scholarly journals Quorum sensing in Bacillus subtilis slows down biofilm formation by enabling sporulation bet hedging

2019 ◽  
Author(s):  
Mihael Spacapan ◽  
Tjaša Danevčič ◽  
Polonca Štefanic ◽  
Ines Mandic-Mulec
Keyword(s):  
Bacillus Subtilis ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Bet Hedging ◽  
Wild Type ◽  
Master Regulator ◽  
Matrix Components ◽  
Sporulation Initiation ◽  
Biofilm Development ◽  
Biofilm Matrix

1.2ABSTRACTThe ComQXPA quorum sensing (QS) system of Bacillus subtilis, a Gram-positive, industrially relevant, endospore forming bacterium, promotes surfactin production. This lipopeptide increases transcription of several genes involved in biofilm matrix synthesis via the Spo0A-P master regulator. We hypothesized that the inactivation of the QS system will therefore result in decreased rates of floating biofilm formation. We find that this is not the case and that the QS deficient mutant forms pellicles with a faster rate and produces more biofilm matrix components than the wild type. As Spo0A-P is the master regulator of sporulation initiation we hypothesized that the ComQXPA dependent signaling promotes sporulation and consequently slows the growth rate of the wild type strain. Indeed, our results confirm that cells with the inactive QS initiate endospore formation in biofilms later and more synchronously than the wild type, as evidenced by spore frequencies and the PspoIIQ promoter activity. We argue, that the QS system acts as a switch that promotes stochastic sporulation initiation and consequently bet hedging behavior. By committing a subpopulation of cells to sporulation early during growth, wild type population grows slower and produces thinner biofilms but also assures better survival under stressful conditions.1.1IMPORTANCEBacillus subtilis is widely employed model organism to study biofilm formation and sporulation in Gram-positive bacteria. The ComQXPA quorum sensing (QS) system indirectly increases the transcription of genes involved in biofilm matrix formation, which predicts a positive role of this QS in biofilm development Here we show that QS mutants actually form more matrix components per pellicle than the wild type and that their pellicles are thicker and form with a faster rate. We explain this, by showing that cells with an inactive QS exhibit a delay in sporulation entry, which is also more synchronous relative to the wild type. We argue, that the ComQXPA QS system acts as a switch that contributes to the stochastic sporulation initiation and though this path promotes bet hedging behavior. This finding is important in terms of “quorum quenching” strategies aiming to down modulate biofilm development through inhibition of QS signaling and underscores the richness of QS regulated phenotypic outcomes among bacterial species.

scholarly journals Quorum-Sensing Regulation of the Biofilm Matrix Genes (pel) of Pseudomonas aeruginosa

2007 ◽  
Vol 189 (14) ◽  
pp. 5383-5386 ◽  
Author(s):  
Yumiko Sakuragi ◽  
Roberto Kolter
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Homoserine Lactone ◽  
Wild Type ◽  
Wild Type Level ◽  
Dna Release ◽  
Biofilm Development ◽  
Development Regulation ◽  
Biofilm Matrix

ABSTRACT Quorum sensing (QS) has been previously shown to play an important role in the development of Pseudomonas aeruginosa biofilms (D. G. Davies et al., Science 280:295-298, 1998). Although QS regulation of swarming and DNA release has been shown to play important roles in biofilm development, regulation of genes directly involved in biosynthesis of biofilm matrix has not been described. Here, transcription of the pel operon, essential for the production of a glucose-rich matrix exopolysaccharide, is shown to be greatly reduced in lasI and rhlI mutants. Chemical complementation of the lasI mutant with 3-oxo-dodecanoyl homoserine lactone restores pel transcription to the wild-type level and biofilm formation ability. These findings thus connect QS signaling and transcription of genes responsible for biofilm matrix biosynthesis.

scholarly journals The ComX Quorum Sensing Peptide of Bacillus subtilis Affects Biofilm Formation Negatively and Sporulation Positively

2020 ◽  
Vol 8 (8) ◽  
pp. 1131 ◽  
Author(s):  
Mihael Špacapan ◽  
Tjaša Danevčič ◽  
Polonca Štefanic ◽  
Michael Porter ◽  
Nicola R. Stanley-Wall ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Quorum Sensing ◽  
Biofilm Formation ◽  
Matrix Protein ◽  
Control Strategies ◽  
Wild Type ◽  
Fibrous Matrix ◽  
Sensing System ◽  
Quorum Sensing System ◽  
Biofilm Development

Quorum sensing (QS) is often required for the formation of bacterial biofilms and is a popular target of biofilm control strategies. Previous studies implicate the ComQXPA quorum sensing system of Bacillus subtilis as a promoter of biofilm formation. Here, we report that ComX signaling peptide deficient mutants form thicker and more robust pellicle biofilms that contain chains of cells. We confirm that ComX positively affects the transcriptional activity of the PepsA promoter, which controls the synthesis of the major matrix polysaccharide. In contrast, ComX negatively controls the PtapA promoter, which drives the production of TasA, a fibrous matrix protein. Overall, the biomass of the mutant biofilm lacking ComX accumulates more monosaccharide and protein content than the wild type. We conclude that this QS phenotype might be due to extended investment into growth rather than spore development. Consistent with this, the ComX deficient mutant shows a delayed activation of the pre-spore specific promoter, PspoIIQ, and a delayed, more synchronous commitment to sporulation. We conclude that ComX mediated early commitment to sporulation of the wild type slows down biofilm formation and modulates the coexistence of multiple biological states during the early stages of biofilm development.

scholarly journals Selective Pressure for Biofilm Formation in Bacillus subtilis: Differential Effect of Mutations in the Master Regulator SinR on Bistability

mBio ◽  
2018 ◽  
Vol 9 (5) ◽  
Author(s):  
Jan Kampf ◽  
Jan Gerwig ◽  
Kerstin Kruse ◽  
Robert Cleverley ◽  
Miriam Dormeyer ◽  
...  
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Biofilm Formation ◽  
Type Strain ◽  
Wild Type Strain ◽  
Selective Pressure ◽  
Wild Type ◽  
Master Regulator ◽  
Form Complex ◽  
Content Type

ABSTRACT Biofilm formation by Bacillus subtilis requires the expression of genes encoding enzymes for extracellular polysaccharide synthesis and for an amyloid-like protein. The master regulator SinR represses all the corresponding genes, and repression of these key biofilm genes is lifted when SinR interacts with its cognate antagonist proteins. The YmdB phosphodiesterase is a recently discovered factor that is involved in the control of SinR activity: cells lacking YmdB exhibit hyperactive SinR and are unable to relieve the repression of the biofilm genes. In this study, we have examined the dynamics of gene expression patterns in wild-type and ymdB mutant cells by microfluidic analysis coupled to time-lapse microscopy. Our results confirm the bistable expression pattern for motility and biofilm genes in the wild-type strain and the loss of biofilm gene expression in the mutant. Moreover, we demonstrated dynamic behavior in subpopulations of the wild-type strain that is characterized by switches in sets of the expressed genes. In order to gain further insights into the role of YmdB, we isolated a set of spontaneous suppressor mutants derived from ymdB mutants that had regained the ability to form complex colonies and biofilms. Interestingly, all of the mutations affected SinR. In some mutants, large genomic regions encompassing sinR were deleted, whereas others had alleles encoding SinR variants. Functional and biochemical studies with these SinR variants revealed how these proteins allowed biofilm gene expression in the ymdB mutant strains. IMPORTANCE Many bacteria are able to choose between two mutually exclusive lifestyles: biofilm formation and motility. In the model bacterium Bacillus subtilis, this choice is made by each individual cell rather than at the population level. The transcriptional repressor SinR is the master regulator in this decision-making process. The regulation of SinR activity involves complex control of its own expression and of its interaction with antagonist proteins. We show that the YmdB phosphodiesterase is required to allow the expression of SinR-repressed genes in a subpopulation of cells and that such subpopulations can switch between different SinR activity states. Suppressor analyses revealed that ymdB mutants readily acquire mutations affecting SinR, thus restoring biofilm formation. These findings suggest that B. subtilis cells experience selective pressure to form the extracellular matrix that is characteristic of biofilms and that YmdB is required for the homeostasis of SinR and/or its antagonists.

scholarly journals Quorum Sensing Influences Burkholderia thailandensis Biofilm Development and Matrix Production

2016 ◽  
Vol 198 (19) ◽  
pp. 2643-2650 ◽  
Author(s):  
Boo Shan Tseng ◽  
Charlotte D. Majerczyk ◽  
Daniel Passos da Silva ◽  
Josephine R. Chandler ◽  
E. Peter Greenberg ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Biofilm Formation ◽  
Bacterial Species ◽  
Matrix Material ◽  
Close Relative ◽  
Wild Type ◽  
Flow Conditions ◽  
Burkholderia Thailandensis ◽  
Content Type ◽  
Biofilm Development

ABSTRACTMembers of the genusBurkholderiaare known to be adept at biofilm formation, which presumably assists in the survival of these organisms in the environment and the host. Biofilm formation has been linked to quorum sensing (QS) in several bacterial species. In this study, we characterizedBurkholderia thailandensisbiofilm development under flow conditions and sought to determine whether QS contributes to this process.B. thailandensisbiofilm formation exhibited an unusual pattern: the cells formed small aggregates and then proceeded to produce mature biofilms characterized by “dome” structures filled with biofilm matrix material. We showed that this process was dependent on QS.B. thailandensishas three acyl-homoserine lactone (AHL) QS systems (QS-1, QS-2, and QS-3). An AHL-negative strain produced biofilms consisting of cell aggregates but lacking the matrix-filled dome structures. This phenotype was rescued via exogenous addition of the three AHL signals. Of the threeB. thailandensisQS systems, we show that QS-1 is required for proper biofilm development, since abtaR1mutant, which is defective in QS-1 regulation, forms biofilms without these dome structures. Furthermore, our data show that the wild-type biofilm biomass, as well as the material inside the domes, stains with a fucose-binding lectin. ThebtaR1mutant biofilms, however, are negative for fucose staining. This suggests that the QS-1 system regulates the production of a fucose-containing exopolysaccharide in wild-type biofilms. Finally, we present data showing that QS ability during biofilm development produces a biofilm that is resistant to dispersion under stress conditions.IMPORTANCEThe saprophyteBurkholderia thailandensisis a close relative of the pathogenic bacteriumBurkholderia pseudomallei, the causative agent of melioidosis, which is contracted from its environmental reservoir. Since most bacteria in the environment reside in biofilms,B. thailandensisis an ideal model organism for investigating questions inBurkholderiaphysiology. In this study, we characterizedB. thailandensisbiofilm development and sought to determine if quorum sensing (QS) contributes to this process. Our work shows thatB. thailandensisproduces biofilms with unusual dome structures under flow conditions. Our findings suggest that these dome structures are filled with a QS-regulated, fucose-containing exopolysaccharide that may be involved in the resilience ofB. thailandensisbiofilms against changes in the nutritional environment.

scholarly journals AbaM Regulates Quorum Sensing, Biofilm Formation and Virulence in Acinetobacter baumannii

2021 ◽  
Author(s):  
Mario López-Martín ◽  
Jean-Frédéric Dubern ◽  
Morgan R. Alexander ◽  
Paul Williams
Keyword(s):  
Quorum Sensing ◽  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Plant Pathogens ◽  
Galleria Mellonella ◽  
Homoserine Lactone ◽  
Infection Model ◽  
Wild Type ◽  
Surface Motility ◽  
Biofilm Development

Acinetobacter baumannii possesses a single divergent luxR/luxI-type quorum sensing (QS) locus named abaR/abaI. This locus also contains a third gene located between abaR and abaI which we term abaM that codes for an uncharacterized member of the RsaM protein family known to regulate N-acylhomoserine lactone (AHL) dependent QS in other β- and γ-proteobacteria. Here we show that disruption of abaM via a T26 insertion in A. baumannii strain AB5075 resulted in increased production of N-(3-hydroxydodecanoyl)-L-homoserine lactone (OHC12) and enhanced surface motility and biofilm formation. In contrast to the wild type and abaI::T26 mutant, the virulence of the abaM::T26 mutant was completely attenuated in a Galleria mellonella infection model. Transcriptomic analysis of the abaM::T26 mutant revealed that AbaM differentially regulates at least 76 genes including the csu pilus operon and the acinetin 505 lipopeptide biosynthetic operon, that are involved in surface adherence, biofilm formation and virulence. A comparison of the wild type, abaM::T26 and abaI::T26 transcriptomes, indicates that AbaM regulates ∼21% of the QS regulon including the csu operon. Moreover, the QS genes (abaI/abaR) were among the most upregulated in the abaM::T26 mutant. A. baumannii lux-based abaM reporter gene fusions revealed that abaM expression is positively regulated by QS but negatively auto-regulated. Overall, the data presented in this work demonstrates that AbaM plays a central role in regulating A. baumannii QS, virulence, surface motility and biofilm formation. Importance Acinetobacter baumanni is a multi-antibiotic resistant pathogen of global healthcare importance. Understanding Acinetobacter virulence gene regulation could aid the development of novel anti-infective strategies. In A. baumannii, the abaR and abaI genes that code for the receptor and synthase components of an N-acylhomoserine (AHL) lactone-dependent quorum sensing system (QS) are separated by abaM. Here we show that although mutation of abaM increased AHL production, surface motility and biofilm development, it resulted in the attenuation of virulence. AbaM was found to control both QS-dependent and QS-independent genes. The significance of this work lies in the identification of AbaM, an RsaM ortholog known to control virulence in plant pathogens, as a modulator of virulence in a human pathogen.

scholarly journals AbaM Regulates Quorum Sensing, Biofilm Formation and Virulence in Acinetobacter baumannii

2020 ◽  
Author(s):  
Mario López-Martín ◽  
Jean-Frédéric Dubern ◽  
Morgan R. Alexander ◽  
Paul Williams
Keyword(s):  
Quorum Sensing ◽  
Acinetobacter Baumannii ◽  
Biofilm Formation ◽  
Plant Pathogens ◽  
Galleria Mellonella ◽  
Homoserine Lactone ◽  
Infection Model ◽  
Wild Type ◽  
Surface Motility ◽  
Biofilm Development

ABSTRACTAcinetobacter baumannii possesses a single divergent luxR/luxI-type quorum sensing (QS) locus named abaR/abaI. This locus also contains a third gene located between abaR and abaI which we term abaM that codes for an uncharacterized member of the RsaM protein family known to regulate N-acylhomoserine lactone (AHL) dependent QS in other β- and γ-proteobacteria. Here we show that disruption of abaM via a T26 insertion in A. baumannii strain AB5075 resulted in increased production of N-(3-hydroxydodecanoyl)-L-homoserine lactone (OHC12) and enhanced surface motility and biofilm formation. In contrast to the wild type and abaI::T26 mutant, the virulence of the abaM::T26 mutant was completely attenuated in a Galleria mellonella infection model. Transcriptomic analysis of the abaM::T26 mutant revealed that abaM differentially regulates at least 76 genes including the csu pilus operon and the acinetin 505 lipopeptide biosynthetic operon, that are involved in surface adherence, biofilm formation and virulence. A comparison of the wild type, abaM::T26 and abaI::T26 transcriptomes, indicates that abaM regulates ~21% of the QS regulon including the csu operon. Moreover, the QS genes (abaI/abaR) were among the most upregulated in the abaM::T26 mutant. A. baumannii lux-based abaM reporter gene fusions revealed that abaM expression is positively regulated by QS but negatively auto-regulated. Overall, the data presented in this work demonstrates that abaM plays a central role in regulating A. baumannii QS, virulence, surface motility and biofilm formation.IMPORTANCEAcinetobacter baumanni is a multi-antibiotic resistant pathogen of global healthcare importance. Understanding Acinetobacter virulence gene regulation could aid the development of novel anti-infective strategies. In A. baumannii, the abaR and abaI genes that code for the receptor and synthase components of an N-acylhomoserine (AHL) lactone-dependent quorum sensing system (QS) are separated by abaM. Here we show that although mutation of abaM increased AHL production, surface motility and biofilm development, it resulted in the attenuation of virulence. abaM was found to control both QS-dependent and QS-independent genes. The significance of this work lies in the identification of abaM, an RsaM ortholog known to control virulence in plant pathogens, as a modulator of virulence in a human pathogen.

Spatio-temporal formation of biofilms and extracellular matrix analysis in Azospirillum brasilense

2020 ◽  
Vol 367 (4) ◽  
Author(s):  
Víctor I Viruega-Góngora ◽  
Iris S Acatitla-Jácome ◽  
Sandra R Reyes-Carmona ◽  
Beatriz E Baca ◽  
Alberto Ramírez-Mata
Keyword(s):  
Extracellular Matrix ◽  
Biofilm Formation ◽  
Azospirillum Brasilense ◽  
Laser Scanning Microscopy ◽  
Extracellular Dna ◽  
Polar Flagellum ◽  
Matrix Components ◽  
Spatio Temporal ◽  
Biofilm Development ◽  
Biofilm Matrix

ABSTRACT Elucidation of biofilm structure formation in the plant growth-promoting rhizobacterium Azospirillum brasilense is necessary to gain a better understanding of the growth of cells within the extracellular matrix and its role in the colonization of plants of agronomic importance. We used immunofluorescence microscopy and confocal laser scanning microscopy to study spatio-temporal biofilm formation on an abiotic surface. Observations facilitated by fluorescence microscopy revealed the presence of polar flagellin, exopolysaccharides, outer major membrane protein (OmaA) and extracellular DNA in the Azospirillum biofilm matrix. In static culture conditions, the polar flagellum disaggregated after 3 days of biofilm growth, but exopolysaccharides were increasing. These findings suggest that the first step in biofilm formation may be attachment, in which the bacterium first makes contact with a surface through its polar flagellum. After attaching to the surface, the long flagella and OmaA intertwine the cells to form a network. These bacterial aggregates initiate biofilm development. The underlying mechanisms dictating how the biofilm matrix components of A. brasilense direct the overall morphology of the biofilm are not well known. The methods developed here might be useful in further studies that analyze the differential spatial regulation of genes encoding matrix components that drive biofilm construction.

scholarly journals Altering the Ratio of Phenazines in Pseudomonas chlororaphis (aureofaciens) Strain 30-84: Effects on Biofilm Formation and Pathogen Inhibition

2008 ◽  
Vol 190 (8) ◽  
pp. 2759-2766 ◽  
Author(s):  
V. S. R. K. Maddula ◽  
E. A. Pierson ◽  
L. S. Pierson
Keyword(s):  
Fungal Pathogen ◽  
Biofilm Formation ◽  
Flow Cell ◽  
Gaeumannomyces Graminis ◽  
Pseudomonas Chlororaphis ◽  
Wild Type ◽  
Initial Attachment ◽  
Pathogen Inhibition ◽  
Biofilm Development ◽  
Derivatives Of

ABSTRACT Pseudomonas chlororaphis strain 30-84 is a plant-beneficial bacterium that is able to control take-all disease of wheat caused by the fungal pathogen Gaeumannomyces graminis var. tritici. The production of phenazines (PZs) by strain 30-84 is the primary mechanism of pathogen inhibition and contributes to the persistence of strain 30-84 in the rhizosphere. PZ production is regulated in part by the PhzR/PhzI quorum-sensing (QS) system. Previous flow cell analyses demonstrated that QS and PZs are involved in biofilm formation in P. chlororaphis (V. S. R. K. Maddula, Z. Zhang, E. A. Pierson, and L. S. Pierson III, Microb. Ecol. 52:289-301, 2006). P. chlororaphis produces mainly two PZs, phenazine-1-carboxylic acid (PCA) and 2-hydroxy-PCA (2-OH-PCA). In the present study, we examined the effect of altering the ratio of PZs produced by P. chlororaphis on biofilm formation and pathogen inhibition. As part of this study, we generated derivatives of strain 30-84 that produced only PCA or overproduced 2-OH-PCA. Using flow cell assays, we found that these PZ-altered derivatives of strain 30-84 differed from the wild type in initial attachment, mature biofilm architecture, and dispersal from biofilms. For example, increased 2-OH-PCA production promoted initial attachment and altered the three-dimensional structure of the mature biofilm relative to the wild type. Additionally, both alterations promoted thicker biofilm development and lowered dispersal rates compared to the wild type. The PZ-altered derivatives of strain 30-84 also differed in their ability to inhibit the fungal pathogen G. graminis var. tritici. Loss of 2-OH-PCA resulted in a significant reduction in the inhibition of G. graminis var. tritici. Our findings suggest that alterations in the ratios of antibiotic secondary metabolites synthesized by an organism may have complex and wide-ranging effects on its biology.

scholarly journals Role of Flagellin-Homologous Proteins in Biofilm Formation by Pathogenic Vibrio Species

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
You-Chul Jung ◽  
Mi-Ae Lee ◽  
Kyu-Ho Lee
Keyword(s):  
Biofilm Formation ◽  
Specific Binding ◽  
Biological Significance ◽  
Wild Type ◽  
Forming Process ◽  
Homologous Proteins ◽  
Content Type ◽  
Pathogenic Vibrio ◽  
Biofilm Matrix

ABSTRACT The pathogenic bacterium Vibrio vulnificus exhibits the ability to form biofilm, for which initiation is dependent upon swimming motility by virtue of a polar flagellum. The filament of its flagellum is composed of multiple flagellin subunits, FlaA, -B, -C, and -D. In V. vulnificus genomes, however, open reading frames (ORFs) annotated by FlaE and -F are also present. Although neither FlaE nor FlaF is involved in filament formation and cellular motility, they are well expressed and secreted to the extracellular milieu through the secretion apparatus for flagellar assembly. In the extrapolymeric matrix of V. vulnificus biofilm, significant levels of FlaEF were detected. Mutants defective in both flaE and flaF formed significantly decreased biofilms compared to the wild-type biofilm. Thus, the potential role of FlaEF during the biofilm-forming process was investigated by exogenous addition of recombinant FlaEF (rFlaEF) to the biofilm assays. The added rFlaE and rFlaF were predominantly incorporated into the biofilm matrix formed by the wild type. However, biofilms formed by a mutant defective in exopolysaccharide (EPS) biosynthesis were not affected by added FlaEF. These results raised a possibility that FlaEF specifically interact with EPS within the biofilm matrix. In vitro pulldown assays using His-tagged rFlaEF or rFlaC revealed the specific binding of EPS to rFlaEF but not to rFlaC. Taken together, our results demonstrate that V. vulnificus FlaEF, flagellin-homologous proteins (FHPs), are crucial for biofilm formation by directly interacting with the essential determinant for biofilm maturation, EPS. Further analyses performed with other pathogenic Vibrio species demonstrated both the presence of FHPs and their important role in biofilm formation. IMPORTANCE Flagellar filaments of the pathogenic Vibrio species, including V. vulnificus, V. parahaemolyticus, and V. cholerae, are composed of multiple flagellin subunits. In their genomes, however, there are higher numbers of the ORFs encoding flagellin-like proteins than the numbers of flagellin subunits required for filament assembly. Since these flagellin-homologous proteins (FHPs) are well expressed and excreted to environments via a flagellin transport channel, their extracellular role in the pathogenic Vibrio has been enigmatic. Their biological significance, which is not related with flagellar functions, has been revealed to be in maturation of biofilm structures. Among various components of the extracellular polymeric matrix produced in the V. vulnificus biofilms, the exopolysaccharides (EPS) are dominant constituents and crucial in maturation of biofilms. The enhancing role of the V. vulnificus FHPs in biofilm formation requires the presence of EPS, as indicated by highly specific interactions among two FHPs and three EPS.

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.

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