scholarly journals Massetolide A Biosynthesis in Pseudomonas fluorescens

2007 ◽  
Vol 190 (8) ◽  
pp. 2777-2789 ◽  
Author(s):  
I. de Bruijn ◽  
M. J. D. de Kock ◽  
P. de Waard ◽  
T. A. van Beek ◽  
J. M. Raaijmakers
Keyword(s):  
In Silico Analysis ◽  
Gene Clusters ◽  
Site Directed Mutagenesis ◽  
Pcr Analysis ◽  
Exponential Growth Phase ◽  
Swarming Motility ◽  
Acylhomoserine Lactone ◽  
Cyclic Lipopeptide ◽  
Peptide Moiety ◽  
Peptide Synthetase

ABSTRACT Massetolide A is a cyclic lipopeptide (CLP) antibiotic produced by various Pseudomonas strains from diverse environments. Cloning, sequencing, site-directed mutagenesis, and complementation showed that massetolide A biosynthesis in P. fluorescens SS101 is governed by three nonribosomal peptide synthetase (NRPS) genes, designated massA, massB, and massC, spanning approximately 30 kb. Prediction of the nature and configuration of the amino acids by in silico analysis of adenylation and condensation domains of the NRPSs was consistent with the chemically determined structure of the peptide moiety of massetolide A. Structural analysis of massetolide A derivatives produced by SS101 indicated that most of the variations in the peptide moiety occur at amino acid positions 4 and 9. Regions flanking the mass genes contained several genes found in other Pseudomonas CLP biosynthesis clusters, which encode LuxR-type transcriptional regulators, ABC transporters, and an RND-like outer membrane protein. In contrast to most Pseudomonas CLP gene clusters known to date, the mass genes are not physically linked but are organized in two separate clusters, with massA disconnected from massB and massC. Quantitative real-time PCR analysis indicated that transcription of massC is strongly reduced when massB is mutated, suggesting that these two genes function in an operon, whereas transcription of massA is independent of massBC and vice versa. Massetolide A is produced in the early exponential growth phase, and biosynthesis appears not to be regulated by N-acylhomoserine lactone-based quorum sensing. Massetolide A production is essential in swarming motility of P. fluorescens SS101 and plays an important role in biofilm formation.

scholarly journals Modulation of covR Expression in Streptococcus mutans UA159

2008 ◽  
Vol 190 (13) ◽  
pp. 4478-4488 ◽  
Author(s):  
Patrick Chong ◽  
Laura Drake ◽  
Indranil Biswas
Keyword(s):  
Dental Caries ◽  
Streptococcus Mutans ◽  
Growth Phase ◽  
Response Regulator ◽  
Transcriptional Start Site ◽  
Site Directed Mutagenesis ◽  
Start Codon ◽  
Pcr Analysis ◽  
Exponential Growth Phase ◽  
Mobility Shift

ABSTRACT The biofilm-forming Streptococcus mutans is a gram-positive bacterium that resides in the human oral cavity and is considered to be the primary etiological agent in the formation of dental caries. The global response regulator CovR, which lacks a cognate sensor kinase, is essential for the pathogenesis and biofilm formation of this bacterium, but it is not clear how covR expression is regulated in S. mutans. In this communication, we present the results of our studies examining various factors that regulate the expression of covR in S. mutans UA159. The results of Southern hybridization and PCR analysis indicated that CovR is an orphan response regulator in various isolates of S. mutans. The transcriptional start site for covR was found to be 221 base pairs upstream of the ATG start codon, and site-directed mutagenesis of the upstream TATAAT box confirmed our findings. The expression of covR is growth phase dependent, with maximal expression observed during exponential-growth phase. While changes to the growth temperature did not significantly affect the expression of covR, increasing the pH or the concentration of Mg2+ in the growth medium leads to an increase in covR expression. The results of semiquantitative reverse transcriptase PCR analysis and in vivo transcriptional-fusion reporter assays indicated that CovR autoregulates its own expression; this was verified by the results of electrophoretic mobility shift assays and DNase I protection assays, which demonstrated direct binding of CovR to the promoter region. Apparently, regulation by Mg2+ and the autoregulation of covR are not linked. A detailed analysis of the regulation of CovR may lead to a better understanding of the pathogenesis of S. mutans, as well as providing further insight into the prevention of dental caries.

scholarly journals Cyclic Lipopeptide Production by Plant-Associated Pseudomonas spp.: Diversity, Activity, Biosynthesis, and Regulation

2006 ◽  
Vol 19 (7) ◽  
pp. 699-710 ◽  
Author(s):  
Jos M. Raaijmakers ◽  
Irene de Bruijn ◽  
Maarten J. D. de Kock
Keyword(s):  
Amino Acids ◽  
Fatty Acid ◽  
Structural Diversity ◽  
Main Mode ◽  
Pseudomonas Spp ◽  
Peptide Synthetases ◽  
Cyclic Lipopeptide ◽  
Peptide Moiety ◽  
Peptide Synthetase ◽  
Signature Sequences

Cyclic lipopeptides (CLPs) are versatile molecules produced by a variety of bacterial genera, including plant-associated Pseudomonas spp. CLPs are composed of a fatty acid tail linked to a short oligopeptide, which is cyclized to form a lactone ring between two amino acids in the peptide chain. CLPs are very diverse both structurally and in terms of their biological activity. The structural diversity is due to differences in the length and composition of the fatty acid tail and to variations in the number, type, and configuration of the amino acids in the peptide moiety. CLPs have received considerable attention for their antimicrobial, cytotoxic, and surfactant properties. For plant-pathogenic Pseudomonas spp., CLPs constitute important virulence factors, and pore formation, followed by cell lysis, is their main mode of action. For the antagonistic Pseudomonas sp., CLPs play a key role in antimicrobial activity, motility, and biofilm formation. CLPs are produced via nonribosomal synthesis on large, multifunctional peptide synthetases. Both the structural organization of the CLP synthetic templates and the presence of specific domains and signature sequences within peptide synthetase genes will be described for both pathogenic and antagonistic Pseudomonas spp. Finally, the role of various genes and regulatory mechanisms in CLP production by Pseudomonas spp., including two-component regulation and quorum sensing, will be discussed in detail.

scholarly journals Regulation of Cyclic Lipopeptide Biosynthesis in Pseudomonas fluorescens by the ClpP Protease

2008 ◽  
Vol 191 (6) ◽  
pp. 1910-1923 ◽  
Author(s):  
I. de Bruijn ◽  
J. M. Raaijmakers
Keyword(s):  
Pseudomonas Fluorescens ◽  
Biofilm Formation ◽  
Site Directed Mutagenesis ◽  
Transcriptional Level ◽  
Two Component System ◽  
Swarming Motility ◽  
Cyclic Lipopeptides ◽  
Peptide Synthetases ◽  
Cyclic Lipopeptide ◽  
Casamino Acids

ABSTRACT Cyclic lipopeptides produced by Pseudomonas species exhibit potent surfactant and broad-spectrum antibiotic properties. Their biosynthesis is governed by large multimodular nonribosomal peptide synthetases, but little is known about the genetic regulatory network. This study provides, for the first time, evidence that the serine protease ClpP regulates the biosynthesis of massetolides, cyclic lipopeptides involved in swarming motility, biofilm formation, and antimicrobial activity of Pseudomonas fluorescens SS101. The results show that ClpP affects the expression of luxR(mA), the transcriptional regulator of the massetolide biosynthesis genes massABC, thereby regulating biofilm formation and swarming motility of P. fluorescens SS101. Transcription of luxR(mA) was significantly repressed in the clpP mutant, and introduction of luxR(mA) restored, in part, massetolide biosynthesis and swarming motility of the clpP mutant. Site-directed mutagenesis and expression analyses indicated that the chaperone subunit ClpX and the Lon protease are not involved in regulation of massetolide biosynthesis and are transcribed independently of clpP. Addition of Casamino Acids enhanced the transcription of luxR(mA) and massABC in the clpP mutant, leading to a partial rescue of massetolide production and swarming motility. The results further suggested that, at the transcriptional level, ClpP-mediated regulation of massetolide biosynthesis operates independently of regulation by the GacA/GacS two-component system. The role of amino acid metabolism and the putative mechanisms underlying ClpP-mediated regulation of cyclic lipopeptide biosynthesis, swarming motility, and growth in P. fluorescens are discussed.

scholarly journals Diversity and Functional Analysis of LuxR-Type Transcriptional Regulators of Cyclic Lipopeptide Biosynthesis in Pseudomonas fluorescens

2009 ◽  
Vol 75 (14) ◽  
pp. 4753-4761 ◽  
Author(s):  
I. de Bruijn ◽  
J. M. Raaijmakers
Keyword(s):  
Pseudomonas Fluorescens ◽  
Response Regulator ◽  
Phylogenetic Analyses ◽  
Regulatory Gene ◽  
Transcriptional Regulators ◽  
Site Directed Mutagenesis ◽  
Acylhomoserine Lactone ◽  
Cyclic Lipopeptide ◽  
Surface Motility

ABSTRACT Cyclic lipopeptides (CLPs) are produced by many Pseudomonas species and have several biological functions, including a role in surface motility, biofilm formation, virulence, and antimicrobial activity. This study focused on the diversity and role of LuxR-type transcriptional regulators in CLP biosynthesis in Pseudomonas species and, specifically, viscosin production by Pseudomonas fluorescens strain SBW25. Phylogenetic analyses showed that CLP biosynthesis genes in Pseudomonas strains are flanked by LuxR-type regulators that contain a DNA-binding helix-turn-helix domain but lack N-acylhomoserine lactone-binding or response regulator domains. For SBW25, site-directed mutagenesis of the genes coding for either of the two identified LuxR-type regulators, designated ViscAR and ViscBCR, strongly reduced transcript levels of the viscABC biosynthesis genes and resulted in a loss of viscosin production. Expression analyses further showed that a mutation in either viscAR or viscBCR did not substantially (change of <2.5-fold) affect transcription of the other regulator. Transformation of the ΔviscAR mutant of SBW25 with a LuxR-type regulatory gene from P. fluorescens strain SS101 that produces massetolide, a CLP structurally related to viscosin, restored transcription of the viscABC genes and viscosin production. The results further showed that a functional viscAR gene was required for heterologous expression of the massetolide biosynthesis genes of strain SS101 in strain SBW25, leading to the production of both viscosin and massetolide. Collectively, these results indicate that the regulators flanking the CLP biosynthesis genes in Pseudomonas species represent a unique LuxR subfamily of proteins and that viscosin biosynthesis in P. fluorescens SBW25 is controlled by two LuxR-type transcriptional regulators.

scholarly journals Specialized Metabolites from Ribosome Engineered Strains of Streptomyces clavuligerus

Metabolites ◽  
2021 ◽  
Vol 11 (4) ◽  
pp. 239
Author(s):  
Arshad Ali Shaikh ◽  
Louis-Felix Nothias ◽  
Santosh K. Srivastava ◽  
Pieter C. Dorrestein ◽  
Kapil Tahlan
Keyword(s):  
In Silico Analysis ◽  
Cost Effective ◽  
Gene Clusters ◽  
Streptomyces Clavuligerus ◽  
Strain Engineering ◽  
Ribosome Recycling Factor ◽  
Metabolic Potential ◽  
Putative Gene ◽  
Specialized Metabolites ◽  
Streptomyces Spp

Bacterial specialized metabolites are of immense importance because of their medicinal, industrial, and agricultural applications. Streptomyces clavuligerus is a known producer of such compounds; however, much of its metabolic potential remains unknown, as many associated biosynthetic gene clusters are silent or expressed at low levels. The overexpression of ribosome recycling factor (frr) and ribosome engineering (induced rpsL mutations) in other Streptomyces spp. has been reported to increase the production of known specialized metabolites. Therefore, we used an overexpression strategy in combination with untargeted metabolomics, molecular networking, and in silico analysis to annotate 28 metabolites in the current study, which have not been reported previously in S. clavuligerus. Many of the newly described metabolites are commonly found in plants, further alluding to the ability of S. clavuligerus to produce such compounds under specific conditions. In addition, the manipulation of frr and rpsL led to different metabolite production profiles in most cases. Known and putative gene clusters associated with the production of the observed compounds are also discussed. This work suggests that the combination of traditional strain engineering and recently developed metabolomics technologies together can provide rapid and cost-effective strategies to further speed up the discovery of novel natural products.

scholarly journals Comprehensive Assessment of Copy Number Alterations Uncovers Recurrent AIFM3 and DLK1 Copy Gain in Medullary Thyroid Carcinoma

Cancers ◽  
2021 ◽  
Vol 13 (2) ◽  
pp. 218
Author(s):  
Aline Neves Araujo ◽  
Cléber Pinto Camacho ◽  
Thais Biude Mendes ◽  
Susan Chow Lindsey ◽  
Lais Moraes ◽  
...  
Keyword(s):  
Thyroid Carcinoma ◽  
Medullary Thyroid Carcinoma ◽  
Copy Number ◽  
Snp Array ◽  
In Silico Analysis ◽  
Pcr Analysis ◽  
Copy Number Alterations ◽  
Medullary Thyroid ◽  
Thyroid C Cells ◽  
Ajcc Staging

Medullary thyroid carcinoma (MTC) is a malignant tumor originating from thyroid C-cells that can occur either in sporadic (70–80%) or hereditary (20–30%) form. In this study we aimed to identify recurrent copy number alterations (CNA) that might be related to the pathogenesis or progression of MTC. We used Affymetrix SNP array 6.0 on MTC and paired-blood samples to identify CNA using PennCNV and Genotyping Console software. The algorithms identified recurrent copy number gains in chromosomes 15q, 10q, 14q and 22q in MTC, whereas 4q cumulated losses. Coding genes were identified within CNA regions. The quantitative PCR analysis performed in an independent series of MTCs (n = 51) confirmed focal recurrent copy number gains encompassing the DLK1 (14q32.2) and AIFM3 (22q11.21) genes. Immunohistochemistry confirmed AIFM3 and DLK1 expression in MTC cases, while no expression was found in normal thyroid tissues and few MTC samples were found with normal copy numbers. The functional relevance of CNA was also assessed by in silico analysis. CNA status correlated with protein expression (DLK1, p = 0.01), tumor size (DLK1, p = 0.04) and AJCC staging (AIFM3p = 0.01 and DLK1p = 0.05). These data provide a novel insight into MTC biology, and suggest a common CNA landscape, regardless of if it is sporadic or hereditary MTC.

scholarly journals Antimicrobials Inspired by Nonribosomal Peptide Synthetase Gene Clusters

2017 ◽  
Vol 139 (4) ◽  
pp. 1404-1407 ◽  
Author(s):  
Xavier Vila-Farres ◽  
John Chu ◽  
Daigo Inoyama ◽  
Melinda A. Ternei ◽  
Christophe Lemetre ◽  
...  
Keyword(s):  
Gene Clusters ◽  
Nonribosomal Peptide Synthetase ◽  
Nonribosomal Peptide ◽  
Peptide Synthetase

scholarly journals Inhibition of Quorum Sensing and Biofilm Formation of Esculetin on Aeromonas Hydrophila

2021 ◽  
Vol 12 ◽  
Author(s):  
Bing Sun ◽  
Huaizhi Luo ◽  
Huan Jiang ◽  
Zhennan Wang ◽  
Aiqun Jia
Keyword(s):  
Quorum Sensing ◽  
Aeromonas Hydrophila ◽  
Biofilm Formation ◽  
Laser Scanning ◽  
Laser Scanning Microscopy ◽  
Confocal Laser ◽  
Pcr Analysis ◽  
Swarming Motility ◽  
Gene Expression Levels ◽  
Good Agreement

Quorum sensing (QS) and biofilm formation inhibition activity of esculetin on Aeromonas hydrophila SHAe 115 were evaluated. Exposure to esculetin at 25, 50, and 100μg/ml significantly inhibited the production of protease and hemolysin, the formation of biofilms and attenuated the swarming motility of A. hydrophila SHAe 115. Biofilm forming inhibition was also observed through confocal laser scanning microscopy and scanning electron microscope. Quantitative real-time PCR analysis indicated that genes positively related to QS and biofilm formation were downregulated to varying degrees, while gene (litR) negatively related to biofilm formation was significantly upregulated. The phenotypic results were in good agreement with gene expression levels. These results indicated that esculetin would be a potential QS inhibitor for A. hydrophila.

scholarly journals The Draft Genome Sequence of The Endophytic Streptomyces Strain VITGV156

2021 ◽  
Author(s):  
Veilumuthu P ◽  
Nagarajan T ◽  
Sasikumar S ◽  
Siva R ◽  
J Godwin Christopher
Keyword(s):  
Secondary Metabolites ◽  
Draft Genome ◽  
Gc Content ◽  
Gene Clusters ◽  
Dominant Group ◽  
Protein Coding ◽  
Peptide Synthetase ◽  
The Family ◽  
Modified Peptides ◽  
Endophytic Streptomyces

Abstract Streptomyces species is one among the dominant group of bacteria in the family Actinobacteria with a rich repertoire of secondary metabolites. Secondary metabolites with antimicrobial activity and plant growth promotor have been isolated from various Streptomyces sp. Here in this investigation, we present the draft genome of a new species, Streptomyces sp. VITGV156 isolated from healthy tomato plant (Lycopersicon esculentum) which has some rare antimicrobial secondary metabolites, like coelichelin, fluostatins, vicenistatin, nystatin, sipanmycin, and informatipeptin. The genome is 8.18 Mb in size with 6,259 protein coding genes. The average GC content of the genome is 72.61 %. Preliminary analysis with antiSMASH 6.0 revealed the presence of 29 biosynthetic gene clusters for the synthesis of potential secondary metabolites. These includes 4 NRPS (non – ribosomal peptide synthetase), 7 PKS (Polyketide Synthases), 2 RiPP (Ribosomally synthesized and post-translationally modified peptides) clusters. When we look into genes associated with secondary metabolites, 406 genes are present which includes 184 genes for cofactor and vitamins, 72 genes for terpenoids and polyketides, 70 genes for xenobiotics and 80 genes for other metabolites are present. Comparative genome analysis of VITGV156 with its closest neighbor Streptomyces luteus strain TRM45540 revealed ANI 91.22% and dDDH value 44.00%.

scholarly journals Inhibition of Quorum Sensing in Serratia marcescens AS-1 by Synthetic Analogs of N-Acylhomoserine Lactone

2007 ◽  
Vol 73 (20) ◽  
pp. 6339-6344 ◽  
Author(s):  
Tomohiro Morohoshi ◽  
Toshitaka Shiono ◽  
Kiyomi Takidouchi ◽  
Masashi Kato ◽  
Norihiro Kato ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Serratia Marcescens ◽  
Biofilm Formation ◽  
Acyl Chain ◽  
Regulatory System ◽  
Homoserine Lactone ◽  
Signal Molecule ◽  
Swarming Motility ◽  
Gram Negative ◽  
Acylhomoserine Lactone

ABSTRACT Quorum sensing is a regulatory system for controlling gene expression in response to increasing cell density. N-Acylhomoserine lactone (AHL) is produced by gram-negative bacteria, which use it as a quorum-sensing signal molecule. Serratia marcescens is a gram-negative opportunistic pathogen which is responsible for an increasing number of serious nosocomial infections. S. marcescens AS-1 produces N-hexanoyl homoserine lactone (C6-HSL) and N-(3-oxohexanoyl) homoserine lactone and regulates prodigiosin production, swarming motility, and biofilm formation by AHL-mediated quorum sensing. We synthesized a series of N-acyl cyclopentylamides with acyl chain lengths ranging from 4 to 12 and estimated their inhibitory effects on prodigiosin production in AS-1. One of these molecules, N-nonanoyl-cyclopentylamide (C9-CPA), had a strong inhibitory effect on prodigiosin production. C9-CPA also inhibited the swarming motility and biofilm formation of AS-1. A competition assay revealed that C9-CPA was able to inhibit quorum sensing at four times the concentration of exogenous C6-HSL and was more effective than the previously reported halogenated furanone. Our results demonstrated that C9-CPA was an effective quorum-sensing inhibitor for S. marcescens AS-1.

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