these cells. Most properties of the 5'-nucleotidase produced in E. coli, that is, its requirements for Cl- and Mg2+, substrate specificity, and inhibition by Zn2+, were similar to those observed in V. parahaemolyticus, but some alterations in properties were observed: The 5'-nucleotidase was partially inducible in V. parahaemolyticus, but its expression in E. coli was completely constitutive. The specific activity of the 5'-nucleotidase in membrane vesicles of E. coli harboring the plasmid was 30 times that observed in whole cells, whereas the specific activities in membrane vesicles and in whole cells of V. parahaemolyticus were almost the same. A new, dense band of protein with an apparent molecular mass of 63 kDa was detected when membrane proteins of E. coli harboring the plasmid were analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

哈氏弧菌dam基因的克隆与分析

利用兼并PCR的方法克隆得到哈氏弧菌T4的DNA腺嘌呤甲基化酶(dam)基因,序列分析表明该基因编码279个氨基酸,与其它已知弧菌的Dam具有较高的同源性,其中与副溶血弧菌Dam的相同性达95%。功能检验表明所克隆的dam基因在大肠杆菌中具有DNA腺嘌呤甲基化酶活性,能够甲基化大肠杆菌染色体DNA GATC序列中的腺嘌呤。运用染色体步移法获得dam基因上游的3251 bp DNA,发现该区域含有3个基因,其与dam在染色体上的相对排列顺序为:莽草酸激酶-脱氢奎尼酸合成酶-damX-dam。对dam上游DNA序列研究发现位于翻译起点ATG上游的78bp、112bp和477bpDNA片段皆具有启动子活性,但前者的活性明显高于后二者。     

Serum resistance in Haemophilus parasuis SC096 strain requires outer membrane protein P2 expression

Haemophilus parasuis outer membrane protein P2 (OmpP2), the most abundant protein in the outer membrane, has been identified as an antigenic protein and a potential virulence factor. To study the precise function of OmpP2, an ompP2-deficient mutant (ΔompP2) of a H.?parasuis serovar 4 clinical strain SC096 was constructed by a modified natural transformation system. Compared with the wild-type SC096 strain, the ΔompP2 mutant showed a pronounced growth defect and exhibited significantly greater sensitivity to the bactericidal action of porcine and rabbit sera, whereas the complemented strain could restore the growth and serum resistance phenotypes. The results indicated that H.?parasuis OmpP2 from SC096 strain is an important surface protein involved in serum resistance.     

Characterization of the antiseptic-resistance gene qacEΔ1 isolated from clinical and environmental isolates of Vibrio parahaemolyticus and Vibrio cholerae non-O1

The nucleotide sequence and mechanism of action were examined on the antiseptic-resistance gene qacE delta 1 that had been isolated from Pseudomonas aeruginosa, Vibrio parahaemolyticus and Vibrio cholerae non-O1. The nucleotide sequences of qacE delta 1 genes isolated from environmental isolates of V. cholerae non-O1 and V. parahaemolyticus differed by one base from that of the gene from P. aeruginosa. Escherichia coli C600 that harbored qacE delta 1 genes from several strains of Vibrio spp. exhibited low-level resistance to intercalating dyes. The resistance of E. coli cells with these genes to intercalating dyes, such as ethidium bromide, was mediated by an efflux system. Moreover, the activity of QacE delta 1 was inhibited in the presence of calcium channel blockers but not of calmodulin inhibitors. These results indicate that the qacE delta 1 gene can be function in E. coli and that the gene mediates resistance in a similar manner to the antiseptic-resistance gene smr.     

Catabolite repression of the H(+)-translocating ATPase in Vibrio parahaemolyticus.

Cells of Vibrio parahaemolyticus grown in the presence of glucose showed reduced (by about 40%) oxidative phosphorylation. With this observation as a basis, we examined the effect of glucose on the level of H(+)-translocating ATPase. The addition of glucose to the growth medium reduced the specific activity and the amount of the H(+)-translocating ATPase in membrane vesicles of V. parahaemolyticus. These reductions were reversed by adding cyclic AMP (cAMP) to the growth medium. We cloned some parts of the unc genes encoding subunits of the H(+)-translocating ATPase of V. parahaemolyticus by means of the polymerase chain reaction. Using an amplified DNA fragment, we carried out Northern (RNA) blot analysis and found that glucose reduced the mRNA level of the H(+)-translocating ATPase gene by about 40% and that cAMP restored it. We determined the DNA sequence of the unc promoter region of V. parahaemolyticus and found a consensus sequence for the cAMP receptor protein-cAMP-binding site. Such a sequence was also found in the promoter region of the unc operon of Vibrio alginolyticus but not in its counterpart in Escherichia coli. We observed a similar reduction in the level of ATPase due to glucose in V. alginolyticus. In E. coli, however, reductions in the ATPase and the unc mRNA levels were not observed. Thus, the unc operon is controlled by cAMP-regulated catabolite repression in V. parahaemolyticus and V. alginolyticus but not in E. coli. Catabolite repression of the unc operon in V. parahaemolyticus is not severe.     

Nucleotide sequence of the thermostable direct hemolysin gene of Vibrio parahaemolyticus.

The gene encoding the thermostable direct hemolysin of Vibrio parahaemolyticus was characterized. This gene (designated tdh) was subcloned into pBR322 in Escherichia coli, and the functional tdh gene was localized to a 1.3-kilobase HindIII fragment. This fragment was sequenced, and the structural gene was found to encode a mature protein of 165 amino acid residues. The mature protein sequence was preceded by a putative signal peptide sequence of 24 amino acids. A putative tdh promoter, determined by its similarity to concensus sequences, was not functional in E. coli. However, a promoter that was functional in E. coli was shown to exist further upstream by use of a promoter probe plasmid. A 5.7-kilobase SalI fragment containing the structural gene and both potential promoters was cloned into a broad-host-range plasmid and mobilized into a Kanagawa phenomenon-negative V. parahaemolyticus strain. In contrast to E. coli, where the hemolysin was detected only in cell lysates, introduction of the cloned gene into V. parahaemolyticus resulted in the production of extracellular hemolysin.     

Growth of Escherichia coli MG1655 on LB medium: monitoring utilization of amino acids, peptides, and nucleotides with transcriptional microarrays

Analysis of gene expression data related to assimilation and biosynthesis of nitrogen-containing compounds amino acids, peptides, and nucleotides was used to monitor availability of these nutrients to Escherichia coli MG1655 growing on Luria-Bertani medium. The data indicate that free amino acids and nucleotides only transiently support the nitrogen requirement for growth and are no longer available by 3.5 h of fermentation. The resulting shortage of available nitrogen sources induces the Ntr response, which involves induction of the glnALG, glnK-amtB, dppABCDF, and oppABCDF operons as well as the genes coding for outer membrane proteins, porins OmpA and OmpC, and proteases OmpP and OmpT. The increased uptake of peptides facilitated by the products of dppABCDF, oppABCDF, ompA, ompC, ompP, and ompT alleviates nitrogen limitation of the growth.     

Effects of pho regulatory mutations and phoA gene amplification on alkaline phosphatase synthesis and release by lky mutants of Escherichia coli K12

lky mutants of Escherichia coli K12 spontaneously released alkaline phosphatase (APase) into the extracellular medium to give up to 300 units ml-1. APase is a phosphate repressible periplasmic enzyme encoded by the gene phoA. With a view to establishing a method of easy purification, we have analysed APase synthesis and release patterns of isogenic lky strains containing either a constitutive pho regulatory mutation, or a hybrid plasmid carrying the structural gene phoA+ and pho regulatory genes, or a transducing phi 80 phoA+ phage. In the presence of the phoS2333 mutation, F- lky strains lysogenized with phi 80 phoBin phoA+ phage and grown in high phosphate medium were able to release eight times more APase activity (2300 units ml-1) than haploid strain 2336 (phoS+ lky) grown in low phosphate medium. Neither protein synthesis, the cell export machinery nor leakage mechanisms were limiting for APase release. Sufficient APase was released into the medium to facilitate its purification.     

Cloning and characterization of a gene encoding a new thermostable hemolysin from Vibrio parahaemolyticus

A new thermostable hemolysin (delta-VPH) gene was cloned from a Kanagawa-negative Vibrio parahaemolyticus strain into vector pBR322 in Escherichia coli K12. The nucleotide and amino acid sequences had no homology with those of the thermostable direct hemolysin (TDH) which causes the Kanagawa phenomenon, and of the thermolabile hemolysin (TLH) of V. parahaemolyticus. The gene was present in all V. parahaemolyticus strains tested and also in one strain of V. damsela.     

Porin OmpP2 of Haemophilus influenzae shows specificity for nicotinamide-derived nucleotide substrates

Haemophilus influenzae has an absolute requirement for NAD (factor V) because it lacks all biosynthetic enzymes necessary for de novo synthesis of that cofactor. Therefore, growth in vitro requires the presence of NAD itself, NMN, or nicotinamide riboside (NR). To address uptake abilities of these compounds, we investigated outer membrane proteins. By analyzing ompP2 knockout mutants, we found that NAD and NMN uptake was prevented, whereas NR uptake was not. Through investigation of the properties of purified OmpP2 in artificial lipid membrane systems, the substrate specificity of OmpP2 for NAD and NMN was determined, with KS values of approximately 8 and 4mm, respectively, in 0.1 m KCl, whereas no interaction was detected for the nucleoside NR and other purine or pyrimidine nucleotide or nucleoside species. Based on our analysis, we assume that an intrinsic binding site within OmpP2 exists that facilitates diffusion of these compounds across the outer membrane, recognizing carbonyl and exposed phosphate groups. Because OmpP2 was formerly described as a general diffusion porin, an additional property of acting as a facilitator for nicotinamide-based nucleotide transport may have evolved to support and optimize utilization of the essential cofactor sources NAD and NMN in H. influenzae.     

Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.     

Genetic and physical characterization of proBA genes of the marine bacterium Vibrio parahaemolyticus.

Intracellular proline pools have been implicated in the halotolerance of many organisms. To examine this relationship in a moderately halotolerant marine bacterium, Vibrio parahaemolyticus, proline biosynthesis genes were cloned in various plasmids. Some genetic and structural properties of those genes were examined. Subcloning showed that about 3.1 kilobases of V. parahaemolyticus DNA could complement proA and proB but not proC mutations of Escherichia coli. The same fragment would also complement some Pro- mutants of V. parahaemolyticus. Gamma-delta insertion mutagenesis of this subcloned fragment indicated that proB and proA genes of V. parahaemolyticus might be transcribed from different promoters. Two other genes, phoE and gpt, which map closely to the proBA genes in E. coli, were also found to be in close proximity to the proBA genes of V. parahaemolyticus.     

Overlapping and separate controls on the phosphate regulon in Escherichia coli K12

The physiological and genetic controls operating on phosphate-regulated promoters were studied in greater detail. This was done by defining the control for three phosphate-regulated genes: phoA, psiE, and psiO. Each is highly inducible by phosphate starvation. Individually, these phosphate-starvation-inducible, psi, genes at the same time show common and differing features in their molecular control. The phoA gene, encoding alkaline phosphatase, is specifically induced by phosphate starvation. It is negatively controlled by phoR as well as by the phosphate-specific transport (PST) system in Escherichia coli. phoA induction is positively controlled by the phoB, M, and R products; it is unaffected by the cAMP and CAP system. The psiE and psiO genes were studied by using strains with lacZ fused to their respective promoters. psiE-lacZ is induced by phosphate-, carbon- or nitrogen-limited growth. Genetically, psiE-lacZ induction is partially phoB and phoR-dependent. However, its expression is phoM-independent. This implies that phoB/phoR coupled control differs from phoB/phoM coupled control. Repression of psiE-lacZ is substantially altered in only some PST mutants, such as phoT. In addition, psiE-lacZ is negatively controlled by the cAMP and CAP system. psiO-lacZ is induced by phosphate-, carbon- or nitrogen-limited growth or by anaerobiosis. Its expression is unaffected by any pho mutation that has been previously described. A cell density-dependent induction of psiO-lacZ is observed in lon mutants. Also, psiO-lacZ is negatively controlled by the cAMP-CAP system. In summary, these results demonstrate that co-ordinately regulated promoters can have some common regulatory elements while, at the same time, not sharing other controlling factors.     

副溶血性弧菌群体感应蛋白CqsA的原核表达

副溶血性孤菌CqsA是一种推测的信号分子合成酶,其合成的信号分子在群体感应系统中可能具有重要的调控作用.从副溶血性弧菌ATCC 17802中克隆cqsA基因,构建重组质粒pET22b-cqsA.测序后转化大肠杆菌B1L21进行IPTG诱导表达,使用SDS-PAGE分析融合蛋白的表达状况,并通过6× His-Tag进行Western blotting检测.结果显示,经0.6 mmol/L IPTG诱导4h,目的基因以包涵体形式高效表达,表达的融合蛋白大小约为49 kD,与理论值相符(437 aa).表明副溶血性弧菌群体感应蛋白CqsA在大肠杆菌中成功表达,为后续寻找副溶血性孤菌群体感应信号分子,进一步探索副溶血性弧菌群体感应系统提供参考.     

Identification of the phoM gene product and its regulation in Escherichia coli K-12.

Plasmids containing the chromosome region of Escherichia coli encoding phoM, whose product is a positive regulator of alkaline phosphatase expression, were isolated from the Clarke and Carbon plasmid bank. A 9.9-kilobase EcoRI fragment of plasmid pLC17-39 (subcloned into pBR322) was able to complement both phoM and thrB mutations. Restriction endonuclease analysis and in vitro mutagenesis of the hybird plasmids enabled the localization of the phoM gene locus to 3 kilobases of the cloned chromosomal fragment. The phoM gene product was identified, with maxicell techniques, as a protein with an approximate molecular weight of 55,000. A phoM-lacZ protein fusion was constructed by using a plasmid carrying the phoM gene and a derivative of phage lambda, lambda plac Mu2. Restriction endonuclease analysis of the plasmid carrying the fusion indicated that phoM is transcribed in a clockwise direction on the circular E. coli chromosome. Analysis of strains bearing the fusion on a multiple-copy plasmid or integrated at the lambda attachment site of the chromosome indicated that the synthesis of the phoM gene product was unaffected by phosphate limitation of growth. The expression of the phoM gene was studied in strains with mutations in genes encoding effectors of the pho regulon. A threefold increase in phoM expression was seen in a phoU strain in comparison with the wild-type strain.