Implication of stringent response in the increase of mutability of the whiG and whiH genes during Streptomyces coelicolor development

In Streptomyces ambofaciens, genetic instability occurring during aerial mycelium development gives rise to white mutant papillae on colonies. Pig-pap mutants deriving from such papillae are unable to sporulate and devoid of the large genome rearrangement usually observed in the other Streptomyces mutants that genetic instability generated. Pig-pap mutants frequently harboured discrete mutations affecting the whiG gene. Furthermore, it has been established that the production of papillae dramatically increased when S. ambofaciens was grown under an amino acid limitation. In this work, we tested the implication of the stringent response, induced during an amino acid limitation, in the production of white papillae in Streptomyces coelicolor, a species which is phylogenetically close to S. ambofaciens. First, we showed that S. coelicolor produced mutant papillae and that this production was increased under an amino acid limitation. Secondly, we showed that the Pig-pap mutants generated both with and without amino acid limitation frequently exhibited mutations in whiH or whiG genes. Finally, we observed that a relA mutant of S. coelicolor, which was unable to elicit the stringent response under an amino acid limitation, was also unable to produce papillae. The restoration of the ability to elicit the stringent response also restored the papillae production. These papillae gave rise to Pig-pap mutants displaying the same characteristics as Pig-pap mutants spontaneously appearing on wild-type colonies. Altogether, these results show that whatever the underlying mechanism, the stringent response is involved in the production of white papillae in S. coelicolor.     

The Streptomyces coelicolor A3(2) bldB region contains at least two genes involved in morphological development

Streptomyces coelicolor A3(2) bldB mutants are blocked in the formation of aerial hyphae. A phage library of wild-type S. coelicolor DNA was used to isolate recombinant phages which restore wild-type morphological development to several bldB mutants. Of several mutations, one, bld-28, previously mapped at bldB was not complemented by the cloned region, indicating that the bldB locus is composed of at least two distinct genes. Partial localization of bldB-complementing activity showed that a 1.5 kb fragment is sufficient for complementation of the bld-15 mutation whereas bld-17 requires the same region as well as additional sequences. Under stringent conditions, genomic DNA hybridizing to the cloned sequences was absent from other Streptomyces species, including the closely related Streptomyces lividans 66. DNA sequences causing marked plasmid structural instability in S. coelicolor, but not in S. lividans, are also located in this region.     

圈卷产色链霉菌分化相关基因——saw1的结构和功能

用双脱氧链终止法进行了分化基因——saw1的双链测序.结果表明在1500bp的DNA片段中有一个完整的开读框架(ORF),其编码区是在419bp至1252bp处.其产物与已知的天蓝色链霉菌whiG的氨基酸序列有89%的同源性.当把1500bp的saw1DNA片段插入到链霉菌表达质粒载体pIJ702后,构建的重组质粒转化天蓝色链霉菌孢子形成缺陷突变株C71,可使C71形成孢子和灰色色素.用基因破坏的策略进一步研究了该基因的生物学功能,结果表明saw1在圈卷产色链霉菌气生菌丝到孢子形成的发育转变中有重要作用,是分化中控制孢子发育起始的一个重要基因.     

Glycosylation of a Streptomyces coelicolor A3(2) cell envelope protein is required for infection by bacteriophage phi C31

Mutants of Streptomyces coelicolor A3(2) J1929 (Delta pglY) were isolated that were resistant to the Streptomyces temperate phage phi C31. These strains could be transfected with phi C31 DNA, but could not act as infective centres after exposure to phage. Thus, it was concluded that infection was blocked at the adsorption/DNA injection step. The mutants fell into three classes. Class I mutants were complemented by a gene, SCE87.05, isolated from the cosmid library of S. coelicolor A3(2). The product of SCE87.05 had good overall homology to a Mycobacterium tuberculosis hypothetical protein and regions with similarity to dolichol phosphate-D-mannose:protein O-D-mannosyltransferases. Concanavalin A (ConA) inhibited phi C31 infection of S. coelicolor J1929, and this could be partially reversed by the addition of the sugar, alpha-D-methyl-pyranoside. Moreover, glycosylated proteins from J1929, but not from the class I mutant DT1017, were detected using ConA as a probe in Western blots. Class I and II mutants were sensitive to phi C31hc, a previously isolated phage exhibiting an extended host range phenotype, conferred by h. A phage with the same phenotype, phi DT4002, was isolated independently, and a missense mutation was found in a putative tail gene. It is proposed that the phi C31 receptor is a cell wall glycoprotein, and that the phi C31h mutation compensates for the lack of glycosylation of the receptor.     

High-level expression of whiG——A key gene for Streptomyces differentiation in Escherichia coli

Six nucleotides located in the region of translation start site of whiG were changed. whiG was amplified by PCR technique. Reformed sequences were determined. This gene was directly subcloned into expression vector pET11c containing strong T7 promoter, and the recombinant plasmid was introduced into E. coli BL21(DE3), which could be induced by IPTG to produce T7 RNA polymerase. The SDS-PAGE result showed that whiG highly expressed in E. coli BL21(DE3), and the yield of whiG product was about 20% of insoluble proteins in cell. whiG product (σwhiG) was further identified by Western blot hybridization after making its antibody. whiG gene was subcloned into Streptomyces plasmid pIJ6021, and then it was introduced into sporulation deficient mutant C71 from Streptomyces coelicolor. The result showed that C71 could restore sporulation and σwhiG has biological functions.     

天蓝色链霉菌孢子形成的关键基因——whiG的诱导表达

与链霉菌分化有关的whiG结构基因已亚克隆到链霉菌表达载体pAK203的tipA启动子下游.该启动子能被硫链丝菌素诱导.whiG基因的诱导表达不仅可使天蓝色链霉菌孢子形成缺陷突变株C71恢复产生孢子的能力,而且也能使天蓝色链霉菌野生型菌株J1501和变铅青链霉菌TK54的孢子形成更为丰满.Western杂交也进一步证实了诱导表达后whiG基因产物——σ~(whiG)产量的增加.这为依赖于σ~(whiG)RNA多聚酶的发育调控启动子体外转录的研究提供了有利条件.     

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.     

WhiA, a protein of unknown function conserved among gram-positive bacteria, is essential for sporulation in Streptomyces coelicolor A3(2)

The whiA sporulation gene of Streptomyces coelicolor A3(2), which plays a key role in switching aerial hyphae away from continued extension growth and toward sporulation septation, was cloned by complementation of whiA mutants. DNA sequencing of the wild-type allele and five whiA mutations verified that whiA is a gene encoding a protein with homologues in all gram-positive bacteria whose genome sequence is known, whether of high or low G+C content. No function has been attributed to any of these WhiA-like proteins. In most cases, as in S. coelicolor, the whiA-like gene is downstream of other conserved genes in an operon-like cluster. Phenotypic analysis of a constructed disruption mutant confirmed that whiA is essential for sporulation. whiA is transcribed from at least two promoters, the most downstream of which is located within the preceding gene and is strongly up-regulated when colonies are undergoing sporulation. The up-regulation depends on a functional whiA gene, suggesting positive autoregulation, although it is not known whether this is direct or indirect. Unlike the promoters of some other sporulation-regulatory genes, the whiA promoter does not depend on the sporulation-specific sigma factor encoded by whiG.     

New Sporulation Loci in Streptomyces coelicolor A3(2)

Sporulation mutants of Streptomyces coelicolor appear white because they are defective in the synthesis of the grey polyketide spore pigment, and such white (whi) mutants had been used to define eight sporulation loci, whiA, whiB, whiD, whiE, whiG, whiH, whiI, and whiJ (K. F. Chater, J. Gen. Microbiol. 72:9-28, 1972; N. J. Ryding, Ph.D. thesis, University of East Anglia, 1995). In an attempt to identify new whi loci, we mutagenized S. coelicolor M145 spores with nitrosoguanidine and identified 770 mutants with colonies ranging from white to medium grey. After excluding unstable strains, we examined the isolates by phase-contrast microscopy and chose 115 whi mutants with clear morphological phenotypes for further study. To exclude mutants representing cloned whi genes, self-transmissible SCP2*-derived plasmids carrying whiA, whiB, whiG, whiH, or whiJ (but not whiD, whiE, or whiI) were introduced into each mutant by conjugation, and strains in which the wild-type phenotype was restored either partially or completely by any of these plasmids were excluded from further analysis. In an attempt to complement some of the remaining 31 whi mutants, an SCP2* library of wild-type S. coelicolor chromosomal DNA was introduced into 19 of the mutants by conjugation. Clones restoring the wild-type phenotype to 12 of the 19 strains were isolated and found to represent five distinct loci, designated whiK, whiL, whiM, whiN, and whiO. Each of the five loci was located on the ordered cosmid library: whiL, whiM, whiN, and whiO occupied positions distinct from previously cloned whi genes; whiK was located on the same cosmid overlap as whiD, but the two loci were shown by complementation to be distinct. The phenotypes resulting from mutations at each of these new loci are described.     

Characterization of Temperate Actinophage φC31 Isolated from Streptomyces coelicolor A3(2)

Actinophage phiC31 isolated from Streptomyces coelicolor A3(2), the only strain among actinomycetes for which a genetic map had been constructed, appears to be a typical temperate phage. After phiC31 infection, true lysogenic cultures arose which liberated phage and were immune to infection with homologous phage after repeated single-colony isolations and treatment with phage-specific antiserum. Clear-plaque (c) mutants were derived from phiC31 phage which failed to lysogenize sensitive cultures. Actinophage phiC31 has a temperature-sensitive stage of reproduction. A phage which reproduces with the same effectiveness at high (37 C) and low (28 C) temperatures has also been obtained. Heat-inducible (ct) mutants were isolated from this phage which were able to lysogenize sensitive cultures at 28 C but failed to do so at 37 C. Properties of ct mutants suggest that ct mutations involve a gene controlling maintenance of the lysogenic state in actinomycetes and synthesizing repressor, which may become heat-sensitive as a result of mutation.     

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.     

Cloning of DNA involved in sporulation of Streptomyces griseus

Twenty-two bald mutants of Streptomyces griseus were isolated and classified into four phenotypic groups, two of which showed conditional sporulation. A 3-kilobase fragment of DNA was cloned in a high-copy-number vector and detected by its ability to restore sporulation to one class of conditionally bald mutants. Analysis of subclones demonstrated that the sporulation property was contained within a 2.5-kilobase fragment. Hybridization studies and restriction analysis indicated that this DNA fragment was present in several Streptomyces species and was distinct from DNA that has been shown to complement afsA mutants of S. bikiniensis and bldA mutants of S. coelicolor.     

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.     

abaA, a new pleiotropic regulatory locus for antibiotic production in Streptomyces coelicolor.

Production of the blue-pigmented antibiotic actinorhodin is greatly enhanced in Streptomyces lividans and Streptomyces coelicolor by transformation with a 2.7-kb DNA fragment from the S. coelicolor chromosome cloned on a multicopy plasmid. Southern analysis, restriction map comparisons, and map locations of the cloned genes revealed that these genes were different from other known S. coelicolor genes concerned with actinorhodin biosynthesis or its pleiotropic regulation. Computer analysis of the DNA sequence showed five putative open reading frames (ORFs), which were named ORFA, ORFB, and ORFC (transcribed in one direction) and ORFD and ORFE (transcribed in the opposite direction). Subcloning experiments revealed that ORFB together with 137 bp downstream of it is responsible for antibiotic overproduction in S. lividans. Insertion of a phi C31 prophage into ORFB by homologous recombination gave rise to a mutant phenotype in which the production of actinorhodin, undecylprodigiosin, and the calcium-dependent antibiotic (but not methylenomycin) was reduced or abolished. The nonproducing mutants were not affected in the timing or vigor or sporulation. A possible involvement of ORFA in antibiotic production in S. coelicolor is not excluded. abaA constitutes a new locus which, like the afs and abs genes previously described, pleiotropically regulates antibiotic production. DNA sequences that hybridize with the cloned DNA are present in several different Streptomyces species.