Mark J. Buttner

RsrA, an anti‐sigma factor regulated by redox change

SigR (σR) is a sigma factor responsible for inducing the thioredoxin system in response to oxidative stress in the antibiotic‐producing, Gram‐positive bacterium Streptomyces coelicolor A3(2). Here we identify a redox‐sensitive, σR‐specific anti‐sigma factor, RsrA, which binds σR and inhibits σR‐directed transcription in vitro only under reducing conditions. Exposure to H2O2 or to the thiol‐specific oxidant diamide caused the dissociation of the σR–RsrA complex, thereby allowing σR‐dependent transcription. This correlated with intramolecular disulfide bond formation in RsrA. Thioredoxin was able to reduce oxidized RsrA, suggesting that σR, RsrA and the thioredoxin system comprise a novel feedback homeostasis loop that senses and responds to changes in the intracellular thiol–disulfide redox balance.

σR, an RNA polymerase sigma factor that modulates expression of the thioredoxin system in response to oxidative stress in Streptomyces coelicolor A3(2)

We have identified an RNA polymerase sigma factor, σR, that is part of a system that senses and responds to thiol oxidation in the Gram‐positive, antibiotic‐producing bacterium Streptomyces coelicolor A3(2). Deletion of the gene (sigR) encoding σR caused sensitivity to the thiol‐specific oxidant diamide and to the redox cycling compounds menadione and plumbagin. This correlated with reduced levels of disulfide reductase activity and an inability to induce this activity on exposure to diamide. The trxBA operon, encoding thioredoxin reductase and thioredoxin, was found to be under the direct control of σR. trxBA is transcribed from two promoters, trxBp1 and trxBp2, separated by 5–6 bp. trxBp1 is transiently induced at least 50‐fold in response to diamide treatment in a sigR‐dependent manner. Purified σR directed transcription from trxBp1 in vitro, indicating that trxBp1 is a target for σR. Transcription of sigR itself initiates at two promoters, sigRp1 and sigRp2, which are separated by 173 bp. The sigRp2 transcript was undetectable in a sigR‐null mutant, and purified σR could direct transcription from sigRp2 in vitro, indicating that sigR is positively autoregulated. Transcription from sigRp2 was also transiently induced (70‐fold) following treatment with diamide. We propose a model in which σR induces expression of the thioredoxin system in response to cytoplasmic disulfide bond formation. Upon reestablishment of normal thiol levels, σR activity is switched off, resulting in down‐regulation of trxBA and sigR. We present evidence that the σR system also functions in the actinomycete pathogen Mycobacterium tuberculosis.

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, /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 σWhiG‐dependent promoters and EnWhiG 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.

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 σ70 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 σB of Bacillus subtilis. However, while σB is involved in stationary‐phase gene expression and in the general stress response in B. subtilis, σF 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. σF is proposed to control the late stages of spore development in Streptomyces.

A putative two-component signal transduction system regulates sigmaE, a sigma factor required for normal cell wall integrity in Streptomyces coelicolor A3(2).

The extracytoplasmic function (ECF) sigma factor, σE, is required for normal cell wall integrity in Streptomyces coelicolor. We have investigated the regulation of σE through a transcriptional and mutational analysis of sigE and the surrounding genes. Nucleotide sequencing identified three genes located downstream of sigE ; orf202, cseB and cseC (cse, control of sigE ). cseB and cseC encode a putative response regulator and a putative transmembrane sensor histidine protein kinase respectively. Although most sigE transcription appeared to be monocistronic, sigE was also transcribed as part of a larger operon, including at least orf202. sigE null mutants are sensitive to cell wall lytic enzymes, have an altered peptidoglycan muropeptide profile, and on medium deficient in Mg2+ they overproduce actinorhodin, sporulate poorly and form crenellated colonies. A constructed cseB null mutant appeared to have the same phenotype as a sigE null mutant, which was accounted for by the observed absolute dependence of the sigE promoter on cseB. It is likely that the major role of cseB is to regulate sigE transcription because expression of sigE alone from a heterologous promoter suppressed the cseB mutation. Mg2+ suppresses the CseB/SigE phenotype, probably by stabilizing the cell envelope, and sigE transcript levels were consistently higher in Mg2+‐deficient cultures than in high Mg2+‐grown cultures. We propose a model in which the CseB/CseC two‐component system modulates activity of the sigE promoter in response to signals from the cell envelope.

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 StreptomycesσE cognate promoter, and purified EσE holoenzyme transcribed the phsA promoter in vitro. However, the phsA promoter was still active in an S. antibioticussigE 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.

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.

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.

Genetic analysis of the φC31 ‐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 φC31 homo‐immune phages, and to be absent from the closely related strain Streptomyces Iividans. 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 PgltsS. 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.