Using the previously established two-plasmid system for the identification of promoters recognized by a particular sigma factor, we identified two positive DNA fragments that were active only after induced sigG, encoding sigma factor sigmaG of Streptomyces coelicolor A3(2). High-resolution S1-nuclease mapping in the Escherichia coli two-plasmid system identified potential promoters, PG45 and PG54, whose sequences were similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor sigmaB. However, both putative sigmaG-dependent promoters were not active in S. coelicolor. Sequence analysis of the regions potentially governed by the promoters revealed a gene encoding a hypothetical protein SCO5555 and the rrnE gene encoding rRNA operon. To confirm that sigG encodes sigma factor, the sigmaG protein was overproduced in E. coli and purified. In an in vitro transcription assay, sigmaG, after complementation with S. coelicolor core RNA polymerase, was able to recognize both sigmaG-dependent promoters and initiate transcription.

• MeSH
• bakteriální geny MeSH
• bakteriální proteiny chemie   genetika   izolace a purifikace   metabolismus   MeSH
• DNA footprinting MeSH
• Escherichia coli genetika   MeSH
• genetická transkripce * MeSH
• geny rRNA MeSH
• klonování DNA MeSH
• molekulární sekvence - údaje MeSH
• plazmidy MeSH
• promotorové oblasti (genetika) MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie nukleových kyselin MeSH
• sekvenční seřazení MeSH
• sigma faktor chemie   genetika   izolace a purifikace   metabolismus   MeSH
• Streptomyces coelicolor fyziologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• sigma faktor MeSH

The gene corresponding to the recently identified whiB-paralogous gene wblE in S. coelicolor was found after sequencing the downstream region of the stress-response sporulation-specific sigma-factor gene, sigH, in S. coelicolor A3(2). Sequence analysis has revealed an ORF exhibiting high similarity to sporulation transcription factors WhiB and WhiD. A stable null mutant of the wblE gene was obtained by integrative transformation, via double cross-over. Disruption of the S. coelicolor wblE gene appeared to have no obvious effect on growth, morphology, differentiation, and production of the pigmented antibiotics actinorhodin and undecylprodigiosin. Expression of the wblE gene was investigated during differentiation by S1 nuclease mapping, using RNA prepared from S. coelicolor A3(2) and its isogenic sigF and sigH mutants grown to various developmental stages. A single promoter was identified upstream of the wblE coding region. The wblEp promoter was induced at the beginning of aerial mycelium formation and its activity decreased later in differentiation. No differences in expression of the wblEp promoter were detected in S. coelicolor A3(2) mutants in sigF and sigH genes for sporulation-specific sigma factors. Sequence of the wblEp promoter showed partial similarity to the consensus sequence of the extracytoplasmic sigma factors.

• MeSH
• anthrachinony metabolismus   MeSH
• bakteriální geny * MeSH
• bakteriální RNA izolace a purifikace   MeSH
• DNA bakterií genetika   izolace a purifikace   MeSH
• inzerční mutageneze MeSH
• molekulární sekvence - údaje MeSH
• prodigiosin analogy a deriváty   metabolismus   MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u bakterií MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční homologie MeSH
• spory bakteriální * MeSH
• Streptomyces cytologie   genetika   růst a vývoj   metabolismus   MeSH
• transkripční faktory genetika   fyziologie   MeSH
• vývojová regulace genové exprese MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• actinorhodin MeSH Prohlížeč
• anthrachinony MeSH
• bakteriální RNA MeSH
• DNA bakterií MeSH
• prodigiosin MeSH
• transkripční faktory MeSH
• undecylprodigiosin MeSH Prohlížeč

A gapR gene, encoding a protein similar to the AraC/XylS family of bacterial transcriptional regulators, was previously identified upstream of the gap gene, coding for glyceraldehyde-3-phosphate dehydrogenase in Streptomyces aureofaciens. The GapR protein overproduced in Escherichia coli was shown to bind to the gap-P promoter region. Using the gel mobility shift assay with cell-free protein extracts from different developmental stages of S. aureofaciens, we identified several other proteins, in addition to GapR, that specifically bound to the S. aureofaciens gap-P promoter region. When cell-free extracts from S. aureofaciens cultivated in liquid medium with glucose were analyzed, only one complex corresponding to GapR was detected. A new protein interacting with the gap-P promoter was detected in stationary culture of S. aureofaciens grown in the presence of mannitol as carbon sources. The GapR protein was partially purified from S. aureofaciens cultivated in liquid medium containing glucose and used for binding studies. DNA footprinting analysis revealed an identical protected region as previously identified for the GapR protein overproduced from Escherichia coli. The direct role of the GapR protein in the regulation of gap expression in S. aureofaciens in vivo was confirmed but regulation of gap expression seems to be more complex, possibly involving other regulatory protein(s), depending on the developmental stage of S. aureofaciens.

• MeSH
• bakteriální geny * MeSH
• bakteriální proteiny genetika   metabolismus   MeSH
• DNA bakterií genetika   MeSH
• DNA vazebné proteiny genetika   metabolismus   MeSH
• Escherichia coli genetika   MeSH
• glyceraldehyd-3-fosfátdehydrogenasy genetika   MeSH
• molekulární sekvence - údaje MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese enzymů MeSH
• regulace genové exprese u bakterií MeSH
• rekombinantní proteiny genetika   metabolismus   MeSH
• restrikční mapování MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• Streptomyces aureofaciens genetika   růst a vývoj   metabolismus   MeSH
• trans-aktivátory genetika   metabolismus   MeSH
• vazebná místa genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA bakterií MeSH
• DNA vazebné proteiny MeSH
• GapR protein, Streptomyces aureofaciens MeSH Prohlížeč
• glyceraldehyd-3-fosfátdehydrogenasy MeSH
• rekombinantní proteiny MeSH
• trans-aktivátory MeSH

Using the gel mobility-shift assay with protein fractions from different developmental stages of solid-grown Streptomyces aureofaciens, we identified two different proteins specifically bound to the whiH promoter region. Only one protein (RwhA) was detected in young substrate mycelium cultivated in liquid medium. On comparing the mobility of the resulting complexes, one of the bound proteins present in substrate mycelium and in early stages of aerial mycelium seemed to be identical with the RwhA. The other detected protein with a higher mobility (RwhB) was present in all developmental stages except for mature spores. DNA footprinting analysis localized the binding site of RwhB to nucleotides -23 to +40 relative to the transcription start point of the PwhiH promoter. RwhA from young substrate mycelium protected the DNA fragment from -106 to -77 in coding strand and -126 to -82 in noncoding strand. WhiH has homology to a large family of metabolism-related repressors and seems to regulate negatively its own expression. These observations (and the results of transcription analysis of the whiH gene obtained earlier) suggest that two different proteins influence the expression of whiH gene in S. aureofaciens. The putative repressor-like RwhA protein protects expression of whiH in substrate mycelium either in liquid medium or during differentiation. The other detected protein, RwhB, which binds to the whiH promoter region during differentiation, may represent two forms of WhiH, one with a repressor role at the beginning of differentiation and second with the role of activator at the time of sporulation.

• MeSH
• bakteriální proteiny analýza   metabolismus   MeSH
• DNA footprinting MeSH
• DNA vazebné proteiny analýza   metabolismus   MeSH
• molekulární sekvence - údaje MeSH
• promotorové oblasti (genetika) MeSH
• regulace genové exprese u bakterií * MeSH
• represorové proteiny genetika   MeSH
• retardační test MeSH
• sekvence nukleotidů MeSH
• Streptomyces chemie   genetika   růst a vývoj   MeSH
• transkripční faktory genetika   MeSH
• vazebná místa MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• DNA vazebné proteiny MeSH
• represorové proteiny MeSH
• transkripční faktory MeSH
• whiH protein, Streptomyces MeSH Prohlížeč

Using the method for the identification of promoters recognized by the sporulation specific sigma factor (sigma F), we identified a positive 950 bp Sau3AI DNA fragment in Streptomyces coelicolor A3(2). High-resolution S1-nuclease mapping identified a potential promoter, PF35, in the E. coli two-plasmid system similar to the consensus sequence of Bacillus subtilis promoters recognized by the general stress-response sigma factor (sigma B). However, the putative sigF-dependent promoter, PF35, was inactive in S. coelicolor in the course of differentiation, and it was located divergently in the promoter region directing expression of the chiC gene encoding chitinase. Sequence analysis of the region potentially governed by PF35 revealed two translationally coupled genes encoding proteins similar to bacterial two-component regulatory systems, and with the highest similarity to the two-component system chiS, chiR, regulating chitinase activity in Streptomyces thermoviolaceus. However, the genes had a divergent orientation with respect to the PF35 promoter. Disruption of the S. coelicolor chiR gene appeared to have no obvious effect on growth, morphology, differentiation, and production of pigmented antibiotic actinorhodin and undecylprodigiosin. Moreover, the chiR disruption did not affect the overall chitinase activity.

• MeSH
• bakteriální proteiny chemie   genetika   metabolismus   MeSH
• chitinasy genetika   metabolismus   MeSH
• delece genu MeSH
• klonování DNA * MeSH
• molekulární sekvence - údaje MeSH
• operon MeSH
• promotorové oblasti (genetika) MeSH
• proteinkinasy * MeSH
• regulace genové exprese u bakterií genetika   MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sigma faktor MeSH
• Streptomyces enzymologie   genetika   růst a vývoj   MeSH
• transkripční faktory * MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• ChiR protein, Streptomyces MeSH Prohlížeč
• ChiS protein, Streptomyces thermoviolaceus MeSH Prohlížeč
• chitinase C-1 MeSH Prohlížeč
• chitinasy MeSH
• FliA protein, Bacteria MeSH Prohlížeč
• proteinkinasy * MeSH
• sigma faktor MeSH
• transkripční faktory * MeSH

A Streptomyces aureofaciens gene, gap, encoding glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was previously identified. Hybridization studies suggested the presence of a second gap gene in S. aureofaciens. To clone the gene, S. aureofaciens subgenomic library was screened with an oligonucleotide probe encoding a peptide motif conserved in all GAPDH. 3352 bp positive BamHI fragment was identified, the length of which correlated with the hybridization signal. The nucleotide sequence of the fragment was determined, and analysis of the sequence revealed the presence of three open reading frames (ORF). However, none of the genes coded for GAPDH. All three genes formed an operon, consisting of gene orf251, with a high homology to a conserved gene present only in archaeabacteria, and the aldA and adhA genes homologous to various eukaryotic and prokaryotic aldehyde- and alcohol-dehydrogenases, with maximum homology to the phenylacetaldehyde dehydrogenases and arylalcohol dehydrogenases, respectively.

• MeSH
• aldehyddehydrogenasa chemie   genetika   metabolismus   MeSH
• klonování DNA * MeSH
• molekulární sekvence - údaje MeSH
• sekvence aminokyselin MeSH
• sekvence nukleotidů MeSH
• sekvenční analýza DNA MeSH
• sekvenční seřazení MeSH
• Streptomyces aureofaciens enzymologie   genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• aldehyddehydrogenasa MeSH