Your email was sent successfully. Check your inbox.

An error occurred while sending the email. Please try again.

Proceed reservation?

Export
  • 1
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The promoter region of the agarase gene (dagA) of Streptomyces coelicolor A3(2) is complex; it consists of four distinct promoters with different -10 and -35 regions. We report the isolation of a form of RNA polymerase that mediates transcription in vitro from the dag Ap4 promoter. The core components of this RNA polymerase are associated with a polypeptide of c. 66 kDa; holoenzyme reconstitution experiments show that the 66 kDa polypeptide functions as a sigma factor that directs transcription from the dag Ap4 and Bacillus subtilis veg promoters in vitro. Alignment of the DNA sequences of these two promoters shows that they have bases in common in the -10 and -35 regions and that these sequences are similar to those observed for the major RNA polymerases of other bacteria. N-terminal amino acid sequence analysis of the 66 kDa polypeptide revealed it to be the product of the hrdB gene. Previous experiments showed that the predicted amino acid sequence of the hrdB gene product is very similar to the major sigma factors of other bacteria and suggested that disruption of the hrdB gene is lethal. These observations together lead to the conclusion that we have isolated the major RNA polymerase of Streptomyces coelicolor A3(2).We have developed an improved protocol for the renaturation of sigma factors that have been Isolated by preparative sodium dodecyl sulphate/polyacrylamide gel electrophoresis (SDS-PAGE). This method involves renaturing the polypeptide in the presence of the bacterial chaperonin GroEL. We expect this protocol to find general application for renaturation of other polypeptides that have been subjected to SDS-PAGE.
    Type of Medium: Electronic Resource
    Signatur Availability
    BibTip Others were also interested in ...
  • 2
    ISSN: 1617-4623
    Keywords: Streptomyces ; Promoter-probes ; Transcription ; Gene expression ; fd terminator
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary Several versatile, multi-copy, promoter-probe plasmid vectors have been constructed that replicate in a wide range of Streptomyces species. Transcriptional activity is detected by the expression of a promoter-less aminoglycoside phosphotransferase gene (neo) derived from the transposon Tn5; expression of this gene confers kanamycin and neomycin resistance on Streptomyces lividans. An efficient transcriptional terminator from E. coli phage fd has been inserted upstream of the neo coding region to prevent significant transcriptional read-through from vector promoters. A translational stop codon situated downstream from the site(s) used for cloning and preceding and in frame with the ATG start codon of the neo gene ensures the detection of transcriptional, rather than translational, fusions. Relative promoter strengths can be determined by gradient plate assays of kanamycin resistance, by measuring the amount of aminoglycoside phosphotransferase produced or by estimating neo mRNA synthesised. The high copy number of the vectors facilitates the rapid isolation and characterisation of promoter-active fragments and convenient restriction sites are available for DNA sequencing and S1 mapping of cloned inserts. Some derivatives contain a poly-linker that facilitates the insertion, excision and analysis of cloned fragments and which enhances the use of these plasmids as general cloning vectors.
    Type of Medium: Electronic Resource
    Signatur Availability
    BibTip Others were also interested in ...
  • 3
    ISSN: 1617-4623
    Keywords: Streptomyces coelicolor A3(2) ; Glucose kinase ; Glucose repression ; Agarase
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Abstract The glucose kinase gene (glkA-ORF3) of Streptomyces coelicolor A3(2) plays an essential role in glucose utilisation and in glucose repression of a variety of genes involved in the utilisation of alternative carbon sources. These genes include dagA, which encodes an extracellular agarase that permits agar utilisation. Suppressor mutants of glkA-ORF3 deletion strains capable of utilising glucose (Glc+) arise at a frequency of about 10−5 on prolonged incubation. The Glc+ phenotype of the mutants is reversible (at a frequency of about 10−3) and reflects either the activation of a normally silent glucose kinase gene or the modification of an existing sugar kinase. Although the level of glucose kinase activity in the Glc+ supressor mutants is similar to that in the glkA + parental strain, glucose repression of dagA remains defective. Expression of the glucose kinase gene of Zymomonas mobilis in glkA-ORF3 mutants restored glucose utilisation, but not glucose repression of dagA. Over-expression of glkA-ORF3 on a high-copy-number plasmid failed to restore glucose repression of dagA in glkA-ORF3 mutants and led to loss of glucose repression of dagA in a glkA + strain. These results suggest that glucose phosphorylation itself is not sufficient for glucose repression and that glkA-ORF3 plays a specific regulatory role in triggering glucose repression in S. coelicolor A3(2).
    Type of Medium: Electronic Resource
    Signatur Availability
    BibTip Others were also interested in ...
  • 4
    ISSN: 1617-4623
    Keywords: Promoters ; S1 mapping ; In vitro transcription ; Protein secretion ; Signal peptide
    Source: Springer Online Journal Archives 1860-2000
    Topics: Biology
    Notes: Summary The DNA sequence of a 1.77 kb region of the Streptomyces coelicolor chromosome containing the coding and regulatory regions of the extracellular agarase (dagA) gene was determined. The sequence predicts a primary translation product of 309 amino acids and Mr 35132. Comparison of the N-terminal sequence determined for the mature extracellular protein with that of the primary translation product deduced from the DNA sequence predicts the presence of a 30 amino acid signal peptide. Analysis of the transcription of the dagA gene using high resolution S1 mapping, in vitro transcription, dinucleotide-primed in vitro transcription and in vivo promoter probing identified four promoters, initiating transcription approximately 32, 77, 125 and 220 nucleotides upstream of the coding sequence.
    Type of Medium: Electronic Resource
    Signatur Availability
    BibTip Others were also interested in ...
  • 5
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Vancomycin is the front-line therapy for treating problematic infections caused by methicillin-resistant Staphylococcus aureus (MRSA), and the spread of vancomycin resistance is an acute problem. Vancomycin blocks cross-linking between peptidoglycan intermediates by binding to the d-Ala-d-Ala termini of bacterial cell wall precursors, which are the substrate of transglycosylase/transpeptidase. We have characterized a cluster of seven genes (vanSRJKHAX) in Streptomyces coelicolor that confers inducible, high-level vancomycin resistance. vanHAX are orthologous to genes found in vancomycin-resistant enterococci that encode enzymes predicted to reprogramme peptidoglycan biosynthesis such that cell wall precursors terminate in d-Ala-d-Lac rather than d-Ala-d-Ala. vanR and vanS encode a two-component signal transduction system that mediates transcriptional induction of the seven van genes. vanJ and vanK are novel genes that have no counterpart in previously characterized vancomycin resistance clusters from pathogens. VanK is a member of the Fem family of enzymes that add the cross-bridge amino acids to the stem pentapeptide of cell wall precursors, and vanK is essential for vancomycin resistance. The van genes are organized into four transcription units, vanRS, vanJ, vanK and vanHAX, and these transcripts are induced by vancomycin in a vanR-dependent manner. To develop a sensitive bioassay for inducers of the vancomycin resistance system, the promoter of vanJ was fused to a reporter gene conferring resistance to kanamycin. All the inducers identified were glycopeptide antibiotics, but teicoplanin, a membrane-anchored glycopeptide, failed to act as an inducer. Analysis of mutants defective in the vanRS and cseBC cell envelope signal transduction systems revealed significant cross-talk between the two pathways.
    Type of Medium: Electronic Resource
    Signatur Availability
    BibTip Others were also interested in ...
  • 6
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: Whereas in Bacillus subtilis, a general stress response stimulon under the control of a single sigma factor (SigB) is induced by different physiological and environmental stresses (heat, salt or ethanol shock), in Streptomyces coelicolor, these environmental stresses induce independent sets of proteins, and its genome encodes nine SigB paralogues. To investigate possible functions of multiple sigB-like genes in S. coelicolor, Pctc, a promoter routinely used to assay SigB activity in vivo, was analysed as a heterologous reporter. The fact that Pctc was activated by osmotic shock, but not by heat or ethanol, confirmed that stress response system(s) could operate independently in S. coelicolor. Pctc was also induced transiently during growth of liquid cultures, presumably by nutritional signals. We purified an RNA polymerase holoenzyme from crude extracts that catalysed specific transcription of Pctc in vitro. Its sigma subunit was identified as a product of the sigH gene, which is co-transcribed downstream of a putative antisigma factor gene (prsH). Although the sigH function was not needed for normal colony morphology, prsH was conditionally required for both aerial hyphae formation and regulation of antibiotic biosynthesis. Levels of two different sigH-encoded proteins were growth phase dependent but not significantly changed by osmotic stress, implying the important roles of post-translational regulatory elements such as PrsH. In addition, synthesis of three other SigH-like proteins was induced by osmotic stress, but not by ethanol or heat. Two of them were genetically assigned to sigH homologous loci sigI and sigJ and shown to be independently regulated. This family of SigH-like proteins displayed different osmotic response kinetics. Thus, in contrast to many other bacteria, S. coelicolor uses an osmotic sensory system that can co-ordinate the activity of multiple paralogues to control the relative activity of promoters within the same stress stimulon. Such specialized stress response systems may reflect adaptive functions needed for colonial differentiation.
    Type of Medium: Electronic Resource
    Signatur Availability
    BibTip Others were also interested in ...
  • 7
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: BldD is a transcription factor required for aerial hyphae formation in the filamentous bacterium Streptomyces coelicolor. Three targets of BldD regulation were discovered by a number of means, including examination of bld gene interdependence, selective enrichment of chromosomal DNA fragments bound by BldD and searching the promoter regions of known developmental genes for matches to a previously characterized BldD binding site. The three BldD targets identified were the developmental sigma factor genes, whiG and bldN, and a previously uncharacterized gene, designated bdtA, encoding a putative transcription factor. In each target gene, the sequences bound by BldD were characterized by electrophoretic mobility shift and DNase I footprinting assays, and their alignment suggested AGTgA (n)m TCACc as a consensus BldD operator. The in vivo effect of mutation in bldD on the expression of these three target genes was assessed using S1 nuclease protection assays. In each case, target gene expression was upregulated during early colony development in the bldD background, suggesting that, in the wild type, BldD acts to repress premature expression of whiG, bldN and bdtA during vegetative growth.
    Type of Medium: Electronic Resource
    Signatur Availability
    BibTip Others were also interested in ...
  • 8
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: whiK was one of five new whi loci identified in a recent screen of NTG-induced whi mutants and was defined by three mutants, R273, R318 and R655. R273 and R318 produce long, tightly coiled aerial hyphae with frequent septation. In contrast, R655 shows a more severe phenotype; it produces straight, undifferentiated aerial hyphae with very rare short chains of spores. Subcloning and sequencing showed that whiK encodes a member of the FixJ subfamily of response regulators, with a C-terminal helix–turn–helix DNA-binding domain and an apparently typical N-terminal phosphorylation pocket. Unexpectedly, a constructed whiK null mutant failed to form aerial mycelium, showing that different alleles of this locus can arrest Streptomyces coelicolor development at very distinct stages. As a consequence of the null mutant phenotype, whiK was renamed bldM. The bldM null mutant fits into the extracellular signalling cascade proposed for S. coelicolor and is a member of the bldD extracellular complementation group. The three original NTG-induced mutations that defined the whiK/bldM locus each affected the putative phosphorylation pocket. The mutations in R273 and in R318 were the same, replacing a highly conserved glycine (G-62) with aspartate. The more severe mutant, R655, carried a C-7Y substitution adjacent to the highly conserved DD motif at positions 8–9. However, although BldM has all the highly conserved residues associated with the phosphorylation pocket of conventional response regulators, aspartate-54, the putative site of phosphorylation, is not required for BldM function. Constructed mutant alleles carrying either D-54N or D-54A substitutions complemented the bldM null mutant in single copy in trans, and strains carrying the D-54N or the D-54A substitution at the native chromosomal bldM locus sporulated normally. BldM was not phosphorylated in vitro with either of the small-molecule phosphodonors acetyl phosphate or carbamoyl phosphate under conditions in which a control response regulator protein, NtrC, was labelled efficiently.
    Type of Medium: Electronic Resource
    Signatur Availability
    BibTip Others were also interested in ...
  • 9
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: The extracytoplasmic function (ECF) sigma factor, σE, is required for normal cell wall integrity in Streptomyces coelicolor. We have investigated the regulation of σE through a transcriptional and mutational analysis of sigE and the surrounding genes. Nucleotide sequencing identified three genes located downstream of sigE ; orf202, cseB and cseC (cse, control of sigE ). cseB and cseC encode a putative response regulator and a putative transmembrane sensor histidine protein kinase respectively. Although most sigE transcription appeared to be monocistronic, sigE was also transcribed as part of a larger operon, including at least orf202. sigE null mutants are sensitive to cell wall lytic enzymes, have an altered peptidoglycan muropeptide profile, and on medium deficient in Mg2+ they overproduce actinorhodin, sporulate poorly and form crenellated colonies. A constructed cseB null mutant appeared to have the same phenotype as a sigE null mutant, which was accounted for by the observed absolute dependence of the sigE promoter on cseB. It is likely that the major role of cseB is to regulate sigE transcription because expression of sigE alone from a heterologous promoter suppressed the cseB mutation. Mg2+ suppresses the CseB/SigE phenotype, probably by stabilizing the cell envelope, and sigE transcript levels were consistently higher in Mg2+-deficient cultures than in high Mg2+-grown cultures. We propose a model in which the CseB/CseC two-component system modulates activity of the sigE promoter in response to signals from the cell envelope.
    Type of Medium: Electronic Resource
    Signatur Availability
    BibTip Others were also interested in ...
  • 10
    ISSN: 1365-2958
    Source: Blackwell Publishing Journal Backfiles 1879-2005
    Topics: Biology , Medicine
    Notes: whiH is one of several known loci specifically needed for the orderly multiple sporulation septation of aerial hyphae of Streptomyces coelicolor A3(2) and for the expression of at least some late sporulation genes. DNA complementing whiH mutants was located immediately upstream of hrdB, which encodes the principal σ factor of S. coelicolor. Sequencing revealed a gene whose disruption gave rise to a typical whiH mutant phenotype. Four whiH mutants contained base changes or a frameshift in this gene. The deduced product of whiH is related to a large family of bacterial regulatory proteins, the most similar being several repressors (such as GntR of Bacillus subtilis) responsive to carboxylate-containing intermediates in carbon metabolism. Transcription of whiH was initiated at a single promoter, PwhiH. Levels of whiH mRNA were developmentally regulated, increasing sharply when aerial mycelium was present, and reaching a maximum approximately when spores were first detectable. Transcript levels were markedly increased in a whiH mutant, indicating the possible involvement of WhiH in negative regulation of its own production. PwhiH was directly dependent on the σ factor encoded by another sporulation gene, whiG, as shown by in vivo and in vitro transcription analysis.
    Type of Medium: Electronic Resource
    Signatur Availability
    BibTip Others were also interested in ...
Close ⊗
This website uses cookies and the analysis tool Matomo. More information can be found here...