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
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ABSTRACT |
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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.
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INTRODUCTION |
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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 (414 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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METHODS |
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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 (2095%, v/v) over a 30 min period at a flow rate of 2 ml min-1 and monitored at 210 nm. Six fractions (F1F6) 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 manufacturers 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, Queens 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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RESULTS |
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The crude F113G22 DCM extract was separated into six fractions (F1F6) 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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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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DISCUSSION |
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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.
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ACKNOWLEDGEMENTS |
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Received 11 April 2000;
revised 20 July 2000;
accepted 27 July 2000.