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1 Department of Pathology, Faculty of Medicine-Ramathibodi Hospital, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
2 Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Sciences, University of Nottingham, Nottingham NG7 2RD, UK
3 Department Laboratory of Biotechnology, Chulabhorn Research Institute, Lak Si, Bangkok, 10210, Thailand
4 Department of Biochemistry, Faculty of Sciences, Mahidol University, Rama VI Road, Bangkok 10400, Thailand
Correspondence
Mongkol Kunakorn
ramkn{at}mahidol.ac.th
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). The organism can be isolated from soil and water. Human infections occur mainly through skin abrasions and inhalation of contaminated aerosols. Frequent relapse has been observed after apparent cure and serological studies have shown that a significant proportion of individuals in endemic areas can be infected asymptomatically (Cheng & Currie, 2005). In vitro studies have demonstrated that B. pseudomallei can survive and multiply inside phagocytes (Jones et al., 1996). To survive inside the phagolysosome, the organism has to endure both acid and oxidative stress. DNA-binding protein from starved cells (Dps) is an abundant protein in stationary-phase Escherichia coli cells (Almiron et al., 1992). Although Dps was originally described as a non-specific DNA-binding protein involved in resistance to oxidative stress, it is actually a bacterioferritin and there are examples of Dps proteins which both bind DNA and sequester iron (Martinez & Kolter, 1997; Stillman et al., 2005). These are thought to protect DNA from damage both as a physical shield and by inhibiting oxyradical formation catalysed by the Fenton reaction. Recently, the crystal structures of two Dps proteins (DpsA and DpsB) from Lactococcus lactis have been described; both proteins were demonstrated to bind DNA via an N-terminal 
-helix (Stillman et al., 2005)
In B. pseudomallei, DpsA has been shown to protect DNA from damage by both acid and oxidative stress (Loprasert et al., 2004). The dpsA gene in the B. pseudomallei genome is located downstream of katG, which encodes a bifunctional enzyme with both catalase and peroxidase activities. Although the mechanism by which DpsA is regulated is not well understood, it is known that expression increases in response to oxidative stress through increased transcription of the katG (catalase peroxidase) promoter, which is OxyR-dependent (Loprasert et al., 2004). Furthermore, dpsA can also be transcribed from its own promoter in an OxyR-independent manner (Loprasert et al., 2004).
Quorum sensing (QS) is a term used to describe the phenomenon where bacteria coordinate the production of a diverse array of phenotypic behaviours in accordance with their cell population size via production of diffusible cell-to-cell signal molecules (Swift et al., 2001; Cámara et al., 2002). Once a threshold concentration has been reached, a response is triggered that leads to changes in gene expression and consequently the phenotype of the cells. In Gram-negative bacteria, the most intensively studied QS systems rely upon the interaction of N-acylhomoserine lactone (AHL) signal molecules, synthesized via LuxI-type AHL synthases, with LuxR-type transcriptional regulator proteins. Together, the LuxR-type protein and its cognate AHL then activate the expression of specific target genes (Swift et al., 2001). Many Gram-negative bacteria possess more than one LuxR and/or LuxI gene and produce multiple AHLs. For example, the opportunistic pathogen Pseudomonas aeruginosa contains two LuxRI systems which operate in a hierarchical manner to regulate an arsenal of virulence determinants and secondary metabolites (Cámara et al., 2002; Lazdunski et al., 2004).
In B. pseudomallei, a LuxRI AHL-dependent QS system termed BpsRI was first described in 2002 by P. Lumjiaktase and co-workers (GenBank accession no. AF501236). Subsequently, Valade et al. (2004) reported that the PmlI-PmlR QS system is required for full virulence in B. pseudomallei strain 008 as a pmlI mutant was significantly less virulent than the parental strain in a murine infection model. The PmlI protein exhibits 98 % sequence identity to BpsI (Valade et al., 2004). In B. pseudomallei strain KHW, a LuxRI pair closely related to BpsI-BpsR was described by Song et al. (2005), who reported that it positively regulated phospholipase C but negatively regulated siderophore production. Both bpsI and bpsR mutants were attenuated in a Caenorhabditis elegans virulence assay (Song et al., 2005). Using HPLC and bioassays of B. pseudomallei spent culture supernatants, Valade et al. (2004) tentatively identified N-decanoylhomoserine lactone (C10-HSL), which they attributed to PmlI although they did not examine the supernatant of the pmlI mutant or express pmlI in E. coli to establish whether PmlI was indeed responsible for C10-HSL synthesis. Song et al. (2005) expressed bpsI in E. coli and, by HPLC, tentatively identified N-octanoylhomoserine lactone (C8-HSL) but did not examine the AHL profile of a B. pseudomallei bpsI mutant. Recently, three LuxRI pairs together with two additional LuxR homologues have been identified in B. pseudomallei DD503 (Ulrich et al., 2004a). DD503 was reported to produce at least five AHLs, including C8-HSL, C10-HSL, N-(3-hydroxyoctanoyl)homoserine lactone (3-hydroxy-C8-HSL), N-3-hydroxydecanoyl homoserine lactone (3-hydroxy-C10-HSL) and N-3-oxotetradecanoyl homoserine lactone (3-oxo-C14-HSL). Mutation of individual B. pseudomallei luxI homologues was reported to have no effect on the AHL profile (Ulrich et al., 2004a).
The regulation of dpsA expression in Burkholderia is poorly understood but one possibility is that it is regulated via AHL-dependent QS since there is a lux box motif located within its promoter region. Here we define the nature of the AHLs synthesized by B. pseudomallei PP844 and show that dpsA expression and resistance to oxidative stress is dependent on QS via BpsIR and C8-HSL.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Unless otherwise stated, bacteria were cultured using Luria–Bertani (LB) broth or agar with appropriate antibiotics at 37 °C. In the case of mixed cultures, e.g. conjugations, incubations were at 30 °C. Pseudomonas agar base supplemented with SR 103E (cetrimide, fucidin and cephaloridine) from Oxoid was used, after conjugation, as a selective medium to inhibit growth of E. coli. M9 minimal medium with 2 % (w/v) glucose was used for 
-galactosidase activity assays. Antibiotics were used at the following concentrations when required: ampicillin 100 µg ml–1, trimethoprim 200 µg ml–1 for B. pseudomallei and 100 µg ml–1 for E. coli, spectinomycin 800 µg ml–1 for B. pseudomallei and 200 µg ml–1 for E. coli, tetracycline 60 µg ml–1 and chloramphenicol 40 µg ml–1.
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Construction of B. pseudomallei bpsI and bpsR knockout and complemented mutants.
bpsI (PKI5), bpsR (PKR7) and bpsIR (KBIR5) mutants were constructed in B. pseudomallei strain PP844. Briefly, A 298 bp fragment of the bpsI gene was amplified from PP844 using primers BIPF (5'-GTCACGCCGATCAGTTGCTT-3') and BIPR (5'-AGTACGATCGCGACGATACC-3'). The blunt-ended product was ligated into the suicide vector pKNOCK-Tc to create pKBI, which was then mobilized from E. coli S17-1 
pir into PP844 by conjugation. Single-crossover insertion mutants were selected on pseudomonas base agar containing 60 µg tetracycline ml–1. A blunt-ended 323 bp fragment of the bpsR gene was amplified using primers BRPF (5'-CGACACCTATCCGAACGGCT-3') and BRPR (5'-AACGGCTCATCAGCGAGTGC-3'). The resulting fragment was ligated into pKNOCK-Cm to create pKBR. After conjugation into PP844, mutants were selected on pseudomonas base agar containing 40 µg chloramphenicol ml–1. Finally, the double bpsIR mutant KBIR5 was created by conjugating pKBR into the PKI5 mutant and selecting on pseudomonas base agar containing 60 µg tetracycline ml–1 and 40 µg chloramphenicol ml–1. For complementation of the knockout strain, pBBR-R2 was conjugated into PKR7 to create PKR7+R, which was selected on pseudomonas base agar containing 800 µg spectinomycin ml–1 and 40 µg chloramphenicol ml–1. Plasmid pBBR-IR3 was conjugated into KBIR5 to create KBIR5+IR, which was selected on pseudomonas base agar containing 800 µg spectinomycin ml–1, 60 µg tetracycline ml–1 and 40 µg chloramphenicol ml–1.
Construction of dpsA : : lacZ transcriptional fusion strains.
TnpD is a mini-transposon vector containing the dpsA promoter fused to lacZ and maintained in E. coli CC118 (CpUT) as described in previous studies (Loprasert et al., 2004). Integration of the dpsA promoter : : lacZ transcriptional fusion into the chromosome of B. pseudomallei PP844, the QS mutants PKI5, PKR7 and KBIR5 and their corresponding complemented strains was achieved by conjugation of TnpD on plates containing trimethoprim (200 µg ml–1). In order to express the DpsA protein in KBIR5, pDps (Loprasert et al., 2004) was transformed into this mutant to create KBIR5+dpsA. To determine whether BpsR regulated dpsA directly, pBBR-R2 was introduced into E. coli CpUT to generate E. coli CpUT+R. The bacteria were selected on agar containing trimethoprim (100 µg ml–1) and spectinomycin (200 µg ml–1).
Assay for -galactosidase activity.
Cell lysates taken from different phases of growth of B. pseudomallei strains grown in MM9 medium with 0.5 µM NaCl and with or without C8-HSL (200 nM) at 37 °C were prepared using bacterial protein extraction reagent (Pierce) and assayed for -galactosidase activity in Miller units using o-nitrophenyl--D-galactoside as a substrate (Miller, 1972). Similar assays were undertaken for E. coli CpUT and CpUT+R grown in the absence or presence of C8-HSL, 3-oxo-C8-HSL, 3-hydroxy-C8-HSL, C10-HSL, 3-hydroxy-C10-HSL and 3-hydroxy-C12-HSL (100 nM).
Growth on oxidant agar plates.
Bacterial cultures were grown overnight in M9 low-glucose medium, adjusted to OD600 1.0 and 10-fold serially diluted. Ten microlitres of each dilution was spotted onto LB agar containing 150 µM tert-butyl hydroperoxide (t-BOOH) and the extent of growth was observed after 24 h incubation at 37 °C (Loprasert et al., 2004).
Growth inhibition zone assay.
Bacterial cultures grown overnight in M9 low-glucose medium were adjusted to OD600 1.0 and added to 3 ml warm top LB agar. The mixtures were overlaid onto LB agar plates. Paper discs containing t-BOOH (250 µM) were placed on the cell lawn. The diameters of growth inhibition zones were measured after 24 h incubation (Loprasert et al., 2004).
Synthesis of AHLs.
A range of AHLs with acyl side chains from C4 to C14 in length, with or without 3-oxo or 3-hydroxy substituents, were synthesized as described by Chhabra et al. (1993, 2003).
AHL extraction and LC MS/MS analysis.
B. pseudomallei strains were grown to OD600 1.6 in 2 l tryptic soy broth at 37 °C with shaking at 250 r.p.m. Cells were removed by centrifugation and the supernatant was extracted twice with equal volumes of acidified ethyl acetate (100 µl glacial acetic acid per litre of ethyl acetate) and concentrated by rotary evaporation at 40–45 °C. The residue was resuspended in 50 µl methanol prior to liquid chromatography tandem mass spectrometry (LC-MS/MS). AHLs were separated by reverse-phase chromatography (RP-HPLC) using an Exsil Pure C18 MS 5µ column (250x2.1 mm; Alltech Associates) coupled to a tandem mass spectrometer (Applied Biosystems 4000 Q-TRAP) and eluted with a 35–70 % w/v acetonitrile/water gradient as described by Yates et al. (2002). Enhanced product trap experiments (EPI) were triggered by precursor ion (m/z 102) scanning for the m/z range 150–350. The precursor ion m/z 102 is characterisitic of the homoserine lactone ring moeity. EPI spectra (m/z range 80–400) containing an ion at m/z 102 were compared with the product mass spectra of the corresponding synthetic AHL standard.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Six AHLs were unequivocally identified by comparison of their retention times, and their molecular and principal fragment ions with synthetic standards. These AHLs were C8-HSL, 3-oxo-C8-HSL, 3-hydroxy-C8-HSL, C10-HSL, 3-hydroxy-C10-HSL and 3-hydroxy-C12-HSL.
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).
To evaluate the impact of bpsI and bpsR mutations on the AHL profile of B. pseudomallei PP844, we constructed bpsI (PKI5) and bpsR (PKR7) mutants. Table 2 compares the AHL profiles derived from LC MS/MS analysis of the corresponding spent culture supernatants. In both mutants, the only compounds absent are C8-HSL and 3-oxo-C8-HSL, a finding which indicates that bpsI is responsible for their synthesis in B. pseudomallei PP844 and that the BpsIR system does not affect the expression of the other AHL synthases present in this organism. Table 2 also shows that bpsI is regulated by BpsR, since mutation of bpsR results in the loss of C8-HSL and 3-oxo-C8-HSL synthesis.
Expression of dpsA in B. pseudomallei is BpsIR/C8-HSL-dependent
In the promoter region (–74 to –55) of dpsA, we identified a 20 bp sequence (GCATCCCGcATCGGGcATGC) as a lux box motif characteristic of genes which are regulated via LuxRI/AHL-dependent QS. Without the lower-case c, this motif will be perfectly palindromic. Nevertheless, this motif matches the consensus sequence for the Vibrio fischeri luxI lux box at 11 out of 21 positions as well as the P. aeruginosa rhlI lux box (12/20 bases). To assess whether QS is involved in regulating the response of B. pseudomallei to oxidative stress, we first introduced a dpsA : : lacZ transcriptional fusion via TnpD onto the chromosome of B. pseudomallei PP844, the isogenic bpsI (PKI5), bpsR (PKR7) mutants and the bpsIR double mutant (KBIR5) as well as the corresponding complemented strains.
Fig. 1 shows that dpsA expression is induced in the late exponential phase of growth (6 h post-inoculation). The bpsI, bpsR and bpsIR mutants all exhibited substantially reduced levels of -galactosidase activity throughout growth when compared to the PP844 wild-type, indicating that dpsA is regulated via bpsIR. Provision of exogenous, synthetic C8-HSL to the bpsI mutant (PKI5) or genetic complementation of the bpsR (PKR7) and bpsIR (KBIR5) mutants completely restored dpsA expression (Fig. 1), suggesting that dpsA is directly or indirectly regulated by the bpsIR QS system. Fig. 1 also demonstrates that mutations in the bpsIR QS system have no adverse effects on the growth of B. pseudomallei under these culture conditions and that exogenous synthetic C8-HSL is unable to overcome the growth-phase dependency of dpsA expression.
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). To determine the sensitivity of the bpsI, bpsR and bpsIR mutants to oxidative stress, each strain was grown on oxidant agar plates containing 150 µM t-BOOH. Each of the mutants was more sensitive to t-BOOH than the wild-type or the corresponding complemented strains (Fig. 2a). The wild-type and complemented strains grew when diluted to 10–6–10–7 c.f.u. ml–1; however, in contrast, the QS mutants only grew when diluted to 10–3–10–4 c.f.u. ml–1. This suggests that PKI5, PKR7 and KBIR5 are 1000–10 000 times more sensitive to hydroperoxide stress.
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). Fig. 2(b) shows that the wild-type grew to the dilution of 10–7 c.f.u. ml–1 and KBIR5 to 10–2 c.f.u. ml–1, whereas KBIR5+dpsA grew to 10–6 c.f.u. ml–1. Taken together, these results demonstrate that the bpsI and bpsR QS mutants are more sensitive to oxidative stress and this is likely to be due to a reduction in dpsA expression and hence DpsA production. The growth inhibition zone assay (Fig. 2c) further confirmed that both wild-type and complemented B. pseudomallei strains were more resistant to t-BOOH than were the QS mutants on LB agar. The dpsA-complemented strain also showed more resistance to t-BOOH, as expected.
BpsR and C8-HSL are required for maximum expression of dpsA in E. coli
To determine whether BpsR directly regulated the expression of dpsA in the presence or absence of AHLs, we used E. coli CpUT habouring the dpsA : : lacZ transcriptional fusion plasmid, TnpD, together with pBBR-R2 to give E. coli strain CpUT+R. Fig. 3 shows that dpsA promoter activity in E. coli CpUT is 
800 Miller units ml–1 and remains unchanged on introducing bpsR (E. coli CpUT+R). Exogenous provision of C8-HSL to E. coli CpUT+R but not E. coli CpUT increased dpsA expression approximately threefold (to 2200 Miller units ml–1). None of the other AHLs produced by B. pseudomallei strain PP844 enhanced dpsA expression.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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), Rhizobium leguminosarum (Lithgow et al., 2000) and Yersinia pseudotuberculosis (Atkinson et al., 1999), B. pseudomallei possesses several LuxI homologues and produces multiple AHL QS signal molecules. B. pseudomallei PP844 is an extremely virulent strain isolated from a patient who died from the most severe clinical manifestation of melioidosis (Utaisincharoen et al., 2001). PP844 produces six AHLs with C8, C10 and C12 acyl side chains with or without C-3 position substituents. Of these, 3-oxo-C8-HSL and 3-hydroxy-C12-HSL have not previously been identified in B. pseudomallei while C8-HSL, 3-hydroxy-C8-HSL, C10-HSL and 3-hydroxy-C10-HSL were previously reported by Ulrich et al. (2004a) in B. pseudomallei strain DD03. This strain also made 3-oxo-C14-HSL, an AHL which was not present in B. pseudomallei PP844 culture supernatants. In bacteria which possess multiple LuxRI homologues, these QS systems are usually interdependent. In B. pseudomallei DD03, mutation of any of the three individual AHL synthase genes had no effect on the AHL profile apart from the pmlI1 mutant, which did not produce 3-hydroxy-C14-HSL. These data do not however define which AHLs are synthesized by which LuxI homologue and suggest that there is substantial redundancy in the system. Here we have shown that mutation of bpsI results in the specific loss of two AHLs, C8-HSL and 3-oxo-C8-HSL, from the AHL profile of the parental PP844 strain. To confirm these data, bpsI was expressed in E. coli. However, only C8-HSL was synthesized, suggesting either that E. coli is unable to synthesize 3-oxo-C8-HSL via BpsI or that 3-oxo-C8-HSL is produced via a different AHL synthase, the expression of which depends on the presence of C8-HSL. When expressed in a heterologous host, LuxI homologues do not always generate the same AHL profile as in the original bacterium (Atkinson et al., 1999) and this is the most likely explanation for our observation. Our unequivocal demonstration that BpsI directs the synthesis of C8-HSL is consistent with the HPLC and bioassay data reported by Song et al. (2005) for B. pseudomallei strain KHW. However, it is not possible to conclude that C10-HSL is the main AHL produced via PmlI (the equivalent gene to bpsI) in B. pseudomallei strain 088 since the authors only examined culture supernatants from the parent strain (Valade et al., 2004), which produces multiple AHLs.
In B. pseudomallei and the closely related obligate animal pathogen Burkholderia mallei, QS mutants are highly attenuated in experimental animal infection models (Ulrich et al., 2004a, b). Although B. mallei appears to possess only two luxI homologues, nevertheless it also produces C8-HSL, 3-hydroxy-C8-HSL, C10-HSL and 3-hydroxy-C10-HSL (Ulrich et al., 2004b) whereas the non-pathogenic Burkholderia thailandensis does not produce any of the 3-hydroxy or 3-oxo compounds although it does synthesize C6-HSL, C8-HSL and C10-HSL (Ulrich et al., 2004c). It is therefore possible that the QS systems employing the substituted AHLs are more closely associated with the regulation of virulence.
Mutation of bpsR in PP844 also resulted in the loss of C8-HSL and 3-oxo-C8-HSL synthesis, indicating that BpsR is required for the synthesis of these two AHLs, presumably by controlling bpsI expression. Indeed, Song et al. (2005) have shown that C8-HSL is required to activate transcription of both bpsI and bpsR. Our data also indicate that the bpsIR system does not control the expression of the two other luxI homologue systems present in B. pseudomallei although it remains possible that the other LuxR proteins and AHLs may influence bpsIR expression.
The organic hydroperoxide t-BOOH has been shown to cause DNA damage in mammalian cells because it reacts with metals to generate tert-butoxyl radicals (Altman et al., 1994). DpsA-type proteins have previously been demonstrated to prevent iron-dependent hydroxy radical formation (Yamamoto et al., 2002) and in B. pseudomallei, DpsA conferred protection against t-BOOH (Loprasert et al., 2004). As bpsI and bpsR mutants exhibit reduced expression of dpsA, we thought it likely that they would show increased sensitivity to t-BOOH. This was indeed the case, with both mutants being more sensitive to t-BOOH. This defect could be complemented by provision of C8-HSL to the bpsI mutant or by genetic complementation of the bpsR and bpsRI mutants. In addition, the viability of the QS mutants was reduced in the presence of t-BOOH when compared with the parent strain cultured under similar conditions. Protection against t-BOOH could also be achieved in the bpsRI mutants by increasing the expression of dpsA. The data suggest that the increased sensitivity to t-BOOH observed in the bpsRI mutants is due specifically to a reduction in dpsA expression. Thus the response of the B. pseudomallei wild-type to oxidative stress is partially controlled in a cell population density dependent manner through QS as demonstrated in this study, perhaps reflecting the need to protect DNA from oxidative damage in high-density ‘overcrowded’ stationary-phase cultures. In P. aeruginosa, the response to oxidative stress imposed by hydrogen peroxide and the 
-generating agent phenazine methosulphate is also QS controlled since sodA, sodB and katA are regulated by both the las and rhl QS systems (Hassett et al., 1999).
B. pseudomallei can resist phagocytic intracellular killing (Egan & Gordon, 1996) and remain dormant within a host for many years (Nathan et al., 2005). It has evolved a variety of mechanisms to protect its DNA from oxidative damage from either cellular metabolism or the environment, and under such conditions will produce high levels of the non-specific DNA-binding protein DpsA, which effectively protects DNA against oxidants (Almiron et al., 1992; Loprasert et al., 2004). In B. pseudomallei (and also Burkholderia cenocepacia strain J2315) dpsA is located adjacent to katG. In the former, the two genes are co-transcribed during oxidative stress but under conditions where katG is not highly induced, dpsA is transcribed from a second promoter within the katG–dpsA intergenic region (Loprasert et al., 2003). This region also contains a lux box motif and here we have shown that dpsA expression is positively controlled by the BpsRI QS system. In B. cepacia ATCC 25416, Aguilar et al. (2003) identified a genomic clone (P80) that was activated in an E. coli strain carrying CepR when supplied with C8-HSL. Although they were unable to identify the target gene(s) regulated by CepR in ATCC 25416, from the sequence data obtained they noted that a DpsA homologue was present 200 bp downstream of the identified sequence in B. cenocepacia J2315, a strain whose genome has been sequenced. Although no direct evidence was presented, it is possible that the response of B. cepacia complex to oxidative stress may also be QS controlled.
In B. pseudomallei, dpsA expression is not completely dependent on bpsRI since -galactosidase activities of 450 Miller units ml–1 are observed in the QS mutants (Fig. 1). It is therefore likely that dpsA expression is also subject to control by a number of regulatory systems where QS provides the population density signal required to trigger dpsA expression in combination with other environmental signals. This is a characteristic of many AHL-dependent QS systems (Withers et al., 2001). Furthermore, it is noteworthy that dpsA expression was not advanced in B. pseudomallei by provision of exogenous C8-HSL and remained population and growth-phase dependent. This phenomenon has also been noted in P. aeruginosa, where provision of exogenous AHLs at the start of growth does not induce early induction of QS-dependent virulence determinants (Diggle et al., 2002, 2003).
In conclusion, we show that (a) B. pseudomallei PP844 synthesizes six AHLs, two of which (3-oxo-C8-HSL and 3-hydroxy-C12-HSL) have not previously been identified in B. pseudomallei; (b) BpsI directs the synthesis of C8-HSL and 3-oxo-C8-HSL; and (c) BpsR, in conjunction with C8-HSL, contributes to the oxidative stress response by positively regulating dpsA expression.
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ACKNOWLEDGEMENTS |
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Received 16 June 2006;
revised 24 August 2006;
accepted 25 August 2006.
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