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 Stress Tolerance and Environmental Fitness of Pseudomonas fluorescens A506, Which Has a Mutation in rpoS

2009 ◽  
Vol 99 (6) ◽  
pp. 679-688 ◽  
Author(s):  
Mary J. Hagen ◽  
Virginia O. Stockwell ◽  
Cheryl A. Whistler ◽  
Kenneth B. Johnson ◽  
Joyce E. Loper
Keyword(s):  
Biological Control ◽  
Stress Response ◽  
Pseudomonas Fluorescens ◽  
Biological Control Agent ◽  
Environmental Stresses ◽  
Control Agent ◽  
Full Length ◽  
Site Directed Mutagenesis ◽  
Plant Surfaces

Establishment of suppressive populations of bacterial biological control agents on aerial plant surfaces is a critical phase in biologically based management of floral diseases. Periodically, biocontrol agents encounter inhospitable conditions for growth on plants; consequently, tolerance of environmental stresses may contribute to their fitness. In many gram-negative bacteria, including strains of Pseudomonas spp., the capacity to survive environmental stresses is influenced by the stationary phase sigma factor RpoS. This study focused on the role of RpoS in stress response and epiphytic fitness of Pseudomonas fluorescens A506, a well-studied bacterial biological control agent. We detected a frameshift mutation in the rpoS of A506 and demonstrated that the mutation resulted in a truncated, nonfunctional RpoS. Using site-directed mutagenesis, we deleted a nucleotide from rpoS, which then encoded a full-length, functional RpoS. We compared the stress response and epiphytic fitness of A506 with derivative strains having the functional full-length RpoS or a disrupted, nonfunctional RpoS. RpoS had little effect on stress response of A506 and no consistent influence on epiphytic population size of A506 on pear or apple leaves or flowers. Although the capacity of strain A506 to withstand exposure to environmental stresses was similar to that of other fluorescent pseudomonads, this capacity was largely independent of rpoS.

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 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 The MarR-Type Regulator PA3458 Is Involved in Osmoadaptation Control in Pseudomonas aeruginosa

2021 ◽  
Vol 22 (8) ◽  
pp. 3982
Author(s):  
Karolina Kotecka ◽  
Adam Kawalek ◽  
Kamil Kobylecki ◽  
Aneta Agnieszka Bartosik
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Binding Sites ◽  
Transcriptional Profiling ◽  
Mrna Level ◽  
Transcriptional Regulators ◽  
Resistance Mechanisms ◽  
Chronic Infections ◽  
Environmental Signals ◽  
Regulatory Systems

Pseudomonas aeruginosa is a facultative human pathogen, causing acute and chronic infections that are especially dangerous for immunocompromised patients. The eradication of P. aeruginosa is difficult due to its intrinsic antibiotic resistance mechanisms, high adaptability, and genetic plasticity. The bacterium possesses multilevel regulatory systems engaging a huge repertoire of transcriptional regulators (TRs). Among these, the MarR family encompasses a number of proteins, mainly acting as repressors, which are involved in response to various environmental signals. In this work, we aimed to decipher the role of PA3458, a putative MarR-type TR from P. aeruginosa. Transcriptional profiling of P. aeruginosa PAO1161 overexpressing PA3458 showed changes in the mRNA level of 133 genes; among them, 100 were down-regulated, suggesting the repressor function of PA3458. Concomitantly, ChIP-seq analysis identified more than 300 PA3458 binding sites in P. aeruginosa. The PA3458 regulon encompasses genes involved in stress response, including the PA3459–PA3461 operon, which is divergent to PA3458. This operon encodes an asparagine synthase, a GNAT-family acetyltransferase, and a glutamyl aminopeptidase engaged in the production of N-acetylglutaminylglutamine amide (NAGGN), which is a potent bacterial osmoprotectant. We showed that PA3458-mediated control of PA3459–PA3461 expression is required for the adaptation of P. aeruginosa growth in high osmolarity. Overall, our data indicate that PA3458 plays a role in osmoadaptation control in P. aeruginosa.

scholarly journals Long non-coding RNA LINC00665 promotes gemcitabine resistance of Cholangiocarcinoma cells via regulating EMT and stemness properties through miR-424-5p/BCL9L axis

2021 ◽  
Vol 12 (1) ◽  
Author(s):  
Min Lu ◽  
Xinglei Qin ◽  
Yajun Zhou ◽  
Gang Li ◽  
Zhaoyang Liu ◽  
...  
Keyword(s):  
Acquired Resistance ◽  
Transcriptional Regulators ◽  
First Line ◽  
Protein Coding ◽  
Gemcitabine Resistance ◽  
Non Coding Rna ◽  
Sphere Formation ◽  
Long Non Coding Rna ◽  
Line Chemotherapy

AbstractGemcitabine is the first-line chemotherapy drug for cholangiocarcinoma (CCA), but acquired resistance has been frequently observed in CCA patients. To search for potential long noncoding RNAs (lncRNAs) involved in gemcitabine resistance, two gemcitabine resistant CCA cell lines were established and dysregulated lncRNAs were identified by lncRNA microarray. Long intergenic non-protein coding RNA 665 (LINC00665) were found to rank the top 10 upregulated lncRNAs in our study, and high LINC00665 expression was closely associated with poor prognosis and chemoresistance of CCA patients. Silencing LINC00665 in gemcitabine resistant CCA cells impaired gemcitabine tolerance, while enforced LINC00665 expression increased gemcitabine resistance of sensitive CCA cells. The gemcitabine resistant CCA cells showed increased EMT and stemness properties, and silencing LINC00665 suppressed sphere formation, migration, invasion and expression of EMT and stemness markers. In addition, Wnt/β-Catenin signaling was activated in gemcitabine resistant CCA cells, but LINC00665 knockdown suppressed Wnt/β-Catenin activation. B-cell CLL/lymphoma 9-like (BCL9L), the nucleus transcriptional regulators of Wnt/β-Catenin signaling, plays a key role in the nucleus translocation of β-Catenin and promotes β-Catenin-dependent transcription. In our study, we found that LINC00665 regulated BCL9L expression by acting as a molecular sponge for miR-424-5p. Moreover, silencing BCL9L or miR-424-5p overexpression suppressed gemcitabine resistance, EMT, stemness and Wnt/β-Catenin activation in resistant CCA cells. In conclusion, our results disclosed the important role of LINC00665 in gemcitabine resistance of CCA cells, and provided a new biomarker or therapeutic target for CCA treament.

scholarly journals Site-directed mutagenesis of tyrosine hydroxylase. Role of serine 40 in catalysis.

1992 ◽  
Vol 267 (36) ◽  
pp. 25754-25758
Author(s):  
J Wu ◽  
D Filer ◽  
A.J. Friedhoff ◽  
M Goldstein
Keyword(s):  
Tyrosine Hydroxylase ◽  
Site Directed Mutagenesis ◽  
Directed Mutagenesis
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