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K F Chater

Publications and source records attributed to K F Chater.

At least 73 records · Page 4Linked to original sources
PubMed1989

Multilevel regulation of Streptomyces differentiation.

Streptomyces species are unusual among bacteria in their extensive colony differentiation, which involves the development of spore-bearing aerial hyphae on mycelial colonies. This process, which is usually accompanied by antibiotic production, is controlled by factors as diverse as pheromones, a transfer RNA and a sigma factor dedicated to differentiation, and perhaps by storage compounds and substances related to antibiotics.

Amino Acid Sequence↗
PubMed1988

The SalI (SalGI) restriction-modification system of Streptomyces albus G.

The salIR and salM genes of Streptomyces albus G specify the SalGI (SalI) restriction enzyme and its cognate methyltransferase, respectively. These enzymes are responsible for restriction and modification of bacteriophages. Some phages carry genes that interfere with SalI-specific modification. The sal genes have been cloned in a Streptomyces host-vector system. Use of the cloned DNA as a hybridization probe reveals that sal mutants frequently arise from transposition of a DNA segment of approx. 1 kb into the sal genes. Some, but not all, other bacteria that produce SalGI isoschizomers contain nucleotide sequences that hybridize with sal DNA.

Bacterial Proteins↗
PubMed1988

Structure and regulation of controlling sequences for the Streptomyces coelicolor glycerol operon.

The pathway for glycerol catabolism in Streptomyces coelicolor is determined by the gylABX operon. The sequence of about 1500 base-pairs (bp) preceding the structural genes of the operon has been determined, and related to a detailed transcriptional analysis of this region. The gylABX operon contains two major promoters, gylP1 and gylP2, separated by 50 bp. Both promoters are glycerol-inducible and glucose-repressible. A 900-base transcription unit, gylR, is situated immediately upstream of the gylABX promoter region and contains an open reading frame for a 27,600 Mr protein. The predicted sequence of this protein contains a region that is similar to the helix-turn-helix domains of certain DNA-binding proteins. Transcription of gylR is also glycerol-inducible, but is only weakly glucose-repressible, and initiates predominantly from a single promoter, gylRp. The three promoters, gylRP, gylP1 and gylP2, each resemble the "typical" prokaryotic consensus promoter sequence. The DNA sequence of the gylR and gylABX promoter regions share some striking features. These include almost identical operator-like elements (segments of which are tandemly repeated around gylRP) and tracts of alternating purine-pyrimidine residues.

Base Sequence↗
PubMed1988

Cloning and transcription analysis of the entire glycerol utilization (gylABX) operon of Streptomyces coelicolor A3(2) and identification of a closely associated transcription unit.

The entire glycerol utilization (gylABX) operon of Streptomyces coelicolor A3(2) was cloned and its transcriptional organization and regulation was analyzed by Northern blotting, S1 nuclease mapping and transcriptional fusions. Transcription of the operon is glycerol-inducible and glucose-repressible; glyA (presumptively encoding glycerol kinase), gylB (encoding sn-glycerol-3-phosphate dehydrogenase) and gylX (a non-essential 1.1 kb sequence) are transcribed consecutively to give a 5.4 kb mRNA. Two alternative transcription termination or gyl mRNA processing sites are located within the operon; one (a discrete site) lies between gylB and gylX and the other (a heterogeneous site) positioned 3 kb into the operon, may correspond to the gylA-gylB intercistronic region. A 0.9 kb glycerol-inducible transcription unit is located immediately upstream of gylABX. Transcriptional fusion studies employing an attP site-deleted phage vector provided complementary evidence for the organization of the operon.

Chromosome Mapping↗
PubMed1987

Nucleotide sequence of IS110, an insertion sequence of Streptomyces coelicolor A3(2).

The nucleotide sequences of the Streptomyces transposable element IS110 and its insertion site in the DNA of a derivative of the temperate phage luminal diameter C31 were determined. The element is inserted about 460 bp from the right-hand end of luminal diameter C31 DNA, in a region of apparently non-coding DNA. The target site (in a run of seven C residues) is within an 11 bp sequence homologous with one end of IS110. The inserted element is flanked by runs of 11 and 15 C residues which form part of more extensive regions of homology between the left and right junction regions. Imperfect inverted repeats (10 matches out of 15 bp) are present near (but not at) the ends of IS110. The whole IS110 element contains about 1550 bp of which 71% are G-C bp. One major potentially protein-coding region (ORF 1215) was detected, of 1215 bp, the product of which, a presumptively soluble protein of MR 43,563, was not overtly related to any entry in a protein sequence database. A smaller open reading frame (ORF 330) was tentatively identified in the opposite strand of the ORF 1215 region.

Amino Acid Sequence↗
PubMed1987

The glucose kinase gene of Streptomyces coelicolor and its use in selecting spontaneous deletions for desired regions of the genome.

A number of deletions in the glucose kinase (glk) region of the Streptomyces coelicolor chromosome were found among spontaneous glk mutants. The deletions were identified by probing Southern blots of chromosomal DNA from glk mutants with cloned glk DNA. The deletions ranged in size from 0.3 kb to greater than 2.9 kb. When cloned glk DNA was introduced on a phi C31 phage vector into a glk mutant that contained a deletion of the entire homologous chromosomal glk region, glucose kinase activity was detected in extracts of these cells. The entire coding information for at least a subunit of glucose kinase is therefore present on the cloned glk DNA. The 0.3 kb glk chromosomal deletion was used to demonstrate that transfer of chromosomal glk mutations on to the phi C31::glk phage could occur by recombination in vivo. Since glk mutations frequently arise from deletion events, a method was devised for inserting the cloned glk DNA at sites in the chromosome for which cloned DNA is available, and thus facilitating the isolation of deletions in those DNA regions. phi C31::glk vectors containing a deletion of the phage att site cannot lysogenize S. coelicolor recipients containing a deletion of the glk chromosomal gene unless these phages contain S. coelicolor chromosomal DNA. In such lysogens, the glk gene becomes integrated into the chromosome by homologous recombination directed by the chromosomal insert on the phage DNA. In appropriate selective conditions, mutants which contain deletions of the glk gene that extend into the adjacent host DNA can be easily isolated. This method was used to insert glk into the methylenomycin biosynthetic genes, and isolate derivatives with deletions of host DNA from within the prophage into the adjacent host DNA. Phenotypic and Southern blot analysis of the deletions showed that there are no genes essential for methylenomycin biosynthesis for at least 13 kb to the left of a region concerned with negative regulation of methylenomycin biosynthesis. Many of the deletions also removed part of the phi C31 prophage.

Chromosome Deletion↗
PubMed1987

Cloning of Streptomyces griseus and Streptomyces lividans genes for glycerol dissimilation.

Streptomyces lividans gyl DNA (for glycerol utilisation) was cloned by complementation of a Streptomyces coelicolor gyl mutant. Restriction mapping showed that the cloned DNA was highly homologous (perhaps 99%) to S. coelicolor gyl DNA. Using phage-mediated mutational cloning, an internal fragment of the S. coelicolor gyl operon was used to generate a gyl mutant of S. lividans, which subsequently served as recipient in the cloning of gyl DNA from S. griseus. A 7.5-kb SstI-generated fragment of S. griseus DNA was obtained which, as judged by analysis of restriction sites, was only perhaps 87% homologous with the S. coelicolor gyl operon. The cloned S. griseus DNA appears to contain intact gylA and gylB genes and probably also an upstream gene related to the putative gyl regulatory '0.9-kb' gene of S. coelicolor. Cloning of the fragment on a high-copy-number vector in S. lividans did not lead to high levels of the enzymes encoded by gylA and gylB. The S. griseus gylA and gylB genes were not detectably expressed in Escherichia coli glp mutants.

Cloning, Molecular↗
PubMed1987

Nucleotide sequence analysis reveals similarities between proteins determining methylenomycin A resistance in Streptomyces and tetracycline resistance in eubacteria.

Previous studies had localised the gene (mmr) for resistance to methylenomycin A (Mm) to a 2.5-kb PstI fragment in the middle of a cluster of Mm biosynthetic genes from the Streptomyces coelicolor plasmid SCP1. In this paper, the gene has been more precisely located by sub-cloning, and the nucleotide sequence of the whole fragment has been determined. The predicted mmr-specified protein (Mr 49238) would be hydrophobic, with some homology at the amino acid level to tetracycline-resistance proteins from both Gram-positive and Gram-negative bacteria. Comparisons of hydropathy plots of the amino acid sequences reinforces the idea that the proteins are similar. It is suggested that Mm resistance may be conferred by a membrane protein, perhaps controlling efflux of the antibiotic. No significant homology was detected by hybridisation analysis between mmr and a cloned oxytetracycline (OTc)-resistance gene (tetB) of the OTc producer Streptomyces rimosus, and no cross-resistance was conferred by these genes. Sequences on both sides of mmr appear to encode proteins. The direction of translation in each case would be opposite to that of mmr translation. This suggests that mmr is transcribed as a monocistronic mRNA from a bidirectional promoter. An extensive inverted repeat sequence between the stop codons of mmr and the converging gene may function as a bidirectional transcription terminator.

Amino Acid Sequence↗
PubMed1987

Streptomyces albus G produces an antibiotic complex identical to paulomycins A and B.

An antibiotic complex active against multiply resistant strains of staphylococci and other Gram-positive bacteria was isolated from cultures of Streptomyces albus G. Silica gel and Sephadex LH-20 column chromatography gave two congeners with Mr values of 786 and 772, which differed by one -CH2-group. The two homologues contained an isothiocyanate group, and proved to be identical with paulomycins A and B produced by Streptomyces paulus; the FAB mass spectra, in addition, proved the same two congeners to be present in proceomycin obtained from Streptomyces alboniger.

Anti-Bacterial Agents↗
PubMed1987

Pleiotropic morphological and antibiotic deficiencies result from mutations in a gene encoding a tRNA-like product in Streptomyces coelicolor A3(2).

In Streptomyces coelicolor, bldA mutants are defective in antibiotic production and the development of aerial hyphae and spores. Subcloning analysis showed that sequences spanning an NcoI site in cloned bldA+ DNA were needed to allow complementation of a bldA mutant. Nucleotide sequencing revealed a tRNA-like sequence 9 bp downstream from the NcoI site. Five independent bldA mutations all fell in a 16-bp region in the tRNA-like sequence, one of them changing the putative anticodon. In RNA dot-blot analysis, hybridization was detected with a probe specific for the tRNA-like transcript but not with a probe for "anti-tRNA-like" transcripts. The transcripts detected were all in the salt-soluble RNA fraction and accumulated relatively late in growth. It is postulated that bldA specifies a tRNA that would recognize the codon UUA (for leucine). This codon is very rare in Streptomyces genes [which generally contain greater than 70 mole% (G + C)], suggesting a possible role for bldA in translational control of development.

Base Sequence↗
PubMed1987

Cloning of whiG, a gene critical for sporulation of Streptomyces coelicolor A3(2).

In whiG mutants of Streptomyces coelicolor A3(2), aerial hyphae do not show any sign of sporulation. A library of S. coelicolor DNA was prepared in a phi C31 temperate phage vector (KC516), and one recombinant phage (KC750) that could restore the wild-type phenotype to a collection of whiG mutants when integrated into their genomes was found. Subcloning experiments with low- and high-copy-number Streptomyces plasmid vectors allowed partial localization of whiG in the cloned DNA and revealed that hypersporulation was associated with the presence of extra copies of whiG.

Cloning, Molecular↗
PubMed1986

The expression of the Escherichia coli lacZ gene in Streptomyces.

The Escherichia coli lacZ gene was stably introduced into phi C31-based phage cloning vectors in Streptomyces lividans. However, lacZ could not be stably introduced into S. lividans on the plasmid vectors pIJ702 or pIJ41. Studies of the expression of lacZ in S. lividans and S. coelicolor were facilitated by the use of mutants and/or growth conditions in which endogenous beta-galactosidase activity was low or absent. Plaques and lysogens of phi C31::lacZ constructs involving transcriptional fusions to the pBR322 tet gene contained beta-galactosidase activity. Activities were markedly higher in S. lividans than in S. coelicolor. Insertion of the major transcriptional terminator of coliphage fd between the tet promoter and lacZ reduced lacZ expression more in lysogens than in lytically infected cultures, suggesting the existence of a phage-specified anti-termination function. Several phi C31 derivatives suitable for the construction of different kinds of transcriptional and translational fusions in Streptomyces were derived. The expression of lacZ in one transcriptional fusion vector (KC659) was increased both in plaques and in lysogens by the appropriately positioned insertion of a previously characterized promoter from the aph gene of S. fradiae. When phi C31 DNA fragments were inserted into KC659, at least one recombinant phage gave high beta-galactosidase activity in lytic infections, but not in lysogens. The potential usefulness of lacZ fusions in Streptomyces is discussed.

Bacterial Proteins↗
PubMed1986

Properties of in vitro recombinant derivatives of pJV1, a multi-copy plasmid from Streptomyces phaeochromogenes.

The 10.8 kb plasmid pJV1, isolated from Streptomyces phaeochromogenes, has a high copy number (about 150) and a broad host range among Streptomyces spp. Several pJV1 derivatives carrying the thiostrepton resistance gene (tsr) of S. azureus were made. One derivative, pWOR191, was shown to promote its own transfer and to mobilize chromosomal markers in S. lividans. Another derivative, pWOR109, was non-transmissible. Deletion in vitro of a segment of pWOR109 gave pWOR120 (5.6 kb), which has single BamHI and Bg/II sites shown to be capable of accepting 'foreign' DNA such as a previously cloned S. antibioticus DNA fragment encoding tyrosinase, giving vectors (pWOR125, pWOR126) with properties resembling the well-established multicopy vector pIJ702. Shuttle vectors capable of functioning in both S. lividans and Escherichia coli were also constructed. The region of pJV1 essential for replication and maintenance was localized to a 2.5 kb segment. Stable maintenance of pWOR109 and pWOR120 was observed in the presence of derivatives of pIJ101, the progenitor of pIJ702.

Chromosomes, Bacterial↗
PubMed1985

Resistance, regulatory and production genes for the antibiotic methylenomycin are clustered.

At least 17 kb of DNA from the large unisolatable Streptomyces coelicolor A3(2) plasmid SCP1 are concerned with methylenomycin biosynthesis. Mutational cloning analysis, using insert-directed integration of att site deleted phage vectors into an SCP1-containing host, provided evidence of two large transcription units, of at least 6.6 kb and 9.5 kb. At the leftmost apparent end of the larger (left-hand) transcription unit is a region apparently involved in negative regulation of methylenomycin biosynthesis: when fragments from this region were used to direct phage integration, marked overproduction of methylenomycin resulted. The methylenomycin resistance determinant is located at the rightmost end of this same transcription unit. Hybridisation analysis with 13 kb of the cloned mmy region showed that it was closely similar to a segment of pSV1, a plasmid that specifies methylenomycin biosynthesis in S. violaceus-ruber SANK 95570.

Anti-Bacterial Agents↗
PubMed1985

Physical and genetic analysis of IS110, a transposable element of Streptomyces coelicolor A3(2).

On at least three independent occasions a 1.6 kb segment of Streptomyces coelicolor DNA was detected in apparently the same location in an attP-deleted derivative of the temperate phage phiC31 that carried a selectable viomycin resistance gene. This sequence (termed IS110) allowed integration of the phage (giving viomycin-resistant transductants) at homologous sequences (detected by Southern hybridisation) at several locations in the S. coelicolor genome. The inserted prophages facilitated genetic mapping of two IS110 copies in the chromosomal linkage map. A third copy did not exhibit simple segregation with chromosomal markers, and there appeared to be a frequent DNA rearrangement close to this copy. Some variation in the number of copies of IS110 and their location has taken place in the pedigree of S. coelicolor derivatives. IS110 did not hybridise to any known S. coelicolor plasmid, nor to any of several other IS-like elements previously described in other Streptomyces plasmids or phages. It hybridised strongly to DNA from only a small minority of other Streptomyces species and was absent from S. lividans, a close relative of S. coelicolor.

Bacteriophages↗
PubMed1985

New derivatives of the Streptomyces temperate phage phi C31 useful for the cloning and functional analysis of Streptomyces DNA.

The thiostrepton resistance gene (tsr) of Streptomyces azureus, and a synthetic oligonucleotide adapter sequence, were introduced into the DNA of attP-site-deleted phage phi C31-based cloning vectors. The DNA of two of the new derivatives, KC515 and KC516, contains single sites for the enzymes BamHI, BglII, PstI, PvuII, SstI (two sites close together) and XhoI, available for the insertion of DNA of up to 4 kb. The two vectors also contain a cloned, promoterless viomycin phosphotransferase gene (vph) from Streptomyces vinaceus. When an internal segment of the Streptomyces coelicolor glycerol (gyl) operon was inserted at the appropriate position and in the correct orientation next to vph, it could bring about in vivo recombination leading to fusion of vph of the chromosomally located gyl operon, resulting in glycerol-regulated expression of viomycin resistance. Two other new phi C31 derivatives, KC505 and KC518, are PstI and BamHI replacement vectors, respectively, for 2-8-kb DNA fragments, and allow simple screening for the presence of inserted DNA.

Bacteriophages↗
PubMed1985

Phage-mediated cloning of bldA, a region involved in Streptomyces coelicolor morphological development, and its analysis by genetic complementation.

Streptomyces coelicolor bald (bld) mutants form colonies of vegetative substrate mycelium, but do not develop aerial hyphae or spore chains. The bldA strains form none of the four antibiotics known to be produced by the parent strain. With a vector derived from the temperate bacteriophage phi C31, a 5.6-kilobase fragment of wildtype DNA was cloned which restored sporulation to five independent bldA mutants when lysogenized with the recombinant phage. The cloned gene(s) was dominant over the mutant alleles. Phage integration by recombination of the cloned bldA+ DNA with the bldA region of each mutant produced mainly sporulating colonies, presumably heterozygous bldA+/bldA partial diploids for the insert DNA. However, a minority of these primary transductants were bald and were apparently homozygous bldA/bldA mutant partial diploids, formed by some homogenetization process. The phages released from the bald lysogens carried bldA mutations and were used to show that bldA+ sequences had been cloned and that fine mapping of the region could be performed.

Alleles↗