Departments of Microbiology and Immunology1, and Biochemistry and Molecular Biology2, University of Melbourne, Victoria 3010, Australia
Author for correspondence: Helen Billman-Jacobe. Tel: +61 3 8344 5698. Fax: +61 3 9347 1540. e-mail: hbj{at}unimelb.edu.au
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ABSTRACT |
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Keywords: GPL, mycobacteria, methylation
Abbreviations: DIG, digoxigenin; ESI-MS, electrospray ionization mass spectrometry; GPL, glycopeptidolipid; HPTLC, high-performance thin-layer chromatography
a The GenBank accession number for the sequence determined in this work is AY138899.
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INTRODUCTION |
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Several M. smegmatis genes involved in the synthesis of the GPL core have now been characterized, and shown to encode a peptide synthetase (Mps), a rhamnose O-methyltransferase (Mtf1), an acetyltransferase (Atf1) and a potential transporter protein (TmptC) (Billman-Jacobe et al., 1999 ; Patterson et al., 2000
; Recht & Kolter, 2001
; Recht et al., 2000
). Disruption of all these genes results in bacteria with a rough colony morphology. In this paper we describe the M. smegmatis GPL biosynthetic gene cluster and identify the methyltransferase that catalyses the conversion of the fatty acid from the 3-hydroxy to the 3-methoxy form.
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METHODS |
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Disruption of mtf2.
The plasmid, pHBJ45, which was used to make a gene-disruption cassette, was pUC18 containing the entire mtf2 ORF and flanking sequences on a 3·6 kb EcoRIHindIII fragment (Patterson et al., 2000 ). To disrupt mtf2, the plasmid was linearized by digestion with BglII and treated with T4 DNA polymerase. A streptomycin-resistance gene was excised from pUC19
(Prentki & Krisch, 1984
) by digestion with HindIII and EcoRI and treated with T4 DNA polymerase. The streptomycin-resistance cassette was ligated to pHBJ45 and the recombinant plasmid was transformed into E. coli. The resulting plasmid, pHBJ370, contained mtf2::str on a 5·6 kb fragment which was then excised by HindIIIEcoRI digestion and cloned into HindIIIEcoRI-digested pK18mobsacB (Schafer et al., 1994
) to create pHBJ371 for gene replacement.
For complementation of mtf2 mutants, a 1·7 kb SalI fragment containing the entire mtf2 ORF was cloned into the SalI site of a mycobacterial shuttle vector, pMV261 (Stover et al., 1991 ). Recombinant plasmids with the insert cloned in both orientations were isolated to test if expression of the cloned gene was independent of the vector-encoded GroEL promoter. The plasmids were named pHBJ389 and pHBJ390 (Table 1
).
Extraction of cell-wall-associated GPLs.
Wild-type, mutant and complemented strains were grown in LB broth at 37 °C and cells were harvested by centrifugation (2000 g, 15 min) when in mid-exponential phase. Cells were washed in phosphate-buffered saline and then extracted in 20 vols of chloroform/methanol (2:1, v/v) for 2 h at room temperature with constant agitation. Cell wall lipids were recovered in the chloroform phase after Folch partitioning (Folch et al., 1957 ). The lipid extract was subjected to alkaline methanolysis (0·2 M NaOH in methanol, 2 h, 40 °C) to cleave ester-linked fatty acids. The alkali-stable GPLs were recovered in the upper 1-butanol phase after biphasic partitioning between water-saturated 1-butanol (4 vols) and water (2 vols).
HPTLC analyses and purification of GPLs.
The GPL extracts were analysed by high-performance thin-layer chromatography (HPTLC) using aluminium-backed silica Gel 60 HPTLC sheets (Merck). The HPTLC sheets were developed in chloroform/methanol (9:1, v/v) for chromatographic separation of the GPLs. Individual GPL species were purified using the same solvent system. Silica bands were scraped and extracted twice in chloroform/methanol (2:1, v/v) with sonication. Glycolipids were visualized by spraying with orcinol/H2SO4 and charring at 100 °C.
Compositional analysis.
Monosaccharide analyses of crude GPL extracts or purified species were performed after hydrolysis in 2 M trifluoroacetic acid (100 °C, 2 h) and conversion of the released monosaccharides into their corresponding alditol acetate derivatives (Billman-Jacobe et al., 1999 ). The alditol acetate derivatives were separated and analysed by GC-MS on a Hewlett Packard HP-1 column using a Hewlett Packard model 6890 gas chromatograph and a model 5973 mass detector.
Electrospray ionization mass spectrometry (ESI-MS).
HPTLC-purified GPL species were analysed with a Bruker Esquire 3000 electrospray ionization mass spectrometer fitted with a nanospray attachment. GPLs were analysed in negative-ion mode.
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RESULTS |
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Genes involved in deoxy sugar synthesis. Genes encoding the four enzymes for rhamnose synthesis (glucose-1-phosphate thymidylyltransferase, dTDP-glucose 4,6-dehydrogenase, dTDP-4-dehydrorhamnose 3,5-epimerase and dTDP dehydrorhamnose reductase) are conserved among bacteria (Stevenson et al., 1994 ). Rhamnose biosynthesis has been described in M. tuberculosis and M. smegmatis and genes encoding the enzymes above have been identified in M. tuberculosis (Cole et al., 1998
; Ma et al., 1997
). Two genes in the GPL locus possibly encode the enzymes for the first two steps of rhamnose biosynthesis. The rmlA gene product is 92% similar to RmlA of M. tuberculosis, which encodes glucose-1-phosphate thymidylyltransferase and has been shown to convert glucose 1-phosphate to dTDP-glucose (Liu & Thorson, 1994
; Ma et al., 1997
). Several glucose-1-phosphate thymidylyltransferases have a conserved motif in the N-terminus which is also observed in the M. smegmatis RmlA polypeptide sequence (LAGGSGTRLHP) (Aguirrezabalaga et al., 2000
; Merson-Davies et al., 1994
; Steffensky et al., 2000
). The second gene that is possibly involved in 6-deoxy sugar synthesis is rmlB. The deduced product of rmlB is proposed to be a dTDP-glucose 4,6-dehydrogenase which would catalyse conversion of dTDP-glucose to dTDP-xylohexulose, the second step in dTDP-deoxy sugar synthesis (Liu & Thorson, 1994
; Ma et al., 1997
). The amino acid sequence is most similar to GepiA (86% similarity) of M. avium; however, it is also similar to several possible dTDP-glucose 4,6-dehydrogenases of M. tuberculosis including RmlB2 (75% similarity). The M. smegmatis RmlB has a NAD cofactor-binding motif (TGGAGFIG) at the N-terminus and a second motif (YAATKLAQE) which is conserved in all members of the short-chain dehydrogenase/reductase family of enzymes, of which nucleotide sugar dehydratases are a subfamily (Baker & Blasco, 1992
; Gerratana et al., 2001
; Jornvall et al., 1995
).
Putative glycosyltransferases. There are three proposed glycosyltransferase genes in the GPL biosynthetic locus. These three ORFs, gtf1, gtf2 and gtf3, are similar to putative M. avium glycosyltransferases, GtfA (7585% similarity), GtfB (7178% similarity) and the authentic rhamnosyltransferase RtfA (7275% similarity) (Eckstein et al., 1998 ). Gft1 is also similar to M. tuberculosis Rv1524 (Cole et al., 1998
).
Acetyltransferase. The gene product of atf1 is thought to be an O-acetyltransferase because an M. smegmatis transposon mutant with a disrupted atf1 gene synthesized nonacetylated GPLs (Recht & Kolter, 2001 ). The deduced protein encoded by atf1 is 70% similar to the putative acetyltransferases of M. avium subsp. paratuberculosis (Bull et al., 2000
) and M. avium AtfA.
Methyltransferases. There are four ORFs that could encode methyltransferases in the GPL cluster. They all have conserved S-adenosylmethionine-binding motifs and, to varying degrees, display motifs conserved in other methyltransferases (Fig. 2) (Malone et al., 1995
). M. smegmatis mtf1 has been shown to encode a 3-O-methyltransferase that adds the first methyl group to the terminal rhamnose on the GPL core (Patterson et al., 2000
). M. smegmatis mtf3 is predicted to encode a protein that is similar to the authentic rhamnosyl O-methyltransferase ElmMIII of Streptomyces olivaceus (Patallo et al., 2001
) and the putative M. avium O-methyltransferases, MtfC and MtfB. M. smegmatis mtf4 is predicted to encode a protein that is similar to authentic rhamnosyl O-methyltransferases of S. olivaceus ElmMI (55% similarity) and ElmMII (52% similarity) (Fig. 2
). These analyses suggest that mtf1, mtf3 and mtf4 are likely to be required for the addition of the three methyl groups to GPL rhamnose or other sugars. This raises the question of the function of the fourth mtf gene, mtf2. This gene is predicted to encode a protein that is similar to two putative M. tuberculosis methyltransferases: Rv2952 (66% similarity) and Rv1523 (65% similarity) (Cole et al., 1998
). It is also similar to RapM (53% similarity) of the polyketide producer Streptomyces hygroscopicus.
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Disruption of mtf2 in M. smegmatis
We decided to disrupt the ORF of mtf2 to determine the role of the enzyme encoded by it. The disruption plasmid, pHBJ371 (see above) contained the mtf2 ORF disrupted by insertion of the 2 kb streptomycin-resistance marker cloned into a vector carrying a kanamycin-resistance marker and the sacB gene encoding levansucrase, but it lacked a mycobacterial origin of replication. M. smegmatis mc2155 was transformed with pHBJ371, and resulting transformants selected with streptomycin and kanamycin were then cultured on media containing streptomycin and sucrose to select for mutants that had undergone mtf2 gene replacement. Four kanamycin-sensitive, streptomycin- and sucrose-resistant transformants were characterized further.
Purified genomic DNA was digested with PstI and EcoRI and then transferred to a nylon membrane for Southern hybridization. The membrane was probed with a DIG-labelled probe generated by PCR amplification of mtf2 using the oligonucleotide primers #109 (5'-AGGCTGAAAGACGACGAC-3') and #239 (5'-GATTCGAGCGCATCGA-3'). Fig. 3 shows that the probe bound to a 4·2 kb fragment in mc2155 as expected. Three putative double crossover mutants each had a hybridizing band of 6·2 kb, indicating that gene replacement had occurred. In the remaining clone, the entire pHBJ371 had integrated through a single homologous recombination. The colony morphology of all the mutants was smooth and indistinguishable from the parent strain. A mutant that had undergone replacement of mtf2, Myco493, was selected for further analysis.
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DISCUSSION |
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Genes encoding the four enzymes for rhamnose synthesis are conserved among bacteria and are arranged in an operon in enteric bacteria (Stevenson et al., 1994 ). In all mycobacteria, including non-GPL-expressing species, rhamnose biosynthesis is of interest because this residue links the arabinogalactan portion of the cell wall core to peptidoglycan (McNeil et al., 1990
). Rhamnose biosynthesis has been delineated in M. tuberculosis and M. smegmatis and genes encoding the key enzymes have been characterized in M. tuberculosis (Cole et al., 1998
; Ma et al., 1997
). Unlike the situation in enteric bacteria the mycobacterial genes for rhamnose biosynthesis are dispersed around the chromosome in a similar fashion to that described in Saccharopolyspora spinosa (Madduri et al., 2001
). Rhamnose is also required for glycosylation of GPLs in M. smegmatis, and genes for glucose-1-phosphate thymidylyltransferase (rmlA) and dTDP glucose 4,6-dehydrogenase (rmlB) occur in the GPL biosynthetic locus. Rhamnose and 6-dTal are derived from identical biosynthetic precursors, with two dTDP-dehydrorhamnose reductases having opposite stereospecificity providing either TDP-rhamnose or TDP-6-dTal (Liu & Thorson, 1994
). The steps catalysed by RmlA and RmlB will therefore lead to synthesis of intermediates that could be converted to either of the sugars characteristic of GPLs.
Four methyltransferase genes occur in the GPL locus. The methyltransferases were tentatively divided into two groups on the basis of the amino acid sequence similarity in four methyltransferase motifs (Fig. 2). In DNA methyltransferases, motifs I and V contact S-adenosylmethionine, the methyl donor, and motifs IV and VI contribute to the structure of the active site (Malone et al., 1995
). Fig. 2
shows an alignment of the amino acid sequences that are conserved among selected authentic and putative methyltransferases. Group 1 includes the M. smegmatis methyltransferases (Mtf1, Mtf3 and Mtf4) which are like the authentic methyltransferases, ElmMI, ElmMII and ElmMIII, that methylate rhamnose during synthesis of the antitumour polyketide elloramycin in S. olivaceus (Patallo et al., 2001
). They are also similar to the putative methyltransferases of the ser2 cluster in M. avium (MtfB, MtfC and MtfD), which may carry out rhamnose modification in GPL synthesis. AveBVII, MycF and TylF methylate the sugar components in the macrolides avermectin, mycinamicin and tylosin, respectively (Fouces et al., 1999
; Ikeda et al., 1999
; Inouye et al., 1994
). If the grouping is significant then it may be used to predict that Mtf3 and Mtf4 are possible rhamnosyl methyltransferases.
In this study, we provide direct evidence that the M. smegmatis mtf2 gene encodes an O-methyltransferase that modifies the hydroxy group of the fatty acids rather than rhamnose. An mtf2 mutant was made by targeted gene replacement. The mutant, Myco493, synthesized GPLs with the same glycopeptide headgroup as the wild-type mc2155 strain. However, HPTLC and ESI-MS analyses revealed that the mutant lacked GPL molecular species with O-methylated fatty acids. Complementation of the Myco493 mutant with mtf2 restored the synthesis of these molecular species. Moreover, overexpression of mtf2 in wild-type bacteria resulted in the increased expression of these molecular species. Collectively, these data suggest that mtf2 encodes an O-methyltransferase that specifically modifies the GPL amide-linked fatty acid. This reaction presumably occurs early in GPL biosynthesis.
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ACKNOWLEDGEMENTS |
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Received 2 April 2002;
revised 10 June 2002;
accepted 11 June 2002.