BACKGROUND
cyanobacteria are among the earliest branching groups on earth, dating back  <dig> - <dig>  billion years, based on the fossil evidence  <cit> .as a taxonomic unit characterized by the ability to execute oxygenic photosynthesis, cyanobacteria constitute a group of species diverse in genome size and ecological habitats, indicating the significance of comparative genome research.
cyanobacteria, with a variation in genome size from  <dig>  mb  to  <dig>  mb , are found in almost every imaginable environment, from ocean to fresh water to bare rock.
cyanobacteria also inhabit in the extreme environments, for example, synechococcus sp.
ja-2-3b'a  and synechococcus sp.
ja-3-3ab were separated from hot spring.
as unicellular and non-nitrogen-fixing cyanobacteria, prochlorococcus sp.
and synechococcus sp.
maintain the smallest genome sizes and account for significant biomass and primary production of marine biosphere  <cit> .
other unicellular species have larger genome sizes, including water bloom forming cyanobacteria , a thylakoids absence cyanobacterium , a nitrogen-fixing cyanobacterium , and an animal-cyanobacterial symbionsis .
while the diazotrophic filamentous cyanobacteria comprise the largest genome size, such as nostoc sp.
pcc  <dig>  anabaena variabilis atcc  <dig>  plant-cyanobacteria symbionsis nostoc punctiforme pcc  <dig> and marine trichodesmium sp.
ims  <dig>
programmed cell death  is a suicide mechanism to promote and maintain genetic stability  <cit> .
pcd was considered as a characteristic of metazoans for a long time before apoptosis markers were found in yeast which indicates multicellularity is not the most important prerequisite <cit> .
recently, pcd mechanism has been observed in all but one of the six/eight major groups of prokaryotes, with the exception of the rhizaria  <cit> .
experimental evidences for pcd in cyanobacteria come from three species, including the freshwater cyanobacterium anabaena spp.
exposed to univalent-cation salts, the bloom-causing cyanobacterium microcystis aeruginosa from st. lucie estuary by treatment with h2o <dig> and trichodesmium sp.
ims  <dig> suffering iron starvation and light irradiance  <cit> .
caspases  are one of the most important and widely researched apoptotic proteins in mammalian pcd.
caspase was initially thought to be limited to metazoans, and no one had managed to identify caspase homologues, either in plants or bacteria.
then uren and his colleagues identified two ancient families of caspase-like proteins, paracaspases and metacaspases in silico  <cit>  and khan and his co-workers demonstrated that a yeast metacaspase  mediates pcd in saccharomyces cerevisiae <cit> .
hereafter, metacaspases were found involved in pcd of yeasts, filamentous fungi, plants, and a variety of bacteria.
most of these metacaspases share sequence homology with caspases, but show different substrate specificity  <cit> .
metacaspases belong to caspase family , which are part of the clan cd, a family of proteases characteristic with their his/cys catalytic dyad  <cit> .
metacaspases process a conserved caspase catalytic subunit p <dig> domain , and share conserved amino acid residues within his- and cys-catalytic sites  <cit> .
interestingly, most of the typical genes encoding in metazoan pcd are missing in bacteria and early-branching eukaryotes, such as cad and p <dig> <cit> .
therefore, the presence of metacaspases suggests a concernful role within pcd evolution.
cyanobacteria maintain a rich metacaspase pool, and many of these genes have been identified in silico  <cit>  was identified in some sequenced cyanobacteria strains, including gloeobacter violaceus pcc  <dig>  thermosynechococcus elongatus bp- <dig>  synechocystis sp.
pcc  <dig>  trichodesmium erythraeum ism  <dig>  nostoc punctiforme pcc  <dig>  nostoc sp.
pcc  <dig>  and anabaena variabilis atcc  <dig>  metacaspases were absent in med <dig>  prochlorococcus marinus mit  <dig>  ss  <dig> , synechococcus sp.
wh  <dig>  synechococcus elongatus pcc  <dig> and synechococcus elongates  <cit> .
with the completion of genome sequencing of several cyanobacterial species, modifications and supplements are needed.
as of november  <dig>   <dig> genomes of unicellular and filamentous cyanobacteria became available, which facilitates cyanobacterial systemic analysis for restriction-modification systems and serine/threonine protein kinases  <cit> .
the comparative genome research on metacaspases and other pcd proteins in filamentous fungi has been documented  <cit> .
besides, koonin and aravind described a clear affinity of bacterial metacaspases and the metazoan caspases by phylogenetic analysis of caspase-like protease superfamily  <cit> .
in this study, we selected five proven metacaspases in marine diatom thalassiosira pseudonana to search for cyanobacterial metacaspases  <cit> .
metacaspases in thalassiosira pseudonana were chosen due to the few metacaspases verified experimentally in cyanobacteria and the close evolutional relationship between cyanobacteria and eukaryotic phytoplankton.
we employed a blastp-plus-phylogeny reconstruction approach  <cit>  to analyze metacaspase sequences in cyanobacteria, and present an overall view of their classifications, structure, phylogeny and evolution.
better understanding of cyanobacterial metacaspases may provide further insights into evolution of pcd.
RESULTS
identification of metacaspase proteins
the  <dig> complete cyanobacterial genomes downloaded from img database were used in this research .
to identify proteins similar to proven metacaspases from thalassiosira pseudonana, we performed blastp searches of the  <dig> cyanobacterial genomes.
cdd  <cit>  and smart  <cit>  analyses with the derived sequences were then carried out to eliminate false positives.
two proteins annotated as caspase catalytic subunit p <dig> in img database, including  <dig>  from trichodesmium erythraeum ism  <dig> and  <dig> from microcystis aeruginosa nies- <dig>  were found to lack catalytic domain and excluded.
of two proteins , casc domain  recognized by cdd was not identified in smart analysis  <cit> , and both of the proteins were excluded.
altogether,  <dig> putative metacaspase sequences were considered in this study .
twenty-six of which were originally annotated as peptidase c <dig>  caspase catalytic subunit p <dig> or protein containing caspase domain.
the remaining  <dig> proteins were accepted as metacaspases in this research, including  <dig> proteins annotated by other additional domains ,  <dig> proteins annotated as hypothetical proteins, and  <dig> proteins annotated as unknown function protein duf <dig>
a: the numbers indicate "img gene object identifier" in img database
amid diverse cyanobacterial genomes, the number of metacaspase genes varies from  <dig> to  <dig>  within unicellular cyanobacteria, symbiont acaryochloris marina mbic  <dig> has  <dig> mcas, much more than other species.
correspondingly, the percentage of metacaspases within total proteins  is highest among unicellular cyanobacteria.
all of the marine synechococcus strains lack mcas except for synechococcus sp.
pcc  <dig>  only one metacaspase gene was found in the synechocystis sp.
pcc  <dig>  four synechococcus strains inhabit in freshwater, land and hot spring contain one metacaspase for each, including pcc  <dig>  pcc  <dig>  ja-2-3b'a, and ja-3-3ab.
water-blooming cyanobacterium microcystis aeruginosa nies- <dig> contains two mcas.
the percentages of mcas within total proteins  in synechocystis sp., synechococcus sp.
and microcystis sp.
strains are the lowest.
the other two unicellular cyanobacterial strains, gloeobacter violaceus pcc  <dig> and cyanothece sp.
atcc  <dig>  maintain  <dig> to  <dig> mcas and have moderate percentages of mcas in total proteins:  <dig> % and  <dig> %, respectively.
compared to unicellular cyanobacteria, filamentous diazotrophic cyanobacteria have more metacaspase genes .
among the filamentous cyanobacteria, nostoc punctiforme pcc  <dig> has the largest genome; however, nonheterocystous cyanobacterium trichodesmium erythraeum ims  <dig> contains the largest number of metacaspases and the highest percentage in total protein  .
one or two mcas containing mutations of conserved catalytic sites  were found in every cyanobacterial strain, exclusive of gloeobacter violaceus pcc  <dig>  in most cases, the his residues are replaced by tyr, and the cys residues mutate into ser, asn or gly.
uniquely, within the metacaspase  from cyanothece sp.
attc  <dig>  gln substitutes for the cys residue .
structure and function
based on structural characteristics, we classify the identified cyanobacterial metacaspases into two families, cbmc α and cbmc β .
cyanobacterial metacaspase family α  includes  <dig> mcas that process none other identifiable domains than p <dig>  this family was further divided into four subfamilies.
subfamily α  processing the casc domain only, contains one mca .
subfamily ii  maintaining a single transmembrane  domain, contains five mcas found in filamentous cyanobacteria anabaena variabilis atcc  <dig>  nostoc punctiforme pcc  <dig>  nostoc sp.
pcc  <dig>  and unicellular cyanobacterium acaryochloris marina mbic  <dig>  subfamily iii  contains  <dig> proteins, which is the majority in cyanobacterial mcas.
these mcas have an unidentified c-terminal domain each, and distribute among all cyanobacterial strains except gloeobacter violaceus pcc  <dig>  mcas attributed to subfamily i to iii process casc in their n-terminal.
while the single mca of subfamily iv ,  <dig> from trichodesmium erythraeum ism  <dig>  holds the casc domain in the c-terminal.
cyanobacterial metacaspase family β  occupying at least one additional domain, comprises  <dig>  metacaspases from all filamentous cyanobacteria and three unicellular species .
the number of additional domains varies from  <dig> to  <dig>  gloeobacter violaceus pcc  <dig> contains the largest number of additional domains, which is  <dig> times than that of the total mcas .
seven unicellular strains lack additional domains, including synechococcus sp.
strains, microcystis aeruginosa nies- <dig> and synechocystis sp.
pcc  <dig>
in total,  <dig> types of additional domains were identified in cyanobacterial mcas: anf-receptor, wd <dig>  gun <dig>  nacht, duf <dig>  chase <dig>  pentapeptide, dexdc, helicc, and ez-heat .
most of these domains are involved in signal transduction, for example chase <dig>  gun <dig>  anf-receptor and wd <dig> .
in addition, prevalent domains with scaffolding or unknown functions in bacteria were identified, such as duf <dig>  ez heat and pentapeptide  <cit> .
two domains within helicases, helicc and dexdc, were found to fuse together in  <dig> of acaryochloris marina mbic  <dig>  interestingly, pcd related domain nacht was also detected in two mcas .
wd <dig> repeats, the most prevalent additional domains identified in  <dig> cyanobacterial species, replicate for  <dig> to  <dig> copies in a single metacaspase protein.
some additional domains were identified exclusively in a particular metacaspase protein, including dexdc and helicc in  <dig> ; ez heat in  <dig> , chase <dig> in  <dig> , and anf-receptor in  <dig> .
phylogenetic analysis
considering the confusion created by additional domains with possibly separate evolutionary histories, the conserved catalytic domains of mcas instead of their whole sequences were used during the phylogenetic study .
the catalytic domains, about  <dig> amino acids in length, were identified using smart and cdd databases  <cit> .
the mca phylogenetic tree was rooted in the human caspase- <dig> and the putative metacaspase of gamma proteobacterium.
the phylogeny tree comprises two clades in general.
mcas from cbmc α belong to clade i  that also includes two bacterial mcas  and seven eukaryotic mcas from t. pseudonana, s. cerevisiae and chlamydomonas reinhardtii.
all of these mcas are orthologues because of obvious evolutionary relationships with high bootstrap value.
clade ii  contains members of cbmc β family with wd <dig>  duf <dig> and signalling domains, which cluster separately according to the additional domains.
those from subfamily cbmc α-tm and eubacteria  attribute to clade ii as well.
in a word, most photosynthetic bacterial mcas cluster with mcas of cyanobacterial family β and the eukaryotic mcas gather with family α. within each clade, the mcas cluster according to the phylogeny of the species.
within clade i, one copy of mca containing the his/cys mutation is found in each cyanobacterium that maintains mca, except for gloeobacter violaceus pcc  <dig>  all of these mcas belong to cbmc α-other and cluster strictly based on the phylogeny of the species.
all of the mutated mcas in clade i are orthologs because of their close evolutionary relationships.
besides, four mutated mcas from one unicellular and three filamentous cyanobacteria gather together in clade ii.
anabaena variabilis atcc  <dig> and nostoc sp.
pcc  <dig>  two filamentous species that show close evolutionary relationships in 16s rrna tree, share  <dig> pairs of mca sequences in clade i. four wd40-containing mcas from gloeobacter violaceus pcc  <dig> form a separate cluster together.
in addition, some mcas adjacent to each other on the chromosome display close evolutionary relationships, including 637717526/ <dig>  637718423/ <dig> of anabaena variabilis atcc  <dig> and 637230642/ <dig> of nostoc sp.
pcc  <dig>  some mcas of subfamily α are flanked with two wd40- or gun4-containing mcas.
discussion
although metacaspases do not cleave caspase substrates  <cit> , several evidences have been given to support their roles in pcd in plants .
for example, when challenged by the plant pathogen, arabidopsis ko lines of metacaspase suppress cell death  <cit> .
arabidopsis metacaspse- <dig> ko lines triggered by uvc or h2o <dig> display reduced cell death  <cit> .
moreover, in the suspensor cells of an embryogenic culture of picea abies, down-regulation of mca leads to a phenotype with a reduced cell death  <cit> .
what makes metacaspases so interesting?
first, most of the metazoan pcd-related genes are lost in unicellular organisms, excepting metacaspases that play vital roles in pcd of eukaryotic planktons and yeasts.
second, compared with caspases and paracaspases identified in higher animals, metacaspases are widespread among bacteria, fungi and plants, which suggest their early evolutionary positions  <cit> .
bidle and falkowski identified cyanobacterial and phytoplankton metacaspases in silico, and explored the evolution deeply  <cit> .
with the completion of genome sequencing of several cyanobacterial species, modifications and supplements are needed.
for example, mca was reported to be absent in synechococcus sp.
pcc  <dig>  but a protein  was found to contain p <dig> domain.
cyanobacterium thermosynechococcus elongatus bp- <dig> was proved to have no metacaspase orthologue in our study.
moreover, with the release of genomic sequence, mcas were identified in synechococcus sp.
ja-3-3ab and synechococcus sp.
ja-2-3b'a.
the distribution of putative metacaspase encoding open reading
frames  in cyanobacteria is an integrated function of the genome sizes and the ecophysiological properties.
most cyanobacteria process proportionate numbers of putative metacaspase genes with genome sizes, except for symbiotic acaryochloris marina mbic  <dig>  though death is not the only way to adapt to environmental changes, for example, cyanobacteria modify their metabolism in response to different stress conditions  <cit> , death is still a direct and drastic cellular response to environmental changes.
thus diverse distributions of metacaspase genes may reflect various environmental selective pressures.
for example, putative mca encoding orfs are not widespread through unicellular cyanobacteria .
all of the prochlorococcus and synechococcus strains lived in the oligotrophic open ocean lack putative metacaspase genes.
while synechococcus strains that inhabit in freshwater and hot spring still maintain one metacaspase encoding orf.
considering the similar genome size, environmental selective pressure may take responsibility for this difference.
parallel conclusion was provided by serine/threonine kinases in cyanobacteria indicating remarkable reduction of signal transduction proteins and environmental stress response systems in the ocean  <cit> .
gene lost is revealed to facilitate these cyanobacteria to acclimatize to the oligotrophic environment.
the major driving force was supposed to be "a selective process favouring the adaptation of these cyanobacteria", which was discussed by alexis dufresne et al.
in detail  <cit> .
filamentous heterocystous cyanobacteria, on the other hand, differentiate heterocysts in response to the absence of combined nitrogen, and exhibit ecological properties including broad symbiotic competence with plants and fungi  <cit> , contain more putative mca encoding orfs even after allowing for their larger genome sizes  <cit> .
the symbol of caspase superfamily is the possession of catalytic p <dig> domain and the conserved cys-his dyad, forming the "specificity pocket"  <cit> .
within cyanobacterial metacaspases, sequence contexts of his and cys are basically the same as those in caspases sghg, and cys:qacg)  <cit> .
the maintaining of the conserved his and cys indicate the importance of these catalytic sites.
interestingly,  <dig> cyanobacterial mcas encode tyr in place of his and ser/asn/gln/gly instead of cys.
likewise, of two metacaspases in t. brucei, tbmca <dig> and tbmca <dig>  ser occupies the site of the putative cys  <cit> .
considering the fact that experimental mutation of the active-site cysteine to serine resulted in inactive of some cysteine peptidases, mutated cyanobacterial metacaspases may be catalytically inactive.
domain fusion provides a chance to recruit related functions in a single protein, especially within bacteria which maintain smaller genomes and compact gene clusters  <cit> .
additional domains of cyanobacterial mcas, such as gun <dig>  <cit>  and wd <dig>  <cit> , illuminate the signalling pathways involved in pcd.
owe to the considerably specific proteolytic activity and proximity-induced activation, the signalling domains may be the target of the metacaspases.
consequently, metacaspases may take a share in signalling mediation instead of mere protein degradation.
two domains within helicases, helicc and dexdc, were found to fuse together in  <dig> of acaryochloris marina mbic  <dig>  which implies the interactions between the caspase proteolytic activity and atp-dependent rna/dna unwinding.
in addition, domains with same functions tend to assemble in a protein  <cit> , therefore the identification of nacht  <cit>  domain reinforces the possibility of metacaspase involving in pcd.
compared with metazoan caspases, bacterial metacaspases may present a minimal set of apoptotic machinery.
additional domains are typically employed as "sensor response modules" and form multi-domain proteins with mcas to participate in signal transduction.
it can be imagined that caspases recruit additional domains or even large motifs to apoptotic complexes in the evolution.
previous classification criterion of mcas is based on the prodomain, and mcas can be classified into two families   <cit> .
however, most cyanobacterial mcas maintain a c-terminal extension without the linker region and have varied additional domains .
therefore a novel categorization standard based on the additional domain was given in this research.
to avoid future confusion of the mca families, the names "type α" and "type β" were used instead of "type i" and "type ii".
the obtained metacaspase phylogenetic tree indicates that mcas display clear-cut relationships, based strictly on structural characteristics and the phylogeny of the species.
moreover, compared to the species phylogeny, structural characteristics play a more critical role.
the tree of catalytic p <dig> domains coincides well with the phylogenies based on 16s rrna, indicating the rare gene gain-and-loss events and the importance of mcas that remain conserved in history.
besides, four wd40-containing mcas from gloeobacter violaceus pcc  <dig> form a separate cluster indicating obvious lineage-specific duplication events.
anabaena variabilis atcc  <dig> and nostoc sp.
pcc  <dig>  two filamentous species that enjoy a very close evolutionary relatedness in 16s rrna tree, share  <dig> pairs of mca sequences in clade i. these nonorthologous mcas may be produced by gene duplication before the divergence of the two species.
most photosynthetic bacterial mcas cluster with mcas of cyanobacterial family β  and the eukaryotic mcas cluster with family α .
the presence of the mutated mcas and their convergences suggest that mcas of family α and β evolve separately.
one possibility is that the recruitment of wd <dig> additional domain occurs later than the divergence of the two clades.
CONCLUSIONS
the availability of cyanobacterial genome sequences facilitates comparative analysis.
metacaspases, sequence homologs to caspases, play key roles in programmed cell death  in several prokaryotes, fungi and plants.
among  <dig> species of cyanobacteria, a total of  <dig> putative metacaspase genes have been identified.
the quantity of metacaspase genes in unicellular and filamentous cyanobacteria depends on the genome size and ecological habitat.
the cys-his dyad of caspase superfamily is conserved in most cyanobacterial mcas, however, tyr and ser/asn/gln/gly residues have also been detected in the sites of his and cys within some metacaspases.
ten types and a total of  <dig> additional domains were identified, most of which may involve in signal recognition.
programmed cell death related nacht domain was also found in cyanobacterial metacaspases.
phylogenetic tree of mca catalytic p <dig> domains coincides well with the phylogenies based on 16s rrna.

 cyanobacteria are among the earliest branching groups on earth, dating back - billion years, based on the fossil evidence.as a taxonomic unit characterized by the ability to execute oxygenic photosynthesis, cyanobacteria constitute a group of species diverse in genome size and ecological habitats, indicating the significance of comparative genome research. cyanobacteria, with a variation in genome size from mb to mb, are found in almost every imaginable environment, from ocean to fresh water to bare rock. cyanobacteria also inhabit in the extreme environments, for example, synechococcus sp. ja-2-3b'a and synechococcus sp. ja-3-3ab were separated from hot spring. as unicellular and non-nitrogen-fixing cyanobacteria, prochlorococcus sp. and synechococcus sp. maintain the smallest genome sizes and account for significant biomass and primary production of marine biosphere. other unicellular species have larger genome sizes, including water bloom forming cyanobacteria, a thylakoids absence cyanobacterium, a nitrogen-fixing cyanobacterium, and an animal-cyanobacterial symbionsis. while the diazotrophic filamentous cyanobacteria comprise the largest genome size, such as nostoc sp. pcc anabaena variabilis atcc plant-cyanobacteria symbionsis nostoc punctiforme pcc and marine trichodesmium sp. ims programmed cell death is a suicide mechanism to promote and maintain genetic stability. pcd was considered as a characteristic of metazoans for a long time before apoptosis markers were found in yeast which indicates multicellularity is not the most important prerequisite. recently, pcd mechanism has been observed in all but one of the six/eight major groups of prokaryotes, with the exception of the rhizaria. experimental evidences for pcd in cyanobacteria come from three species, including the freshwater cyanobacterium anabaena spp. exposed to univalent-cation salts, the bloom-causing cyanobacterium microcystis aeruginosa from st. lucie estuary by treatment with h2o and trichodesmium sp. ims suffering iron starvation and light irradiance. caspases are one of the most important and widely researched apoptotic proteins in mammalian pcd. caspase was initially thought to be limited to metazoans, and no one had managed to identify caspase homologues, either in plants or bacteria. then uren and his colleagues identified two ancient families of caspase-like proteins, paracaspases and metacaspases in silico and khan and his co-workers demonstrated that a yeast metacaspase mediates pcd in saccharomyces cerevisiae. hereafter, metacaspases were found involved in pcd of yeasts, filamentous fungi, plants, and a variety of bacteria. most of these metacaspases share sequence homology with caspases, but show different substrate specificity. metacaspases belong to caspase family, which are part of the clan cd, a family of proteases characteristic with their his/cys catalytic dyad. metacaspases process a conserved caspase catalytic subunit p domain, and share conserved amino acid residues within his- and cys-catalytic sites. interestingly, most of the typical genes encoding in metazoan pcd are missing in bacteria and early-branching eukaryotes, such as cad and p. therefore, the presence of metacaspases suggests a concernful role within pcd evolution. cyanobacteria maintain a rich metacaspase pool, and many of these genes have been identified in silico was identified in some sequenced cyanobacteria strains, including gloeobacter violaceus pcc thermosynechococcus elongatus bp- synechocystis sp. pcc trichodesmium erythraeum ism nostoc punctiforme pcc nostoc sp. pcc and anabaena variabilis atcc metacaspases were absent in med prochlorococcus marinus mit ss, synechococcus sp. wh synechococcus elongatus pcc and synechococcus elongates. with the completion of genome sequencing of several cyanobacterial species, modifications and supplements are needed. as of november genomes of unicellular and filamentous cyanobacteria became available, which facilitates cyanobacterial systemic analysis for restriction-modification systems and serine/threonine protein kinases. the comparative genome research on metacaspases and other pcd proteins in filamentous fungi has been documented. besides, koonin and aravind described a clear affinity of bacterial metacaspases and the metazoan caspases by phylogenetic analysis of caspase-like protease superfamily. in this study, we selected five proven metacaspases in marine diatom thalassiosira pseudonana to search for cyanobacterial metacaspases. metacaspases in thalassiosira pseudonana were chosen due to the few metacaspases verified experimentally in cyanobacteria and the close evolutional relationship between cyanobacteria and eukaryotic phytoplankton. we employed a blastp-plus-phylogeny reconstruction approach to analyze metacaspase sequences in cyanobacteria, and present an overall view of their classifications, structure, phylogeny and evolution. better understanding of cyanobacterial metacaspases may provide further insights into evolution of pcd.  identification of metacaspase proteins the complete cyanobacterial genomes downloaded from img database were used in this research. to identify proteins similar to proven metacaspases from thalassiosira pseudonana, we performed blastp searches of the cyanobacterial genomes. cdd and smart analyses with the derived sequences were then carried out to eliminate false positives. two proteins annotated as caspase catalytic subunit p in img database, including from trichodesmium erythraeum ism and from microcystis aeruginosa nies- were found to lack catalytic domain and excluded. of two proteins, casc domain recognized by cdd was not identified in smart analysis, and both of the proteins were excluded. altogether, putative metacaspase sequences were considered in this study. twenty-six of which were originally annotated as peptidase c caspase catalytic subunit p or protein containing caspase domain. the remaining proteins were accepted as metacaspases in this research, including proteins annotated by other additional domains, proteins annotated as hypothetical proteins, and proteins annotated as unknown function protein duf a: the numbers indicate "img gene object identifier" in img database amid diverse cyanobacterial genomes, the number of metacaspase genes varies from to within unicellular cyanobacteria, symbiont acaryochloris marina mbic has mcas, much more than other species. correspondingly, the percentage of metacaspases within total proteins is highest among unicellular cyanobacteria. all of the marine synechococcus strains lack mcas except for synechococcus sp. pcc only one metacaspase gene was found in the synechocystis sp. pcc four synechococcus strains inhabit in freshwater, land and hot spring contain one metacaspase for each, including pcc pcc ja-2-3b'a, and ja-3-3ab. water-blooming cyanobacterium microcystis aeruginosa nies- contains two mcas. the percentages of mcas within total proteins in synechocystis sp., synechococcus sp. and microcystis sp. strains are the lowest. the other two unicellular cyanobacterial strains, gloeobacter violaceus pcc and cyanothece sp. atcc maintain to mcas and have moderate percentages of mcas in total proteins: % and %, respectively. compared to unicellular cyanobacteria, filamentous diazotrophic cyanobacteria have more metacaspase genes. among the filamentous cyanobacteria, nostoc punctiforme pcc has the largest genome; however, nonheterocystous cyanobacterium trichodesmium erythraeum ims contains the largest number of metacaspases and the highest percentage in total protein. one or two mcas containing mutations of conserved catalytic sites were found in every cyanobacterial strain, exclusive of gloeobacter violaceus pcc in most cases, the his residues are replaced by tyr, and the cys residues mutate into ser, asn or gly. uniquely, within the metacaspase from cyanothece sp. attc gln substitutes for the cys residue. structure and function based on structural characteristics, we classify the identified cyanobacterial metacaspases into two families, cbmc α and cbmc β. cyanobacterial metacaspase family α includes mcas that process none other identifiable domains than p this family was further divided into four subfamilies. subfamily α processing the casc domain only, contains one mca. subfamily ii maintaining a single transmembrane domain, contains five mcas found in filamentous cyanobacteria anabaena variabilis atcc nostoc punctiforme pcc nostoc sp. pcc and unicellular cyanobacterium acaryochloris marina mbic subfamily iii contains proteins, which is the majority in cyanobacterial mcas. these mcas have an unidentified c-terminal domain each, and distribute among all cyanobacterial strains except gloeobacter violaceus pcc mcas attributed to subfamily i to iii process casc in their n-terminal. while the single mca of subfamily iv, from trichodesmium erythraeum ism holds the casc domain in the c-terminal. cyanobacterial metacaspase family β occupying at least one additional domain, comprises metacaspases from all filamentous cyanobacteria and three unicellular species. the number of additional domains varies from to gloeobacter violaceus pcc contains the largest number of additional domains, which is times than that of the total mcas. seven unicellular strains lack additional domains, including synechococcus sp. strains, microcystis aeruginosa nies- and synechocystis sp. pcc in total, types of additional domains were identified in cyanobacterial mcas: anf-receptor, wd gun nacht, duf chase pentapeptide, dexdc, helicc, and ez-heat. most of these domains are involved in signal transduction, for example chase gun anf-receptor and wd. in addition, prevalent domains with scaffolding or unknown functions in bacteria were identified, such as duf ez heat and pentapeptide. two domains within helicases, helicc and dexdc, were found to fuse together in of acaryochloris marina mbic interestingly, pcd related domain nacht was also detected in two mcas. wd repeats, the most prevalent additional domains identified in cyanobacterial species, replicate for to copies in a single metacaspase protein. some additional domains were identified exclusively in a particular metacaspase protein, including dexdc and helicc in; ez heat in, chase in, and anf-receptor in. phylogenetic analysis considering the confusion created by additional domains with possibly separate evolutionary histories, the conserved catalytic domains of mcas instead of their whole sequences were used during the phylogenetic study. the catalytic domains, about amino acids in length, were identified using smart and cdd databases. the mca phylogenetic tree was rooted in the human caspase- and the putative metacaspase of gamma proteobacterium. the phylogeny tree comprises two clades in general. mcas from cbmc α belong to clade i that also includes two bacterial mcas and seven eukaryotic mcas from t. pseudonana, s. cerevisiae and chlamydomonas reinhardtii. all of these mcas are orthologues because of obvious evolutionary relationships with high bootstrap value. clade ii contains members of cbmc β family with wd duf and signalling domains, which cluster separately according to the additional domains. those from subfamily cbmc α-tm and eubacteria attribute to clade ii as well. in a word, most photosynthetic bacterial mcas cluster with mcas of cyanobacterial family β and the eukaryotic mcas gather with family α. within each clade, the mcas cluster according to the phylogeny of the species. within clade i, one copy of mca containing the his/cys mutation is found in each cyanobacterium that maintains mca, except for gloeobacter violaceus pcc all of these mcas belong to cbmc α-other and cluster strictly based on the phylogeny of the species. all of the mutated mcas in clade i are orthologs because of their close evolutionary relationships. besides, four mutated mcas from one unicellular and three filamentous cyanobacteria gather together in clade ii. anabaena variabilis atcc and nostoc sp. pcc two filamentous species that show close evolutionary relationships in 16s rrna tree, share pairs of mca sequences in clade i. four wd40-containing mcas from gloeobacter violaceus pcc form a separate cluster together. in addition, some mcas adjacent to each other on the chromosome display close evolutionary relationships, including 637717526/ 637718423/ of anabaena variabilis atcc and 637230642/ of nostoc sp. pcc some mcas of subfamily α are flanked with two wd40- or gun4-containing mcas. discussion although metacaspases do not cleave caspase substrates, several evidences have been given to support their roles in pcd in plants. for example, when challenged by the plant pathogen, arabidopsis ko lines of metacaspase suppress cell death. arabidopsis metacaspse- ko lines triggered by uvc or h2o display reduced cell death. moreover, in the suspensor cells of an embryogenic culture of picea abies, down-regulation of mca leads to a phenotype with a reduced cell death. what makes metacaspases so interesting? first, most of the metazoan pcd-related genes are lost in unicellular organisms, excepting metacaspases that play vital roles in pcd of eukaryotic planktons and yeasts. second, compared with caspases and paracaspases identified in higher animals, metacaspases are widespread among bacteria, fungi and plants, which suggest their early evolutionary positions. bidle and falkowski identified cyanobacterial and phytoplankton metacaspases in silico, and explored the evolution deeply. with the completion of genome sequencing of several cyanobacterial species, modifications and supplements are needed. for example, mca was reported to be absent in synechococcus sp. pcc but a protein was found to contain p domain. cyanobacterium thermosynechococcus elongatus bp- was proved to have no metacaspase orthologue in our study. moreover, with the release of genomic sequence, mcas were identified in synechococcus sp. ja-3-3ab and synechococcus sp. ja-2-3b'a. the distribution of putative metacaspase encoding open reading frames in cyanobacteria is an integrated function of the genome sizes and the ecophysiological properties. most cyanobacteria process proportionate numbers of putative metacaspase genes with genome sizes, except for symbiotic acaryochloris marina mbic though death is not the only way to adapt to environmental changes, for example, cyanobacteria modify their metabolism in response to different stress conditions, death is still a direct and drastic cellular response to environmental changes. thus diverse distributions of metacaspase genes may reflect various environmental selective pressures. for example, putative mca encoding orfs are not widespread through unicellular cyanobacteria. all of the prochlorococcus and synechococcus strains lived in the oligotrophic open ocean lack putative metacaspase genes. while synechococcus strains that inhabit in freshwater and hot spring still maintain one metacaspase encoding orf. considering the similar genome size, environmental selective pressure may take responsibility for this difference. parallel conclusion was provided by serine/threonine kinases in cyanobacteria indicating remarkable reduction of signal transduction proteins and environmental stress response systems in the ocean. gene lost is revealed to facilitate these cyanobacteria to acclimatize to the oligotrophic environment. the major driving force was supposed to be "a selective process favouring the adaptation of these cyanobacteria", which was discussed by alexis dufresne et al. in detail. filamentous heterocystous cyanobacteria, on the other hand, differentiate heterocysts in response to the absence of combined nitrogen, and exhibit ecological properties including broad symbiotic competence with plants and fungi, contain more putative mca encoding orfs even after allowing for their larger genome sizes. the symbol of caspase superfamily is the possession of catalytic p domain and the conserved cys-his dyad, forming the "specificity pocket". within cyanobacterial metacaspases, sequence contexts of his and cys are basically the same as those in caspases sghg, and cys:qacg). the maintaining of the conserved his and cys indicate the importance of these catalytic sites. interestingly, cyanobacterial mcas encode tyr in place of his and ser/asn/gln/gly instead of cys. likewise, of two metacaspases in t. brucei, tbmca and tbmca ser occupies the site of the putative cys. considering the fact that experimental mutation of the active-site cysteine to serine resulted in inactive of some cysteine peptidases, mutated cyanobacterial metacaspases may be catalytically inactive. domain fusion provides a chance to recruit related functions in a single protein, especially within bacteria which maintain smaller genomes and compact gene clusters. additional domains of cyanobacterial mcas, such as gun and wd, illuminate the signalling pathways involved in pcd. owe to the considerably specific proteolytic activity and proximity-induced activation, the signalling domains may be the target of the metacaspases. consequently, metacaspases may take a share in signalling mediation instead of mere protein degradation. two domains within helicases, helicc and dexdc, were found to fuse together in of acaryochloris marina mbic which implies the interactions between the caspase proteolytic activity and atp-dependent rna/dna unwinding. in addition, domains with same functions tend to assemble in a protein, therefore the identification of nacht domain reinforces the possibility of metacaspase involving in pcd. compared with metazoan caspases, bacterial metacaspases may present a minimal set of apoptotic machinery. additional domains are typically employed as "sensor response modules" and form multi-domain proteins with mcas to participate in signal transduction. it can be imagined that caspases recruit additional domains or even large motifs to apoptotic complexes in the evolution. previous classification criterion of mcas is based on the prodomain, and mcas can be classified into two families. however, most cyanobacterial mcas maintain a c-terminal extension without the linker region and have varied additional domains. therefore a novel categorization standard based on the additional domain was given in this research. to avoid future confusion of the mca families, the names "type α" and "type β" were used instead of "type i" and "type ii". the obtained metacaspase phylogenetic tree indicates that mcas display clear-cut relationships, based strictly on structural characteristics and the phylogeny of the species. moreover, compared to the species phylogeny, structural characteristics play a more critical role. the tree of catalytic p domains coincides well with the phylogenies based on 16s rrna, indicating the rare gene gain-and-loss events and the importance of mcas that remain conserved in history. besides, four wd40-containing mcas from gloeobacter violaceus pcc form a separate cluster indicating obvious lineage-specific duplication events. anabaena variabilis atcc and nostoc sp. pcc two filamentous species that enjoy a very close evolutionary relatedness in 16s rrna tree, share pairs of mca sequences in clade i. these nonorthologous mcas may be produced by gene duplication before the divergence of the two species. most photosynthetic bacterial mcas cluster with mcas of cyanobacterial family β and the eukaryotic mcas cluster with family α. the presence of the mutated mcas and their convergences suggest that mcas of family α and β evolve separately. one possibility is that the recruitment of wd additional domain occurs later than the divergence of the two clades.  the availability of cyanobacterial genome sequences facilitates comparative analysis. metacaspases, sequence homologs to caspases, play key roles in programmed cell death in several prokaryotes, fungi and plants. among species of cyanobacteria, a total of putative metacaspase genes have been identified. the quantity of metacaspase genes in unicellular and filamentous cyanobacteria depends on the genome size and ecological habitat. the cys-his dyad of caspase superfamily is conserved in most cyanobacterial mcas, however, tyr and ser/asn/gln/gly residues have also been detected in the sites of his and cys within some metacaspases. ten types and a total of additional domains were identified, most of which may involve in signal recognition. programmed cell death related nacht domain was also found in cyanobacterial metacaspases. phylogenetic tree of mca catalytic p domains coincides well with the phylogenies based on 16s rrna. 
phylogenetic tree of mca catalytic p domains coincides well with the domain structure and the phylogenies based on 16s rrna. cyanobacteria are an ancient group of photoautotrophic prokaryotes with wide variations in genome size and ecological habitat. the existence and quantity of mca genes in unicellular and filamentous cyanobacteria are a function of the genome size and ecological habitat. mcas have been identified in several prokaryotes, fungi and plants; however, knowledge about cyanobacterial metacaspases still remains obscure. a total of putative mcas were identified, which are abundant in filamentous diazotrophic cyanobacteria and acaryochloris marina mbic and absent in all prochlorococcus and marine synechococcus strains, except synechococcus sp. ten types and a total of additional domains were identified, most of which involves in signal transduction. pcc the cys-his dyad of caspase superfamily is conserved, while mutations are also detected in some cyanobacteria. apoptotic related nacht domain was also found in two cyanobacterial mcas. mcas of family α and β seem to evolve separately and the recruitment of wd additional domain occurs later than the divergence of the two families. metacaspases are cysteine proteinases that have sequence homology to caspases and play essential roles in programmed cell death.
 cyanobacteria are an ancient group of photoautotrophic prokaryotes with wide variations in genome size and ecological habitat. metacaspases are cysteine proteinases that have sequence homology to caspases and play essential roles in programmed cell death. mcas have been identified in several prokaryotes, fungi and plants; however, knowledge about cyanobacterial metacaspases still remains obscure. with the availability of sequenced genomes of cyanobacteria, we perform a comparative analysis of metacaspases and explore their distribution, domain structure and evolution.  a total of putative mcas were identified, which are abundant in filamentous diazotrophic cyanobacteria and acaryochloris marina mbic and absent in all prochlorococcus and marine synechococcus strains, except synechococcus sp. pcc the cys-his dyad of caspase superfamily is conserved, while mutations are also detected in some cyanobacteria. mcas can be classified into two major families based on the additional domain structure. ten types and a total of additional domains were identified, most of which involves in signal transduction. apoptotic related nacht domain was also found in two cyanobacterial mcas. phylogenetic tree of mca catalytic p domains coincides well with the domain structure and the phylogenies based on 16s rrna.  the existence and quantity of mca genes in unicellular and filamentous cyanobacteria are a function of the genome size and ecological habitat. mcas of family α and β seem to evolve separately and the recruitment of wd additional domain occurs later than the divergence of the two families. in this study, a general framework of sequence-structure-function connections for the metacaspases has been revealed, which may provide new targets for function investigation. 