scholarly journals Timing and Localization of Rhamnolipid Synthesis Gene Expression in Pseudomonas aeruginosa Biofilms

2005 ◽  
Vol 187 (1) ◽  
pp. 37-44 ◽  
Author(s):  
Yannick Lequette ◽  
E. P. Greenberg
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Promoter Activity ◽  
Fluorescent Dyes ◽  
Homoserine Lactone ◽  
Biofilm Growth ◽  
Neutral Site ◽  
Biofilm Architecture ◽  
Biofilm Development

ABSTRACT Pseudomonas aeruginosa biofilms can develop mushroom-like structures with stalks and caps consisting of discrete subpopulations of cells. Self-produced rhamnolipid surfactants have been shown to be important in development of the mushroom-like structures. The quorum-sensing-controlled rhlAB operon is required for rhamnolipid synthesis. We have introduced an rhlA-gfp fusion into a neutral site in the P. aeruginosa genome to study rhlAB promoter activity in rhamnolipid-producing biofilms. Expression of the rhlA-gfp fusion in biofilms requires the quorum-sensing signal butanoyl-homoserine lactone, but other factors are also required for expression. Early in biofilm development rhlA-gfp expression is low, even in the presence of added butanoyl-homoserine lactone. Expression of the fusion becomes apparent after microcolonies with a depth of >20 μm have formed and, as shown by differential labeling with rfp or fluorescent dyes, rhlA-gfp is preferentially expressed in the stalks rather than the caps of mature mushrooms. The rhlA-gfp expression pattern is not greatly influenced by addition of butanoyl-homoserine lactone to the biofilm growth medium. We propose that rhamnolipid synthesis occurs in biofilms after stalks have formed but prior to capping in the mushroom-like structures. The differential expression of rhlAB may play a role in the development of normal biofilm architecture.

scholarly journals Quorum-Sensing Genes in Pseudomonas aeruginosa Biofilms: Their Role and Expression Patterns

2001 ◽  
Vol 67 (4) ◽  
pp. 1865-1873 ◽  
Author(s):  
Teresa R. De Kievit ◽  
Richard Gillis ◽  
Steve Marx ◽  
Chris Brown ◽  
Barbara H. Iglewski
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Fluorescent Protein ◽  
Expression Patterns ◽  
Homoserine Lactone ◽  
Lower Percentage ◽  
Spatial Studies ◽  
Green Fluorescent ◽  
Biofilm Development

ABSTRACT Acylated homoserine lactone molecules are used by a number of gram-negative bacteria to regulate cell density-dependent gene expression by a mechanism known as quorum sensing (QS). InPseudomonas aeruginosa, QS or cell-to-cell signaling controls expression of a number of virulence factors, as well as biofilm differentiation. In this study, we investigated the role played by the las and rhl QS systems during the early stages of static biofilm formation when cells are adhering to a surface and forming microcolonies. These studies revealed a marked difference in biofilm formation between the PAO1 parent and the QS mutants when glucose, but not citrate, was used as the sole carbon source. To further elucidate the contribution of lasI andrhlI to biofilm maturation, we utilized fusions to unstable green fluorescent protein in concert with confocal microscopy to perform real-time temporal and spatial studies of these genes in a flowing environment. During the course of 8-day biofilm development,lasI expression was found to progressively decrease over time. Conversely, rhlI expression remained steady throughout biofilm development but occurred in a lower percentage of cells. Spatial analysis revealed that lasI andrhlI were maximally expressed in cells located at the substratum and that expression decreased with increasing biofilm height. Because QS was shown previously to be involved in biofilm differentiation, these findings have important implications for the design of biofilm prevention and eradication strategies.

scholarly journals Microarray Analysis of Pseudomonas aeruginosa Quorum-Sensing Regulons: Effects of Growth Phase and Environment

2003 ◽  
Vol 185 (7) ◽  
pp. 2080-2095 ◽  
Author(s):  
Victoria E. Wagner ◽  
Daniel Bushnell ◽  
Luciano Passador ◽  
Andrew I. Brooks ◽  
Barbara H. Iglewski
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Expression Patterns ◽  
Antibiotic Sensitivity ◽  
Opportunistic Pathogen ◽  
Homoserine Lactone ◽  
Medium Composition ◽  
Logarithmic Phase ◽  
Significant Differential Expression ◽  
Biofilm Development

ABSTRACT Bacterial communication via quorum sensing (QS) has been reported to be important in the production of virulence factors, antibiotic sensitivity, and biofilm development. Two QS systems, known as the las and rhl systems, have been identified previously in the opportunistic pathogen Pseudomonas aeruginosa. High-density oligonucleotide microarrays for the P. aeruginosa PAO1 genome were used to investigate global gene expression patterns modulated by QS regulons. In the initial experiments we focused on identifying las and/or rhl QS-regulated genes using a QS signal generation-deficient mutant (PAO-JP2) that was cultured with and without added exogenous autoinducers [N-(3-oxododecanoyl) homoserine lactone and N-butyryl homoserine lactone]. Conservatively, 616 genes showed statistically significant differential expression (P ≤ 0.05) in response to the exogenous autoinducers and were classified as QS regulated. A total of 244 genes were identified as being QS regulated at the mid-logarithmic phase, and 450 genes were identified as being QS regulated at the early stationary phase. Most of the previously reported QS-promoted genes were confirmed, and a large number of additional QS-promoted genes were identified. Importantly, 222 genes were identified as being QS repressed. Environmental factors, such as medium composition and oxygen availability, eliminated detection of transcripts of many genes that were identified as being QS regulated.

scholarly journals Pseudomonas aeruginosa Displays Multiple Phenotypes during Development as a Biofilm

2002 ◽  
Vol 184 (4) ◽  
pp. 1140-1154 ◽  
Author(s):  
Karin Sauer ◽  
Anne K. Camper ◽  
Garth D. Ehrlich ◽  
J. William Costerton ◽  
David G. Davies
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Protein Identification ◽  
Electrophoretic Analysis ◽  
Cell Protein ◽  
Biofilm Growth ◽  
Alginate Production ◽  
Multiple Phenotypes ◽  
Reporter Gene Analysis ◽  
Biofilm Development

ABSTRACT Complementary approaches were employed to characterize transitional episodes in Pseudomonas aeruginosa biofilm development using direct observation and whole-cell protein analysis. Microscopy and in situ reporter gene analysis were used to directly observe changes in biofilm physiology and to act as signposts to standardize protein collection for two-dimensional electrophoretic analysis and protein identification in chemostat and continuous-culture biofilm-grown populations. Using these approaches, we characterized five stages of biofilm development: (i) reversible attachment, (ii) irreversible attachment, (iii) maturation-1, (iv) maturation-2, and (v) dispersion. Biofilm cells were shown to change regulation of motility, alginate production, and quorum sensing during the process of development. The average difference in detectable protein regulation between each of the five stages of development was 35% (approximately 525 proteins). When planktonic cells were compared with maturation-2 stage biofilm cells, more than 800 proteins were shown to have a sixfold or greater change in expression level (over 50% of the proteome). This difference was higher than when planktonic P. aeruginosa were compared with planktonic cultures of Pseudomonas putida. Las quorum sensing was shown to play no role in early biofilm development but was important in later stages. Biofilm cells in the dispersion stage were more similar to planktonic bacteria than to maturation-2 stage bacteria. These results demonstrate that P. aeruginosa displays multiple phenotypes during biofilm development and that knowledge of stage-specific physiology may be important in detecting and controlling biofilm growth.

scholarly journals The RhlR quorum-sensing receptor controls Pseudomonas aeruginosa pathogenesis and biofilm development independently of its canonical homoserine lactone autoinducer

2017 ◽  
Vol 13 (7) ◽  
pp. e1006504 ◽  
Author(s):  
Sampriti Mukherjee ◽  
Dina Moustafa ◽  
Chari D. Smith ◽  
Joanna B. Goldberg ◽  
Bonnie L. Bassler
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Homoserine Lactone ◽  
Biofilm Development

scholarly journals Two GacA-Dependent Small RNAs Modulate the Quorum-Sensing Response in Pseudomonas aeruginosa

2006 ◽  
Vol 188 (16) ◽  
pp. 6026-6033 ◽  
Author(s):  
Elisabeth Kay ◽  
Bérénice Humair ◽  
Valérie Dénervaud ◽  
Kathrin Riedel ◽  
Stéphanie Spahr ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Small Rnas ◽  
Homoserine Lactone ◽  
Two Component System ◽  
Bond Forming ◽  
Small Regulatory Rnas ◽  
Regulatory Rnas ◽  
Chitin Binding Protein ◽  
Biofilm Development

ABSTRACT In Pseudomonas aeruginosa, the GacS/GacA two-component system positively controls the quorum-sensing machinery and the expression of extracellular products via two small regulatory RNAs, RsmY and RsmZ. An rsmY rsmZ double mutant and a gacA mutant were similarly impaired in the synthesis of the quorum-sensing signal N-butanoyl-homoserine lactone, the disulfide bond-forming enzyme DsbA, and the exoproducts hydrogen cyanide, pyocyanin, elastase, chitinase (ChiC), and chitin-binding protein (CbpD). Both mutants showed increased swarming ability, azurin release, and early biofilm development.

scholarly journals ThePseudomonas aeruginosaOrphan Quorum Sensing Signal Receptor QscR Regulates Global Quorum Sensing Gene Expression by Activating a Single Linked Operon

mBio ◽  
2018 ◽  
Vol 9 (4) ◽  
Author(s):  
Fengming Ding ◽  
Ken-Ichi Oinuma ◽  
Nicole E. Smalley ◽  
Amy L. Schaefer ◽  
Omar Hamwy ◽  
...  
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Transcription Factor ◽  
Quorum Sensing ◽  
Cell Communication ◽  
Homoserine Lactone ◽  
Acyl Homoserine Lactone ◽  
Null Mutant ◽  
Content Type ◽  
Single Operon

ABSTRACTPseudomonas aeruginosauses two acyl-homoserine lactone signals and two quorum sensing (QS) transcription factors, LasR and RhlR, to activate dozens of genes. LasR responds toN-3-oxo-dodecanoyl-homoserine lactone (3OC12-HSL) and RhlR toN-butanoyl-homoserine lactone (C4-HSL). There is a thirdP. aeruginosaacyl-homoserine-lactone-responsive transcription factor, QscR, which acts to dampen or delay activation of genes by LasR and RhlR by an unknown mechanism. To better understand the role of QscR inP. aeruginosaQS, we performed a chromatin immunoprecipitation analysis, which showed this transcription factor bound the promoter of only a single operon of three genes linked toqscR, PA1895 to PA1897. Other genes that appear to be regulated by QscR in transcriptome studies were not direct targets of QscR. Deletion of PA1897 recapitulates the early QS activation phenotype of a QscR-null mutant, and the phenotype of a QscR-null mutant was complemented by PA1895-1897 but not by PA1897 alone. We conclude that QscR acts to modulate quorum sensing through regulation of a single operon, apparently raising the QS threshold of the population and providing a “brake” on QS autoinduction.IMPORTANCEQuorum sensing, a cell-cell communication system, is broadly distributed among bacteria and is commonly used to regulate the production of shared products. An important consequence of quorum sensing is a delay in production of certain products until the population density is high. The bacteriumPseudomonas aeruginosahas a particularly complicated quorum sensing system involving multiple signals and receptors. One of these receptors, QscR, downregulates gene expression, unlike the other receptors inP. aeruginosa. QscR does so by inducing the expression of a single operon whose function provides an element of resistance to a population reaching a quorum. This finding has importance for design of quorum sensing inhibitory strategies and can also inform design of synthetic biological circuits that use quorum sensing receptors to regulate gene expression.

Transcriptome analysis of Pseudomonas aeruginosa biofilm development: anaerobic respiration and iron limitation

Biofilms ◽  
2005 ◽  
Vol 2 (1) ◽  
pp. 37-61 ◽  
Author(s):  
M. Hentzer ◽  
L. Eberl ◽  
M. Givskov
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Antimicrobial Agents ◽  
Expression Profiles ◽  
Gene Expression Profiles ◽  
Complex Surface ◽  
Homoserine Lactone ◽  
Iron Limitation ◽  
Form Complex ◽  
Biofilm Development

In nature, bacteria are able to form complex surface-attached communities called biofilms. Microbial biofilms pose a particular problem in many human infections because of an inherent tolerance to antimicrobial agents and host immune killing and clearance. We have used complementary DNA (cDNA) microarray technology to identify Pseudomonas aeruginosa genes that are differentially expressed in growing and developing biofilms. Our study shows that, when compared with planktonic bacteria, gene expression profiles of biofilm cells have the highest resemblance to the profiles of stationary-phase cells. We suggest that the process of biofilm development involves a series of adaptive responses including those to anaerobic and iron-limitation stresses, rather than being associated with a unique biofilm developmental program. Mapping of quorum-sensing regulated genes in a P. aeruginosa biofilm identified a set of N-acyl homoserine lactone (AHL)-dependent genes that are exclusively expressed in sessile cells. One of these genes, pvdQ, encodes an AHL acylase that degrades long-acyl but not short-acyl AHLs. This result may provide an explanation for the previous finding that the level of long-acyl AHLs is greatly reduced in P. aeruginosa biofilm cells as compared with their planktonic counterparts. Furthermore, we present evidence that quorum sensing is participating in the control of iron-limitation responses in the biofilm cells.

scholarly journals Advancing the Quorum in Pseudomonas aeruginosa: MvaT and the Regulation of N-Acylhomoserine Lactone Production and Virulence Gene Expression

2002 ◽  
Vol 184 (10) ◽  
pp. 2576-2586 ◽  
Author(s):  
Stephen P. Diggle ◽  
Klaus Winzer ◽  
Andrée Lazdunski ◽  
Paul Williams ◽  
Miguel Cámara
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Growth Phase ◽  
Virulence Gene ◽  
Homoserine Lactone ◽  
Logarithmic Phase ◽  
Signal Molecules ◽  
Virulence Gene Expression ◽  
Acylhomoserine Lactone

ABSTRACT Pseudomonas aeruginosa regulates the production of many exoproteins and secondary metabolites via a hierarchical quorum-sensing cascade through LasR and RhlR and their cognate signal molecules N-(3-oxododecanoyl)-l-homoserine lactone (3O-C12-HSL) and N-(butanoyl)-l-homoserine lactone (C4-HSL). In this study, we found that transcription of the quorum sensing-regulated genes lecA (coding for PA-IL lectin), lasB (coding for elastase), and rpoS appeared to be growth phase dependent and their expression could not be advanced to the logarithmic phase in cells growing in batch culture by the addition of exogenous C4-HSL and 3O-C12-HSL. To identify novel regulators responsible for this growth phase dependency, a P. aeruginosa lecA::lux reporter strain was subjected to random transposon mutagenesis. A number of mutants affected in lecA expression were found that exhibited altered production of multiple quorum sensing-dependent phenotypes. While some mutations were mapped to new loci such as clpA and mvaT and a putative efflux system, a number of mutations were also mapped to known regulators such as lasR, rhlR, and rpoS. MvaT was identified as a novel global regulator of virulence gene expression, as a mutation in mvaT resulted in enhanced lecA expression and pyocyanin production. This mutant also showed altered swarming ability and production of the LasB and LasA proteases, 3O-C12-HSL, and C4-HSL. Furthermore, addition of exogenous 3O-C12-HSL and C4-HSL to the mvaT mutant significantly advanced lecA expression, suggesting that MvaT is involved in the growth phase-dependent regulation of the lecA gene.

scholarly journals Identification, Timing, and Signal Specificity of Pseudomonas aeruginosa Quorum-Controlled Genes: a Transcriptome Analysis

2003 ◽  
Vol 185 (7) ◽  
pp. 2066-2079 ◽  
Author(s):  
Martin Schuster ◽  
C. Phoebe Lostroh ◽  
Tomoo Ogi ◽  
E. P. Greenberg
Keyword(s):  
Gene Expression ◽  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Stationary Phase ◽  
Transcriptome Analysis ◽  
Homoserine Lactone ◽  
Logarithmic Phase ◽  
Acyl Homoserine Lactone ◽  
Global Regulators ◽  
Signaling Systems

ABSTRACT There are two interrelated acyl-homoserine lactone quorum-sensing-signaling systems in Pseudomonas aeruginosa. These systems, the LasR-LasI system and the RhlR-RhlI system, are global regulators of gene expression. We performed a transcriptome analysis to identify quorum-sensing-controlled genes and to better understand quorum-sensing control of P. aeruginosa gene expression. We compared gene expression in a LasI-RhlI signal mutant grown with added signals to gene expression without added signals, and we compared a LasR-RhlR signal receptor mutant to its parent. In all, we identified 315 quorum-induced and 38 quorum-repressed genes, representing about 6% of the P. aeruginosa genome. The quorum-repressed genes were activated in the stationary phase in quorum-sensing mutants but were not activated in the parent strain. The analysis of quorum-induced genes suggests that the signal specificities are on a continuum and that the timing of gene expression is on a continuum (some genes are induced early in growth, most genes are induced at the transition from the logarithmic phase to the stationary phase, and some genes are induced during the stationary phase). In general, timing was not related to signal concentration. We suggest that the level of the signal receptor, LasR, is a critical trigger for quorum-activated gene expression. Acyl-homoserine lactone quorum sensing appears to be a system that allows ordered expression of hundreds of genes during P. aeruginosa growth in culture.

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