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.

An aberrant protein synthesis activity is linked with antibiotic overproduction in rpsL mutants of Streptomyces coelicolor A3(2)

Microbiology ◽  
2003 ◽  
Vol 149 (11) ◽  
pp. 3299-3309 ◽  
Author(s):  
Yoshiko Okamoto-Hosoya ◽  
Takeshi Hosaka ◽  
Kozo Ochi
Keyword(s):  
Protein Synthesis ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Translation System ◽  
In The Wild ◽  
Synthesis Activity ◽  
Aberrant Protein ◽  
Ribosomal Protein S12 ◽  
High Level ◽  
Actinorhodin Production

Certain mutations in the rpsL gene (encoding the ribosomal protein S12) activate or enhance antibiotic production in various bacteria. K88E and P91S rpsL mutants of Streptomyces coelicolor A3(2), with an enhanced actinorhodin production, were found to exhibit an aberrant protein synthesis activity. While a high level of this activity (as determined by the incorporation of labelled leucine) was detected at the late stationary phase in the mutants, it decreased with age of the cells in the wild-type strain. In addition, the aberrant protein synthesis was particularly pronounced when cells were subjected to amino acid shift-down, and was independent of their ability to accumulate ppGpp. Ribosomes of K88E and P91S mutants displayed an increased accuracy in protein synthesis as demonstrated by the poly(U)-directed cell-free translation system, but so did K43N, K43T, K43R and K88R mutants, which were streptomycin resistant but showed no effect on actinorhodin production. This eliminates the possibility that the increased accuracy level is a cause of the antibiotic overproduction in the K88E and P91S mutants. The K88E and P91S mutant ribosomes exhibited an increased stability of the 70S complex under low concentrations of magnesium. The authors propose that the aberrant activation of protein synthesis caused by the increased stability of the ribosome is responsible for the remarkable enhancement of antibiotic production in the K88E and P91S mutants.

scholarly journals Phosphorylated AbsA2 Negatively Regulates Antibiotic Production in Streptomyces coelicolor through Interactions with Pathway-Specific Regulatory Gene Promoters

2007 ◽  
Vol 189 (14) ◽  
pp. 5284-5292 ◽  
Author(s):  
Nancy L. McKenzie ◽  
Justin R. Nodwell
Keyword(s):  
Response Regulator ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Gene Clusters ◽  
Negative Regulator ◽  
Gene Promoters ◽  
Promoter Regions ◽  
Calcium Dependent

ABSTRACT The AbsA two-component signal transduction system, comprised of the sensor kinase AbsA1 and the response regulator AbsA2, acts as a negative regulator of antibiotic production in Streptomyces coelicolor, for which the phosphorylated form of AbsA2 (AbsA2∼P) is the agent of repression. In this study, we used chromatin immunoprecipitation to show that AbsA2 binds the promoter regions of actII-ORF4, cdaR, and redZ, which encode pathway-specific activators for actinorhodin, calcium-dependent antibiotic, and undecylprodigiosin, respectively. We confirm that these interactions also occur in vitro and that the binding of AbsA2 to each gene is enhanced by phosphorylation. Induced expression of actII-ORF4 and redZ in the hyperrepressive absA1 mutant (C542) brought about pathway-specific restoration of actinorhodin and undecylprodigiosin production, respectively. Our results suggest that AbsA2∼P interacts with as many as four sites in the region that includes the actII-ORF4 promoter. These data suggest that AbsA2∼P inhibits antibiotic production by directly interfering with the expression of pathway-specific regulators of antibiotic biosynthetic gene clusters.

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 Role of GntR Family Regulatory Gene SCO1678 in Gluconate Metabolism in Streptomyces coelicolor M145

2017 ◽  
Vol 2017 ◽  
pp. 1-9 ◽  
Author(s):  
Olga Tsypik ◽  
Roman Makitrynskyy ◽  
Agnieszka Bera ◽  
Lijiang Song ◽  
Wolfgang Wohlleben ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Functional Characterization ◽  
Bioinformatic Analysis ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Putative Operon

Here we report functional characterization of the Streptomyces coelicolor M145 gene SCO1678, which encodes a GntR-like regulator of the FadR subfamily. Bioinformatic analysis suggested that SCO1678 is part of putative operon (gnt) involved in gluconate metabolism. Combining the results of SCO1678 knockout, transcriptional analysis of gnt operon, and Sco1678 protein-DNA electromobility shift assays, we established that Sco1678 protein controls the gluconate operon. It does so via repression of its transcription from a single promoter located between genes SCO1678 and SCO1679. The knockout also influenced, in a medium-dependent manner, the production of secondary metabolites by S. coelicolor. In comparison to the wild type, on gluconate-containing minimal medium, the SCO1678 mutant produced much less actinorhodin and accumulated a yellow-colored pigment, likely to be the cryptic polyketide coelimycin. Possible links between gluconate metabolism and antibiotic production are discussed.

scholarly journals Identification of a Novel Lincomycin Resistance Mutation Associated with Activation of Antibiotic Production in Streptomyces coelicolor A3(2)

2016 ◽  
Vol 61 (2) ◽  
Author(s):  
Guojun Wang ◽  
Masumi Izawa ◽  
Xiaoge Yang ◽  
Dongbo Xu ◽  
Yukinori Tanaka ◽  
...  
Keyword(s):  
Novel Mutation ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Resistance Mutation ◽  
Gene Replacement ◽  
Sequencing Analysis ◽  
Hybrid Gene ◽  
Content Type ◽  
High Level ◽  
Lincomycin Resistance

ABSTRACT Comparative genome sequencing analysis of a lincomycin-resistant strain of Streptomyces coelicolor A3(2) and the wild-type strain identified a novel mutation conferring a high level of lincomycin resistance. Surprisingly, the new mutation was an in-frame DNA deletion in the genes SCO4597 and SCO4598, resulting in formation of the hybrid gene linR. SCO4597 and SCO4598 encode two histidine kinases, which together with SCO4596, encoding a response regulator, constitute a unique two-component system. Sequence analysis indicated that these three genes and their arrangement patterns are ubiquitous among all Streptomyces genomes sequenced to date, suggesting these genes play important regulatory roles. Gene replacement showed that this mutation was responsible for the high level of lincomycin resistance, the overproduction of the antibiotic actinorhodin, and the enhanced morphological differentiation of this strain. Moreover, heterologous expression of the hybrid gene linR in Escherichia coli conferred resistance to lincomycin in this organism. Introduction of the hybrid gene linR in various Streptomyces strains by gene engineering technology may widely activate and/or enhance antibiotic production.

scholarly journals A Streptomyces coelicolor Antibiotic Regulatory Gene, absB, Encodes an RNase III Homolog

1999 ◽  
Vol 181 (19) ◽  
pp. 6142-6151 ◽  
Author(s):  
Brenda Price ◽  
Trifon Adamidis ◽  
Renqui Kong ◽  
Wendy Champness
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Open Reading Frame ◽  
Dependent Manner ◽  
Rnase Iii ◽  
Reading Frame ◽  
E Coli ◽  
Lines Of Evidence ◽  
Global Defect

ABSTRACT Streptomyces coelicolor produces four genetically and structurally distinct antibiotics in a growth-phase-dependent manner.S. coelicolor mutants globally deficient in antibiotic production (Abs− phenotype) have previously been isolated, and some of these were found to define the absB locus. In this study, we isolated absB-complementing DNA and show that it encodes the S. coelicolor homolog of RNase III (rnc). Several lines of evidence indicate that theabsB mutant global defect in antibiotic synthesis is due to a deficiency in RNase III. In marker exchange experiments, the S. coelicolor rnc gene rescued absB mutants, restoring antibiotic production. Sequencing the DNA of absB mutants confirmed that the absB mutations lay in thernc open reading frame. Constructed disruptions ofrnc in both S. coelicolor 1501 andStreptomyces lividans 1326 caused an Abs−phenotype. An absB mutation caused accumulation of 30S rRNA precursors, as had previously been reported for E. coli rncmutants. The absB gene is widely conserved in streptomycetes. We speculate on why an RNase III deficiency could globally affect the synthesis of antibiotics.

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 Identification and characterization of the Streptomyces globisporus 1912 regulatory gene lndYR that affects sporulation and antibiotic production

Microbiology ◽  
2011 ◽  
Vol 157 (4) ◽  
pp. 1240-1249 ◽  
Author(s):  
Bohdan Ostash ◽  
Yuriy Rebets ◽  
Maksym Myronovskyy ◽  
Olga Tsypik ◽  
Iryna Ostash ◽  
...  
Keyword(s):  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Functional Characterization ◽  
Signal Features ◽  
Abc Transporter Genes ◽  
Identification And Characterization

Here, we report the identification and functional characterization of the Streptomyces globisporus 1912 gene lndYR, which encodes a GntR-like regulator of the YtrA subfamily. Disruption of lndYR arrested sporulation and antibiotic production in S. globisporus. The results of in vivo and in vitro studies revealed that the ABC transporter genes lndW–lndW2 are targets of LndYR repressive action. In Streptomyces coelicolor M145, lndYR overexpression caused a significant increase in the amount of extracellular actinorhodin. We suggest that lndYR controls the transcription of transport system genes in response to an as-yet-unidentified signal. Features that distinguish lndYR-based regulation from other known regulators are discussed.

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