scholarly journals Changes in the Extracellular Proteome Caused by the Absence of the bldA Gene Product, a Developmentally Significant tRNA, Reveal a New Target for the Pleiotropic Regulator AdpA in Streptomyces coelicolor

2005 ◽  
Vol 187 (9) ◽  
pp. 2957-2966 ◽  
Author(s):  
Dae-Wi Kim ◽  
Keith Chater ◽  
Kye-Joon Lee ◽  
Andy Hesketh
Keyword(s):  
Stationary Phase ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Morphological Differentiation ◽  
Secreted Proteins ◽  
Two Dimensional Gel Electrophoresis ◽  
Extracellular Proteome ◽  
Peptide Mass ◽  
Subtilisin Inhibitor

ABSTRACT The extracellular proteome of Streptomyces coelicolor grown in a liquid medium was analyzed by using two-dimensional gel electrophoresis and matrix-assisted laser desorption ionization-time of flight peptide mass fingerprint analysis. Culture supernatants became protein rich only after rapid growth had been completed, supporting the idea that protein secretion is largely a stationary phase phenomenon. Out of about 600 protein spots observed, 72 were characterized. The products of 47 genes were identified, with only 11 examples predicted to be secreted proteins. Mutation in bldA, previously known to impair the stationary phase processes of antibiotic production and morphological differentiation, also induced changes in the extracellular proteome, revealing even greater pleiotropy in the bldA phenotype than previously known. Four proteins increased in abundance in the bldA mutant, while the products of 11 genes, including four secreted proteins, were severely down-regulated. Although bldA encodes the only tRNA capable of efficiently translating the rare UUA (leucine) codon, none of the latter group of genes contains an in-frame TTA. SCO0762, a serine-protease inhibitor belonging to the Streptomyces subtilisin inhibitor family implicated in differentiation in other streptomycetes, was completely absent from the bldA mutant. This dependence was shown to be mediated via the TTA-containing regulatory gene adpA, also known as bldH, a developmental gene that is responsible for the effects of bldA on differentiation. Mutation of the SCO0762 gene abolished detectable trypsin-protease inhibitory activity but did not result in any obvious morphological defects.

scholarly journals Secreted-Protein Response to σU Activity in Streptomyces coelicolor

2007 ◽  
Vol 190 (3) ◽  
pp. 894-904 ◽  
Author(s):  
Nadria D. Gordon ◽  
Geri L. Ottaviano ◽  
Sarah E. Connell ◽  
Gregory V. Tobkin ◽  
Crystal H. Son ◽  
...  
Keyword(s):  
Sigma Factor ◽  
Fluorescent Protein ◽  
Cell Envelope ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Aerial Mycelium ◽  
Secreted Proteins ◽  
Wild Type ◽  
Extracellular Proteome ◽  
Peptide Mass

ABSTRACT The filamentous bacterium Streptomyces coelicolor forms an aerial mycelium as a prerequisite to sporulation, which occurs in the aerial hyphae. Uncontrolled activity of the extracytoplasmic function sigma factor σU blocks the process of aerial mycelium formation in this organism. Using a green fluorescent protein transcriptional reporter, we have demonstrated that sigU transcription is autoregulated. We have defined a σU-dependent promoter sequence and used this to identify 22 likely σU regulon members in the S. coelicolor genome. Since many of these genes encode probable secreted proteins, we characterized the extracellular proteome of a mutant with high σU activity caused by disruption of rsuA, the presumed cognate anti-sigma factor of σU. This mutant secreted a much greater quantity and diversity of proteins than the wild-type strain. Peptide mass fingerprinting was used to identify 79 proteins from the rsuA mutant culture supernatant. The most abundant species, SCO2217, SCO0930, and SCO2207, corresponded to secreted proteins or lipoproteins of unknown functions whose genes are in the proposed σU regulon. Several unique proteases were also detected in the extracellular proteome of the mutant, and the levels of the protease inhibitor SCO0762 were much reduced compared to those of the wild type. Consequently, extracellular protease activity was elevated about fourfold in the rsuA mutant. The functions of the proteins secreted as a result of σU activity may be important for combating cell envelope stress and modulating morphological differentiation in S. coelicolor.

scholarly journals Identification of a Gene Negatively Affecting Antibiotic Production and Morphological Differentiation in Streptomyces coelicolor A3(2)

2006 ◽  
Vol 188 (24) ◽  
pp. 8368-8375 ◽  
Author(s):  
Wencheng Li ◽  
Xin Ying ◽  
Yuzheng Guo ◽  
Zhen Yu ◽  
Xiufen Zhou ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Morphological Differentiation ◽  
Aerial Mycelium ◽  
Protein Domain ◽  
Chromosomal Dna ◽  
Reading Frame ◽  
Directed Mutation ◽  
In The Wild

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.

scholarly journals Repression of Antibiotic Production and Sporulation in Streptomyces coelicolor by Overexpression of a TetR Family Transcriptional Regulator

2010 ◽  
Vol 76 (23) ◽  
pp. 7741-7753 ◽  
Author(s):  
Delin Xu ◽  
Nicolas Seghezzi ◽  
Catherine Esnault ◽  
Marie-Joelle Virolle
Keyword(s):  
Target Genes ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Morphological Differentiation ◽  
Promoter Sequence ◽  
Inhibitory Effect ◽  
Binding Motif

ABSTRACT The overexpression of a regulatory gene of the TetR family (SCO3201) originating either from Streptomyces lividans or from Streptomyces coelicolor was shown to strongly repress antibiotic production (calcium-dependent antibiotic [CDA], undecylprodigiosin [RED], and actinorhodin [ACT]) of S. coelicolor and of the ppk mutant strain of S. lividans. Curiously, the overexpression of this gene also had a strong inhibitory effect on the sporulation process of S. coelicolor but not on that of S. lividans. SCO3201 was shown to negatively regulate its own transcription, and its DNA binding motif was found to overlap its −35 promoter sequence. The interruption of this gene in S. lividans or S. coelicolor did not lead to any obvious phenotypes, indicating that when overexpressed SCO3201 likely controls the expression of target genes of other TetR regulators involved in the regulation of the metabolic and morphological differentiation process in S. coelicolor. The direct and functional interaction of SCO3201 with the promoter region of scbA, a gene under the positive control of the TetR-like regulator, ScbR, was indeed demonstrated by in vitro as well as in vivo approaches.

Inactivation or amplification of the spa2 gene, encoding a potential stationary-phase regulator, affects differentiation in Streptomyces ambofaciens

1997 ◽  
Vol 43 (12) ◽  
pp. 1118-1125 ◽  
Author(s):  
Martine Aubert ◽  
Elisabeth Weber ◽  
Brigitte Gintz ◽  
Bernard Decaris ◽  
Keith F. Chater
Keyword(s):  
Stationary Phase ◽  
Antibiotic Production ◽  
Morphological Differentiation ◽  
Aerial Mycelium ◽  
Detectable Effect ◽  
Multicopy Plasmid ◽  
Gene Encoding ◽  
Wild Type Phenotype ◽  
Streptomyces Ambofaciens ◽  
Mycelial Development

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.

Effects of growth phase and the developmentally significant bldA-specified tRNA on the membrane-associated proteome of Streptomyces coelicolor

Microbiology ◽  
2005 ◽  
Vol 151 (8) ◽  
pp. 2707-2720 ◽  
Author(s):  
Dae-Wi Kim ◽  
Keith F. Chater ◽  
Kye-Joon Lee ◽  
Andy Hesketh
Keyword(s):  
Minimal Medium ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Transmembrane Helix ◽  
Morphological Differentiation ◽  
Hypothetical Proteins ◽  
Membrane Proteome ◽  
Protein Mass ◽  
Associated Proteins ◽  
Transporter Systems

Previous proteomic analyses of Streptomyces coelicolor by two-dimensional electrophoresis and protein mass fingerprinting focused on extracts from total cellular material. Here, the membrane-associated proteome of cultures grown in a liquid minimal medium was partially characterized. The products of some 120 genes were characterized from the membrane fraction, with 70 predicted to possess at least one transmembrane helix. A notably high proportion of ABC transporter systems was represented; the specific types detected provided a snapshot of the nutritional requirements of the mycelium. The membrane-associated proteins did not change very much in abundance in different phases of growth in liquid minimal medium. Identification of gene products not expected to be present in membrane protein extracts led to a reconsideration of the genome annotation in two cases, and supplemented scarce information on 11 hypothetical/conserved hypothetical proteins of unknown function. The wild-type membrane proteome was compared with that of a bldA mutant lacking the only tRNA capable of efficient translation of the rare UUA (leucine) codon. Such mutants are unaffected in vegetative growth but are defective in many aspects of secondary metabolism and morphological differentiation. There were a few clear changes in the membrane proteome of the mutant. In particular, two hypothetical proteins (SCO4244 and SCO4252) were completely absent from the bldA mutant, and this was associated with the TTA-containing regulatory gene SCO4263. Evidence for the control of a cluster of function-unknown genes by the SCO4263 regulator revealed a new aspect of the pleiotropic bldA phenotype.

scholarly journals A Novel Insertion Mutation in Streptomyces coelicolor Ribosomal S12 Protein Results in Paromomycin Resistance and Antibiotic Overproduction

2008 ◽  
Vol 53 (3) ◽  
pp. 1019-1026 ◽  
Author(s):  
Guojun Wang ◽  
Takashi Inaoka ◽  
Susumu Okamoto ◽  
Kozo Ochi
Keyword(s):  
Transcriptional Activation ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Translation System ◽  
Insertion Mutation ◽  
Resistance Level ◽  
Double Mutation ◽  
Paromomycin Resistance ◽  
High Level

ABSTRACT We identified a novel paromomycin resistance-associated mutation in rpsL, caused by the insertion of a glycine residue at position 92, in Streptomyces coelicolor ribosomal protein S12. This insertion mutation (GI92) resulted in a 20-fold increase in the paromomycin resistance level. In combination with another S12 mutation, K88E, the GI92 mutation markedly enhanced the production of the blue-colored polyketide antibiotic actinorhodin and the red-colored antibiotic undecylprodigiosin. The gene replacement experiments demonstrated that the K88E-GI92 double mutation in the rpsL gene was responsible for the marked enhancement of antibiotic production observed. Ribosomes with the K88E-GI92 double mutation were characterized by error restrictiveness (i.e., hyperaccuracy). Using a cell-free translation system, we found that mutant ribosomes harboring the K88E-GI92 double mutation but not ribosomes harboring the GI92 mutation alone displayed sixfold greater translation activity relative to that of the wild-type ribosomes at late growth phase. This resulted in the overproduction of actinorhodin, caused by the transcriptional activation of the pathway-specific regulatory gene actII-orf4, possibly due to the increased translation of transcripts encoding activators of actII-orf4. The mutant with the K88E-GI92 double mutation accumulated a high level of ribosome recycling factor at late stationary phase, underlying the high level of protein synthesis activity observed.

scholarly journals The global role of ppGpp synthesis in morphological differentiation and antibiotic production in Streptomyces coelicolor A3(2)

2007 ◽  
Vol 8 (8) ◽  
pp. R161 ◽  
Author(s):  
Andrew Hesketh ◽  
Wenqiong Chen ◽  
Jamie Ryding ◽  
Sherman Chang ◽  
Mervyn Bibb
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation

scholarly journals BldG and SCO3548 Interact Antagonistically To Control Key Developmental Processes in Streptomyces coelicolor

2009 ◽  
Vol 191 (8) ◽  
pp. 2541-2550 ◽  
Author(s):  
Archana Parashar ◽  
Kimberley R. Colvin ◽  
Dawn R. D. Bignell ◽  
Brenda K. Leskiw
Keyword(s):  
Sigma Factor ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Affinity Purification ◽  
Regulatory System ◽  
Direct Interaction ◽  
Morphological Differentiation ◽  
Sigma Factors ◽  
Developmental Processes ◽  
Two Hybrid

ABSTRACT The similarity of BldG and the downstream coexpressed protein SCO3548 to anti-anti-sigma and anti-sigma factors, respectively, together with the phenotype of a bldG mutant, suggests that BldG and SCO3548 interact as part of a regulatory system to control both antibiotic production and morphological differentiation in Streptomyces coelicolor. A combination of bacterial two-hybrid, affinity purification, and far-Western analyses demonstrated that there was self-interaction of both BldG and SCO3548, as well as a direct interaction between the two proteins. Furthermore, a genetic complementation experiment demonstrated that SCO3548 antagonizes the function of BldG, similar to other anti-anti-sigma/anti-sigma factor pairs. It is therefore proposed that BldG and SCO3548 form a partner-switching pair that regulates the function of one or more sigma factors in S. coelicolor. The conservation of bldG and sco3548 in other streptomycetes demonstrates that this system is likely a key regulatory switch controlling developmental processes throughout the genus Streptomyces.

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