scholarly journals Regulation of the Biosynthesis of the Macrolide Antibiotic Spiramycin in Streptomyces ambofaciens

2010 ◽  
Vol 192 (21) ◽  
pp. 5813-5821 ◽  
Author(s):  
Fatma Karray ◽  
Emmanuelle Darbon ◽  
Hoang Chuong Nguyen ◽  
Josette Gagnat ◽  
Jean-Luc Pernodet
Keyword(s):  
Expression Analysis ◽  
Wild Type Strain ◽  
Macrolide Antibiotic ◽  
Regulatory Gene ◽  
Biosynthetic Gene Cluster ◽  
Wild Type ◽  
Rt Pcr ◽  
Reverse Transcription Pcr ◽  
Streptomyces Ambofaciens ◽  
In The Wild

ABSTRACT Streptomyces ambofaciens synthesizes the macrolide antibiotic spiramycin. The biosynthetic gene cluster for spiramycin has been characterized for S. ambofaciens. In addition to the regulatory gene srmR (srm22), previously identified (M. Geistlich et al., Mol. Microbiol. 6:2019-2029, 1992), three putative regulatory genes had been identified by sequence analysis. Gene expression analysis and gene inactivation experiments showed that only one of these three genes, srm40, plays a major role in the regulation of spiramycin biosynthesis. The disruption of srm22 or srm40 eliminated spiramycin production while their overexpression increased spiramycin production. Expression analysis was performed by reverse transcription-PCR (RT-PCR) for all the genes of the cluster in the wild-type strain and in the srm22 (srmR) and srm40 deletion mutants. The results from the expression analysis, together with the ones from the complementation experiments, indicated that Srm22 is required for srm40 expression, Srm40 being a pathway-specific activator that controls most, if not all, of the spiramycin biosynthetic genes.

scholarly journals Improvement of Nemadectin Production by Overexpressing the Regulatory Gene nemR and Nemadectin Biosynthetic Gene Cluster in Streptomyces Cyaneogriseus

2021 ◽  
Author(s):  
Yuan-Jie Wu ◽  
Song-Bai Yang ◽  
Zheng-Yu Zhang ◽  
Shao-Xin Chen
Keyword(s):  
Gene Cluster ◽  
Wild Type Strain ◽  
Regulatory Gene ◽  
Anthelmintic Activity ◽  
Biosynthetic Gene Cluster ◽  
Wild Type ◽  
Transcription Level ◽  
Positive Role ◽  
C 23 ◽  
In The Wild

AbstractNemadectin, a 16-member macrocyclic lactone antiparasitic antibiotic, is produced by Streptomyces cyaneogriseus subspecies noncyanogenus. Moxidectin, a C-23 oximate derivative of nemadectin, is widely used as a pesticide due to its broad-spectrum, highly efficient, and safe anthelmintic activity. NemR, a LAL family regulator, is encoded by nemR and is involved in nemadectin biosynthesis in S. cyaneogriseus. In this report, gene disruption and complementation experiments showed that nemR plays a positive role in the biosynthesis of nemadectin. The transcription level of nemadectin biosynthetic genes in the nemR knockout strain was significantly decreased compared with those in the wild-type strain MOX-101. However, overexpression of nemR under the control of native or strong constitutive promoters resulted in the opposite, increasing the production of nemadectin by 56.5 or 73.5%, respectively, when compared with MOX-101. In addition, the gene cluster of nemadectin biosynthesis was further cloned and overexpressed using a CRISPR method, which significantly increase nemadectin yield by 108.6% (509 mg/L) when compared with MOX-101.

scholarly journals Discovery of new antibacterial accramycins from a genetic variant of the soil bacterium, Streptomyces sp. MA37

2020 ◽  
Author(s):  
Fleurdeliz Maglangit ◽  
Yuting Zhang ◽  
Kwaku Kyeremeh ◽  
Hai Deng
Keyword(s):  
Wild Type Strain ◽  
Biosynthetic Gene Cluster ◽  
Multidrug Resistant ◽  
Regulatory Genes ◽  
Negative Regulator ◽  
Clinical Isolates ◽  
Wild Type ◽  
In The Wild ◽  
A Minor ◽  
New Metabolites

AbstractContinued mining of natural products from the strain Streptomyces sp. MA37 in our laboratory led to the discovery of a minor specialised metabolite (SM) called accramycin A. Owing to its low yield (0.2mg/L) in the wild type strain, we investigated the roles of regulatory genes in the corresponding biosynthetic gene cluster (acc BGC) through gene inactivation with the aim of improving the titre of this compound. One of the resulting mutants (ΔaccJ) dramatically upregulated the production of accramycin A 1 by 330-fold (66mg/L). Furthermore, ten new metabolites, accramycins B-K 2-11, were discovered, together with two known compounds, naphthacemycin B112 and fasamycin C 13 from the mutant extract. This suggested that accJ, annotated as Multiple Antibiotic Resistance Regulator (MarR), is a negative regulator gene in the accramycin biosynthesis. Compounds 1-13 inhibited the Gram-positive pathogens (S. aureus, E. faecalis) and clinical isolates, E. faecium (K59-68 and K60-39), and S. haemolyticus with minimal inhibitory concentration (MIC) values in the range of 1.5-12.5µg/mL. Remarkably, compounds 1-13 displayed superior activity against K60-39 (MIC = 3.1-6.3µg/mL) than ampicillin (MIC = 25µg/mL), and offer promising potential for the development of accramycin-based antibiotics that target multidrug-resistant Enterococcus clinical isolates. Our results highlight the importance of identifying the roles of regulatory genes in natural product discovery.

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 Identification by Gene Deletion Analysis of a Regulator, VmsR, That Controls Virginiamycin Biosynthesis in Streptomyces virginiae

2000 ◽  
Vol 182 (21) ◽  
pp. 6259-6263 ◽  
Author(s):  
Ryu Kawachi ◽  
Usamas Wangchaisoonthorn ◽  
Takuya Nihira ◽  
Yasuhiro Yamada
Keyword(s):  
Gene Deletion ◽  
Wild Type Strain ◽  
Gene Clusters ◽  
Receptor Protein ◽  
Wild Type ◽  
Specific Receptor ◽  
Biosynthetic Gene Clusters ◽  
Reverse Transcription Pcr ◽  
Streptomyces Virginiae ◽  
In The Wild

ABSTRACT Virginiae butanolide (VB)-BarA of Streptomyces virginiae is one of the newly discovered pairs of a butyrolactone autoregulator and a corresponding receptor protein ofStreptomyces species and regulates the production of the antibiotic virginiamycin (VM) in S. virginiae. The genevmsR was found to be situated 4.7 kbp upstream of thebarA gene, which encodes the VB-specific receptor. ThevmsR product was predicted to be a regulator of VM biosynthesis based on its high homology to someStreptomyces pathway-specific transcriptional regulators for the biosynthetic gene clusters of polyketide antibiotics, such asStreptomyces peucetius DnrI (47.5% identity, 84.3% similarity), which controls daunorubicin biosynthesis. AvmsR deletion mutant was created by homologous recombination. Neither virginiamycin M1 nor virginiamycin S was produced in the vmsR mutant, while amounts of VB and BarA similar to those produced in the wild-type strain were detected. Reverse transcription-PCR analyses confirmed that the vmsRdeletion had no deleterious effects on the transcription of thevmsR-surrounding genes, indicating that VmsR is a positive regulator of VM biosynthesis in S. virginiae.

scholarly journals Roles offkbNin Positive Regulation andtcs7in Negative Regulation of FK506 Biosynthesis in Streptomyces sp. Strain KCTC 11604BP

2012 ◽  
Vol 78 (7) ◽  
pp. 2249-2255 ◽  
Author(s):  
SangJoon Mo ◽  
Young Ji Yoo ◽  
Yeon Hee Ban ◽  
Sung-Kwon Lee ◽  
Eunji Kim ◽  
...  
Keyword(s):  
Type Strain ◽  
Wild Type Strain ◽  
Regulatory Protein ◽  
Strain Improvement ◽  
Transcriptional Regulators ◽  
Biosynthetic Gene Cluster ◽  
Wild Type ◽  
Biosynthetic Genes ◽  
Content Type ◽  
In The Wild

ABSTRACTFK506 is an important 23-member polyketide macrolide with immunosuppressant activity. Its entire biosynthetic gene cluster was previously cloned fromStreptomycessp. strain KCTC 11604BP, and sequence analysis identified three putative regulatory genes,tcs2,tcs7, andfkbN, which encode proteins with high similarity to the AsnC family transcriptional regulators, LysR-type transcriptional regulators, and LAL family transcriptional regulators, respectively. Overexpression and in-frame deletion oftcs2did not affect the production of FK506 or co-occurring FK520 compared to results for the wild-type strain, suggesting thattcs2is not involved in their biosynthesis.fkbNoverexpression improved the levels of FK506 and FK520 production by approximately 2.0-fold, and a deletion offkbNcaused the complete loss of FK506 and FK520 production. Although the overexpression oftcs7decreased the levels of FK506 and FK520 production slightly, a deletion oftcs7caused 1.9-fold and 1.5-fold increases in FK506 and FK520 production, respectively. Finally,fkbNoverexpression in thetcs7deletion strain resulted in a 4.0-fold (21 mg liter−1) increase in FK506 production compared to that by the wild-type strain. This suggests thatfkbNencodes a positive regulatory protein essential for FK506/FK520 biosynthesis and that the gene product oftcs7negatively regulates their biosynthesis, demonstrating the potential of exploiting this information for strain improvement. Semiquantitative reverse transcription-PCR (RT-PCR) analyses of the transcription levels of the FK506 biosynthetic genes in the wild-type and mutant strains proved that most of the FK506 biosynthetic genes are regulated byfkbNin a positive manner and negatively bytcs7.

scholarly journals Carbazole-Degradative IncP-7 Plasmid pCAR1.2 Is Structurally Unstable in Pseudomonas fluorescens Pf0-1, Which Accumulates Catechol, the Intermediate of the Carbazole Degradation Pathway

2009 ◽  
Vol 75 (12) ◽  
pp. 3920-3929 ◽  
Author(s):  
Yurika Takahashi ◽  
Masaki Shintani ◽  
Li Li ◽  
Hisakazu Yamane ◽  
Hideaki Nojiri
Keyword(s):  
Wild Type Strain ◽  
Regulatory Gene ◽  
Degradation Pathway ◽  
Wild Type ◽  
Reverse Transcription Pcr ◽  
Chromosomal Genes ◽  
The Stability ◽  
Genetic Structures ◽  
Delayed Growth ◽  
Selection Of

ABSTRACT We determined the effect of the host on the function and structure of the nearly identical IncP-7 carbazole-degradative plasmids pCAR1.1 and pCAR1.2. We constructed Pseudomonas aeruginosa PAO1(pCAR1.2) and P. fluorescens Pf0-1Km(pCAR1.2) and compared their growth on carbazole- and succinate-containing media with that of P. putida KT2440(pCAR1.1). We also assessed the stability of the genetic structures of the plasmids in each of the three hosts. Pf0-1Km(pCAR1.2) showed dramatically delayed growth when carbazole was supplied as the sole carbon source, while the three strains grew at nearly the same rate on succinate. Among the carbazole-grown Pf0-1Km(pCAR1.2) cells, two types of deficient strains appeared and dominated the population; such dominance was not observed in the other two strains or for succinate-grown Pf0-1Km(pCAR1.2). Genetic analysis showed that the two deficient strains possessed pCAR1.2 derivatives in which the carbazole-degradative car operon was deleted or its regulatory gene, antR, was deleted by homologous recombination between insertion sequences. From genomic information and quantitative reverse transcription-PCR analyses of the genes involved in carbazole mineralization by Pf0-1Km(pCAR1.2), we found that the cat genes on the chromosome of Pf0-1Km, which are necessary for the degradation of catechol (a toxic intermediate in the carbazole catabolic pathway), were not induced in the presence of carbazole. The resulting accumulation of catechol may have enabled the strain that lost its carbazole-degrading ability to have overall higher fitness than the wild-type strain. These results suggest that the functions of the chromosomal genes contributed to the selection of plasmid derivatives with altered structures.

Isolation and characterization of a mutator strain of Streptomyces ambofaciens ATCC23877 exhibiting an increased level of genetic instability

1996 ◽  
Vol 42 (6) ◽  
pp. 562-570 ◽  
Author(s):  
Dominique Vandewiele ◽  
Jean-Nicolas Volff ◽  
Bertrand Aigle ◽  
Jean-Marc Simonet ◽  
Bernard Decaris
Keyword(s):  
Genetic Instability ◽  
Type Strain ◽  
Wild Type Strain ◽  
Genomic Analysis ◽  
Wild Type ◽  
Mutator Strain ◽  
Isolation And Characterization ◽  
Streptomyces Ambofaciens ◽  
In The Wild

In Streptomyces ambofaciens ATCC23877, 0.7% of pigment-defective mutants (Pig−) can be observed in the progeny of wild-type colonies. A mutator (Mut−) strain was isolated from the offspring of the wild-type strain. The Mut− strain produced colonies that sported nonpigmented papillae. Furthermore, the frequency of Pig− colonies obtained in the progeny of this strain was fivefold higher than in the wild-type strain. This strain showed the same level of sensitivity to ultraviolet light and mitomycin C as the wild-type strain. This Mut− phenotype was found to be reversible at high frequency (3 × 10−3). Genomic analysis using pulsed-field gel electrophoresis (PFGE) showed that the Pig− mutants arisen from the Mut− strain were less frequently rearranged (32% were deleted) compared with the mutants arising from the wild type (59% were deleted). Moreover, the Pig− papillae mutants possessed no visible rearrangement as revealed by PFGE analyses.Key words: Streptomyces, genetic instability, mutator strain, papillae.

scholarly journals Effects of Mutations of RAD50, RAD51, RAD52, and Related Genes on Illegitimate Recombination in Saccharomyces cerevisiae

Genetics ◽  
1996 ◽  
Vol 142 (2) ◽  
pp. 383-391 ◽  
Author(s):  
Yasumasa Tsukamoto ◽  
Jun-ichi Kato ◽  
Hideo Ikeda
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Quantitative Analysis ◽  
Structural Analysis ◽  
Recombination Rate ◽  
Type Strain ◽  
Negative Selection ◽  
Wild Type Strain ◽  
Illegitimate Recombination ◽  
Wild Type ◽  
In The Wild

Abstract To examine the mechanism of illegitimate recombination in Saccharomyces cerevisiae, we have developed a plasmid system for quantitative analysis of deletion formation. A can1 cyh2 cell carrying two negative selection markers, the CAN1 and CYH2 genes, on a YCp plasmid is sensitive to canavanine and cycloheximide, but the cell becomes resistant to both drugs when the plasmid has a deletion over the CAN1 and CYH2 genes. Structural analysis of the recombinant plasmids obtained from the resistant cells showed that the plasmids had deletions at various sites of the CAN1-CYH2 region and there were only short regions of homology (1-5 bp) at the recombination junctions. The results indicated that the deletion detected in this system were formed by illegitimate recombination. Study on the effect of several rad mutations showed that the recombination rate was reduced by 30-, 10-, 10-, and 10-fold in the rad52, rad50, mre11, and xrs2 mutants, respectively, while in the rud51, 54, 55, and 57 mutants, the rate was comparable to that in the wild-type strain. The rad52 mutation did not affect length of homology at junction sites of illegitimate recombination.

scholarly journals The Cloning and Molecular Analysis of pawn-B in Paramecium tetraurelia

Genetics ◽  
2000 ◽  
Vol 155 (3) ◽  
pp. 1105-1117 ◽  
Author(s):  
W John Haynes ◽  
Kit-Yin Ling ◽  
Robin R Preston ◽  
Yoshiro Saimi ◽  
Ching Kung
Keyword(s):  
Genomic Library ◽  
Open Reading Frame ◽  
Paramecium Tetraurelia ◽  
Wild Type ◽  
Rt Pcr ◽  
Reading Frame ◽  
Close Proximity ◽  
In The Wild ◽  
Dna Fragment ◽  
Alternative Sites

Abstract Pawn mutants of Paramecium tetraurelia lack a depolarization-activated Ca2+ current and do not swim backward. Using the method of microinjection and sorting a genomic library, we have cloned a DNA fragment that complements pawn-B (pwB/pwB). The minimal complementing fragment is a 798-bp open reading frame (ORF) that restores the Ca2+ current and the backward swimming when expressed. This ORF contains a 29-bp intron and is transcribed and translated. The translated product has two putative transmembrane domains but no clear matches in current databases. Mutations in the available pwB alleles were found within this ORF. The d4-95 and d4-96 alleles are single base substitutions, while d4-662 (previously pawn-D) harbors a 44-bp insertion that matches an internal eliminated sequence (IES) found in the wild-type germline DNA except for a single C-to-T transition. Northern hybridizations and RT-PCR indicate that d4-662 transcripts are rapidly degraded or not produced. A second 155-bp IES in the wild-type germline ORF excises at two alternative sites spanning three asparagine codons. The pwB ORF appears to be separated from a 5′ neighboring ORF by only 36 bp. The close proximity of the two ORFs and the location of the pwB protein as indicated by GFP-fusion constructs are discussed.

scholarly journals Expression and function of the cdgD gene, encoding a CHASE–PAS-DGC-EAL domain protein, in Azospirillum brasilense

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
José Francisco Cruz-Pérez ◽  
Roxana Lara-Oueilhe ◽  
Cynthia Marcos-Jiménez ◽  
Ricardo Cuatlayotl-Olarte ◽  
María Luisa Xiqui-Vázquez ◽  
...  
Keyword(s):  
Azospirillum Brasilense ◽  
Type Strain ◽  
Wild Type Strain ◽  
Soil Conditions ◽  
Wild Type ◽  
Gene Encoding ◽  
Wheat Roots ◽  
Genes Encoding ◽  
In The Wild ◽  
Plant Growth Promoting

AbstractThe plant growth-promoting bacterium Azospirillum brasilense contains several genes encoding proteins involved in the biosynthesis and degradation of the second messenger cyclic-di-GMP, which may control key bacterial functions, such as biofilm formation and motility. Here, we analysed the function and expression of the cdgD gene, encoding a multidomain protein that includes GGDEF-EAL domains and CHASE and PAS domains. An insertional cdgD gene mutant was constructed, and analysis of biofilm and extracellular polymeric substance production, as well as the motility phenotype indicated that cdgD encoded a functional diguanylate protein. These results were correlated with a reduced overall cellular concentration of cyclic-di-GMP in the mutant over 48 h compared with that observed in the wild-type strain, which was recovered in the complemented strain. In addition, cdgD gene expression was measured in cells growing under planktonic or biofilm conditions, and differential expression was observed when KNO3 or NH4Cl was added to the minimal medium as a nitrogen source. The transcriptional fusion of the cdgD promoter with the gene encoding the autofluorescent mCherry protein indicated that the cdgD gene was expressed both under abiotic conditions and in association with wheat roots. Reduced colonization of wheat roots was observed for the mutant compared with the wild-type strain grown in the same soil conditions. The Azospirillum-plant association begins with the motility of the bacterium towards the plant rhizosphere followed by the adsorption and adherence of these bacteria to plant roots. Therefore, it is important to study the genes that contribute to this initial interaction of the bacterium with its host plant.

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