BIOMERIT Research Centre, Microbiology Department and Biosciences Institute, National University of Ireland, Cork, Ireland
Correspondence
Fergal O'Gara
f.ogara{at}ucc.ie
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession numbers for the genes reported in this paper are: pasA, DQ088968; olsA, AY876048; patB, AY876049; hdtS, AF286536.
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INTRODUCTION |
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Phospholipid biosynthesis has been most intensively studied in Escherichia coli, which has been a model for studying the genetic and biochemical aspects of lipid metabolism for over 40 years. E. coli has a relatively simple phospholipid composition, consisting mainly of phosphatidylethanolamine (PE), phosphatidylglycerol (PG) and cardiolipin (CL) (DiRusso et al., 1999). In this bacterium, the outer membrane consists of approximately 25 % dry weight phospholipid, while the inner membrane is composed of approximately 40 % (DiRusso et al., 1999
). Phospholipid biosynthesis occurs primarily by de novo synthesis on the inner membrane of the cell envelope using glycerol 3-phosphate (G3P) and fatty acids as the main substrates (Cronan & Rock, 1996
). Sequentially, two acyltransferase reactions transfer acyl groups from acyl-acyl carrier protein (acyl-ACP) or acyl-coenzyme A (acyl-CoA) to G3P. The first acyltransferase reaction results in the transfer of an acyl group to the sn-1 position of G3P to produce lysophosphatidic acid (LPA), and in E. coli this reaction is catalysed by the enzyme encoded by the plsB gene (Larson et al., 1980
; Lightner et al., 1983
; Rock & Cronan, 1981
). The second step transfers an acyl group to the sn-2 position of LPA to produce phosphatidic acid (PA) and is catalysed by the enzyme LPA acyltransferase, encoded by plsC in E. coli (Coleman, 1990
). PA is then converted to PE, PG and CL. Variation in the membrane phospholipid composition comes from both the diversity of phospholipid head groups, and from differences in the chain length and saturation of the acyl groups in the sn-1 and sn-2 positions. When grown at 37 °C, the phospholipids of E. coli contain mainly saturated fatty acids at the sn-1 position (C16 : 0) and unsaturated fatty acid chains (C16 : 1 or C18 : 1) at the sn-2 position (Cronan & Rock, 1996
). It has been proposed that the different substrate specificities of G3P acyltransferase (PlsB) and LPA acyltransferase (PlsC) partly account for the acyl chain distribution on the glycerol backbone of membrane phospholipids (Cronan & Rock, 1987
). Although in E. coli, a single essential gene, plsC, encodes LPA acyltransferase, in Neisseria meningitidis, two LPA acyltransferases have been identified and characterized (Shih et al., 1999
; Swartley et al., 1995
). Designated NlaA and NlaB, both proteins share homology to PlsC, possess specific LPA acyltransferase activity in vitro, and can complement a temperature-sensitive E. coli plsC mutant (Shih et al., 1999
; Swartley et al., 1995
). It has been proposed that N. meningitidis possesses more than one LPA acyltransferase to facilitate a greater diversity of membrane phospholipids (Shih et al., 1999
). Based on these findings and the importance of phospholipid structure and composition to environmental adaptation, we have investigated the possible occurrence of multiple LPA acyltransferases in P. fluorescens F113. This strain of P. fluorescens has potential as a biocontrol agent and is capable of inhibiting the growth of Pythium ultimum in vitro and in natural ecosystems (Fenton et al., 1992
; Shanahan et al., 1992
). In this study, three putative LPA acyltransferases, termed hdtS, olsA and patB, were identified from P. fluorescens F113 and characterized. These genes were localized to different regions in the P. fluorescens F113 chromosome and all shared homology with E. coli PlsC. Analysis of these genes showed they possessed different but related functions. We discuss the implications of these data for the environmental adaptability of P. fluorescens.
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METHODS |
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Cloning of P. fluorescens F113 olsA and patB.
Using online databases, the region surrounding the olsA gene in several Pseudomonas species was analysed and, based on sequence similarity, primers MC12F and MC12R were designed. Following PCR analysis on P. fluorescens F113 genomic DNA, the resulting 2·7 kb PCR product was gel-purified and introduced into pCR2.1TOPO using the TA cloning kit. The PCR product was subsequently sequenced using primers M13F, M13R, MC12F, MC12R, NlaF, NlaR, Nla2F and Nla2R to obtain the full sequence of olsA and the upstream gene. The GenBank accession number for olsA is AY876048. Cloning of the patB gene was carried out as follows: based on sequence similarity, primers MC26F and MC26R were designed to amplify the region containing the putative patB gene. The resulting 740 bp PCR product was gel purified and introduced into pCR2.1TOPO using the TA cloning kit to form plasmid pCR2.1patB. The patB ORF was subsequently sequenced using the M13F and M13R primer pair. The GenBank accession number for patB is AY876049.
Plasmid constructions.
To construct plasmid pBBR1MCShdtS, an 815 bp region surrounding the hdtS ORF was amplified using primers MC15F and MC7R. Primer MC15F contains an artificial ribosome-binding site at the 5' end while MC7R contains an Asp718 site. The resultant PCR product was cloned into the pCR2.1TOPO vector to form pCR2.1hdtS, retrieved by digestion with EcoRV and Asp718, and cloned into EcoRV/Asp718-cut pBBR1MCS. Plasmid pBBR1MCSolsA was obtained by subcloning the olsA gene into the multiple cloning site of pBBR1MCS as follows: the olsA ORF was PCR-amplified from P. fluorescens F113 DNA using primers MCER and MCKF, which contain EcoRV and Asp718 restriction enzyme sites, respectively. The 1·05 kb PCR product obtained was cloned into the pCR2.1TOPO vector to form pCR2.1olsA, retrieved by digestion with EcoRV and Asp718, and cloned into EcoRV/Asp718-cut pBBR1MCS. To construct plasmid pBBR1MCSpatB, a 733 bp region surrounding the patB ORF was amplified using primers MC32F and MC32R, which contain EcoRV and Asp718 restriction enzyme sites, respectively. Primer MC32F also contains an artificial ribosome-binding site at the 5' end. The resultant PCR product was digested with the appropriate enzyme and cloned into EcoRV/Asp718-cut pBBR1MCS. pBBR1MCSplsC was obtained by subcloning the plsC gene from E. coli using primers MC22F and MC22R. The resultant 844 bp PCR product was cloned into the pCR2.1TOPO vector, retrieved by digestion with EcoRV and Asp718, and cloned into EcoRV/Asp718-cut pBBR1MCS.
P. fluorescens F113 mutant constructions.
The P. fluorescens F113 hdtS mutant was obtained as follows: a 397 bp fragment of the hdtS ORF was PCR-amplified using primers MC9F and MC9R, which contain XbaI and PstI restriction enzyme sites, respectively. The PCR product was cloned into XbaI/PstI-cut pK18mob to form pK18mobhdtS and the vector introduced into P. fluorescens F113 by triparental mating, selecting for a single crossover event. The P. fluorescens F113 patB mutant was obtained as follows: a 402 bp fragment of the patB ORF was PCR-amplified using primers MC30F and MC30R. The PCR product obtained was cloned into the pCR2.1TOPO vector and excised with EcoRI. The resultant fragment was cloned into alkaline phosphatase (Promega)-treated EcoRI-cut pK18mob to form pK18mobpatB and the vector introduced into P. fluorescens F113 by triparental mating, selecting for a single crossover event. All single mutants were complemented via introduction of the appropriate gene in trans.
Fatty acid methyl ester (FAME) analysis.
Alterations in the fatty acid compositions of the membrane phospholipids from P. fluorescens F113 and associated mutants were measured via FAME analysis, carried out by Microcheck Inc.
Motility analysis.
Swimming ability was analysed on tryptone swim plates [1 % (w/v) tryptone, 0·5 % (w/v) NaCl, 0·3 % (w/v) agar]. These plates were inoculated using a sterile toothpick with bacteria grown overnight on LB agar. Motility was assessed qualitatively by examining the circular turbid zone formed by bacterial cells migrating from the inoculation point at 24 h post-inoculation.
E. coli plsC complementation experiments.
Plasmids pBBR1MCShdtS, pBBR1MCSolsA and pBBR1MCSpatB were transformed into the E. coli plsC mutant strain JC201. To test complementation on plates, the E. coli parent strain (JC200), JC201 and JC201 harbouring each of the three P. fluorescens genes were grown on LB plates overnight at 42 °C. For liquid complementation assays, the strains were grown in LB media with shaking for 1 h at 30 °C before shifting to the non-permissive temperature of 42 °C, at which the growth rate was measured spectrophotometrically for a further 8 h.
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RESULTS |
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DISCUSSION |
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The evidence establishing HdtS as an LPA acyltransferase, functionally analogous to PlsC in E. coli, is substantial. The two most compelling pieces of data are the following: first, hdtS and plsC genes can cross-complement and functionally replace each other; and second, a hdtS mutant shows alterations in cellular fatty acid composition consistent with an alteration in LPA acyltransferase activity. The hdtS gene was previously postulated to encode an N-acyl homoserine lactone (HSL) synthase (Laue et al., 2000), but our data suggest that this is not its physiological function in P. fluorescens. In addition to the experiments mentioned above, we analysed HSL production in the hdtS mutant and found that this mutant makes normal levels of C6-HSL (data not shown). We also failed to detect HSLs in E. coli expressing the construct used in this study. Based on the combined evidence, it seems likely that the previous findings reflected non-specific acyltransferase activity in E. coli under the particular growth conditions used in that study. A similar conclusion has been reached with studies of the homologous gene, PA0005, in P. aeruginosa (C. Baysse, unpublished data). As LPA acyltransferase activity is an essential activity, and as the hdtS gene is not essential, it follows that another protein(s) must be capable of carrying out the function, analogous to NlaA and NlaB in N. meningitidis. In silico analysis of complete genome sequences identified OlsA and PatB as the only likely candidates for this role. Bioinformatic analysis suggests that olsA encodes a lyso-ornithine lipid acyltransferase. Although absolute confirmation that this is the true function of this enzyme requires further experimental evidence, the findings that an olsA mutant showed no changes in membrane fatty acid profiles (data not shown) and that the olsA gene cannot complement an E. coli plsC mutant are consistent with this designation. In contrast, the patB gene partially complements an E. coli plsC mutation, and our inability to make the hdtSpatB mutant suggests that in the absence of HdtS, PatB carries out the LPA acyltransferase reaction. Thus, PatB is an LPA acyltransferase, although this may not be its primary function or activity.
An obvious question is why does P. fluorescens have two enzymes to carry out a reaction that is catalysed by one in E. coli? Following several previous studies, it has been suggested that the rationale for multiple LPA acyltransferases is to permit the synthesis of a greater, or perhaps regulated, range of membrane phospholipids (Brown et al., 2002; Shih et al., 1999
). In the case of N. meningitidis, the data suggest that NlaA may preferentially specify shorter acyl side chains at the sn-2 position, whereas NlaB would specify longer chain acyl groups. In fact, data from our study indicate that the opposite situation prevails in Pseudomonas: HdtS appears to favour shorter (C16) side chains over longer (C18) side chains. The precise role of patB remains to be determined, as analysis of fatty acid profiles in mutant strains did not establish its function. One explanation might be that these genes are differentially expressed under certain environmental conditions and our single-condition assay failed to represent this. Although we did establish by RT-PCR that all three genes are expressed in P. fluorescens (data not shown), comprehensive expression studies will be required to address this issue.
Although all three putative acyltransferases possess conserved domains, OlsA displays some divergence from HdtS and PatB. Both HdtS and PatB contain the peptide sequences NHQS and PEGTR, whereas OlsA contains the peptide sequences NHVS and PEGTT at these two conserved sites (Fig. 1). Similar variation was previously noted for NlaA and NlaB in N. meningitidis: NlaA containing the peptide sequences NHVS and PEART at the two conserved sites, and NlaB containing the sequences KHQS and PEGTR. Although it remains to be determined whether these particular sequence changes are responsible for altered substrate specificity, it has been found that slight changes in the amino acid sequence of LPA acyltransferase proteins are sufficient to alter activity or substrate specificity (Nagiec et al., 1993
; West et al., 1997
). This may explain some apparent anomalies, such as why N. meningitidis NlaA can complement the E. coli plsC mutant but P. fluorescens OlsA cannot.
The potential significance of phospholipid biosynthesis in plant-associated bacteria has previously been highlighted. In particular, phosphatidylcholine, which is a significant component of Pseudomonas membranes, has been proposed to play an important role in plantmicrobe associations (Lopez-Lara et al., 2003). Of a range of in vitro phenotypes assessed, impaired growth in the hdtS and patB mutants, and reduced motility in the hdtS mutant were ecologically relevant phenotypes observed. Furthermore, it is also important to recognize that bacteria in natural ecosystems will be exposed a wider range of stresses and environmental changes than was assessed in this study. The demonstration that P. fluorescens possesses at least two LPA acyltransferases is the first indication that this bacterium may have regulatory mechanisms to vary one aspect of membrane phospholipid structure. It will be very interesting to determine whether other mechanisms also exist, and what role they play in environmental adaptation. Indeed, the finding that P. fluorescens also possesses an enzyme specifying the synthesis of ornithine-containing lipids suggests an additional mode of varying membrane structure. With specific regard to this study, some intriguing questions remain to be answered. Perhaps foremost is the relationship between the LPA acyltransferases, their genetic regulation, and their substrate specificity. Exploring gene regulation is complicated by the observation that in P. fluorescens, as in N. meningitidis, these genes are located in operons with genes that appear to be functionally unlinked (Shih et al., 1999
; Swartley & Stephens, 1995
). In addition, it is curious that, although PatB bears highest homology to E. coli PlsC, HdtS appears to be the functional equivalent of this protein in P. fluorescens F113. With the growing realization of the potential significance of membrane structure for bacterial cell function, answering these questions will provide an opportunity to explore this issue in the context of an ecologically relevant and diverse bacterial species, P. fluorescens. The potential for regulated alteration of phospholipids and other membrane components is suggestive of a novel bacterial strategy for environmental adaptation and interaction with plants. Future studies will address the biology of this as well as potential applications for environmental biotechnology.
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ACKNOWLEDGEMENTS |
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Received 9 February 2005;
revised 1 June 2005;
accepted 6 June 2005.
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