Biomedical subjects

M J Buttner

Publications and source records attributed to M J Buttner.

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

Characterization of the rpoC gene of Streptomyces coelicolor A3(2) and its use to develop a simple and rapid method for the purification of RNA polymerase.

The Streptomyces coelicolor rpoC gene, that encodes the beta' subunit of RNA polymerase, was isolated using the Escherichia coli rpoC gene as a hybridization probe. Comparison of the predicted amino acid sequence of the S. coelicolor beta' subunit to those characterized from other bacteria revealed three distinct subfamilies of beta' subunits, one of which consists of the S. coelicolor subunit and those from Mycobacterium leprae and Mycoplasma genitalium. Using site-directed mutagenesis, the carboxy terminus of the S. coelicolor beta' subunit was modified to contain six histidine residues. The histidine-tagged gene, rpoCHIS, was used to replace the wild-type allele in the chromosome of S. coelicolor and S. lividans. These strains were unaffected in growth and sporulation, demonstrating that the histidine-tagged RNA polymerase was competent to carry out all essential in-vivo functions. During a 1-day procedure, highly purified RNA polymerase was obtained by nickel-NTA agarose affinity chromatography followed by heparin-sepharose chromatography. Using in-vitro run-off transcription assays, the affinity purified RNA polymerase was shown to initiate transcription correctly from the S. lividans galP1 and galP2 promoters, and the Bacillus subtilus veg and ctc promoters. An extension of this procedure yielded highly-purified core RNA polymerase. To facilitate introduction of the rpoCHIS allele into other genetic backgrounds, a mutation in the adjacent gene, rpoB (rifA), conferring rifampin-resistance, was isolated in S. coelicolor to provide a genetic marker to follow transfer of the rpoCHIS allele. The use of this affinity chromatography procedure, in combination with the ability to introduce the rpoCHIS allele into different Streptomyces strains by transformation, will greatly facilitate the in-vitro analysis of transcription in members of this genus.

Amino Acid Sequence↗

PubMed1997

Sigma-E is required for the production of the antibiotic actinomycin in Streptomyces antibioticus.

The phsA gene encodes phenoxazinone synthase (PHS), which catalyses the penultimate step in the pathway for actinomycin biosynthesis in Streptomyces antibioticus. The phsA promoter strikingly resembles a putative Streptomyces sigma E cognate promoter, and purified E sigma E holoenzyme transcribed the phsA promoter in vitro. However, the phsA promoter was still active in an S. antibioticus sigE null mutant and the level of PHS activity was unaffected. Despite this, disruption of sigE blocked actinomycin production completely. The loss of actinomycin production correlated with a 10-fold decrease in the activity of actinomycin synthetase I, the enzyme which catalyses the activation of the precursor of the actinomycin chromophore.

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

A new RNA polymerase sigma factor, sigma F, is required for the late stages of morphological differentiation in Streptomyces spp.

A gene (sigF) encoding a new sigma factor was isolated from Streptomyces aureofaciens using a degenerate oligonucleotide probe designed from the GLI(KDNE)A motif lying within the well-conserved region 2.2 of the eubacterial sigma 70 family. Homologues were present in other Streptomyces spp., and that of the genetically well studied Streptomyces coelicolor A3(2) was also cloned. The nucleotide sequences of the two sigF genes were determined and shown to encode primary translation products of 287 (S. coelicolor) and 295 (S. aureofaciens) amino acid residues, both showing greatest similarity to sigma B of Bacillus subtilis. However, while sigma B is involved in stationary-phase gene expression and in the general stress response in B. subtilis, sigma F affects morphological differentiation in Streptomyces. Disruption of sigF did not affect vegetative growth but did cause a whi mutant phenotype. Microscopic examination showed that the sigF mutant produced spores that were smaller and deformed compared with those of the wild type, that the spore walls were thinner and sensitive to detergents and that in sigF mutant spores the chromosome failed to condense. sigma F is proposed to control the late stages of spore development in Streptomyces.

Amino Acid Sequence↗

PubMed1995

Deletion of DNA lying close to the glkA locus induces ectopic sporulation in Streptomyces coelicolor A3(2).

Streptomyces coelicolor A3(2) J1668 sporulated ectopically in the substrate hyphae (the Esp phenotype) with the same time course as sporulation in the aerial hyphae. Examination of related strains implied that the Esp phenotype was caused by the deletion of DNA that lies close to, but is distinct from, the glucose kinase gene (glkA). Co-transduction of the Esp phenotype with the deletion present in J1668 confirmed this hypothesis. The size of the deletion was found to be 7.4 kb. Construction of a strain carrying both the J1668 deletion and a whiG mutation demonstrated that the Esp phenotype depends on at least one of the genes required for the differentiation of aerial hyphae into spores.

Bacterial Proteins↗

PubMed1995

Two genes involved in the phase-variable phi C31 resistance mechanism of Streptomyces coelicolor A3(2).

The phage growth limitation (Pgl) system of Streptomyces coelicolor confers resistance to phi C31 and its homoimmune phages. The positions of the pgl genes within a 16-kb clone of S. coelicolor DNA were defined by subcloning, insertional inactivation, and deletion mapping. Nucleotide sequencing and functional analysis identified two genes, pglY and pglZ, required for the Pgl+ (phage-resistant) phenotype. pglY and pglZ, which may be translationally coupled, are predicted to encode proteins with M(r)S of 141,000 and 104,000, respectively. Neither protein shows significant similarity to other known proteins, but PglY has a putative ATP/GTP binding motif. The pglY and pglZ genes are cotranscribed from a single promoter which appears to be constitutive and is not induced by phage infection.

Amino Acid Sequence↗

PubMed1995

Mapping of genes involved in macromolecular synthesis on the chromosome of Streptomyces coelicolor A3(2).

The genes for the beta, beta', and seven sigma factor subunits of RNA polymerase, for elongation factors EF-Tu1 and EF-Tu3, and for six rRNA operons were mapped on the combined genetic and physical map of the Streptomyces coelicolor chromosome. Like the previously mapped tRNA genes, the RNA polymerase and rRNA genes map to scattered positions. The lack of rRNA operons in the immediate vicinity of the origin of replication (oriC) and the absence of tRNA genes in any of the rRNA operons are novel features of the Streptomyces chromosome.

Chromosome Mapping↗

PubMed1994

Analysis of the Streptomyces coelicolor sigE gene reveals the existence of a subfamily of eubacterial RNA polymerase sigma factors involved in the regulation of extracytoplasmic functions.

sigma E, an RNA polymerase sigma factor of apparent M(r) 28,000, was previously identified by its ability to direct transcription from the P2 promoter of the agarose gene (dagA) of Streptomyces coelicolor. A degenerate oligonucleotide probe, designed from the N-terminal sequence of purified sigma E, was used to isolate the sigma E gene (sigE). The predicted sequence of sigma E shows greatest similarity to sequences of seven other proteins: Myxococcus xanthus CarQ, Pseudomonas aeruginosa AlgU, Pseudomonas syringae HrpL, Escherichia coli sigma E, Alcaligenes eutrophus CnrH, E. coli FecI, and Bacillus subtilis SigX, a protein of unknown function. These eight proteins define a subfamily of eubacterial RNA polymerase factors sufficiently different from other sigma s that, in many cases, they are not identified by standard similarity searching methods. Available information suggests that all of them regulate extracytoplasmic functions and that they function as effector molecules responding to extracytoplasmic stimuli. A. eutrophus CnrH appears to be a plasmid-encoded factor.

Amino Acid Sequence↗

PubMed1994

Glucose repression in Streptomyces coelicolor A3(2): a likely regulatory role for glucose kinase.

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).

Blotting, Southern↗

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↗

PubMed1992

Isolation and characterization of the major vegetative RNA polymerase of Streptomyces coelicolor A3(2); renaturation of a sigma subunit using GroEL.

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 dagAp4 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 dagAp4 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.

Amino Acid Sequence↗

PubMed1992

Construction and characterization of Streptomyces coelicolor A3(2) mutants that are multiply deficient in the nonessential hrd-encoded RNA polymerase sigma factors.

Previous studies showed that Streptomyces coelicolor A3(2) has four genes (hrdA, hrdB, hrdC, and hrdD) that appear to encode RNA polymerase sigma factors very similar to the sigma 70 subunit of Escherichia coli and that hrdC and hrdD could be individually disrupted without causing obvious phenotypic defects. Here, hrdA was cloned and stable null hrdA and hrdD mutants were constructed by gene replacement. These two mutants and a previously constructed hrdC null mutant were used in crosses to generate hrdAC, hrdAD, hrdCD, and hrdACD strains. The inability to synthesize one, two, or all three of the nonessential hrd-encoded sigma factors had no obvious phenotypic consequences.

Alleles↗

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

Characterization of a gene conferring bialaphos resistance in Streptomyces coelicolor A3(2).

A gene (bar) was identified adjacent to the hrdD sigma factor gene in Streptomyces coelicolor A3(2). The predicted bar product showed 32.2% and 30.4% identity to those of the pat and bar genes of the bialaphos (Bp) producers Streptomyces viridochromogenes and Streptomyces hygroscopicus, respectively; these genes encode phosphinothricin (PPT) N-acetyltransferases that function as enzymes in the Bp biosynthetic pathway and as resistance determinants. The S. coelicolor bar gene conferred high-level resistance to Bp when cloned in S. coelicolor on a high-copy-number vector. Enzymic assay showed that the S. coelicolor bar gene product inactivates PPT by transfer of acetyl groups from acetyl CoA. The S. coelicolor bar gene appears to be expressed from two promoters (p1 and p2) and is divergently transcribed with respect to hrdD. The downstream (barp2) transcript overlaps the hrdDp1 transcript and the upstream (barp1) transcript overlaps both the hrdDp1 and hrdDp2 transcripts. Inactivation of hrdD did not prevent transcription from either bar promoter, indicating that sigma hrdD is not essential for recognition of these sequences.

Acetyltransferases↗

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↗