Influence of Quorum Sensing and Iron on Twitching Motility and Biofilm Formation in Pseudomonas aeruginosa

ABSTRACT Reducing iron (Fe) levels in a defined minimal medium reduced the growth yields of planktonic and biofilm Pseudomonas aeruginosa, though biofilm biomass was affected to the greatest extent and at FeCl3 concentrations where planktonic cell growth was not compromised. Highlighting this apparently greater need for Fe, biofilm growth yields were markedly reduced in a mutant unable to produce pyoverdine (and, so, deficient in pyoverdine-mediated Fe acquisition) at concentrations of FeCl3 that did not adversely affect biofilm yields of a pyoverdine-producing wild-type strain. Concomitant with the reduced biofilm yields at low Fe concentrations, P. aeruginosa showed enhanced twitching motility in Fe-deficient versus Fe-replete minimal media. A mutant deficient in low-Fe-stimulated twitching motility but normal as regards twitching motility on Fe-rich medium was isolated and shown to be disrupted in rhlI, whose product is responsible for synthesis of the N-butanoyl homoserine lactone (C4-HSL) quorum-sensing signal. In contrast to wild-type cells, which formed thin, flat, undeveloped biofilms in Fe-limited medium, the rhlI mutant formed substantially developed though not fully mature biofilms under Fe limitation. C4-HSL production increased markedly in Fe-limited versus Fe-rich P. aeruginosa cultures, and cell-free low-Fe culture supernatants restored the twitching motility of the rhlI mutant on Fe-limited minimal medium and stimulated the twitching motility of rhlI and wild-type P. aeruginosa on Fe-rich minimal medium. Still, addition of exogenous C4-HSL did not stimulate the twitching motility of either strain on Fe-replete medium, indicating that some Fe-regulated and RhlI/C4-HSL-dependent extracellular product(s) was responsible for the enhanced twitching motility (and reduced biofilm formation) seen in response to Fe limitation.

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

Journal of Bacteriology ◽

10.1128/jb.00137-07 ◽

2007 ◽

Vol 189 (14) ◽

pp. 5383-5386 ◽

Cited By ~ 187

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.67.4.1865-1873.2001 ◽

2001 ◽

Vol 67 (4) ◽

pp. 1865-1873 ◽

Cited By ~ 341

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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A role for rhamnolipid in biofilm dispersion

Biofilms ◽

10.1017/s147905050400119x ◽

2004 ◽

Vol 1 (2) ◽

pp. 91-99 ◽

Cited By ~ 48

Author(s):

S. R. Schooling ◽

U. K. Charaf ◽

D. G. Allison ◽

P. Gilbert

Keyword(s):

Surface Tension ◽

Quorum Sensing ◽

Biofilm Formation ◽

Culture Medium ◽

High Cell Density ◽

Culture Media ◽

Homoserine Lactone ◽

Wild Type ◽

In The Wild ◽

Biofilm Dispersion

Biofilms are often considered as localized zones of high cell density. Quorum sensing provides a means for control of population processes and has been implicated in the regulation of biofilm activities. We present a role for quorum sensing in programmed detachment and dispersal processes. Biofilms of Pseudomonas aeruginosa PAO1 and its isogenic homoserine lactone (HSL) mutant P. aeruginosa PAO-JP2 were grown in batch culture on glass substrata; differences were found in the rate and extent of formation of biofilm. Climax communities were observed for PAO1 at 24 h. These were later accompanied by foaming, a drop in the surface tension of culture media and dispersal of the biofilm, after which no subsequent biofilm accretion occurred. PAO-JP2 cultures reformed biofilm post-detachment and did not foam. Prevention of biofilm reformation in the wild type was related to some component excreted into the culture medium. Rhamnolipid, a biosurfactant regulated by quorum sensing, was detected in PAO1 cultures. When rhamnolipid was added to freshly inoculated substrata, biofilm formation was inhibited. At 20 h, PAO1 biofilms were transferred to medium with added rhamnolipid: biofilm was relatively unaffected. Biofilm events were also studied in medium supplemented with N-butyryl-L-homoserine lactone, which is involved in the regulation of rhamnolipid synthesis. Both strains exhibited similar trends of rapid biofilm formation and dramatic changes in the rate and extent of biofilm accretion. In both cases, there was premature foaming, lowered surface tension and elevated rhamnolipid levels. A role for HSLs in maintenance of biofilm and events leading to dispersion of cells is proposed. This role would encompass dispersion but not necessarily detachment of cells from biofilm and supports a new function for rhamnolipid in pathogenesis, whereby rhamnolipid would promote the dissemination of cells from a nidus of infection.

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Differential Regulation of Twitching Motility and Elastase Production by Vfr in Pseudomonas aeruginosa

Journal of Bacteriology ◽

10.1128/jb.184.13.3605-3613.2002 ◽

2002 ◽

Vol 184 (13) ◽

pp. 3605-3613 ◽

Cited By ~ 127

Author(s):

Scott A. Beatson ◽

Cynthia B. Whitchurch ◽

Jennifer L. Sargent ◽

Roger C. Levesque ◽

John S. Mattick

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Binding Pocket ◽

Receptor Protein ◽

Twitching Motility ◽

Wild Type ◽

Type Iv ◽

Allelic Deletion ◽

Pyocyanin Production ◽

Null Mutants

ABSTRACT Vfr, a homolog of Escherichia coli cyclic AMP (cAMP) receptor protein, has been shown to regulate quorum sensing, exotoxin A production, and regA transcription in Pseudomonas aeruginosa. We identified a twitching motility-defective mutant that carries a transposon insertion in vfr and confirmed that vfr is required for twitching motility by construction of an independent allelic deletion-replacement mutant of vfr that exhibited the same phenotype, as well as by the restoration of normal twitching motility by complementation of these mutants with wild-type vfr. Vfr-null mutants exhibited severely reduced twitching motility with barely detectable levels of type IV pili, as well as loss of elastase production and altered pyocyanin production. We also identified reduced-twitching variants of quorum-sensing mutants (PAK lasI::Tc) with a spontaneous deletion in vfr (S. A. Beatson, C. B. Whitchurch, A. B. T. Semmler, and J. S. Mattick, J. Bacteriol., 184:3598-3604, 2002), the net result of which was the loss of five residues (EQERS) from the putative cAMP-binding pocket of Vfr. This allele (VfrΔEQERS) was capable of restoring elastase and pyocyanin production to wild-type levels in vfr-null mutants but not their defects in twitching motility. Furthermore, structural analysis of Vfr and VfrΔEQERS in relation to E. coli CRP suggests that Vfr is capable of binding both cAMP and cyclic GMP whereas VfrΔEQERS is only capable of responding to cAMP. We suggest that Vfr controls twitching motility and quorum sensing via independent pathways in response to these different signals, bound by the same cyclic nucleotide monophosphate-binding pocket.

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Quorum-Sensing Systems in the Plant Growth-Promoting Bacterium Paraburkholderia phytofirmans PsJN Exhibit Cross-Regulation and Are Involved in Biofilm Formation

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-17-0008-r ◽

2017 ◽

Vol 30 (7) ◽

pp. 557-565 ◽

Cited By ~ 16

Author(s):

Ana Zúñiga ◽

Raúl A. Donoso ◽

Daniela Ruiz ◽

Gonzalo A. Ruz ◽

Bernardo González

Keyword(s):

Quorum Sensing ◽

Biofilm Formation ◽

Type Strain ◽

Wild Type Strain ◽

Homoserine Lactone ◽

Wild Type ◽

Transcript Levels ◽

Sensing Systems ◽

Plant Growth Promoting ◽

Growth Promoting

Quorum-sensing systems play important roles in host colonization and host establishment of Burkholderiales species. Beneficial Paraburkholderia species share a conserved quorum-sensing (QS) system, designated BraI/R, that controls different phenotypes. In this context, the plant growth-promoting bacterium Paraburkholderia phytofirmans PsJN possesses two different homoserine lactone QS systems BpI.1/R.1 and BpI.2/R.2 (BraI/R-like QS system). The BpI.1/R.1 QS system was previously reported to be important to colonize and produce beneficial effects in Arabidopsis thaliana plants. Here, we analyzed the temporal variations of the QS gene transcript levels in the wild-type strain colonizing plant roots. The gene expression patterns showed relevant differences in both QS systems compared with the wild-type strain in the unplanted control treatment. The gene expression data were used to reconstruct a regulatory network model of QS systems in P. phytofirmans PsJN, using a Boolean network model. Also, we examined the phenotypic traits and transcript levels of genes involved in QS systems, using P. phytofirmans mutants in homoserine lactone synthases genes. We observed that the BpI.1/R.1 QS system regulates biofilm formation production in strain PsJN and this phenotype was associated with the lower expression of a specific extracytoplasmic function sigma factor ecf26.1 gene (implicated in biofilm formation) in the bpI.1 mutant strain.

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Mutation of luxS Affects Biofilm Formation in Streptococcus mutans

Infection and Immunity ◽

10.1128/iai.71.4.1972-1979.2003 ◽

2003 ◽

Vol 71 (4) ◽

pp. 1972-1979 ◽

Cited By ~ 185

Author(s):

Justin Merritt ◽

Fengxia Qi ◽

Steven D. Goodman ◽

Maxwell H. Anderson ◽

Wenyuan Shi

Keyword(s):

Quorum Sensing ◽

Streptococcus Mutans ◽

Biofilm Formation ◽

Microscopic Analysis ◽

Homoserine Lactone ◽

Good Evidence ◽

Genome Project ◽

Bacterial Biofilm ◽

Wild Type

ABSTRACT Quorum sensing is a bacterial mechanism for regulating gene expression in response to changes in population density. Many bacteria are capable of acyl-homoserine lactone-based or peptide-based intraspecies quorum sensing and luxS-dependent interspecies quorum sensing. While there is good evidence about the involvement of intraspecies quorum sensing in bacterial biofilm, little is known about the role of luxS in biofilm formation. In this study, we report for the first time that luxS-dependent quorum sensing is involved in biofilm formation of Streptococcus mutans. S. mutans is a major cariogenic bacterium in the multispecies bacterial biofilm commonly known as dental plaque. An ortholog of luxS for S. mutans was identified using the data available in the S. mutans genome project (http://www.genome.ou.edu/smutans.html ). Using an assay developed for the detection of the LuxS-associated quorum sensing signal autoinducer 2 (AI-2), it was demonstrated that this ortholog was able to complement the luxS negative phenotype of Escherichia coli DH5α. It was also shown that AI-2 is indeed produced by S. mutans. AI-2 production is maximal during mid- to late-log growth in batch culture. Mutant strains devoid of the luxS gene were constructed and found to be defective in producing the AI-2 signal. There are also marked phenotypic differences between the wild type and the luxS mutants. Microscopic analysis of in vitro-grown biofilm structure revealed that the luxS mutant biofilms adopted a much more granular appearance, rather than the relatively smooth, confluent layer normally seen in the wild type. These results suggest that LuxS-dependent signal may play an important role in biofilm formation of S. mutans.

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Role of Quorum Sensing and Antimicrobial Component Production by Serratia plymuthica in Formation of Biofilms, Including Mixed Biofilms with Escherichia coli

Applied and Environmental Microbiology ◽

10.1128/aem.01708-06 ◽

2006 ◽

Vol 72 (11) ◽

pp. 7294-7300 ◽

Cited By ~ 40

Author(s):

Pieter Moons ◽

Rob Van Houdt ◽

Abram Aertsen ◽

Kristof Vanoirbeek ◽

Yves Engelborghs ◽

...

Keyword(s):

Escherichia Coli ◽

Quorum Sensing ◽

Biofilm Formation ◽

Homoserine Lactone ◽

Broth Culture ◽

Confocal Laser ◽

Wild Type ◽

Serratia Plymuthica ◽

E Coli

ABSTRACT We have previously characterized the N-acyl-l-homoserine lactone-based quorum-sensing system of the biofilm isolate Serratia plymuthica RVH1. Here we investigated the role of quorum sensing and of quorum-sensing-dependent production of an antimicrobial compound (AC) on biofilm formation by RVH1 and on the cocultivation of RVH1 and Escherichia coli in planktonic cultures or in biofilms. Biofilm formation of S. plymuthica was not affected by the knockout of splI or splR, the S. plymuthica homologs of the luxI or luxR quorum-sensing gene, respectively, or by the knockout of AC production. E. coli grew well in mixed broth culture with RVH1 until the latter reached 8.5 to 9.5 log CFU/ml, after which the E. coli colony counts steeply declined. In comparison, only a very small decline occurred in cocultures with the S. plymuthica AC-deficient and splI mutants. Complementation with exogenous N-hexanoyl-l-homoserine lactone rescued the wild-type phenotype of the splI mutant. The splR knockout mutant also induced a steep decline of E. coli, consistent with its proposed function as a repressor of quorum-sensing-regulated genes. The numbers of E. coli in 3-day-old mixed biofilms followed a similar pattern, being higher with S. plymuthica deficient in SplI or AC production than with wild-type S. plymuthica, the splR mutant, or the splI mutant in the presence of N-hexanoyl-l-homoserine lactone. Confocal laser scanning microscopic analysis of mixed biofilms established with strains producing different fluorescent proteins showed that E. coli microcolonies were less developed in the presence of RVH1 than in the presence of the AC-deficient mutant.

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Inhibition of Quorum Sensing in Pseudomonas aeruginosa by N-Acyl Cyclopentylamides

Applied and Environmental Microbiology ◽

10.1128/aem.02233-06 ◽

2007 ◽

Vol 73 (10) ◽

pp. 3183-3188 ◽

Cited By ~ 114

Author(s):

Takenori Ishida ◽

Tsukasa Ikeda ◽

Noboru Takiguchi ◽

Akio Kuroda ◽

Hisao Ohtake ◽

...

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Virulence Factors ◽

Biofilm Formation ◽

Homoserine Lactone ◽

Ring Structure ◽

Response Regulators ◽

Maximal Inhibition ◽

Sensing Systems ◽

Reporter Assays

ABSTRACT N-Octanoyl cyclopentylamide (C8-CPA) was found to moderately inhibit quorum sensing in Pseudomonas aeruginosa PAO1. To obtain more powerful inhibitors, a series of structural analogs of C8-CPA were synthesized and examined for their ability to inhibit quorum sensing in P. aeruginosa PAO1. The lasB-lacZ and rhlA-lacZ reporter assays revealed that the chain length and the ring structure were critical for C8-CPA analogs to inhibit quorum sensing. N-Decanoyl cyclopentylamide (C10-CPA) was found to be the strongest inhibitor, and its concentrations required for half-maximal inhibition for lasB-lacZ and rhlA-lacZ expression were 80 and 90 μM, respectively. C10-CPA also inhibited production of virulence factors, including elastase, pyocyanin, and rhamnolipid, and biofilm formation without affecting growth of P. aeruginosa PAO1. C10-CPA inhibited induction of both lasI-lacZ by N-(3-oxododecanoyl)-l-homoserine lactone (PAI1) and rhlA-lacZ by N-butanoyl-l-homoserine lactone (PAI2) in the lasI rhlI mutant of P. aeruginosa PAO1, indicating that C10-CPA interferes with the las and rhl quorum-sensing systems via inhibiting interaction between their response regulators (LasR and RhlR) and autoinducers.

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

Journal of Bacteriology ◽

10.1128/jb.00635-20 ◽

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.

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Positive Autoregulation of an Acyl-Homoserine Lactone Quorum-Sensing Circuit Synchronizes the Population Response

mBio ◽

10.1128/mbio.01079-17 ◽

2017 ◽

Vol 8 (4) ◽

Cited By ~ 10

Author(s):

Rebecca L. Scholz ◽

E. Peter Greenberg

Keyword(s):

Pseudomonas Aeruginosa ◽

Quorum Sensing ◽

Biological Significance ◽

Population Level ◽

Homoserine Lactone ◽

Acyl Homoserine Lactone ◽

Wild Type ◽

Positive Signal ◽

Signal Production ◽

Engineered Strain

ABSTRACTMany proteobacteria utilize acyl-homoserine lactone quorum-sensing signals. At low population densities, cells produce a basal level of signal, and when sufficient signal has accumulated in the surrounding environment, it binds to its receptor, and quorum-sensing-dependent genes can be activated. A common characteristic of acyl-homoserine lactone quorum sensing is that signal production is positively autoregulated. We have examined the role of positive signal autoregulation inPseudomonas aeruginosa. We compared population responses and individual cell responses in populations of wild-typeP. aeruginosato responses in a strain with the signal synthase gene controlled by an arabinose-inducible promoter so that signal was produced at a constant rate per cell regardless of cell population density. At a population level, responses of the wild type and the engineered strain were indistinguishable, but the responses of individual cells in a population of the wild type showed greater synchrony than the responses of the engineered strain. Although sufficient signal is required to activate expression of quorum-sensing-regulated genes, it is not sufficient for activation of certain genes, the late genes, and their expression is delayed until other conditions are met. We found that late gene responses were reduced in the engineered strain. We conclude that positive signal autoregulation is not a required element in acyl-homoserine lactone quorum sensing, but it functions to enhance synchrony of the responses of individuals in a population. Synchrony might be advantageous in some situations, whereas a less coordinated quorum-sensing response might allow bet hedging and be advantageous in other situations.IMPORTANCEThere are many quorum-sensing systems that involve a transcriptional activator, which responds to an acyl-homoserine lactone signal. In all of the examples studied, the gene coding for signal production is positively autoregulated by the signal, and it has even been described as essential for a quorum-sensing response. We have used the opportunistic pathogenPseudomonas aeruginosaas a model to show that positive autoregulation is not required for a robust quorum-sensing response. We also show that positive autoregulation of signal production enhances the synchrony of the response. This information enhances our general understanding of the biological significance of how acyl-homoserine lactone quorum-sensing circuits are arranged.

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