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Regulation of autoinducer 2 production and luxS expression in a pathogenic Edwardsiella tarda strain

¹ Institute of Oceanology, Chinese Academy of Sciences, Qingdao 266071, PR China
² Graduate University of the Chinese Academy of Sciences, Beijing 100049, PR China

Correspondence
Li Sun
lsun{at}ms.qdio.ac.cn

	ABSTRACT

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Edwardsiella tarda is a bacterial pathogen that can infect both humans and animals. TX1, an Ed. tarda strain isolated from diseased fish, was found to produce autoinducer 2 (AI-2)-like activity that was growth phase dependent and modulated by growth conditions. The gene coding for the AI-2 synthase was cloned from TX1 and designated luxS_Et. LuxS_Et was able to complement the AI-2 mutant phenotype of Escherichia coli strain DH5. Expression of luxS_Et correlated with AI-2 activity and was increased by glucose and decreased by elevated temperature. The effect of glucose was shown to be mediated through the cAMP-CRP complex, which repressed luxS_Et expression. Overexpression of luxS_Et enhanced AI-2 activity in TX1, whereas disruption of luxS_Et expression by antisense RNA interference (i) reduced the level of AI-2 activity, (ii) impaired bacterial growth under various conditions, (iii) weakened the expression of genes associated with the type III secretion system and biofilm formation, and (iv) attenuated bacterial virulence. Addition of exogenous AI-2 was able to complement the deficiencies in the expression of TTSS genes and biofilm production but failed to rescue the growth defects. Our results (i) demonstrated that the AI-2 activity in TX1 is controlled at least in part at the level of luxS_Et expression, which in turn is regulated by growth conditions, and that the temporal expression of luxS_Et is essential for optimal bacterial infection and survival; and (ii) suggested the existence in Ed. tarda of a LuxS/AI-2-mediated signal transduction pathway that regulates the production of virulence-associated elements.

The GenBank/EMBL/DDBJ accession number for the sequence of the luxS_Et region is EU070919.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Quorum sensing (QS) is a complex process by which cell–cell communication is achieved in bacteria (Gonzalez & Keshavan, 2006). For Gram-negative bacteria, QS systems can be classified into several categories based on the type of autoinducers and relaying molecules involved. One of these, as exemplified by that observed with Vibrio harveyi in the regulation of bioluminescence production (Bassler et al. 1993; Mok et al., 2003), employs autoinducer-2 (AI-2) as the signalling molecule. The V. harveyi AI-2 is produced from S-adenosylhomocysteine (SAH) via enzymic steps involving the nucleosidase Pfs, which converts SAH to S-ribosylhomocysteine (SRH), and LuxS, which converts SRH to 4,5-dihydroxy-2,3-pentanedione, the immediate precursor of AI-2 (De Keersmaecker et al., 2006; Lewis et al., 2001; Schauder et al., 2001). Unlike AI-1, which is generally species-specific and is responded to only by cells of kindred genetic lineages, AI-2 has been discovered to exist in diverse bacteria and the AI-2 molecules produced by one bacterial species are responded to by bacteria of different species and genera. Consistent with its proposed role as an interspecies communication signal, AI-2-mediated signal transduction circuits have been found to regulate biological processes that coordinate the behaviour of cells as a community to best adapt to various environmental situations. Such biological processes include biofilm formation, conjugation, sporulation, and production of virulence elements (Gonzalez Barrios et al., 2006; McNab et al., 2003; Rickard et al., 2006; Surette et al., 1999); the last has been linked to the LuxS/AI-2 quorum-sensing system via, most often, mutational studies of the luxS gene (Lyon et al., 2001; Marouni & Sela, 2003; Ohtani et al., 2002; Stroeher et al., 2003; Winzer et al., 2002b; Zhu et al., 2002).

Edwardsiella tarda is a Gram-negative bacterium that can be pathogenic to a broad range of host, including humans. Currently the major concern raised by this bacterium is its ability to cause edwardsiellosis, a systemic disease that has been reported to occur in different parts of the world. Although recognized as one of the leading pathogens that pose a serious threat to the development of aquaculture industries worldwide, Ed. tarda has not been studied in a scope merited by its importance, and many fundamental processes, especially those concerning bacterial infection and survival, remain to be elucidated. Recently Morohoshi et al. (2004) identified the LuxI/LuxR homologues EdwI/EdwR in Ed. tarda, and suggested that, as in many other pathogens, the EdwI/EdwR system probably regulates the production of certain virulence factors.

We present in this report the study of AI-2 activity and luxS expression in Ed. tarda strain TX1, a fish pathogen. Our results indicate that the AI-2 activity in TX1 is controlled at the level of luxS expression, which is modulated by growth conditions. By using antisense RNA interference we obtained evidence supporting the idea that there exists in TX1 an active LuxS/AI-2-mediated QS pathway that is involved in bacterial pathogenicity.

	METHODS

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Bacterial strains and growth conditions.
The bacterial strains used in this study are listed in Table 1. Unless otherwise indicated, all strains were grown in Luria–Bertani (LB) medium at 37 °C (for Escherichia coli) or 28 °C (for all others) (Zhang & Sun, 2007). Appropriate antibiotics and supplements were added at the following concentrations: ampicillin, 100 µg ml^–1; kanamycin, 50 µg ml^–1; tetracycline, 15 µg ml^–1; X-Gal, 40 µg ml^–1. The mean generation time (g) of bacterial growth was calculated as described by Eagon (1962).

Plasmid constructions.
The plasmids used in this study are listed in Table 1; primers are listed in Table 2. To construct pBT, the rrnB transcription terminator of pTrcHis (Invitrogen) was ligated into pBR322 between the EcoRV and BsaBI sites, resulting in plasmid pBRB. The Ptrc promoter of pTrcHis was amplified by PCR with primers TrcF2/TrcR1 and inserted into pBRB between the EcoRI and BamHI sites, yielding plasmid pBT. pBTES was generated by inserting luxS_Et (amplified by PCR with primers F19/R17) into the SmaI site of pBT. To create pSC11, an EcoRI–EcoRV–BamHI linker was inserted into pSC6 between the EcoRI and BamHI sites, resulting in pSC7, which was then cut with EcoRV/PvuII and ligated to a promoterless lacZ gene amplified from TOP10RS65 with primers LacF2/LacR3. To construct pSC100, the P_luxS-containing 100 bp DNA (named D100) immediately upstream of the translational start of luxS_Et was amplified by PCR with primers F11/R9 and the PCR products were ligated into pSC11 at the SwaI site. Similarly, pSC100M was created by inserting D100 containing the mutated P_luxS (amplified by PCR with primers F17/R9) into pSC11 at the SwaI site. pJRSN was created by first inserting the bla gene of pBR322 into pDN18 at the EcoRI site, resulting in plasmid pJRA, which was then digested with EcoRV and ligated to the SwaI fragment carrying the P_trc-luxS_Et fusion of pBTES. pJR18 was constructed by inserting the antisense RNA of luxS_Et (amplified by PCR with primers R18/F19) into the SmaI site of pBT, resulting in pBTSR; the SwaI fragment of pBTSR containing the P_trc-luxS antisense RNA coding region was inserted into the EcoRV site of pJRA, yielding pJR18. pJZS was created by first inserting the promoterless lacZ gene into pACYC177 (New England BioLabs) at the SmaI site, resulting in plasmid p178, which was then digested with SmaI and ligated to P_luxS (generated by PCR with primers F10/R9), resulting in plasmid p178LS; the SmaI fragment of p178LS containing the P_luxS-lacZ fusion was inserted into pJRA at the EcoRV site. To create pJZL, the luciferase gene (luc) of pGL3-Basic (Promega) was inserted into pET28 (Novagen), resulting in pGL28; luc with the ribosome-binding site was amplified by PCR from pGL28 and inserted into pSC7 between the EcoRV and PvuII sites, yielding pSC17, which was then digested with SwaI and ligated to P_luxS, resulting in pJZL.

Real-time reverse transcriptase (RT) PCR.
Total RNA was extracted from fish organs and from bacterial cells grown in appropriate medium to OD₆₀₀ 1 by using the SV total RNA isolation system (Promega). Real-time RT-PCR was carried out in an ABI 7300 Real-time Detection System (Applied Biosystems) by using the SYBR ExScript RT-PCR kit (Takara, China). Each assay was performed in triplicate with 16S rRNA as a control. Dissociation analysis of amplification products was performed at the end of each PCR to confirm that only one PCR product was amplified and detected. The comparative CT (2^–CT) method was used to analyse the mRNA level. All data are given in terms of relative mRNA expressed as means±SEM. Statistical analyses were performed by using the two-tailed t-test.

Preparation of recombinant CRP.
The E. coli crp gene was amplified by PCR with primers CRPF1/CRPR1 and the PCR products were ligated into pET258 between the NdeI and XhoI sites, resulting in pECRP, which was introduced into the E. coli BL21(DE3) by transformation. The recombinant CRP was purified from BL21(DE3)/pECRP by using nickel-NTA beads as described previously (Zhang & Sun, 2007).

Gel electrophoresis mobility shift assay (EMSA).
The 288 bp DNA fragment containing P_luxS was generated by PCR with primers F10/R9 and labelled with carboxyfluorescein. The labelled DNA was mixed with the purified recombinant CRP and incubated at 37 °C for 20 min in CRP binding buffer (Sun et al., 2004) with or without 400 nM cAMP. After the reaction, the samples were run on a nondenaturing 8 % polyacrylamide gel. As a negative control the 200 bp DNA region upstream of the EcoRI site of pBR322 was included in the assay.

AI-2 assay.
The AI-2 assay was performed essentially as described by Surette & Bassler (1999). To prepare cell-free culture fluids, overnight cultures of cells grown in LB medium at 28 °C were diluted 1 : 100 in fresh LB medium; 2 ml of cell culture was taken every 30 min and the cell-free supernatant was obtained by centrifugation followed by filtering through a 0.22 µm filter (Millipore). For measurement of bioluminescence induction, an overnight culture of V. harveyi strain BB170 grown in AB medium at 28 °C was diluted 1 : 5000 in fresh AB medium supplemented with cell-free culture fluids (10 %) of the tested strains or with growth medium (as the control). Growth was continued and light production was measured by using a Glomax luminometer (Promega).

Complementation by exogenous AI-2.
Ed. tarda TX1 was cultured in LB medium to OD₆₀₀ 1 and cell-free culture fluids (prepared as above) were added at a concentration of 10 % to the growth medium of the cells under examination.

Biofilm development assay.
Biofilm formation on a polystyrene surface was determined exactly as described by Xu et al. (2006).

Bacterial conjugation.
pJRA and its variants were introduced into E. coli S17-1pir (Biomedal) by transformation. The transformants and Ed. tarda TX1 were grown in LB medium to OD₆₀₀ 1 and mixed in a 1 : 1 ratio. The mixed cells were washed and resuspended in 10 mM MgSO₄ and dropped onto an LB plate. After incubation at 28 °C for 12 h, the growth on the plate was scraped off and resuspended in 1 ml LB, from which 100 µl was taken and spread onto a LB plate supplemented with ampicillin and tetracycline. The plate was incubated at 28 °C for 48 h and the colonies that appeared were verified to be authentic transconjugants by PCR and sequence analysis of the PCR products.

β-Galactosidase assay.
This was performed as described by Sun et al. (1998).

Animal model study.
Japanese flounders (Paralichthys olivaceus) (14 g) were divided randomly into several groups (40 fish/group). Each group was injected intraperitoneally (i.p.) with the test bacterium that had been cultured to OD₆₀₀ 0.5 in LB medium, washed, and resuspended in phosphate-buffered saline (PBS). The animals were monitored for mortality in the 10 days post-infection and the accumulated mortalities were calculated. To examine bacterial dissemination, the blood and organs of the infected fish were removed under sterile conditions. The organs were homogenized with glass homogenizers and plated on LB plates containing selective antibiotics. The blood was plated directly. The plates were incubated at 28 °C for 48 h and the colonies that emerged were examined for TX1 harbouring or not harbouring plasmid by PCR using TX1-, pJRA- and pJR18-specific primers and subsequent sequencing of the PCR products. Statistical analyses were performed by the two-tailed t-test.

Database searching and nucleotide sequence accession number.
Database searching was conducted using the BLAST programs at the NCBI (National Center for Biotechnology Information). The nucleotide sequence of the luxS_Et region has been deposited in the GenBank database under accession number EU070919.

	RESULTS

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Cell-free culture fluids of TX1 exhibit AI-2-like activity which is growth phase dependent and modulated by growth conditions
To investigate whether Ed. tarda strain TX1 produced active AI-2, the cell-free culture fluids of this strain, which was isolated from diseased fish, was analysed for the ability to stimulate light production in V. harveyi strain BB170, a reporter of AI-2 signalling (Bassler et al., 1993). As shown in Fig. 1(a), TX1 possessed AI-2 activity that, up to the early exponential phase, increased with the progress of the growth in a manner similar to that observed with BB120, the wild-type strain from which BB170 is derived. After the cell density exceeded OD₆₀₀ 0.75, however, the AI-2 activity in BB120 declined sharply whereas that in TX1 continued to increase until the cell density reached OD₆₀₀ 1, where the AI-2 activity peaked. The maximum fold induction of light production effected by the culture fluids of TX1 was 35 % lower than that effected by BB120, suggesting that either the amount of active AI-2 present in TX1 is less than that in BB120 or the AI-2 of TX1 is less effective in the activation of the specific QS pathway in BB170. To determine whether the AI-2 activity of TX1 was affected by growth conditions, TX1 was cultured at 28 °C in LB with or without 0.5 % glucose to OD₆₀₀ 0.95 or in LB medium at 37 °C to OD₆₀₀ 0.65, which was close to the maximum cell density under the specific growth condition. Subsequent AI-2 assay indicated that the AI-2 activity was significantly augmented by glucose and reduced by high temperature (Fig. 1b).

View larger version (21K): [in this window] [in a new window]   Fig. 1. (a) AI-2 activity in Ed. tarda TX1 and in E. coli DH5 carrying pBT variants. Cell-free culture fluids of TX1, DH5 harbouring pBT or pBTES, and V. harveyi BB120 were taken at various growth points and assayed for AI-2 activity. DH5/pBT showed no AI-2 activity (not plotted). (b) AI-2 activity in TX1 under different growth conditions. TX1 was grown in LB medium at 28 °C (left) or in LB medium at 37 °C (right) or at 28 °C in LB supplemented with 0.5 % glucose (centre). The cells used for AI-2 assay were harvested at OD600 0.95 except for those grown at 37 °C, which were harvested at OD600 0.65. In both (a) and (b), AI-2 activities are presented as fold induction over the control. Data are representative of at least three independent experiments and presented as the means±SEM. **, P<0.01.

TX1 possesses a luxS homologue that can complement the AI-2-defective phenotype of E. coli DH5

Identification of the luxS_Et promoter
On the chromosome, luxS_Et is preceded by an ORF designated gcl; as there is a putative rho-independent transcriptional terminator (RITT) between gcl and luxS_Et (Fig. 2), we speculated that the promoter of luxS_Et was probably located downstream of the RITT. Sequence inspection identified a putative ⁷⁰-dependent promoter (named P_luxS) located 31 bp downstream of the RITT (Fig. 2). P_luxS was cloned into the promoter-probe plasmid pSC11, a low-copy-number plasmid with a pSC101 replication origin and a promoterless lacZ gene as the reporter of heterologous promoter activity. The recombinant plasmid, pSC100, was introduced into DH5 by transformation. The transformant gave blue colonies on X-Gal plates and produced detectable β-galactosidase activity (44 Miller units), suggesting that P_luxS was an active promoter. In support of this, DH5 transformed with pSC100M, which is identical to pSC100 except that the first T in the –10 region of P_luxS is ‘down’-mutated to G, produced no detectable β-galactosidase activity.

View larger version (24K): [in this window] [in a new window]   Fig. 2. Nucleotide sequence of the intergenic region between gcl and luxSEt. The translation stop and start codons of gcl and luxSEt, respectively, are in bold capital letters; the putative RITT is italicized; the putative CRP-binding site is underlined; the –10 and –35 elements of PluxS are capitalized and indicated.

View larger version (17K): [in this window] [in a new window]   Fig. 3. Expression of luxSEt in relation to growth phase (a) and growth conditions (b). (a) Total RNA was extracted from Ed. tarda TX1 grown to different growth phases and used for real-time RT-PCR. (b) Total RNA was extracted from strain TX1 grown to OD600 0.9 in LB medium at 28 °C or in LB with the indicated modifications (i.e. at 37 °C, in the presence of 0.5 % glucose, 5 mM cAMP, or glucose plus cAMP) and used for real-time RT-PCR. The luxSEt mRNA level was normalized to that of the 16S rRNA. Data are presented as the means±SEM. **, P<0.01; *, P<0.05.

The effect of glucose upon luxS_Et expression is mediated through the cAMP–CRP complex
Since glucose can decrease the cellular cAMP level and thus affect the activity of the cAMP receptor protein (CRP), we determined whether cAMP had any effect upon luxS_Et expression by real-time RT-PCR. The results showed that the presence of cAMP (5 mM) alone significantly repressed luxS_Et expression whereas cAMP combined with glucose completely abolished the stimulating effect of glucose (Fig. 3b). A similar cAMP effect was also observed with AI-2 activity (data not shown). In line with these observations, a promoter activity assay indicated that DH5 harbouring pJZL, in which P_luxS directs the expression of a promoterless luciferase gene, exhibited luciferase activity that was approximately ninefold higher in the absence than in the presence of cAMP, suggesting that the E. coli CRP repressed transcription from P_luxS. Consistent with this conclusion, in vitro EMSA demonstrated that the purified recombinant E. coli CRP could bind specifically to the DNA fragment containing P_luxS (Fig. 4). Sequence inspection revealed a potential CRP-binding site that partially matches the consensus CRP-binding sequence, TGTGAN₆TCACA, 13 bp downstream of P_luxS (Fig. 2). Taken together, these results indicated that glucose upregulates luxS_Et expression by inactivating CRP, which represses transcription from P_luxS.

View larger version (35K): [in this window] [in a new window]   Fig. 4. Specific binding of CRP to the promoter region of luxSEt. EMSA was performed in CRP binding buffer containing the carboxyfluorescein-labelled DNA fragment harbouring PluxS and purified recombinant E. coli CRP in the presence or absence of cAMP. After the reaction, the samples were run on a nondenaturing 8 % polyacrylamide gel. The DNA in lanes 5 and 6 served as controls.

Effect on growth.
Growth pattern studies showed that, compared with TX1/pJRA, TX1/pJR18 displayed a slower generation time (g) and lower maximum cell densities under all the conditions examined, which included growth in LB medium at 28 °C, under iron depletion caused by the iron chelator 2,2'-dipyridyl, in LB medium at 37 °C, and in 0.6 M NaCl (2216E medium) at 28 °C (Fig. 5); under these conditions, the differences in g between TX1/pJRA and TX1/pJR18 were 8.6, 10.3, 30.4 and 9.5 %, respectively, while the differences in maximum cell density between TX1/pJRA and TX1/pJR18 were 12.1, 25.3, 78.9 and 44 %, respectively. LuxS is known to function in two cellular aspects, one in cell–cell signalling (via AI-2) and the other in the activated methyl cycle; complementation of a physiological defect by exogenous AI-2 can be an indicator that the particular defect is due to impairment in the former function of LuxS. In the case of TX1/pJR18, addition of exogenous AI-2 in the form of AI-2-containing culture supernatant of TX1 failed to rescue the growth defects (data not shown), suggesting that these defects are probably not the result of changes in AI-2-mediated signalling process but more likely that of impairment in central metabolism.

View larger version (18K): [in this window] [in a new window]   Fig. 5. Growth patterns of Ed. tarda TX1/pJRA and TX1/pJR18 under various conditions. (a) Cells were cultured at 28 °C in LB medium with or without 100 µM 2,2'-dipyridyl (DP). (b) Cells were cultured in LB at 37 °C or in 2216E medium at 28 °C. g, mean generation time.

View larger version (16K): [in this window] [in a new window]   Fig. 6. Bacterial dissemination in the kidney (a) and blood (b) of infected fish. Flounders were i.p. injected with Ed. tarda TX1/pJR18 or TX1/pJRA and the blood and kidneys of at least three fish were taken at various time points post-infection. The homogenized organs and the blood were plated on selective LB plates and the number of plasmid-bearing TX1 was counted. Data are presented as the means±SEM. **, P<0.01.

Effect on the expression of virulence-associated factors.
To find out the potential genetic mechanisms underlying the attenuated virulence observed with TX1/pJR18, we examined the production/expression in TX1/pJR18 of known virulence elements: the type III secretion system (TTSS), type VI secretion system, Eth haemolysin system and biofilm formation (Hirono et al., 1997; Tan et al., 2005; Zheng & Leung, 2007). The TTSS genes are located in two chromosomal regions, region 1 and region 2; real-time RT-PCR analysis demonstrated that luxS_Et antisense RNA had no effect on the expression of eseB and eseD, which are in region 1, but reduced the expression of esrA and orf26 (Fig. 7a), which are in region 2. To determine whether luxS_Et antisense RNA had any effect on biofilm production, TX1/pJRA and TX1/pJR18 were cultured to OD₆₀₀ 0.8 and placed into a 96-well polystyrene plate; after incubation at 28 °C for 24 h, the development of biofilm was examined. Biofilm production was reduced threefold in TX1/pJR18 compared with that in TX1/pJRA (Fig. 7b). Addition of AI-2-containing cell-free culture supernatant of TX1 restored both biofilm production and orf26/esrA expression in TX1/pJR18 to a level approaching that in TX1/pJRA but had no effect on the expression of eseB and eseD (Fig. 7). These results suggested that LuxS_Et modulates the expression of orf26/esrA and biofilm development probably through the action of AI-2, which implies the existence in TX1 of a functional LuxS/AI-2-mediated signalling system. To determine whether there was any difference in orf26/esrA expression between TX1/pJR18 and TX1/pJRA during infection, real-time RT PCR was carried out to analyse the expression of orf26/esrA using RNA extracted from the spleens and livers taken 24 h after the fish were infected with TX1/pJR18 and TX1/pJRA. The results showed that the expression levels of orf26 and esrA were, respectively, 5.7- and 4-fold lower in TX1/pJR18-infected fish than those in TX1/pJRA-infected fish (data not shown); hence interference with luxS_Et expression had a significant effect on the expression of orf26 and esrA during infection.

View larger version (25K): [in this window] [in a new window]   Fig. 7. Comparison of the expression of TTSS genes (a) and biofilm formation (b) in Ed. tarda TX1/pJRA and TX1/pJR18. (a) Real-time RT-PCR was performed using total RNA extracted from cells grown to OD600 0.8 in LB medium supplemented with or without AI-2-containing cell-free culture fluids of strain TX1 (10 %). 16S rRNA was used as a control. For each gene, the mRNA level of TX1/pJRA was set as 1. (b) Biofilm formation on a polystyrene surface was determined for TX1/pJRA and TX1/pJR18 in the presence or absence of AI-2 supplemented as above. Data are presented as the means±SEM. **, P<0.01.

	DISCUSSION

TOP ABSTRACT INTRODUCTION METHODS RESULTS DISCUSSION REFERENCES

 
Surette & Bassler (1999) observed that in Salmonella typhimurium AI-2 production is affected by environmental factors such as glucose and pH, which boost the AI-2 level. In our study we found that in Ed. tarda TX1 both AI-2 activity and luxS_Et expression were influenced positively by glucose and negatively by high temperature. Studies by Wang et al. (2005) revealed that in E. coli AI-2 production and luxS expression are subject to catabolite repression through the cAMP–CRP complex, which indirectly inhibits the expression of luxS and directly stimulates the expression of lsr, which encodes an ATP-binding cassette transporter that functions as an AI-2 uptake system (Taga et al., 2001, 2003; Xavier & Bassler, 2005). In the case of TX1, glucose modulated luxS_Et expression via cAMP–CRP, which directly repressed luxS_Et expression, probably by blocking the formation of the DNA–RNA polymerase complex, considering the location of the putative CRP-binding site that is downstream of P_luxS. Although Lsr-like transporting systems have not yet been discovered in Ed. tarda, it is possible that in Ed. tarda TX1 the glucose-associated increase of AI-2 activity is the combined result of both enhanced production, due to augmented expression of luxS_Et, and reduced clearance of the signalling molecule.

In our study we observed a rough correlation between AI-2 activity and luxS_Et expression, which suggested that in strain TX1 production of AI-2 activity is at least in part controlled at the level of luxS_Et expression. Interference with the regulated expression of luxS_Et and AI-2 synthesis appeared to have severe consequences upon certain physiological processes that manifested, in one form, as growth defects. The failure of the growth defects to be complemented by exogenous AI-2 rules out, to a large extent, the involvement of AI-2-mediated signal transduction. As one of the enzymes participating in the activated methyl cycle, LuxS plays an important role in cellular metabolism (Doherty et al., 2006; Vendeville et al., 2005; Winzer et al., 2002a); hence interruption of luxS expression has a direct effect on fundamental cellular processes (Kendall et al., 2007), which probably accounts for the growth deficiencies observed with TX1/pJR18 under various conditions.

It is well documented that LuxS is involved in the regulation of virulence development in diverse bacteria (Coulthurst et al., 2004, 2007; Day & Maurelli, 2001; Joyce et al., 2004). In E. coli, V. harveyi and Streptococcus pyogenes LuxS has been associated with the production of virulence factors such as the TTSS, extracellular proteases and biofilm development (Henke & Bassler, 2004a, b; Herzberg et al., 2006; Li et al., 2007; Lyon et al., 2001; Sircili et al., 2004). In TX1 we found that interference with luxS_Et expression affected biofilm production and the expression of TTSS-encoding genes located in DNA region 2. Given the fact that luxS_Et expression was not entirely blocked off but only reduced by the antisense RNA, the null effect observed with eseB/eseD, evpA/evpB and ethA/ethB cannot rule out the possibility that these genes are subject to LuxS_Et regulation but to a lesser degree than that observed with orf26/esrA and hence undetectable under our experimental conditions. Since both the TTSS and biofilm formation have been related to bacterial pathogenicity, the reduced production of these elements may have contributed to the attenuated virulence observed with TX1/pJR18. This hypothesis is consistent with the observation that interference with luxS_Et expression significantly altered the expression of orf26 and esrA during infection. In addition, the effect of luxS_Et antisense RNA on growth may play a part in mitigating the bacterial virulence of TX1/pJR18.

In conclusion, our study has demonstrated that in Ed. tarda TX1 AI-2 activity correlates to a certain degree with the expression of luxS_Et, which is in turn regulated by growth phase and growth conditions. Our results favour the notion that LuxS_Et plays a dual role, one in cellular metabolism and the other in AI-2-mediated signalling; interruption of the first role of LuxS_Et leads to a growth defect whereas interruption of the second leads to aberrations in the development of certain biological characteristics that are required for full bacterial virulence.

	ACKNOWLEDGEMENTS

 
This work was supported by 973 Project of China grant 2006CB101807 and the National Natural Science Foundation of China (NSFC) grants 40676090 and 40576071.

Edited by: P. Cornelis

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Received 5 February 2008; revised 9 April 2008; accepted 9 April 2008.

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