Amplification of the housekeeping sigma factor in Pseudomonas fluorescens CHA0 enhances antibiotic production and improves biocontrol abilities.

Pseudomonas fluorescens CHA0 produces a variety of secondary metabolites, in particular the antibiotics pyoluteorin and 2,4-diacetylphloroglucinol, and protects various plants from diseases caused by soilborne pathogenic fungi. The rpoD gene encoding the housekeeping sigma factor sigma 70 of P. fluorescens was sequenced. The deduced RpoD protein showed 83% identity with RpoD of Pseudomonas aeruginosa and 67% identity with RpoD of Escherichia coli. Attempts to inactivate the single chromosomal rpoD gene of strain CHA0 were unsuccessful, indicating an essential role of this gene. When rpoD was carried by an IncP vector in strain CHA0, the production of both antibiotics was increased severalfold and, in parallel, protection of cucumber against disease caused by Pythium ultimum was improved, in comparison with strain CHA0.     

Principal sigma subunit of the Caulobacter crescentus RNA polymerase.

We have identified the gene encoding the Caulobacter crescentus principal sigma subunit, RpoD. The rpoD gene codes for a polypeptide of 653 amino acids with a predicted molecular mass of 72,623 Da (sigma 73). The C. crescentus sigma subunit has extensive amino acid sequence homology with the principal sigma factors of a number of divergent procaryotes. In particular, the segments designated region 2 that are involved in core polymerase binding and promoter recognition were identical among these bacteria despite the fact that the -10 region recognized by the C. crescentus sigma 73 differs significantly from that of the other bacteria. Thus, it appears that additional sigma factor regions must be involved in -10 region recognition. This conclusion was strengthened by a heterologous complementation assay in which C. crescentus sigma 73 was capable of complementing the Escherichia coli rpoD285 temperature-sensitive mutant. Furthermore, C. crescentus sigma 73 conferred new specificity on the E. coli RNA polymerase, allowing the expression of C. crescentus promoters in E. coli. Thus, the C. crescentus sigma 73 appears to have a broader specificity than does the sigma 70 of the enteric bacteria.     

荧光假单胞菌M18 rpoD克隆及其对抗生素合成的影响

荧光假单胞菌M18对多种植物病原真菌具有显著的抑制作用。荧光假单胞菌(Pseuclomones fluo-rescens)M18能同时合成吩嗪-1-羧酸(PCA)和藤黄绿菌素(P1t)两种抗生素。从M18的基因组中克隆了rpoD基因,其相应的氨基酸序列与荧光假单胞菌CHAO中RpoD蛋白的氨基酸序列完全相同。利用基因重组技术和大肠杆菌-荧光假单胞菌穿梭质粒,pME6032,将rpoD置于强启动子Ptac的控制下,导入M18菌株。发现经重组质粒转化的M18,与对照相比,培养基中PCA和Plt开始累积的时间分别提前4h和8h,积累量提高1倍和6倍.     

Isolation and characterization of the gene coding for the major sigma factor of Rickettsia prowazekii DNA-dependent RNA polymerase.

The gene coding for the major sigma factor of Rickettsia prowazekii, an obligate intracellular parasitic bacterium, has been isolated utilizing an oligodeoxyribonucleotide as a probe to a conserved region of major sigma factors. Nucleotide sequence analysis revealed an open reading frame of 1905 bp that could encode a protein of 635 amino acids (aa) with a calculated molecular size of 73 kDa (sigma 73). R. prowazekii sigma 73 displayed extensive homology with major sigma factors from a variety of eubacteria. Comparison of the major sigma factors from Escherichia coli and R. prowazekii revealed 44.9% aa identity. R. prowazekii sigma 73 produced in E. coli minicells migrated as a 85-kDa protein when analyzed by sodium dodecyl sulfate-polyacrylamide-gel electrophoresis. This anomalous migration is characteristic of eubacterial major sigma factors and agrees with the migration noted for the purified rickettsial sigma protein. Despite a similarity to the E. coli sigma 70 encoded by rpoD, R. prowazekii sigma 73 did not complement E. coli rpoD temperature-sensitive mutants.     

Isolation of the rpoD gene encoding the principal sigma factor of the deep-sea piezophilic bacterium Shewanella violacea strain DSS12 and its overexpression in Escherichia coli

The gene encoding the principal a factor (rpoD) of the piezophilic bacterium Shewanella violacea was cloned and sequenced. The rpoD gene was found to encode a polypeptide consisting of 614 amino acid residues, showing 75.6 and 64.3% identity to those of Escherichia coli and Pseudomonas putida, respectively. Comparison with E. coli sigma70 and P. putida sigma70 showed that significant similarity exists in four conserved regions known to be required for promoter recognition and core binding. Using an expression plasmid harboring the rpoD gene, the S. violacea sigma70 factor was overexpressed in E. coli and successfully purified to near homogeneity.     

Direct evidence for active segregation of oriC regions of the Bacillus subtilis chromosome and co-localization with the Spo0J partitioning protein

We have cloned the rpoD gene coding for the major sigma factor of Bordetella pertussis . The deduced amino acid sequence reveals a protein of 733 residues which has extensive amino acid homology with the principal σ factors of a number of divergent prokaryotes. It is larger than most σ factors identified to date, having a molecular mass of 81.3 kDa. We have designated this factor sigma 80. In a heterologous complementation assay, B. pertussis rpoD was able to complement the Escherichia coli rpoD temperature-sensitive mutant UQ285. Furthermore, B. pertussis rpoD conferred better specificity to the E. coli RNA polymerase, allowing increased expression of the B. pertussis virulence-associated fha promoter, but could not activate the ptx and cya promoters in the E. coli UQ285 strains carrying the B. pertussis bvg locus. We discuss the implications of these results on the mechanisms involved in the activation of virulence-associated promoters.     

Evolutionary coorigin of the RNA polymerase sigma factor and the lambda repressor inferred from gene nucleotide sequence homologies

The lambda phage cI gene and E. coli rpoD gene encoding the lambda repressor and sigma factor, respectively, were aligned with each other based on the internal homologies found in the rpoD gene. Statistical evaluations for these intragenic and intergenic base sequence homologies in the corresponding alignments have conclusively demonstrated that the rpoD gene must have evolved by repeated gene duplications from a primitive gene closely similar to and co-ancestral to the cI gene.     

Cloning and DNA sequence of the gene coding for the major sigma factor from Myxococcus xanthus.

The gene for a sigma factor (rpoD) was cloned from Myxococcus xanthus, a soil bacterium which differentiates to form fruiting bodies upon starvation for nutrients. The DNA sequence of the gene was determined, and an open reading frame encoding a polypeptide of 708 amino acid residues (Mr = 80,391) was identified. Except for the amino-terminal sequence consisting of 100 residues, the M. xanthus sigma factor (sigma-80) showed extensive similarity with Escherichia coli sigma-70 as well as Bacillus subtilis sigma-43. In particular, the carboxy-terminal sequence of 242 residues that is known to be required for promoter recognition and core recognition showed 78 and 72% amino acid sequence identity with the E. coli and B. subtilis sigma factors, respectively. The putative RpoD protein was detected at the position of an apparent molecular weight of 86,000 by Western blot (immunoblot) analysis by using antiserum against B. subtilis sigma-43, which agreed well with the position of a vegetative sigma factor of M. xanthus previously identified by Rudd and Zusman (K. Rudd and D. R. Zusman, J. Bacteriol. 151:89-105, 1982).     

The rpoD1 gene of Synechococcus sp. strain PCC 7942 encodes the principal sigma factor of RNA polymerase

RNA polymerase was purified from the unicellular cyanobacterium, Synechococcus sp. strain PCC 7942, and found to be associated with a 52 kilodalton (kDa) polypeptide. The determined N-terminal sequence of the polypeptide was identical to the predicted amino-acid sequence of the rpoD1 gene product. Furthermore, the rpoD1 gene is suggested to be indispensable for viability by the inability to disrupt the gene. These results indicate that the rpoD1 gene product is the principal sigma factor of RNA polymerase.     

Cloning and analysis of the gene (rpoDA) for the principal sigma factor of Pseudomonas aeruginosa

A gene (rpoDA) of Pseudomonas aeruginosa whose gene product has a homologous function and structure with the principal sigma factor of Escherichia coli was cloned and sequenced. The DNA region corresponding to one of the two hybridization signals found in P. aeruginosa DNA with a synthetic oligonucleotide probe (rpoD probe) was shown to be able to complement a temperature sensitive mutation of Escherichia coli rpoD gene. The amino acid sequence deduced from the nucleotide sequence of rpoDA showed an extensive homology with that of the principal sigma factor of E. coli throughout the entire region, which indicates that the two gene products have an essentially identical domain structure. A common basic structure observed among principal sigma factors of different eubacterial strains was proposed. RpoDA protein was identified in the extract of the cell carrying a plasmid clone with the rpoDA gene insert by Western blot analysis.     

Identification of multiple RNA polymerase sigma factor homologs in the cyanobacterium Anabaena sp. strain PCC 7120: cloning, expression, and inactivation of the sigB and sigC genes.

The sigA gene of Anabaena sp. strain PCC 7120, encoding the principal RNA polymerase sigma factor, and the complement of the rpoD oligonucleotide (K. Tanaka, T. Shiina, and H. Takahashi, Science 242:1040-1042, 1988) were used as probes to isolate two genes, sigB and sigC, which encode two putative sigma factors exhibiting high degrees of similarity to SigA, to HrdA, -B, -C, and -D of Streptomyces coelicolor, and to KatF of Escherichia coli. sigB and sigC code for polypeptides of 332 and 416 amino acids with predicted molecular weights of 38,431 and 47,459, respectively. sigB and sigC mRNAs are detectable only under nitrogen-limiting conditions. Insertional inactivation of sigB and sigC indicates that neither gene alone is essential for nitrogen fixation or heterocyst differentiation.     

Nucleotide sequence and organization of Bacillus subtilis RNA polymerase major sigma (sigma 43) operon.

The gene coding for Bacillus subtilis RNA polymerase major sigma 43, rpoD, was cloned together with its neighboring genes in a 7 kb EcoRI fragment. The complete nucleotide sequence of a 5 kb fragment including the entire rpoD gene revealed the presence of two other genes preceding rpoD in the order P23-dnaE-rpoD. The dnaE codes for DNA primase while the function of P23 remains unknown. The three genes reside in an operon that is similar in organization to the E. coli RNA polymerase major sigma 70 operon, which is composed of genes encoding small ribosome protein S21 (rpsU), DNA primase (dnaG), and RNA polymerase sigma 70 (rpoD). There is a relatively high degree of base and amino acid homology between the DNA primase and sigma genes. The most significant differences between the two operons are observed in the molecular size of the first genes (P23 and rpsU), the complete lack of amino acid homology between P23 and S21, the molecular weights of the two rpoD genes, the size of the intercistronic region between the first two genes, and the regulatory elements of the operon.