假单胞菌株M18 psrA突变株的构建及其对吩嗪-1-羧酸和藤黄绿菌素合成的调控

【目的】假单胞菌M18是一株能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素(Plt)两种抗生素的植物根际促生细菌。PsrA为细菌TetR家族转录调控因子。为了研究PsrA对PCA与Plt生物合成的影响,从M18菌株基因组中扩增psrA基因。【方法】通过同源重组技术,构建庆大霉素抗性片段置换psrA的突变菌株M18psrA。利用基因互补、lacZ报告基因融合分析实验,验证PsrA对抗生素合成基因的调控作用。【结果】在PPM和KMB培养基中,分别比较野生型菌株M18和突变菌株M18psrA的PCA与Plt产量,突变菌株M18psrA的PCA产量显著下降;Plt产量显著升高,为野生型菌株的10-15倍。基因互补、lacZ报告基因融合分析,进一步证明了psrA正调控PCA的phz2合成基因簇,负调控Plt的合成基因簇。【结论】PsrA区别性调控抗生素PCA与Plt的生物合成。     

Regulation of rpoS gene expression in Pseudomonas: involvement of a TetR family regulator

The rpoS gene encodes the sigma factor which was identified in several gram-negative bacteria as a central regulator during stationary phase. rpoS gene regulation is known to respond to cell density, showing higher expression in stationary phase. For Pseudomonas aeruginosa, it has been demonstrated that the cell-density-dependent regulation response known as quorum sensing interacts with this regulatory response. Using the rpoS promoter of P. putida, we identified a genomic Tn5 insertion mutant of P. putida which showed a 90% decrease in rpoS promoter activity, resulting in less RpoS being present in a cell at stationary phase. Molecular analysis revealed that this mutant carried a Tn5 insertion in a gene, designated psrA (Pseudomonas sigma regulator), which codes for a protein (PsrA) of 26.3 kDa. PsrA contains a helix-turn-helix motif typical of DNA binding proteins and belongs to the TetR family of bacterial regulators. The homolog of the psrA gene was identified in P. aeruginosa; the protein showed 90% identity to PsrA of P. putida. A psrA::Tn5 insertion mutant of P. aeruginosa was constructed. In both Pseudomonas species, psrA was genetically linked to the SOS lexA repressor gene. Similar to what was observed for P. putida, a psrA null mutant of P. aeruginosa also showed a 90% reduction in rpoS promoter activity; both mutants could be complemented for rpoS promoter activity when the psrA gene was provided in trans. psrA mutants of both Pseudomonas species lost the ability to induce rpoS expression at stationary phase, but they retained the ability to produce quorum-sensing autoinducer molecules. PsrA was demonstrated to negatively regulate psrA gene expression in Pseudomonas and in Escherichia coli as well as to be capable of activating the rpoS promoter in E. coli. Our data suggest that PsrA is an important regulatory protein of Pseudomonas spp. involved in the regulatory cascade controlling rpoS gene regulation in response to cell density.     

Structures of genes nasA and nasB, encoding assimilatory nitrate and nitrite reductases in Klebsiella pneumoniae M5al.

Klebsiella pneumoniae can use nitrate and nitrite as sole nitrogen sources during aerobic growth. Assimilatory nitrate and nitrite reductases convert nitrate through nitrite to ammonium. We report here the molecular cloning of the nasA and nasB genes, which encode assimilatory nitrate and nitrite reductase, respectively. These genes are tightly linked and probably form a nasBA operon. In vivo protein expression and DNA sequence analysis revealed that the nasA and nasB genes encode 92- and 104-kDa proteins, respectively. The NASA polypeptide is homologous to other prokaryotic molybdoenzymes, and the NASB polypeptide is homologous to eukaryotic and prokaryotic NADH-nitrite reductases. The narL gene product positively regulates expression of the structural genes for respiratory nitrate reductase, narGHJI. Surprisingly, we found that the nasBA operon is tightly linked to the narL-narGHJI region in K. pneumoniae, even though the nitrate assimilatory and respiratory enzymes serve different physiological functions.     

Molybdenum cofactor requirement for biotin sulfoxide reduction in Escherichia coli.

The bisC gene of Escherichia coli is tentatively identified as the structural gene for biotin sulfoxide reductase by the isolation of bisC(Ts) mutants that make thermolabile enzyme. The products of four other E. coli genes (chlA, chlB, chlE and chlG) are also needed for enzymatic activity. Mutations previously assigned to the bisA, bisB, and bisD genes belong to genes chlA, chlE, and chlG, respectively. The biotin sulfoxide reductase deficiency of a chlG, mutant is partially reversed by the addition of 10 mM molybdate to the growth medium. Mutational inactivation of the chlD gene reduces the specific activity of biotin sulfoxide reductase about twofold. This effect is reversed by the addition of 1 mM molybdate to the growth medium. The specific activity of biotin sulfoxide reductase is decreased about 30-fold by the presence of tungstate in the growth medium, an effect that has been observed previously with nitrate reductase and other molybdoenzymes. The specific activity of biotin sulfoxide reductase is not elevated in a lysate prepared by derepressing a lambda cI857 chlG prophage. Whereas biotin sulfoxide reductase prepared by sonic extraction of growing cells is almost completely dependent on the presence of a small heat-stable protein resembling thioredoxin, much of the enzyme obtained from lysates of thermoinduced lambda cI857 lysogens does not require this factor.     

Cytochrome oxidase genes from Thermus thermophilus. Nucleotide sequence and analysis of the deduced primary structure of subunit IIc of cytochrome caa3.

Cytochrome caa3, a cytochrome c oxidase from Thermus thermophilus, is a two-subunit enzyme containing the four canonical metal centers of cytochrome c oxidases (cytochromes a and a3; copper centers CuA and CuB) and an additional cytochrome c. The smaller subunit contains heme C and was termed the C-protein. We have cloned the genes encoding the subunits of the oxidase and determined the nucleotide sequence of the C-protein gene. The gene and deduced primary amino acid sequences establish that both the gene and the protein are fusions with a typical subunit II sequence and a characteristic cytochrome c sequence; we now call this subunit IIc. The protein thus appears to represent a covalent joining of substrate (cytochrome c) to its enzyme (cytochrome c oxidase). In common with other subunits II, subunit IIc contains two hydrophobic segments of amino acids near the amino terminus that probably form transmembrane helices. Variability analysis of the Thermus and other subunit II sequences suggests that the two putative transmembrane helices in subunit II may be located on the surface of the hydrophobic portion of the intact cytochrome oxidase protein complex. Also in common with other subunits II is a relatively hydrophilic intermembrane domain containing a set of conserved amino acids (2 cysteines and 2 histidines) which have previously been proposed by others to serve as ligands to the CuA center. We compared the subunit IIc sequence with that of related proteins. N2O reductase of Pseudomonas stutzeri, a multi-copper protein that appears to contain a CuA site (Scott, R.A., Zumft, W.G., Coyle, C.L., and Dooley, D.M. (1989) Proc. Natl. Acad. Sci. U.S.A. 86, 4082-4086), contains a 59-residue sequence element that is homologous to the "CuA sequence motif" found in cytochrome oxidase subunits II, including all four putative copper ligands. By contrast, subunit II of the Escherichia coli quinol oxidase, cytochrome bo, also contains a region homologous to the CuA motif, but it lacks the proposed metal binding histidine and cysteine residues; this is consistent with the apparent absence of CuA from cytochrome bo.     

Roles of the narJ and narI gene products in the expression of nitrate reductase in Escherichia coli

Nitrate reductase, released and purified from membrane fractions of Escherichia coli, is composed of three subunits. Formation of the enzyme depends on induction of the nar operon, narGHJI, which is composed of four open reading frames (ORF). Previous studies established that the first two genes in the operon narG and narH encode the alpha and beta subunits, respectively, while formation of the gamma subunit, cytochrome bNR, depends on expression of the promoter distal genes. The narJ and narI genes were subcloned separately into plasmids where each was under the control of the nar promoter. Expression of these plasmids in a mutant which forms only alpha and beta subunits revealed that expression of the narI gene is sufficient to restore normal levels of cytochrome bNR, but expression of both genes is required for assembly of fully active, membrane-bound nitrate reductase. The amino acid composition, the N-terminal sequence, and the sequence of cyanogen bromide fragments derived from the isolated gamma subunit corresponds to that expected for a protein produced by the narI ORF. A protein corresponding to the narJ ORF did not appear to be associated with the purified nitrate reductase complex or with the complex immunoprecipitated from Triton X-100-solubilized membrane preparations. We conclude that narI encodes the gamma subunit (cytochrome bNR) and that while the product of the narJ gene is required for assembly of fully active membrane-bound enzyme it is not tightly associated with the active enzyme.     

Cloning and expression of the genes of two fumarate reductase subunits from Wolinella succinogenes

The fumarate reductase complex of the anaerobic bacterium Wolinella succinogenes catalyzes the electron transfer from menaquinol to fumarate. Two structural genes coding for subunits of the enzyme have been cloned in Escherichia coli. The genes were isolated from a lambda EMBL3 phage gene bank by immunological screening and subcloned in an expression vector. The genes frdA and frdB, which encode the FAD protein (Frd A, Mr 79,000) and the iron-sulfur protein (Frd B, Mr 31,000) of the fumarate reductase complex, were cloned together with a W. succinogenes promoter. The gene order was promoter-frdA-frdB. The FAD protein and the iron-sulfur protein were expressed in the correct molar mass in E. coli from the clones. The identity of the frdA gene and the suggested polarity were confirmed by comparing the amino-terminal sequence of the Frd A protein with that predicted from the 5'-terminal nucleotide sequence of frdA. The frdA and frdB genes are present only once in the genome. A region downstream of frdB, possibly a gene encoding cytochrome b of the fumarate reductase complex, hybridizes with a second site in the genome.     

A three-component dicamba O-demethylase from Pseudomonas maltophilia, strain DI-6: gene isolation, characterization, and heterologous expression

Dicamba O-demethylase is a multicomponent enzyme from Pseudomonas maltophilia, strain DI-6, that catalyzes the conversion of the widely used herbicide dicamba (2-methoxy-3,6-dichlorobenzoic acid) to DCSA (3,6-dichlorosalicylic acid). We recently described the biochemical characteristics of the three components of this enzyme (i.e. reductase(DIC), ferredoxin(DIC), and oxygenase(DIC)) and classified the oxygenase component of dicamba O-demethylase as a member of the Rieske non-heme iron family of oxygenases. In the current study, we used N-terminal and internal amino acid sequence information from the purified proteins to clone the genes that encode dicamba O-demethylase. Two reductase genes (ddmA1 and ddmA2) with predicted amino acid sequences of 408 and 409 residues were identified. The open reading frames encode 43.7- and 43.9-kDa proteins that are 99.3% identical to each other and homologous to members of the FAD-dependent pyridine nucleotide reductase family. The ferredoxin coding sequence (ddmB) specifies an 11.4-kDa protein composed of 105 residues with similarity to the adrenodoxin family of [2Fe-2S] bacterial ferredoxins. The oxygenase gene (ddmC) encodes a 37.3-kDa protein composed of 339 amino acids that is homologous to members of the Phthalate family of Rieske non-heme iron oxygenases that function as monooxygenases. Southern analysis localized the oxygenase gene to a megaplasmid in cells of P. maltophilia. Mixtures of the three highly purified recombinant dicamba O-demethylase components overexpressed in Escherichia coli converted dicamba to DCSA with an efficiency similar to that of the native enzyme, suggesting that all of the components required for optimal enzymatic activity have been identified. Computer modeling suggests that oxygenase(DIC) has strong similarities with the core alphasubunits of naphthalene 1,2-dioxygenase. Nonetheless, the present studies point to dicamba O-demethylase as an enzyme system with its own unique combination of characteristics.     

Wolinella succinogenes fumarate reductase contains a dihaem cytochrome b

The fumarate reductase operon of Wolinella succinogenes is made up of three structural genes (frd-CAB). The frdC gene was located next to the promoter region and identified as the cytochrome b structural gene encoding 256 amino acid residues. The N-terminal amino acid sequences of seven fragments derived from the cytochrome b moiety of the enzyme all mapped within the frdC gene. This suggested that the enzyme contained only one species of cytochrome b. Re-evaluation of earlier measurements of subunit composition, haem B content and molecular weight led to the conclusion that the enzyme contained one molecule of cytochrome b with two haem B groups. The hydropathy plot of the amino acid sequence predicted five membrane-spanning hydrophobic segments, the first four of which contained a single histidine residue each. These residues could form the axial ligands to the two haem B groups. FrdC was found to be homologous with the cytochrome b (SdhC) of the Bacillus subtilis succinate dehydrogenase, but not with the hydrophobic subunits of the fumarate reductase or succinate dehydrogenase of Escherichia coli.     

Structure of an ATPase operon of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius

The nucleotide sequence of the operon of the ATPase complex of an acidothermophilic archaebacterium, Sulfolobus acidocaldarius, has been determined. In addition to the three previously reported genes for the alpha, beta, and c (proteolipid) subunits of the ATPase complex (Denda, K., Konishi, J., Oshima, T., Date, T., and Yoshida, M. (1989) J. Biol. Chem. 264, 7119-7121), the operon contained three other genes encoding hydrophilic proteins with molecular masses 25, 13, and 7 kDa. The 25-kDa protein is the third largest subunit (gamma), the 13-kDa protein is most likely the fourth subunit (delta), and the 7-kDa protein may correspond to an unknown subunit of the ATPase, tentatively named as epsilon subunit. They do not have significant sequence similarity to subunits in F0F1-ATPases and eukaryotic V-type ATPases, whereas the other three subunits, alpha, beta, and c, have homologous counterparts in F0F1- and V-type ATPases. The order of the genes in the operon was delta alpha beta gamma epsilon c. The S. acidocaldarius ATPase operon differed from the eucabacterial F0F1-ATPase operon in that the former contains only one gene for a hydrophobic subunit at the most downstream part of the operon whereas the latter has three hydrophobic F0 genes preceding five hydrophilic F1 genes.     

Two distinct heterodisulfide reductase-like enzymes in the sulfate-reducing archaeon Archaeoglobus profundus.

Heterodisulfide reductase (Hdr) is a unique disulfide reductase that plays a key role in the energy metabolism of methanogenic archaea. Two types of Hdr have been identified and characterized from distantly related methanogens. Here we show that the sulfate-reducing archaeon Archaeoglobus profundus cultivated on H2/sulfate forms enzymes related to both types of Hdr. From the membrane fraction of A. profundus, a two-subunit enzyme (HmeCD) composed of a b-type cytochrome and a hydrophilic iron-sulfur protein was isolated. The amino-terminal sequences of these subunits revealed high sequence identities to subunits HmeC and HmeD of the Hme complex from A. fulgidus. HmeC and HmeD in turn are closely related to subunits HdrE and HdrD of Hdr from Methanosarcina spp. From the soluble fraction of A. profundus a six-subunit enzyme complex (Mvh:Hdl) containing Ni, iron-sulfur clusters and FAD was isolated. Via amino-terminal sequencing, the encoding genes were identified in the genome of the closely related species A. fulgidus in which these genes are clustered. They encode a three-subunit [NiFe] hydrogenase with high sequence identity to the F420-nonreducing hydrogenase from Methanothermobacter spp. while the remaining three polypeptides are related to the three-subunit heterodisulfide reductase from Methanothermobacter spp. The oxidized enzyme exhibited an unusual EPR spectrum with gxyz = 2.014, 1.939 and 1.895 similar to that observed for oxidized Hme and Hdr. Upon reduction with H2 this signal was no longer detectable.