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Journal of Bacteriology, February 2006, p. 1451-1461, Vol. 188, No. 4
0021-9193/06/$08.00+0     doi:10.1128/JB.188.4.1451-1461.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Quorum Sensing in Yersinia enterocolitica Controls Swimming and Swarming Motility

Steve Atkinson,1* Chien-Yi Chang,1 R. Elizabeth Sockett,2 Miguel Cámara,2 and Paul Williams1

Institute of Infection, Immunity and Inflammation, Centre for Biomolecular Science, University of Nottingham, Nottingham NG7 2UH, United Kingdom,1 Institute of Genetics, University of Nottingham, Queen's Medical Centre, Nottingham NG7 2UH, United Kingdom2

Received 12 July 2005/ Accepted 22 November 2005


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ABSTRACT
 
The Yersinia enterocolitica LuxI homologue YenI directs the synthesis of N-3-(oxohexanoyl)homoserine lactone (3-oxo-C6-HSL) and N-hexanoylhomoserine lactone (C6-HSL). In a Y. enterocolitica yenI mutant, swimming motility is temporally delayed while swarming motility is abolished. Since both swimming and swarming are flagellum dependent, we purified the flagellin protein from the parent and yenI mutant. Electrophoresis revealed that in contrast to the parent strain, the yenI mutant grown for 17 h at 26°C lacked the 45-kDa flagellin protein FleB. Reverse transcription-PCR indicated that while mutation of yenI had no effect on yenR, flhDC (the motility master regulator) or fliA (the flagellar sigma factor) expression, fleB (the flagellin structural gene) was down-regulated. Since 3-oxo-C6-HSL and C6-HSL did not restore swimming or swarming in the yenI mutant, we reexamined the N-acylhomoserine lactone (AHL) profile of Y. enterocolitica. Using AHL biosensors and mass spectrometry, we identified three additional AHLs synthesized via YenI: N-(3-oxodecanoyl)homoserine lactone, N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL), and N-(3-oxotetradecanoyl)homoserine lactone. However, none of the long-chain AHLs either alone or in combination with the short-chain AHLs restored swarming or swimming in the yenI mutant. By investigating the transport of radiolabeled 3-oxo-C12-HSL and by introducing an AHL biosensor into the yenI mutant we demonstrate that the inability of exogenous AHLs to restore motility to the yenI mutant is not related to a lack of AHL uptake. However, both AHL synthesis and motility were restored by complementation of the yenI mutant with a plasmid-borne copy of yenI.


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INTRODUCTION
 
Yersinia enterocolitica is a mammalian enteropathogen which can present in humans as enteritis, enterocolitis, mesenteric lymphadenitis, or terminal ileitis normally after the consumption of contaminated food or through direct inoculation following a blood transfusion. Y. enterocolitica exhibits a biphasic lifestyle which facilitates an existence in both terrestrial/aquatic and mammalian environments and multiple Y. enterocolitica virulence factors have been identified, including the Yersinia outer proteins, invasins (inv), and flagellum-mediated motility (6).

The expression of Yersinia virulence genes is tightly regulated by a range of environmental factors with temperature playing a major role. In addition, all three human pathogenic Yersinia spp. produce N-acylhomoserine lactone (AHL) quorum-sensing signal molecules which, in many different gram-negative bacteria are involved in the cell population density-dependent regulation of virulence, secondary metabolite production, and biofilm maturation (41, 42).

Homologues of the LuxI (AHL synthase) and LuxR (response regulator) protein families have been identified in Y. pseudotuberculosis, and Y. pestis as well as Y. enterocolitica (3, 41, 43). However, Y. enterocolitica in contrast to Y. pseudotuberculosis and Y. pestis has only one LuxRI pair (YenRI) compared to the two LuxRI pairs found in the other Yersinia species which are pathogenic to humans. YenI is responsible for the synthesis of two AHLs, N-hexanoylhomoserine lactone (C6-HSL) and N-(3-oxohexanoyl)homoserine lactone (3-oxo-C6-HSL), respectively (46). Both AHLs are produced in an approximately 1:1 ratio in Y. enterocolitica and when yenI is expressed in either Escherichia coli (46) or in tobacco plants (11).

The production of 3-oxo-C6-HSL and C6-HSL in both virulent (90/54; pYV+) and avirulent (10460; pYV–) Y. enterocolitica strains is abolished following mutation of yenI. 3-oxo-C6-HSL but not C6-HSL has been detected in the tissues of mice infected with mouse virulent Y. enterocolitica O:8 serotype strains indicating that the quorum sensing circuitry is functional during the disease process (15). However, the contribution of AHL-dependent quorum sensing to the virulence of Y. enterocolitica or indeed for any other pathogenic Yersinia species is not yet clear. In Y. pseudotuberculosis, which contains two luxR/I pairs termed ypsR/I and ytbR/I, a comparison of the parent with the corresponding ypsR and ypsI mutants indicated that the mutants exhibited a temperature-dependent clumping phenotype (ypsR mutant), the overexpression of a major flagellin subunit protein (ypsR mutant) and increased motility (both ypsR and ypsI mutants) (3, 4). For Y. enterocolitica, while there were no obvious differences in Yop protein profiles when the wild type was compared with the isogenic yenI mutant, two-dimensional sodium dodecyl sulfate (SDS)- polyacrylamide gel electrophoresis (PAGE) revealed that numerous cellular proteins were differentially regulated by quorum sensing in this strain (46).

Although it has not been experimentally established, flagellar gene expression in Y. enterocolitica and Y. pseudotuberculosis is likely to be similar to that in other members of the Enterobacteriaceae and to follow a hierarchical cascade consisting of three major flagellar gene classes, I, II, and III (52). An analysis of the first pass annotation of the Y. enterocolitica genome suggests that all the key regulatory elements of the flagellum cascade are present including flhDC, fliA, flgM, mot, and che genes (sequence data produced by the Y. enterocolitica Sequencing Group at the Sanger Institute and can be obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/ye/). The flhDC operon (class I) is at the top of the hierarchy and is required for the expression of the class II genes. These code for structural and accessory proteins required for assembly of the flagellum basal body and hook and also fliA and flgM which respectively encode the sigma factor, {sigma}28 and the anti-{sigma}28, FlgM protein. Class III genes code for the proteins involved in maturation of the flagellum and chemosensory system which are transcribed from {sigma}28-dependent promoters (52).

In the present paper we show that a yenI mutant is unable to swim or swarm and establish that quorum sensing regulates motility at least in part, at the level of the flagellin structural gene, fleB. We also show that YenI is responsible for directing the synthesis of three long-chain AHLs in addition to the short-chain AHLs previously characterized.


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MATERIALS AND METHODS
 
Bacterial strains and growth conditions. The strains used in this study were Y. enterocolitica 90/54 (serotype O:9, pYV+), the corresponding Y. enterocolitica yenI insertion-deletion mutant described by Throup et al. (46) and E. coli JM109 (50). Bacteria were grown in LB Lennox broth (21) with the appropriate antibiotics where required. Agar plates contained 15 g/liter no. 1 agar (Oxoid). Minimal swarm motility plates contained 10 g/liter tryptone, 5 g/liter NaCl and 0.6% (wt/vol) Bacto agar (Difco Laboratories) and 10 mM glucose which was filter sterilized into the medium immediately before pouring once the swarm agar had been sterilized by autoclaving and cooled to 50°C. For some experiments, surfactin (1 µg/ml, Sigma) was added to the swarm plates (22).

Minimal swim motility agar plates contained 10 g/liter tryptone, 5 g/liter NaCl and 0.3% (wt/vol) Bacto agar as previously described (3). 1 µl of overnight seed cultures grown at 30°C were inoculated into (swim) or onto (swarm) agar plates respectively and incubated at 26°C. To determine whether exogenous AHLs restored swimming or swarming motility, C6-HSL, 3-oxo-C6-HSL, N-octanoylhomoserine lactone (C8-HSL), N-(3-oxodecanoyl)homoserine lactone (3-oxo-C10-HSL), N-(3-oxododecanoyl)homoserine lactone (3-oxo-C12-HSL), N-(3-oxotetradecanoyl)homoserine lactone (3-oxo-C14-HSL), or AHL extracts prepared from spent culture supernatants from the parent were added to each plate either individually or in combination at concentrations ranging from 0 to 1 µM.

AHL synthesis. AHLs were synthesized as previously described (8, 7) for use as standards for thin-layer chromatography (TLC), high-pressure liquid chromatography (HPLC), HPLC-mass spectrometry (LC-MS), and the swimming and swarming restoration assays. Tritiated 3-oxo-C12-HSL (N-[3-oxo-[6,7-3H2] dodecanoyl]-L-homoserine lactone) was custom synthesized by Amersham Biosciences United Kingdom by titration of the unsaturated precursor, N-[(E)-3-oxo-6-dodecenoyl]-L-homoserine lactone by the procedure described previously (16). The unsaturated precursor was obtained by the condensation of (E)-4-decenoic acid with Meldrum's acid followed by amidation with L-homoserine lactone (7).

DNA manipulations. Plasmid minipreps were performed using the Promega Wizard system, agarose gel electrophoresis and standard methods for the preparation of competent cells and electroporation were performed as previously described (35). Purification of DNA fragments from agarose gels used Qiaquick DNA purification columns (QIAGEN Ltd.). Restriction endonucleases, DNA ligase and other DNA modification enzymes were used according to the manufacturers' instructions (Promega). Ligations were dialyzed against sterile HPLC-grade water by applying to floating Millipore 0.0250µm filters (Millipore Ltd.) before electroporation to reduce the risk of arcing.

Purification and sequencing of flagellar proteins. Y. enterocolitica parent and yenI mutant flagellar proteins were isolated from liquid cultures after overnight incubation for 17 or 24 h (with shaking) at 26°C and analyzed by SDS-PAGE as previously described (39). Briefly, bacteria in liquid culture were passed 20 times through a 0.5-mm diameter, 200-mm-long cannula attached at both ends to a 10-ml syringe. During this process flagella are sheared from the bacterial surface, the debris and bacterial cells are then removed by centrifugation, the flagella are pelleted at 41,000 x g (1 h) and resuspended in a suitable volume of phosphate-buffered saline prior to analysis by SDS-PAGE.

To identify the 42-kDa protein band in the parent at 17 h and the parent and yenI mutant at 24 h, the relevant bands were excised from the polyacrylamide gel, and after an in-gel tryptic digest, the resulting peptide mixture was subjected to matrix-assisted laser desorption ionization-time of flight (MALDI-TOF)-MS (Waters Corp.) operating at a resolution of >10,000 full width at half maximum in reflectron mode. Peptide peak list files were generated both manually and automatically. Background peaks resulting from trypsin autolysis and commonly observed keratin contamination were excluded from database searching. Peak lists were entered into a MASCOT PMF database search engine, search parameters included a peptide mass accuracy tolerance of 0.2 Da and searches allowed for modifications such as alkylation of cysteine during the tryptic digest procedure and the possible formation of methionine sulfoxide.

Complementation assays and PCR. The Y. enterocolitica strain 90/54 is naturally ampicillin resistant as is the published yenI clone, pYeI7 (46). We were therefore unable to trans-complement the yenI mutant with pYeI7 and therefore we isolated yenI from pYeI7 on a HindIII-EcoRI fragment (these restriction sites are present in the multicloning site of the pT7T3 backbone vector of pYeI7) and cloned it into the chloramphenicol-resistant vector pSU18 (4) to give pSUyenI. Both pSU18 and pSUyenI were introduced into the yenI mutant by electroporation.

Using lux-based AHL sensors in conjunction with plasmid and cosmid libraries prepared from genomic DNA isolated from the yenI mutant in trans complementation was performed to screen for further luxI homologues as described previously (43, 46). Cosmid libraries were prepared using the Strategene Giga Pack II packaging extract following the manufacturers instructions. PCR, using primers designed to amplify luxI homologues from Y. pseudotuberculosis (3), was undertaken in a further attempt to amplify a second luxI homologue from Y. enterocolitica.

RT-PCR. RNA was purified from cells grown in LB at 26°C for 14, 17, 20, and 24 h using the Roche Highpure RNA isolation kit (Roche Diagnostics) following the manufacturers instructions. For the expression of yenR in the yenI mutant RNA was isolated from cells grown under the same conditions as above after 6, 12, 18, and 24 h. cDNA was then synthesized from the RNA templates using the Amersham First Strand cDNA synthesis kit (Amersham Pharmacia). The newly synthesized first-strand cDNA was used as a template for PCR using the following primers designed to the coding regions of the yenR, flhDC, fliA, and one of the three flagellin structural genes, fleB: for yenR, YenR1 (GGCCTATGGTGATCTTCG) and YenR2 (CCTGACAACATTGCCAAT); forflhDC: Mot14 (CAGCGATGTTTCGTCTCG) and Mot15 (CTAACCTGCTCATCCAGC); for fliA: Mot16 (GCTGTAGCGGATTAGCG) and Mot17 (GAGGCATTGCGTTTGCAG); and for fleB: Mot18 (GGTGCCATTCAACACGTC) and Mot19 (CCTGTCTCTGCTGACTC). As a control for contamination of the RNA by DNA, PCR was carried out using the newly synthesized RNA as a template prior to first strand cDNA synthesis. At each time point, immediately before centrifugation and harvesting for RNA isolation, the bacterial cells were examined for motility by phase contrast microscopy.

Identification, purification and characterization of AHLs. Y. enterocolitica parent, isogenic yenI mutant and Escherichia coli[pYeI7] strains were grown at 26°C or 37°C respectively in LB Lennox buffered with 50 mM MOPS (3-[N-morpholino] propanesulfonic acid) pH 6.8 (LBmops), to ensure the supernatant did not rise above pH 7.0 and promote lactonolysis (51). Late-exponential-phase, cell-free culture supernatants were extracted twice with dichloromethane as described before (46, 3) and assayed for AHLs using the biosensors Chromobacterium violaceum CV026 and E. coli JM109 lasRI'::luxCDABE (pSB1075), which respond to short-chain (CV026) or long-chain (E. coli[pSB1075]) AHLs, respectively, by producing the purple pigment violacein or bioluminescence.

Bioluminescence was detected using a Berthold LB980 photon video camera (E.G. and G Berthold U.K. Ltd., Milton Keynes, U.K). AHLs were detected by using the biosensors as agar overlays either on LB agar plates or on thin-layer chromatograms as described before (19, 26, 48, 52). To purify long-chain AHLs from spent culture supernatants, samples were separated on an aluminum backed RP2 TLC plates and the active spots/streaks identified and reextracted from the silica matrix as previously described (3). The extracts were fractionated on an RP8 semipreparative HPLC column (Kromasil KR 100-5C8 [, 250 by 7 mm column; Hichrom) by elution with a linear gradient of acetonitrile (20 to 100%) in water over a 32-min period and held at 100% for a further 10 min using a flow rate of 1.62 ml/min and monitoring at A200.

Seven fractions were collected over a 42-min period and assayed for activity using E. coli[pSB1075] in overlay plate assays. Fractions exhibiting the highest activity were further purified using an isocratic mobile phase, acetonitrile/water (60:40, vol/vol). The final active subfractions were collected, pooled and reanalyzed using an HPLC linked to a photodiode array (PDA) system (Waters 996 PDA system operating with a Millennium Chromatography Manager; Waters, Watford, England). Both the retention time and spectral properties were compared to a series of synthetic standards. The major active fractions were then subjected to HPLC-mass spectrometry (LC-MS) (Waters Micromass Quattro Ultra in conjunction with an Agilent 1100 LC system) using the same isocratic conditions mobile phase (acetonitrile in water, 60:40% [vol/vol]) and the spectra obtained were compared with those of the synthetic standards subjected to the same LC-MS conditions.

Intracellular accumulation of AHLs. To determine whether exogenously supplied AHLs are internalized by the yenI mutant, two approaches were taken. First, radiolabel experiments were carried out essentially as described by Pearson et al. (31). Briefly, 10 ml overnight cultures of the Y. enterocolitica parent, yenI mutant and E. coli[pSB401] (grown at 22°C) were harvested and resuspended in 250 µl of KG buffer (10 mM potassium phosphate[pH 7.0], 0.03% glycerol). Tritiated 3-oxo-C12-HSL (2.70 TBq/mmol) was added to a final concentration of 100 nM in a final volume of 300 µl. The bacteria were incubated at 22°C for 20 min and then centrifuged through 75 µl of Nyosil (Nye Lubricants, New Bedford, Mass.) containing 25% trichloroacetic acid and 10% glycerol. The supernatant was removed and the bacterial pellet was resuspended in 150 µl of KG buffer. Equal volumes of both the supernatant and the pellet fractions were counted in a Topcount NXT microplate scintillation and luminescence counter (Packard Bioscience United Kingdom).

Second, the AHL-negative yenI mutant was transformed with the AHL biosensor pSB401 (48). If AHLs accumulate intracellularly in Y. enterocolitica, then this strain which does not bioluminesce should respond by emitting light. To explore the response of Y. enterocolitica yenI[pSB401] to AHLs, the organism was grown overnight at 26°C, the culture diluted 1 in 50 and grown for 3 to 4 h before being inoculated in 96 well microtiter plates at an initial A600 of 0.05 in the absence or presence of 3-oxo-C6-HSL or 3-oxo-C12-HSL (1 µM). The plates were incubated for 12 h at 26°C in a Lucy Anthos II combined spectrophotometer/luminometer. Readings were taken at timed intervals and relative light output was plotted as a function of cell population density (A492).


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RESULTS
 
Quorum-sensing controls swimming motility, swarming motility and temporally controls flagellar synthesis in Y. enterocolitica. Swimming motility was analyzed at 26°C in both the Y. enterocolitica parent and the yenI mutant which revealed that after 17 h, the mutant failed to swim when compared with the parent but began to swim after 24 h (Fig. 1A). On swarm plates the yenI mutant in contrast to the parent strain was unable to swarm (Fig. 2A).


Figure 1
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  		FIG. 1. Analysis of swimming motility in Y. enterocolitica and the isogenic yenI mutant on 0.3% swim agar plates which were point-inoculated and grown at 26°C. (A) The parent and the yenI mutant after 17 and 24 h showing that the yenI mutant is unable to swim when compared to the parent but begins to swim after 24 h. (B) The complemented yenI mutant containing either pSU18 or pSUyenI after 17 and 24 h showing that swimming motility is restored to parental levels in the yenI mutant when a functional copy of yenI is supplied in trans.


Figure 2
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  		FIG. 2. Analysis of swarming motility in Y. enterocolitica and the isogenic yenI mutant on 0.6% swarm agar plates grown at 26°C and monitored after 24 h. (A) The yenI mutant is unable to swarm compared to the parent. (B) When the yenI mutant is complemented with a functional copy of yenI in trans (pSUyenI) swarming motility is restored to parental levels whereas the yenI mutant complemented with the vector alone remains nonmotile.

To determine whether the loss of motility was due to the absence of flagella, Y. enterocolitica and the corresponding isogenic yenI mutant were grown in liquid cultures for 17 h at 26°C. When these cultures were examined by phase contrast microscopy the yenI mutant was nonmotile whereas the parent was vigorously swimming (data not shown). Flagellar proteins were isolated from these strains after 17 h of growth and examined by SDS-PAGE. An approximately 45-kDa band was visible for the parent but was absent from the yenI mutant (Fig. 3). If the cultures were grown for 24 h then the yenI mutant also produced the flagellin protein (albeit in significantly smaller amounts, data not shown) consistent with the late onset of motility observed in the swimming plate assays.


Figure 3
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  		FIG. 3. SDS-PAGE of flagellar proteins isolated from Y. enterocolitica and the isogenic yenI mutant after 17 h of growth in liquid culture. (A) The ~45-kDa band is present in flagellar extracts taken from the parent but absent from the yenI mutant. (B) There is no band visible for the yenI mutant, indicating that the mutant is incapable of producing flagellin.

The identity of the 42-kDa band was determined using MALDI-TOF MS which demonstrated that the profile for the 42-kDa band closely matched (P < 0.05) that of FleB for flagellar extracts from the parent at 17 h and 24 h and the parent and yenI mutant at 24 h (data not shown).

Quorum sensing controls motility at the level of fleB transcription. Since the late-onset motility phenotype of the yenI mutant appeared to be associated with the appearance of flagellin, we sought to determine whether mutation of yenI influenced expression of the master flagellar regulator fhlDC, the alternative sigma factor fliA, or fleB, one of the three Y. enterocolitica flagellin structural genes. RT-PCR analysis revealed that flhDC (Fig. 4A) and fliA (Fig. 4B) are transcribed similarly in both the parent and yenI mutant. flhDC transcripts (Fig. 4A) are clearly present at 17 h when ~80% of the wild-type cells but very few yenI mutant cells are motile. flhDC expression also continues through to 20 and 24 h (data not shown). In contrast, the fliA transcript was present in both the parent and mutant after 14 h but absent after 17 h of growth (Fig. 4B). For fleB, however, there is clearly differential regulation. A much lower level of fleB transcription is apparent in the yenI mutant at both time points (Fig. 4C, compare lanes 4 and 5).


Figure 4
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  		FIG. 4. RT-PCR analysis of the presence of flhDC (A), fliA (B) and fleB (C) transcripts in the Y. enterocolitica parent and yenI mutant grown in liquid culture at 26°C for 14 and 17 h, respectively. In each case lane 1 is the positive control, lanes 2 and 3 are the PCR using RNA templates isolated from the parent and yenI mutant, respectively, prior to first-strand cDNA synthesis, lanes 4 and 5 are the PCR using the newly synthesized cDNA templates isolated from the parent and yenI mutant, respectively. The size of the PCR products from primers Mot14 and Mot15 (flhDC) is 832 bp, that of Mot16 and Mot17 (fliA) is 435 bp, and that of Mot18 and Mot19 (fleB) is 778 bp.

yenI and yenR are convergently transcribed and overlapping on opposite strands and we therefore felt it unlikely that the insertion deletion in yenI would influence the transcription of yenR. To confirm that the observed phenotypes were specifically to the yenI mutation, the expression of yenR was examined in the parent and yenI mutant using RT-PCR using primers designed to the internal sequence of yenR. At 6, 12, 18, and 24 h yenR was expressed in both the parent and the yenI mutant (data not shown)

Exogenous provision of C6-HSL or 3-oxo-C6-HSL does not restore swimming or swarming motility. A characteristic feature of many AHL-dependent quorum-sensing systems is that loss of a specific phenotype through mutation of the corresponding AHL synthase can be restored by exogenous provision of the cognate AHL(s) (10, 24, 48). To determine whether swimming and swarming could be restored in Y. enterocolitica by the provision of exogenous AHLs, the yenI mutant was assayed on swim and swarm plates supplemented with a range of concentrations of either C6-HSL or 3-oxo-C6-HSL or both (from 100 pM to 100 nM). Neither swimming nor swarming motility was restored.

In Serratia marcescens and Pseudomonas aeruginosa, AHL-dependent quorum-sensing controls swarming motility indirectly by regulating biosurfactant production (9, 10, 22, 47). To determine whether this was also the case for Y. enterocolitica, we incorporated a bacterial biosurfactant into the swarm agar plates. However the biosurfactant had no effect on either the swarming behavior of the wild type or the lack of swarming motility in the yenI mutant under these conditions (data not shown).

YenI directs the synthesis of long-chain as well as short-chain AHLs. Since neither 3-oxo-C6-HSL nor C6-HSL restored swimming or swarming in the yenI mutant, we examined Y. enterocolitica spent culture supernatants for the presence of additional AHLs using TLC in conjunction with the AHL biosensors E. coli[pSB1075] and C. violaceum CV026, which respond preferentially to long (most sensitively to 3-oxo-C12-HSL) and short-chain AHLs, respectively (26, 49). Spent culture supernatants were extracted with dichloromethane and chromatographed on reverse phase (RP2 or RP18) TLC plates prior to being overlaid with the E. coli[pSB1075] or the C. violaceum biosensor as described in the Materials and Methods. Figure 5A shows that Y. enterocolitica produces multiple long-chain AHLs (lane 2). This activity is absent from the yenI mutant indicating that their synthesis is YenI dependent (lane 3).


Figure 5
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  		FIG. 5. TLC chromatograms of the AHL profiles from supernatant extracts of Y. enterocolitica strains grown at 26°C for 3 h. (A) Using the AHL biosensor E. coli[pSB1075], lane 1, synthetic standards (from bottom) 3.0 x 10–12 mol of 3-oxo-C14-HSL, 3.3 x 10–13 mol of 3-oxo-C12-HSL, and 3.7 x 10–12 mol of 3-oxo-C10-HSL. Lanes 2 to 4, AHLs extracted from Y. enterocolitica, the yenI mutant, and the yenI mutant complemented with pSUyenI, respectively. (B) Using the C. violaceum CV026 AHL biosensor, lane 1, synthetic standard 4.7 x 10–10 mol of 3-oxo-C6-HSL. Lane 2, synthetic standards (from bottom) 4.4 x 10–9 mol of C8-HSL and 5.0 x 10–11 mol of C6-HSL. Lanes 3 to 5, AHLs extracted from Y. enterocolitica, the yenI mutant, and the yenI mutant complemented with pSUyenI, respectively. Migration of AHLs can vary by as much as ±5% compared to synthetic standards.

After transformation of the yenI mutant with either pSU18 or pSUyenI, spent culture supernatant extracts were prepared from each strain grown overnight in LBmops. Figure 5A (lane 4) shows that long-chain AHL synthesis is restored in the yenI complemented mutant.

A similar pattern emerges when the parent and the isogenic yenI mutant and yenI mutant containing pSUyenI are assayed for short-chain AHL synthesis using CV026. Figure 5B (lane 4) shows that the yenI mutant is deficient for short-chain AHLs, the synthesis of which are restored on complementation with pSUyenI (Fig. 5B, lane 5). By comparing the bioluminescence intensities, as determined using the plot facility of the Berthold Luminograph, for the long-chain AHLs with the intensity of the purple spots for the short-chain AHLs and in comparison with synthetic AHL standards at known concentrations, we estimated that the short-chain AHLs are produced in larger amounts than the long-chain molecules at a ratio of approximately 3:1.

To demonstrate that YenI is directly responsible for the synthesis of the new long-chain AHLs, the AHL profile of E. coli expressing yenI was examined after TLC using the E. coli[pSB1075] biosensor. Active spots and streaks were observed which migrated to the same position on RP2 TLC plates as the putative long-chain AHLs produced by the Y. enterocolitica wild type strains (Fig. 5A, lane 4). To establish the identities of the long-chain AHLs generated via YenI, the culture supernatant extracts prepared from E. coli(pYeI7) (46) were fractionated by preparative HPLC. Each fraction was collected and reanalyzed using the E. coli[pSB1075] AHL biosensor and the retention times and spectral properties of components within the active fractions compared with synthetic AHL standards. These data indicated the presence of three compounds, 3-oxo-C10-HSL, 3-oxo-C12-HSL, and 3-oxo-C14-HSL. Their identities were unequivocally confirmed using LC-MS which revealed the presence of AHLs with molecular ions [M+H] of 269, 297, and 325, respectively (Fig. 6). Thus, YenI is responsible for generating at least five different AHLs under the growth conditions employed


Figure 6
Figure 6
Figure 6
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  		FIG. 6. Mass spectra of three long-chain AHLs purified from spent culture supernatants of E. coli JM109 transformed with yenI on pYeI7 which correspond with those of synthetic (A) 3-oxo-C10-HSL ([M+H] 269), (B) 3-oxo-C12-HSL ([M+H] 298), and (C) 3-oxo-C14-HSL ([M+H] 326).

Genetic complementation but not exogenous provision of AHLs restores swimming and swarming motility. 3-Oxo-C10-HSL, 3-oxo-C12-HSL, and 3-oxo-C14-HSL, provided at a range of different concentrations (from 0 to 1 µM), either alone or in combination were examined for their ability to restore swimming and swarming motility. Neither swimming nor swarming motility could be restored to the levels seen in the parent (data not shown). Furthermore, neither the inclusion of short-chain AHLs nor the use of supernatant extracts prepared from the parent Y. enterocolitica strains grown into late exponential/stationary phase restored motility to the yenI mutant. However, complementation of the yenI mutant with pSUyenI but not the backbone vector pSU18 restored swimming on motility agar plates and in broth cultures (Fig. 1B and data not shown). Swarming was also restored to parental levels in the complemented yenI mutant (Fig. 2C).

To determine whether the inability of exogenous AHLs to restore motility in the yenI mutant is a consequence of the inability of the AHLs to gain intracellular access, both the yenI mutant and parent were incubated with tritiated 3-oxo-C12 HSL and the supernatant and cell contents assayed for the radiolabeled AHL. Figure 7A shows that similar levels of the radiolabeled 3-oxo-C12-HSL accumulated in both the yenI mutant and wild type indicating that the Yersinia cell envelope is permeable to AHLs. These data were confirmed by transforming the yenI mutant with the AHL biosensor pSB401 (48) and assayed for light production in the presence of exogenously added 3-oxo-C6 or 3-oxo-C12 HSL. Both AHLs activated the yenI[pSB401] strain, indicating that both long and short-chain AHLs are capable of entering Y. enterocolitica and are functional, as indicated by the activation of LuxR-dependent gene expression (Fig. 7B).


Figure 7
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  		FIG. 7. (A) Uptake of tritiated 3-oxo-C12-HSL by the Y. enterocolitica parent, yenI mutant, and E. coli JM109[pSB401]. (B) Induction of bioluminescence after 8 h in the Y. enterocolitica yenI mutant containing the AHL biosensor pSB401 when supplied with an exogenous source of 3-oxo-C6 HSL or 3-oxo-C12 HSL.


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DISCUSSION
 
Quorum sensing within bacterial populations can promote pathogenesis, symbiosis, cellular dissemination or dispersal, DNA transfer, microbial biofilm development, as well as the production of antibiotics and other secondary metabolites (41). One factor important for colonization and pathogenesis is bacterial surface translocation, which can be achieved by swimming, swarming, or sliding motility. For example, swarming motility in Proteus mirabilis has been implicated in uropathogenesis since mutants unable to swarm cannot ascend from the bladder to the kidney (1). In Y. enterocolitica, flagella have been suggested to play an important role during the early stages of infection. In particular, the expression of flagellar genes in Y. enterocolitica influences invasion of eukaryotic cells, probably by ensuring that the bacterium can migrate to, and initiate host cell contact (52, 53). While swimming and swarming are both flagellum-dependent, sliding is the term used to describe the flagellum-independent migration of bacterial cells over a surface. In several different gram-negative bacteria, swarming (Serratia liquefaciens, Pseudomonas aeruginosa and Burkholderia cepacia (10, 14, 18) sliding (Serratia marcescens; (13) and swimming (Y. pseudotuberculosis, V. fischeri, and Pseudomonas syringae [3, 23, 34]) have each been demonstrated to be regulated, at least in part, by AHL-dependent quorum sensing. Here we show that in Y. enterocolitica both swimming and swarming are regulated by AHL-dependent quorum sensing since mutation of yenI results in the loss of both types of motility.

Compared with the Y. enterocolitica parent strain, swimming motility in the yenI mutant is severely temporally delayed. This contrasts with Y. pseudotuberculosis where mutation of the corresponding AHL synthase (ypsI) results in the early induction of motility where swimming at 22°C is induced after 6 h in the ypsI mutant but delayed for a further 20 h in the parent (3). Quorum sensing is thus an important environmental cue involved in controlling the onset of swimming motility in different Yersinia species but exerts temporally opposite effects. However Y. pseudotuberculosis contains a second LuxI homologue (YtbI) which also impacts on swimming on motility agar (Atkinson, Chang, Buckley, Sockett, Cámara, and Williams, unpublished data). This is because mutation of ytbI in a ypsI genetic background abolishes the early-onset motility phenotype observed in the single ypsI mutant.

Despite exhaustive efforts to clone a second Y. enterocolitica AHL synthase using PCR with primers designed to amplify the ytbI/R locus from Y. pseudotuberculosis or by complementing the AHL biosensor E. coli[pSB401] with both plasmid and cosmid Y. enterocolitica gene libraries, no additional AHL synthase was identified. Analysis of the completed but unpublished genome sequence of Y. enterocolitica strain 8081 (NCTC 13174) suggests that YenI is the only LuxI homologue present (sequence data produced by the Y. enterocolitica Sequencing Group at the Sanger Institute and can be obtained from ftp://ftp.sanger.ac.uk/pub/pathogens/ye/). These data indicate that the quorum sensing-dependent regulation of swimming motility in Y. enterocolitica and Y. pseudotuberculosis is mechanistically distinct since, in the latter, both LuxRI pairs are required for activation and repression of swimming motility on motility plates at 22°C (Atkinson, Chang, Buckley, Sockett, Cámara, and Williams, unpublished data).

MALDI-TOF-MS and the subsequent sequence analysis revealed the identity of the 42-kDa protein to be that of FleB. We believe, in the light of the flagellar protein extractions, sequence data and through a preliminary study of the unfinished first pass annotated Y. enterocolitica genome that FleB rather than FleA, -B, and -C represents the major flagellin in this organism and is homologous with FliC from other species such as E. coli and Salmonella spp.

Since mutation of yenI in Y. enterocolitica severely delays production of flagellar, and hence the onset of motility, it is possible that AHL-dependent quorum sensing controls motility at the level of flhDC since flhDC is essential for motility in Y. enterocolitica (55). However, RT-PCR analysis revealed no obvious differences in flhDC expression after 17 h of growth even though 80% of the parent cells but only 1% of the yenI mutants were motile at this time point. This is also consistent with the presence of the major flagellin protein in the parent but not the mutant after 17 h. Interestingly, even though FlhDC has been reported to be essential for motility in Y. enterocolitica (55), no flhDC transcript was present after 14 h of growth even though ~60% of the cells were motile. This would suggest that sufficient FlhDC protein is present in the stationary-phase cells used as the inoculum to drive flagellar synthesis during the early stages of growth. Additional flhDC expression after 17 h of growth probably reflects a requirement for additional FlhDC protein for continued flagellar synthesis and to facilitate the broader role of FlhDC as a regulator of genes outside of the motility cascade (5, 32, 33). Our data therefore suggest that quorum sensing is acting at a later stage of flagellar synthesis than flhDC.

We therefore used RT-PCR to examine expression of the alternative sigma factor, fliA which is required for class III gene expression. Again we observed no obvious differences in fliA expression when comparing parent and yenI mutant using RT-PCR. Interestingly, fliA transcripts are present in both the Y. enterocolitica parent and mutant after 14 h of growth but absent after 17 h even though the parent is highly motile at this time point. By analogy with E. coli, this is probably because the anti-sigma factor FlgM negates the effects of FliA such that it is only when intracellular levels of FlgM protein fall that FliA is capable of activating the class III genes (25). Thus, in Y. enterocolitica after 14 h, it is possible that once the intracellular levels of FlgM falls the intracellular levels of FliA are sufficient to trigger the class III motility genes without the need for any further fliA transcription.

Since quorum sensing does not regulate swimming motility via flhDC or fliA, we examined transcription of one of the major Y. enterocolitica flagellin structural proteins, fleB after 14 and 17 h. These data revealed that, compared with the parent strain, fleB is expressed at a much lower level after 14 h and is absent after 17 h of growth in the yenI mutant. These data are again consistent with the presence of the major flagellin protein in the parent and its absence in the mutant and suggest that quorum sensing may be acting at both the transcriptional and posttranscriptional levels (since some fleB transcript but not flagellin protein is present at 14 h) Although this finding of quorum-sensing-dependent regulation at the level of fleB was unexpected, there is a possible biological explanation.

Young et al. (54) have shown that the nascent flagellar apparatus can secrete a series of proteins termed Flagella Outer Proteins (FOPS) before the flagellar filament is formed. One of these proteins is YplA, a phospholipase virulence determinant which helps Y. enterocolitica colonize tissues, promotes a more vigorous inflammatory response, and is expressed in a growth phase-dependent manner (36, 37). It is therefore possible that the incomplete flagellar apparatus continues to function as a secretory apparatus for FOPS for a prolonged period in the yenI mutant. Regulation at the level of fleB through AHL-dependent quorum sensing may therefore represent a more efficient mechanism for reciprocally controlling FOP secretion and motility in conjunction with cell population density rather than control via class I or II regulators. Further work will be required to define in more detail the mechanism by which quorum sensing regulates swimming motility in Y. enterocolitica.

In common with swimming, swarming motility also requires an intact flagellum and although the yersiniae are, to our knowledge, the only bacteria in which AHL-dependent quorum sensing has been shown to control swimming, swarming is known to be AHL-dependent in several different gram negatives. In S. liquefaciens, P. aeruginosa, and B. cepacia, mutation of the genes coding for the AHL synthases swrI, rhlI, and cepI, respectively, renders each organism unable to swarm since they are unable to produce the surfactants (e.g., serrawettin in S. liquefaciens and rhamnolipids in P. aeruginosa) necessary for swarm colony development which lower surface tension so reducing friction between the bacterial cells and surfaces. Swarming in these AHL synthase mutants can be restored by genetic complementation, by supplementing the swarm plate with a biosurfactant, or by provision of the cognate AHL(s).

For the Y. enterocolitica yenI mutant, swarming motility could be restored by complementation with a plasmid-borne copy of yenI, which confirms that there is clearly a link between the quorum sensing system and swarming motility. However, the incorporation of a biosurfactant into the swarm agar failed to restore swarming motility which indicates that the inability of yenI mutants to swarm is not just a consequence of the loss of biosurfactant production but also reflects the requirement for an intact flagellum. However, since the Y. enterocolitica yenI mutant exhibits delayed rather than abolished flagellin synthesis and as it fails to swarm after prolonged incubation in the presence of a surfactant, this suggests that quorum sensing in Y. enterocolitica must control genes other than those required for flagellar synthesis and biosurfactant production.

Among pathogenic and nonpathogenic species of yersinia, (3, 40, 47) AHL dependent quorum sensing systems are highly conserved, although the molecular details vary. The three human pathogenic Yersinia spp. all produce 3-oxo-C6-HSL and C6-HSL (3, 40, 46) while Y. pseudotuberculosis also produces N-octanoyl homoserine lactone (C8-HSL). Using a long-chain AHL biosensor followed by HPLC and LC-MS we have now identified three additional AHLs produced via YenI. These are 3-oxo-C10-HSL, 3-oxo-C12-HSL, and 3-oxo-C14-HSL. Thus, Y. enterocolitica produces AHLs with acyl side chains varying in length from six to fourteen carbons. Long-chain AHLs have not previously been identified in the yersiniae. Until recently, the only other gram-negative bacterium reported to produce 3-oxo-C12-HSL was P. aeruginosa (29), although Vibrio vulnificus has also now been shown to produce this quorum-sensing signal molecule (28, 29).

Long-chain AHLs and in particular, 3-oxo-C12-HSL, have been considered to function as a virulence determinants as a consequence of their potent proinflammatory and immune-modulatory activities and capacity for stimulating vasodilatation in isolated blood vessels (20, 38, 45). 3-Oxo-C12-HSL also exerts a profound effect on heart rate, inducing bradycardia in live conscious rats (12). In addition both 3-oxo-C10-HSL and 3-oxo-C14-HSL also exhibit similar albeit reduced immune-modulatory activity (7). In P. aeruginosa, 3-oxo-C12-HSL is considered to play a role not only in regulating virulence gene expression but also in the manipulation of eukaryotic cells and tissues to maximize the provision of nutrients via the bloodstream while down regulating host defense mechanisms. It is therefore conceivable that the long-chain AHLs may contribute directly to Y. enterocolitica pathogenicity by modulating the host response.

Although complementation of the yenI mutant with a plasmid borne copy of the intact gene restores swimming and swarming motility, the provision of long and short AHLs either individually or in combination does not. This finding was unexpected since most AHL synthase mutants respond to exogenously supplied AHLs. For example, swarming in S. liquefaciens swrI and P. aeruginosa rhlI mutants (8, 10) can be restored by provision of the cognate AHL. Furthermore, experiments with radiolabeled AHLs indicate that V. fischeri, E. coli, and P. aeruginosa are all permeable to AHLs (17, 30, 31). By examining the uptake of tritiated 3-oxo-C12-HSL and the response of an AHL biosensor transformed into the yenI mutant, we have shown that exogenous short and long-chain AHLs enter into Y. enterocolitica. Consequently the inability of exogenous AHLs to restore motility in the yenI mutant cannot be due to a lack of intracellular uptake.

Since Y. enterocolitica produces at least five different short and long-chain AHLs, there may be a requirement for the specific AHLs or AHL combinations at specific concentrations and times which were not reproduced in our experiments. Furthermore, for some LuxR proteins, notably TraR, the N-terminal AHL binding site is completely buried within the protein such that prefolded TraR cannot bind to its AHL ligand (N-[3-oxooctanoyl]homoserine lactone; 3-oxo-C8-HSL). Instead, 3-oxo-C8-HSL must be available to assist TraR activation during protein synthesis (56). For Y. enterocolitica, it is therefore possible that the timing of exogenous AHL provision within the yenI swarming and swimming experiments does not provide for the optimal activation of YenR. However, since we have been unable to construct a yenR mutant, the contribution of YenR to swimming and swarming in Y. enterocolitica is currently not known.

Finally, not all AHL synthase mutants respond to exogenous AHLs. For example, a cviI mutant of Chromobacterium violaceum which no longer produces the purple pigment violacein does not respond to the cognate AHL, C6-HSL (26, 44) By subjecting C. violaceum to mutagenesis using two different transposons, an AHL negative mutant (CV026) was obtained which responded to C6-HSL by producing violacein (26). In this strain, the transposon insertions were mapped to the AHL synthase gene cviI and to a repressor which appears to be a regulatory RNA (26, 44). Mutation of the repressor gene alone resulted in copious overproduction of violacein (44). It is therefore possible that a similar mechanism is involved in controlling quorum sensing and hence the response of Y. enterocolitica to exogenous AHLs.


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ACKNOWLEDGMENTS
 
We thank Mavis Daykin, Ram Chhabra, Chris Harty, and Alex Truman for AHL synthesis, Catherine Ortori for the LC-MS analysis, and David Pritchard for the tritiated 3-oxo-C12-HSL.

This work was supported by a grant from the Biotechnology and Biological Sciences Research Council, United Kingdom, which is gratefully acknowledged.


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FOOTNOTES
 
* Corresponding author. Mailing address: Institute of Infections, Immunity and Inflammation, Centre for Biomolecular Science, University of Nottingham, Nottingham NG7 2RD, United Kingdom. Phone: 44-115-9515089. Fax: 44-115-8467951. E-mail: steve.atkinson{at}nottingham.ac.uk. Back


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Journal of Bacteriology, February 2006, p. 1451-1461, Vol. 188, No. 4
0021-9193/06/$08.00+0     doi:10.1128/JB.188.4.1451-1461.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.



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