scholarly journals Identification of a gene from Streptomyces rimosus M527 negatively affecting antibiotic biosynthesis and morphological differentiation

2020 ◽  
Author(s):  
Zheng Ma ◽  
Zhangqing Song ◽  
Jie Xu ◽  
Andreas Bechthold ◽  
Xiaoping Yu
Keyword(s):  
Plant Pathogens ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Negative Regulator ◽  
Antibiotic Biosynthesis ◽  
Pcr Analysis ◽  
Streptomyces Rimosus ◽  
Over Expression ◽  
Reverse Transcription Pcr ◽  
Fungal Plant Pathogens

Abstract Background: The polyene macrolide rimocidin, produced by Streptomyces rimosus M527, was found to be highly effective against a broad range of fungal plant pathogens. Current understanding of the regulatory mechanism of rimocidin biosynthesis and morphological differentiation in S. rimosus M527 is limited. NsdA is considered as a negative regulator involved in morphological differentiation and biosynthesis of secondary metabolites in some Streptomyces.Results: In this study, nsdAsr was cloned from S. rimosus M527. The role of nsdAsr in rimocidin biosynthesis and morphological differentiation was investigated by gene deletion, complementation, and over-expression. A ΔnsdAsr mutant was obtained using CRISPR/Cas9. The mutant produced more rimocidin (46%) and generated more spores than the wild-type strain. Over-expression of nsdAsr led to a decrease in rimocidin production and impairment of sporulation. Quantitative reverse transcription-PCR (qRT-PCR) analysis revealed that transcription of rim genes responsible for rimocidin biosynthesis was up-regulated in the ΔnsdAsr mutant but down-regulated in the nsdAsr over-expression strain. Similar effects have been described for Streptomyces coelicolor M145 and the industrial toyocamycin-producing strain Streptomyces diastatochromogenes 1628.Conclusion: NsdAsr is identified as a negative regulator of sporluation and antibiotic biosynthesis as well as the transcription of biosynthetic genes both in its host S. rimosus M527 and in model strain S. coelicolor and industrial producer strain S. diastatochromogenes 1628. This work will provide further information for understanding regulatory mechanisms controlling rimocidin biosynthesis in S. rimosus M527.

scholarly journals Characterization of rrdA, a TetR Family Protein Gene Involved in the Regulation of Secondary Metabolism in Streptomyces coelicolor

2009 ◽  
Vol 75 (7) ◽  
pp. 2158-2165 ◽  
Author(s):  
Xijun Ou ◽  
Bo Zhang ◽  
Lin Zhang ◽  
Guoping Zhao ◽  
Xiaoming Ding
Keyword(s):  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Transcript Level ◽  
Biosynthetic Gene Cluster ◽  
Pcr Analysis ◽  
Reverse Transcription Pcr ◽  
Family Protein ◽  
Severe Reduction ◽  
High Level

ABSTRACT Streptomyces not only exhibits complex morphological differentiation but also produces a plethora of secondary metabolites, particularly antibiotics. To improve our general understanding of the complex network of undecylprodigiosin (Red) biosynthesis regulation, we used an in vivo transposition system to identify novel regulators that influence Red production in Streptomyces coelicolor M145. Using this screening system, we obtained 25 Red-deficient mutants. Twenty-four of these mutants had a transposon inserted in the previously described Red biosynthetic gene cluster and produced different amounts of another secondary metabolite, actinorhodin (Act). One mutant was shown to have an insertion in a different region of the chromosome upstream of the previously uncharacterized gene rrdA (regulator of redD, sco1104), which encodes a putative TetR family transcription factor. Compared with wild-type strain M145, the rrdA null mutant exhibited increased Red production and decreased Act production. A high level of rrdA expression resulted in a severe reduction in Red production and Act overproduction. Reverse transcription-PCR analysis showed that RrdA negatively regulated Red production by controlling redD mRNA abundance, while no change was observed at the transcript level of the Act-specific activator gene, actII-orf4. The effects on Act biosynthesis might arise from competition for precursors that are common to both pathways.

scholarly journals Characterization of the Noncanonical Regulatory and Transporter Genes in Atratumycin Biosynthesis and Production in a Heterologous Host

Marine Drugs ◽  
2019 ◽  
Vol 17 (10) ◽  
pp. 560 ◽  
Author(s):  
Zhijie Yang ◽  
Xin Wei ◽  
Jianqiao He ◽  
Changli Sun ◽  
Jianhua Ju ◽  
...  
Keyword(s):  
Gene Cluster ◽  
Streptomyces Coelicolor ◽  
Regulatory Gene ◽  
Regulatory Protein ◽  
Biosynthetic Gene Cluster ◽  
Negative Regulator ◽  
Over Expression ◽  
Lead Compounds ◽  
Mycobacteria Tuberculosis ◽  
Abc Transporter Genes

Atratumycin is a cyclodepsipeptide with activity against Mycobacteria tuberculosis isolated from deep-sea derived Streptomyces atratus SCSIO ZH16NS-80S. Analysis of the atratumycin biosynthetic gene cluster (atr) revealed that its biosynthesis is regulated by multiple factors, including two LuxR regulatory genes (atr1 and atr2), two ABC transporter genes (atr29 and atr30) and one Streptomyces antibiotic regulatory gene (atr32). In this work, three regulatory and two transporter genes were unambiguously determined to provide positive, negative and self-protective roles during biosynthesis of atratumycin through bioinformatic analyses, gene inactivations and trans-complementation studies. Notably, an unusual Streptomyces antibiotic regulatory protein Atr32 was characterized as a negative regulator; the function of Atr32 is distinct from previous studies. Five over-expression mutant strains were constructed by rational application of the regulatory and transporter genes; the resulting strains produced significantly improved titers of atratumycin that were ca. 1.7–2.3 fold greater than wild-type (WT) producer. Furthermore, the atratumycin gene cluster was successfully expressed in Streptomyces coelicolor M1154, thus paving the way for the transfer and recombination of large DNA fragments. Overall, this finding sets the stage for understanding the unique biosynthesis of pharmaceutically important atratumycin and lays the foundation for generating anti-tuberculosis lead compounds possessing novel structures.

scholarly journals The RNA Polymerase Omega Factor RpoZ Is Regulated by PhoP and Has an Important Role in Antibiotic Biosynthesis and Morphological Differentiation in Streptomyces coelicolor

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 ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Response Regulator ◽  
Antibiotic Production ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Luciferase Reporter ◽  
Antibiotic Biosynthesis ◽  
Binding Assays ◽  
Content Type ◽  
Phosphate Depletion

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.

scholarly journals Molecular Characterization of an Endopolygalacturonase from Fusarium oxysporum Expressed during Early Stages of Infection

2001 ◽  
Vol 67 (5) ◽  
pp. 2191-2196 ◽  
Author(s):  
Fé I. Garcı́a-Maceira ◽  
A. Di Pietro ◽  
M. Dolores Huertas-González ◽  
M. Carmen Ruiz-Roldán ◽  
M. Isabel G. Roncero
Keyword(s):  
Fusarium Oxysporum ◽  
Plant Pathogens ◽  
Detectable Effect ◽  
Calculated Molecular Mass ◽  
Reverse Transcription Pcr ◽  
Fungal Plant Pathogens ◽  
Heterologous System ◽  
Tomato Roots ◽  
Pectinolytic Enzymes

ABSTRACT The tomato vascular wilt pathogen Fusarium oxysporumf. sp. lycopersici produces an array of pectinolytic enzymes that may contribute to penetration and colonization of the host plant. Here we report the isolation of pg5, encoding a novel extracellular endopolygalacturonase (endoPG) that is highly conserved among different formae speciales of F. oxysporum. The putative mature pg5 product has a calculated molecular mass of 35 kDa and a pI of 8.3 and is more closely related to endoPGs from other fungal plant pathogens than to PG1, the major endoPG of F. oxysporum. Overexpression ofpg5 in a bacterial heterologous system produced a 35-kDa protein with endoPG activity. Accumulation of pg5transcript is induced by citrus pectin and d-galacturonic acid and repressed by glucose. As shown by reverse transcription-PCR,pg5 is expressed by F. oxysporum in tomato roots during the initial stages of infection. Targeted inactivation of pg5 has no detectable effect on virulence toward tomato plants.

scholarly journals Regulation of the Streptomyces coelicolor Calcium-Dependent Antibiotic by absA, Encoding a Cluster-Linked Two-Component System

2002 ◽  
Vol 184 (3) ◽  
pp. 794-805 ◽  
Author(s):  
N. Jamie Ryding ◽  
Todd B. Anderson ◽  
Wendy C. Champness
Keyword(s):  
Genomic Sequence ◽  
Sequence Data ◽  
Streptomyces Coelicolor ◽  
Negative Regulation ◽  
Negative Regulator ◽  
Antibiotic Biosynthesis ◽  
Two Component System ◽  
Component System ◽  
Calcium Dependent ◽  
Two Component

ABSTRACT The Streptomyces coelicolor absA two-component system was initially identified through analysis of mutations in the sensor kinase absA1 that caused inhibition of all four antibiotics synthesized by this strain. Previous genetic analysis had suggested that the phosphorylated form of AbsA2 acted as a negative regulator of antibiotic biosynthesis in S. coelicolor (T. B. Anderson, P. Brian, and W. C. Champness, Mol. Microbiol. 39:553–566, 2001). Genomic sequence data subsequently provided by the Sanger Centre (Cambridge, United Kingdom) revealed that absA was located within the gene cluster for production of one of the four antibiotics, calcium-dependent antibiotic (CDA). In this paper we have identified numerous transcriptional start sites within the CDA cluster and have shown that the original antibiotic-negative mutants used to identify absA exhibit a stronger negative regulation of promoters upstream of the proposed CDA biosynthetic genes than of promoters in the clusters responsible for production of actinorhodin and undecylprodigiosin. The same antibiotic-negative mutants also showed an increase in transcription from a promoter divergent to that of absA, upstream of a putative ABC transporter, in addition to an increase in transcription of absA itself. Interestingly, the negative regulation of the biosynthetic transcripts did not appear to be mediated by transcriptional regulation of cdaR (a gene encoding a homolog of the pathway-specific regulators of the act and red clusters) or by any other recognizable transcriptional regulator associated with the cluster. The role of absA in regulating the expression of the diverse antibiotic biosynthesis clusters in the genome is discussed in light of its location in the cda cluster.

scholarly journals The relA/spoT-Homologous Gene inStreptomyces coelicolor Encodes Both Ribosome-Dependent (p)ppGppSynthesizing and -Degrading Activities

1998 ◽  
Vol 180 (16) ◽  
pp. 4123-4132 ◽  
Author(s):  
Oscar H. Martínez-Costa ◽  
Miguel A. Fernández-Moreno ◽  
Francisco Malpartida
Keyword(s):  
Amino Acids ◽  
Deletion Mutant ◽  
Streptomyces Coelicolor ◽  
Stringent Response ◽  
Morphological Differentiation ◽  
Antibiotic Biosynthesis ◽  
Homologous Gene ◽  
E Coli

ABSTRACT Streptomyces coelicolor (p)ppGpp synthetase (Rel protein) belongs to the RelA and SpoT (RelA/SpoT) family, which is involved in (p)ppGpp metabolism and the stringent response. The potential functions of the rel gene have been examined.S. coelicolor Rel has been shown to be ribosome associated, and its activity in vitro is ribosome dependent. Analysis in vivo of the active recombinant protein in well-defined Escherichia coli relA and relA/spoT mutants provides evidence thatS. coelicolor Rel, like native E. coli RelA, is functionally ribosome associated, resulting in ribosome-dependent (p)ppGpp accumulation upon amino acid deprivation. Expression of anS. coelicolor C-terminally deleted Rel, comprised of only the first 489 amino acids, catalyzes a ribosome-independent (p)ppGpp formation, in the same manner as the E. colitruncated RelA protein (1 to 455 amino acids). An E. coli relA spoT double deletion mutant transformed with S. coelicolor rel gene suppresses the phenotype associated with (p)ppGpp deficiency. However, in such a strain, arel-mediated (p)ppGpp response apparently occurs after glucose depletion, but only in the absence of amino acids. Analysis of ppGpp decay in E. coli expressing the S. coelicolor rel gene suggests that it also encodes a (p)ppGpp-degrading activity. By deletion analysis, the catalytic domains of S. coelicolor Rel for (p)ppGpp synthesis and degradation have been located within its N terminus (amino acids 267 to 453 and 93 to 397, respectively). In addition,E. coli relA in an S. coelicolor reldeletion mutant restores actinorhodine production and shows a nearly normal morphological differentiation, as does the wild-typerel gene, which is in agreement with the proposed role of (p)ppGpp nucleotides in antibiotic biosynthesis.

scholarly journals Characterization of γ-Butyrolactone Autoregulatory Signaling Gene Homologs in the Angucyclinone Polyketide WS5995B Producer Streptomyces acidiscabies

2009 ◽  
Vol 191 (15) ◽  
pp. 4786-4797 ◽  
Author(s):  
Frank G. Healy ◽  
Kevin P. Eaton ◽  
Prajit Limsirichai ◽  
Joel F. Aldrich ◽  
Alaina K. Plowman ◽  
...  
Keyword(s):  
Dna Sequences ◽  
Type Strain ◽  
Wild Type Strain ◽  
Morphological Differentiation ◽  
Pcr Analysis ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Morphological Defects ◽  
In The Wild ◽  
Streptomyces Acidiscabies

ABSTRACT Organisms belonging to the genus Streptomyces produce numerous important secondary metabolites and undergo a sophisticated morphological differentiation program. In many instances these processes are under the control of γ-butyrolactone (GBL) autoregulatory systems. Streptomyces acidiscabies strain 84.104 produces the secondary metabolite aromatic angucyclinone polyketide WS5995B. In order to explore the role of GBL regulatory circuitry in WS5995B production and morphogenesis in S. acidiscabies, a gene cluster encoding GBL autoregulatory signaling homologs was identified and characterized. Two GBL receptor homologs, sabR and sabS, were found flanking a GBL synthase homolog sabA. Strains carrying mutations in sabS produced elevated levels of WS5995B and displayed conditional morphological defects reminiscent of defects seen in Streptomyces bldA mutants. Notably, sabS possesses a TTA codon predicted to be recognized by tRNAleu. sabA mutants produced higher levels of WS5995B than the wild-type strain but to a lesser extent than the levels of WS5995B seen in sabS mutants. Purified recombinant SabR and SabS were tested for their abilities to bind predicted AT-rich autoregulatory element (ARE) boxes within the sabRAS region. SabS did not bind any DNA sequences in this region, while SabR bound an ARE box in the region upstream of sabS. Quantitative reverse transcription-PCR analysis revealed higher levels of sabS transcript in sabR mutants than in the wild-type strain, suggesting that sabS expression is repressed by SabR. Based on these data, we propose that the S. acidiscabies sabRAS genes encode components of a signaling pathway which participates in the regulation of WS5995B production and morphogenesis.

scholarly journals The Genomes of the Non-Clearing-Zone-Forming and Natural-Rubber- Degrading Species Gordonia polyisoprenivorans and Gordonia westfalica Harbor Genes Expressing Lcp Activity in Streptomyces Strains

2008 ◽  
Vol 74 (8) ◽  
pp. 2288-2297 ◽  
Author(s):  
Daniel Bröker ◽  
David Dietz ◽  
Matthias Arenskötter ◽  
Alexander Steinbüchel
Keyword(s):  
Streptomyces Coelicolor ◽  
Pcr Amplification ◽  
Gel Permeation Chromatography ◽  
Amino Acid Identity ◽  
Pcr Analysis ◽  
Genomic Libraries ◽  
Reverse Transcription Pcr ◽  
Degrading Bacterium ◽  
Degrading Bacteria ◽  
Gordonia Polyisoprenivorans

ABSTRACT The latex-clearing protein (LcpK30) from the rubber-degrading bacterium Streptomyces sp. strain K30 is involved in the cleavage of poly(cis-1,4-isoprene), yielding isoprenoid aldehydes and ketones. Lcp homologues have so far been detected in all investigated clearing-zone-forming rubber-degrading bacteria. Internal degenerated oligonucleotides derived from lcp genes of Streptomyces sp. strain K30 (lcp K30), Streptomyces coelicolor strain A3(2), and Nocardia farcinica strains IFM10152 and E1 were applied in PCR to investigate whether lcp homologues occur also in the non-clearing-zone-forming rubber-utilizing bacteria Gordonia polyisoprenivorans strains VH2 and Y2K, Gordonia alkanivorans strain 44187, and Gordonia westfalica strain Kb1, which grow adhesively on rubber. The 1,230- and 1,224-bp lcp-homologous genes from G. polyisoprenivorans strain VH2 (lcp VH2) and G. westfalica strain Kb1 (lcp Kb1) were obtained after screening genomic libraries by degenerated PCR amplification, and their translational products exhibited 50 and 52% amino acid identity, respectively, to LcpK30. Recombinant lcp VH2 and lcp Kb1 harboring cells of the non-rubber-degrading Streptomyces lividans strain TK23 were able to form clearing zones and aldehydes on latex overlay-agar plates, thus indicating that lcp VH2 and lcp Kb1 encode functionally active proteins. Analysis by gel permeation chromatography demonstrated lower polymer concentrations and molecular weights of the remaining polyisoprenoid molecules after incubation with these recombinant S. lividans strains. Reverse transcription-PCR analysis demonstrated that lcp VH2 was transcribed in cells of G. polyisoprenivorans strain VH2 cultivated in the presence of poly(cis-1,4-isoprene) but not in the presence of sodium acetate. Anti-LcpK30 immunoglobulin Gs, which were raised in this study, were rather specific for LcpK30 and did not cross-react with LcpVH2 and LcpKb1. A lcp VH2 disruption mutant was still able to grow with poly(cis-1,4-isoprene) as sole carbon source; therefore, lcp VH2 seems not to be essential for rubber degradation in G. polyisoprenivorans.

scholarly journals Roles of Aconitase in Growth, Metabolism, and Morphological Differentiation of Streptomyces coelicolor

2001 ◽  
Vol 183 (10) ◽  
pp. 3193-3203 ◽  
Author(s):  
P. H. Viollier ◽  
K. T. Nguyen ◽  
W. Minas ◽  
M. Folcher ◽  
G. E. Dale ◽  
...  
Keyword(s):  
Citrate Synthase ◽  
Streptomyces Coelicolor ◽  
Morphological Differentiation ◽  
Tca Cycle ◽  
Antibiotic Biosynthesis ◽  
Complete Medium ◽  
Iron Regulatory Proteins ◽  
Early Growth Phase

ABSTRACT The studies of aconitase presented here, along with those of citrate synthase (P. H. Viollier, W. Minas, G. E. Dale, M. Folcher, and C. J. Thompson, J. Bacteriol. 183:3184–3192, 2001), were undertaken to investigate the role of the tricarboxylic acid (TCA) cycle in Streptomyces coelicolor development. A single aconitase activity (AcoA) was detected in protein extracts of cultures during column purification. The deduced amino acid sequence of the cloned acoA gene constituted the N-terminal sequence of semipurified AcoA and was homologous to bacterial A-type aconitases and bifunctional eukaryotic aconitases (iron regulatory proteins). The fact that an acoA disruption mutant (BZ4) did not grow on minimal glucose media in the absence of glutamate confirmed that this gene encoded the primary vegetative aconitase catalyzing flux through the TCA cycle. On glucose-based complete medium, BZ4 had defects in growth, antibiotic biosynthesis, and aerial hypha formation, partially due to medium acidification and accumulation of citrate. The inhibitory effects of acids and citrate on BZ4 were partly suppressed by buffer or by introducing a citrate synthase mutation. However, the fact that growth of an acoA citA mutant remained impaired, even on a nonacidogenic carbon source, suggested alternative functions of AcoA. Immunoblots revealed that AcoA was present primarily during substrate mycelial growth on solid medium. Transcription ofacoA was limited to the early growth phase in liquid cultures from a start site mapped in vitro and in vivo.

scholarly journals CodY-Dependent Regulation of Sporulation in Clostridium difficile

2016 ◽  
Vol 198 (15) ◽  
pp. 2113-2130 ◽  
Author(s):  
Kathryn L. Nawrocki ◽  
Adrianne N. Edwards ◽  
Nadine Daou ◽  
Laurent Bouillaut ◽  
Shonna M. McBride
Keyword(s):  
Clostridium Difficile ◽  
Nutrient Limitation ◽  
Negative Regulator ◽  
Pcr Analysis ◽  
Content Type ◽  
Reverse Transcription Pcr ◽  
Sporulation Initiation ◽  
The Impact ◽  
The Relationship

ABSTRACTClostridium difficilemust form a spore to survive outside the gastrointestinal tract. The factors that trigger sporulation inC. difficileremain poorly understood. Previous studies have suggested that a link exists between nutritional status and sporulation initiation inC. difficile. In this study, we investigated the impact of the global nutritional regulator CodY on sporulation inC. difficilestrains from the historical 012 ribotype and the current epidemic 027 ribotype. Sporulation frequencies were increased in both backgrounds, demonstrating that CodY represses sporulation inC. difficile. The 027codYmutant exhibited a greater increase in spore formation than the 012codYmutant. To determine the role of CodY in the observed sporulation phenotypes, we examined several factors that are known to influence sporulation inC. difficile. Using transcriptional reporter fusions and quantitative reverse transcription-PCR (qRT-PCR) analysis, we found that two loci associated with the initiation of sporulation,oppandsinR, are regulated by CodY. The data demonstrate that CodY is a repressor of sporulation inC. difficileand that the impact of CodY on sporulation and expression of specific genes is significantly influenced by the strain background. These results suggest that the variability of CodY-dependent regulation is an important contributor to virulence and sporulation in current epidemic isolates. This report provides further evidence that nutritional state, virulence, and sporulation are linked inC. difficile.IMPORTANCEThis study sought to examine the relationship between nutrition and sporulation inC. difficileby examining the global nutritional regulator CodY. CodY is a known virulence and nutritional regulator ofC. difficile, but its role in sporulation was unknown. Here, we demonstrate that CodY is a negative regulator of sporulation in two different ribotypes ofC. difficile. We also demonstrate that CodY regulates known effectors of sporulation, Opp and SinR. These results support the idea that nutrient limitation is a trigger for sporulation inC. difficileand that the response to nutrient limitation is coordinated by CodY. Additionally, we demonstrate that CodY has an altered role in sporulation regulation for some strains.

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