1 Unité de Génétique Moléculaire Bactérienne, Institut Pasteur, 28 Rue du Docteur Roux, 75725 Paris Cedex 15, France
2 Plate-Forme 4 Intégration et analyse génomiques, Génopole, Institut Pasteur, 28 Rue du Docteur Roux, 75725 Paris Cedex 15, France
3 Australian Bacterial Pathogenesis Program, Department of Microbiology, Monash University, Clayton, 3800, Australia
Correspondence
Timothy P. Stinear
tim.stinear{at}med.monash.edu.au
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ABSTRACT |
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The GenBank/EMBL/DDBJ accession number for the sequence of pMUM001 and its annotation reported in this paper is BX649209.
A summary of the 81 predicted CDS in Mycobacterium ulcerans pMUM001 is shown in Supplementary Table S1, available with the online version of this paper at http://mic.sgmjournals.org.
Present address: Department of Biochemistry and Molecular Biology, Monash University, Clayton, Australia.
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INTRODUCTION |
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We recently reported the presence in M. ulcerans of a 174 kb circular plasmid, named pMUM001 (Stinear et al., 2004). More than half of the plasmid is composed of three highly unusual polyketide synthase (PKS) genes that are required for the synthesis of the polyketide toxin mycolactone. There is a precedent for plasmid-borne genes involved in secondary metabolite biosynthesis. The pSLA2-L plasmid from Streptomyces rochei is rich in genes encoding type I and type II PKS clusters, and non-ribosomal peptide synthetases (Mochizuki et al., 2003
). The three mycolactone PKS genes (mlsA1, mlsA2 and mlsB) stand out for two reasons. First, they encode some of the largest proteins ever reported (MLSA1, 1·8 MDa; MLSA2, 0·26 MDa; and MLSB, 1·2 MDa); second, there is an extreme level of nucleotide and amino acid sequence conservation (>97 % nucleotide identity) among the various functional domains of the 18 modules that comprise the three synthases. This extended level of sequence conservation is unprecedented and points to the very recent evolution of this locus.
Large plasmids harbouring factors that confer key adaptations to new niche environments are a recurring theme amongst bacterial pathogens and allow the host to make large evolutionary leaps over small evolutionary time scales. Well-known examples among human bacterial pathogens include the pXO plasmids of Bacillus anthracis (Okinaka et al., 1999), the pYV plasmids of Yersinia pestis (Parkhill et al., 2001
), and the pINV plasmids of Shigella (Buchrieser et al., 2000
). Each of these species is highly clonal. They share a nearly identical genome structure and sequence with other species in their genera, and via plasmid acquisition, sometimes in concert with other more subtle genome changes; they have evolved adaptive advantages for new environments, adaptations that may also present a pathogenic phenotype. Recent evolution by horizontal transfer fits well with the hypothesis that M. ulcerans is a clonal derivative of Mycobacterium marinum, a hypothesis formed from multi-locus sequence typing and hybridization analyses that showed a high level of DNA sequence conservation and gene synteny between these phenotypically diverse species (Stinear et al., 2000
).
Plasmids have been widely reported among many mycobacterial species (Pashley & Stoker, 2000). However, until the discovery of pMUM001, mycobacterial plasmids have never been directly linked to virulence, and the absence of plasmids among members of the Mycobacterium tuberculosis complex has led researchers to believe that plasmid-mediated lateral gene transfer is not an important factor for mycobacterial pathogenesis. Very few mycobacterial plasmids have been characterized, with complete DNA sequences available for only three mycobacterial episomes: pAL5000, a 4·8 kb circular element from Mycobacterium fortuitum (Rauzier et al., 1988
); pCLP, a 23 kb linear element from Mycobacterium celatum (Le Dantec et al., 2001
); and pVT2, a 12·9 kb element from Mycobacterium avium (Kirby et al., 2002
). There are very few reports of functions being assigned to mycobacterial plasmids, although several studies have suggested that genes involved in different forms of hydrocarbon metabolism are plasmid borne (Coleman & Spain, 2003
; Guerin & Jones, 1988
; Waterhouse et al., 1991
).
There are 81 predicted protein-coding sequences (CDS) on pMUM001 and we have previously described in detail the six CDS that are involved with the synthesis of mycolactone (Stinear et al., 2004). In this present study, the remaining 75 CDS are described with a functional study of the plasmid replication region.
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METHODS |
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Nucleic acid techniques.
General methods for DNA manipulation were as described by Sambrook et al. (1989). For Southern hybridization experiments, DNA was extracted from mycobacteria as described by Boddinghaus et al. (1990)
. Approximately 1 µg DNA was digested with NsiI and the resulting fragments were separated by agarose gel electrophoresis. The DNA was then transferred to Hybond-N+ membranes by alkaline capillary transfer in the presence of 0·4 M NaOH. A DNA probe based on the repA gene was prepared by PCR-mediated incorporation of Digoxigenin dUTP into the 413 bp repA amplification product. This product was obtained using the primer sequences RepA-F, 5'-CTACGAGCTGGTCAGCAATG-3' (position 665684) and RepA-R, 5'-ATCGACGCTCGCTACTTCTG-3' (position 10771058). Genomic DNA from MUAgy99 was used as template. Southern hybridization conditions were as described previously (Stinear et al., 1999
).
Construction of the shuttle plasmid pMUDNA2.1.
As part of the M. ulcerans genome sequencing project (http://genopole.pasteur.fr/Mulc/BuruList.html), a whole-genome shotgun clone library of M. ulcerans strain Agy99 was prepared in E. coli using the vector pCDNA2.1 (Invitrogen) (Stinear et al., 2004). In this library were several E. coli shotgun clones that contained M. ulcerans sequences overlapping the predicted origin of replication (ori) of pMUM001. One such clone called mu0260E04 with an insert of 6 kb was selected for further study. To permit selection in a mycobacterial background, the apramycin resistance gene aac(3)-IV was cloned into mu0260E04 (Paget & Davies, 1996
). This was achieved by PCR amplification and modification of the aac(3)-IV cassette using the oligonucleotides ApraF-SpeI (5'-GGACTAGTCCCGGGTTCATGTGCAGCTC-3') and ApraR-SpeI (5'-GGACTAGTCCCGGGCATTGAGCGTCAGCAT-3') to incorporate flanking SpeI sites (underlined). The resultant PCR product was digested with SpeI and then cloned into the unique XbaI site of mu0260E04, resulting in the hybrid vector pMUDNA2.1 (see Fig. 1
). The deletion constructs pMUDNA2.1-1 and pMUDNA2.1-3 were prepared by double restriction-endonuclease digestion of pMUDNA2.1 with HpaI/SpeI and EcoRV/SpeI, respectively. Two mycobacteria/E. coli shuttle vectors were used as a positive controls in all transformation experiments. These were (i) the autonomously replicating vector pMV261, which is based on the pAL5000 replicon and confers resistance to kanamycin (Snapper et al., 1990
) and (ii) an integrating vector pJKD8003, based on mycobacteriophage L5 (Hatfull & Sarkis, 1993
) and containing the same aac(3)-IV apramycin resistance gene as used for the construction of pMUDNA2.1. Conditions for the preparation and electroporation of M. smegmatis were as previously described (Snapper et al., 1990
). For electroporation of other mycobacteria, cells were harvested at room temperature from late-exponential-phase cultures, washed twice in sterile water, then once in sterile 10 % glycerol (v/v) and finally resuspended in 0·01 volume of 10 % glycerol (v/v). In all experiments a 200 µl aliquot of freshly prepared cells was used for each electroporation with a BTX electroporator (Genetronics) at 2·5 kV, 25 µF and 1000
. After pulsing, 1 ml Middlebrook 7H9 medium was added to the cells and they were incubated overnight at 30 °C with shaking before plating on Middlebrook 7H10 agar containing the appropriate antibiotic. The following quantities of plasmid DNA were used in each transformation in a final volume of 5 µl: pAL5000, 150 ng; pJKD8003, 55 ng; pMUDNA2.1, 780 ng; pMUDNA2.1-1, 360 ng; pMUDNA2.1-3, 230 ng. Transformation experiments were conducted in triplicate (i.e. three biological repeats from different preparations of competent cells). The efficiency of transformation (EOT) was expressed as the mean number of transformants±SD per µg plasmid DNA.
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Bioinformatic analysis.
Sequence analysis and annotation of the plasmid were managed using ARTEMIS, release 5 (http://www.sanger.ac.uk/Software). Potential CDS with apppropriate G+C content, correlation scores and codon usage were compared with sequences present in public databases using FASTA (Pearson & Lipman, 1988), BLAST (Altschul et al., 1990
) and CLUSTAL W (Thompson et al., 1994
). Additional functional insight was gleaned using the Prosite (Hulo et al., 2004
) and Pfam (Bateman et al., 2002
) databases, and the TMHMM program (Sonnhammer et al., 1998
) was used to predict transmembrane helices. IS family designations were made after reference to the IS database (http://www-is.biotoul.fr/). The sequence of pMUM001 and its annotation have been previously deposited in the GenBank/EMBL/DDBJ databases under accession no BX649209 (Stinear et al., 2004
).
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RESULTS AND DISCUSSION |
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Analysis of the sequence 1 to 600 bp of repA revealed several features suggestive of an iteron-containing origin of replication. Iterons are direct repeat sequences that bind RepA and exert control over plasmid replication. A single pair of 16 bp iterons was identified in the region 180550 bp upstream of the repA initiation codon (Fig. 3). The spacing between iterons is usually a multiple of 11, a distance reflecting the helical periodicity of dsDNA, implying that the binding sites for RepA are on the same face of the DNA (del Solar et al., 1998
). The spacing for the iteron identified in pMUM001 is 143 bp, a multiple of 11. Low plasmid copy number is a characteristic of iteron plasmids. It has been proposed that as copy number increases, the RepA molecules bound to the iteron of one origin begin to interact with similar complexes generated on other origins, generating a so-called hand-cuffed state that suppresses replication (del Solar et al., 1998
). Other features commonly associated with iteron-containing replicons are multiple inverted repeats of partial-iteron sequences. These are generally situated immediately upstream of the repA start codon in the repA promoter region (del Solar et al., 1998
). In pMUM001, the situation appears somewhat different. A single 12 bp partial inverted repeat of the iteron sequence was detected in the region between the iterons. No obvious promoter elements were found in these upstream sequences; however, the region 1 to 261 bp of the repA ATG shares a very high identity with the same region in pJAZ38 (75 % nucleotide identity) and a 69 bp subsection of this region is highly conserved among mycobacterial plasmids (Picardeau et al., 2000
) (Fig. 3
), suggesting that this region plays an important but as-yet-unidentified role for plasmid replication.
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To test the hypothesis that this region contains a functional replication origin, a small-insert (36 kb) E. coli shotgun library of pMUM001 was screened and a clone with a 6 kb fragment was selected. This fragment spanned the region from position 172 467 to 4190 that encompassed the 5 end of MUP081 and the putative ori, repA and parA genes. The clone, named pmu0260E04, was modified by the insertion of aac(3)-IV, a gene conferring resistance to apramycin and thus permitting selection in a mycobacterial background (Paget & Davies, 1996). This construct, named pMUDNA2.1 (Fig. 1
), was used to try and transform M. smegmatis, M. fortuitum and M. marinum. Transformants were only obtained for M. marinum (Table 1
). The efficiency of transformation (EOT) of M. marinum transformed with pMUDNA2.1 was close to the EOT obtained using the pAL5000-based shuttle plasmid pMV261 (Table 1
). Deletion studies were then conducted to try and define the minimum region of pMUM001 required for replication. Two deletion constructs of pMUDNA2.1 were made. The first construct (pMUDNA2.1-1) was made by removing the 1300 bp region between the unique SpeI and HpaI sites. This region spans the entire parA gene and 372 bp of upstream sequence (Fig. 1
). The second construct (pMUDNA2.1-3) was made by deleting the 2610 bp region between the unique SpeI and EcoRV sites. This 2610 bp segment spanned all of the pMUDNA2.1-1 deletion plus the predicted CDS MUP003 and MUP004. Both these constructs were capable of transformation of M. marinum with an EOT equivalent to that obtained from pMUDNA2.1 (data not shown). This result suggests that the 3327 bp of pMUM001 sequence spanning MUP002, repA, oriM and the partial sequence of MUP081 is sufficient to support replication.
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Membrane-associated proteins
Significant amounts of mycolactone can be detected in an M. ulcerans culture supernatant, suggesting that there may be active transport of the molecule out of the bacterial cell. Lipid export in other mycobacteria is known to involve large transmembrane proteins, such as the MmpLs, which in M. tuberculosis are found clustered with genes involved in lipid metabolism, including type I PKS (Tekaia et al., 1999). Analysis of the pMUM001 sequence revealed no mmpL-like genes. Ten hypothetical proteins that may play a role in mycolactone export were identified, as they contained features such as membrane-spanning domains, signal sequences, lipoprotein attachment sites or hydrophobic N-terminal sequences (see Supplementary Table S1). Whatever their function, the 10 CDS listed in Supplementary Table S1 may encode surface-exposed antigens and, given the absence of orthologues in available databases, they may be interesting candidates for testing as M. ulcerans-specific antigens with potential application in serodiagnosis or vaccine development.
Insertion sequences
Based on the presence of characteristic transposase sequences, 26 copies of various IS or IS-like sequences were identified on pMUM001. They are distributed throughout pMUM001 and interspersed among defined functional CDS clusters (e.g. replication, maintenance, toxin production). Twelve IS were copies of the known M. ulcerans elements, IS2404 and IS2606 (Stinear et al., 1999), and the remaining 14 were previously unreported (Fig. 2
, Table 2
). Transposase sequence comparisons revealed related proteins in other actinomycetes and in more distant genera. There were three copies of a putative IS belonging to the IS4 family (MUP025, MUP028, MUP037). However, each copy of this element had been disrupted by insertion of another element (IS2404 for MUP028, and IS2606 for MUP025 and MUP037), thus precluding delineation of this IS. The sequences bounded by the ends of the loading module domains of mlsA1 and mlsB and extending through to MUP035 and MUP043 represent 8 kb of identical nucleotide sequence (Fig. 1
). This region also contains three different pairs of putative IS (MUP033 and MUP041, MUP034 and MUP042, MUP035 and MUP043). Since the flanking sequences for these IS are also identical, the IS boundaries could not be determined. There is remarkably little distance (90 bp) between the initiation codons of the PKS genes mlsB and mlsA1 and the transposase genes (MUP033 and MUP041) that precede each of them. This raises the possibility that the promoter region for the two PKS genes lies within these IS elements.
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BLASTN analysis of the 26 IS sequences against the draft M. ulcerans genome sequence did not reveal any paralogous elements on the M. ulcerans chromosome, with the exception of IS2404 and IS2606.
IS2404 and IS2606 have been previously reported as high-copy-number elements associated with M. ulcerans (Stinear et al., 1999). Four copies of IS2404 were identified on pMUM001. The original description of IS2404 reported an element of 1274 bp, with 12 bp inverted repeats, that encodes a putative transposase of 348 aa and produces 6 bp target-site duplications. It is now apparent that IS2404 exists in at least two forms (IS2404a and IS2404b), both forms 94 bp longer than previously described. There was one copy of IS2404a, an element of 1368 bp, containing 41 bp perfect inverted repeats (sequence 5'-CAGGGCTCCGGCGTTGTTGATTAGCAGGCTTGTGAGCTGGG-3') and possibly producing 10 bp target-site duplications.
Both forms of IS2404 are predicted to encode a single transposase of 348 aa. IS2404b is the same as IS2404a in all respects, except that it contains an internal stop codon, resulting in predicted transposase fragments of 234 aa and 113 aa. However, there is probably read-through of this stop codon, as there are three copies of IS2404b, suggesting that the element may still be capable of transposition.
Eight copies of the element IS2606 were also identified. It, too, was found to be larger than the 1406 bp initially reported (Stinear et al., 1999). It has a size of 1438 bp, with 31 bp imperfect inverted repeats, producing target-site duplications of 7 bp and encoding a putative transposase of 444 aa. One copy contained a frame-shift mutation (MUP060 and MUP061) within the transposase region.
Concluding comments
Megaplasmids (50500 kb) are widespread across many bacterial genera and represent a major resource for lateral gene transfer within microbial communities. Genetic mosaicism has emerged as a common structural theme for these elements (Molbak et al., 2003) and is particularly evident in pMUM001, which is similar in size to certain mycobacteriophages such as Bxz1 that also display a mosaic arrangement (Pedulla et al., 2003
). In part, the mosaic arrangement may stem from the large number of IS elements carried by pMUM001. These are present in both direct and inverted orientations, and recombination between these repeats is expected to contribute to variation in both plasmid size and function. An example of this has already been reported (Stinear et al., 2004
).
In this study, we have identified the Rep locus, required for replication, and demonstrated functionality. The resultant shuttle plasmid, pMUDNA2.1, should be useful for genetic analysis of both M. marinum and M. ulcerans. Furthermore, the replicon of pMUM001 may even facilitate the production of mycolactone in a heterologous host although, given the large size of the PKS genes and their repetitive nature, this may be technically challenging. Nonetheless, heterologous expression would represent an important step forward in the functional analysis of mycolactone biosynthesis and even open new prophylactic avenues for preventing BU.
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ACKNOWLEDGEMENTS |
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Received 5 October 2004;
revised 3 December 2004;
accepted 16 December 2004.
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