BACKGROUND
planktonic cyanobacteria often form heavy scums or blooms in freshwater lakes, ponds and reservoirs worldwide  <cit> .
cyanobacterial blooms constitute a health-risk for human beings via recreational or drinking water through the production of a range of hepatotoxins and neurotoxins  <cit> .
microcystins are a diverse group of low molecular weight cyclic heptapeptides and are the most common hepatotoxins produced by cyanobacteria.
they are potent inhibitors of eukaryotic protein phosphatases  <dig> and 2a  <cit>  and are linked to the deaths of wild animals and livestock worldwide  <cit> .
there are over  <dig> structural variants of microcystins differing in modifications to the peptide backbone or the type of amino acids incorporated into the microcystin  <cit> .
the general structure of microcystins can be summarized as cyclo-d-ala1-x2-d-measp3-z4-adda5-d-glu6-mdha7where x and z are variable l-amino acids .
many of these microcystin variants are synthesized simultaneously by the producing cyanobacterium  <cit> .
structural variation has been encountered at all seven positions, but the highest degree of structural variation is found at the x and z positions .
the two most common microcystin variants, microcystin-lr and microcystin-rr, contain l-leu or l-arg at the x position and l-arg at the z position in the final cyclic heptapeptide.
however, microcystins may also contain other proteinogenic, non-proteinogenic and dicarboxylic acids at these positions  <cit> .
structural variants of microcystin do not have the same toxicities and microcystin-lr is an order of magnitude more toxic than microcystin-rr  <cit> .
microcystins are mainly produced by planktonic strains of the distantly related cyanobacterial genera anabaena, microcystis and planktothrix  <cit> .
microcystin production is also known from a small number of planktonic, benthic and terrestrial strains of the genera nostoc  <cit> , hapalosiphon  <cit> , and phormidium  <cit> .
insertional mutagenesis has demonstrated that all microcystin variants produced by microcystis aeruginosa s- <dig>  k- <dig> and pcc  <dig> are synthesized by an enzyme complex encoded in a single 55-kb gene cluster  <cit> .
the enzyme complex which directs the biosynthesis of microcystins includes peptide synthetases, polyketide synthases, mixed peptide synthetases-polyketide synthases, and tailoring enzymes  <cit> .
phylogenetic analyses suggest that the microcystin synthetase gene cluster was present in the last common ancestor of all present-day producer organisms  <cit> .
the sporadic distribution of microcystin synthetase gene clusters among cyanobacteria is proposed to be the result of gene loss rather than recent horizontal gene transfer  <cit> .
many important antibiotics, siderophores and toxins are synthesized on nrps enzyme complexes  <cit> .
nrpss possess a highly conserved modular structure with each module being comprised of catalytic domains responsible for the adenylation, thioester formation and in most cases condensation of specific amino acids  <cit> .
the arrangement of these domains within the multifunctional enzymes determines the number and order of the amino acid constituents of the peptide product  <cit> .
additional domains for the modification of amino acid residues such as epimerization, heterocyclisation, oxidation, formylation, reduction or n-methylation may also be included in the module  <cit> .
the modular structure of nrpss allows the rational design of novel peptides by targeted replacement of these catalytic domains  <cit> .
the adenylation domain appears to be the primary determinant of substrate selectivity in nrpss .
high structural conservation of the adenylation domain allows prediction of amino acids lining the putative binding pocket which determines substrate specificity  <cit> .
however, recent studies predict an editing function for the condensation domain suggesting that condensation and adenylation domains in artificial junctions may be incompatible and block peptide synthesis  <cit> .
this finding lead to the hypothesis that in nature condensation and adenylation domains may act as an inseparable couple and be transferred together during natural rearrangements of nrps gene clusters  <cit> .
the amino acids incorporated at the x and z positions in structural variants of microcystin are recognized and activated by the mcyb <dig> and mcyc adenylation domains .
recombination between the adenylation domains of mcyb <dig> and mcyc is linked to the production of microcystin-rr by strains of the genus microcystis  <cit> .
recombination is thought to be an important factor contributing to the genetic diversity of the microcystin synthetase gene cluster in strains of the genus microcystis  <cit> .
however, it is not clear how widespread this phenomenon is in other microcystin producers or if the condensation domain is also transferred with the adenylation domain.
here we undertake a multigene phylogenetic study in order to investigate the number and timing of recombination events during the evolution of the microcystin synthetase gene cluster in a variety of microcystin producing cyanobacteria.
we show clear evidence for the recurrent exchange and replacement of the adenylation domain without the concomitant transfer of the condensation domain in a broad range of microcystin producing cyanobacteria.
RESULTS
structural characterization of the identified microcystins
we documented the simultaneous production of  <dig> to  <dig> microcystin variants in these strains .
the microcystin variants produced by these strains differed in the methylation of the α-amino group of mdha, the β-carboxyl of d-measp and the c <dig> hydroxyl of adda.
however, most structural differences lay in the type of amino acid incorporated at the x position.
most strains produced microcystins that contained l-leu at the x position .
the strains included in this study also produced microcystins which contained l-arg, l-hil, l-hph, l-hty, l-phe, l-try, l-tyr, or l-val at the x position .
most strains produced microcystins that contained l-arg at the z position .
however, almost half of the microcystin variants contained l-har at the z-position in nostoc sp.
<dig> .
almost all variants produced in hapalosiphon hibernicus bz-3- <dig> contained l-ala at the z-position but this strain also produced minor microcystins variants in trace amounts that contained l-leu or l-val at this position .
the strains included in this study produced a wide range of common and rare microcystins.
a large number of minor microcystin variants were identified through characteristic uv spectra.
however, in most cases the low amounts of microcystins produced prevented characterization of the total structures.
recombination breakpoints in mcyb <dig> and mcyc
phylogenetic-compatibility analysis indicated extensive incongruence between the adenylation and condensation domains of mcyb <dig> and mcyc .
analysis of the nucleotide sequences of mcyb <dig> and mcyc identified recombination breakpoints in the adenylation and thiolation domains using six different methods to detect recombination .
the recombination area extended across the entire adenylation domains spanning conserved core motifs a1– <dig> into the middle of the thiolation domain .
additional sets of breakpoints were identified within the adenylation domain in anabaena sp.
18b <dig> replacing the adenylation domain elements a2–a <dig> .
in the case of microcystis aeruginosa pcc <dig> a second set of breakpoints were also identified spanning the substrate conferring portions of the adenylation domain between the core motifs a3–a <dig> .
phylogenetic analysis of mcyb <dig> and mcyc condensation and adenylation domains
maximum-likelihood trees based over  <dig>  bp of nucleotide data from  <dig> housekeeping genes and  <dig> microcystin synthetase genes were congruent and each topology received robust bootstrap support .
maximum-likelihood trees based on the amino acid sequence of the condensation domain from mcyb <dig> and mcyc are also broadly congruent with the phylogeny of the producer organism .
mcyb <dig> condensation domains were monophyletic and grouped together with condensations domains with a d-peptidyl donor .
interestingly the mcyc condensation domains were also monophyletic but grouped with condensation domains with an l-peptidyl donor  despite having d-measp as the donor amino acid.
maximum-likelihood trees based on the amino acid sequence of the condensation domains and the a3–a <dig> substrate conferring portion of the adenylation domain of mcyb <dig> and mcyc differed considerably .
the mcyb <dig> and mcyc adenylation domain nucleotide sequences of anabaena spp.
<dig>  18b <dig>  66a and hapalosiphon hibernicus bz-3- <dig> were all more similar to one another than they were to other mcyb <dig> or mcyc sequences .
the nucleotide sequence similarity between each of these pairs of condensation domains was very low and ranged from  <dig> to 28% .
however, the nucleotide sequence similarity between each of these pairs of adenylation domains was very high and ranged from  <dig> to 97% .
there was no clear evidence for such recent recombination between mcyb <dig> and mcyc in microcystis viridis nies  <dig>  planktothrix agardhii niva 126/ <dig>   <dig> or nostoc sp.
<dig> .
the sequence divergence between mcyb <dig> and mcyc from these strains is higher than the sequence divergence between housekeeping genes and other microcystin synthetase genes within these genera  <cit> .
in the case of nostoc sp.
io-102-i and microcystis aeruginosa pcc <dig> the amino acid sequence of the mcyb <dig> adenylation domain differed considerably from other mcyb <dig> adenylation domains.
this region of dissimilarity extended across the entire adenylation domain  in nostoc sp.
io-102-i but was limited to the a3–a <dig> region of the adenylation domain in microcystis aeruginosa pcc <dig>  this is reflected in the phylogenetic position of these two adenylation domains in maximum-likelihood trees based on the a3–a <dig> portions of the adenylation domain .
sequence similarities between nucleotide sequence of the condensation domains from mcyb <dig> and mcyc and adenylation domains from mcyb <dig> and mcyc from the same strain based on uncorrected p distances converted to percentage similarity.
substrate specificities of the mcyb <dig> and mcyc adenylation domains
the l-asp residue at position  <dig> and the l-lys residue at  <dig> which interact with the α-amino and the carboxyl groups, respectively, to lock orientation of the l-α-amino acid upon activation  <cit>  were conserved in all strains included in this study .
the mcyb <dig> and mcyc adenylation domain binding pockets differed between  <dig> and  <dig> amino acids in pairwise comparisons in most strains .
however, the amino acids lining the putative binding pockets of mcyb <dig> and mcyc in anabaena sp.
18b <dig> and hapalosiphon hibernicus bz-3- <dig> were identical .
specificity codes inferred from the protein sequence of the mcyb <dig> and mcyc adenylation domains through pairwise alignment against grsa  <cit> .
the principal amino acid present at the x and z positions in microcystins analyzed with the lc-ms method.
discussion
we did not find separate congruent clusters of mcyb <dig> and mcyc adenylation domain sequences  as might have been anticipated under an evolutionary scenario in which all microcystin synthetase genes share the same evolutionary history  <cit> .
instead we found intermixed clusters of mcyb <dig> and mcyc adenylation domains .
in some instances we identified very low levels of sequence divergence in pairwise comparisons of the nucleotide sequences of mcyb <dig> and mcyc adenylation domain from the same strain .
this discordance together with the low levels of sequence divergence is consistent with multiple recent independent recombination events.
recombination would lead to the overwriting of the mcyb <dig> and mcyc adenylation domains contributing to sequence homogenization and explain the low divergence of the mcyb <dig> and mcyc adenylation domains relative to the mcyb <dig> and mcyc condensation domains in anabaena spp.
<dig>  18b <dig>  66a and hapalosiphon hibernicus bz-3- <dig> .
however, in addition to these recent recombination events our phylogenetic analysis reveals evidence for replacement of the mcyb <dig> adenylation domain in nostoc sp.
io-102-i and microcystis aeruginosa pcc <dig> .
the high sequence divergence between the adenylation domains of mcyb <dig> in these two strains and other mcyb <dig> adenylation domain sequences included in this study  could be explained by two independent replacement events involving a non-homologous adenylation domain from another peptide synthetase gene cluster.
a recombination event has been proposed to replace the adenylation domain of mcyb <dig> and mcyc of planktothrix agardhii niva 126/ <dig>  <cit> .
however, the mcyb <dig> and mcyc adenylation domains of planktothrix agardhii niva 126/ <dig> and  <dig> as well as nostoc sp.
<dig> clustered separately suggesting that the recombination event precedes the divergence of these two genera.
together, our results indicate that these two adenylation domains are recombination hotspots within the microcystin peptide synthetase gene cluster.
the recombination events at the mcyb <dig> and mcyc are limited to the adenylation domain and the condensation domains in mcyb <dig> and mcyc are highly divergent and group in separate clusters .
recombination breakpoints are all limited to the adenylation domain .
the phylogenetic discordance between the adenylation and condensation domains is inconsistent with the hypothesis that adenylation and condensation domains are transferred together as a unit.
two rounds of peptide chain elongation are catalyzed by mcyb, which typically activates and condenses l-leu and d-measp into the growing peptide chain  <cit> .
this protein directs the transfer of d-peptidyl intermediates involving a carboxy-terminal epimerase domain of mcya and the condensation domain of mcyb <dig>  <cit> .
peptide bond formation is achieved between the α-amino group of d-ala and the α-carboxyl group of l-leu  <cit> .
in keeping with this the mcyb <dig> condensation domain clusters with domains involved in d-l peptide bonds .
the final condensation reaction is performed between the β-carboxyl group of β-measp and the α-amino group of l-arg by mcyc prior to cyclisation and the resulting peptide bond is atypical  <cit> .
interestingly, the condensation domain of mcyc does not group with previously described d-l condensation domains but group instead with condensation domains with l-peptidyl amino acids as donors .
however, the mcyc condensation domain also lacks the typical hhxxxdg his motif in its active site typically present in the condensation domains with d- and l-peptidyl donors  <cit> .
although adenylation domains are the primary determinants of substrate specificity in nrpss condensation domains are also reported to exhibit moderate to high substrate selectivity  <cit> .
it may be that differences in the substrate specificities of the condensation domains from mcyb <dig> and mcyc mean that the co-transfer of the adenylation and condensation domains would result in a non-functional peptide synthetase.
non-compatible adenylation and condensation domains are predicted to cause a drastic reduction of catalytic competence or even a complete failure to synthesize the desired peptide by the engineered nrps  <cit> .
replacement of condensation domains in mcyb <dig> and mcyc may lead to a disruption of the overall integrity of the peptide assembly process, in particular the order and timing of condensation reactions.
the simultaneous production of the microcystin -lr and -rr variants has been interpreted as a lack of specificity at the mcyb <dig> adenylation domain  <cit> .
we predicted the  <dig> amino acids lining the putative binding pocket in the adenylation domain of mcyb <dig> and mcyc though alignment against the grsa adenylation domain  <cit> .
the  <dig> amino acids lining the binding pocket which interact with the side chain and functional group and were highly variable in mcyb <dig> and to a lesser extent mcyc .
single amino acid changes in the amino acids lining the putative binding pocket of the adenylation domain are known to have an effect on the type of amino acid that is recognized and activated by the adenylation domain  <cit> .
single amino acid change  in mcyc could be responsible for shifting the incorporation of l-arg towards l-har in nostoc sp.
<dig> .
similarly a single amino acid change in  in mcyb <dig> could be responsible for shifting the incorporation of l-arg towards l-hty in anabaena sp.
66a.
experimental expression and mutation of the adenylation domains in each case could verify this hypothesis.
determining the amino acids lining the substrate conferring portions of the adenylation domains of non-ribosomal peptide synthetase gene clusters can yield invaluable predictions of substrate specificities of unknown peptide synthetases  <cit> .
the putative binding pocket of the adenylation domains of mcyb <dig> and mcyc in hapalosiphon hibernicus bz-3- <dig> are identical .
however, this strain incorporated 91% l-leu at the x position and 99% l-ala at the z position .
our results suggest that caution should be taken when inferring substrate specificity given the general lack of knowledge about how widespread adenylation domain replacement is in nature.
many important antibiotics, antimicrobial compounds, siderophores and toxins are synthesized on non-ribosomal peptide synthetase enzyme complexes  <cit> .
there is much current interest in engineering non-ribosomal peptide synthetases in order to create new peptides with potential biological activities  <cit> .
it has been suggested that peptide synthetase would gain most effectively through transfer of entire modules  <cit> .
some artificial combinations of adenylation and condensation domains result in non-functional products  <cit> .
this led to the hypothesis that non-ribosomal peptide synthetase modules evolve as a unit  <cit> .
here we have clear evidence for the exchange and replacement of the adenylation domain without the concomitant transfer of the condensation domain.
CONCLUSIONS
our results demonstrate that the mcyb <dig> and mcyc adenylation domains are recombination hotspots in the microcystin synthetase gene cluster.
we show clear evidence for the recurrent exchange and replacement of the adenylation domain in a broad range of microcystin producing cyanobacteria.
our results show that functional peptide synthetases can be created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains.

 planktonic cyanobacteria often form heavy scums or blooms in freshwater lakes, ponds and reservoirs worldwide. cyanobacterial blooms constitute a health-risk for human beings via recreational or drinking water through the production of a range of hepatotoxins and neurotoxins. microcystins are a diverse group of low molecular weight cyclic heptapeptides and are the most common hepatotoxins produced by cyanobacteria. they are potent inhibitors of eukaryotic protein phosphatases and 2a and are linked to the deaths of wild animals and livestock worldwide. there are over structural variants of microcystins differing in modifications to the peptide backbone or the type of amino acids incorporated into the microcystin. the general structure of microcystins can be summarized as cyclo-d-ala1-x2-d-measp3-z4-adda5-d-glu6-mdha7where x and z are variable l-amino acids. many of these microcystin variants are synthesized simultaneously by the producing cyanobacterium. structural variation has been encountered at all seven positions, but the highest degree of structural variation is found at the x and z positions. the two most common microcystin variants, microcystin-lr and microcystin-rr, contain l-leu or l-arg at the x position and l-arg at the z position in the final cyclic heptapeptide. however, microcystins may also contain other proteinogenic, non-proteinogenic and dicarboxylic acids at these positions. structural variants of microcystin do not have the same toxicities and microcystin-lr is an order of magnitude more toxic than microcystin-rr. microcystins are mainly produced by planktonic strains of the distantly related cyanobacterial genera anabaena, microcystis and planktothrix. microcystin production is also known from a small number of planktonic, benthic and terrestrial strains of the genera nostoc, hapalosiphon, and phormidium. insertional mutagenesis has demonstrated that all microcystin variants produced by microcystis aeruginosa s- k- and pcc are synthesized by an enzyme complex encoded in a single 55-kb gene cluster. the enzyme complex which directs the biosynthesis of microcystins includes peptide synthetases, polyketide synthases, mixed peptide synthetases-polyketide synthases, and tailoring enzymes. phylogenetic analyses suggest that the microcystin synthetase gene cluster was present in the last common ancestor of all present-day producer organisms. the sporadic distribution of microcystin synthetase gene clusters among cyanobacteria is proposed to be the result of gene loss rather than recent horizontal gene transfer. many important antibiotics, siderophores and toxins are synthesized on nrps enzyme complexes. nrpss possess a highly conserved modular structure with each module being comprised of catalytic domains responsible for the adenylation, thioester formation and in most cases condensation of specific amino acids. the arrangement of these domains within the multifunctional enzymes determines the number and order of the amino acid constituents of the peptide product. additional domains for the modification of amino acid residues such as epimerization, heterocyclisation, oxidation, formylation, reduction or n-methylation may also be included in the module. the modular structure of nrpss allows the rational design of novel peptides by targeted replacement of these catalytic domains. the adenylation domain appears to be the primary determinant of substrate selectivity in nrpss. high structural conservation of the adenylation domain allows prediction of amino acids lining the putative binding pocket which determines substrate specificity. however, recent studies predict an editing function for the condensation domain suggesting that condensation and adenylation domains in artificial junctions may be incompatible and block peptide synthesis. this finding lead to the hypothesis that in nature condensation and adenylation domains may act as an inseparable couple and be transferred together during natural rearrangements of nrps gene clusters. the amino acids incorporated at the x and z positions in structural variants of microcystin are recognized and activated by the mcyb and mcyc adenylation domains. recombination between the adenylation domains of mcyb and mcyc is linked to the production of microcystin-rr by strains of the genus microcystis. recombination is thought to be an important factor contributing to the genetic diversity of the microcystin synthetase gene cluster in strains of the genus microcystis. however, it is not clear how widespread this phenomenon is in other microcystin producers or if the condensation domain is also transferred with the adenylation domain. here we undertake a multigene phylogenetic study in order to investigate the number and timing of recombination events during the evolution of the microcystin synthetase gene cluster in a variety of microcystin producing cyanobacteria. we show clear evidence for the recurrent exchange and replacement of the adenylation domain without the concomitant transfer of the condensation domain in a broad range of microcystin producing cyanobacteria.  structural characterization of the identified microcystins we documented the simultaneous production of to microcystin variants in these strains. the microcystin variants produced by these strains differed in the methylation of the α-amino group of mdha, the β-carboxyl of d-measp and the c hydroxyl of adda. however, most structural differences lay in the type of amino acid incorporated at the x position. most strains produced microcystins that contained l-leu at the x position. the strains included in this study also produced microcystins which contained l-arg, l-hil, l-hph, l-hty, l-phe, l-try, l-tyr, or l-val at the x position. most strains produced microcystins that contained l-arg at the z position. however, almost half of the microcystin variants contained l-har at the z-position in nostoc sp.. almost all variants produced in hapalosiphon hibernicus bz-3- contained l-ala at the z-position but this strain also produced minor microcystins variants in trace amounts that contained l-leu or l-val at this position. the strains included in this study produced a wide range of common and rare microcystins. a large number of minor microcystin variants were identified through characteristic uv spectra. however, in most cases the low amounts of microcystins produced prevented characterization of the total structures. recombination breakpoints in mcyb and mcyc phylogenetic-compatibility analysis indicated extensive incongruence between the adenylation and condensation domains of mcyb and mcyc. analysis of the nucleotide sequences of mcyb and mcyc identified recombination breakpoints in the adenylation and thiolation domains using six different methods to detect recombination. the recombination area extended across the entire adenylation domains spanning conserved core motifs a1– into the middle of the thiolation domain. additional sets of breakpoints were identified within the adenylation domain in anabaena sp. 18b replacing the adenylation domain elements a2–a. in the case of microcystis aeruginosa pcc a second set of breakpoints were also identified spanning the substrate conferring portions of the adenylation domain between the core motifs a3–a. phylogenetic analysis of mcyb and mcyc condensation and adenylation domains maximum-likelihood trees based over bp of nucleotide data from housekeeping genes and microcystin synthetase genes were congruent and each topology received robust bootstrap support. maximum-likelihood trees based on the amino acid sequence of the condensation domain from mcyb and mcyc are also broadly congruent with the phylogeny of the producer organism. mcyb condensation domains were monophyletic and grouped together with condensations domains with a d-peptidyl donor. interestingly the mcyc condensation domains were also monophyletic but grouped with condensation domains with an l-peptidyl donor despite having d-measp as the donor amino acid. maximum-likelihood trees based on the amino acid sequence of the condensation domains and the a3–a substrate conferring portion of the adenylation domain of mcyb and mcyc differed considerably. the mcyb and mcyc adenylation domain nucleotide sequences of anabaena spp. 18b 66a and hapalosiphon hibernicus bz-3- were all more similar to one another than they were to other mcyb or mcyc sequences. the nucleotide sequence similarity between each of these pairs of condensation domains was very low and ranged from to 28%. however, the nucleotide sequence similarity between each of these pairs of adenylation domains was very high and ranged from to 97%. there was no clear evidence for such recent recombination between mcyb and mcyc in microcystis viridis nies planktothrix agardhii niva 126/ or nostoc sp.. the sequence divergence between mcyb and mcyc from these strains is higher than the sequence divergence between housekeeping genes and other microcystin synthetase genes within these genera. in the case of nostoc sp. io-102-i and microcystis aeruginosa pcc the amino acid sequence of the mcyb adenylation domain differed considerably from other mcyb adenylation domains. this region of dissimilarity extended across the entire adenylation domain in nostoc sp. io-102-i but was limited to the a3–a region of the adenylation domain in microcystis aeruginosa pcc this is reflected in the phylogenetic position of these two adenylation domains in maximum-likelihood trees based on the a3–a portions of the adenylation domain. sequence similarities between nucleotide sequence of the condensation domains from mcyb and mcyc and adenylation domains from mcyb and mcyc from the same strain based on uncorrected p distances converted to percentage similarity. substrate specificities of the mcyb and mcyc adenylation domains the l-asp residue at position and the l-lys residue at which interact with the α-amino and the carboxyl groups, respectively, to lock orientation of the l-α-amino acid upon activation were conserved in all strains included in this study. the mcyb and mcyc adenylation domain binding pockets differed between and amino acids in pairwise comparisons in most strains. however, the amino acids lining the putative binding pockets of mcyb and mcyc in anabaena sp. 18b and hapalosiphon hibernicus bz-3- were identical. specificity codes inferred from the protein sequence of the mcyb and mcyc adenylation domains through pairwise alignment against grsa. the principal amino acid present at the x and z positions in microcystins analyzed with the lc-ms method. discussion we did not find separate congruent clusters of mcyb and mcyc adenylation domain sequences as might have been anticipated under an evolutionary scenario in which all microcystin synthetase genes share the same evolutionary history. instead we found intermixed clusters of mcyb and mcyc adenylation domains. in some instances we identified very low levels of sequence divergence in pairwise comparisons of the nucleotide sequences of mcyb and mcyc adenylation domain from the same strain. this discordance together with the low levels of sequence divergence is consistent with multiple recent independent recombination events. recombination would lead to the overwriting of the mcyb and mcyc adenylation domains contributing to sequence homogenization and explain the low divergence of the mcyb and mcyc adenylation domains relative to the mcyb and mcyc condensation domains in anabaena spp. 18b 66a and hapalosiphon hibernicus bz-3-. however, in addition to these recent recombination events our phylogenetic analysis reveals evidence for replacement of the mcyb adenylation domain in nostoc sp. io-102-i and microcystis aeruginosa pcc. the high sequence divergence between the adenylation domains of mcyb in these two strains and other mcyb adenylation domain sequences included in this study could be explained by two independent replacement events involving a non-homologous adenylation domain from another peptide synthetase gene cluster. a recombination event has been proposed to replace the adenylation domain of mcyb and mcyc of planktothrix agardhii niva 126/. however, the mcyb and mcyc adenylation domains of planktothrix agardhii niva 126/ and as well as nostoc sp. clustered separately suggesting that the recombination event precedes the divergence of these two genera. together, our results indicate that these two adenylation domains are recombination hotspots within the microcystin peptide synthetase gene cluster. the recombination events at the mcyb and mcyc are limited to the adenylation domain and the condensation domains in mcyb and mcyc are highly divergent and group in separate clusters. recombination breakpoints are all limited to the adenylation domain. the phylogenetic discordance between the adenylation and condensation domains is inconsistent with the hypothesis that adenylation and condensation domains are transferred together as a unit. two rounds of peptide chain elongation are catalyzed by mcyb, which typically activates and condenses l-leu and d-measp into the growing peptide chain. this protein directs the transfer of d-peptidyl intermediates involving a carboxy-terminal epimerase domain of mcya and the condensation domain of mcyb. peptide bond formation is achieved between the α-amino group of d-ala and the α-carboxyl group of l-leu. in keeping with this the mcyb condensation domain clusters with domains involved in d-l peptide bonds. the final condensation reaction is performed between the β-carboxyl group of β-measp and the α-amino group of l-arg by mcyc prior to cyclisation and the resulting peptide bond is atypical. interestingly, the condensation domain of mcyc does not group with previously described d-l condensation domains but group instead with condensation domains with l-peptidyl amino acids as donors. however, the mcyc condensation domain also lacks the typical hhxxxdg his motif in its active site typically present in the condensation domains with d- and l-peptidyl donors. although adenylation domains are the primary determinants of substrate specificity in nrpss condensation domains are also reported to exhibit moderate to high substrate selectivity. it may be that differences in the substrate specificities of the condensation domains from mcyb and mcyc mean that the co-transfer of the adenylation and condensation domains would result in a non-functional peptide synthetase. non-compatible adenylation and condensation domains are predicted to cause a drastic reduction of catalytic competence or even a complete failure to synthesize the desired peptide by the engineered nrps. replacement of condensation domains in mcyb and mcyc may lead to a disruption of the overall integrity of the peptide assembly process, in particular the order and timing of condensation reactions. the simultaneous production of the microcystin -lr and -rr variants has been interpreted as a lack of specificity at the mcyb adenylation domain. we predicted the amino acids lining the putative binding pocket in the adenylation domain of mcyb and mcyc though alignment against the grsa adenylation domain. the amino acids lining the binding pocket which interact with the side chain and functional group and were highly variable in mcyb and to a lesser extent mcyc. single amino acid changes in the amino acids lining the putative binding pocket of the adenylation domain are known to have an effect on the type of amino acid that is recognized and activated by the adenylation domain. single amino acid change in mcyc could be responsible for shifting the incorporation of l-arg towards l-har in nostoc sp.. similarly a single amino acid change in in mcyb could be responsible for shifting the incorporation of l-arg towards l-hty in anabaena sp. 66a. experimental expression and mutation of the adenylation domains in each case could verify this hypothesis. determining the amino acids lining the substrate conferring portions of the adenylation domains of non-ribosomal peptide synthetase gene clusters can yield invaluable predictions of substrate specificities of unknown peptide synthetases. the putative binding pocket of the adenylation domains of mcyb and mcyc in hapalosiphon hibernicus bz-3- are identical. however, this strain incorporated 91% l-leu at the x position and 99% l-ala at the z position. our results suggest that caution should be taken when inferring substrate specificity given the general lack of knowledge about how widespread adenylation domain replacement is in nature. many important antibiotics, antimicrobial compounds, siderophores and toxins are synthesized on non-ribosomal peptide synthetase enzyme complexes. there is much current interest in engineering non-ribosomal peptide synthetases in order to create new peptides with potential biological activities. it has been suggested that peptide synthetase would gain most effectively through transfer of entire modules. some artificial combinations of adenylation and condensation domains result in non-functional products. this led to the hypothesis that non-ribosomal peptide synthetase modules evolve as a unit. here we have clear evidence for the exchange and replacement of the adenylation domain without the concomitant transfer of the condensation domain.  our results demonstrate that the mcyb and mcyc adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. we show clear evidence for the recurrent exchange and replacement of the adenylation domain in a broad range of microcystin producing cyanobacteria. our results show that functional peptide synthetases can be created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains. 
our findings demonstrate that the mcyb and mcyc adenylation domains are recombination hotspots in the microcystin synthetase gene cluster. our results provide support for complex evolutionary processes taking place at the mcyb and mcyc adenylation domains which recognize and activate the amino acids found at x and z positions. we also find clear evidence for independent adenylation domain conversion of mcyb by unrelated peptide synthetase modules in strains of the genera nostoc and microcystis. a recombination event between the first module of mcyb and mcyc in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus microcystis. the recombination events replace only the adenylation domain in each case and the condensation domains of mcyb and mcyc are not transferred together with the adenylation domain. this study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains. many structural variants of microcystins are produced simulatenously.
 microcystins are small cyclic heptapeptide toxins produced by a range of distantly related cyanobacteria. microcystins are synthesized on large nrps-pks enzyme complexes. many structural variants of microcystins are produced simulatenously. a recombination event between the first module of mcyb and mcyc in the microcystin synthetase gene cluster is linked to the simultaneous production of microcystin variants in strains of the genus microcystis.  here we undertook a phylogenetic study to investigate the order and timing of recombination between the mcyb and mcyc genes in a diverse selection of microcystin producing cyanobacteria. our results provide support for complex evolutionary processes taking place at the mcyb and mcyc adenylation domains which recognize and activate the amino acids found at x and z positions. we find evidence for recent recombination between mcyb and mcyc in strains of the genera anabaena, microcystis, and hapalosiphon. we also find clear evidence for independent adenylation domain conversion of mcyb by unrelated peptide synthetase modules in strains of the genera nostoc and microcystis. the recombination events replace only the adenylation domain in each case and the condensation domains of mcyb and mcyc are not transferred together with the adenylation domain. our findings demonstrate that the mcyb and mcyc adenylation domains are recombination hotspots in the microcystin synthetase gene cluster.  recombination is thought to be one of the main mechanisms driving the diversification of nrpss. however, there is very little information on how recombination takes place in nature. this study demonstrates that functional peptide synthetases are created in nature through transfer of adenylation domains without the concomitant transfer of condensation domains. 