Center for Marine Biotechnology and Biomedicine, Marine Biology Research Division, Scripps Institution of Oceanography, University of California, San Diego, La Jolla, CA 92093-0202, USA1
Author for correspondence: Douglas H. Bartlett. Tel: +1 858 534 5233. Fax: +1 858 534 7313. e-mail: dbartlett{at}ucsd.edu
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ABSTRACT |
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Keywords: eicosapentaenoic acid, pfa genes, hydrostatic pressure, polyketide synthase, ribonuclease protection assay
Abbreviations: ACP, acyl carrier protein; AT, acyl CoA:ACP transacylase; CLF, chain length factor; DHA, docosahexaenoic acid; DH/I, dehydratase/isomerase; EPA, eicosapentaenoic acid; ER, enoyl reductase; FAS, fatty acid synthase; KR, ß-ketoacyl-ACP reductase; KS, ß-ketoacyl-ACP synthase; PKS, polyketide synthase; PPTase, phosphopantetheinyl transferase; PUFA, polyunsaturated fatty acid; RPA, ribonuclease protection assay
The GenBank accession numbers for the sequences reported in this paper are AF409100 and AF467805.
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INTRODUCTION |
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PUFAs were once thought to be absent in bacterial membranes (Erwin & Bloch, 1964 ), but numerous bacterial species of marine origin have now been shown to produce very-long-chain PUFAs such as EPA and DHA. Such isolates have been found to be particularly prevalent in high-pressure, low-temperature deep-sea habitats and permanently cold marine environments (DeLong & Yayanos, 1986
; Nichols et al., 1993
; Yano et al., 1997
). The enrichment of PUFA-producing strains from these environments has led to speculation that PUFA synthesis is an important adaptation for countering the effects of elevated hydrostatic pressure and low temperature on membrane fluidity or phase. In strains which have been analysed, PUFA synthesis undergoes temperature-dependent and, for deep-sea isolates, pressure-dependent regulation. Typically, as cultivation temperature is decreased, and/or pressure increased, PUFA incorporation into membrane phospholipids is enhanced. This modulation is thought to maintain appropriate membrane physical structure (Russell & Nichols, 1999
). However, for at least one psychrotolerant piezophilic (high-pressure-adapted) deep-sea bacterium, Photobacterium profundum strain SS9, growth at high pressure and low temperature does not depend upon PUFA synthesis (Allen et al., 1999
).
A variety of bacterial fatty acid biosynthetic mechanisms exist which vary with taxonomic identity and class of fatty acid product (Cronan & Rock, 1996 ; Fujii & Fulco, 1977
; Rawlings, 1998
). Some reports have suggested bacterial omega-3 PUFA production to be mediated by undefined desaturases (Russell & Nichols, 1999
; Tanaka et al., 1999
; Watanabe et al., 1997
). However, sequence studies of bacterial genes required for PUFA biosynthesis have gradually led to a reappraisal of this view. Initial insight into the genetics of bacterial PUFA synthesis was gained by the cloning and analysis of a 38 kbp genomic fragment from the EPA producer Shewanella sp. strain SCRC-2738 (Yazawa, 1996
). Five Shewanella genes, designated ORFs 2, 5, 6, 7 and 8, were shown to be necessary for recombinant EPA synthesis in Escherichia coli and in the marine cyanobacterium Synechococcus sp. (Takeyama et al., 1997
; Yazawa, 1996
). A subsequent analysis of the predicted amino acid sequences of the products of these genes indicated that they are most related to microbial polyketide synthase (PKS) complexes and fatty acid synthase (FAS) enzymes (Metz et al., 2001
). PKS enzymes catalyse the synthesis of a wide array of complex natural products by the repetitive condensation and processing of simple monomeric substrates in a process resembling fatty acid synthesis (Hopwood & Sherman, 1990
). In addition to the Shewanella sp. SCRC-2738 sequences, related genes partially responsible for PUFA production have been analysed from the DHA-producing bacterium Moritella marina strain MP-1 (formerly Vibrio marinus) (Tanaka et al., 1999
) and from a DHA-producing thraustochytrid marine protist belonging to the genus Schizochytrium (Metz et al., 2001
).
Recently, Metz et al. (2001) reported biochemical analyses of PUFA production in E. coli strains harbouring Shewanella sp. SCRC-2738 DNA and in the Schizochytrium species. Consistent with the examination of enzyme domains, isotopic labelling studies provided compelling support for a PKS-like pathway of PUFA synthesis in both systems studied (Metz et al., 2001
). However, whereas considerable advances have been made towards a mechanistic understanding of microbial PUFA production, very little is known about the regulation of PUFA synthesis. The present study reports the cloning and molecular analysis of genes responsible for EPA synthesis, herein referred to as pfa (polyunsaturated fatty acid) genes, from the deep-sea bacterium P. profundum strain SS9. Transcriptional regulation of the SS9 pfaAD genes was analysed as a function of varying temperature and hydrostatic pressure, and SS9 mutants containing polar insertions in two pfa genes were used to verify gene function and to help delineate the transcriptional organization of the pfa operon. Furthermore, an SS9 mutant that overproduces EPA was characterized and found to upregulate pfa gene transcription.
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METHODS |
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DNA hybridizations and fosmid clone manipulations.
Fosmid library clones were replicated onto MagnaCharge (MSI, Westboro, MA, USA) nylon filters and hybridized to DNA probes obtained from internal fragments of SS9 pfa genes using standard protocols (Sambrook et al., 1989 ). Previously we reported the cloning of an 885 bp internal fragment of the SS9 pfaA gene (designated ORF 3/4 in the previous report) making use of arbitrary primers derived from the Shewanella sp. SCRC-2738 EPA gene sequence (GenBank accession no. U73935) (Allen et al., 1999
). An internal fragment of SS9 pfaD was subsequently obtained using primers ORF9-3 (5'-CGTTGAAGCATCAGCTTTCTT-3') and ORF9-2 (5'-TACGCCCATCTCGAACATATC-3') derived from SCRC-2738 EPA gene sequence. The resultant 571 bp PCR product contained a high degree of similarity, 79% identity at the DNA level, to the SCRC-2738 pfaD homologue (designated ORF7; Yazawa, 1996
)). Filters were initially probed using the internal fragment of SS9 pfaA, stripped of bound probe, and reprobed with the pfaD probe. Of the 42 clones to which both probes hybridized, fosmid 8E1 (the clone with the smallest insert size of 33·1 kbp) was selected for further analysis and sequencing. Fosmid DNA was purified using the Qiagen Plasmid Midi kit and digested with NotI to excise the cloned insert. A subclone library of fosmid 8E1 was prepared by digesting the NotI insert with Sau3A, size selecting for 12 kbp fragments, and ligation into BamHI-digested pUC18. Plasmid minipreparations of the fosmid 8E1:pUC18 subclones were prepared and sequenced using pUC18-specific primers flanking the cloned inserts.
DNA sequencing and analysis.
Double-stranded DNA sequencing reactions were performed using the ABI Prism BigDye Terminator Cycle Sequencing Ready Reaction kit (Applied Biosystems) and run on an Applied Biosystems model 373 DNA sequencing system. Initial sequence analysis and contig assembly was performed using Sequencher 3.1 software (Gene Codes Corp.). Additional sequence needed to fill in contig gaps was obtained using sequence-specific oligonucleotide primers and sequencing of PCR products. Global similarity searches were performed using the BLAST network service (Altschul et al., 1997 ). Multiple alignments were performed using ClustalW (Higgins & Sharp, 1988
) in conjunction with GeneDoc software (Nicholas & Nicholas, 1997
). Domain arrangement analyses of the predicted amino acid sequences of the pfa genes were conducted using the ProDom database of protein domain families (Corpet et al., 1999
), the Conserved Domain Database with Reverse Position Specific BLAST (Altschul et al., 1997
) and the ISREC ProfileScan server (http://hits.isb-sib.ch/cgi-bin/hits_motifscan).
Insertional inactivation mutagenesis.
Following previously published procedures (Allen & Bartlett, 2000 ; Allen et al., 1999
), insertional inactivation mutagenesis was performed targeting the SS9 pfaD gene. Briefly, an internal fragment of pfaD was PCR-amplified using primers ORF9-3 and ORF9-2 (sequences listed above; amplified region corresponding to 2370824278 of GenBank accession no. AF409100), cloned into the pCR2.1-TOPO vector (Invitrogen) and subcloned into the mobilizable suicide vector pMUT100 (Brahamsha, 1996
). The pfaD:pMUT100 construct was introduced into SS9 from E. coli by conjugal transfer as described by Chi & Bartlett (1993)
. Kanamycin-resistant exconjugants arose from plasmid integration into the SS9 chromosome in a single crossover event yielding strain EA50, with pfaD insertionally inactivated. The site of plasmid insertion was verified by PCR amplification of a portion of the pfaD gene using primers located upstream of the insertion site together with pMUT100-specific primers.
Cloning of phosphopantetheinyl transferase (PPTase) genes.
The Bacillus subtilis sfp gene (GenBank accession no. X63158) was isolated from B. subtilis by PCR amplification of the complete gene using primers 5'-TGCTGAATTATGCTGTGGCAAGGC-3' and 5'-GCTTCTCGAAATGATGTTCCCCGG-3'. In attempts to isolate PUFA synthase PPTase gene sequences, degenerate PCR primers were designed to conserved PPTase motifs found by alignment of known PPTase protein sequences including the PUFA synthase PPTase of Shewanella sp. strain SCRC-2738 (ORF2; GenBank accession no. U73935). Template DNA isolated from a variety of EPA-producing bacterial strains was employed; however, only DNA from the psychrotolerant, moderate piezophile Shewanella sp. strain SC2A (DeLong et al., 1997 ) yielded an amplification product of the expected size relative to the SCRC-2738 sequence. Using forward primer 5'-GGCGATAAAGGYAARCCK-3' and reverse primer 5'-CAACGHTCRATRTCWCCACC-3' a 212 bp product was sequenced whose deduced amino acid sequence showed 48% identity and 61% similarity over 72 amino acids to the SCRC-2738 ORF2 product. In order to obtain flanking DNA sequence, primers internal to the SC2A PPTase sequence were designed for inverse PCR (Ochman et al., 1990
) and an SC2A cosmid library (Chilukuri & Bartlett, 1997
) was screened using the SC2A PPTase internal fragment. Colony hybridizations were performed according to standard protocols (Sambrook et al., 1989
). Plasmid DNA was isolated from positively hybridizing clones and sequenced. The complete sequence of the Shewanella sp. strain SC2A PPTase is deposited under GenBank accession no. AF467805.
Fatty acid analyses.
Extraction and analysis of fatty acid methyl ester preparations via combined gas chromatography-mass spectroscopy were performed as previously described (Allen et al., 1999 ). Fatty acids are denoted as number of carbon atoms:number of double bonds.
RNA isolation and ribonuclease protection assay (RPA) analyses.
Total RNA was extracted from mid-exponential-phase P. profundum strains grown at various temperatures and pressures using the RNAzol B method (Tel-Test, Friendswood, TX, USA). [-32P]UTP-labelled RNA probes were synthesized using the T7 RNA polymerase MAXIscript in vitro transcription kit (Ambion, Austin, TX) and RPAs were performed using the RPA III kit according to the manufacturers protocols (Ambion). Probe template preparations involved the PCR amplification of fragments of SS9 pfa genes, cloning of PCR products into the pCR2.1-TOPO vector and subsequent subcloning of inserts into the pDP18 transcription vector (Ambion). The sizes of full-length probes and protected fragments were as follows (the positions of pfa RPA probes are indicated with reference to GenBank accession no. AF409100): pfaA 377/268 bp (94229689), pfaB 465/322 bp (1533215653), pfaC 477/344 bp (1913319476), pfaD 477/344 bp (2338523728), pfaA/B 520/264 bp (1502015283), pfaB/C 516/260 bp (1712917388), pfaC/D 511/255 bp (2305223306). RNA probes were purified from denaturing acrylamide continuous gels, co-precipitated with 10 µg total RNA and hybridized overnight at 45 °C. Following RNase treatment, protected fragments were separated on denaturing acrylamide gels (5% acrylamide/8 M urea) against undigested probe and appropriate controls. For detection of probes and protected fragments, gels were transferred to filter paper and exposed to X-ray film overnight. [
-32P]UTP-labelled RNA Century Markers (Ambion) were used as size standards.
Primer extension analysis.
Primer extension analysis of the SS9 pfaAD genes was performed using the Primer Extension System-AMV Reverse Transcriptase kit (Promega). RNA was isolated from SS9 strain DB110 using the RNAzol B method. Multiple pfa extension primers were tested; however, only pfaA primers 5'-GCCATGCCAACAATCGCAAT-3' (position 75297548) and 5'-GTTGCGATTAGGCAACTGGTGA-3' (position 73797400) yielded extension products. Primers were end-labelled using [-32P]ATP and T4 polynucleotide kinase. Labelled primers were annealed to 40 µg SS9 RNA and extended using AMV-RT. For DNA sequencing, pfaA plasmid templates were constructed using DNA amplified with primers 5'-AACCTCTTGCTCCAGTGATTG-3' and 5'-TATCACGGTCGTATGTTTCCG-3' (amplified fragment position 70677856), cloned into pCR2.1, and sequenced using the labelled primers used for primer extension in conjunction with the fmol DNA Cycle Sequencing System Kit (Promega). DNA fragments were resolved on 8 M urea/8% polyacrylamide gels.
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RESULTS |
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Of the 19 ORFs identified, seven did not possess any significant homology to GenBank sequences and were noted as hypothetical proteins. Upstream of the SS9 pfa genes is a putative methyl-accepting chemotaxis protein (similar to Vibrio cholerae protein VCA093) and a cluster of four genes which appear to be arranged in an operon structure. Included in this cluster is a putative fabH paralogue. We denote this gene as a putative paralogue because a homologue of fabH has previously been cloned and sequenced from SS9, located within a distinct cluster of genes involved in saturated and monounsaturated fatty acid synthesis, an organization found in numerous -proteobacteria (our unpublished results). In addition, putative haloalkane dehalogenase, acyl-CoA ligase, and steroid dehydrogenase genes were identified within this upstream cluster. Based on BLAST searches, each of these four genes displays the highest similarity to genes in the plant pathogen Xylella fastidiosa (GenBank accession no. AE003849). Unlike SS9, however, none of these genes are linked in the Xylella genome. Downstream of the SS9 pfa cluster numerous ORFs of unknown function were identified as well as an alkaline serine protease (homologue of Vibrio metschnikovii vapT) and a formyltetrahydrofolate deformylase gene (homologue of Pseudomonas aeruginosa purU) involved in purine biosynthesis.
Analysis of SS9 EPA biosynthetic enzymes
Similarity searches of the SS9 pfa gene products revealed significant matches to numerous multifunctional enzyme complexes involved in such processes as polyketide antibiotic synthesis (Hopwood & Sherman, 1990 ; Pfeifer & Khosla, 2001
), eukaryotic fatty acid synthesis (Beaudoin et al., 2000
; Parker-Barnes et al., 2000
) and heterocyst glycolipid synthesis (Campbell et al., 1997
). Domain analyses within individual pfa gene products also revealed numerous enzyme domains characteristic of functions present in bacterial type II fatty acid synthesis. This type of organization is similar to type I PKSs, multifunctional enzymes containing sets of FAS-related activities for successive rounds of polyketide chain elongation and derivatization (Rawlings, 2001
). Fig. 2
shows the domain organization of the SS9, Shewanella and Moritella pfaAD deduced amino acid sequences. Seven enzyme domains were identified within the pfa products: ß-ketoacyl-ACP synthase (KS), acyl CoA-ACP transacylase (AT), acyl carrier protein (ACP), ß-ketoacyl-ACP reductase (KR), chain length factor (CLF; possible malonyl-ACP decarboxylase activity Bisang et al., 1999
), ß-hydroxyacyl-ACP dehydratase/isomerase (DH/I) and enoyl reductase (ER).
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Interestingly, growth of SS9 in the presence of the fungal antibiotic cerulenin, a potent irreversible inhibitor of fatty acid biosynthetic condensing enzymes such as KAS I and KAS II (Omura, 1981 ), has no effect on EPA production (Allen et al., 1999
). This resistance could result from blocked access of cerulenin to the Pfa KS active sites, reflecting structural differences between the Pfa KS domains and type II KAS enzymes.
EPA synthesis in SS9 and overproducing strain EA2
The percentage composition of EPA present in SS9 membranes undergoes temperature- and pressure-dependent modulation (Allen et al., 1999 ). An increase in cultivation pressure from 0·1 MPa to 28 MPa [0·1 MPa=1 atm=1 bar] results in nearly a fourfold increase in EPA percentage composition (Fig. 3
). Similarly, EPA percentage composition undergoes moderate increase in response to reduced cultivation temperature, i.e. 15 °C to 4 °C (Allen et al., 1999
). Fig. 3
shows the percentage composition of EPA as a function of varying hydrostatic pressure in wild-type SS9 and an SS9 mutant strain found to overproduce EPA. This strain, designated EA2, was isolated as an oleic acid (18:1n-9)-requiring auxotrophic chemical mutant (Allen et al., 1999
). Strain EA2 constitutively produces EPA at a level nearly fivefold that of wild-type SS9 grown at atmospheric pressure (Fig. 3
).
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Introduction of PPTase genes into E. coli harbouring SS9 pfaAD
In an attempt to achieve recombinant EPA production in E. coli we sought to obtain a gene encoding the last remaining enzyme activity needed: PPTase activity. PPTase genes were recovered from both the EPA-producing deep-sea Shewanella sp. strain SC2A (DeLong et al., 1997 ) and B. subtilis. Based on BLAST analyses, the SC2A putative PPTase shares 55% identity (89/161 amino acids) with the Shewanella sp. strain SCRC-2738 PUFA synthase PPTase (ORF2) shown to be required for recombinant EPA production in E. coli host strains (Metz et al., 2001
). Introduction of either the SC2A PPTase gene or the sfp gene, the surfactin-synthetase-activating PPTase of B. subtilis (Nakano et al., 1992
), into E. coli strains expressing SS9 pfaAD from pFOS8E1 consistently failed to yield recombinant EPA synthesis.
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DISCUSSION |
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The ability to introduce multiple double bonds into a single acyl chain in the absence of desaturation reactions likely arises from the activities of the DH/I domains present in the microbial PUFA synthases (bacterial PfaC homologues and Schizochytrium ORF C). Such dehydration/isomerization reactions would be analogous to those catalysed by FabA (ß-hydroxydecanoyl-ACP dehydratase) in bacterial monounsaturated fatty acid synthesis (Cronan & Rock, 1996 ). By dehydration of the ß-hydroxyacyl-ACP substrate, product of ß-ketoacyl-ACP synthase condensation and subsequent reduction of the ß-ketoester by ß-ketoacyl-ACP reductase, a trans double bond is introduced into the growing acyl chain. In selective rounds of acyl chain elongation these double bonds are either preserved by isomerization to the cis form to form an unsaturated acyl-ACP or reduced by an enoyl reductase to a saturated acyl-ACP. Metz et al. (2001)
propose a hypothetical pathway for EPA synthesis in Shewanella sp. SCRC-2738 wherein position-specific isomerases (trans-2,cis-3 and trans-2,cis-2) are involved in trans/cis double bond isomerization. Such a mechanism may be consistent with two DH/I domains being present in PfaC homologues (Fig. 2
). Alternatively, the two DH/I domains could be analogous to the FabA and FabZ ß-hydroxyacyl-ACP dehydratase isozymes found in E. coli which differ in reactivity and specificity (Heath & Rock, 1996
).
Unique to microbial PUFA synthases is the presence of clustered repetitive ACP domains (Fig. 2): SS9 pfaA possesses five ACP domains, Shewanella sp. has six, Moritella has five, and Schizochytrium has nine (Metz et al., 2001
; Tanaka et al., 1999
). Intermediates in the biosynthetic process are presumably bound to these ACP domains as thioesters with AT domains being required for the loading of the starter and extender units. The significance of the disparity in the number of ACP repeats among the PUFA synthase systems is unknown.
Currently, we have been unable to achieve recombinant EPA synthesis in E. coli with the introduced SS9 pfaAD genes. This shortcoming arises from the need for an additional gene whose product is required for the post-translational modification of the constituent ACP domains present in PfaA. This activity is achieved by a PPTase which converts apo-ACP to its active holo-form by transfer of a 4'-phosphopantetheinyl moiety from coenzyme A to ACP (Lambalot et al., 1996 ). In Shewanella sp. SCRC-2738 a fifth gene, designated ORF2, required for recombinant EPA synthesis in E. coli has been identified as a PPTase and is located within close proximity of the pfaAD operons (Metz et al., 2001
). Unlike SCRC-2738, the PUFA synthase PPTase is unlinked from the other pfa genes in SS9 and Moritella. We have been unable to clone the ORF2 homologue from either SS9 or Moritella. During attempts to obtain recombinant EPA synthesis, we introduced two PPTase genes into E. coli harbouring SS9 pfaAD, B. subtilis sfp and a Shewanella ORF2 homologue. The B. subtilis sfp gene, involved in surfactin biosynthesis, encodes a PPTase with a broad substrate recognition spectrum (Nakano et al., 1992
; Reuter et al., 1999
). In addition, a PPTase homologue which contained a high degree of identity to Shewanella sp. SCRC-2738 ORF2 was cloned and sequenced from the EPA producer Shewanella sp. strain SC2A. Expression of either of these genes in E. coli harbouring SS9 pfaAD failed to yield recombinant EPA synthesis, suggesting a high degree of specificity of individual PPTases to their cognate ACPs.
While substantial progress has been made towards a mechanistic understanding of microbial PUFA synthesis, very little information exists regarding the regulation of bacterial PUFA production. In those organisms that have been studied, modulation of PUFA percentage composition occurs during changes in cultivation temperature or pressure. For example, growth of SS9 at a hydrostatic pressure of 28 MPa results in an approximately fourfold increase in EPA percentage composition relative to growth at 0·1 MPa (Fig. 3). At the outset of our studies, one possibility was that this modulation was the result of transcriptional regulation of the EPA biosynthetic genes.
RPA analyses performed on each of the SS9 pfa genes using RNA extracted from SS9 cells cultivated at various temperatures and pressures revealed that the pfa genes are not transcriptionally regulated in an adaptive manner in response to these parameters (Fig. 4). The observed reduction in pfa transcript abundance at elevated pressure is confounding and could result from diminished transcription initiation or increased transcript turnover at high pressure. Numerous prokaryotic species regulate percentage composition of particular membrane fatty acids in response to cultivation parameters. In E. coli increased cis-vaccenic acid (18:1n-11) composition at low temperature is an intrinsic property of the fatty acid biosynthetic enzyme KAS II (ß-ketoacyl-ACP synthase II), product of the fabF gene, and a similar regulatory mechanism may account for increased cis-vaccenic acid composition at high pressure in SS9 (Allen & Bartlett, 2000
; Cronan & Rock, 1996
). In both bacteria, fabF is not transcriptionally regulated and, at least for the E. coli enzyme, it is the relative activity of the enzyme at different temperatures that is responsible for the increased production of 18:1 at low temperature. Hence, the possibility exists that PUFA synthases exhibit temperature/pressure-responsive characteristics.
Transcriptional analyses indicate that the pfa gene cluster is organized into two operons, pfaAC and pfaD (Figs 4 and 5
). Evidence in support of this conclusion includes the presence of overlapping start/stop codons of adjacent genes, RPA results with probes spanning intergenic regions, and transcript analyses of a strain containing a polar insertion within pfaA. The transcriptional start of pfaA has been mapped to 169 bp upstream of the translational start (Fig. 6
).
Results from SS9 suggest that the pathway for PUFA synthesis is separate and distinct from the type II FAS producing monounsaturated and saturated fatty acids (Allen & Bartlett, 2000 ; Allen et al., 1999
). Many of the type II FAS genes have been cloned and sequenced from SS9 and Moritella (Allen & Bartlett, 2000
; Tanaka et al., 1999
). Metz et al. (2001)
reported a probable PUFA synthetic mechanism reliant on malonyl-CoA derived from acetate as would be expected for the type II FAS system. An interesting question is the cross-talk that exists between the two systems with regard to coordinated expression and lipid incorporation. Some initial insight into this interplay has been provided by pfa transcript analysis of an SS9 mutant strain, designated EA2, originally isolated as an oleic acid auxotrophic chemical mutant that overproduces EPA nearly fivefold compared to wild-type SS9 (Fig. 3
). In addition, this strain greatly underproduces monounsaturated fatty acids (MUFAs) (Allen et al., 1999
). pfa transcript analyses in this strain reveal substantial pfaAD overexpression relative to wild-type SS9 (Fig. 4
). While the nature of the mutation in this strain has yet to be resolved, two opposing hypotheses can be proposed. Either this strain harbours a lesion resulting in decreased MUFA production which results in compensatory increases in pfa transcription and EPA synthesis, or the mutation results in overexpression of both pfa operons and the cellular response is decreased MUFA synthesis. Both models require the presence of a transcription factor that modulates pfa gene expression.
The high degree of sequence similarity between the bacterial (Shewanella sp SCRC-2738, M. marina and SS9) and the eukaryotic microbe Schizochytrium pfa genes suggests the possible involvement of horizontal gene transfer in the acquisition of the pfa gene clusters in the marine environment. However, among the three bacterial strains whose pfa gene clusters have been cloned and sequenced no sequence conservation flanking the pfa clusters is observed with the exception of a single undefined ORF located upstream of pfaA in SS9 and Moritella. Furthermore, there is no apparent GC bias among the pfaAD genes nor is there indication of flanking genes possessing functions which could facilitate horizontal transfer.
Located upstream of the SS9 pfa cluster resides an intriguing cluster of four genes which appear to be organized into a possible operon (Fig. 1). Included in this cluster is a putative fabH (3-oxoacyl-ACP synthase III; KAS III) paralogue. This fabH paralogue is distinct from the fab cluster fabH homologue, involved in type II fatty acid biosynthesis initiation, which we have cloned and sequenced from SS9 (our unpublished results). Multiple fabH-like sequences have been identified in a few bacterial species including B. subtilis (yjaX, GenBank accession no. F69842, and yhfB, Y14083) and V. cholerae (GenBank accession nos A82423 and H82128). Within this cluster also reside a putative haloalkane dehalogenase, a probable acyl-CoA ligase, and a putative steroid dehydrogenase/isomerase. Proteins of the hydroxysteroid dehydrogenase/isomerase family (Labrie et al., 1992
) are unusual in prokaryotic organisms, with only a single other bacterial homologue having been identified in the plant pathogen Xylella fastidiosa (GenBank accesssion no. AE004004). The sequence and possible operon structure of this gene cluster suggest that their products could function in a common metabolic process including some aspect of fatty acid physiology. Curiously, upstream of the M. marina pfa gene cluster lie two genes presumably involved in fatty acid metabolism as well, a 3-ketoacyl-CoA thiolase/acetyl-CoA acetyltransferase and a probable lipid A acyltransferase (ORFs 1 and 3, GenBank accession no. AB025342).
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ACKNOWLEDGEMENTS |
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Received 29 October 2001;
revised 18 January 2002;
accepted 4 February 2002.
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