• Journal of Microbiology
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• v.45 no.6
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• pp.534-540
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• 2007

The Streptomyces coelicolor A3(2) genome contains six operons (rrnA to F) for ribosomal RNA synthesis. Transcription from rrnD occurs from four promoters (p1 to p4). We found that transcripts from the p1 and p3 promoters were most abundant in vivo in the early exponential phase. However, at later phases of exponential and stationary growth, transcripts from the p1 promoter decreased drastically, with the p3 and p4 transcripts constituting the major forms. Partially purified RNA polymerase supported transcription from the p3 and p4 promoters, whereas pure reconstituted RNA polymerase with core enzyme (E) and the major vegetative sigma factor ${\sigma}^{HrdB}$ ($E{\cdot}{\sigma}^{HrdB}$) did not. In order to assess any potential requirement for additional factor(s) that allow transcription from the p3 and p4 promoters, we fractionated a partially purified RNA polymerase preparation by denaturing gel filtration chromatography. We found that transcription from the p3 and p4 promoters required factor(s) of about 30-35 kDa in addition to RNAP holoenzyme ($E{\cdot}{\sigma}^{HrdB}$). Therefore, transcription from the p3 and p4 promoters, which contain a consensus -10 region but no -35 for ${\sigma}^{HrdB}$ recognition, are likely to be regulated by transcription factor(s) that modulate RNA polymerase holoenzyme activity in S. coelicolor.

• Journal of Microbiology and Biotechnology
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• v.8 no.3
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• pp.280-283
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• 1998

We have cloned the RNA polymerase sigma-like factors from a wide range of actinomycetes by using specific primers with the polymerase chain reaction (PCR). The specific oligonucleotide primers were designed on the basis of amino acid sequences of conserved regions from HrdA, B, D of Streptomyces griseus as well as from the rpoD box of many eubacteria. The consensus sequences were from the rpoD box and helix-turn-helix motif involved in -35 recognition. The designed primers were successfully applied to amplify the DNA fragments of the hrd homolog genes from 8 different strains of actinomycetes which produce a wide variety of important antibiotics. The 480 bp of the DNA fragment was amplified from all 8 strains, and it was identified as a part of hrdA and hrdB as we designed. The deduced amino acid sequence of PCR-amplified DNA fragments were highly homologous to those of other known RNA polymerase sigma factors of S. griseus and Streptomyces aureofaciens. Therefore, this study with specifically designed primers will support rapid cloning of the RNA polymerase sigma factors which recognize different classes of promoters from actinomycetes, and it will also be helpful in understanding the relationship of promoters and sigma factors leading to heterogeneity of RNA polymerases in actinomycetes.

• Journal of Microbiology
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• v.42 no.2
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• pp.103-110
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• 2004

In order to understand the molecular basis of selective expression of stationary-phase genes by RNA polymerase containing$\sigma$$\^$38/ (E$\sigma$$\^$38/) in Escherichia coli, we examined transcription from the stationary-phase promoters, katEP, bo1AP, hdeABP, csgBAP, and mcbP, in vivo and in vitro. Although these pro-moters are preferentially recognized in vivo by E$\sigma$$\^$38/, they are transcribed in vitro by both E$\sigma$$\^$38/ and E$\sigma$$\^$70/ containing the major exponential $\sigma$, $\sigma$$\^$70/. In the presence of high concentrations of glutamate salts, how-ever, oldy E$\sigma$$\^$38/ was able to efficiently transcribe from these promoters, which supports the concept that the promoter selectivity of $\sigma$$\^$38/-containing RNA polymerase is observed only under specific reaction con-ditions. The examination of 6S RNA, which is encoded by the ssr1 gene in vivo, showed that it reduced E$\sigma$$\^$70/ activity during the stationary phase, but this reduction of activity did not result in the elevation of E$\sigma$$\^$38/ activity. Thus, the preferential expression of stationary-phase genes by E$\sigma$$\^$38/ is unlikely the con-sequence of selective inhibition of E$\sigma$$\^$70/ by 6S RNA.

• BMB Reports
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• v.43 no.3
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• pp.176-181
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• 2010

The primary sigma factor ($\sigma^{A}$) of Staphylococcus aureus, a potential drug target, was little investigated at the structural level. Using an N-terminal histidine-tagged $\sigma^{A}$ (His-$\sigma^{A}$), here we have demonstrated that it exits as a monomer in solution, possesses multiple domains, harbors primarily $\alpha$-helix and efficiently binds to a S. aureus promoter DNA in the presence of core RNA polymerase. While both N- and C-terminal ends of His-$\sigma^{A}$ are flexible in nature, two Trp residues in its DNA binding region are buried. Upon increasing the incubation temperature from 25$^{\circ}$ to 40$^{\circ}C$, $\sim$60% of the input His-$\sigma^{A}$ was cleaved by thermolysin. Aggregation of His-$\sigma^{A}$ was also initiated rapidly at 45$^{\circ}C$. From the equilibrium unfolding experiment, the Gibbs free energy of stabilization of His-$\sigma^{A}$ was estimated to be +0.70 kcal $mol^{-1}$. The data together suggest that primary sigma factor of S. aureus is an unstable protein. Core RNA polymerase however stabilized $\sigma^{A}$ appreciably.

• BMB Reports
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• v.31 no.6
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• pp.607-610
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• 1998

Transcription of mitochondrial DNA in the yeast S. cerevisiae depends on recognition of a consensus nonanucleotide promoter sequence by mitochondrial RNA polymerase specificity factor, which is a 43 kDa polypeptide encoded by the nuclear MTF1 gene. Mtf1p has only limited amino acid sequence homology to bacterial sigma factors, but functions in many ways like sigma in that it is required for promoter recognition and initiation of transcription. To analyze the corebinding region of Mtf1p, monoclonal antibodies to this protein were prepared. Recombinant Mtf1p overproduced in E. coli was purified to near homogeneity and used to raise monoclonal antibodies (mAbs). From fused cells screened for Mtf1p mAbs by immunodot blot analysis, 19 positive clones were initially isolated. Further analysis of positive clones by Western blotting resulted in 4 mAbs of Mtf1p.

• Korean Journal of Microbiology
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• v.32 no.4
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• pp.264-270
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• 1994

A gene coding for a novel putative $\sigma$ factor of RNA polymerase has been identified from Streptomyces coelicolor A3(2) using Escherichia coli rpoS gene fragment as a probe. The 486 bp rpoS gene fragment was amplified from E. coli genomic DNA by PCR with two synthetic oligonucleotides, the sequences of which were deduced from the amino acid sequences in the regions 2.3 and 4.2 conserved among various bacterial factors. When E. coli genomic DNA fragments were hybridized with cloned rpoS probe, only one band corresponding to rpoS gene (3.2 kb PvuII fragment or 2.3 kb KpnI fragment) was detected. In S. coelicolor, however, two bands were detected both in PvuII digested DNA and SalI digested DNA. 3.5 kb PvuII fragment which binds the rpoS gene probe was cloned (pMS1) from the sublibrary, and the nucleotide sequences of 1.0 kb BamH'/HincII subclone (pBH2) was partially determined. The nucleotide sequences revealed extensive similarity to other $\sigma$ factor genes of S. coelicolor (hrdA, hrdB, hrdC, hrdD), S. aureofaciens (hrdA, hrdB, hrdC, hrdD), Synechococcus species, Pseudomonas aeruginosa, Stigmatella aurantiaca, and Anabaena species. The nucleotide sequences in regions 1.2 and 4 were compared with the corresponding regions of 5 known ${\sigma}$ factor genes of S. coelicolor by multiple alignment. It turned out that the cloned gene is most closely related to hrdA showing 88% amino acid similarity in region 1.2 and 75% in region 4.

• The Microorganisms and Industry
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• v.14 no.1
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• pp.17-20
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• 1988

A genetic analysis of the complex Bacillus subtilis transcriptional apparatus is essential to understand the function, regulation, and interaction of the transcriptase components during growth and sporulation. This approach in Escherichia coli has uncovered fundamental mechanisms regulating gene expression Cole and Nomura, 1986; Lindahl and Zengel, 1986) and an analysis of the B. subtilis transcriptase will allow comoparison of the E.coli system to another bacterium that has evolved under different selective pressures. To this end we used antibody probes to isolate the alpha, beta, and beta' core subunit genes from a .lambda.gtill expression vector library. To address the question of function ans regulation of the minor sigma factors that confer promoter specifity on the polymerase core (Losick et al., 1986), we used the same approach to isolate the gene for the 37,000 dalton sigma factor, sigma-37.

• Journal of Life Science
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• v.30 no.1
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• pp.88-95
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• 2020

Using a random arbitrarily primed polymerase chain reaction, messenger RNA expression levels were assessed after exposure to 10% (v/v) toluene for 8 hr in solvent-tolerant Pseudomonas sp. BCNU 106. Among the 100 up-expressed products, 50 complementary DNA fragments were confirmed to express repeatedly; these were cloned and then sequenced. Blast analysis revealed that toluene stimulated an adaptive increase in the gene expression level in association with transcriptions such as LysR family of transcriptional regulators and RNA polymerase factor sigma-32. The expression of catalase and Mn2+/Fe2+ transporter genes functionally associated with inorganic ion transport and metabolism increased, and the increased expression of type IV pilus assembly PilZ and multi-sensor signal transduction histidine kinase genes, functionally categorized into signal transduction and mechanisms, was also demonstrated under toluene stress. The gene expression level of beta-hexosaminidase in association with carbohydrate transport and metabolism increased, and those of DNA polymerase III subunit epsilon, DNA-3-methyladenine glycosylase II, DEAD/DEAH box helicase domain-containing protein, and ABC transporter also increased after exposure to toluene in DNA replication, recombination, and repair, and even in defense mechanism. In particular, the RNAs corresponding to the ABC transporter, Mn2+/Fe2+ transporter, and the β-hexosaminidase gene were confirmed to be markedly induced in the presence of 10% toluene. Thus, defense mechanism, cellular ion homeostasis, and biofilm formation were shown as essential for toluene tolerance in Pseudomonas sp. BCNU 106.

• Microbiology and Biotechnology Letters
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• v.32 no.2
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• pp.190-194
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• 2004

The gene product of dctD (DctD) activates transcription from the dctA promoter regulatory region by the $\sigma^{54}$ -holoenzyme form ofRNA polymerase ($E\sigma^{54}$ ) in Rhizobium meliloti and R. leguminosarum. The Escherichia coli integration host factor (IHF) stimulated DctD-mediated activation from the dctA promoter regulatory region of R. leguminosarum but not R. meliloti. In the absence of UAS, IHF inhibited DctD-mediated activation from both of these promoter regulatory regions. IHF also inhibited activation from R. leguminosarum dctA by nitrogen regulatory protein C (NtrC), another activator of $E\sigma^{54}$ but not by one which lacks a specific binding site in this promoter regulatory region. IHF, however, stimulated NtrC-mediated activation from the R. meliloti dctA promoter. Upon removal of the UAS, IHF inhibited NtrC-mediated transcription activation from the R. meliloti dctA promoter regulatory region. These data suggest that IHF likely faciliates productive contacts between the activators NtrC or DctD and $E\sigma^{54}$ to stimulate activation from dctA promoter.

• Journal of fish pathology
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• v.30 no.2
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• pp.79-88
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• 2017

At the present, fish farms are suffering a lot of economic losses due to infectious diseases caused by various pathogens including aeromonad. Aeromonad is ubiquitous bacteria that causes infectious diseases. At least 26 species in the genus Aeromonas have been reported to cause fatal infections not only in salmonid fishes, but also in other freshwater and seawater fishes. Molecular techniques based on nucleic acid sequences of 16S rDNA and housekeeping genes can be used to identify the Aeromonas species. In this study, The genus Aeromonas was isolated from salmonid fishes of sixteen fish farms in Gangwon-Do, Korea and phylogenetically identified based on the sequences of 16S rDNA and housekeeping genes for Aeromonad, i.e. RNA polymerase sigma factor ${\sigma}^{70}$ (rpoD) or DNA gyrase subunit B (gyrB). Consequently, 96 strains were collected from Atlantic salmon (Salmo salar), coho salmon (Oncorhynchus kisutch), masou salmon (Oncorhynchus masou) and rainbow trout (Oncorhynchus mykiss), and 36 isolates were identified as the genus Aeromonas by 16S rDNA analysis. Thirty six Aeromonad isolates were further analysed based on rpoD or gyrB gene sequences and found Aeromonas salmonicida (24 isolates), A. sobria (10 isolates), A. media (1 isolates) and A. popoffii (1 isolates), indicating that A. salmonicida is a main infectious bacteria in Salmonid fishes in Gangwon-Do. It was also proved that the phylogenetic identification of Aeromonas species based on the sequences of housekeeping gene is more precise than the 16S rDNA sequence.