The Leeuwenhoek Lecture, 1987 - Towards an understanding of gene switching in Streptomyces , the basis of sporulation and antibiotic production

1988 ◽  
Vol 235 (1279) ◽  
pp. 121-138 ◽  

Streptomycetes are soil bacteria that differ from the genetically well-known Escherichia coli in two striking characteristics. (1) Instead of consisting of an alternation of growth and fission of morphologically simple, undifferentiated rods, the streptomycete life cycle involves the formation of a system of elongated, branching hyphae which, after a period of vegetative growth, respond to specific signals by producing specialized spore-bearing structures. (2) The streptomycetes produce an unrivalled range of chemically diverse ‘secondary metabolites’, which we recognize as antibiotics, herbicides and pharmacologically active molecules, and which presumably play an important role in the streptomycete life cycle in nature. This ‘physiological’ differentiation is often tem­porally associated with the morphological differentiation of sporulation and there are common elements in the regulation of the two sets of processes. In the model system provided by Streptomyces coelicolor A3(2), the isolation of several whole clusters of linked antibiotic biosynthetic pathway genes, and some key regulatory genes involved in sporulation, has made it possible to study the basis for the switching on and off of particular sets of genes during morphological and ‘physiological’ differen­tiation. Genetic analysis clearly reveals a regulatory cascade operating at several levels in a ‘physiological’ branch of the differentiation control system. At the lowest level, within individual clusters of antibiotic biosynthesis genes are genes with a role as activators of the structural genes for the pathway enzymes, and also resistance genes. It is attractive to speculate that the latter play a dual role: protecting the organism from self-destruction by its own potentially lethal product, and forming an essential component of a regulatory circuit that activates the biosyn­thetic genes, thus ensuring that resistance is established before any antibiotic is made. A next higher level of regulation is revealed by the isolation of mutations in a gene ( afsB ) required for expression (probably at the level of transcription) of all five known secondary metabolic pathways in the organism. At a higher level still, the bldA gene, whose product seems to be a tRNA essential to translate the rare (in high [G + C] Streptomyces DNA) TTA leucine codon, controls or influences the whole gamut of morphological and ‘physiological’ differentiation, because bldA mutants fail to produce either secondary metabolites or aerial mycelium and spores, while growing normally in the vegetative phase. Thus a decision to switch from vegetative growth to the secondary phase of colonial development may be taken at the level of translation. In the ‘morphological’ branch of the proposed regulatory cascade, a key gene is whiG whose product, essential for the earliest known step in the metamorphosis of aerial hyphae into spore chains, appears to be an RNA polymerase sigma factor which is not needed for transcription of vegetative genes, but seems to control, at the level of transcription, the decision to sporulate.

1997 ◽  
Vol 43 (12) ◽  
pp. 1118-1125 ◽  
Author(s):  
Martine Aubert ◽  
Elisabeth Weber ◽  
Brigitte Gintz ◽  
Bernard Decaris ◽  
Keith F. Chater

The deduced product of the spa2 gene of Streptomyces ambofaciens is a homologue of RspA, involved in stationary-phase σs factor regulation in Escherichia coli. This suggests that Spa2 could play a part in stationary-phase-associated differentiation in S. ambofaciens. The disruption of spa2 led to reductions in aerial mycelial development and associated spore pigmentation. The mutant phenotype reverted to the wild-type phenotype when the disruption construct spontaneously excised. The spa2 disruption had no detectable effect on growth rates in different media or antibiotic production and resistance. When spa2 was placed on a multicopy plasmid, a severe defect in formation and pigmentation of aerial mycelium resulted. These results strongly suggest that Spa2 is involved in a complex manner in the morphological differentiation process.Key words: Streptomyces, differentiation, stationary-phase regulator.


2011 ◽  
Vol 77 (21) ◽  
pp. 7586-7594 ◽  
Author(s):  
Fernando Santos-Beneit ◽  
Mónica Barriuso-Iglesias ◽  
Lorena T. Fernández-Martínez ◽  
Miriam Martínez-Castro ◽  
Alberto Sola-Landa ◽  
...  

ABSTRACTThe RNA polymerase (RNAP) omega factor (ω) forms a complex with the α2ββ′ core of this enzyme in bacteria. We have characterized therpoZgene ofStreptomyces coelicolor, which encodes a small protein (90 amino acids) identified as the omega factor. Deletion of therpoZgene resulted in strains with a slightly reduced growth rate, although they were still able to sporulate. The biosynthesis of actinorhodin and, particularly, that of undecylprodigiosin were drastically reduced in the ΔrpoZstrain, suggesting that expression of these secondary metabolite biosynthetic genes is dependent upon the presence of RpoZ in the RNAP complex. Complementation of the ΔrpoZmutant with the wild-typerpoZallele restored both phenotype and antibiotic production. Interestingly, therpoZgene contains a PHO box in its promoter region. DNA binding assays showed that the phosphate response regulator PhoP binds to such a region. Since luciferase reporter studies showed thatrpoZpromoter activity was increased in a ΔphoPbackground, it can be concluded thatrpoZis controlled negatively by PhoP, thus connecting phosphate depletion regulation with antibiotic production and morphological differentiation inStreptomyces.


2014 ◽  
Vol 70 (a1) ◽  
pp. C214-C214
Author(s):  
Jun Ohtsuka ◽  
Ming Dong Yao ◽  
Koji Nagata ◽  
Ken-ichi Miyazono ◽  
Yuehua Zhi ◽  
...  

AdpA is the central transcriptional factor in the A-factor regulatory cascade of Streptomyces griseus and activates hundreds of genes required for both secondary metabolism and morphological differentiation, leading to onset of streptomycin biosynthesis as well as aerial mycelium formation and sporulation. It has been shown that AdpA binds to over 500 operator regions with the loosely conserved consensus sequence, 5'-TGGCSNGWWY-3' (S: G or C; W: A or T; Y: T or C; and N: any nucleotide). However, it is still obscure how AdpA can control hundreds of genes. To reveal the molecular basis of the low nucleotide sequence specificity, we have determined the crystal structure of the complex of DNA-binding domain of AdpA and a 14-mer duplex DNA with two-nucleotide overhangs at 5'-ends at 2.95-Å resolution. The crystal structure and electrophoretic mobility-shift assays showed that only two arginine residues, Arg262 and Arg266, are involved in the sequence recognition and determine the nucleotide specificity/preference of continuous five base-pairs of positions 1–5 in the consensus sequence. These results partially explain how AdpA directly controls hundreds of genes.


2016 ◽  
Vol 82 (18) ◽  
pp. 5661-5672 ◽  
Author(s):  
Steffen Sigle ◽  
Nadja Steblau ◽  
Wolfgang Wohlleben ◽  
Günther Muth

ABSTRACTAlthough anionic glycopolymers are crucial components of the Gram-positive cell envelope, the relevance of anionic glycopolymers for vegetative growth and morphological differentiation ofStreptomyces coelicolorA3(2) is unknown. Here, we show that the LytR-CpsA-Psr (LCP) protein PdtA (SCO2578), a TagV-like glycopolymer transferase, has a dual function in theS. coelicolorA3(2) life cycle. Despite the presence of 10 additional LCP homologs, PdtA is crucial for proper sporulation. The integrity of the spore envelope was severely affected in apdtAdeletion mutant, resulting in 34% nonviable spores.pdtAdeletion caused a significant reduction in the polydiglycosylphosphate content of the spore envelope. Beyond that, apical tip extension and normal branching of vegetative mycelium were severely impaired on high-salt medium. This growth defect coincided with the mislocalization of peptidoglycan synthesis. Thus, PdtA itself or the polydiglycosylphosphate attached to the peptidoglycan by the glycopolymer transferase PdtA also has a crucial function in apical tip extension of vegetative hyphae under stress conditions.IMPORTANCEAnionic glycopolymers are underappreciated components of the Gram-positive cell envelope. They provide rigidity to the cell wall and position extracellular enzymes involved in peptidoglycan remodeling. AlthoughStreptomyces coelicolorA3(2), the model organism for bacterial antibiotic production, is known to produce two distinct cell wall-linked glycopolymers, teichulosonic acid and polydiglycosylphosphate, the role of these glycopolymers in theS. coelicolorA3(2) life cycle has not been addressed so far. This study reveals a crucial function of the anionic glycopolymer polydiglycosylphosphate for the growth and morphological differentiation ofS. coelicolorA3(2). Polydiglycosylphosphate is attached to the spore wall by the LytR-CpsA-Psr protein PdtA (SCO2578), a component of theStreptomycesspore wall-synthesizing complex (SSSC), to ensure the integrity of the spore envelope. Surprisingly, PdtA also has a crucial role in vegetative growth under stress conditions and is required for proper peptidoglycan incorporation during apical tip extension.


2006 ◽  
Vol 188 (24) ◽  
pp. 8368-8375 ◽  
Author(s):  
Wencheng Li ◽  
Xin Ying ◽  
Yuzheng Guo ◽  
Zhen Yu ◽  
Xiufen Zhou ◽  
...  

ABSTRACT SC7A1 is a cosmid with an insert of chromosomal DNA from Streptomyces coelicolor A3(2). Its insertion into the chromosome of S. coelicolor strains caused a duplication of a segment of ca. 40 kb and delayed actinorhodin antibiotic production and sporulation, implying that SC7A1 carried a gene negatively affecting these processes. The subcloning of SC7A1 insert DNA resulted in the identification of the open reading frame SCO5582 as nsdA, a gene n egatively affecting S treptomyces d ifferentiation. The disruption of chromosomal nsdA caused the overproduction of spores and of three of four known S. coelicolor antibiotics of quite different chemical types. In at least one case (that of actinorhodin), this was correlated with premature expression of a pathway-specific regulatory gene (actII-orf4), implying that nsdA in the wild-type strain indirectly repressed the expression of the actinorhodin biosynthesis cluster. nsdA expression was up-regulated upon aerial mycelium initiation and was strongest in the aerial mycelium. NsdA has DUF921, a Streptomyces protein domain of unknown function and a conserved SXR site. A site-directed mutation (S458A) in this site in NsdA abolished its function. Blast searching showed that NsdA homologues are present in some Streptomyces genomes. Outside of streptomycetes, NsdA-like proteins have been found in several actinomycetes. The disruption of the nsdA-like gene SCO4114 had no obvious phenotypic effects on S. coelicolor. The nsdA orthologue SAV2652 in S. avermitilis could complement the S. coelicolor nsdA-null mutant phenotype.


2004 ◽  
Vol 186 (11) ◽  
pp. 3570-3577 ◽  
Author(s):  
Amy M. Gehring ◽  
Stephanie T. Wang ◽  
Daniel B. Kearns ◽  
Narie Yoo Storer ◽  
Richard Losick

ABSTRACT Filamentous soil bacteria of the genus Streptomyces carry out complex developmental cycles that result in sporulation and production of numerous secondary metabolites with pharmaceutically important activities. To further characterize the molecular basis of these developmental events, we screened for mutants of Streptomyces coelicolor that exhibit aberrant morphological differentiation and/or secondary metabolite production. On the basis of this screening analysis and the subsequent complementation analysis of the mutants obtained we assigned developmental roles to a gene involved in methionine biosynthesis (metH) and two previously uncharacterized genes (SCO6938 and SCO2525) and we reidentified two previously described developmental genes (bldA and bldM). In contrast to most previously studied genes involved in development, the genes newly identified in the present study all appear to encode biosynthetic enzymes instead of regulatory proteins. The MetH methionine synthase appears to be required for conversion of aerial hyphae into chains of spores, SCO6938 is a probable acyl coenzyme A dehydrogenase that contributes to the proper timing of aerial mycelium formation and antibiotic production, and SCO2525 is a putative methyltransferase that influences various aspects of colony growth and development.


New antibiotics can still be discovered by the development of novel screening procedures. Notable successes over the last few years include the monobactams, β- lactamase inhibitors (clavulanic acid) and new glycopeptides in the antibacterial held; antiparasitic agents such as avermectins; and herbicidal antibiotics like bialaphos. In the future we can expect the engineering of genes from ‘difficult’ pathogens, including mycobacteria and fungi, and cancer cells, to provide increasingly useful in vitro targets for the screening of antibiotics that can kill pathogens and tumours. There will also be a greater awareness of the need to reveal the full potential for antibiotic production on the part of microorganisms by the physiologial and/or genetic awakening of ‘silent’ genes. Nevertheless, the supply of natural antibiotics for direct use or chemical modification is not infinite and there will be increasing scope for widening the range of available antibiotics by genetic engineering. ‘Hybrid’ antibiotics have been shown to be generated by the transfer of genes on suitable vectors between strains producing chemically related compounds. More exciting is the possibility of generating novelty by the genetic engineering of the synthases that determine the basic structure of antibiotics belonging to such classes as the β-lactams and polyketides. Research in this area will certainly yield knowledge of considerable scientific interest and probably also of potential applicability. In the improvement of antibiotic titre in actinomycetes, protoplast fusion between divergent selection lines has taken a place alongside random mutation and screening. In some cases the cloning of genes controlling metabolic ‘bottlenecks’ in fungi and actinomycetes will give an immediate benefit in the conversion of accumulated biosynthetic intermediates to the desired end product. However, the main impact of genetic engineering in titre improvement will probably come only after a further use of this technology to understand and manipulate the regulation of antibiotic biosynthesis as a facet of the general challenge of understanding differential gene expression. Streptomyces offers a particularly fertile field for such research, following the isolation of DNA segments that carry groups of closely linked operons for the biosynthesis of and resistance to particular antibiotics, and of genes with pleiotropic effects on morphological differentiation and secondary metabolite formation.


2021 ◽  
Author(s):  
Toshiki Nagakubo ◽  
Tatsuya Yamamoto ◽  
Shumpei Asamizu ◽  
Masanori Toyofuku ◽  
Nobuhiko Nomura ◽  
...  

Abstract Extracellular contractile injection systems (eCISs) are structurally similar to headless phages and are versatile nanomachines conserved among diverse classes of bacteria. Herein, Streptomyces species, which comprise filamentous Gram-positive bacteria and are ubiquitous in soil, were shown to produce Streptomyces phage tail-like particles (SLPs) from eCIS-related genes that are widely conserved among Streptomyces species. In some Streptomyces species, these eCIS-related genes are regulated by a key regulatory gene, which is essential for Streptomyces life cycle and is involved in morphological differentiation and antibiotic production. Deletion mutants of S. lividans of the eCIS-related genes appeared phenotypically normal in terms of morphological differentiation and antibiotic production, suggesting that SLPs are involved in other aspects of Streptomyces life cycle. Using co-culture method, we found that colonies of SLP-deficient mutants of S. lividans were more severely invaded by fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. In addition, microscopic and transcriptional analyses demonstrated that SLP expression was elevated upon co-culture with S. pombe. In contrast, co-culture with Bacillus subtilis markedly decreased SLP expression and increased antibiotic production. Our findings demonstrate that in Streptomyces, eCIS-related genes affect microbial competition, and the patterns of SLP expression can differ depending on the competitor species.


2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Toshiki Nagakubo ◽  
Tatsuya Yamamoto ◽  
Shumpei Asamizu ◽  
Masanori Toyofuku ◽  
Nobuhiko Nomura ◽  
...  

AbstractExtracellular contractile injection systems (eCISs) are structurally similar to headless phages and are versatile nanomachines conserved among diverse classes of bacteria. Herein, Streptomyces species, which comprise filamentous Gram-positive bacteria and are ubiquitous in soil, were shown to produce Streptomyces phage tail-like particles (SLPs) from eCIS-related genes that are widely conserved among Streptomyces species. In some Streptomyces species, these eCIS-related genes are regulated by a key regulatory gene, which is essential for Streptomyces life cycle and is involved in morphological differentiation and antibiotic production. Deletion mutants of S. lividans of the eCIS-related genes appeared phenotypically normal in terms of morphological differentiation and antibiotic production, suggesting that SLPs are involved in other aspects of Streptomyces life cycle. Using co-culture method, we found that colonies of SLP-deficient mutants of S. lividans were more severely invaded by fungi, including Saccharomyces cerevisiae and Schizosaccharomyces pombe. In addition, microscopic and transcriptional analyses demonstrated that SLP expression was elevated upon co-culture with the fungi. In contrast, co-culture with Bacillus subtilis markedly decreased SLP expression and increased antibiotic production. Our findings demonstrate that in Streptomyces, eCIS-related genes affect microbial competition, and the patterns of SLP expression can differ depending on the competitor species.


2002 ◽  
Vol 184 (23) ◽  
pp. 6417-6423 ◽  
Author(s):  
Ikuo Kojima ◽  
Kano Kasuga ◽  
Masayuki Kobayashi ◽  
Akira Fukasawa ◽  
Satoshi Mizuno ◽  
...  

ABSTRACT The occurrence of pleiotropic mutants that are defective in both antibiotic production and aerial mycelium formation is peculiar to streptomycetes. Pleiotropic mutant KSB was isolated from wild-type Streptomyces kasugaensis A1R6, which produces kasugamycin, an antifungal aminoglycoside antibiotic. A 9.3-kb DNA fragment was cloned from the chromosomal DNA of strain A1R6 by complementary restoration of kasugamycin production and aerial hypha formation to mutant KSB. Complementation experiments with deletion plasmids and subsequent DNA analysis indicated that orf5, encoding 90 amino acids, was responsible for the restoration. A protein homology search revealed that orf5 was a homolog of rpoZ, the gene that is known to encode RNA polymerase subunit omega (ω), thus leading to the conclusion that orf5 was rpoZ in S. kasugaensis. The pleiotropy of mutant KSB was attributed to a 2-bp frameshift deletion in the rpoZ region of mutant KSB, which probably resulted in a truncated, incomplete ω of 47 amino acids. Furthermore, rpoZ-disrupted mutant R6D4 obtained from strain A1R6 by insertion of Tn5 aphII into the middle of the rpoZ-coding region produced neither kasugamycin nor aerial mycelia, similar to mutant KSB. When rpoZ of S. kasugaensis and Streptomyces coelicolor, whose deduced products differed in the sixth amino acid residue, were introduced into mutant R6D4 via a plasmid, both transformants produced kasugamycin and aerial hyphae without significant differences. This study established that rpoZ is required for kasugamycin production and aerial mycelium formation in S. kasugaensis and responsible for pleiotropy.


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