The biocontrol strain Pseudomonas fluorescens F113 produces the Rhizobium small bacteriocin, N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone, via HdtS, a putative novel N-acylhomoserine lactone synthase

Bridget E. Laue1, Yan Jiang1, Siri Ram Chhabra1, Sinead Jacob2, Gordon S. A. B. Stewartb,1, Andrea Hardman1, J. Allan Downie3, Fergal O’Gara2 and Paul Williams1

School of Pharmaceutical Sciences, University of Nottingham, Nottingham NG7 2RD, UK1
Biomerit Research Centre, Department of Microbiology, National University of Ireland, Cork, Ireland2
John Innes Centre, Colney, Norwich NR4 7UH, UK3

Author for correspondence: Paul Williams. Tel: +44 115 9515047. Fax: +44 115 8466296. e-mail: paul.williams{at}nottingham.ac.uk


   ABSTRACT
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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Several different species of Pseudomonas produce N-acylhomoserine lactones (AHLs), quorum-sensing signal molecules which are involved in the cell-density-dependent control of secondary metabolite and virulence gene expression. When Pseudomonas fluorescens F113 was cross-streaked against AHL biosensors capable of sensitively detecting either short (C4–C8) or long (C10–C14) acyl chain AHLs, no activity was detectable. However, by extracting cell-free stationary-phase culture supernatants with dichloromethane followed by reverse-phase HPLC, three distinct fractions were obtained capable of activating the AHL biosensors. Three AHLs were subsequently characterized using high-resolution MS and chemical synthesis. These were (i) N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone (3OH,C14:1-HSL), a molecule previously known as the Rhizobium leguminosarum small bacteriocin as a consequence of its growth inhibitory properties, (ii) N-decanoylhomoserine lactone (C10-HSL) and (iii) N-hexanoylhomoserine lactone (C6-HSL). A gene (hdtS) capable of directing synthesis of all three P. fluorescens AHLs in Escherichia coli was cloned and sequenced. In vitro transcription/translation of hdtS yielded a protein of approximately 33 kDa capable of directing the synthesis of 3OH,C14:1-HSL, C10-HSL and C6-HSL in E. coli. HdtS does not belong to either of the known AHL synthase families (LuxI or LuxM) and is related to the lysophosphatidic acid acyltransferase family. HdtS may therefore constitute a member of a third protein family capable of AHL biosynthesis.

Keywords: Pseudomonas fluorescens, N-acylhomoserine lactone synthase, Rhizobium, N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone, acyltransferase

Abbreviations: AHL, N-acylhomoserine lactone; DCM, dichloromethane; C6-HSL, N-hexanoylhomoserine lactone; C10-HSL, N-decanoylhomoserine lactone; 3OH,C14:1-HSL, N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone; PDA, photodiode array

The GenBank accession number for the sequence determined in this work is AF286536.

b Deceased.


   INTRODUCTION
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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Apart from direct cell–cell contact, the production of small, diffusible signal molecules constitutes one of the few mechanisms through which bacteria can communicate with each other. The perception of such signal molecules enables an individual bacterial cell to sense ‘self’ or others, and so to adapt to environmental conditions in concert with population density. This cell–cell communication mechanism is now generally referred to as ‘quorum sensing’ and regulates a variety of physiological processes, including bioluminescence, swarming, swimming and twitching motility, antibiotic biosynthesis, biofilm differentiation, conjugation and the production of virulence determinants in plant and animal pathogens (for reviews, see Swift et al., 1996 ; Dunny & Winans, 1999 ; England et al., 1999 ; Williams et al., 2000 ).

Quorum sensing relies upon the interaction of a diffusible signal molecule with either a sensor or a transcriptional regulator protein to couple gene expression with cell population density. Several chemically distinct families of quorum-sensing signal molecules have now been identified, amongst which the N-acylhomoserine lactones (AHLs) have probably been the most intensively investigated (Swift et al., 1996 , 1999 ; Fuqua et al., 1996 ). AHLs are produced by bacteria belonging to many different Gram-negative genera and vary mainly with respect to the length (4–14 carbons) and substituent (H, O or OH) at the 3-position of their N-acyl side chains. Accumulation of the AHL molecule above a threshold concentration, through the activity of a signal generator protein (usually a member of the LuxI family; Moré et al., 1996 ; Schaefer et al., 1996 ; Jiang et al., 1998 ) renders the population quorate. Appropriate target gene(s) are then activated via the action of a member of the LuxR family of transcriptional activators in concert with the AHL signal molecule (Swift et al., 1996 ; Fuqua et al., 1996 ).

Among the species belonging to the genus Pseudomonas, Pseudomonas aeruginosa, Pseudomonas aureofaciens (now Pseudomonas chlororaphis), Pseudomonas fluorescens and Pseudomonas syringae have all been reported to produce AHLs (Bainton et al., 1992 ; Pearson et al., 1994 , 1995 ; Winson et al., 1995 ; Shaw et al., 1997 ; Wood et al., 1997 ). P. aeruginosa, for example, possesses a sophisticated regulatory hierarchy consisting of two separate but linked quorum-sensing circuits (Latifi et al., 1995 , 1996 ; Pesci et al., 1997 ). These are termed the las and rhl circuits, and each possesses a LuxI homologue (LasI or RhlI) and a LuxR homologue (LasR or RhlR). The AHLs that signal within the las and rhl systems are N-(3-oxododecanoyl)-L-homoserine lactone (3O,C12-HSL) and N-butanoyl-L-homoserine lactone (C4-HSL), respectively. Together, the two systems constitute a hierarchical cascade that co-ordinates the production of virulence factors (Latifi et al., 1995 , 1996 ; Pesci et al., 1997 ), the xcp general secretion apparatus (Chapon-Hervé et al., 1997 ), twitching motility (Glessner et al., 1999 ) and stationary-phase genes (via the alternative sigma factor, RpoS; Latifi et al., 1996 ). Although C4-HSL and 3O,C12-HSL have not been found in other pseudomonads, AHLs including N-hexanoyl-homoserine lactone (C6-HSL, P. aureofaciens; Wood et al., 1997 ), N-(3-oxohexanoyl)-L-homoserine lactone (3O,C6-HSL, P. syringae group; Shaw et al., 1997 ; Cha et al., 1998 ) and the 3-hydroxy-forms of N-hexanoyl-, N-octanoyl- and N-decanoyl-homoserine-L-lactones (P. fluorescens; Shaw et al., 1997 ) have been identified as the dominant AHLs produced in some, but not all, strains of each Pseudomonas species tested.

As many root-colonizing fluorescent pseudomonads can efficiently control diseases caused by soil-borne pathogens, there is considerable interest in exploiting their potential as crop protectants (Rainey, 1999 ). Their biocontrol capabilities reside largely in their capacity to produce a range of antifungal secondary metabolites, including hydrogen cyanide, phenazines, pyoluteorin and 2,4-diacetylphloroglucinol. P. aureofaciens 3-84, for example, produces three phenazine antibiotics which suppress take-all disease of wheat by inhibiting the causative agent, Gaeumannomyces graminis var. tritici (Wood et al., 1997 ). Phenazine synthesis is controlled, in part, via the LuxR homologue PhzR and C6-HSL such that mutation of the luxI homologue phzI leads to loss of phenazine production and hence biocontrol activity (Wood et al., 1997 ). Similarly, 2,4-diacetylphloroglucinol production by P. fluorescens F113 inhibits growth of Pythium ultimum in vitro and protects sugar beet seedlings from damping-off disease caused by P. ultimum in soil microcosms (Fenton et al., 1992 ; Shanahan et al., 1992 ). Since 2,4-diacetylphloroglucinol is produced at high cell densities, it is possible that synthesis of this potent antifungal may be under quorum-sensing-dependent control (Delany et al., 2000 ). However, although a gene cluster involved in phloroglucinol synthesis and regulation has been cloned and sequenced from P. fluorescens F113, no luxI or luxR homologues have been identified (Delany et al., 2000 ).

To begin to explore the possibility that AHLs may be involved in regulating the biocontrol properties of P. fluorescens F113, we sought to (i) identify and chemically characterize any AHLs produced and (ii) clone and sequence any P. fluorescens genes involved in AHL biosynthesis. In this paper, we (a) show that F113 makes at least three different AHLs including N-(3-hydroxy-7-cis-tetradecenoyl) homoserine-lactone 3OH,C14:1-HSL), an AHL previously associated exclusively with growth inhibition in Rhizobium leguminosarum, and (b) identify a gene (hdtS) which does not belong to the LuxI or LuxM family of AHL synthases but which, nevertheless, when introduced into Escherichia coli, is capable of directing the synthesis of the P. fluorescens AHLs.


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ABSTRACT
INTRODUCTION
METHODS
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DISCUSSION
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Bacterial strains, plasmids and growth media.
Bacterial strains and plasmids used in this study are listed in Table 1. P. fluorescens F113 or the 2,4 diacetylphloroglucinol-negative Tn5 derivative P. fluorescens F113G22 (Fenton et al., 1992 ) was grown with shaking at 28 °C in sucrose asparagine (SA) medium (Scher & Baker, 1982 ) supplemented with 100 µM FeCl3. E. coli JM109 was grown at 37 °C in LB broth or on LB agar containing, where required, ampicillin (100 µg ml-1). For blue/white screening of recombinants, IPTG and X-Gal were added to a final concentration of 0·1 mM and 40 µg ml-1, respectively.


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Table 1. Bacterial strains and plasmids used in this study

 
AHL reporter plate assays.
To detect AHL production on agar plates, in spent culture supernatants or in HPLC fractions, biosensors (Table 1) which respond to exogenous AHLs by either producing the purple pigment violacein (Chromobacterium violaceum CV026; McLean et al., 1997 ) or by emitting light [E. coli(pSB401) or E. coli(pSB1075); Winson et al., 1998 ] were employed. C. violaceum CV026 responds most sensitively to AHLs with 4–6 carbon acyl side chains irrespective of the substituent at the C-3 position of the acyl chain; E. coli(pSB401) detects compounds with 6–8 carbon side chains and E. coli(pSB1075) is preferentially activated by AHLs with long (10–14 carbon) side chains. Cross-streaks of P. fluorescens or recombinant E. coli against the AHL biosensors were carried out on LB agar as described by Latifi et al. (1995 ) and Swift et al. (1997) . For analysis of spent culture supernatants and HPLC fractions, 3 ml vols molten semi-solid LB agar (0·5%, w/v) were seeded with 100 µl of an overnight culture of the appropriate AHL biosensor and poured immediately over the surface of pre-warmed LB agar plates. Wells were punched in the agar and filled with either spent culture supernatant or HPLC fractions. Synthetic AHL standards were used as positive controls and sterile LB broth as a negative control. After overnight incubation, plates were examined for zones of purple pigmentation (C. violaceum CV026) or for light output [E. coli(pSB401) or E. coli(pSB1075)] using a Berthold LB980 photon video camera.

Rhizobium growth inhibition assay.
Bioassay plates were prepared by overlaying TY agar plates with 3 ml TY soft agar (0·65%, w/v, agar) containing 100 µl of a stationary-phase culture of R. leguminosarum A34 (Rodelas et al., 1999 ). HPLC fractions or synthetic AHLs were added to wells punched into the agar. Synthetic 3OH,C14:1-HSL (1 µg ml-1) was used as a positive control. Plates were incubated at 28 °C for 48 h and examined for zones of growth-inhibitory activity around the wells.

Purification and chemical characterization of AHLs produced by P. fluorescens F113.
Cell-free stationary-phase culture supernatants were extracted with dichloromethane (DCM) using 70:30 supernatant/DCM as described previously (McClean et al., 1997 ; Cámara et al., 1998 ). DCM was removed by rotary evaporation and the residue reconstituted in acetonitrile for fractionation by HPLC using a C8 reverse-phase preparative HPLC column (Kromasil KR100-5C8; 250 mmx8 mm). Fractions were eluted with a linear gradient of acetonitrile in water (20–95%, v/v) over a 30 min period at a flow rate of 2 ml min-1 and monitored at 210 nm. Six fractions (F1–F6) were collected, each covering a 5 min interval, and assayed for activity using the AHL assays described above. Active fractions were pooled and, for short-chain AHLs, rechromatographed using 35% acetonitrile in water, then analysed by TLC on reverse-phase RP-18 F254s plates (BDH) with a solvent system of methanol/water (60:40, v/v), essentially as described previously using C. violaceum CV026 as the indicator organism (McClean et al., 1997 ; Shaw et al., 1997 ). For long-chain AHL molecules, active fractions were rechromatographed using 60% acetonitrile in water and then analysed by TLC on normal-phase silica gel 60 F254 plates (BDH) with a solvent system of acetone/hexane (55:45, v/v). These plates were overlaid with E. coli(pSB1075) as the AHL indicator organism. After overnight incubation of TLC plates at 30 °C, AHLs were located as purple spots on a white background (for C. violaceum CV026) or as bright spots on a dark background (for the E. coli lux-based AHL biosensors) when viewed with a Berthold LB980 photon video camera.

Active samples were also analysed on an analytical HPLC column attached to a photodiode array (PDA) system (Waters 996 PDA system operating with a Millenium 2010 Chromatography Manager), and both retention time and PDA spectral profiles were compared with those of synthetic AHL standards. Following preparative HPLC, the final active subfractions were analysed by HPLC-MS (Micromass Instruments). This technique couples the resolving power of C8 reverse-phase HPLC directly with MS such that the mass of the molecular ion [M+H] and its major component fragments can be determined for a compound with a given retention time. Samples eluting from the HPLC column were ionized by positive ion electrospray (ES)-MS and the spectra obtained were compared with those of the synthetic AHL standard subjected to the same HPLC-MS conditions.

AHL synthesis.
N-Hexanoylhomoserine-L-lactone (C6-HSL), N-(3-oxohexanoyl)-L-homoserine lactone (3O,C6-HSL), N-octanoylhomoserine-L-lactone (C8-HSL), N-decanoyl-L-homoserine lactone (C10-HSL), N-(3-oxotetradecanoyl)-L-homoserine lactone (3O,C14-HSL) and N-(3-hydroxy-7-cis-tetradecenoyl)-L-homoserine lactone (3OH,C14:1-HSL), were synthesized essentially as previously described by Chhabra et al. (1993) . Each synthetic AHL was purified to homogeneity by preparative HPLC and its structure confirmed by MS and proton NMR spectroscopy.

DNA manipulation and sequencing.
DNA was manipulated by standard methods (Sambrook et al., 1989 ). Restriction enzymes (Promega) were used according to the manufacturer’s instructions. Agarose gel electrophoresis and Southern blot transfer were performed essentially as described by Sambrook et al. (1989 ). DNA probes were labelled with digoxigenin and detected using the DIG Luminescent Detection kit supplied by Boehringer Mannheim. Oligonucleotide synthesis and DNA sequencing were performed at the Biopolymer Synthesis and Analysis Unit, University of Nottingham, Queen’s Medical Centre, Nottingham. Automated non-radioactive sequencing reactions were carried out using the BigDye terminator cycle sequencing kit in conjunction with a 373A automated sequencer (Perkin Elmer Applied Biosystems).

Screening of a P. fluorescens genomic library for AHL synthases.
The method described by Swift et al. (1993) and by Throup et al. (1995 ) for cloning luxI homologues by complementation of AHL biosensor strains was used. A genomic P. fluorescens library of random BamHI/PstI fragments was constructed in pBluescript II SK(+) (Stratagene) and introduced by electroporation into E. coli JM109(pSB401). The resulting clones were examined for light output using the Berthold LB980 photon video camera.

Identification of the hdtS gene product.
The hdtS gene was amplified with Taq DNA polymerase (Promega) using pBL59 as a template with the primers 5'-GATGTCGATATTGCAGGCCATC and 5'-CTCAAACAGCGAGCTGGTCGG. The amplified DNA fragment (782 bp) was cloned into the blunt-ended vector pT7Blue-2 as described by the supplier (Novagen). The recombinant pT7Blue-2 plasmid containing hdtS was termed pBLYJ3.1. E. coli JM109 was transformed by electroporation and the transformants were grown and screened on X-Gal/IPTG indicator plates containing ampicillin. To identify the hdtS gene product, the STP3-Biotin non-radioactive transcription/translation kit (Novagen) was used. Prior to performing an in vitro transcription/translation assay, a DNA template incorporating the T7 promoter and the hdtS insert with primers R-20mer and U-19mer (Novagen) was generated from pBLYJ3.1 by PCR. The in-vitro-generated biotinylated translation products were analysed by SDS-PAGE and Western blotting. Western blots were developed with a Streptavidin AP LumiBlot kit (Novagen).

DNA and protein sequence analysis.
The GenBank database (release 116) was searched using the BLAST suite of programs (Altschul et al., 1997 ). Sequences were extracted from GenBank using the ACNUC retrieval software (Gouy et al., 1985 ). Sequences were aligned and compared using CLUSTAL W (Thompson et al., 1994 ).


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ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
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Chemical characterization of AHLs produced by P. fluorescens F113: C6-HSL and C10-HSL
When P. fluorescens F113 was cross-streaked on agar plates against either C. violaceum CV026 or the lux-based E. coli AHL biosensors, neither stimulation of violacein production nor bioluminescence was observed. Similarly, no activity was observed when spent culture supernatants were added to wells cut into agar and overlaid with an AHL biosensor (data not shown). Since this may have been due to the production of secondary metabolites such as 2,4-diacetylphloroglucinol, we also examined the response of the AHL biosensors to P. fluorescens F113G22, which carries a Tn5 insertion in genes determining 2,4-diacetylphloroglucinol biosynthesis (Fenton et al., 1992 ). Again, no stimulation of any of the AHL biosensors was observed. Since this may have been due to other secondary metabolites which are toxic for the biosensors, we sought to separate any AHLs produced by DCM extraction of cell-free culture supernatants followed by HPLC.

The crude F113G22 DCM extract was separated into six fractions (F1–F6) by preparative reverse-phase HPLC using a linear gradient of acetonitrile in water as described in Methods and by Cámara et al. (1998) . When assayed by TLC overlaid with C. violaceum CV026, fraction F3 stimulated violacein production and contained a compound with an RF value similar to that of the synthetic C6-HSL standard. Further subfractionation of F3 using an isocratic mobile phase of 35% acetonitrile in water yielded a compound which eluted with the same retention time (17 min) and PDA spectrum as synthetic C6-HSL [Fig. 1(a) and data not shown]. To unequivocally confirm the identity of this AHL, F3 was subjected to HPLC-MS. The ES-MS spectrum obtained revealed the presence of a molecular ion [M+H] of m/z 200 and a profile of breakdown products including the [M+H] 102 fragment (characteristic of the HSL moeity), indistinguishable from the synthetic C6-HSL standard (data not shown).



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Fig. 1. Structures of (a) N-hexanoyl-L-homoserine lactone (C6-HSL), (b) N-decanoyl-L-homoserine lactone (C10-HSL), (c) N-(3-oxotetradecanoyl)-L-homoserine lactone (3O,C14-HSL), and (d) N-(3-hydroxytetradecenoyl)-L-homoserine lactone (3OH,C14:1-HSL).

 
Although F5 did not stimulate C. violaceum CV026 to produce pigment, it was able to stimulate light emission from E. coli(pSB1075). This AHL biosensor preferentially responds to AHLs with acyl side chains of 10–14 carbons in length. Thus the results suggest that P. fluorescens F113 makes AHLs of short and long chain lengths. Subfractionation of F5 yielded a compound which eluted in 70% acetonitrile in water with a retention time of 8·5 min and possessed a PDA spectrum identical to that of C10-HSL. The HPLC-mass spectrum of the compound present in F5 revealed a molecular ion [M+H] of m/z 256 (Fig. 2a) and is identical to that of synthetic C10-HSL (Figs 2b, 1b).



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Fig. 2. HPLC-mass spectra of the natural C10-HSL isolated from P. fluorescens F113G22 supernatants (a) and synthetic C10-HSL (b) recorded under positive ion mode atmospheric pressure ionization using (above) low cone voltage (~15 kV) showing m/z at 256 for [M+H] and (below) high cone voltage (~25 kV) showing the expected fragmentation products, for example m/z 155 (C10 acyl side chain) and 102 (HSL-moiety).

 
P. fluorescens F113 produces 3OH,C14:1-HSL, the small bacteriocin of R. leguminosarum
HPLC subfractionation of fraction F5 using different acetonitrile gradients also yielded a peak of activity with a different retention time and PDA spectrum from that of C10-HSL. Using a mobile phase of 60% acetonitrile in water, this compound eluted with a retention time of approximately 13 min. The HPLC-mass spectrum of this compound revealed a molecular ion [M+H] of m/z 326 and the characteristic fragmentation products of m/z 308, 225 and 102 (Fig. 3). This pattern of fragmentation is consistent with the presence of a homoserine lactone ring (m/z102) and a C14 acyl chain (m/z 225), suggesting that this compound could be either N-(3-oxotetradecanoyl)-L-homoserine lactone (3O,C14-HSL; Fig. 1c) or N-(3-hydroxytetradecenoyl)-L-homoserine lactone (3OH,C14:1-HSL; Fig. 1d). The [M+H] 308 fragment, however, indicates that it is likely to be the latter compound since hydroxylated AHLs characteristically lose water during MS analysis (Shaw et al., 1997 ). These data therefore suggested that this P. fluorescens AHL is related to N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone (3OH,C14:1-HSL), the small bacteriocin of R. leguminosarum (Gray et al., 1996 , Schripsema et al., 1996 ). To determine whether this P. fluorescens AHL was indeed the Rhizobium small bacteriocin, we synthesized both 3OH,C14:1-HSL and 3O,C14-HSL. Both the natural and synthetic 3OH,C14:1-HSL compounds, but not 3O,C14-HSL (or C10-HSL or C6-HSL), inhibited the growth of the small-sensitive R. leguminosarum strain A34, which harbours the sym plasmid pRL1JI (Rodelas et al., 1999 ). In addition, the chromatographic properties and both the PDA spectrum (data not shown) and HPLC-mass spectrum (Fig. 3b) of the molecule isolated from P. fluorescens were indistinguishable from that of the synthetic 3OH,C14:1-HSL. Based on these properties, the position and stereochemistry of the double bond is tentatively assigned to be 7Z, i.e. the P. fluorescens molecule has a 7–8 cis double bond, thus validating that the P. fluorescens molecule is N-(3-hydroxy-7-cis-tetradecenoyl)homoserine lactone (Fig. 1d).



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Fig. 3. HPLC-mass spectra of the natural 3OH,C14:1-HSL isolated from P. fluorescens F113G22 supernatants (a) and synthetic 3OH,C14:1-HSL (b) recorded under positive ion mode atmospheric pressure ionization using (above) low cone voltage (~15 kV) showing m/z at 325·8 for [M+H] and (below) high cone voltage (~25 kV) showing the expected fragmentation products, for example m/z 308 [M-H2O], 225 [14-C acyl side chain], 207 [side chain-H2O] and 102 [HSL moiety].

 
Identification of a novel genetic locus responsible for AHL production in P. fluorescens
To identify the gene(s) responsible for AHL production in P. fluorescens F113, we used a previously described strategy for the isolation of genes, the products of which belong to the LuxI family of AHL synthases. (Swift et al., 1993 ; Throup et al., 1995 ). The basis of selection is the in trans complementation of an E. coli AHL biosensor deleted for luxI such as pSB401, which is based on the Vibrio(Photobacterium) fischeri luxR::luxI'luxCDABE (Throup et al., 1995 ; Winson et al., 1998 ). A BamHI/PstI digest of P. fluorescens F113 chromosomal DNA was cloned into similarly digested pBluescript II SK(+). The ligation was used to transform E. coli JM109(pSB401). A number of bioluminescent colonies were obtained and the brightest was selected for further analysis. This clone, designated pBL59, contained a 2·4 kb DNA insert. DCM extracts of spent culture supernatants and HPLC fractions F3 and F5 of E. coli(pBL59) were subjected to both reverse-phase (RP18) and normal-phase (silica) TLC as described in Methods and overlaid with either C. violaceum or E. coli(pSB1075), respectively. The results obtained showed that pBL59 directs the synthesis of C6-HSL and a compound(s) migrating with a similar RF value to that of synthetic C10-HSL and 3OH,C14:1-HSL standards (Fig. 4). These data confirm that pBL59 contains a gene capable of directing AHL synthesis.



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Fig. 4. Thin layer chromatograms of the AHLs purified from spent culture supernatants by HPLC as described in Methods from E. coli JM109(pBL59) and E. coli JM109(pBLYJ3.1). In (a), the silica TLC plate was overlaid after chromatography with the long-chain AHL biosensor E. coli(pSB1075) and in (b) a reverse-phase RP18 plate was overlaid after chromatography with the short-chain AHL biosensor C. violaceum CV026. In (a) bioluminescence was detected using a Berthold photon video imaging camera and in (b) a purple spot is indicative of the AHL-mediated activation of violacein production. (a) Lanes: 1, C10-HSL; 2, 3OH,C14:1-HSL; 3, crude DCM extract from E. coli JM109(pBL59); 4, HPLC fraction F5 from JM109(pBL59); 5, crude DCM extract from E. coli JM109(pBLYJ3.1); 6, HPLC fraction F5 from JM109(pBLYJ3.1). (b) Lanes: 1, 3O,C6-HSL plus C8-HSL; 2, C6-HSL; 3, HPLC fraction F3 from JM109(pBL59); 4, HPLC fraction F3 from E. coli JM109(pBLYJ3.1). The direction of solvent migration is marked with an arrow.

 
DNA sequence analysis of the pBL59 2·4 kb insert revealed two complete and two partial ORFs (Fig. 5a). ORF1, which is incomplete at the 5' end, is a homologue of glyS, which encodes glycine tRNA synthetase ß-subunit and is 43% identical to the E. coli K-12 GlyS protein (GenBank accession no. AE000433). Database sequence comparisons of ORF4, which is also 5'-incomplete, revealed that the gene product exhibits some similarity to the histidine kinase sensor proteins of two-component response regulator systems. However, since both ORF1 and ORF4 are incomplete, the AHL synthase was presumed to be either ORF2 or ORF3. Surprisingly, database sequence analysis indicated that neither ORF was a member of the established LuxI or LuxM families of AHL synthases. The product of ORF2 is related to the imidazoleglycerol-phosphate dehydratase/histidinol-phosphatases, as encoded by hisB genes found in Synechocystis (GenBank accession no. D64004), Haemophilus influenzae (U32744) and E. coli (hisB, AE000293 and yaeD, AE000129). Database analysis of ORF3 indicated that the product of this gene exhibits similarity to numerous entries indicated to encode lysophosphatidic acid acyltransferases (or 1-acyl-sn-glycerol-3-phosphate acyltransferases). ORF3 is 32% identical to the Neisseria meningitidis nlaBgene product and 34% identical to the translated Rickettsia prowazekii gene plsC. The plsC name has been allocated to the R. prowazekii gene via the genome project on the basis of its similarity to the E. coli plsC, but since the similarity to nlaB is higher, the R. prowazekii gene is probably not the orthologue of plsC. Thus, the P. fluorescens ORF3 shares only about 20% identity with the E. coli plsC gene product.



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Fig. 5. (a) Map of the 2·4 kb P. fluorescens F113 DNA insert in pBL59. The complete (ORF2 and ORF3) and partial (ORF1 and ORF4) ORFs corresponding to the glyS homologue (ORF1), hisB homologue (ORF2), hdtS (ORF3) and the sensor kinase homologue (ORF4) are shown as open arrows. The sizes of the two complete genes are shown above the arrows. The dotted arrows indicate the positions of the regions deleted to construct pBL70 (deletion 1, which removed partial ORF1 and 277 bp of the 5' end of ORF2) and pBL71 (deletion 2, which removed 145 bp of the 3' end of ORF3 and partial ORF4). (b) Light emission from E. coli(pSB401) transformed with pBL59 (1), pBL70 (2), pBluescript II SK(+) (3) or pBL71 (4) and patch-streaked onto L agar plates. Bioluminescence was recorded after overnight growth using a Berthold LB980 photon video camera. Only E. coli(pSB401)(pBL59) and E. coli(pSB401)(pBL70) are strongly bioluminescent.

 
To determine which of these ORFs was involved in directing AHL biosynthesis, two deletion mutations were constructed in pBL59 (Fig. 5a). In the first, 277 bp of the 5' end of ORF2 was deleted together with partial ORF1. This plasmid (termed pBL70) when transformed into E. coli(pSB401) retained its ability to induce light emission (Fig. 5b). In contrast, a deletion of the 3' end of the 2·4 kb insert which removed 145 bp of ORF3 together with partial ORF4 (plasmid pBL71; Fig. 5a) disrupted light output, i.e. no bioluminescence was observed when pBL71 was transformed into E. coli(pSB401) (Fig. 5b). These data implied that the AHL synthase activity resided within ORF3, which we termed hdtS (see Discussion).

HdtS directs the synthesis of C6-HSL, C10-HSL and 3OH,C14:1-HSL in E. coli
To identify the hdtS gene product and to determine whether HdtS could direct the synthesis of the P. fluorescens AHLs, the gene was amplified from pBL59 by PCR and cloned into the expression plasmid pT7Blue-2, to give pBLYJ3.1. Using a non-radioactive transcription/translation assay, the HtdS protein was identified after SDS-PAGE and Western blotting as a protein of ~33 kDa (Fig. 6), which is in good agreement with the predicted size of the encoded protein.



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Fig. 6. Western blot analysis of the hdtS gene product by in vitro transcription/translation as described in Methods. Lanes: 1, positive control template, ß-galactosidase (119 kDa); 2 and 3, negative control (no DNA template); 4 and 5, pBLYJ3.1 as the DNA template. The ~33 kDa hdtS product is clearly visible in lanes 4 and 5. Lane 6 shows the molecular mass markers.

 
To determine which AHL(s) was produced by E. coli JM109(pBLYJ3.1), stationary-phase cell-free culture supernatants were extracted with DCM, subjected to either reverse-phase or normal-phase TLC, and then overlaid with C. violaceum CV026 or E. coli(pSB1075), respectively, as described in Methods. Fig. 4 shows that HdtS can direct the synthesis of both short-chain and long-chain AHLs. A compound migrating with the same RF value as C6-HSL could be clearly observed on the CV026 TLC overlay (Fig. 4b). On normal-phase silica TLC plates, at least one active spot capable of activating pSB1075 is clearly apparent (Fig. 4a). We therefore subjected E. coli JM109(pBLYJ3.1) supernatants to DCM extraction, HPLC, HPLC-MS and the Rhizobium small bacteriocin assay. Three compounds with molecular ions [M+H] of m/z 200, 256 and 326, corresponding, respectively, to C6-HSL, C10-HSL and 3OH,C14:1-HSL, were clearly identified, indicating that HdtS directed synthesis of each of the three P. fluorescens AHLs. None of these compounds was present in spent supernatants from E. coli JM109(pT7Blue-2). Furthermore, the compound identified as 3OH,C14:1-HSL inhibited the growth of the small-bacteriocin-sensitive strain of R. leguminosarum A34, but did not inhibit the growth of a derivative of A34 known to be resistant to small bacteriocin, as would be expected for growth inhibition by 3OH,C14:1-HSL (data not shown).


   DISCUSSION
TOP
ABSTRACT
INTRODUCTION
METHODS
RESULTS
DISCUSSION
REFERENCES
 
In this study, we have shown that P. fluorescens F113 produces at least three different AHLs. Although two of these compounds, namely C6-HSL and 3OH,C14:1-HSL, are known to be produced by other Gram-negative bacteria, C10-HSL has not previously been found as a naturally occurring AHL. AHLs with a C10 acyl side chain, namely N-(3-hydroxydecanoyl)homoserine lactone (3OH,C10-HSL) and N-(3-oxodecanoyl)homoserine lactone (3O,C10-HSL), have been unequivocally chemically characterized in P. fluorescens 2-79 (Shaw et al., 1997 ) and in the fish pathogen Vibrio (now Listonella) anguillarum (Milton et al., 1997 ), respectively. A compound migrating on TLC plates with a similar RFvalue to that of 3O,C10-HSL has also been reported to occur in the Agrobacterium tumefaciens strain Bo542 (Cha et al., 1998 ). In contrast, C6-HSL is produced by a number of different Gram-negative genera, including Aeromonas (Swift et al., 1997 ), Chromobacterium (McClean et al., 1997 ), Pseudomonas (Winson et al., 1995 ), Rhizobium (Rodelas et al., 1999 ), Serratia (Eberl et al., 1996 ), Vibrio (Kuo et al., 1994 ) and Yersinia (Throup et al., 1995 ; Atkinson et al., 1999 ). Interestingly, none of the three AHLs made by P. fluorescens F113 and described in the present paper have yet been identified in other P. fluorescens strains. Shaw et al. (1997) reported that P. fluorescens 2-79 produced up to five putative AHL signal molecules, three of which were identified as N-(3-hydroxyhexanoyl)homoserine lactone (3OH,C6-HSL), N-(3-hydroxyoctanoyl)homoserine lactone (3OH,C8-HSL) and 3OH,C10-HSL respectively. However, no AHLs were detected in the culture supernatants of either P. fluorescens 1855.344 (Cha et al., 1998 ) or P. fluorescens NCIMB 10586 (Holden et al., 1999 ). Given the differences in AHL profiles of the P. fluorescens strains examined to date, it will clearly be of interest to determine whether there is any correlation between AHL profile and strain habitat. In this context, the discovery that P. fluorescens F113 synthesizes 3OH,C14:1-HSL, a molecule which, prior to its chemical characterization, was known as the Rhizobium small bacteriocin (Hirsch, 1979 ; Wijffelman et al., 1983 ) is particularly interesting. Small is produced by most strains of R. leguminosarum and was considered to be a bacteriocin because it produces zones of growth inhibition when assayed using a sensitive strain of R. leguminosarum as a lawn in an agar plate (Hirsch, 1979 ; Wijffelman et al., 1983 ).

AHL-mediated inhibition of bacterial growth is unusual and Gray et al. (1996) concluded that in R. leguminosarum, 3OH,C14:1-HSL converted exponentially growing cells into stationary-phase cells, arresting further growth even though the cell density remained low. Subsequently, Thorne & Williams (1999) showed that R. leguminosarum cells which enter stationary phase at low cell density (e.g. due to carbon limitation) do not have the same prolonged survival characteristics as those that enter stationary phase at high cell density. However, addition of exogenous 3OH,C14:1-HSL induced long-term survival characteristics in cells of R. leguminosarum that entered stationary phase at a low cell density (Thorne & Williams, 1999 ). The addition of AHL-containing spent culture supernatants from the 2,4-diacetylphloroglucinol-negative mutant F113G22 to the wild-type F113 strain had no effect on growth or on 2,4-diacetylphloroglucinol production (data not shown). Whether 3OH,C14:1-HSL produced by P. fluorescens is employed to control growth of Rhizobium (or other bacteria) within the soil or rhizosphere environment is not yet known. Interestingly, the sensitivity of R. leguminosarum to 3OH,C14:1-HSL, but not its synthesis, is mediated by a genetic locus on Sym plasmids such as pRL1JI since strains cured of the plasmid are not susceptible to growth inhibition (Hirsch 1979 ; Wijffelman et al., 1983 ; Gray et al., 1996 ).

Using a functional complementation assay which has been successfully employed to clone members of the LuxI family of AHL synthases from many different Gram-negative bacteria (Atkinson et al., 1999 ; Eberl et al., 1996 ; Milton et al., 1997 ; Swift et al., 1993 , 1997 ; Throup et al., 1995 ), we identified a P. fluorescens F113 gene termed hdtS which, when introduced into E. coli, results in the synthesis of the same AHLs as those produced by P. fluorescens. HdtS is clearly neither a member of the LuxI family nor a member of the LuxM family of AHL synthases and was so called since it directs the synthesis of AHLs with acyl side chains of six (hexa-), ten (deca-) and fourteen (tetradeca-) carbons in length. Thus, in common with the LuxI protein family, HdtS is capable of directing the synthesis of more than one AHL. LuxI homologues, such as RhlI from P. aeruginosa (Winson et al., 1995 ) and AhyI from A. hydrophila (Swift et al., 1997 ), in both homologous and heterologous (E. coli) genetic backgrounds, produce both a major (N-butanoylhomoserine lactone; C4-HSL) and a minor (C6-HSL) AHL. Other homologues, such as YenI and YpsI from Yersinia enterocolitica (Throup et al., 1995 ) and Yersinia pseudotuberculosis (Atkinson et al., 1999 ), respectively, produce a 50:50 mixture of C6-HSL and 3O,C6-HSL, whilst RhiI from R. leguminosarum makes four AHLs, two of which have been identified as C6-HSL and C8-HSL (Rodelas et al., 1999 ). Furthermore, 3OH,C14:1-HSL in R. leguminosarum and the closely related N-(7-cis-tetradecenoyl)homoserine lactone (C14:1-HSL) in Rhodobacter sphaeroides are also synthesized via LuxI homologues, in this case CinI (Lithgow et al., 2000 ) and CerI (Puskas et al., 1997 ), respectively.

Apart from the LuxI family of AHL synthases, of which there are now over 20 homologues in the sequence databases, a second type of AHL synthase has been identified in both Vibrio harveyi (Bassler et al., 1993 ) and V. fischeri (Gilson et al., 1995 ). These have been termed LuxLM and AinS, respectively, and are responsible for the synthesis of N-(3-hydroxybutanoyl)homoserine lactone (3OH,C4-HSL) and C8-HSL, respectively. Interestingly, both AHL synthase families employ S-adenosylmethionine (SAM) as the source of the homoserine lactone moiety, whilst the acyl chain is supplied by either the appropriately charged acyl-acyl carrier protein (acyl-ACP) or acyl-coenzyme A (acyl-CoA) (Hanzelka & Greenberg, 1996 ; Jiang et al., 1998 ; Moré et al., 1996 ; Schaefer et al., 1996 ; Val & Cronan, 1998 ). For RhlI (Jiang et al., 1998 ; Parsek et al., 1999 ) and AinS (Hanzelka et al., 1999 ), butanoyl-CoA and octanoyl-CoA, respectively, have also been shown to be capable of supplying the acyl side chain. For both AHL synthases, it is probable that SAM binds to the enzyme followed by the acyl-ACP (or acyl-CoA), such that an amide bond is formed between SAM and the acyl group, followed by cyclization of the acyl-SAM and then the release of the AHL and 5'-methylthioadenosine (Moré et al., 1996 ; Hanzelka et al., 1999 ; Parsek et al., 1999 ).

In this study, we have described the identification and characterization of HdtS, a putative third class of AHL synthases. Database comparisons suggest that HdtS is most closely related to the lysophosphatidic acid (LPA) acyltransferase family, which includes NlaB from N. meningitidis (Shih et al., 1999 ) and PlsC from E. coli (Rock et al., 1996 ). However, neither of these Gram-negative bacteria have been shown to produce any AHLs (Swift et al., 1999 ). In E. coli, the LPA acyltransferase, PlsC, catalyses the transfer of an acyl chain from either acyl-CoA or acyl-ACP onto LPA to produce phosphatidic acid, which, like LPA, is a critical phospholipid intermediate in cell membrane biosynthesis. In this respect, HdtS possesses two motifs, NHQS and PEGTR, which are highly conserved in prokaryotic and in eukaryotic LPA acyltransferases (West et al., 1997 ). These may constitute an acyl-CoA/acyl-ACP binding site, although amino acid residues outside these motifs have been identified which alter the activity or specificity of these acyltransferases (Shih et al., 1999 ). It is therefore possible that HdtS is an acyltransferase which transfers acyl chains onto a substrate such as SAM to generate AHLs. However, it is also possible that HdtS is involved in the synthesis of substrates required for AHL formation via another enzyme. Further in vitro work using the purified HdtS protein will therefore be required to define its enzymic function and substrate specificity. In addition, the target P. fluorescens structural gene(s) whose expression is controlled via the AHLs identified in this paper, and which may also influence the biocontrol characteristics of P. fluorescens F113, await the construction of an hdtS-negative mutant.


   ACKNOWLEDGEMENTS
 
We thank Mavis Daykin and Catherine Ortori (School of Pharmaceutical Sciences, University of Nottingham) for their assistance with the HPLC and MS, respectively. We are grateful to Paul Sharp (Institute of Genetics, University of Nottingham) for analysis of the gene sequences. This work was supported by a contract from the European Union (BIO4-CT96-0181) and by a grant from the Biotechnology and Biological Sciences Research Council (UK).


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Received 11 April 2000; revised 20 July 2000; accepted 27 July 2000.