Department of Molecular Microbiology and Biotechnology, Tel-Aviv University, Ramat Aviv 69978, Israel1
Author for correspondence: David L. Gutnick. Tel: +972 3 6409834. Fax: +972 3 6409407. e-mail: davidg{at}post.tau.ac.il
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ABSTRACT |
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Keywords: emulsan, biosurfactant, amphipathic biopolymer, polysaccharide biosynthesis
Abbreviations: Cm, chloramphenicol; Km, kanamycin; TLU, translucent; TMR, transmembrane helical region
The GenBank/EMBL accession number for the sequence analysis of the eight fragments determined in this work is AJ243431.
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INTRODUCTION |
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METHODS |
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Isolation of translucent (TLU) mutants.
Emulsan-defective A. lwoffii RAG-1 mutants were obtained following mini-Tn10Km transposon mutagenesis (Leahy et al., 1993 ). TLU mutants were selected by plating the cell suspension onto ethanol-minimal salts medium containing Km and Cm (to counterselect the donor). TLU mutants are visually less opaque then the wild-type and have previously been shown to be emulsan-defective (Bayer et al., 1983
; Pines & Gutnick, 1981
).
Emulsifying activity assay.
Emulsan was assayed functionally on the basis of its ability to form an oil/water emulsion from a mixture of hexadecane and 2-methylnaphthalene in 7·5 ml 20 mM Tris buffer (pH 7·0) containing 10 mM MgSO4 as previously described (Rosenberg et al., 1979 ). One unit of emulsan is that amount which gives rise to a turbidity of 100 Klett units in the standard assay. Typically, purified emulsan exhibits a specific activity of 150180 U (mg biopolymer)-1.
Analysis of TLU mutants.
TLU mutants were analysed by Southern hybridization (Southern, 1975 ). The gene encoding Km resistance was excised from the plasmid pLOFKm with NotI, labelled using the DIG system (DIG-High prime DNA labelling kit; Roche Molecular Biochemicals) and used as a probe in Southern hybridization assays. Genomic DNA was isolated as described by Hopwood et al. (1985)
. EcoRI-digested genomic DNA was used as template for Southern hybridization. To obtain the flanking sequence of the insertion, an EcoRI mini-library of the approximate size of the fragment that reacted with the Km probe was inserted into pUC18. The clones containing the Km resistance gene were selected on plates and the isolated plasmids sequenced using an ABI 377 DNA sequencing apparatus (Perkin-Elmer). The orientation of the fragments with respect to each other was predicted according to the sequence analysis and confirmed by PCR (Eppendorf Master Cycler model 5330 plus) using appropriate primers. Sequence databases were searched with the National Center for Biotechnology Information BLAST network server (Altschul et al., 1990
). The TMHMM server was used to predict transmembrane helices of the proteins (Sonnhammer et al., 1998
).
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RESULTS AND DISCUSSION |
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Predicting the biosynthetic route of emulsan based on homologies to the genes involved in emulsan biosynthesis
Using computer-assisted BLAST searches, putative homologues were identified which suggested an involvement in (a) production of nucleotide sugar precursors, (b) transglycosylation reactions for synthesis of the membrane-bound trisaccharide repeat unit, (c) transacetylation, (d) repeat unit polymerization and polysaccharide transport. We employ the BPGN (bacterial polysaccharide gene nomenclature) scheme (Reeves et al., 1996 ) for naming polysaccharide biosynthetic genes according to the three letter w** system and refer to the emulsan biosynthetic cluster(s) as the wee (exopolysaccharide; emulsan) region. In accordance with this system, genes which we consider are likely to be pathway-specific have been labelled weeAweeK, respectively. Other genes (wza, wzb, wzc, wzx, wzy) in the emulsan wee cluster were found to share sequence homology with a variety of more general saccharide-processing genes encoding functions common to a variety of capsular biosynthetic clusters, and were therefore assigned the same commonly used name (Reeves et al., 1996
). In addition, sequence analysis revealed five ORFs exhibiting similarities to sugar pathway gene products, Pgi, Pgm (partially sequenced), GalU, GalE and Ugd. The putative proteins encoded by the wee cluster along with the relevant homologues and their sequence identity and similarity values are shown in Table 2
. Based on these homologies and their functions we propose a hypothetical biosynthetic pathway (Fig. 3
). The subsequent verification of these proposed reactions by suitable in vitro analyses is currently in progress. The basis for this proposal is presented below. Reference is made both to Fig. 2
and to Table 2
, which can be used for clarification.
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Homologues of Pgm have previously been shown to be bifunctional enzymes catalysing both phosphoglucomutase and phosphomannomutase activity (Ye et al., 1994 ). GalU homologues show UTP-glucose-1-phosphate uridylyltransferase activity (Crater et al., 1995
; Weissborn et al., 1994
), whilst GalE homologues exhibit UDP-glucose 4-epimerase activity (Lee et al., 1995
; Scupham & Triplett, 1997
). To obtain one of the nucleotide sugar precursors of emulsan, UDP-N-acetyl-D-galactosamine, from UDP-N-acetyl-D-glucosamine, the 4-epimerase activity is needed. We propose that GalE is responsible for this conversion (Fig. 3
).
RkpK of Sinorhizobium meliloti, a Ugd homologue, exhibits UDP-glucose dehydrogenase activity (Kereszt et al., 1998 ). It should be noted that the similarity of Ugd to its homologues is significantly lower than the similarity of the other sugar pathway genes to their homologues. It is possible, therefore, that in A. lwoffii RAG-1 the Ugd homologue may use a different substrate. No putative roles were assigned for Pgi, Pgm GalU and Ugd in the biosynthetic pathway of emulsan. The reason A. lwoffii LN303 is emulsan-negative could be explained by a polar effect on galE downstream of ugd.
WeeA and WeeB are closely related to WecB and WecC of the enterobacterial common antigen (ECA) cluster of E. coli (Table 2). WecB is an UDP-N-acetylglucosamine 2-epimerase (Meier-Dieter et al., 1990
; Sala et al., 1996
) while WecC was found to exhibit UDP-N-acetyl-mannosaminuronic acid dehydrogenase activity (Meier-Dieter et al., 1990
). On the basis of the backbone structure of emulsan and in analogy with known enzymic interconversions of nucleoside-diphosphate-linked sugars, we propose that WeeA and WeeB are involved in the biosynthesis of UDP-N-acetyl-L-galactosaminuronic acid (Singh et al., 1990
). Accordingly, WeeA would convert UDP-N-acetyl-D-glucosamine into UDP-N-acetylmannosamine. Subsequently, WeeB would oxidize the UDP-N-acetylmannosamine into UDP-N-acetylmannosaminuronic acid (Fig. 3
). The third enzyme, which would be needed to obtain UDP-N-acetyl-L-galactosaminuronic acid, is a 3,5-epimerase and could be WeeE or WeeF, for which no putative functions have been assigned.
WeeJ is similar to several putative perosamine synthetases (Table 2), which show high similarity to a large number of pyridoxal-binding proteins (Stroeher et al., 1995
). The WeeK protein (Table 2
) is similar to several dTDP-glucose 4,6-dehydratases and UDP-glucose 4-epimerases (Comstock et al., 1996
; Dean et al., 1999
; Skurnik et al., 1995
). Analysis of the protein sequence of WeeK predicted four transmembrane helical regions (TMRs) within the N-terminal portion of the protein. If WeeK is similar to a dTDP-glucose 4,6-dehydratase, it may be responsible for the conversion of UDP-D-glucosamine into UDP-4-keto-6-deoxy-D-glucosamine. WeeJ could subsequently catalyse the formation of diamino 2,4-diamino-6-deoxy-D-glucosamine, a component of the repeat unit, from UDP-4-keto-6-deoxy-D-glucosamine (Fig. 3
).
Synthesis and acylation of the repeat unit
Three ORFs, WeeD, WeeG and WeeH, show significant similarity to bacterial glycosyltransferases (Table 2). The WeeH protein is similar to the C-terminal portion of WbaP, which is the galactosyl transferase responsible for transferring the first galactose 1-phosphate from GDP-galactose to the undecaprenyl phosphate (UndPP) during the biosynthesis of the O-antigen of Salmonella enterica (Wang et al., 1996
). In this regard, analysis of the protein sequence of WeeH predicts one TMR, which is similar to the C-terminal portion of WbaP (Wang et al., 1996
). These three gene products, weeH, D and G, might be involved in the transfer of the three activated nucleotide sugars of emulsan to UndPP (Fig. 3
).
WeeC and WeeI show significant similarity to acetyltransferases from other bacteria. These proteins all contain a sequence of 50 amino acids exhibiting similarity to a conserved region of the NodL-LacA-CysE acetyltransferase family (Lin et al., 1994 ). These proteins might also be involved in transacylation of the polysaccharide backbone with other longer-chain-length fatty acids, thereby conferring amphipathic characteristics on the water-soluble emulsan polysaccharide (Fig. 3
). Thus far we have found no homologues which might be involved in the transamidation reactions which might generate amide linkages with longer-chain-length fatty acids on the amino sugars.
Polymerization and polymer transport
The Wzx protein exhibits similarity to polysaccharide exporter proteins (PST), which are highly divergent (Table 2), but share a similar predicted topology containing 12 TMRs (Paulsen et al., 1997
). Wzx is considered to catalyse the translocation of the membrane-bound repeat unit so that rather than facing the cytoplasm, the repeat unit now faces the periplasm (Liu et al., 1996
). A second integral membrane protein with 12 predicted TMRs, Wzy, shows similarity to several proteins which have been implicated in polymerization of the repeat unit on the periplasmic side of the cytoplasmic membrane (Whitfield, 1995
). Three ORFs (wza, wzb and wzc) comprise that region of the wee cluster, which is transcribed in the opposite direction. This conserved region of genes is prevalent in clusters for expression of E. coli group I K antigens and in capsular clusters of several other bacteria (Rahn et al., 1999
). In E. coli strains carrying mutations in wza and wzc, capsular polysaccharide is still partially polymerized but not assembled on the cell surface, suggesting that these proteins are involved in translocation of the polysaccharide out of the periplasm into the environment (Drummelsmith & Whitfield, 1999
). Homologues of Wza (Table 2
) are putative outer-membrane lipoproteins with a predicted ß-barrel structure and are members of the outer-membrane auxiliary (OMA) family (Paulsen et al., 1997
). It has been suggested that Wza is involved in forming a channel for surface polysaccharide secretion (Drummelsmith & Whitfield, 2000
). Proteins similar to Wzc of A. lwoffii RAG-1 are grouped in the cytoplasmic membrane periplasmic auxiliary (MPA1) family (Paulsen et al., 1997
). Sequence analysis of Wzc of A. lwoffii RAG-1 predicts two TMRs and an ATP-binding motif in the C-terminal tail, which is similar to members of the MPA1 protein family. Additionally, Wzc of A. lwoffii RAG-1 shows high similarity to Ptk of Acinetobacter johnsonnii, Etk of E. coli, and Wzc of E. coli, all autophosphorylating protein tyrosine kinases (Grangeasse et al., 1997
; Ofir et al., 1999
; Vincent et al., 1999
). Proteins similar to Wzb are predicted to be acid phosphatases. Dephosphorylation of the phosphorylated protein tyrosine kinase was demonstrated with Ptp of A. johnsonnii and Wzb of E. coli (Grangeasse et al., 1997
; Vincent et al., 1999
). These activities have recently been determined in A. lwoffii RAG-1 and the results will be presented separately.
No putative functions have been assigned to ORFs WeeE and WeeF. In the case of WeeF no homologues were identified. ORF WeeE encodes a putative 712 aa protein. The first N-terminal half of the translated sequence shows low similarity to sugar dehydrogenases, while the C-terminal half shows low similarity to three different enzymes of no obvious function for emulsan biosynthesis.
Interestingly, the G+C content of four ORFs, weeD (a glycosyltransferase), wzx, wzy and weeC, is lower than that of the remainder of the cluster (Fig. 1). Sequence analysis revealed a preferential usage of the A+T-rich codons for leucine, isoleucine, lysine, asparagine and phenylalanine. This has previously been reported for several genes involved in polysaccharide biosynthesis, including that of S. enterica (Reeves, 1993
). This unusually low G+C content supports the hypothesis that the polysaccharide gene clusters were likely to have been assembled from several sources and that these genes have evolved in species with accordingly low G+C content (Reeves, 1993
).
Thus far, A. lwoffii RAG-1 is the only natural isolate that produces the emulsan biopolymer. It is somewhat surprising, therefore, that almost all of the biosynthetic genes are at least partially homologous to known proteins involved in polysaccharide biosynthesis in other organisms. Capsular gene clusters have been cloned from a number of Gram-negative bacteria although the most studied to date are those of E. coli, which may be regarded as a paradigm for Gram-negative bacteria (Roberts, 1996 ). A new system for classification recognizes four groups of E. coli polysaccharide capsules, based on genetic and biosynthetic criteria (Whitfield & Roberts, 1999
). The occurrence of homologues in the wee cluster to proteins involved in biosynthesis of group 1 K antigens in E. coli, coupled with the fact that the emulsan biopolymer is a high molecular mass (apparent molecular mass
106) surface polysaccharide during exponential growth suggests that emulsan is a member of the group 1 family (Paulsen et al., 1997
; Whitfield & Roberts, 1999
). In contrast, the wee cluster apparently contains neither JUMPstart nor ops sequences, which are conserved in group 1 clusters (Rahn et al., 1999
). In addition, group 1 capsules are generally not considered to contain amino sugars (Roberts, 1996
). According to this criterion, emulsan might be thought of as a member of the group 4 family of polysaccharides. The two biosynthetic pathways (group 1 and group 4) appear to differ by the presence of a WbaP homologue, which functions in the initial transglycosylation reaction in the biosynthesis of group 1 K antigens (Wang et al., 1996
).
As a working hypothesis, and in analogy to the pathway for biosynthesis of type I E. coli capsules (Whitfield & Roberts, 1999 ), we propose that emulsan biosynthesis starts with the conversion of L-fructose-6-phosphate into the three nucleotide sugar precursors (Fig. 3
). The first amino sugar is then transferred to undecaprenyl phosphate by a WbaP homologue. Subsequently, other glycosyltransferases add the remaining two amino sugars of the repeat unit. The membrane-bound repeat unit is then transferred across the plasma membrane by the Wzx homologue. Polymerization is believed to occur at the periplasmic face of the plasma membrane and is catalysed by the polymerase Wzy. Accordingly, the nascent polymer grows at the reducing terminus, one repeat unit at a time. Wza and Wzc are putatively involved in the translocation of the mature polysaccharide through the outer membrane. Despite the strong similarity of the organization of the wee cluster to clusters of other capsular biosynthetic genes, the functional emulsan bioemulsifier is relatively unusual. This may be due to the amphipathicity conferred on the apoemulsan backbone by specific transacetylases (or transacylases) within the cluster. For example, cleavage of about 40% of the ester linkages resulted in a dramatic reduction of emulsan activity and hydrocarbon substrate specificity (Shabtai & Gutnick, 1985
). In addition, hydrocarbon substrate specificity and full emulsifying activity have also been shown to depend on non-covalent protein(s) associated with the amphipathic polysaccharide during its release into the culture broth. Nonetheless, removal of the emulsan-associated protein, yielding apoemulsan, retains many of the original properties of emulsan itself.
Experiments to characterize the gene products and their enzymic activities in vitro are currently in progress, along with studies on the regulation of pathway expression.
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ACKNOWLEDGEMENTS |
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Received 30 October 2000;
revised 9 March 2001;
accepted 23 March 2001.