scholarly journals Acyl-group specificity of AHL synthases involved in quorum-sensing in Roseobacter group bacteria

2018 ◽  
Vol 14 ◽  
pp. 1309-1316 ◽  
Author(s):  
Lisa Ziesche ◽  
Jan Rinkel ◽  
Jeroen S Dickschat ◽  
Stefan Schulz
Keyword(s):  
Quorum Sensing ◽  
Chain Length ◽  
Acyl Chain ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Incubation Experiments ◽  
Dinoroseobacter Shibae ◽  
A Cell ◽  
Acyl Coenzyme A

N-Acylhomoserine lactones (AHLs) are important bacterial messengers, mediating different bacterial traits by quorum sensing in a cell-density dependent manner. AHLs are also produced by many bacteria of the marine Roseobacter group, which constitutes a large group within the marine microbiome. Often, specific mixtures of AHLs differing in chain length and oxidation status are produced by bacteria, but how the biosynthetic enzymes, LuxI homologs, are selecting the correct acyl precursors is largely unknown. We have analyzed the AHL production in Dinoroseobacter shibae and three Phaeobacter inhibens strains, revealing strain-specific mixtures. Although large differences were present between the species, the fatty acid profiles, the pool for the acyl precursors for AHL biosynthesis, were very similar. To test the acyl-chain selectivity, the three enzymes LuxI1 and LuxI2 from D. shibae DFL-12 as well as PgaI2 from P. inhibens DSM 17395 were heterologously expressed in E. coli and the enzymes isolated for in vitro incubation experiments. The enzymes readily accepted shortened acyl coenzyme A analogs, N-pantothenoylcysteamine thioesters of fatty acids (PCEs). Fifteen PCEs were synthesized, varying in chain length from C4 to C20, the degree of unsaturation and also including unusual acid esters, e.g., 2E,11Z-C18:2-PCE. The latter served as a precursor of the major AHL of D. shibae DFL-12 LuxI1, 2E,11Z-C18:2-homoserine lactone (HSL). Incubation experiments revealed that PgaI2 accepts all substrates except C4 and C20-PCE. Competition experiments demonstrated a preference of this enzyme for C10 and C12 PCEs. In contrast, the LuxI enzymes of D. shibae are more selective. While 2E,11Z-C18:2-PCE is preferentially accepted by LuxI1, all other PCEs were not, except for the shorter, saturated C10–C14-PCEs. The AHL synthase LuxI2 accepted only C14 PCE and 3-hydroxydecanoyl-PCE. In summary, chain-length selectivity in AHLs can vary between different AHL enzymes. Both, a broad substrate acceptance and tuned specificity occur in the investigated enzymes.

scholarly journals PapRIV, a BV-2 microglial cell activating quorum sensing peptide

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Yorick Janssens ◽  
Nathan Debunne ◽  
Anton De Spiegeleer ◽  
Evelien Wynendaele ◽  
Marta Planas ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Cell Model ◽  
Dependent Manner ◽  
Communication Signals ◽  
Gram Positive Bacteria ◽  
A Cell ◽  
Gastro Intestinal Tract

AbstractQuorum sensing peptides (QSPs) are bacterial peptides produced by Gram-positive bacteria to communicate with their peers in a cell-density dependent manner. These peptides do not only act as interbacterial communication signals, but can also have effects on the host. Compelling evidence demonstrates the presence of a gut-brain axis and more specifically, the role of the gut microbiota in microglial functioning. The aim of this study is to investigate microglial activating properties of a selected QSP (PapRIV) which is produced by Bacillus cereus species. PapRIV showed in vitro activating properties of BV-2 microglia cells and was able to cross the in vitro Caco-2 cell model and reach the brain. In vivo peptide presence was also demonstrated in mouse plasma. The peptide caused induction of IL-6, TNFα and ROS expression and increased the fraction of ameboid BV-2 microglia cells in an NF-κB dependent manner. Different metabolites were identified in serum, of which the main metabolite still remained active. PapRIV is thus able to cross the gastro-intestinal tract and the blood–brain barrier and shows in vitro activating properties in BV-2 microglia cells, hereby indicating a potential role of this quorum sensing peptide in gut-brain interaction.

scholarly journals Non-native autoinducer analogs capable of modulating the SdiA quorum sensing receptor inSalmonella entericaserovar Typhimurium

2018 ◽  
Vol 14 ◽  
pp. 2651-2664 ◽  
Author(s):  
Matthew J Styles ◽  
Helen E Blackwell
Keyword(s):  
Quorum Sensing ◽  
Small Molecules ◽  
Homoserine Lactone ◽  
Interspecies Interactions ◽  
Host Colonization ◽  
Serovar Typhimurium ◽  
Coordinate Group ◽  
A Cell ◽  
Production Host

Quorum sensing (QS) allows many common bacterial pathogens to coordinate group behaviors such as virulence factor production, host colonization, and biofilm formation at high population densities. This cell–cell signaling process is regulated byN-acyl L-homoserine lactone (AHL) signals, or autoinducers, and LuxR-type receptors in Gram-negative bacteria. SdiA is an orphan LuxR-type receptor found inEscherichia, Salmonella, Klebsiella, and Enterobactergenera that responds to AHL signals produced by other species and regulates genes involved in several aspects of host colonization. The inhibition of QS using non-native small molecules that target LuxR-type receptors offers a non-biocidal approach for studying, and potentially controlling, virulence in these bacteria. To date, few studies have characterized the features of AHLs and other small molecules capable of SdiA agonism, and no SdiA antagonists have been reported. Herein, we report the screening of a set of AHL analogs to both uncover agonists and antagonists of SdiA and to start to delineate structure–activity relationships (SARs) for SdiA:AHL interactions. Using a cell-based reporter of SdiA inSalmonella entericaserovar Typhimurium, several non-natural SdiA agonists and the first set of SdiA antagonists were identified and characterized. These compounds represent new chemical probes for exploring the mechanisms by which SdiA functions during infection and its role in interspecies interactions. Moreover, as SdiA is highly stable when produced in vitro, these compounds could advance fundamental studies of LuxR-type receptor:ligand interactions that engender both agonism and antagonism.

scholarly journals Disrupting quorum sensing alters social interactions in Chromobacterium violaceum

2021 ◽  
Vol 7 (1) ◽  
Author(s):  
Sonia Mion ◽  
Nathan Carriot ◽  
Julien Lopez ◽  
Laure Plener ◽  
Annick Ortalo-Magné ◽  
...  
Keyword(s):  
Quorum Sensing ◽  
Social Interactions ◽  
Antibiotic Production ◽  
Quorum Quenching ◽  
Homoserine Lactone ◽  
Chromobacterium Violaceum ◽  
Dependent Manner ◽  
Gram Negative ◽  
A Cell ◽  
Microbial Population Dynamics

AbstractQuorum sensing (QS) is a communication system used by bacteria to coordinate a wide panel of biological functions in a cell density-dependent manner. The Gram-negative Chromobacterium violaceum has previously been shown to use an acyl-homoserine lactone (AHL)-based QS to regulate various behaviors, including the production of proteases, hydrogen cyanide, or antimicrobial compounds such as violacein. By using combined metabolomic and proteomic approaches, we demonstrated that QS modulates the production of antimicrobial and toxic compounds in C. violaceum ATCC 12472. We provided the first evidence of anisomycin antibiotic production by this strain as well as evidence of its regulation by QS and identified new AHLs produced by C. violaceum ATCC 12472. Furthermore, we demonstrated that targeting AHLs with lactonase leads to major QS disruption yielding significant molecular and phenotypic changes. These modifications resulted in drastic changes in social interactions between C. violaceum and a Gram-positive bacterium (Bacillus cereus), a yeast (Saccharomyces cerevisiae), immune cells (murine macrophages), and an animal model (planarian Schmidtea mediterranea). These results underscored that AHL-based QS plays a key role in the capacity of C. violaceum to interact with micro- and macroorganisms and that quorum quenching can affect microbial population dynamics beyond AHL-producing bacteria and Gram-negative bacteria.

scholarly journals Pseudomonas aeruginosa Las quorum sensing autoinducer suppresses growth and biofilm production in Legionella species

Microbiology ◽  
2009 ◽  
Vol 155 (6) ◽  
pp. 1934-1939 ◽  
Author(s):  
Soichiro Kimura ◽  
Kazuhiro Tateda ◽  
Yoshikazu Ishii ◽  
Manabu Horikawa ◽  
Shinichi Miyairi ◽  
...  
Keyword(s):  
Pseudomonas Aeruginosa ◽  
Quorum Sensing ◽  
Legionella Pneumophila ◽  
Bacterial Species ◽  
Cell Signalling ◽  
Homoserine Lactone ◽  
Alcaligenes Faecalis ◽  
Suppressive Effect ◽  
Dependent Manner ◽  
A Cell

Bacteria commonly communicate with each other by a cell-to-cell signalling mechanism known as quorum sensing (QS). Recent studies have shown that the Las QS autoinducer N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL) of Pseudomonas aeruginosa performs a variety of functions not only in intraspecies communication, but also in interspecies and interkingdom interactions. In this study, we report the effects of Pseudomonas 3-oxo-C12-HSL on the growth and suppression of virulence factors in other bacterial species that frequently co-exist with Ps. aeruginosa in nature. It was found that 3-oxo-C12-HSL, but not its analogues, suppressed the growth of Legionella pneumophila in a dose-dependent manner. However, 3-oxo-C12-HSL did not exhibit a growth-suppressive effect on Serratia marcescens, Proteus mirabilis, Escherichia coli, Alcaligenes faecalis and Stenotrophomonas maltophilia. A concentration of 50 μM 3-oxo-C12-HSL completely inhibited the growth of L. pneumophila. Additionally, a significant suppression of biofilm formation was demonstrated in L. pneumophila exposed to 3-oxo-C12-HSL. Our results suggest that the Pseudomonas QS autoinducer 3-oxo-C12-HSL exerts both bacteriostatic and virulence factor-suppressive activities on L. pneumophila alone.

scholarly journals Role of the C-Terminal Domain of the Alpha Subunit of RNA Polymerase in LuxR-Dependent Transcriptional Activation of the lux Operon during Quorum Sensing

2002 ◽  
Vol 184 (16) ◽  
pp. 4520-4528 ◽  
Author(s):  
Angela H. Finney ◽  
Robert J. Blick ◽  
Katsuhiko Murakami ◽  
Akira Ishihama ◽  
Ann M. Stevens
Keyword(s):  
Quorum Sensing ◽  
Rna Polymerase ◽  
Transcriptional Activation ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Α Subunit ◽  
Terminal Domain

ABSTRACT During quorum sensing in Vibrio fischeri, the luminescence, or lux, operon is regulated in a cell density-dependent manner by the activator LuxR in the presence of an acylated homoserine lactone autoinducer molecule [N-(3-oxohexanoyl) homoserine lactone]. LuxR, which binds to the lux operon promoter at a position centered at −42.5 relative to the transcription initiation site, is thought to function as an ambidextrous activator making multiple contacts with RNA polymerase (RNAP). The specific role of the α-subunit C-terminal domain (αCTD) of RNAP in LuxR-dependent transcriptional activation of the lux operon promoter has been investigated. The effects of 70 alanine substitution variants of the α subunit were determined in vivo by measuring the rate of transcription of the lux operon via luciferase assays in recombinant Escherichia coli. The mutant RNAPs from strains exhibiting at least twofold-increased or -decreased activity in comparison to the wild type were further examined by in vitro assays. Since full-length LuxR has not been purified, an autoinducer-independent N-terminally truncated form of LuxR, LuxRΔN, was used for in vitro studies. Single-round transcription assays were performed using reconstituted mutant RNAPs in the presence of LuxRΔN, and 14 alanine substitutions in the αCTD were identified as having negative effects on the rate of transcription from the lux operon promoter. Five of these 14 α variants were also involved in the mechanisms of both LuxR- and LuxRΔN-dependent activation in vivo. The positions of these residues lie roughly within the 265 and 287 determinants in α that have been identified through studies of the cyclic AMP receptor protein and its interactions with RNAP. This suggests a model where residues 262, 265, and 296 in α play roles in DNA recognition and residues 290 and 314 play roles in α-LuxR interactions at the lux operon promoter during quorum sensing.

scholarly journals The Complex Quorum Sensing Circuitry of Burkholderia thailandensis Is Both Hierarchically and Homeostatically Organized

mBio ◽  
2017 ◽  
Vol 8 (6) ◽  
Author(s):  
Servane Le Guillouzer ◽  
Marie-Christine Groleau ◽  
Eric Déziel
Keyword(s):  
Quorum Sensing ◽  
Regulatory Network ◽  
Target Genes ◽  
Wide Spectrum ◽  
Homoserine Lactone ◽  
Growth Stages ◽  
Dependent Manner ◽  
Burkholderia Thailandensis ◽  
Bacterial Communication ◽  
A Cell

ABSTRACT The genome of the bacterium Burkholderia thailandensis encodes three complete LuxI/LuxR-type quorum sensing (QS) systems: BtaI1/BtaR1 (QS-1), BtaI2/BtaR2 (QS-2), and BtaI3/BtaR3 (QS-3). The LuxR-type transcriptional regulators BtaR1, BtaR2, and BtaR3 modulate the expression of target genes in association with various N-acyl-l-homoserine lactones (AHLs) as signaling molecules produced by the LuxI-type synthases BtaI1, BtaI2, and BtaI3. We have systematically dissected the complex QS circuitry of B. thailandensis strain E264. Direct quantification of N-octanoyl-homoserine lactone (C8-HSL), N-3-hydroxy-decanoyl-homoserine lactone (3OHC10-HSL), and N-3-hydroxy-octanoyl-homoserine lactone (3OHC8-HSL), the primary AHLs produced by this bacterium, was performed by liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS) in the wild-type strain and in QS deletion mutants. This was compared to the transcription of btaI1, btaI2, and btaI3 using chromosomal mini-CTX-lux transcriptional reporters. Furthermore, the levels of expression of btaR1, btaR2, and btaR3 were monitored by quantitative reverse transcription-PCR (qRT-PCR). We observed that C8-HSL, 3OHC10-HSL, and 3OHC8-HSL are differentially produced over time during bacterial growth and correlate with the btaI1, btaI2, and btaI3 gene expression profiles, revealing a successive activation of the corresponding QS systems. Moreover, the transcription of the btaR1, btaR2, and btaR3 genes is modulated by cognate and noncognate AHLs, showing that their regulation depends on themselves and on other QS systems. We conclude that the three QS systems in B. thailandensis are interdependent, suggesting that they cooperate dynamically and function in a concerted manner in modulating the expression of QS target genes through a successive regulatory network. IMPORTANCE Quorum sensing (QS) is a widespread bacterial communication system coordinating the expression of specific genes in a cell density-dependent manner and allowing bacteria to synchronize their activities and to function as multicellular communities. QS plays a crucial role in bacterial pathogenicity by regulating the expression of a wide spectrum of virulence/survival factors and is essential to environmental adaptation. The results presented here demonstrate that the multiple QS systems coexisting in the bacterium Burkholderia thailandensis, which is considered the avirulent version of the human pathogen Burkholderia pseudomallei and thus commonly used as an alternative study model, are hierarchically and homeostatically organized. We found these QS systems to be finely integrated into a complex regulatory network, including transcriptional and posttranscriptional interactions, and further incorporating growth stages and temporal expression. These results provide a unique, comprehensive illustration of a sophisticated QS network and will contribute to a better comprehension of the regulatory mechanisms that can be involved in the expression of QS-controlled genes, in particular those associated with the establishment of host-pathogen interactions and acclimatization to the environment. IMPORTANCE Quorum sensing (QS) is a widespread bacterial communication system coordinating the expression of specific genes in a cell density-dependent manner and allowing bacteria to synchronize their activities and to function as multicellular communities. QS plays a crucial role in bacterial pathogenicity by regulating the expression of a wide spectrum of virulence/survival factors and is essential to environmental adaptation. The results presented here demonstrate that the multiple QS systems coexisting in the bacterium Burkholderia thailandensis, which is considered the avirulent version of the human pathogen Burkholderia pseudomallei and thus commonly used as an alternative study model, are hierarchically and homeostatically organized. We found these QS systems to be finely integrated into a complex regulatory network, including transcriptional and posttranscriptional interactions, and further incorporating growth stages and temporal expression. These results provide a unique, comprehensive illustration of a sophisticated QS network and will contribute to a better comprehension of the regulatory mechanisms that can be involved in the expression of QS-controlled genes, in particular those associated with the establishment of host-pathogen interactions and acclimatization to the environment.

scholarly journals The AinS autoinducer synthase and LitR master regulator of quorum sensing regulate N-3-hydroxy-decanoyl-homoserine-lactone production and motility in Aliivibrio wodanis 06/09/139

2019 ◽  
Author(s):  
Amudha Deepalakshmi Maharajan ◽  
Hilde Hansen ◽  
Nils Peder Willassen
Keyword(s):  
Quorum Sensing ◽  
Cell Density ◽  
Homoserine Lactone ◽  
Temperature Tolerance ◽  
Dependent Manner ◽  
Secretion Systems ◽  
Master Regulator ◽  
Density Dependent ◽  
Ulcer Disease ◽  
A Cell

Abstract Background Quorum Sensing (QS) is a cell to cell communication system, in which bacteria synthesize and respond to signaling molecules called autoinducers (AI). QS is cell density dependent and known to be involved in regulating virulence, motility and secretion systems to interact with the host or other bacteria. Aliivibrio wodanis is frequently isolated together with Moritella viscosa from the infected Atlantic salmon during outbreaks of the winter ulcer disease. M. viscosa is the main causative agent of the disease while the presence of A. wodanis is still unclear. It is hypothesized that A. wodanis might influence the progression of winter ulcer. The genome of A. wodanis 06/09/139 encodes two autoinducer synthase genes (ainS and luxS) and a master regulator litR. LitR homologs in other aliivibrios have been shown to regulate several phenotypes in a cell density dependent manner. Moreover, a previous study has shown that A. wodanis 06/09/139 produces only one AHL N-3-hydroxy-decanoyl-homoserine-lactone (3OHC10-HSL). Hence, in this work, we have studied the QS system in A. wodanis 06/09/139 by knocking out QS genes ainS and litR. The effects of the deletions were studied with regard to growth, AHL production and motility at different temperatures. Results By using HPLC-MS/MS, we found that the deletion of ainS in A. wodanis 06/09/139 resulted in the loss of 3OHC10-HSL production. The 3OHC10-HSL production in A. wodanis 06/09/139 increased with increase in cell density and more 3OHC10-HSL was produced at 6°C than at 12, 16 and 20°C. The litR mutant demonstrated a ~20% reduction in the production of 3OHC10-HSL relative to the wild type at the stationary phase. Compared to the wildtype and the ainS mutant strains, the litR mutant resulted in a strain with improved temperature tolerance. The motility in mutants (∆litR and ∆ainS) were significantly higher than that of the wildtype. Conclusions Our study shows that AinS in A. wodanis 06/09/139 is the AHL synthase responsible for 3OHC10-HSL production, where the production is both cell density and temperature dependent. Our data also shows that LitR regulates 3OHC10-HSL production only to a minor extent and both LitR and AinS are negative regulators of motility.

scholarly journals Specificity of Acyl-Homoserine Lactone Synthases Examined by Mass Spectrometry

2006 ◽  
Vol 188 (2) ◽  
pp. 773-783 ◽  
Author(s):  
Ty A. Gould ◽  
Jake Herman ◽  
Jessica Krank ◽  
Robert C. Murphy ◽  
Mair E. A. Churchill
Keyword(s):  
Mass Spectrometry ◽  
Acyl Carrier Protein ◽  
Acyl Chain ◽  
Internal Standard ◽  
Binding Pocket ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Pantoea Stewartii ◽  
Liquid Chromatography Tandem Mass ◽  
A Cell

ABSTRACT Many gram-negative bacteria produce a specific set of N-acyl-l-homoserine-lactone (AHL) signaling molecules for the purpose of quorum sensing, which is a means of regulating coordinated gene expression in a cell-density-dependent manner. AHLs are produced from acylated acyl-carrier protein (acyl-ACP) and S-adenosyl-l-methionine by the AHL synthase enzyme. The appearance of specific AHLs is due in large part to the intrinsic specificity of the enzyme for subsets of acyl-ACP substrates. Structural studies of the Pantoea stewartii enzyme EsaI and AHL-sensitive bioassays revealed that threonine 140 in the acyl chain binding pocket directs the enzyme toward production of 3-oxo-homoserine lactones. Mass spectrometry was used to examine the range of AHL molecular species produced by AHL synthases under a variety of conditions. An AHL selective normal-phase chromatographic purification with addition of a deuterated AHL internal standard was followed by reverse-phase liquid chromatography-tandem mass spectrometry in order to obtain estimates of the relative amounts of different AHLs from biological samples. The AHLs produced by wild-type and engineered EsaI and LasI AHL synthases show that intrinsic specificity and different cellular conditions influence the production of AHLs. The threonine at position 140 in EsaI is important for the preference for 3-oxo-acyl-ACPs, but the role of the equivalent threonine in LasI is less clear. In addition, LasI expressed in Escherichia coli produces a high proportion of unusual AHLs with acyl chains consisting of an odd number of carbons. Furthermore, these studies offer additional methods that will be useful for surveying and quantitating AHLs from different sources.

scholarly journals Control of Acetic Acid Fermentation by Quorum Sensing via N-Acylhomoserine Lactones in Gluconacetobacter intermedius

2008 ◽  
Vol 190 (7) ◽  
pp. 2546-2555 ◽  
Author(s):  
Aya Iida ◽  
Yasuo Ohnishi ◽  
Sueharu Horinouchi
Keyword(s):  
Acetic Acid ◽  
Quorum Sensing ◽  
Parental Strain ◽  
Gluconic Acid ◽  
Acyl Chain ◽  
Homoserine Lactone ◽  
Transcriptional Analysis ◽  
Dependent Manner ◽  
Acid Fermentation ◽  
Gram Negative

ABSTRACT A number of gram-negative bacteria regulate gene expression in a cell density-dependent manner by quorum sensing via N-acylhomoserine lactones (AHLs). Gluconacetobacter intermedius NCI1051, a gram-negative acetic acid bacterium, produces three different AHLs, N-decanoyl-l-homoserine lactone, N-dodecanoyl-l-homoserine lactone, and an N-dodecanoyl-l-homoserine lactone with a single unsaturated bond in its acyl chain, as determined by liquid chromatography-tandem mass spectrometry. Two genes encoding an AHL synthase and a cognate regulator were cloned from strain NCI1051 and designated ginI and ginR, respectively. Disruption of ginI or ginR abolished AHL production, indicating that NCI1051 contains a single set of quorum-sensing genes. Transcriptional analysis showed that ginI is activated by GinR, which is consistent with the finding that there is an inverted repeat whose nucleotide sequence is similar to the sequence bound by members of the LuxR family at position −45 with respect to the transcriptional start site of ginI. A single gene, designated ginA, located just downstream of ginI is transcribed by read-through from the GinR-inducible ginI promoter. A ginA mutant, as well as the ginI and ginR mutants, grew more rapidly in medium containing 2% (vol/vol) ethanol and accumulated acetic acid at a higher rate with a greater final yield than parental strain NCI1051. In addition, these mutants produced larger amounts of gluconic acid than the parental strain. These data demonstrate that the GinI/GinR quorum-sensing system in G. intermedius controls the expression of ginA, which in turn represses oxidative fermentation, including acetic acid and gluconic acid fermentation.

scholarly journals Quorum sensing regulates dpsA and the oxidative stress response in Burkholderia pseudomallei

Microbiology ◽  
2006 ◽  
Vol 152 (12) ◽  
pp. 3651-3659 ◽  
Author(s):  
Putthapoom Lumjiaktase ◽  
Stephen P. Diggle ◽  
Suvit Loprasert ◽  
Sumalee Tungpradabkul ◽  
Mavis Daykin ◽  
...  
Keyword(s):  
Oxidative Stress ◽  
Quorum Sensing ◽  
Burkholderia Pseudomallei ◽  
Dna Binding Protein ◽  
Homoserine Lactone ◽  
Dependent Manner ◽  
Acylhomoserine Lactone ◽  
Organic Hydroperoxides ◽  
Genes Encoding ◽  
A Cell

Burkholderia pseudomallei is the causative agent of melioidosis, a fatal human tropical disease. The non-specific DNA-binding protein DpsA plays a key role in protecting B. pseudomallei from oxidative stress mediated, for example, by organic hydroperoxides. The regulation of dpsA expression is poorly understood but one possibility is that it is regulated in a cell population density-dependent manner via N-acylhomoserine lactone (AHL)-dependent quorum sensing (QS) since a lux-box motif has been located within the dpsA promoter region. Using liquid chromatography and tandem mass spectrometry, it was first established that B. pseudomallei strain PP844 synthesizes AHLs. These were identified as N-octanoylhomoserine lactone (C8-HSL), N-(3-oxooctanoyl)homoserine lactone (3-oxo-C8-HSL), N-(3-hydroxyoctanoyl)-homoserine lactone (3-hydroxy-C8-HSL), N-decanoylhomoserine lactone (C10-HSL), N-(3-hydroxydecanoyl) homoserine lactone (3-hydroxy-C10-HSL) and N-(3-hydroxydodecanoyl)homoserine lactone (3-hydroxy-C12-HSL). Mutation of the genes encoding the LuxI homologue BpsI or the LuxR homologue BpsR resulted in the loss of C8-HSL and 3-oxo-C8-HSL synthesis, demonstrating that BpsI was responsible for directing the synthesis of these AHLs only and that bpsI expression and hence C8-HSL and 3-oxo-C8-HSL production depends on BpsR. In bpsI, bpsR and bpsIR mutants, dpsA expression was substantially down-regulated. Furthermore, dpsA expression in Escherichia coli required both BpsR and C8-HSL. bpsIR-deficient mutants exhibited hypersensitivity to the organic hydroperoxide tert-butyl hydroperoxide by displaying a reduction in cell viability which was restored by provision of exogenous C8-HSL (bpsI mutant only), by complementation with the bpsIR genes or by overexpression of dpsA. These data indicate that in B. pseudomallei, QS regulates the response to oxidative stress at least in part via the BpsR/C8-HSL-dependent regulation of DpsA.

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