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1 Howard Hughes Medical Institute, Department of Microbiology and Immunology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461, USA
2 School of Biosciences, University of Birmingham, Edgbaston, Birmingham B15 2TT, UK
Correspondence
William R. Jacobs, Jr
jacobsw{at}hhmi.org
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ABSTRACT |
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 TOP ABSTRACT INTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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; Puzo, 1990). Sulfolipids are one of the major M. tuberculosis cell wall lipids, of which sulfolipid-I (SL-I) is the most abundant (Goren, 1970a, b; Goren et al., 1976; Middlebrook et al., 1959). The presence of sulfolipids in virulent strains led to the speculation that this class of polar lipids plays an important role in pathogenesis. However, there have been mixed reports about the role of sulfolipids in virulence, with various modulatory effects being observed with purified sulfolipids (Okamoto et al., 2006; Pabst et al., 1988; Zhang et al., 1988), but no attenuation observed with mutant strains in mouse and guinea pig models of infection (Rousseau et al., 2003). SL-I consists of a tetra-acylated trehalose sulfate (Fig. 1): of the four acyl groups that are esterified to trehalose, one is palmitate while the remaining three are complex methyl-branched fatty acids, termed phthioceranic and hydroxyphthioceranic acids (Goren et al., 1971). The sulfation of trehalose is presumed to be the first step in SL-I assembly and is catalysed by the sulfotransferase Stf0 (Mougous et al., 2004). The palmitate is fatty acid synthase-I (FAS-I)-derived, the methyl-branched moieties, phthioceranic and hydroxyphthioceranic acids, are synthesized by a polyketide synthase, Pks2 (Sirakova et al., 2001). Present in the same cluster as pks2 is mmpL8, a gene encoding a transmembrane protein involved in transport (Converse et al., 2003; Domenech et al., 2004). Though the genes involved in the biosynthesis of individual components of SL-I are well studied, not much is known about the assembly of SL-I, vis-à-vis the components and sequence of events involved in acylation of trehalose-2-sulfate to SL-I. Located immediately downstream of pks2 is Rv3824c (papA1; Fig. 2a), which encodes a protein that belongs to a subfamily of acyltransferases associated with mycobacterial polyketide synthases [polyketide synthase associated proteins (PAPs)]. The presence of a characteristic HX3DX14Y acyltransferase motif suggested a role for PapA1 in the acylation steps of SL-I assembly. Indeed, PapA5, another PAP, has been shown to be an acyltransferase involved in the esterification of phthiocerol with methyl-branched mycocerosic fatty acids to form the virulence lipid phthiocerol dimycocerosate (PDIM) (Onwueme et al., 2004; Trivedi et al., 2005). In general, pks clusters in M. tuberculosis are associated with only one PAP-encoding gene (Cole et al., 1998). Interestingly, Rv3820c (papA2), a gene located 5.8 kb downstream of papA1 (Fig. 2a), also encodes a protein with an acyltransferase motif, indicating that SL-I biosynthesis may involve more than one PAP. A similar arrangement of homologues of pks2, papA1 and papA2 is also seen in the genome of the bovine pathogen Mycobacterium bovis (Garnier et al., 2003), although the M. bovis-derived vaccine strain BCG does not synthesize SL-I (Rivera-Marrero et al., 2002). In these studies we have used a genetic approach to investigate whether papA1 and papA2 play a role in SL-I biosynthesis. Individual null mutants of H37Rv papA1 and papA2 failed to produce SL-I. However, no intermediates were observed in either of the mutants, indicating an essential role for each acyltransferase in assembly of complete SL-I.
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METHODS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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. Mycobacterium tuberculosis H37Rv was cultured in 7H9 broth (Difco) containing 10 % Middlebrook OADC enrichment and 0.05 % Tween 80, or on Middlebrook 7H10 agar (Difco); Escherichia coli strains were cultured in LB broth. The concentrations of antibiotics used were 75 µg hygromycin ml–1 and 20 µg kanamycin ml–1 for H37Rv, and 150 µg hygromycin ml–1 and 40 µg kanamycin ml–1 for E. coli.
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1 kb sequences flanking the left and right of the M. tuberculosis H37Rv papA1 gene were PCR amplified from genomic DNA using the primer pairs papA1L1 (5'-CGCACTAGTGTGTTCTCCAGCAGCAACGG-3')/papA1L2 (5'-AGCAGATCTCGAGGTGTACTCGTGCTGCC-3') and papA1R1 (5'-TGCTCTAGAGAAAGGATGACATGGCGGTC-3')/papA1R2 (5'-AGACTTAAGGCAACGAGGCGCTGTGCAAC-3') respectively. Following cloning into pCR2.1-TOPO and sequencing, the cloned PCR fragments were excised using the primer-introduced restriction sites and cloned into the allelic-exchange plasmid vector pJSC347 (Table 1
). The resultant plasmid, pYUB2418, was then packaged into the temperature-sensitive phage phAE159 (J. Kriakov & W. R. Jacobs Jr., unpublished results) as described previously (Bardarov et al., 2002) to yield the papA1-knockout phage phAE405. In a similar fashion the primer pairs papA2L1 (5'-CGGACTAGTCAGCTTATGCGCACCAC-3')/papA2L2 (5'-AATCTCGAGCGGTGATGCGTGCCAAC-3') and papA2R1 (5'-CGGTCTAGAGTCGCCAATTACATCGCG-3')/papA2R2 (5'-AATGGTACCAGTGGCGTCTGGACGACG-3') were used to PCR amplify 1 kb sequences flanking the papA2 gene. Following sequence confirmation, the flanks were used to construct the allelic-exchange plasmid pYUB2419, which was then packaged into phAE159 to yield the papA2-knockout phage phAE406. The phages were used to generate allelic-exchange mutants using specialized transduction as described previously (Bardarov et al., 2002).
Complementation of mutant strains.
The primers papA1-F (5'-CATGAATTCGTGCGAATAGGACC-3') and papA1-R (5'-GGCGTTAACCTAAGCTTCTCTATC-3') were used to PCR amplify the papA1 gene from H37Rv genomic DNA. Following cloning into pCR2.1-TOPO and sequencing, the cloned PCR fragment was excised by digestion of the primer-introduced EcoRI and HpaI sites and cloned downstream of the hsp60 promoter in the integrative vector pMV361 (Stover et al., 1991). The resultant plasmid pMV361papA1 was introduced into 
papA1 by electroporation to yield the complemented strain papA1 : : pMV361papA1. Similarly, the primers papA2-F (5'-CTAAGCTTGTGTTTAGCATTACAAC-3') and papA2-R (5'-CGATCGATTCATGTGCCTGGTTTAAG-3') were used to PCR amplify the papA2 gene. Following subcloning and sequence confirmation, the PCR product was excised and cloned as a HindIII–ClaI fragment into pMV361 to yield the complementing plasmid pMV361papA2, which was then electroporated into papA2 to yield the complemented strain papA2 : : pMV361papA2.
The acyltransferase motif of papA1 was mutated using the Stratagene Quik Change site-directed mutagenesis kit, with pMV361papA1 as template DNA and the primer pairs papA1HA-F (5'-CAGCATCGATGCTCTGCATGCG-3')/papA1HA-R (5'-CGCATGCAGAGCATCGATGCTG-3') or papA1DA-F (5'-TCTGCATGCGGCCGGTCAGTTCG-3')/papA1DA-R (5'-CGAACTGACCGGCCGCATGCAGA-3'). The resultant plasmids, pMV361A1-HA or pMV361A1-DA, were then electroporated into papA1. In a similar fashion, the plasmids pMV361A2-HA and pMV361A2-DA were constructed using the primer pairs papA2HA-F (5'-GAGTATCGCTGCTCTCTGTGTC-3')/papA2HA-R (5'-GACACAGAGAGCAGCGATACTC-3') and papA2DA-F (5'-CTCTGTGTCGCCCCGATGATTG-3')/papA2DA-R (5'-CAATCATCGGGGCGACACAGAG-3') respectively, and then electroporated into papA2.
Biochemical analysis of mutant strains.
Labelling of M. tuberculosis cultures with [14C]acetate, [14C]propionate or Na235SO4 was done as described previously (Sirakova et al., 2001). Total lipids were extracted from strains using previously described protocols (Dobson et al., 1985) and separated on a TLC plate using chloroform/methanol/water (100 : 14 : 0.8, by vol.) in the first dimension and chloroform/acetone/methanol/water (50 : 60 : 2.5 : 3, by vol.) in the second dimension. The TLC plates were exposed to X-ray films for 24 h.
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RESULTS |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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) to replace papA1 in the M. tuberculosis H37Rv genome with a hygromycin resistance marker (hyg) (Fig. 2a). Genomic DNA obtained from HygR M. tuberculosis H37Rv colonies following transduction with phAE405 was digested with KpnI and analysed by Southern blotting (Fig. 2b). The band pattern obtained confirmed that papA1 was replaced by hyg. Similarly, the knockout phage phAE406 was used to obtain a papA2 null mutant of H37Rv (Fig. 2c). The papA1 and papA2 null mutants are referred to as papA1 and papA2 respectively.
SL-I is not synthesized in papA1
To determine changes in the lipid profiles of the papA1 mutant, we first monitored differences in the [14C]acetate-labelled lipids from wild-type and papA1 strains by TLC. In a solvent system designed to separate M. tuberculosis glycolipids (Dobson et al., 1985), a 14C-labelled lipid, which was clearly present in the wild-type but missing in the mutant strain, migrated in a manner similar to an authentic SL-I standard (Fig. 3a). Three of the four acyl components of SL-I are methyl-branched fatty acids (phthioceranate and hydroxyphthioceranate) which can be labelled using [14C]propionate. Indeed, the lipid missing in papA1, but present in WT showed high incorporation of 14C label when propionate was used as the radiolabel (Fig. 3b). Finally, this lipid also showed incorporation of 35S from Na235SO4, confirming that the species was indeed SL-I (Fig. 3b). Introduction of pMV361papA1, a single copy integrative vector containing papA1 cloned downstream of the hsp60 promoter, into papA1 fully restored SL-I biosynthesis, indicating that the observed phenotype in papA1 was solely due to deletion of papA1 (Fig. 3a, b). These results demonstrated that papA1 function was necessary for SL-I biosynthesis.
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papA1 strain was followed. An initial analysis of total lipids extracted from cultures of H37Rv and papA2 labelled with [14C]acetate indicated that SL-I was missing in the papA2 strain (Fig. 4a). This was then confirmed using [14C]propionate or Na235SO4 as the labelling reagents (Fig. 4b). SL-I biosynthesis could be restored in papA2 following complementation with pMV361papA2, a single-copy integrative vector containing papA2 cloned downstream of the hsp60 promoter (Fig. 4a, b). Thus, like papA1, papA2 was also essential for SL-I biosynthesis.
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; Lewendon et al., 1988). To demonstrate that papA1 and papA2 do encode acyltransferases, we tested the ability of mutant alleles of the genes containing alterations in either of these key residues to restore SL-I biosynthesis in the mutant strain. First, single-copy-integrative plasmids containing two mutant alleles of papA1 containing separate point mutations in the HX3DX14Y motif were constructed using site-directed mutagenesis (Fig. 5). The mutant constructs pMV361A1-HA (His-171
Ala-171) and pMV361A1-DA (Asp-175Ala-175) were then introduced separately into papA1 by electroporation and the resultant strains, papA1 : : pMV361A1-HA and papA1 : : pMV361A1-DA, were tested for functional complementation. Analysis of [14C]propionate- or 35S-labelled total lipids from the transformed strains revealed that neither construct reconstituted SL-I biosynthesis in papA1 (Fig. 3b). Similarly, the plasmids pMV361A2-HA (His-166Ala-166) and pMV361A2-DA (Asp-170Ala-170) failed to restore SL-I biosynthesis in papA2 (Fig. 4b). Thus, the key residues in the putative acyltransferase motifs of PapA1 and PapA2 were essential for functional complementation of the corresponding SL-I deficient mutants, indicating that PapA1 and PapA2 are acyltransferases involved in SL-I biosynthesis.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMETHODSRESULTSDISCUSSIONREFERENCES |
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). Based on the presence of an acyltransferase motif, it was speculated that PAPs may be acyltransferases involved in the assembly of complex lipid metabolites synthesized by the associated pks cluster. Experimental proof for this came first from in vitro experiments using purified PapA5, a PAP involved in PDIM biosynthesis (Onwueme et al., 2004; Trivedi et al., 2005). PDIM is made up of two mycocerosic acid residues esterified to the hydroxyl groups of a diol (phthiocerol). Using purified proteins and artificial substrates, Onwueme et al. (2004) first showed that PapA5 could catalyse esterification of an alcohol with an acyl group. Trivedi et al. (2005) then demonstrated that purified PapA5 catalysed trans-esterification of mycocerosic acid synthase-bound mycocerosic acids to dodecanol.
Of all the pks genes linked to PAPs, pks2 is the only one located next to two pap genes (papA3 and pks3/4 are involved in the biosynthesis of polyacylated trehalose, and the metabolite synthesized by papA4 and pks5 has not yet been identified). Deletion of pks2 resulted in loss of the principal sulfolipid SL-I in M. tuberculosis H37Rv (Sirakova et al., 2001). The absence of SL-I in papA1 and in papA2, the two mutants generated in this study, demonstrated that PapA1 and PapA2 were indeed functionally associated with Pks2. Phthioceranic acid and hydroxyphthioceranic acid, the methyl-branched products of Pks2, are found esterified to positions 3, and 6 and 6', respectively, of sulfated trehalose in the principal sulfolipid SL-I, while position 2 is substituted with FAS-I-derived palmitate. It was thus tempting to speculate that PapA1 and PapA2 may differ in their substrate specificities, with one PAP involved in the transfer of the methyl-branched acyl chains, and the other in that of palmitate. Another possibility was that one of the pks2-associated PAPs was specific for transfer of pthioceranic acid and the other for hydroxyphthioceranic acid, with the transfer of palmitate being carried out by an unknown acyltransferase. In either case, if the acylation reactions were sequential, partially acylated trehalose sulfate intermediates would be expected to accumulate in either papA1 or papA2. While both papA1 and papA2 did not synthesize SL-I, neither mutant strain showed accumulation of any partially acylated intermediate. This result indicated that in one of the mutants, feedback inhibition of the initial step may have prevented the accumulation of the intermediate. Alternatively, instead of a sequential addition of different acyl groups to sulfated trehalose by PapA1 and PapA2, concurrent presence of both proteins may be required for complete assembly of a tetracylated trehalose sulfate molecule. Some indication that this might be a possibility comes from recent work on the components of the PDIM biosynthetic cluster. Jain & Cox (2005) showed that polyketide synthase PpsE directly interacted with the transmembrane protein and PDIM transporter MmpL7. This suggested that not only was MmpL7 involved in transport, but that it also probably acted as a scaffold in a tightly integrated system that couples the biosynthesis of PDIM to its secretion from the bacterial cell. In light of these findings it is plausible that MmpL8, the transporter associated with the pks2 cluster, may play a similar ‘scaffolding’ role in SL-I assembly through interactions with Pks2, PapA1 and Pap2, wherein PapA1 and PapA2 may carry out localized and simultaneous acylation reactions. In the papA1 or papA2 mutant, loss of one PAP would result in non-functional SL-I assembly machinery and complete loss of SL-I biosynthesis (rather than accumulation of a partially acylated intermediate). However, two independent reports of a M. tuberculosis mmpL8 null mutant showed that the loss of MmpL8 resulted in the accumulation of a diacylated trehalose sulfate intermediate (Converse et al., 2003; Domenech et al., 2004). Nevertheless, we did not detect accumulation of such an intermediate in either the papA1 or the papA2 mutant. Though the absence of any partially acylated intermediates in either papA1 or papA2 made it difficult to assign putative substrate specificities (if any) to PapA1 and PapA2, our results have clearly demonstrated that these two distinct acyltransferases are involved in the biosynthesis of the major sulfolipid SL-I in M. tuberculosis.
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ACKNOWLEDGEMENTS |
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Edited by: S. V. Gordon
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Received 3 October 2006;
revised 18 October 2006;
accepted 30 October 2006.
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32P]dCTP-labelled probes were derived from 
-resolvase site; hyg, hygromycin resistance gene. (b) Southern blot of KpnI-digested genomic DNA from H37Rv and 
