BACKGROUND
mycolactone is a polyketide-derived, secondary metabolite and a major virulence factor of the human pathogen mycobacterium ulcerans , the causative agent of buruli ulcer.
at picogram concentrations mycolactone has immunosuppressive properties and at higher concentrations it is cytotoxic for mammalian cells  <cit> .
the molecule is composed of an invariant core comprising a 12-membered macrolactone and side-chain that is esterified to a highly unsaturated acyl side chain, the latter structure varying amongst different mu strains   <cit> .
mu strains from africa, australia and china produce variants named mycolactones a/b, c, and d, respectively whilst mycobacterium liflandii , a pathogen of frogs, produces mycolactone e, and the fish pathogens  and others) produce mycolactone f  <cit>  .
despite the multiple species names given to mycolactone-producing mycobacteria , multi locus sequence analysis  of all these strains indicates they share greater than 98% nucleotide identity  <cit> .
the mpm appear to have evolved from a common m. marinum ancestor by acquisition of a large circular plasmid that conferred the ability to make mycolactones and then spread throughout the world, occupying different hosts  <cit> .
in mu strain agy <dig>  the only strain for which a genome sequence is currently available, a  <dig> kb megaplasmid named pmum <dig> has three very large genes    <cit>  that encode the modular type i pkss required for mycolactone synthesis.
the plasmid also has three putative accessory genes .
mlsa <dig> and mlsa <dig> form a nine-extension module complex that synthesises the mycolactone core, whilst mlsb is a single polypeptide, comprising seven extension modules that are required for the synthesis of the side chain.
bacterial type i pks are modular multi-enzymes and act as molecular assembly lines for the formation of polyketides  <cit> .
these enzymes function in a sequential manner where each pks module is responsible for one round of chain elongation via the addition of  either acetate or propionate, supplied to the pks as an activated malonyl or methylmalonyl-coa thioester.
within each pks module are a series of covalently linked enzymatic domains that process the growing polyketide chain before passing it downstream to the next module in the system  <cit> .
the minimal set of enzymatic domains required for pks activity includes ketosynthase , acyltransferase  and an acyl carrier protein  domain  <cit> .
ketoreductase , dehydratase  and enoylreductase  domains are also commonly found in modules and form a so-called reductive loop, providing reducing enzyme activities that modify the two or three-carbon unit being added to the polyketide  <cit> .
the mycolactone pks  exhibits a number of unusual features that distinguish it from other type i pks complexes.
firstly, the mls pkss are exceptionally large with a total predicted monomeric size of ~ <dig>  mda, placing them amongst the largest known cellular enzymes  <cit> .
secondly, there is an unprecedented level of genetic identity amongst the enzymatic domains of all mls modules.
for other type i pkss, functionally identical domains from the same pks generally share  <dig> – 70% amino acid  sequence identity  <cit> , however, identity between domains of the mls locus ranges from  <dig>  – 100%  <cit> .
the extreme sequence homology within the mls locus might be expected to provide a rich substrate for homologous recombination.
indeed, mycolactone negative mutants frequently arise among laboratory passaged mu strains; caused by partial deletion of the mls genes  <cit> .
mycolactone d produced by an mu strain from china differs from mycolactone a/b by the substitution of a methylene at c2' of the acyl side chain .
in this strain, the final extension module of mlsb possesses an at domain with propionate rather than acetate specificity, suggesting natural recombination within mlsb  <cit> .
however, mycolactone structural variations are quite restricted.
mpm recovered from around the world for  <dig> years all produce mycolactones with an absolutely conserved core and all variations occur within the fatty-acyl side-chain .
the role mycolactones play in the survival of mpm and why variation is only tolerated  in the side-chain is unknown.
in this study we investigated the genetic basis for production of these variant structures, in particular for the frog pathogen m. liflandii.
we determined the complete dna sequence of the  <dig> kb megaplasmid  from mycobacterium liflandii 128fxt and the partial sequence of pmum replicons from mycobacterium marinum dl <dig> and m. ulcerans japan  <dig>  we also employed lc-ms/ms structural analysis of their respective mycolactones.
our results show that mycolactones produced by different mpm are caused by genetic rearrangements between homologous pks domains of the mls locus, highlighting the plasticity of this region and its potential for combinatorial polyketide biochemistry.
RESULTS
overview of pmum <dig> from m. liflandii 128fxt
assembly of the complete dna sequence of pmum <dig> from four overlapping bac clones , revealed a  <dig>  bp circular element containing  <dig> predicted cds.
a summary of the main features of pmum <dig> compared with pmum <dig> is shown in table  <dig> and an overview of its cds distribution is shown in figure 2b.
* percentage based on predicted size of plasmid
plasmid pmum <dig> encodes  <dig> of its cds  on the reverse strand and has a g+c content of  <dig> %, which is similar to pmum <dig>   <cit> .
five genes, spanning  <dig>  kb  of the plasmid, are predicted to be involved in mycolactone biosynthesis, with the same arrangement as those present on pmum <dig>  <cit> .
these genes include the three type i polyketide synthases , a type ii thioesterase  and a fabh-like type iii ketosynthase .
sequencing of the plasmid confirmed the previously reported absence of the cytochrome p <dig> hydroxylase gene cyp140a <dig>  <cit> , which is responsible for the production of mycolactones a/b and d via hydroxylation at the c' <dig> position of the acyl side chain of these variants  <cit> .
overview of pmum <dig> from m. marinum dl240490
three bac clones from a dl genomic library were predicted to span all of pmum <dig>  and these clones were selected for further analyses.
based on the sizes of these bacs as determined by pulsed field gel electrophoresis  and the results of end-sequencing, a map of pmum <dig> was constructed indicating it is a circular replicon with an estimated size of ~ <dig> kb .
due to the complex and time-consuming nature of sequencing mycolactone pks loci caused by the extreme sequence repetition, only the non-pks region of pmum <dig> was fully sequenced for this study.
the bac clone 0412d spanned this region of the plasmid from the 5' end of mlsb to the 3' end of mlsa <dig>  and the complete sequence of 0412d was  <dig>  bp with  <dig> predicted cds.
the two other selected bac clones  were used in pcr, sequencing and southern blot analyses to confirm the domain and module composition of specific regions of the pmum <dig> mls genes .
comparison of pmum plasmids confirms their common origin
both pmum <dig> and pmum <dig> share the same overall organization as pmum <dig> with a core set of  <dig> cds common to all three plasmids .
a complete list of cds from pmum <dig> and the non-pks region of pmum <dig> is presented and discussed in additional files  <dig> and  <dig>  the pmum <dig> gene mup <dig> encodes a putative fabh-like type iii ketosynthase gene that is essential for mycolactone production  <cit>  and may have acyltransferase activity, linking the mycolactone core and acyl-side chain.
this gene is present in all three plasmids  and they share very high sequence identity; their predicted protein products differing by only one amino acid, suggesting the importance of the gene for mycolactone biosynthesis.
there were also several regions of difference observed between the plasmids that included deletions, insertions and other genetic rearrangements.
these alterations are presumably mediated by the many ise found in all pmum plasmids.
for example, the absence of cyp140a <dig> in pmum <dig> &  <dig>  encoding a p <dig> hydroxylase that modifies mycolactone, is most likely explained by an is2606-mediated deletion .
overall, it appears that the main function of the pmum plasmids is for the production of mycolactones with no other obvious virulence or virulence-associated genes present.
the pmum <dig> mls module and domain arrangements correspond with the structure of mycolactone e
the complete sequencing of the mls locus of pmum <dig> from ml revealed an extraordinarily high degree of similarity to the mls locus of pmum <dig> with a module and domain arrangement in near-perfect agreement with predictions based on the proposed structure of mycolactone e  <cit> .
the core macrocyclic lactone of mycolactone e is synthesised by mlsa <dig> and mlsa <dig>  comprising a loading module and nine extension modules, terminating in a putative integral c-terminal thioesterase .
similarly the acyl-side chain is synthesised by mlsb and comprises a load module with six extension modules .
mlsb from pmum <dig> is  <dig>  aa, which is  <dig>  aa shorter than mlsb from pmum <dig> .
the size difference is due to the absence in pmum <dig> of the equivalent of module  <dig> from pmum <dig>  an absence that corresponds precisely with the absence of a ch = ch moiety in the acyl-side chain of mycolactone e  <cit>  .
there was also perfect agreement between observed structure and the pattern of acetate and propionate incorporation predicted from sequence analysis of the at domains.
in particular, the at domain of the mlsb load module is methylmalonyl-coa-specific , indicating that ml uses a propionate starter unit which corresponds perfectly with structure-based predictions  <cit> .
these results provide another example of a "swappable" domain location as the equivalent at domain in pmum <dig> uses an acetate starter unit .
the oxidation state predicted from the mls sequence after each stage of chain extension also aligns very closely with mycolactone e structure.
however, like pmum <dig>  extension module  <dig> of mlsa <dig> contains apparently inactive dh and er domains  <cit> .
to reinforce the veracity of the above findings and to resolve an outstanding discrepancy regarding the correct structure of mycolactone e  <cit> , lc-ms/ms analysis was used to analyse mycolactones detected in lipid extracts from two additional ml strains .
these analyses revealed an identical ion trace and fragmentation pattern for m/z  <dig>  as reported for ml 128fxt .
thus, the combination of these concordant structural data from multiple ml strains together with the sequence analysis of the mls genes from ml 128fxt provide compelling evidence that the mycolactone structure first reported by hong et al  <cit>  is correct.
alterations in pmum <dig> mlsb module and domain arrangement correspond with the structure of mycolactone f
although the complete sequence of the mls locus from pmum <dig> was not determined in this study, the two bac clones 048f and 051b that spanned mlsa and mlsb respectively  permitted some investigations into the genetic basis for the production of mycolactone f from dl.
using bac clone 051b as template, pcr and sequencing of the load module of mlsb identified that this module has an at-i  domain, indicating an acetate starter unit for mycolactone f side-chain synthesis.
this arrangement, which is supported by structural data  <cit>  is the same as pmum <dig> but different to ml which has an at-iii  domain .
the recent total synthesis of mycolactone f showed that the hydroxyl groups at c11' and c13' of its acyl side chain have the opposite stereochemistry to other mycolactones  <cit> .
kr domains control the geometry of the hydroxyls and two types of this domain have been noted , with each type responsible for a specific stereochemistry  <cit> .
as the c11' and c13' side-chain hydroxyl arrangement reported for mycolactone f had not been observed in other mycolactones, it suggested that a switch from a-type to b-type kr domains had occurred within the first two extension modules of mlsb from pmum <dig>  to test this hypothesis, we performed southern hybridisation analysis of bacs containing either mlsa or mlsb from pmum <dig>  pmum <dig> and pmum <dig> with probes for either the kr-a or kr-b domain, and as predicted, southern analysis confirmed that there are no a-type kr domains in mlsb of pmum <dig> , consistent with our hypothesis that the a-type kr domains have been replaced.
this result also suggests the presence of a previously unidentified mls module type consisting of a combination of ks, at-ii  and b-type kr domains .
mlsb module  <dig> is different between mu strains from china and japan
the above examples of module and domain rearrangement prompted a closer inspection of the mycolactones produced by mu strains from japan and china.
mu strains from these countries are genetically very closely related with only one discriminating allele by mlsa and identical indel patterns  <cit> .
previous investigations had revealed that strain mu <dig> from china makes a modified mycolactone  due to the presence of an at-iii  domain in extension module  <dig> of mlsb  <cit> .
however, the only published report of the structure of mycolactones from japan suggested that japanese strains made mycolactone a/b  <cit>  and not mycolactone d as might have been expected.
we used pcr and sequencing of mlsb module  <dig> from the japanese mu strain  <dig> to check the domain arrangement and found that it has an at-ii  domain , which is different to that of mu <dig> china but which is the expected arrangement for mycolactone a/b synthesis .
analysis of lipid extracts from mu <dig> japan by lc-ms/ms confirmed that this strain does make mycolactone a/b, not mycolactone d   <cit> .
sequence comparisons of this region from mu <dig> with the same region among mu <dig> and muagy <dig> africa shows that module arrangements and the corresponding mycolactone produced does not necessarily correlate with strain relatedness , indicating that remodelling of the mls genes is occurring at a frequency independent of single nucleotide mutation and highlighting the dynamic nature of this locus.
ongoing replacement, duplication and deletion of the highly conserved mls modules and domains
as observed in pmum <dig>  the pmum <dig> mls dna is highly repetitive and exhibits extreme sequence conservation between domains with an identical catalytic function.
for example, the  <dig> ks domains share >  <dig> % nucleotide identity over  <dig> nts, which translates to only nine variable amino acid residues among  <dig> aa.
there are three types of domains  that have 100% intra-species nucleotide identity within both pmum <dig> and pmum <dig>  the extreme sequence identity is also conserved between strains and falls only marginally to  <dig>  –  <dig> % for these domains .
the most variable of the enzymatic domains between species are the acp-i domains .
when modules of identical domain arrangement were compared, nucleotide identity ranged from  <dig> %  to  <dig> % .
phylogenetic analysis of the domains and modules across pmum <dig> and pmum <dig> emphasise the high degree of relatedness between these genetic loci but also show that they cluster by strain, suggesting that evolution of the locus is occurring vertically rather than via horizontal exchanges between strains .
* comparisons of plasmid and chromosomal sequences are taken from mulit-locus sequence analysis of concatenated dna sequences of  <dig> nts and  <dig> nts respectively  <cit> .
the previously reported domain swap in mlsb of mu <dig> china suggested either replacement of domains, combinations of domains, or entire modules, possibly by a gene conversion mechanism.
a detailed comparison of modules with an identical domain configuration revealed that identity between one or more domains does not equate with identity over the whole module .
for example, when an interspecies nucleotide comparison is performed across the region from the at-ii domain to the kr-a domain of module  <dig> of mlsa and modules  <dig> and  <dig> of mlsb, there is 100% nucleotide identity over  <dig> nts.
however, the ks and acp domains of these modules have only  <dig> % and  <dig> % identity, respectively.
when other modules of identical organisation are analysed in this manner, similar patterns are seen .
another example of this is the presence of an active dh domain in the lm of mlsb of pmum <dig>  which follows an at-iii domain.
in pmum <dig>  the two domains seen in these positions are a dh domain, with a predicted inactive catalytic site, and an at-i domain .
in no part of mlsa <dig>  mlsa <dig> or mlsb of pmum <dig> or pmum <dig> is an inactive dh paired with an at-iii domain.
as the dh and kr domains are functionally redundant within the lm, this does not indicate a functional constraint imposed on this pairing, but does suggest that the at-iii and dh domains 'move' as a group.
this may also be the case for the er and kr-a domains that always appear in modules with the same domain organization.
this is further highlighted by an intra-species comparison of the domain structure of the load modules of mlsa and mlsb.
individual domains of both the mlsa and mlsb load modules of pmum <dig> are identical, with the exception of the at-iii and active dh domains .
the simplest explanation for the presence of these domains in otherwise identical sequences is introduction via recombination with the same domain cluster from a neighbouring donor module.
due to the high degree of sequence homology between like domains, any of eight potential modules may have been the donor for the introduced at-iii/dh domain pair .
to gauge the extent of mls sequence homology among different mycolactone-producing mycobacteria  we took advantage of a single nucleotide polymorphism  identified within the ks domain during sequencing of pmum <dig>  sequencing of the mls locus of pmum <dig> revealed a hindiii restriction site in the ks domain of all  <dig> modules that comprise mlsa <dig>  mlsa <dig> and mlsb.
these hindiii sites are at the same position within every ks domain and are introduced by a synonymous c → t transition.
curiously, this polymorphism is not present in any of the  <dig> ks domains of pmum <dig>  the presence of this site within the ks domains of the mycolactone e pks represented a convenient tool for screening other mpm.
oligonucleotides were designed to the 5' and 3' ends of the ks domain and pcr was used to amplify all ks domains present in a strain.
subsequent restriction digestion with hindiii of the pcr product obtained from each mpm revealed the presence of this polymorphism in all the ks domains of the south american human mu strains and animal  mpm, with the same profile as ml.
however this variation was absent from all the ks domains of african and australian mu strains, and present in only a proportion of the ks domains from the japanese strain mu <dig> .
one conclusion from these data is that ongoing intra-strain domain replacement  is purifying the mls sequences and resulting in the unusual pan-locus nucleotide homogeneity.
discussion
we present here the first detailed description of two recently discovered megaplasmids, harboured by mycolactone-producing mycobacteria isolated from frogs and fish, named pmum <dig>  and pmum <dig>  respectively.
sequence analysis revealed that both replicons are highly related to pmum <dig> from mu agy <dig>  with the same overall architecture and at least half their coding potential spanned by the three, large mls genes that are required for mycolactone synthesis .
uncovering the genetic basis for the synthesis of mycolactone structural variants by mpm was a major objective of this study.
the complete sequence of pmum <dig> permitted a thorough analysis of its mls locus and an examination of the concordance between module arrangement and the structure of mycolactone e. there was perfect correspondence between the domain and module arrangement for mlsb in pmum <dig> and our earlier lc-ms/ms structural predictions that included the presence of a propionate starter unit and only six extender modules .
further support for this structure was also obtained by lc-ms/ms analysis of lipid extracts from two other ml strains and observing the same ion trace and ms/ms fragmentation pattern.
similarly, analysis of a bac clone that spanned mlsb from pmum <dig> confirmed mycolactone f structure-based predictions on module and domain composition that included an at-ii  domain within the load module and the absence of any a-type kr domains; the latter observation explaining the switched stereochemistry of the c11' and c13' side-chain hydroxyls of mycolactone f  <cit> .
furthermore, the combination of sequencing and lc-ms/ms analysis has shown conclusively that geographically and genetically close mu strains from japan and china produce different mycolactones .
alternative mycolactone production in all of these strains is due to the highly mutable nature of the mycolactone pks locus, facilitated by the high levels of nucleotide identity amongst domains, and presumably supporting homologous recombination.
despite the very high inter-strain nucleotide identity, the mls sequences from pmum <dig> and pmum <dig> form distinct phylogenetic clusters , a separation that correlates with genetic comparisons based on chromosomal genes sequences that show mpm fall into two distinct lineages; the so-called ancestral lineage that includes mpm predominantly infecting fish and frogs and the modern lineage that includes most of the mpm that infect humans  <cit> .
the discrete strain-dependent clustering of the pmum <dig> and pmum <dig> mls domains also suggests that intra- and not inter-strain exchange of homologous domain sequences is the mechanism that has generated metabolite diversity.
in fact, the most parsimonious explanation for the mls domain and module clustering patterns and the distribution of the hindiii polymorphism amongst mpm is the evolution of domains or modules in concert and mutations occurring in repeated domains that then become fixed throughout the genes due to gene conversion events  <cit> .
it was originally suggested that the evolution of the multimodular structure of the pkss is due to repeated rounds of gene duplication from a single ancestor module  <cit> , a notion that has been supported by phylogenetic analysis of ks domains amongst streptomycetes and other pks pathways  <cit> .
intragenic and intergenic recombination appears to have generated diversity within other pks clusters, such as the microcystin pks gene cluster of microcystis sp.
<cit>  and the avermectin and rapamycin clusters of streptomyces sp.
<cit> .
a major stumbling block for combinatorial polyketide biochemistry for production of non-natural products has been the apparent incompatibility of certain domain combinations  <cit> .
in this study we have shown that there is considerable natural tolerance among the mls domains for a variety of polyketide precursors.
for example, by studying the genetic basis for naturally occurring mycolactone variants we have revealed that different at domains within starter units specify either acetate or propionate, addition or deletion of modules accounts for altered chain length, and the stereochemistry of hydroxyl groups results from a replacement of kr domains, all in accord with the modular pks paradigm  <cit> .
furthermore, the high identity among ks domains suggests that they must accept different extender units and many varieties of growing polyketide chain as substrates.
in agreement with this, a close comparison of mls sequences between pmum <dig> & pmum <dig> shows that the subtle alterations in ks sequence do not correlate with the type of substrate.
also, domains or modules that perform the same synthesis reactions in different strains are not necessarily the most closely related, again suggesting the ks domains are permissive.
for example, the ks domains of mlsa that accept the same mycolactone core substrates in mu agy <dig> and ml are not most closely related to each other, as might be predicted if even small changes in the ks alter function, but are more closely related to other ks domains from that strain, which accept different substrates .
exceptions to this are the ks domains from modules  <dig> and  <dig> of mlsa of pmum <dig>  which are most closely related to their counterparts in pmum <dig>  it is also noteworthy that domain swapping occurs in pairs.
the reasons for this are unclear, but one possible explanation is that a pair of adjacent domains provides more favourable conditions for homologous recombination than a single domain.
CONCLUSIONS
in this study dna sequencing and comparative analysis of pmum megaplasmids have been used to document the genetic differences in the toxin-coding dna, and sophisticated mass spectrometry has been used to assign the differences in their chemical structure.
the results confirm predictions, and show that highly specific changes in the modular polyketide synthase genes, akin to strategies used in the laboratory to engineer production of altered polyketide antibiotics, account for the differences.
given their uniquely repetitive structure, the genes for these assembly-line multienzymes appear to represent a natural chemistry set that might be harnessed in different modular combinations to create novel polyketides as potential drug leads.
comparative analysis of the repetitive gene structure has also provided clues to the evolutionary events, particularly recombination and gene conversion, that continue to shape these remarkable systems.

 mycolactone is a polyketide-derived, secondary metabolite and a major virulence factor of the human pathogen mycobacterium ulcerans, the causative agent of buruli ulcer. at picogram concentrations mycolactone has immunosuppressive properties and at higher concentrations it is cytotoxic for mammalian cells. the molecule is composed of an invariant core comprising a 12-membered macrolactone and side-chain that is esterified to a highly unsaturated acyl side chain, the latter structure varying amongst different mu strains. mu strains from africa, australia and china produce variants named mycolactones a/b, c, and d, respectively whilst mycobacterium liflandii, a pathogen of frogs, produces mycolactone e, and the fish pathogens and others) produce mycolactone f. despite the multiple species names given to mycolactone-producing mycobacteria, multi locus sequence analysis of all these strains indicates they share greater than 98% nucleotide identity. the mpm appear to have evolved from a common m. marinum ancestor by acquisition of a large circular plasmid that conferred the ability to make mycolactones and then spread throughout the world, occupying different hosts. in mu strain agy the only strain for which a genome sequence is currently available, a kb megaplasmid named pmum has three very large genes that encode the modular type i pkss required for mycolactone synthesis. the plasmid also has three putative accessory genes. mlsa and mlsa form a nine-extension module complex that synthesises the mycolactone core, whilst mlsb is a single polypeptide, comprising seven extension modules that are required for the synthesis of the side chain. bacterial type i pks are modular multi-enzymes and act as molecular assembly lines for the formation of polyketides. these enzymes function in a sequential manner where each pks module is responsible for one round of chain elongation via the addition of either acetate or propionate, supplied to the pks as an activated malonyl or methylmalonyl-coa thioester. within each pks module are a series of covalently linked enzymatic domains that process the growing polyketide chain before passing it downstream to the next module in the system. the minimal set of enzymatic domains required for pks activity includes ketosynthase, acyltransferase and an acyl carrier protein domain. ketoreductase, dehydratase and enoylreductase domains are also commonly found in modules and form a so-called reductive loop, providing reducing enzyme activities that modify the two or three-carbon unit being added to the polyketide. the mycolactone pks exhibits a number of unusual features that distinguish it from other type i pks complexes. firstly, the mls pkss are exceptionally large with a total predicted monomeric size of ~ mda, placing them amongst the largest known cellular enzymes. secondly, there is an unprecedented level of genetic identity amongst the enzymatic domains of all mls modules. for other type i pkss, functionally identical domains from the same pks generally share – 70% amino acid sequence identity, however, identity between domains of the mls locus ranges from – 100%. the extreme sequence homology within the mls locus might be expected to provide a rich substrate for homologous recombination. indeed, mycolactone negative mutants frequently arise among laboratory passaged mu strains; caused by partial deletion of the mls genes. mycolactone d produced by an mu strain from china differs from mycolactone a/b by the substitution of a methylene at c2' of the acyl side chain. in this strain, the final extension module of mlsb possesses an at domain with propionate rather than acetate specificity, suggesting natural recombination within mlsb. however, mycolactone structural variations are quite restricted. mpm recovered from around the world for years all produce mycolactones with an absolutely conserved core and all variations occur within the fatty-acyl side-chain. the role mycolactones play in the survival of mpm and why variation is only tolerated in the side-chain is unknown. in this study we investigated the genetic basis for production of these variant structures, in particular for the frog pathogen m. liflandii. we determined the complete dna sequence of the kb megaplasmid from mycobacterium liflandii 128fxt and the partial sequence of pmum replicons from mycobacterium marinum dl and m. ulcerans japan we also employed lc-ms/ms structural analysis of their respective mycolactones. our results show that mycolactones produced by different mpm are caused by genetic rearrangements between homologous pks domains of the mls locus, highlighting the plasticity of this region and its potential for combinatorial polyketide biochemistry.  overview of pmum from m. liflandii 128fxt assembly of the complete dna sequence of pmum from four overlapping bac clones, revealed a bp circular element containing predicted cds. a summary of the main features of pmum compared with pmum is shown in table and an overview of its cds distribution is shown in figure 2b. * percentage based on predicted size of plasmid plasmid pmum encodes of its cds on the reverse strand and has a g+c content of %, which is similar to pmum. five genes, spanning kb of the plasmid, are predicted to be involved in mycolactone biosynthesis, with the same arrangement as those present on pmum. these genes include the three type i polyketide synthases, a type ii thioesterase and a fabh-like type iii ketosynthase. sequencing of the plasmid confirmed the previously reported absence of the cytochrome p hydroxylase gene cyp140a, which is responsible for the production of mycolactones a/b and d via hydroxylation at the c' position of the acyl side chain of these variants. overview of pmum from m. marinum dl240490 three bac clones from a dl genomic library were predicted to span all of pmum and these clones were selected for further analyses. based on the sizes of these bacs as determined by pulsed field gel electrophoresis and the results of end-sequencing, a map of pmum was constructed indicating it is a circular replicon with an estimated size of ~ kb. due to the complex and time-consuming nature of sequencing mycolactone pks loci caused by the extreme sequence repetition, only the non-pks region of pmum was fully sequenced for this study. the bac clone 0412d spanned this region of the plasmid from the 5' end of mlsb to the 3' end of mlsa and the complete sequence of 0412d was bp with predicted cds. the two other selected bac clones were used in pcr, sequencing and southern blot analyses to confirm the domain and module composition of specific regions of the pmum mls genes. comparison of pmum plasmids confirms their common origin both pmum and pmum share the same overall organization as pmum with a core set of cds common to all three plasmids. a complete list of cds from pmum and the non-pks region of pmum is presented and discussed in additional files and the pmum gene mup encodes a putative fabh-like type iii ketosynthase gene that is essential for mycolactone production and may have acyltransferase activity, linking the mycolactone core and acyl-side chain. this gene is present in all three plasmids and they share very high sequence identity; their predicted protein products differing by only one amino acid, suggesting the importance of the gene for mycolactone biosynthesis. there were also several regions of difference observed between the plasmids that included deletions, insertions and other genetic rearrangements. these alterations are presumably mediated by the many ise found in all pmum plasmids. for example, the absence of cyp140a in pmum & encoding a p hydroxylase that modifies mycolactone, is most likely explained by an is2606-mediated deletion. overall, it appears that the main function of the pmum plasmids is for the production of mycolactones with no other obvious virulence or virulence-associated genes present. the pmum mls module and domain arrangements correspond with the structure of mycolactone e the complete sequencing of the mls locus of pmum from ml revealed an extraordinarily high degree of similarity to the mls locus of pmum with a module and domain arrangement in near-perfect agreement with predictions based on the proposed structure of mycolactone e. the core macrocyclic lactone of mycolactone e is synthesised by mlsa and mlsa comprising a loading module and nine extension modules, terminating in a putative integral c-terminal thioesterase. similarly the acyl-side chain is synthesised by mlsb and comprises a load module with six extension modules. mlsb from pmum is aa, which is aa shorter than mlsb from pmum. the size difference is due to the absence in pmum of the equivalent of module from pmum an absence that corresponds precisely with the absence of a ch = ch moiety in the acyl-side chain of mycolactone e. there was also perfect agreement between observed structure and the pattern of acetate and propionate incorporation predicted from sequence analysis of the at domains. in particular, the at domain of the mlsb load module is methylmalonyl-coa-specific, indicating that ml uses a propionate starter unit which corresponds perfectly with structure-based predictions. these results provide another example of a "swappable" domain location as the equivalent at domain in pmum uses an acetate starter unit. the oxidation state predicted from the mls sequence after each stage of chain extension also aligns very closely with mycolactone e structure. however, like pmum extension module of mlsa contains apparently inactive dh and er domains. to reinforce the veracity of the above findings and to resolve an outstanding discrepancy regarding the correct structure of mycolactone e, lc-ms/ms analysis was used to analyse mycolactones detected in lipid extracts from two additional ml strains. these analyses revealed an identical ion trace and fragmentation pattern for m/z as reported for ml 128fxt. thus, the combination of these concordant structural data from multiple ml strains together with the sequence analysis of the mls genes from ml 128fxt provide compelling evidence that the mycolactone structure first reported by hong et al is correct. alterations in pmum mlsb module and domain arrangement correspond with the structure of mycolactone f although the complete sequence of the mls locus from pmum was not determined in this study, the two bac clones 048f and 051b that spanned mlsa and mlsb respectively permitted some investigations into the genetic basis for the production of mycolactone f from dl. using bac clone 051b as template, pcr and sequencing of the load module of mlsb identified that this module has an at-i domain, indicating an acetate starter unit for mycolactone f side-chain synthesis. this arrangement, which is supported by structural data is the same as pmum but different to ml which has an at-iii domain. the recent total synthesis of mycolactone f showed that the hydroxyl groups at c11' and c13' of its acyl side chain have the opposite stereochemistry to other mycolactones. kr domains control the geometry of the hydroxyls and two types of this domain have been noted, with each type responsible for a specific stereochemistry. as the c11' and c13' side-chain hydroxyl arrangement reported for mycolactone f had not been observed in other mycolactones, it suggested that a switch from a-type to b-type kr domains had occurred within the first two extension modules of mlsb from pmum to test this hypothesis, we performed southern hybridisation analysis of bacs containing either mlsa or mlsb from pmum pmum and pmum with probes for either the kr-a or kr-b domain, and as predicted, southern analysis confirmed that there are no a-type kr domains in mlsb of pmum, consistent with our hypothesis that the a-type kr domains have been replaced. this result also suggests the presence of a previously unidentified mls module type consisting of a combination of ks, at-ii and b-type kr domains. mlsb module is different between mu strains from china and japan the above examples of module and domain rearrangement prompted a closer inspection of the mycolactones produced by mu strains from japan and china. mu strains from these countries are genetically very closely related with only one discriminating allele by mlsa and identical indel patterns. previous investigations had revealed that strain mu from china makes a modified mycolactone due to the presence of an at-iii domain in extension module of mlsb. however, the only published report of the structure of mycolactones from japan suggested that japanese strains made mycolactone a/b and not mycolactone d as might have been expected. we used pcr and sequencing of mlsb module from the japanese mu strain to check the domain arrangement and found that it has an at-ii domain, which is different to that of mu china but which is the expected arrangement for mycolactone a/b synthesis. analysis of lipid extracts from mu japan by lc-ms/ms confirmed that this strain does make mycolactone a/b, not mycolactone d. sequence comparisons of this region from mu with the same region among mu and muagy africa shows that module arrangements and the corresponding mycolactone produced does not necessarily correlate with strain relatedness, indicating that remodelling of the mls genes is occurring at a frequency independent of single nucleotide mutation and highlighting the dynamic nature of this locus. ongoing replacement, duplication and deletion of the highly conserved mls modules and domains as observed in pmum the pmum mls dna is highly repetitive and exhibits extreme sequence conservation between domains with an identical catalytic function. for example, the ks domains share > % nucleotide identity over nts, which translates to only nine variable amino acid residues among aa. there are three types of domains that have 100% intra-species nucleotide identity within both pmum and pmum the extreme sequence identity is also conserved between strains and falls only marginally to – % for these domains. the most variable of the enzymatic domains between species are the acp-i domains. when modules of identical domain arrangement were compared, nucleotide identity ranged from % to %. phylogenetic analysis of the domains and modules across pmum and pmum emphasise the high degree of relatedness between these genetic loci but also show that they cluster by strain, suggesting that evolution of the locus is occurring vertically rather than via horizontal exchanges between strains. * comparisons of plasmid and chromosomal sequences are taken from mulit-locus sequence analysis of concatenated dna sequences of nts and nts respectively. the previously reported domain swap in mlsb of mu china suggested either replacement of domains, combinations of domains, or entire modules, possibly by a gene conversion mechanism. a detailed comparison of modules with an identical domain configuration revealed that identity between one or more domains does not equate with identity over the whole module. for example, when an interspecies nucleotide comparison is performed across the region from the at-ii domain to the kr-a domain of module of mlsa and modules and of mlsb, there is 100% nucleotide identity over nts. however, the ks and acp domains of these modules have only % and % identity, respectively. when other modules of identical organisation are analysed in this manner, similar patterns are seen. another example of this is the presence of an active dh domain in the lm of mlsb of pmum which follows an at-iii domain. in pmum the two domains seen in these positions are a dh domain, with a predicted inactive catalytic site, and an at-i domain. in no part of mlsa mlsa or mlsb of pmum or pmum is an inactive dh paired with an at-iii domain. as the dh and kr domains are functionally redundant within the lm, this does not indicate a functional constraint imposed on this pairing, but does suggest that the at-iii and dh domains'move' as a group. this may also be the case for the er and kr-a domains that always appear in modules with the same domain organization. this is further highlighted by an intra-species comparison of the domain structure of the load modules of mlsa and mlsb. individual domains of both the mlsa and mlsb load modules of pmum are identical, with the exception of the at-iii and active dh domains. the simplest explanation for the presence of these domains in otherwise identical sequences is introduction via recombination with the same domain cluster from a neighbouring donor module. due to the high degree of sequence homology between like domains, any of eight potential modules may have been the donor for the introduced at-iii/dh domain pair. to gauge the extent of mls sequence homology among different mycolactone-producing mycobacteria we took advantage of a single nucleotide polymorphism identified within the ks domain during sequencing of pmum sequencing of the mls locus of pmum revealed a hindiii restriction site in the ks domain of all modules that comprise mlsa mlsa and mlsb. these hindiii sites are at the same position within every ks domain and are introduced by a synonymous c → t transition. curiously, this polymorphism is not present in any of the ks domains of pmum the presence of this site within the ks domains of the mycolactone e pks represented a convenient tool for screening other mpm. oligonucleotides were designed to the 5' and 3' ends of the ks domain and pcr was used to amplify all ks domains present in a strain. subsequent restriction digestion with hindiii of the pcr product obtained from each mpm revealed the presence of this polymorphism in all the ks domains of the south american human mu strains and animal mpm, with the same profile as ml. however this variation was absent from all the ks domains of african and australian mu strains, and present in only a proportion of the ks domains from the japanese strain mu. one conclusion from these data is that ongoing intra-strain domain replacement is purifying the mls sequences and resulting in the unusual pan-locus nucleotide homogeneity. discussion we present here the first detailed description of two recently discovered megaplasmids, harboured by mycolactone-producing mycobacteria isolated from frogs and fish, named pmum and pmum respectively. sequence analysis revealed that both replicons are highly related to pmum from mu agy with the same overall architecture and at least half their coding potential spanned by the three, large mls genes that are required for mycolactone synthesis. uncovering the genetic basis for the synthesis of mycolactone structural variants by mpm was a major objective of this study. the complete sequence of pmum permitted a thorough analysis of its mls locus and an examination of the concordance between module arrangement and the structure of mycolactone e. there was perfect correspondence between the domain and module arrangement for mlsb in pmum and our earlier lc-ms/ms structural predictions that included the presence of a propionate starter unit and only six extender modules. further support for this structure was also obtained by lc-ms/ms analysis of lipid extracts from two other ml strains and observing the same ion trace and ms/ms fragmentation pattern. similarly, analysis of a bac clone that spanned mlsb from pmum confirmed mycolactone f structure-based predictions on module and domain composition that included an at-ii domain within the load module and the absence of any a-type kr domains; the latter observation explaining the switched stereochemistry of the c11' and c13' side-chain hydroxyls of mycolactone f. furthermore, the combination of sequencing and lc-ms/ms analysis has shown conclusively that geographically and genetically close mu strains from japan and china produce different mycolactones. alternative mycolactone production in all of these strains is due to the highly mutable nature of the mycolactone pks locus, facilitated by the high levels of nucleotide identity amongst domains, and presumably supporting homologous recombination. despite the very high inter-strain nucleotide identity, the mls sequences from pmum and pmum form distinct phylogenetic clusters, a separation that correlates with genetic comparisons based on chromosomal genes sequences that show mpm fall into two distinct lineages; the so-called ancestral lineage that includes mpm predominantly infecting fish and frogs and the modern lineage that includes most of the mpm that infect humans. the discrete strain-dependent clustering of the pmum and pmum mls domains also suggests that intra- and not inter-strain exchange of homologous domain sequences is the mechanism that has generated metabolite diversity. in fact, the most parsimonious explanation for the mls domain and module clustering patterns and the distribution of the hindiii polymorphism amongst mpm is the evolution of domains or modules in concert and mutations occurring in repeated domains that then become fixed throughout the genes due to gene conversion events. it was originally suggested that the evolution of the multimodular structure of the pkss is due to repeated rounds of gene duplication from a single ancestor module, a notion that has been supported by phylogenetic analysis of ks domains amongst streptomycetes and other pks pathways. intragenic and intergenic recombination appears to have generated diversity within other pks clusters, such as the microcystin pks gene cluster of microcystis sp. and the avermectin and rapamycin clusters of streptomyces sp.. a major stumbling block for combinatorial polyketide biochemistry for production of non-natural products has been the apparent incompatibility of certain domain combinations. in this study we have shown that there is considerable natural tolerance among the mls domains for a variety of polyketide precursors. for example, by studying the genetic basis for naturally occurring mycolactone variants we have revealed that different at domains within starter units specify either acetate or propionate, addition or deletion of modules accounts for altered chain length, and the stereochemistry of hydroxyl groups results from a replacement of kr domains, all in accord with the modular pks paradigm. furthermore, the high identity among ks domains suggests that they must accept different extender units and many varieties of growing polyketide chain as substrates. in agreement with this, a close comparison of mls sequences between pmum & pmum shows that the subtle alterations in ks sequence do not correlate with the type of substrate. also, domains or modules that perform the same synthesis reactions in different strains are not necessarily the most closely related, again suggesting the ks domains are permissive. for example, the ks domains of mlsa that accept the same mycolactone core substrates in mu agy and ml are not most closely related to each other, as might be predicted if even small changes in the ks alter function, but are more closely related to other ks domains from that strain, which accept different substrates. exceptions to this are the ks domains from modules and of mlsa of pmum which are most closely related to their counterparts in pmum it is also noteworthy that domain swapping occurs in pairs. the reasons for this are unclear, but one possible explanation is that a pair of adjacent domains provides more favourable conditions for homologous recombination than a single domain.  in this study dna sequencing and comparative analysis of pmum megaplasmids have been used to document the genetic differences in the toxin-coding dna, and sophisticated mass spectrometry has been used to assign the differences in their chemical structure. the results confirm predictions, and show that highly specific changes in the modular polyketide synthase genes, akin to strategies used in the laboratory to engineer production of altered polyketide antibiotics, account for the differences. given their uniquely repetitive structure, the genes for these assembly-line multienzymes appear to represent a natural chemistry set that might be harnessed in different modular combinations to create novel polyketides as potential drug leads. comparative analysis of the repetitive gene structure has also provided clues to the evolutionary events, particularly recombination and gene conversion, that continue to shape these remarkable systems. 
these data reveal how pks module and domain differences affecting mlsb correlate with the production of mycolactones e and f. for mycolactone e these differences from mlsb in m. ulcerans agy include replacement of the at domain of the loading module and the absence of an entire extension module. we report here comparative genomic analysis of pmum the complete dna sequence of a kb megaplasmid from mycobacterium liflandii 128fxt and partial sequence of two additional pmum replicons, combined with liquid chromatography-tandem mass spectrometric analysis. this work has shed light on the evolution of mycolactone biosynthesis among an unusual group of mycobacteria and highlights the potential of the mls locus to become a toolbox for combinatorial pks biochemistry. the mycolactone pks locus on pmum revealed the same large, three-gene structure and extraordinary pattern of near-identical pks domain sequence repetition as observed in pmum with greater than % nucleotide identity among domains of the same function. for mycolactone f there is also a reduction of one extension module but also a swap of ketoreductase domains that explains the characteristic stereochemistry of the two terminal side-chain hydroxyls, an arrangement unique to mycolactone f
 mycolactones are immunosuppressive and cytotoxic polyketides, comprising five naturally occurring structural variants, produced by different species of very closely related mycobacteria including the human pathogen, mycobacterium ulcerans. in m. ulcerans strain agy mycolactone a/b is produced by three highly homologous type i polyketide megasynthases, whose genes are found on a kb plasmid, known as pmum  we report here comparative genomic analysis of pmum the complete dna sequence of a kb megaplasmid from mycobacterium liflandii 128fxt and partial sequence of two additional pmum replicons, combined with liquid chromatography-tandem mass spectrometric analysis. these data reveal how pks module and domain differences affecting mlsb correlate with the production of mycolactones e and f. for mycolactone e these differences from mlsb in m. ulcerans agy include replacement of the at domain of the loading module and the absence of an entire extension module. for mycolactone f there is also a reduction of one extension module but also a swap of ketoreductase domains that explains the characteristic stereochemistry of the two terminal side-chain hydroxyls, an arrangement unique to mycolactone f  the mycolactone pks locus on pmum revealed the same large, three-gene structure and extraordinary pattern of near-identical pks domain sequence repetition as observed in pmum with greater than % nucleotide identity among domains of the same function. intra- and inter-strain comparisons suggest that the extreme sequence homogeneity seen among the mls pks genes is caused by frequent recombination-mediated domain replacement. this work has shed light on the evolution of mycolactone biosynthesis among an unusual group of mycobacteria and highlights the potential of the mls locus to become a toolbox for combinatorial pks biochemistry. 