Biomedical subjects

K F Chater

Publications and source records attributed to K F Chater.

At least 55 records · Page 3Linked to original sources

PubMed1993

Accumulation of bldA-specified tRNA is temporally regulated in Streptomyces coelicolor A3(2).

Deletion of the bldA gene of Streptomyces coelicolor A3(2), which encodes the only tRNA for the rare UUA codon, had no obvious effects on primary growth but interfered with aerial mycelium formation and antibiotic production. To investigate the possible regulatory role of bldA, its transcription start point was identified, and time courses were determined for the appearance of its primary transcript, the processing of the primary transcript to give a mature 5' end, and the apparent efficiency of translation of ampC mRNA, which contains multiple UUA codons. The bldA promoter was active at all times, but processing of the 5' end of the primary transcript was comparatively inefficient in young cultures. This may perhaps involve an antisense RNA, evidence of which was provided by promoter probing and in vitro transcription. The presence of low levels of the processed form of the tRNA in young cultures followed by increased abundance in older cultures contrasted with the pattern observed for accumulation of a different, presumably typical tRNA which was approximately equally abundant throughout growth. The increased accumulation of the 5' processed form of bldA tRNA coincided with more-efficient translation of ampC mRNA in older cultures, supporting the hypothesis that in at least some physiological conditions, bldA may have a regulatory influence on events late in growth, such as morphological differentiation and antibiotic production.

Anti-Bacterial Agents↗

PubMed1993

Genetics of differentiation in Streptomyces.

The use of mutants and molecular genetics has begun to reveal how differentiation is brought about in multicellular, mycelial Streptomyces spp. Alternative pathways to aerial mycelium formation may be activated on different media. In some cases, extracellular signals are transmitted or exchanged. Extracellular proteins may act as morphogens in the erection of aerial hyphae. A rare codon, UUA, is apparently confined to mRNAs from a few genes important only for early stages of differentiation; it is absent from vegetative or developmentally late mRNAs. Sporulation of aerial hyphae involves at least four specific regulatory proteins, including a sigma factor and an unusual small protein. A complex interplay between the sporulation regulatory genes, rather than a simple linear dependence cascade, is emerging.

Amino Acid Sequence↗

PubMed1992

Genetic regulation of secondary metabolic pathways in Streptomyces.

Streptomyces species are (along with the fungi) the best-known antibiotic-producing organisms. Often, they make several different antibiotics. The biosynthesis of each antibiotic is encoded by a complex gene cluster that usually also contains regulatory and resistance genes. Typically, there may be more than one such pathway-specific regulatory gene per cluster. Both activator and repressor genes are known. Some of the regulatory genes for different pathways are related. In S. coelicolor, expression of several such biosynthetic gene clusters also depends on at least 11 globally acting genes, at least one of which is involved in the translation of a rare codon (UUA). A protein phosphorylation cascade also seems to be involved. Gene clusters closely similar to those for the biosynthesis of aromatic polyketide antibiotics determine spore pigment in some species. These genes show different regulation from antibiotic production genes. The evolution of gene clusters for polyketide antibiotics, and the possible adaptive benefits of secondary metabolism, are discussed.

Anti-Bacterial Agents↗

PubMed1992

The Streptomyces coelicolor whiB gene encodes a small transcription factor-like protein dispensable for growth but essential for sporulation.

A non-sporulating mutant (whiB218) of Streptomyces coelicolor A3(2), which proved to contain a deletion of more than 5 kb of DNA including whiB, was complemented by a small cloned DNA fragment, deduced from DNA sequencing to encode a protein of only 87 amino acids. This protein would bear some similarities to transcription factors, including an acidic, somewhat amphipathic alpha-helical region predicted near its N-terminus, and a basic alpha-helical region predicted at its C-terminus. A point mutation (whiB70) giving a phenotype indistinguishable from that of the whiB218 deletion mutant would cause a leucine to proline change at the start of the latter region. The whiB homologue from the closely related species S. lividans differed at only one base from its S. coelicolor counterpart, and would specify an identical polypeptide.

Amino Acid Sequence↗

PubMed1992

Membrane transport proteins: implications of sequence comparisons.

Analyses of the sequences and structures of many transport proteins that differ in substrate specificity, direction of transport and mechanism of transport suggest that they form a family of related proteins. Their sequence similarities imply a common mechanism of action. This hypothesis provides an objective basis for examining their mechanisms of action and relationships to other transporters.

Amino Acid Sequence↗

PubMed1992

Global transcription pattern of phi C31 after induction of a Streptomyces coelicolor lysogen at different growth stages.

Using two complementary strategies for low-resolution S1 mapping, the global pattern of phi C31 transcription was studied after induction of thermoinducible phi C31 lysogens of Streptomyces coelicolor A3(2). A complex pattern of early transcripts was seen, with a peak of abundance at about 10 min post-induction. Nearly all of these transcripts were from DNA located to the right of the c (repressor) gene and to the left of the attP site: a region of about 14 kb. Early transcription was also observed immediately to the left of the c gene. The c gene itself was also induced, with an earlier expression peak (about 5 min post-induction). Primary late transcripts were generally relatively long, but degraded. They apparently corresponded to most of the 18 kb region to the left of the c gene. Some shorter and more persistent late transcripts corresponded to DNA close to or overlapping the cos site. Large late transcripts from a region close to the left-hand end of the phi C31 genome showed evidence of processing to more stable, smaller RNA species. A failure of older cultures (more than 12 h old) to be induced productively was correlated with a much longer period of early transcription, reduced late transcription, failure to synthesize a major virion protein, and failure to package phi C31 DNA. Moreover, heat treatment of the older lysogenic cultures did not result in the phi C31-dependent shut-down of host rRNA transcription previously observed for young cultures (Rodríguez et al., Journal of General Microbiology (1986) 132, 1695-1701; Clayton & Bibb, Molecular Microbiology (1990) 4, 2179-2185).

Bacteriophages↗

PubMed1992

Two promoters for the whiB sporulation gene of Streptomyces coelicolor A3(2) and their activities in relation to development.

Two transcripts corresponding to the whiB sporulation gene of Streptomyces coelicolor A3(2) that differed in length at their 5' ends by 164 nucleotides were identified by S1 mapping. Their presumptive promoters differed from each other; the more downstream, P2, resembled typical prokaryotic promoters (i.e., those recognized by the major form of RNA polymerase) at five of six positions in its -10 and -35 regions; and the more upstream, P1, was comparably similar, instead, to the previously described hrdDp1 promoter (M. J. Buttner, K. F. Chater, and M. J. Bibb, J. Bacteriol. 172:3367-3378, 1990) around -40, -10, and +1. In surface cultures of the wild-type strain, the abundance of transcripts from the weak P1 promoter showed no obvious correlation with the developmental stage, whereas transcripts from P2 were barely detectable until aerial mycelium was present and then became relatively abundant, consistent with the developmental role of whiB. Both types of transcript were detected during and, to a lesser extent, after rapid growth in liquid culture. In addition, both promoters were utilized in vitro by RNA polymerase purified from a liquid culture of S. coelicolor. Transcription from P1 and P2 was observed during surface culture in strains carrying mutations blocking aerial mycelium formation (bldA and bldB) or the formation of spores in aerial mycelium (whiA, whiB, whiG, and whiH). Thus, whiB transcription is not severely dependent on any of these developmental genes, among which whiG is the determinant of a putative sigma factor specific for, and crucial to, sporulation.

Base Sequence↗

PubMed1991

TTA codons in some genes prevent their expression in a class of developmental, antibiotic-negative, Streptomyces mutants.

In Streptomyces coelicolor A3(2) and the related species Streptomyces lividans 66, aerial mycelium formation and antibiotic production are blocked by mutations in bldA, which specifies a tRNA(Leu)-like gene product which would recognize the UUA codon. Here we show that phenotypic expression of three disparate genes (carB, lacZ, and ampC) containing TTA codons depends strongly on bldA. Site-directed mutagenesis of carB, changing its two TTA codons to CTC (leucine) codons, resulted in bldA-independent expression; hence the bldA product is the principal tRNA for the UUA codon. Two other genes (hyg and aad) containing TTA codons show a medium-dependent reduction in phenotypic expression (hygromycin resistance and spectinomycin resistance, respectively) in bldA mutants. For hyg, evidence is presented that the UUA codon is probably being translated by a tRNA with an imperfectly matched anticodon, giving very low levels of gene product but relatively high resistance to hygromycin. It is proposed that TTA codons may be generally absent from genes expressed during vegetative growth and from the structural genes for differentiation and antibiotic production but present in some regulatory and resistance genes associated with the latter processes. The codon may therefore play a role in developmental regulation.

Anti-Bacterial Agents↗

PubMed1991

Isolation and genetic structure of IS112, an insertion sequence responsible for the inactivation of the SalI restriction-modification system of Streptomyces albus G.

IS112 is a transposable element identified in Streptomyces albus G by its frequent mutagenic insertion into the genes for the SalI restriction-modification system. IS112 is present in several copies in the genome of S. albus G. Homologous sequences were detected in other Streptomyces strains. Sequence analysis revealed that IS112 has a length of 883 bp with a GC content of 67.4%. The copy that was isolated contained imperfect inverted repeats (16/20 match) at its ends and was flanked by a 2 bp duplication at the target site, which was located within the gene (salIR) for the SalI endonuclease. A long open reading frame (ORF) encoding a putative polypeptide of 256-253 amino acids spans almost the entire sequence. Significant homology was detected between this polypeptide and that corresponding to ORFB of IS493, an insertion sequence recently isolated from Streptomyces lividans 66.

Amino Acid Sequence↗

PubMed1991

Bidirectional promoter and terminator regions bracket mmr, a resistance gene embedded in the Streptomyces coelicolor A3(2) gene cluster encoding methylenomycin production.

Low- and high-resolution nuclease mapping of in vivo transcripts, and in vitro transcription reactions using purified RNA polymerase, were used to analyse transcription of and around the mmr gene, which specifies resistance of Streptomyces coelicolor A3(2) to methylenomycin (Mm) and is located in the middle of a cluster of Mm-production-encoding genes. Transcription of mmr is from a single major start point (tsp) which is separated by only 81 bp from a divergent tsp. A pattern of direct and inverted repeats in the nucleotide sequence in this region may play a part in regulation of these promoters. The 3' end of the mmr transcript overlaps by 20-30 bp the 3' end of an RNA molecule involved in Mm production. The converging transcripts both terminate at the same large inverted repeat in the DNA. Purified RNA polymerase terminated transcription at this sequence in vitro (albeit only in one orientation).

Anti-Bacterial Agents↗

PubMed1991

Phage vectors that allow monitoring of transcription of secondary metabolism genes in Streptomyces.

We describe a bacteriophage phi C31-based system that permits the transcriptional fusion of the convenient reporter gene xylE to chromosomally located promoters in Streptomyces hosts. Applicability of the system to genes for secondary metabolism is demonstrated in an experiment showing that transcription of genes for actinorhodin production in Streptomyces coelicolor A3(2) depends on a transfer RNA gene (bldA) for the rare UUA codon. Two other phi C31::xylE vectors are described that allow detection of promoter activity away from their natural location, either at single copy in a prophage or during lytic infections in plaques.

Anthraquinones↗

PubMed1991

The use of a rare codon specifically during development?

A range of circumstantial evidence suggests that in Streptomyces spp., genes required for vegetative growth do not contain the leucine codon TTA. Instead, the codon seems to be confined to a few genes necessary during differentiation, when the colonies begin to produce aerial hyphae and antibiotics. Thus, mutations in bldA, the structural gene for tRNATTALeu, do not retard vegetative growth, but they prevent normal aerial mycelium and antibiotic production. Most of the known TTA-containing genes specify regulatory or resistance proteins associated with antibiotic-production clusters. Possibly the ability to translate the UUA codons in mRNA from such genes is confined to late stages of colony development. Factors that might have contributed to the evolution of this unusual situation are discussed.

Base Sequence↗

PubMed1990

The improving prospects for yield increase by genetic engineering in antibiotic-producing Streptomycetes.

Molecular genetics has spawned a dramatic expansion of the biotechnology industry in the direction of the products of single genes. On the other hand, antibiotics--some of the classical products of biotechnology--result from the concerted action of many genes, and it is therefore less straightforward to apply the new techniques to antibiotic production. Studies of cloned genes for antibiotic biosynthesis are now providing information that should allow the application of a combination of traditional and recombinant DNA methodology to the improvement of yield in antibiotic-producing Streptomyces species.

Anti-Bacterial Agents↗

PubMed1990

Discovery of an insertion sequence, IS116, from Streptomyces clavuligerus and its relatedness to other transposable elements from actinomycetes.

We have identified an insertion sequence, IS116, present in Streptomyces clavuligerus at one copy per genome. The element was discovered as a 1.4 kb insertion into the multicopy plasmid pIJ702 after propagation in S. clavuligerus. The nucleotide sequence of IS116 and the flanking sequences from pIJ702 have been determined. The junctions with pIJ702 show no target site duplication and there are no inverted repeats at the ends of the element. One putative coding open reading frame of 1197 bp was identified which would code for a protein product of 399 amino acids. This protein resembles deduced integrase/transposase proteins specified by three other transposable elements of actinomycetes: IS110 and the mini-circle from Streptomyces coelicolor A3(2), and--most particularly--IS900 of Mycobacterium paratuberculosis. Two regions that are relatively conserved among these gene products show features found in similar positions in many reverse transcriptases. IS116 and IS900 are also closely similar in their general organization and (apparently) in their insertion site specificity, whereas IS110 and the mini-circle are quite different in these features.

Actinomycetales↗

PubMed1990

Spore colour in Streptomyces coelicolor A3(2) involves the developmentally regulated synthesis of a compound biosynthetically related to polyketide antibiotics.

Streptomyces coelicolor produces spores whose development of a grey colour requires the activity of the whiE locus. The cloned whiE locus was identified after mobilization into a whiE mutant of a library of S. coelicolor DNA inserted into a transmissible plasmid vector. The whiE region of the cloned DNA was localized both by subcloning and by mutagenesis of the cloned DNA with the Streptomyces transposon Tn4560. Nucleotide sequencing of this region revealed seven open reading frames, of which six show homology at the level of deduced gene products with genes involved in the synthesis of polyketide antibiotics. A previously described S. coelicolor DNA segment encoding biosynthesis of a brown pigment (Horinouchi and Beppu, 1985) corresponds to the cloned whiE DNA. It is proposed that whiE is normally expressed only in the aerial hyphae, where the biosynthetic product is responsible for spore colour.

Amino Acid Sequence↗

PubMed1990

The level of a transcript required for production of a Streptomyces coelicolor antibiotic is conditionally dependent on a tRNA gene.

In Streptomyces coelicolor A3(2), bldA mutants are conditionally defective in aerial mycelium formation and fail to synthesize all four antibiotics produced by bldA+ strains. Previous studies showed that bldA specifies the tRNA for the rarely used leucine codon UUA. Here we describe experiments examining the abundance in a bldA mutant of a transcript involved in antibiotic production. With use of a bacteriophage-based integrative vector, a promotorless xylE reporter gene was inserted into a previously undescribed gene for an early step in biosynthesis of the red antibiotic undecylprodigiosin, located in the red gene cluster. With this transcriptional fusion present at unit copy number in the chromosome, xylE expression in a bldA+ strain was maximal late in growth in a liquid production medium and was virtually absent in a bldA mutant. On plates of a different medium, the bldA mutant was able to produce undecylprodigiosin and to express the red::xylE fusion, but both abilities were repressed by increasing the concentration of phosphate in the medium. These experiments showed that the undecylprodigiosin deficiency of bldA mutants cannot be accounted for by the presence of TTA codons in the red structural genes, but rather that bldA influences red gene mRNA abundance. In low-phosphate conditions, an alternative regulatory pathway can lead to red gene expression.

Anti-Bacterial Agents↗

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

The developmental fate of S. coelicolor hyphae depends upon a gene product homologous with the motility sigma factor of B. subtilis.

In the mycelial prokaryote S. coelicolor, whiG is a gene dispensable for growth but needed for the earliest stages of spore formation in aerial hyphae. Nucleotide sequencing indicates that whiG encodes an RNA polymerase sigma factor highly similar to the motility sigma factor (sigma 28) of B. subtilis. High copy number of an intact whiG gene caused sporulation in vegetative hyphae that are usually fated to lyse without sporulating. However, the introduction of many copies of a sigma 28-dependent promoter from B. subtilis into S. coelicolor reduced sporulation, suggesting partial sequestration of the whiG gene product by the foreign promoter sequences. We propose that the level of whiG sigma factor is crucial in determining the developmental fate of hyphae.

Amino Acid Sequence↗