Identification of Genes Regulated by the cepIR Quorum-Sensing System in Burkholderia cenocepacia by High-Throughput Screening of a Random Promoter Library

ABSTRACT The Burkholderia cenocepacia cepIR quorum-sensing system regulates expression of extracellular proteases, chitinase, and genes involved in ornibactin biosynthesis, biofilm formation, and motility. In a genome-wide screen we identified cepIR-regulated genes by screening a random promoter library of B. cenocepacia K56-2 constructed in a luminescence reporter detection plasmid for differential expression in response to N-octanoyl-l-homoserine lactone (OHL). Eighty-nine clones were identified; in 58 of these clones expression was positively regulated by cepIR, and in 31 expression was negatively regulated by cepIR. The expression profiles of the 89 promoter clones were compared in the cepI mutant K56-dI2 in medium supplemented with 30 pM OHL and K56-2 to confirm that the presence of OHL restored expression to wild-type levels. To validate the promoter library observations and to determine the effect of a cepR mutation on expression of selected genes, the mRNA levels of nine genes whose promoters were predicted to be regulated by cepR were quantitated by quantitative reverse transcription-PCR in the wild type and cepI and cepR mutants. The expression levels of all nine genes were similar in the cepI and cepR mutants and consistent with the promoter-lux reporter activity. The expression of four selected cepIR-regulated gene promoters was examined in a cciIR mutant, and two of these promoters were also regulated by cciIR. This study extends our understanding of genes whose expression is influenced by cepIR and indicates the global regulatory effect of the cepIR system in B. cenocepacia.

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Expression of a luxS Gene Is Not Required for Borrelia burgdorferi Infection of Mice via Needle Inoculation

Infection and Immunity ◽

10.1128/iai.71.5.2892-2896.2003 ◽

2003 ◽

Vol 71 (5) ◽

pp. 2892-2896 ◽

Cited By ~ 44

Author(s):

Anette Hübner ◽

Andrew T. Revel ◽

Dena M. Nolen ◽

Kayla E. Hagman ◽

Michael V. Norgard

Keyword(s):

Quorum Sensing ◽

Borrelia Burgdorferi ◽

Mammalian Host ◽

Wild Type ◽

Sensing System ◽

Autoinducer 2 ◽

Sensing Systems ◽

Quorum Sensing System ◽

Culture Supernatants

ABSTRACT The luxS gene product is an integral component of LuxS/autoinducer-2 (AI-2) quorum-sensing systems in bacteria. A putative luxS gene was expressed at comparable levels by Borrelia burgdorferi strain 297 cultivated either in vitro or in dialysis membrane chambers implanted in rat peritoneal cavities. Although the borrelial luxS gene functionally complemented a LuxS deficiency in Escherichia coli DH5α, AI-2-like activity could not be detected within B. burgdorferi culture supernatants or concentrated cell lysates. Finally, a luxS-deficient mutant of B. burgdorferi was infectious at wild-type levels when it was intradermally needle inoculated into mice, indicating that expression of luxS probably is not required for infectivity but, at the very least, is not essential for mammalian host adaptation. Our findings also challenge the notion that a LuxS/AI-2 quorum-sensing system is operative in B. burgdorferi.

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Inactivation of traP Has No Effect on the Agr Quorum-Sensing System or Virulence of Staphylococcus aureus

Infection and Immunity ◽

10.1128/iai.00491-07 ◽

2007 ◽

Vol 75 (9) ◽

pp. 4519-4527 ◽

Cited By ~ 28

Author(s):

Lindsey N. Shaw ◽

Ing-Marie Jonsson ◽

Vineet K. Singh ◽

Andrej Tarkowski ◽

George C. Stewart

Keyword(s):

Staphylococcus Aureus ◽

Quorum Sensing ◽

Type Strain ◽

Wild Type Strain ◽

Surface Proteins ◽

Virulence Determinants ◽

Wild Type ◽

Expression Levels ◽

Sensing System ◽

Quorum Sensing System

ABSTRACT The success of Staphylococcus aureus as a pathogen can largely be attributed to the plethora of genetic regulators encoded within its genome that temporally regulate its arsenal of virulence determinants throughout its virulence lifestyle. Arguably the most important of these is the two-component, quorum-sensing system agr. Over the last decade, the controversial presence of a second quorum-sensing system (the TRAP system) has been proposed, and it has been mooted to function as the master regulator of virulence in S. aureus by modulating agr. Mutants defective in TRAP are reported to be devoid of agr expression, lacking in hemolytic activity, essentially deficient in the secretion of virulence determinants, and avirulent in infection models. A number of research groups have questioned the validity of the TRAP findings in recent years; however, a thorough and independent analysis of its role in S. aureus physiology and pathogenesis has not been forthcoming. Therefore, we have undertaken such an analysis of the TRAP locus of S. aureus. We found that a traP mutant was equally hemolytic as the wild-type strain. Furthermore, transcriptional profiling found no alterations in the traP mutant in expression levels of agr or in expression levels of multiple agr-regulated genes (hla, sspA, and spa). Analysis of secreted and surface proteins of the traP mutant revealed no deviation in comparison to the parent. Finally, analysis conducted using a murine model of S. aureus septic arthritis revealed that, in contrast to an agr mutant, the traP mutant was just as virulent as the wild-type strain.

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N-Octanoylhomoserine lactone signalling mediated by the BpsI–BpsR quorum sensing system plays a major role in biofilm formation of Burkholderia pseudomallei

Microbiology ◽

10.1099/mic.0.046540-0 ◽

2011 ◽

Vol 157 (4) ◽

pp. 1176-1186 ◽

Cited By ~ 37

Author(s):

Akshamal Mihiranga Gamage ◽

Guanghou Shui ◽

Markus R. Wenk ◽

Kim Lee Chua

Keyword(s):

Quorum Sensing ◽

Biofilm Formation ◽

Burkholderia Pseudomallei ◽

Homoserine Lactone ◽

Tandem Ms ◽

Wild Type ◽

Acylhomoserine Lactone ◽

Sensing System ◽

Sensing Systems ◽

Quorum Sensing System

The genome of Burkholderia pseudomallei encodes three acylhomoserine lactone (AHL) quorum sensing systems, each comprising an AHL synthase and a signal receptor/regulator. The BpsI–BpsR system produces N-octanoylhomoserine lactone (C8HL) and is positively auto-regulated by its AHL product. The products of the remaining two systems have not been identified. In this study, tandem MS was used to identify and quantify the AHL species produced by three clinical B. pseudomallei isolates – KHW, K96243 and H11 – three isogenic KHW mutants that each contain a null mutation in an AHL synthase gene, and recombinant Escherichia coli heterologously expressing each of the three B. pseudomallei AHL synthase genes. BpsI synthesized predominantly C8HL, which accounted for more than 95 % of the extracellular AHLs produced in stationary-phase KHW cultures. The major products of BpsI2 and BpsI3 were N-(3-hydroxy-octanoyl)homoserine lactone (OHC8HL) and N-(3-hydroxy-decanoyl)homoserine lactone, respectively, and their corresponding transcriptional regulators, BpsR2 and BpsR3, were capable of driving reporter gene expression in the presence of these cognate lactones. Formation of biofilm by B. pseudomallei KHW was severely impaired in mutants lacking either BpsI or BpsR but could be restored to near wild-type levels by exogenous C8HL. BpsI2 was not required, and BpsI3 was partially required for biofilm formation. Unlike the bpsI mutant, biofilm formation in the bpsI3 mutant could not be restored to wild-type levels in the presence of OHC8HL, the product of BpsI3. C8HL and OHC8HL had opposite effects on biofilm formation; exogenous C8HL enhanced biofilm formation in both the bpsI3 mutant and wild-type KHW while exogenous OHC8HL suppressed the formation of biofilm in the same strains. We propose that exogenous OHC8HL antagonizes biofilm formation in B. pseudomallei, possibly by competing with endogenous C8HL for binding to BpsR.

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The CepIR quorum-sensing system contributes to the virulence of Burkholderia cenocepacia respiratory infections

Microbiology ◽

10.1099/mic.0.26540-0 ◽

2003 ◽

Vol 149 (12) ◽

pp. 3649-3658 ◽

Cited By ~ 88

Author(s):

P. A. Sokol ◽

U. Sajjan ◽

M. B. Visser ◽

S. Gingues ◽

J. Forstner ◽

...

Keyword(s):

Quorum Sensing ◽

Parent Strain ◽

Respiratory Infections ◽

Virulent Strain ◽

Interleukin 1 ◽

Homoserine Lactone ◽

Burkholderia Cenocepacia ◽

Sensing System ◽

Quorum Sensing System ◽

The Difference

The cepIR genes encode an N-acyl homoserine lactone (AHL)-dependent quorum-sensing system consisting of an AHL synthase that directs the synthesis of N-octanoyl-l-homoserine lactone (ohl) and n-hexanoyl-l-homoserine lactone and a transcriptional regulator. The virulence of cepIR mutants was examined in two animal models. Rats were infected with agar beads containing Burkholderia cenocepacia K56-2, K56-I2 (cepI : : Tpr) or K56-R2 (cepR : : Tn5-OT182). At 10 days post-infection, the extent of lung histopathological changes was significantly lower in lungs infected with K56-I2 or K56-R2 compared to the parent strain. Intranasal infections were performed in Cftr (−/−) mice and their wild-type siblings. K56-2 was more virulent in both groups of mice. K56-I2 was the least virulent strain and was not invasive in the Cftr (−/−) mice. OHL was readily detected in lung homogenates from Cftr (−/−) mice infected with K56-2 but was only detected at levels slightly above background in a few mice infected with K56-I2. Lung homogenates from mice infected with K56-2 had significantly higher levels of the inflammatory mediators murine macrophage inflammatory protein-2, KC/N51, interleukin-1β and interleukin-6 than those from K56-I2-infected animals. These studies indicate that a functional CepIR quorum-sensing system contributes to the severity of B. cenocepacia infections. A zinc metalloprotease gene (zmpA) was shown to be regulated by CepR and may be one of the factors that accounts for the difference in virulence between the cepI mutant and the parent strain.

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The Enterococcus faecalis fsrB Gene, a Key Component of the fsr Quorum-Sensing System, Is Associated with Virulence in the Rabbit Endophthalmitis Model

Infection and Immunity ◽

10.1128/iai.70.8.4678-4681.2002 ◽

2002 ◽

Vol 70 (8) ◽

pp. 4678-4681 ◽

Cited By ~ 59

Author(s):

Eleftherios Mylonakis ◽

Michael Engelbert ◽

Xiang Qin ◽

Costi D. Sifri ◽

Barbara E. Murray ◽

...

Keyword(s):

Quorum Sensing ◽

Enterococcus Faecalis ◽

Deletion Mutant ◽

Wild Type ◽

Sensing System ◽

Quorum Sensing System

ABSTRACT We used a rabbit endophthalmitis model to explore the role of fsrB, a gene required for the function of the fsr quorum-sensing system of Enterococcus faecalis, in pathogenicity. A nonpolar deletion mutant of fsrB had significantly reduced virulence compared to wild type. Complementation of mutation restored virulence. These data corroborate the role of fsrB in E. faecalis pathogenesis and suggest that the rabbit endophthalmitis model can be used to study the in vivo role of quorum sensing.

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A Nonsense Mutation in agrA Accounts for the Defect in agr Expression and the Avirulence of Staphylococcus aureus 8325-4 traP::kan

Infection and Immunity ◽

10.1128/iai.00679-07 ◽

2007 ◽

Vol 75 (9) ◽

pp. 4534-4540 ◽

Cited By ~ 47

Author(s):

Rajan P. Adhikari ◽

Staffan Arvidson ◽

Richard P. Novick

Keyword(s):

Staphylococcus Aureus ◽

Quorum Sensing ◽

Mutant Strain ◽

Nonsense Mutation ◽

Stop Codon ◽

Response Regulator ◽

Wild Type ◽

Sensing System ◽

Quorum Sensing System ◽

Type Sequence

ABSTRACT TraP is a triply phosphorylated staphylococcal protein that has been hypothesized to be the mediator of a second Staphylococcus aureus quorum-sensing system, “SQS1,” that controls expression of the agr system and therefore is essential for the organism's virulence. This hypothesis was based on the loss of agr expression and virulence by a traP mutant of strain 8325-4 and was supported by full complementation of both phenotypic defects by the cloned traP gene in strain NB8 (Y. Gov, I. Borovok, M. Korem, V. K. Singh, R. K. Jayaswal, B. J. Wilkinson, S. M. Rich, and N. Balaban, J. Biol. Chem. 279:14665-14672, 2004), in which the wild-type traP gene was expressed in trans in the 8325-4 traP mutant. We initiated a study of the mechanism by which TraP activates agr and found that the traP mutant strain used for this and other recently published studies has a second mutation, an adventitious stop codon in the middle of agrA, the agr response regulator. The traP mutation, once separated from the agrA defect by outcrossing, had no effect on agr expression or virulence, indicating that the agrA defect accounts fully for the lack of agr expression and for the loss of virulence attributed to the traP mutation. In addition, DNA sequencing showed that the agrA gene in strain NB8 (Gov et al., J. Biol. Chem., 2004), in contrast to that in the agr-defective 8325-4 traP mutant strain, had the wild-type sequence; further, the traP mutation in that strain, when outcrossed, also had no effect on agr expression.

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Autoinduction of Vitreoscilla Hemoglobin Enhanced the Production of Syringomycin from Pseudomonas syringae HS191

Science Letters ◽

10.47262/sl/9.1.132021004 ◽

2021 ◽

Vol 9 (1) ◽

pp. 12-18

Keyword(s):

Quorum Sensing ◽

Pseudomonas Syringae ◽

Oxygen Supply ◽

Expression System ◽

Vitreoscilla Hemoglobin ◽

Logarithmic Phase ◽

Wild Type ◽

High Concentration ◽

Sensing System ◽

Quorum Sensing System

Syringomycin is a cyclic lipodepsipeptide produced by strains of Pseudomonas syringae. The potent herbicidal and fungicidal activities of syringomycin make it a promising compound for fungiostasis and weed control. However, the production of syringomycin from the wild-type strains is low. The discoveries that Pseudomonas syringae is aerobic, and the syringomycin synthetase SyrB2 is an O2-dependent halogenase, led us to establish an autoinducible Vitreoscilla hemoglobin expression system for oxygen supply during fermentation, thereby increasing the yield of syringomycin. By employing the quorum sensing system for the expression of Vitreoscilla hemoglobin gene (vgb), we found that Pseudomonas syringae HS191 that expressed vgb, facilitated the cell growth and the general biomass. Furthermore, syringomycin bioassay showed that the fungal inhibition zones increased from 2.5 mm to 3.2 mm, and HPLC analysis confirmed that the expression of vgb resulted in a 71.1% increase in syringomycin production compared to the wild-type strain. The Vitreoscilla hemoglobin has been widely applied to fermentation optimization; however, in the case of Pseudomonas, increased oxygen supply is only beneficial during the stationary phase, while a high concentration of oxygen inhibited the cell propagation during the logarithmic phase. Here we report the autoinduction of Vitreoscilla hemoglobin by engineering the quorum-sensing system. This synthetic circuit significantly improved the syringomycin production. The Vitreoscilla hemoglobin-autoinduction system not only caters to the dynamic oxygen demand but also avoids inducer supplementation.

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Quorum Sensing Controls Exopolysaccharide Production in Sinorhizobium meliloti

Journal of Bacteriology ◽

10.1128/jb.185.1.325-331.2003 ◽

2003 ◽

Vol 185 (1) ◽

pp. 325-331 ◽

Cited By ~ 148

Author(s):

Melanie M. Marketon ◽

Sarah A. Glenn ◽

Anatol Eberhard ◽

Juan E. González

Keyword(s):

Gene Expression ◽

Quorum Sensing ◽

Sinorhizobium Meliloti ◽

Wild Type Strain ◽

Homoserine Lactone ◽

Symbiotic Relationship ◽

Wild Type ◽

Sensing System ◽

Quorum Sensing System

ABSTRACT Sinorhizobium meliloti is a soil bacterium capable of invading and establishing a symbiotic relationship with alfalfa plants. This invasion process requires the synthesis, by S. meliloti, of at least one of the two symbiotically important exopolysaccharides, succinoglycan and EPS II. We have previously shown that the sinRI locus of S. meliloti encodes a quorum-sensing system that plays a role in the symbiotic process. Here we show that the sinRI locus exerts one level of control through regulation of EPS II synthesis. Disruption of the autoinducer synthase gene, sinI, abolished EPS II production as well as the expression of several genes in the exp operon that are responsible for EPS II synthesis. This phenotype was complemented by the addition of acyl homoserine lactone (AHL) extracts from the wild-type strain but not from a sinI mutant, indicating that the sinRI-specified AHLs are required for exp gene expression. This was further confirmed by the observation that synthetic palmitoleyl homoserine lactone (C16:1-HL), one of the previously identified sinRI-specified AHLs, specifically restored exp gene expression. Most importantly, the absence of symbiotically active EPS II in a sinI mutant was confirmed in plant nodulation assays, emphasizing the role of quorum sensing in symbiosis.

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