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M J Bibb

Publications and source records attributed to M J Bibb.

At least 73 records · Page 4Linked to original sources
PubMed1990

Tandem promoters, tsrp1 and tsrp2, direct transcription of the thiostrepton resistance gene (tsr) of Streptomyces azureus: transcriptional initiation from tsrp2 occurs after deletion of the -35 region.

Nuclease S1 protection experiments indicated that the thiostrepton resistance gene (tsr) of Streptomyces azureus is transcribed from tandem promoters, tsrp1 and tsrp2, that initiate transcription 45 and 173 nucleotides, respectively, upstream of the presumptive translational start codon. The -10 regions of both promoters show similarity to the consensus sequence for the major class of prokaryotic promoters, but the -35 regions do not, although they show some similarity to each other. Replacement of sequences upstream of position -22 relative to the tsrp2 start site with two different DNA segments affected the levels of the tsrp2 transcript but did not alter the tsrp2 initiation site. In vitro transcription assays using RNA polymerase from Streptomyces coelicolor A3(2) also confirmed the location of tsrp2 and identified additional start sites near tsrp2 that were barely detectable with in vivo synthesised RNA. Transcripts corresponding to initiation in vitro at trsp1 could not be detected, suggesting that additional factors are required for utilisation of this promoter.

Amino Acid Sequence↗
PubMed1990

Induction of a phi C31 prophage inhibits rRNA transcription in Streptomyces coelicolor A3(2).

A lysogen of Streptomyces coelicolor A3(2) containing a thermoinducible mutant of the temperate phage phi C31 (phi C31 cts1) was used to obtain synchronous phage development. Filter hybridization experiments indicated a marked reduction in rRNA synthesis after prophage induction. S1 nuclease mapping showed that transcription from each of the four promoters of one rRNA gene set (rrnD) was reduced to approximately the same extent, and that inhibition required protein synthesis. Crude preparations of RNA polymerase from induced lysogens had enhanced transcribing activity for phi C31 DNA which was lost upon further purification. The purified preparations were unimpaired in their ability to transcribe from the rrnD promoters in vitro and apparently unchanged in polypeptide composition. The factor(s) responsible for stimulating phage transcription, and possibly for inhibiting rRNA synthesis, may have been separated from the enzyme during purification.

Bacterial Proteins↗
PubMed1990

Cloning, disruption, and transcriptional analysis of three RNA polymerase sigma factor genes of Streptomyces coelicolor A3(2).

The rpoD gene of Myxococcus xanthus was used as a probe to isolate three Streptomyces coelicolor genes, hrdB, hrdC, and hrdD, which appear to encode RNA polymerase sigma factors extremely similar to the sigma 70 polypeptide of Escherichia coli. Gene disruption experiments suggested that hrdB is essential in S. coelicolor A3(2) but showed that hrdC and hrdD mutants are viable and are apparently unaffected in differentiation, gross morphology, and antibiotic production. S1 nuclease mapping showed that hrdB and hrdD, but not hrdC, were transcribed in liquid culture. The most upstream of two hrdD promoters is internal to an open reading frame (ORF X) on the opposite strand. The predicted product of this gene is homologous to the phosphinothricin acetyltransferases of Streptomyces hygroscopicus and Streptomyces viridochromogenes. The possible significance of the overlapping and divergent transcription of hrdD and ORF X is discussed. A general method for in vivo gene replacement was developed that allowed a positive selection for the desired mutants even in the absence of a mutant phenotype; it was used to isolate a stable hrdC mutant.

Acetyltransferases↗
PubMed1989

Transcriptional analysis of the repressor gene of the temperate Streptomyces phage phi C31.

A 397-bp fragment that contained the 5' end of the coding region of the repressor gene of the temperature Streptomyces phage phi C31 was shown by in vivo promoter-probing to possess bidirectional promoter activity. In vitro runoff transcription experiments, and high resolution transcript mapping of mRNA species produced in vivo using both nuclease S1 and mung bean nuclease, indicated the probable presence of two promoters for the repressor gene with two further promoters oriented in the opposite direction. An inverted repeat sequence is situated 20 bp downstream from the translational stop codon of the repressor gene; high resolution transcript mapping revealed an mRNA endpoint close to this sequence, indicating its likely role as a transcriptional terminator.

Bacteriophages↗
PubMed1989

Structure and deduced function of the granaticin-producing polyketide synthase gene cluster of Streptomyces violaceoruber Tü22.

A 6.5 kb region of DNA from Streptomyces violaceoruber, which contains polyketide synthase (PKS) genes for production of the benzoisochromane quinone moiety of the antibiotic, granaticin, was cloned and sequenced. Of six open reading frames (ORFs) identified, four (ORFs 1-4) would be transcribed in one direction and two (ORFs 5 and 6) divergently from ORFs 1-4. ORF1 and ORF2, which show evidence for translation coupling, encode (deduced) gene products which strongly resemble each other and the Escherichia coli fatty acid ketoacyl synthase (condensing enzyme), FabB. We conclude that ORF1 (which contains a characteristic cysteine residue) functions as a condensing enzyme, possibly as part of a heterodimeric protein including the product of ORF2. The predicted ORF3 gene product strikingly resembles acyl carrier proteins (ACPs) of fatty acid synthase (FAS), particularly in the region of the active site motif, while the predicted ORF5 and ORF6 gene products resemble known oxidoreductases, suggesting that they function as reductive steps required during assembly of the granaticin carbon skeleton. Comparison of the deduced ORF4 gene product with available protein databases failed to elucidate its potential function. The overall conclusion is that the granaticin-producing PKS would consist of at least six separate enzymes involved in carbon chain assembly, thus resembling a Type II, rather than a Type I, FAS.

Amino Acid Sequence↗
PubMed1989

Analysis of the nucleotide sequence of the Streptomyces glaucescens tcmI genes provides key information about the enzymology of polyketide antibiotic biosynthesis.

Key information about the biosynthesis of polyketide metabolites has been uncovered by sequence analysis of the tetracenomycin C polyketide synthase genes (tcml) from Streptomyces glaucescens GLA.0. The sequence data revealed the presence of three complete open reading frames (ORFs). ORF1 and ORF2 appear to be translationally coupled and would encode proteins containing 426 and 405 amino acids, respectively. The two deduced proteins are homologous to known beta-ketoacyl synthases. ORF3 begins 70 nucleotides after the stop codon of ORF2 and would code for an 83 amino acid protein with a strong resemblance to known bacterial, animal and plant acyl-carrier proteins (ACP). The presence of an ACP gene within the tcm gene cluster suggests that different ACPs are used in fatty acid and polyketide biosynthesis in Streptomyces. We conclude from these data and earlier information that polyketide biosynthesis in S. glaucescens, and most likely in other bacteria, involves a multienzyme complex consisting of at least five types of enzymes: acylCoA transferases that load the acyl and 2-carboxyacyl precursors onto the ACP; a beta-ketoacyl synthase that, along with the acylated ACP, forms the poly-beta-ketoacyl intermediates; a poly-beta-ketone cyclase that forms carbocyclic structures from the latter intermediates; a beta-ketoacyl oxidoreductase that forms beta-hydroxyacyl intermediates or reduces ketone groups in fully formed polyketides; and a thioesterase that releases the assembled polyketide from the enzyme.

Amino Acid Sequence↗
PubMed1989

Unusual transcriptional and translational features of the aminoglycoside phosphotransferase gene (aph) from Streptomyces fradiae.

The aminoglycoside phosphotransferase gene (aph) from the neomycin producer Streptomyces fradiae encodes an enzyme (APH) that phosphorylates, and thereby inactivates, the antibiotic neomycin. Two promoters were identified upstream of and oriented toward the aph coding sequence. One promoter (aphp1) initiated transcription at the A of the ATG translational initiation codon, or one to two bases upstream. Mutations made in this promoter region identified functionally important nucleotides and verified that the aphp1 transcript was translated to yield the APH protein, despite the lack of a conventional ribosome binding site. A second aph promoter, aphp2, initiated transcription 315 bp upstream of the translational initiation codon but gave transcripts that appeared to terminate before reaching the coding sequence. Multiple transcriptional initiation sites (pA1-pA5) were identified also in the aph regulatory region oriented in the opposite direction to aph transcription. Promoters for the pA2 and pA4 transcripts overlap with aphp1 such that down-promoter mutations in aphp1 also reduce transcription from the overlapping pA promoters.

Amino Acid Sequence↗
PubMed1988

Cloning, characterisation and regulation of an alpha-amylase gene from Streptomyces venezuelae.

The alpha-amylase gene (aml) of Streptomyces venezuelae ATCC15068 was cloned in Streptomyces lividans TK24 using the plasmid vector pIJ702. Sub-cloning and exonuclease III deletion experiments localised the sequences required for alpha-amylase production to a segment of 2.05 kb. Low-resolution nuclease S1 mapping revealed a aml transcript of approx. 1.7 kb, and the extracellular form of alpha-amylase was estimated by SDS-polyacrylamide gel electrophoresis to be 59 kDa, suggesting that aml mRNA is monocistronic. The nucleotide sequence of aml was determined and high-resolution nuclease S1 mapping experiments identified transcripts that appeared to initiate at a promoter identical to that of the alpha-amylase gene of Streptomyces limosus [Long et al., J. Bacteriol. 169 (1987) 5745-5754]. Transcription of aml in S. venezuelae, and of the cloned gene in Streptomyces coelicolor A3(2), was induced by maltose and repressed by glucose. Glucose repression in S. coelicolor A3(2) depended on a functional glucose kinase gene. The predicted amino acid sequence of the extracellular enzyme was very similar (75% identity) to the alpha-amylase of S. limosus and shared with this enzyme a strong susceptibility to tendamistat, a potent inhibitor of mammalian alpha-amylases. Sequence inspection revealed a putative signal sequence of 28 amino acids that preceded the probable signal peptidase cleavage site.

Amino Acid Sequence↗
PubMed1988

At least three different RNA polymerase holoenzymes direct transcription of the agarase gene (dagA) of Streptomyces coelicolor A3(2).

Using a combination of gel filtration and anion exchange FPLC, three different RNA polymerase holoenzymes from Streptomyces coelicolor A3(2) have been separated. Each holoenzyme transcribes from only one of the four promoters of the S. coelicolor A3(2) dagA gene. Holoenzyme reconstitution experiments identified the sigma factors responsible for recognition of two of the promoters. The previously identified E sigma 49 transcribes from the dagA p3 promoter, whereas a novel species, E sigma 28, recognizes the dagA p2 promoter. Circumstantial evidence suggests that the third holoenzyme, which transcribes from the dagA p4 promoter, is the previously identified E sigma 35. This level of transcriptional complexity supports the idea that RNA polymerase heterogeneity may play a central role in the regulation and coordination of gene expression in this biochemically and morphologically complex bacterium.

Base Sequence↗
PubMed1988

Organisation of the ribosomal RNA genes in Streptomyces coelicolor A3(2).

Using Southern hybridisation of radiolabelled purified ribosomal RNAs to genomic DNA the ribosomal RNA genes of Streptomyces coelicolor A3(2) were shown to be present in six gene sets. Each gene set contains at least one copy of the 5 S, 16 S and 23 S sequences and in at least two cases these are arranged in the order 16 S - 23 S - 5 S. Three gene sets, rrnB, rrnD and rrnF, were isolated by screening a lambda library of S. coelicolor A3(2) DNA. The restriction map of one of these, rrnD, was determined and the nucleotide sequences corresponding to the three rRNAs were localised by Southern hybridisation. The gene order in rrnD is 16 S - 23 S - 5 S.

DNA Restriction Enzymes↗
PubMed1988

The repressor gene (c) of the Streptomyces temperate phage phi c31: nucleotide sequence, analysis and functional cloning.

The nucleotide sequence of the 3.4 kb SphI-G fragment that contained the repressor gene (c) of the temperate Streptomyces phage phi c31 was determined. Analysis of this sequence revealed a large open reading frame with protein coding character and sequence changes in c gene point and deletion mutants identified this as the coding region of the repressor. Two of the mutants studied had undergone deletions of 1.1 kb and 1.4 kb that had occurred across short direct repeats of 6 bp and 11 bp, respectively. Coupled in vitro transcription-translation experiments using the cloned SphI-G fragment and Streptomyces lividans cell free extracts identified a protein product of approximately 72 kDa, in close agreement with that predicted from the nucleotide sequence. A strongly predicted helix-turn-helix motif that may be involved in DNA binding occurred towards the carboxy-terminus of the amino acid sequence. Initial attempts to clone the SphI-G fragment in Streptomyces failed; using information gained from the sequence analysis a smaller segment of this DNA fragment was cloned in S. lividans and conferred immunity to a clear plaque mutant (c1) of phi c31.

Bacteriophages↗
PubMed1988

Cloning, characterization and regulation of an alpha-amylase gene from Streptomyces limosus.

An alpha-amylase gene (aml) of Streptomyces limosus ATCC 19778 was cloned in Streptomyces lividans 66. S1 mapping experiments identified an aml transcript 1.8 kb in length and the extracellular enzyme was estimated to be 59 kD in size, suggesting that aml was transcribed as a monocistronic mRNA species. Expression of the gene was induced by maltose (or maltodextrins) in S. limosus and when aml was cloned in S. lividans or Streptomyces coelicolor A3(2). In S. limosus, mannitol repressed aml expression while glucose had little or no effect; in S. lividans and S. coelicolor the relative effects of the two sugars were reversed. Both induction and carbon-source repression of aml expression appeared to occur at the level of transcriptional initiation. Glucose repression in S. coelicolor was dependent upon a functional glucose kinase gene.

Carbohydrates↗
PubMed1988

Transcriptional analysis of the 16S rRNA gene of the rrnD gene set of Streptomyces coelicolor A3(2).

The nucleotide sequence of 2.5 kb of the Streptomyces coelicolor A3(2) rRNA gene set rrnD, extending from upstream of the 16S rRNA gene to the putative 5' end of the 23S rRNA gene, has been determined (Baylis and Bibb, 1987; this paper). In addition to locating the 5' end of the 16S rRNA gene, nuclease S1 mapping identified seven RNA 5' end-points upstream of the 16S rRNA gene; four of these were coincident with transcriptional initiation points for S. coelicolor A3(2) RNA polymerase in vitro and were consequently regarded as in vivo transcription start points for promoters p1 to p4. One end-point identified by nuclease S1 mapping localized a putative processing site analogous to those found upstream of 16S rRNA genes in other eubacteria. Sequence motifs similar to those discovered in low G+C Gram-positive bacteria were found associated with two of the promoters and the processing site. A probable protein coding region was observed upstream of the promoter region.

Base Sequence↗
PubMed1987

The agarase gene (dagA) of Streptomyces coelicolor A3(2): nucleotide sequence and transcriptional analysis.

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 200 nucleotides upstream of the coding sequence.

Amino Acid Sequence↗
PubMed1987

The agarase gene (dag A) of Streptomyces coelicolor A3(2): affinity purification and characterization of the cloned gene product.

The coding and regulatory sequences of the agarase gene of Streptomyces coelicolor A3(2) were cloned in Streptomyces lividans 66 on the plasmid vector pIJ61, resulting in a several hundred-fold increase in the production of the secreted protein. Subcloning experiments localized the sequences required for agarase production and for the mediation of carbon catabolite repression to a segment of about 1.2 kb. A simple protein purification procedure that uses affinity binding of agarase to agarose beads was developed. Preliminary characterization of the enzyme, together with the results of in vitro transcription-translation studies, suggest that the intracellular form of agarase (about 34 kDa) possesses a signal sequence that is cleaved upon secretion across the cell membrane to produce an extracellular protein of about 29 kDa.

Chromatography, Affinity↗
PubMed1987

alpha-Amylase gene of Streptomyces limosus: nucleotide sequence, expression motifs, and amino acid sequence homology to mammalian and invertebrate alpha-amylases.

The nucleotide sequence of the coding and regulatory regions of the alpha-amylase gene (aml) of Streptomyces limosus was determined. High-resolution S1 mapping was used to locate the 5' end of the transcript and demonstrated that the gene is transcribed from a unique promoter. The predicted amino acid sequence has considerable identity to mammalian and invertebrate alpha-amylases, but not to those of plant, fungal, or eubacterial origin. Consistent with this is the susceptibility of the enzyme to an inhibitor of mammalian alpha-amylases. The amino-terminal sequence of the extracellular enzyme was determined, revealing the presence of a typical signal peptide preceding the mature form of the alpha-amylase.

Amino Acid Sequence↗