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K F Chater

Publications and source records attributed to K F Chater.

At least 37 records · Page 2Linked to original sources
PubMed1998

A developmentally regulated gene encoding a repressor-like protein is essential for sporulation in Streptomyces coelicolor A3(2).

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 on hrdB, which encodes the principal sigma 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 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 sigma factor encoded by another sporulation gene, whiG, as shown by in vivo and in vitro transcription analysis.

Amino Acid Sequence↗
PubMed1998

Developmental regulation of transcription of whiE, a locus specifying the polyketide spore pigment in Streptomyces coelicolor A3 (2)

whiE is a complex locus that specifies the polyketide spore pigment in Streptomyces coelicolor A3(2). Two divergently oriented promoters, whiEP1 and whiEP2, were identified in the whiE gene cluster, and their activities were analyzed during colony development in wild-type and sporulation-deficient strains. Both promoters were developmentally regulated; whiEP1 and whiEP2 transcripts were detected transiently at approximately the time when sporulation septa were observed in the aerial hyphae, and transcription from both promoters depended on each of the six known "early" whi genes required for sporulation septum formation (whiA, -B, -G, -H, -I, and -J). Mutation of the late sporulation-specific sigma factor gene, sigF, had no effect on the activity of whiEP1 but blocked transcription from whiEP2. However, sigmaF-containing holoenzyme was not sufficient to direct transcription of whiEP2 in vitro. The whiEP2 promoter controls expression of whiE ORFVIII, encoding a putative flavin adenine dinucleotide-dependent hydroxylase that catalyzes a late tailoring step in the spore pigment biosynthetic pathway. Disruption of whiE ORFVIII causes a change in spore color, from grey to greenish (T.-W. Yu and D. A. Hopwood, Microbiology 141:2779-2791, 1995). Consistent with these observations, construction of a sigF null mutant of S. coelicolor M145 caused the same change in spore color, showing that disruption of sigF in S. coelicolor changes the nature of the spore pigment rather than preventing its synthesis altogether.

Bacterial Proteins↗
PubMed1997

Denaturation of circular or linear DNA facilitates targeted integrative transformation of Streptomyces coelicolor A3(2): possible relevance to other organisms.

Using Streptomyces coelicolor A3(2) protoplasts, the number of transformants obtained by homologous recombination of incoming double-stranded circular DNA with the recipient chromosome was greatly stimulated by simple denaturation of the donor DNA. This procedure was very effective with inserts over a ca. 100-fold size range, the largest tested being ca. 40-kb inserts in cosmids. These observations led to transformation experiments with linearized cloned DNA and randomly sheared genomic DNA. In both cases, DNA denaturation led to significant levels of transformation. Most of the transformants had resulted from the predicted homologous recombination events. A number of genetic manipulations will be made easier or possible by these procedures.

Bacterial Proteins↗
PubMed1997

A glgC gene essential only for the first of two spatially distinct phases of glycogen synthesis in Streptomyces coelicolor A3(2).

By using a PCR approach based on conserved regions of ADP-glucose pyrophosphorylases, a glgC gene was cloned from Streptomyces coelicolor A3(2). The deduced glgC gene product showed end-to-end relatedness to other bacterial ADP-glucose pyrophosphorylases. The glgC gene is about 1,000 kb from the leftmost chromosome end and is not closely linked to either of the two glgB genes of S. coelicolor, which encode glycogen branching enzymes active in different locations in differentiated colonies. Disruption of glgC eliminated only the first of two temporal peaks of ADP-glucose pyrophosphorylase activity and glycogen accumulation and prevented cytologically observable glycogen accumulation in the substrate mycelium of colonies (phase I), while glycogen deposition in young spore chains (phase II) remained readily detectable. The cloned glgC gene therefore encodes an ADP-glucose pyrophosphorylase essential only for phase I (and it is therefore named glgCI). A second, phase II-specific, glgC gene should also exist in S. coelicolor, though it was not detected by hybridization analysis.

Amino Acid Sequence↗
PubMed1996

Characterization of spaA, a Streptomyces coelicolor gene homologous to a gene involved in sensing starvation in Escherichia coli.

A Streptomyces coelicolor gene, called spaA, homologous to the stationary phase regulatory gene rspA of Escherichia coli [Huisman and Kolter (1994) Science 265, 537-539], was cloned using the Streptomyces ambofaciens rspA homologue spa2 [Schneider et al. (1993) J. Gen. Microbiol. 139, 2559-2567] as a probe. Considerable differences in sequence and in genetic context were detected between spa2 of S. ambofaciens and spaA of S. coelicolor. A cloned internal fragment of spaA was used to direct integration of a phage vector into the spaA gene. The disruption caused delayed antibiotic production (undecylprodigiosin and actinorhodin) and led on further incubation to increased actinorhodin production at high, but not low, cell density. This phenotype was apparent only on the nutritionally poorest of three media tested. The attempted use of an integrating plasmid-based system for gene replacement of spaA gave rise to extensive deletions of adjacent chromosomal DNA.

Amino Acid Sequence↗
PubMed1996

The positions of the sigma-factor genes, whiG and sigF, in the hierarchy controlling the development of spore chains in the aerial hyphae of Streptomyces coelicolor A3(2).

whiG and sigF encode RNA polymerase sigma factors required for sporulation in the aerial hyphae of Streptomyces coelicolor. Their expression was analysed during colony development in wild-type and sporulation-defective whi mutant strains. Each gene was transcribed from a single promoter. Unexpectedly, whiG mRNA was present at all time points, including those taken prior to aerial mycelium formation; this suggests that whiG may be regulated post-transcriptionally. Transcription of whiG did not depend upon any of the six known 'early' whi genes required for sporulation septum formation (whiA, B, G, H, I and J), placing it at the top of the hierarchy of whi loci. sigF expression appeared to be regulated at the level of transcription; sigF transcripts were detected transiently when sporulation septa were observed in the aerial hyphae. Transcription of sigF depended upon all six of the early whi genes, including whiG. The sigF promoter does not resemble the consensus sequence established for sigma WhiG-dependent promoters and E sigma WhiG did not transcribe from the sigF promoter in vitro. Consequently, the genetic dependence of sigF upon whiG is very likely to be indirect. These results show that there is a hierarchical relationship between sigma factors required for Streptomyces sporulation and also that at least five other genes are involved in this transcriptional network.

Bacterial Proteins↗
PubMed1995

Tissue-specific glycogen branching isoenzymes in a multicellular prokaryote, Streptomyces coelicolor A3(2).

In the overtly differentiated colonies of Streptomyces coelicolor A3(2), discrete phases of glycogen synthesis are found at the vegetative/aerial mycelium boundary (phase I) and in the immature spore chains at aerial hyphal tips (phase II). We have characterized two S. coelicolor glgB genes encoding glycogen branching enzyme, which are well separated in the genome. Disruption of glgBl led to the formation of abnormal polyglucan deposits at phase I, with phase II remaining normal, whereas disruption of glgBII interfered specifically with phase II deposits, and not with those of phase I. Thus, each branching enzyme isoform is involved in a different phase of glycogen synthesis. This situation contrasts with that in simple bacteria, which typically have a single set of enzymes for glycogen metabolism, and more closely resembles that in plants.

1,4-alpha-Glucan Branching Enzyme↗
PubMed1994

Sequence of the essential early region of phi C31, a temperate phage of Streptomyces spp. with unusual features in its lytic development.

The temperate phage phi C31 is the most studied bacteriophage infecting Streptomyces spp., and has been used to develop an extensive and widely used series of cloning vectors. The sequence of 10 kb of phi C31 DNA containing most or all of the essential early genes was determined. Among the ORFs, 14 (perhaps 15) appear to be protein-coding, and these have been designated ORF1 to ORF14 and ORFX. Previously mapped transcripts appear to initiate upstream from ORFs 1, 8, 11 and 12, and within ORF3 and ORF12, in each case close to one example of the unusual ('21-mer') sequences that appear to serve as a recognition site for RNA polymerase early in the phi C31 lytic cycle [Ingham et al., Mol. Microbiol. 9 (1993) 1267-1274]. Further copies of the 21-mer are upstream from ORF2 and ORF13. There are four recognisable examples of a conserved inverted repeat sequence motif (CIR) thought to bind phi C31 repressor [Smith and Owen, Mol. Microbiol. 5 (1991) 2833-2844]. Only one CIR is closely associated with a 21-mer sequence, though three are located between known transcription units. Of all 14 ORFs, only one (ORF11) would encode a protein unmistakably resembling other known proteins; its product appears to be a DNA polymerase. Strikingly, two codons, TTA (Leu) and AGG (Arg), are absent from the 14 ORFs.

Amino Acid Sequence↗
PubMed1994

[The effect of a promoter P28-1 of Bacillus subtilis on Streptomyces differentiation].

A 1.1kb promoter P28-1 was inserted into pUC19. After then, the P28-1 was subcloned into the HindIII-EcoRI sites of the high copy number Streptomyces promoter probe plasmid pIJ4083 containing xy1E reporter gene. This recombinant plasmid was designated as pIJ4498. When pIJ4498 was introduced into Streptomyces coelicolor J1501 protoplasts, transformants conferred a white phenotype, whereas the vector pIJ4083 gave rise to colonies of normal, dark grey appearance which is the same as that of J1501 itself. After confirming pIJ4498 structure with some restriction enzymes, it was also introduced into whiG mutant (C71). Crude enzyme extracts were isolated from J1501/pIJ4498, J1501/pIJ4083 and C71/pIJ4498 respectively, the crude enzyme extract from J1501/pIJ4498 could oxidize catechol (colourless) to 2-hydroxy muconic semialdehyde (yellow colour), but crude enzyme extracts from J1501/pIJ4083 and C71/pIJ4498 could not oxidize catechol to 2-hydroxymuconic semialdehyde. The results indicated that P28-1 might be recognised by sigma whiG RNA polymerase, and activated the xylE reporter gene expression and reduced J1501 sporulation.

Bacillus subtilis↗
PubMed1993

Hybridization and DNA sequence analyses suggest an early evolutionary divergence of related biosynthetic gene sets encoding polyketide antibiotics and spore pigments in Streptomyces spp.

The whiE gene cluster of Streptomyces coelicolor, which is related to gene sets encoding the biosynthesis of polycyclic aromatic polyketide antibiotics, determines a spore pigment. Southern blotting using probes from three different parts of the whiE cluster revealed related gene sets in about half of a collection of diverse Streptomyces strains. A 5.2-kb segment of one such cluster, sch, previously shown to determine spore pigmentation in Streptomyces halstedii, was sequenced. Seven open reading frames (ORFs), two of them incomplete, were found. Six of the ORFs resemble the known part of the whiE cluster closely. The derived gene products include a ketosynthase (= condensing enzyme) pair, acyl carrier protein and cyclase, as well as two of unidentified function. The seventh ORF diverges from the main cluster and encodes a protein that resembles a dichlorophenol hydroxylase. Comparison with sequences of related gene sets for the biosynthesis of antibiotics suggests that gene clusters destined to specify pigment production diverged from those destined to specify antibiotics early in the evolution of the Streptomyces genus.

Amino Acid Sequence↗
PubMed1993

Functional and evolutionary implications of a survey of various actinomycetes for homologues of two Streptomyces coelicolor sporulation genes.

In Streptomyces coelicolor A3(2) the whiB and whiG genes are essential for sporulation, their deduced products being a possible transcriptional activator and an RNA polymerase sigma factor, respectively. In a survey of DNA from diverse actinomycetes by Southern blotting, all samples tested hybridized with whiB, but only those representing genera capable of producing sporulating aerial mycelium hybridized with whiG. It is postulated that whiB may play a more intimate role in hyphal fragmentation processes (including sporulation) than whiG. The whiB and whiG homologues (whiB-Stv and whiG-Stv) of Streptoverticillium griseocarneum were cloned and sequenced, and subjected to functional tests in S. coelicolor whiB and whiG mutants. The genes were closely similar, but not identical, to their S. coelicolor counterparts at the DNA and deduced protein levels, and both Stv. griseocarnum gene products could function well in S. coelicolor. However, studies with hybrid transcription units suggested that the promoter region of whiB-Stv is somewhat inefficient in S. coelicolor.

Amino Acid Sequence↗
PubMed1993

Genetic analysis of the phi C31-specific phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2).

The phage growth limitation (Pgl) system of Streptomyces coelicolor A3(2) was shown to be specific to phi C31 homo-immune phages, and to be absent from the closely related strain Streptomyces lividans. A 16 kb fragment of S. coelicolor A3(2) DNA was isolated which complemented the Pgl- phenotype of J1501, a pgl mutant derivative of the Pglts S. coelicolor strain M130. The cloned DNA complemented only half of the available pgl mutants, which therefore represented at least two groups, designated Pgl class A and class B strains. It follows that more than one kind of high-frequency genetic event can lead to the Pgl- phenotype. Crosses between class A and class B strains yielded high frequencies of Pgl+ recombinants. Crosses between strains of the same class gave no Pgl+ recombinants. The cloned DNA was altered by deletion or apparent point mutation upon passage through the two class B strains tested, such that it was no longer capable of complementing class A strains. This accumulation of mutations might suggest that the expression of the cloned DNA is toxic to at least some class B strains. The nature of the genetic instability associated with the Pgl system was not detectable by Southern blot analysis.

Bacteriophages↗
PubMed1993

Multiple novel promoters from the early region in the Streptomyces temperate phage phi C31 are activated during lytic development.

Evidence is presented that transcription of most of the early genes in the Streptomyces coelicolor A3(2) phage phi C31 is from a series of unusual promoters that depend on a function expressed early in the phi C31 lytic cycle. Primer extension analysis on the 5' ends of three early mRNAs, from samples prepared 10 min after induction of a thermosensitive phi C31 lysogen, showed that the 5' ends all mapped close to highly similar sequences, which are proposed to be an important part of phage-specific promoters. In a shotgun cloning experiment, a fragment containing one of these sequences strongly activated transcription of the xyIE reporter gene in plaques of a phi C31-derived promoter-probe vector. Another of the sequences was inserted into a xyIE-containing promoter-probe plasmid vector, and promoted xyIE expression only when the host was supporting the lytic cycle of phi C31. This suggested that a transcription factor needed for activity of the promoters was present only in phi C31-infected cells. Examination of published and unpublished phi C31 sequence data revealed several more sequences that closely resemble the conserved region of the characterized promoters. Most of these are found in positions close to apparent transcription start sites mapped previously by low-resolution S1 mapping. An overall consensus sequence for the conserved region suggests a general organization (though not a primary sequence) resembling that of promoters recognized in other bacteria by the sigma 54 form of RNA polymerase.

Bacteriophages↗
PubMed1993

Complex transcription of an operon encoding the SalI restriction-modification system of Streptomyces albus G.

High-resolution S1 nuclease mapping of mRNA synthesised in vivo, in vitro run-off transcription with RNA polymerase from Streptomyces lividans and gene fusions were used to analyse the transcriptional organization of the SalI restriction-modification system of Streptomyces albus G. The salIR and salIM genes that encode the restriction endonuclease and its cognate methyltransferase constitute an operon which is mainly transcribed from sal-pR1, a promoter located immediately upstream of salIR, with two possible minor promoters further upstream. Another promoter, sal-pM, is within the 3' end of the salIR coding region, and allows expression of the modification gene in the absence of sal-pR1. The sal-pM promoter might be involved in the establishment of modification prior to restriction endonuclease activity. Sequences upstream of the apparent transcriptional start sites for sal-pR1 and sal-pM show similarity with the -10 region of typical vegetatively expressed eubacterial promoters, but appropriately centered -35 regions are absent.

Base Sequence↗
PubMed1993

A hydroxylase-like gene product contributes to synthesis of a polyketide spore pigment in Streptomyces halstedii.

A gene, schC, adjacent to the sch gene cluster encoding the biosynthesis of a polyketide spore pigment in Streptomyces halstedii was sequenced. Its deduced product resembled flavin adenine nucleotide-containing hydroxylases involved in the biosynthesis of polycyclic aromatic polyketide antibiotics and in catabolic pathways of aromatic compounds. When schC was disrupted, the normally green spores of S. halstedii became lilac. An schC-like gene was located in an equivalent position next to a large gene cluster (whiE) known to determine spore pigment in Streptomyces coelicolor A3(2).

Amino Acid Sequence↗
PubMed1993

Two developmentally controlled promoters of Streptomyces coelicolor A3(2) that resemble the major class of motility-related promoters in other bacteria.

Experiments were designed to allow isolation of Streptomyces coelicolor promoters that depend on the whiG sporulation gene, which encodes a putative sigma factor important in the sporulation of aerial hyphae. The strategy, based on earlier evidence that sigma WhiG is limiting for sporulation (K. F. Chater, C. J. Burton, K. A. Plaskitt, M. J. Buttner, C. Méndez, and J. Helmann, Cell 59:133-143, 1989) was to seek DNA fragments that inhibit sporulation in aerial hyphae when present at a high copy number. In a suitable Sau3AI-generated library of DNA from S. coelicolor A3(2), two inserts were found to inhibit sporulation. Both inserts caused expression of the adjacent xylE reporter gene present in the vector in a developmentally normal strain of S. coelicolor, but there was no xylE expression in an otherwise isogenic whiG mutant. S1 nuclease protection experiments were done with RNAs isolated from these plasmid-bearing strains or from the wild-type strain lacking either recombinant plasmid. In each case, an apparent transcription start site was found upstream of an apparent open reading frame (ORF) and just downstream of sequences that resemble consensus features of promoters for motility-related genes in Bacillus subtilis and coliform bacteria. Such promoters depend on sigma factors (sigma D and sigma F, respectively) particularly similar to the deduced whiG gene product. Each of the putative whiG-dependent promoters is within an ORF that is upstream of, and potentially translationally coupled to, the putative whiG-dependent ORF (although use of one of the promoters would necessitate the use of a different start codon, further downstream). Thus, in unknown circumstances, the whiG-dependent ORFs may be expressed from a more remote promoter as part of a complex transcription unit.

Amino Acid Sequence↗