The Mic-PCC7806 genome includes a very large number of DNA sequences containing more than 1000 bases that are repeated at least twice in the genome with more than 90% identity. A comparative analysis of all the cyanobacterial genome sequences available in databases showed that Mic-PCC7806, Mic-NIES843 and Cwa-WH8501 are particularly rich in such DNA repeats. Indeed, they account for 11.7%, 11.7% and 19.8% of the total DNA length, respectively (Figure 2). The cumulative size of the DNA repeated sequences is not strictly a function of genome length as Mic-PCC7806 and Cwa-WH8501 genomes have the highest percentage of DNA repeats, but are of intermediate size relative to the other cyanobacterial genomes (see Additional file 3). In the Mic-PCC7806 genome, 1346 CDSs (25%) are located within these DNA repeats. Among these CDSs, only 256 and 92 belong to the maeru40 and core40 groups, respectively. Most of the CDSs of the core40 group correspond to orthologs that are not located within DNA repeats in other cyanobacterial genomes. This implies that over the course of evolution, resident genes were probably captured by genetic mobile elements. A large number of CDSs (362) are very similar to transposases from the COG database, and 93% of them are located within long DNA repeated sequences. At least 46 transposases correspond to ISMae1A/2/3/4 that had previously been characterized in strain PCC7806 22, but a large majority of the other transposases cannot be clearly associated with any known insertion sequence (only 17 are associated to IS30, 7 to IS1 and 3 to IS5). The genome of Cwa-WH8501 also contains numerous putative transposases. One third of them are associated to IS5, but none to IS30; the DNA repeated sequences are therefore different in each genome, and cannot account for the close phylogenetic relationship between these two strains.

Although Mic-PCC7806 and Mic-NIES843 are very closely related strains (Figure 1), their genomes contain a high number of rearrangements. Moreover, an unexpectedly low level of synteny was also observed between the Microcystis strains and two close relatives, Cwa-WH8501 and Syn-PCC6803 (68% mean CDS similarity). Since the same observation was made for all the cyanobacterial genomes tested, we compared the dynamics of these genomes using a large set of other bacterial genomes chosen on the basis of their sizes and phylogenetic distances. To this end, a synteny score was calculated for a number of genome pairs (see Methods), and then compared to their evolutionary distance based on the 23S-16S rDNA tree. This analysis showed that the synteny scores for cyanobacterial genomes were significantly lower than those obtained for pairs of non-cyanobacterial genomes with similar genome lengths and 23S-16S phylogenetic distances (Figure 3). Similar results were obtained for all the cyanobacterial genomes tested. This means that the low synteny scores observed cannot be related to the long DNA repeated sequences, which occur only in the Mic-PCC7806 and Cwa-WH8501 genomes. These results are in agreement with those of Fang et al. 23, who showed that both persistent and rare genes are significantly clustered in most of the 169 bacterial genomes analyzed. However, in a minority subset of bacterial genomes that includes the cyanobacteria, persistent genes were found to be fairly uniformly distributed throughout the genome.

Interestingly, only 8 clusters with at least 4 CDSs remain syntenic in the genomes of Mic-PCC7806, Cwa-WH8501 and Syn-PCC6803. Four of these clusters correspond to ribosomal proteins. The other clusters are shown in Table 3. Considering the very low level of synteny between cyanobacterial genomes, it is likely that these specific clusters have been subjected to strong positive selection pressure and may play essential roles in these cyanobacteria. Some of these clusters are clearly linked to a specific biological function, such as the transport of phosphate (see Additional file 4) 24, while others consist of conserved proteins with unknown functions. One can thus speculate that these proteins may be involved in the same biological pathway as their close neighbors.

Four groups can clearly be identified among the cyanobacterial genomes studied on the basis of their intergenic distances (Figure 4). The first consists solely of the genome of Ter-IMS101, which harbors exceptionally long intergenic regions. To the best of our knowledge, no data has been published on this genome, which makes it impossible to rule out the possibility that these regions result from the poor quality of the sequence or the syntaxic annotation. The second group includes the genome of Mic-PCC7806 and, among others, those of Cwa-WH8501 and Syn-PCC6803 which have a high proportion of intergenic sequences around 300 bases long; in the case of the Mic-PCC7806 genome, less than 35% of intergenic sequences are shorter than 100 bases. The third group comprises the genomes of Syn-PCC7942, Tel-BP1 and Gvi-PCC7421, which have short intergenic regions, similar in size to those found in a number of other bacterial genomes (see Additional file 5). The fourth group includes some members of the Prochlorococcus genus that have very small genomes with short or no intergenic regions.

The mean length of the intergenic sequences seems to be linked to the genome size of the cyanobacterium, except for the genome of Syn-PCC6803, which is smaller (3.6 Mb) than that of Gvi-PCC7421 (4.6 Mb), but harbors longer intergenic sequences. Although the role of long intergenic sequences in most cyanobacterial genomes remains unclear, we can surmise that they might be involved in the modulation of gene expression, which would allow cells to acclimate to rapid environmental changes.

In order to explore the plasticity of the Mic-PCC7806 genome further, the number of CDSs with an atypical dinucleotide composition was determined using a one-order Markov chain-based methodology 25. This method can identify genes that may have been acquired recently by lateral transfers. In the Mic-PCC7806 genome, a total of 1971 atypical genes were found, including 1402 within 159 clusters of atypical genes (CAGs) that probably correspond to recently acquired foreign genomic elements (Table 4). As expected, more than 98% of Mic-PCC7806 genes belonging to the core40 group were not in CAGs, and 31% of the atypical genes were in the maeru40 group. Moreover, a high percentage (80%) of the transposase genes were in CAGs (16% of the genes present in CAGs encode putative transposases). Compared to seven other cyanobacterial genomes, those of Mic-PCC7806 and Mic-NIES843 harbor the highest percentages of atypical genes (37%) and CAGs (34% and 36%, respectively). These findings may indicate that the Microcystis genomes contain a higher proportion of genes recently acquired by lateral transfers than the other genomes studied.

Blast searches for restriction enzymes and examination of genes surrounding DNA methylases, identified 21 potential restriction enzymes (see Additional file 6), seven of which were found to be co-localized with putative methylases (see Additional file 7) in the Mic-PCC7806 genome. The Mic-NIES843 genome also contains a high number (at least 17) of putative restriction enzymes 21. Blast searches revealed that 14 restriction enzymes are common to both genomes. In contrast, seven and eight restriction enzymes seem specific to Mic-PCC7806 and Mic-NIES843, respectively. The Microcystis aeruginosa strains might thus constitute a rich source of novel restriction enzymes potentially useful in biotechnology. According to Zhao et al. 26, filamentous cyanobacteria (Anabaena, Spirulina and Nostoc strains) contain more restriction and modification genes than unicellular cyanobacteria (Synechocystis, Synechococcus and Prochlorococcus strains). Based on COG annotations, at least as many restriction-modification genes were found in Mic-PCC7806, Mic-NIES843 and Cwa-WH8501 as in filamentous cyanobacteria. Thus, rather than corresponding to a difference between filamentous and unicellular cyanobacteria, the restriction-modification gene content of Microcystis aeruginosa may reflect the potential exposure of the cells to high concentrations of foreign DNA due to the presence of numerous other bacterial cells or viruses associated with Microcystis colonies 27. This exposure to foreign DNA is also consistent with the high number of CAGs putatively acquired by lateral transfers. Whether such a hypothesis might also hold true for planktonic cyanobacteria of the genus Crocosphaera remains an open question.

In bacterial genomes containing a high number of genes for restriction enzymes, short palindromic sequences corresponding to the target sites of these enzymes may be under-represented 28. Since the genomes of Microcystis aeruginosa and Cwa-WH8501 contain a very high number of putative restriction enzymes, there should be a number of under-represented short sequences that correspond to restriction sites. To test this hypothesis, the number of occurrences of each 6-mer was counted, and a frequency distribution calculated for Mic-PCC7806, Mic-NIES843, Cwa-WH8501 and Syn-PCC6803 (Table 5). The under-represented sites in the three first genomes were not found in Syn-PCC6803, a genome devoid of restriction enzymes 29, supporting the idea that these rare 6-mers could indeed correspond to restriction enzyme sites. In total, there are 4096 possible 6-mers, 1.5% of which are palindromes. Fifty-one percent of the rarest 1% of 6-mers in the Mic-PCC7806 genome are palindromes (see Additional file 8). Palindromes are thus over-represented among the rarest 6-mers, further supporting the hypothesis that they could correspond to sites cut by restriction enzymes. The identity of the rarest 1% of 6-mers in the Mic-PCC7806 genome was compared to known restriction sites in other organisms as identified by New England Biolabs 30. We found that 20 of the 41 sites corresponded to sites cut by restriction enzymes in other organisms.

A novel DNA modification system was discovered recently in the Gram-positive bacterium Streptomyces lividans 66 31. This system results in the degradation of DNA in vitro by oxidative, double-stranded, site-specific cleavage during electrophoresis, and is determined by a cluster of five genes (dndA-B-C-D-E). The dnd gene products incorporate sulfur into the DNA backbone as a sequence-selective, stereospecific phosphorothioate modification 32. According to He et al. 33, the resistance of phosphorothiate linkages to a variety of nuclease activities, and the site specific nature of such a modification suggest that phosphorothioates could have a role comparable to that of DNA methylation in protection against nucleases. Although the presence of dndB homologs is not clear in the genomes of cyanobacteria, the rest of the cluster was found in several of them including Mic-PCC7806 (see Additional file 9). Despite the low level of synteny in cyanobacterial genomes (see above), the dndC-D-E genes are still clustered.

During the overwintering benthic phase of their life cycle, Microcystis colonies withstand long periods of darkness. A fermentation pathway has been proposed based on biochemical data 34. All the genes coding for the enzymes required for the various steps in this pathway have been identified in the genome sequence (see Additional file 10). During the benthic phase, Microcystis colonies are exposed to lower temperature and higher pressure. In this respect, it is interesting to note the presence of a gene (mic5251) coding for a protein similar to Hik33 that perceives osmotic stress and cold stress in Syn-PCC6803 35. Another gene, mic5237, is similar to the Ana-PCC7120 orrA gene whose product is involved in osmoregulation 36. A genomic island carrying actM and pfnM, two genes that encode eukaryotic-like proteins, actin and profilin (an actin cognate binding partner), respectively, have been discovered in the Mic-PCC7806 genome. As shown by Guljamow et al. 37, this eukaryotic-like actin forms a shell-like structure that could strengthen cell resistance to hydrostatic and osmotic pressures. Interestingly, these genes are only present in Microcystis cells that inhabit the Braakman water reservoir (The Netherlands), which was cut off from the sea in the 20^th century, and from which the Mic-PCC7806 strain was originally isolated.

Although several different M. aeruginosa morphotypes have been described 38, little is known about their colony formation. The genome sequence of strain Mic-PCC7806 revealed a gene coding for a lectin (mvn; mic3128), which binds specifically to a sugar moiety present on the surface of Mic-PCC7806 cells, and a binding partner has been identified in the lipoplysaccharide fraction 39. A functional correlation between the potent toxin microcystin and this lectin has been demonstrated, with possible implications for the formation of colonial aggregates that are characteristic of different Microcystis morphotypes. Another protein, MrpC (

m

r

p

In natural populations of Microcystis, oxidative stress was shown to induce programmed cell death (PCD) 46. Accordingly, 5 putative eukaryotic caspase-like genes were identified by PSI-Blast in the genome of strain Mic-PCC7806. Three of them (Mic0980, Mic3930 and Mic4051) showed best similarity with Mic-NIES843 proteins that lack caspase-like motifs. Consequently, these three proteins are likely involved in other functions than PCD. In contrast, the Mic1068 protein showed similarity in the caspase-like region with one protein of Mic-NIES843 (MAE24870). The last caspase-like protein of Mic-PCC7806 (Mic5406) is strain-specific. Both mic1068 and mic5406 are expressed, and a cross-reaction with human caspase-3 polyclonal antisera was observed indicating that the proteins are synthesized (data not shown). Alignment of the regions containing the conserved caspase domains of Mic1068, Mic5406, MAE24870 and a yeast metacaspase shows that the Histidine-Cysteine catalytic diad of the key functionnal regions of the capases is conserved (see Additional file 12). PCD might thus be triggered when Microcystis cells are exposed to severe environmental stress conditions, leading to the rapid decline of blooms, as has been suggested by Berman-Frank et al. in the case of Ter-IMS101 47. Mic-PCC7806 and Mic-NIES843 are the only unicellular cyanobacteria known to have genes coding for HstK-like kinases (mic1879 and mic1015), proteins characterized by the presence of both His and Ser/Thr kinase domains 4849. Some of these kinases are implicated in either the iron homeostasis/oxidative stress response or in the differentiation of N₂-fixing cells in filamentous cyanobacteria [4849 and C-C Zhang, unpublished data]. Cell differentiation does not occur in M. aeruginosa, but it would be interesting to test whether these HstK-like protein kinases are involved in iron homeostasis and/or in the control of programmed cell death in response to oxidative stress. It has been proposed that the methionine recycling pathway may contribute to preventing oxidative stress in Bacillus subtilis 5051. Interestingly, all the genes involved in this pathway are present in the Mic-PCC7806 genome (see Additional file 13). One of these genes, mtnW (rbcL_IV), encodes a 2,3-diketo-5-methylthiopentyl-1-phosphate enolase that has been identified in all the Microcystis strains tested including Mic-NIES843 2152, but not in other cyanobacteria for which the genome sequences are available, except Lae-PCC8106 (accession n° ZP_01618990) and Cth-PCC8801 (accession n° ZP_02940034). The putative methionine recycling pathway may thus have a specific role related to the lifestyle or ecological niches inhabited by members of the genera Microcystis, Lyngbya and Cyanothece.

Cyanobacteria are known as prolific producers of natural products, in particular of the nonribosomal peptide and polyketide classes 1553. However, the potential to produce complex secondary metabolites largely varies among the cyanobacterial genera and species, and even among individual strains. Remarkably, the genomes of Mic-PCC7806, Mic-NIES843 and Cwa-WH8501 differ from unicellular cyanobacteria of other genera in that they contain a large number of genes that encode nonribosomal peptide synthetases (NRPS) and polyketide synthases (PKS). Interestingly, such genes in Mic-PCC7806 outnumber those found in Mic-NIES843 and Cwa-WH8501 (Table 6). Apart from the terrestrial filamentous strain Npu-PCC73102, Mic-PCC7806 devotes the largest percentage of its genome (~3.5%) to secondary metabolite production (Table 6) 54.

The strain Mic-PCC7806 is known to produce two isoforms of microcystin 55. The corresponding genes in the bi-directional mcyA-J gene cluster encoding NRPS, PKS and tailoring enzymes 5657 could be re-assigned during the genome sequencing project (Figure 5). Genes for cyanopeptolin biosynthesis (mcn cluster) could be assigned based on the amino acid specificities of the substrate-activating domains of a second NRPS gene cluster that was congruent with the amino acid moieties contained in the cyanopeptolin structure 58 (Figure 5). The mcn genes of Mic-PCC7806 display some similarity to the anabaenopeptilide genes of Anabaena strain 90 59 and to the cyanopeptolin genes of Microcystis wesenbergii 60. In addition, the genome of Mic-PCC7806 harbors three NRPS and PKS gene clusters (Figure 5). One of the clusters displays some similarity to the cluster involved in the production of the protease inhibitor aeruginoside in Planktothrix agardhii Cya 126 61. The genomic data therefore clearly indicate that strain Mic-PCC7806 might be capable of producing a variant of aeruginosin (Figure 5).

The two remaining PKS I gene clusters do not show significant similarity to any known cyanobacterial biosynthetic gene clusters, and may be involved in the production of hitherto unknown compounds (Figure 5 and Table 6). The first gene cluster encodes an iterative PKS I that is similar in both architecture and sequence to the PksE of various actinobacteria, and is accompanied by several tailoring enzymes including three halogenases. The actinobacterial enzyme is involved in the biosynthesis of enedyine type antitumor antibiotics 62. The second PKS gene cluster encodes a modular PKS I complex accompanied by several putative tailoring enzymes, and a PKS III type enzyme that is capable of synthesizing compounds of the chalcone/stilbene family. These biosynthetic enzymes are widespread in plants but have only recently been discovered in bacteria 63. A comparison of the biosynthetic potential of Mic-PCC7806 and Mic-NIES843 reveals that three of the large NRPS/PKS complexes, namely those dedicated to microcystin, cyanopeptolin and aeruginosin production, are encoded on both genomes, whereas some other gene clusters are not shared by both genomes. The biosynthetic versatility of members of the genus Microcystis may thus be larger than expected, since the two strains selected for genome sequencing have similar chemotypes. Beside the NRPS and PKS encoding genes, the genome of Mic-PCC7806 contains a gene cluster similar to the patellamide genes that were recently detected in symbiotic cyanobacterial strains of ascidians 64. Patellamides are a family of cyclic peptides generated from a ribosomally-synthesized precursor. Mic-PCC7806 is the first freshwater cyanobacterium showing the capability to produce patellamide-like peptides. A peptide with striking similarity to the patellamides, microcyclamide, has been reported in M. aeruginosa strain NIES-298 65. Chemical analyses have revealed that the gene cluster discovered in Mic-PCC7806 is indeed dedicated to the production of a microcyclamide-type compound 66. The genome of Mic-PCC7806 could attract further attention, as it also contains gene clusters comprising unique features that have yet to be characterized and which may well produce so-far unidentified natural substances.

Transporter genes are commonly found in the immediate vicinity of the secondary metabolite biosynthetic genes. These secondary metabolites may therefore at least partly function at the surface of Microcystis cells, in the colony-surrounding sheath or in their planktonic environment. Gene clusters involved in the synthesis of secondary metabolites are frequently associated with genes that confer resistance to these metabolites, which would otherwise be toxic to the cells producing them. In Mic-PCC7806, only the transport system associated with the uncharacterized PKS I/PKS III hybrid compound (Figure 5) shows any similarity to typical efflux transporters that potentially confer self-resistance. The compound produced could therefore have an allelopathic or antibacterial role in the environment 67.

Ama-MBIC11017: Acaryochloris marina MBIC11017 (embl: CP000828)

Ana-PCC7120: Anabaena/Nostoc sp. PCC 7120 (embl: BA000019)

Ava-ATCC29413: Anabaena variabilis ATCC 29413 (embl: CP000117)

Cbi-PCC7001: Cyanobium sp. PCC 7001 (gb: 1106012173546)

Cth-ATCC51142: Cyanothece sp. ATCC 51142 (embl: CP000806)

Cth-CCY0110: Cyanothece sp. CCY0110 (gb: 1101676644636–1101676644658)

Cwa-WH8501: Crocosphaera watsonii WH8501 (embl: AADV02000100)

Syn-JA33Ab: Cyanobacteria Yellowstone JA-3-3Ab (embl: CP000239)

Syn-JA23B'a: Cyanobacteria Yellowstone JA-2-3B'a (embl: CP000240)

Gvi-PCC7421: Gloeobacter violaceus PCC 7421 (embl: BA000045)

Lae-PCC8106: Lyngbya aestuari PCC 8106 (gb: 1099428180563–1099428180584)

Mch-PCC7420: Microcoleus chthonoplastes PCC 7420 (gb:1103659003780–1103659003836)

Mic-NIES843: Microcystis aeruginosa NIES-843 (embl: AP009552)

Mic-PCC7806: Microcystis aeruginosa PCC 7806 (embl: AM778843–AM778958)

Nsp-CCY9414: Nodularia spumigena CCY9414 (gb:1099428179735–1099428179797)

Npu-PCC73102: Nostoc punctiforme PCC 73102 (kindly provided by J. C. Meeks) 77

Pro-SS120: Prochlorococcus marinus SS120 (embl: AE017126)

Pro-AS9601: Prochlorococcus marinus AS9601 (embl: CP000551)

Pro-MED4: Prochlorococcus marinus MED4 (embl: BX548174)

Pro-MIT9211: Prochlorococcus marinus MIT9211 (embl: AALP01000001)

Pro-MIT9215: Prochlorococcus marinus MIT9215 (embl: CP000825)

Pro-MIT9301: Prochlorococcus marinus MIT9301 (embl: CP000576)

Pro-MIT9303: Prochlorococcus marinus MIT9303 (embl: CP000554)

Pro-MIT9312: Prochlorococcus marinus MIT9312 (embl: CP000111)

Pro-MIT9313: Prochlorococcus marinus MIT9313 (embl: BX572095)

Pro-MIT9515: Prochlorococcus marinus MIT9515 (embl: CP000552)

Pro-NATL1A: Prochlorococcus marinus NATL1A (embl: CP000553)

Pro-NATL2A: Prochlorococcus marinus NATL2A (embl: CP000095)

Syn-BL107: Synechococcus sp. BL107 (gb: 1099739244347)

Syn-CC9311: Synechococcus sp. CC9311 (embl: CP000435)

Syn-CC9605: Synechococcus sp. CC9605 (embl: CP000110)

Syn-CC9902: Synechococcus sp. CC9902 (embl: CP000097)

Syn-PCC6301: Synechococcus elongatus PCC 6301 (embl: AP008231)

Syn-PCC7002: Synechococcus sp. PCC 7002 (embl: CP000951)

Syn-PCC7335: Synechococcus sp. PCC 7335 (gb: 1103496006889–1103496006899)

Syn-PCC7942: Synechococcus elongatus PCC 7942 (embl: CP000100)

Syn-RCC307: Synechococcus sp. RCC307 (embl: CT978603)

Syn-RS9916: Synechococcus sp. RS9916 (gb: 1100013018508)

Syn-RS9917: Synechococcus sp. RS9917 (gb: 1099465004208)

Syn-WH5701: Synechococcus sp. WH5701 (gb: 1099465003749–1099465003864)

Syn-WH7803: Synechococcus sp. WH7803 (embl: CT971583)

Syn-WH7805: Synechococcus sp. WH7805 (gb: 1099646010155–1099646010157)

Syn-WH8102: Synechococcus sp. WH8102 (gb: BX548020)

Syn-PCC6803: Synechocystis sp. PCC 6803 (embl: BA000022)

Tel-BP1: Thermosynechococcus elongatus BP-1 (embl: BA000039)

Ter-IMS101: Trichodesmium erythreum IMS101 (embl: CP000393)

Mic-PCC7806/Cwa-WH8501

Mic-PCC7806/Syn-PCC6803

Cwa-WH8501/Syn-PCC6803

Lae-PCC8106/Ter-IMS101

Npu-PCC73102/Ava-ATCC29413

Npu-PCC73102/Ana-PCC7120

Npu-PCC73102/Nsp-CCY9414

Nsp-CCY9414/Ana-PCC7120

Ava-ATCC29413/Nsp-CCY9414

Ana-PCC7120/Ava-ATCC29413

Shigella dysenteriae, serovar 1, strain Sd97/Sd197 (CP000034_GR)

Acidovorax avenae subsp. citrulli AAC00-1 (NC_008752)

Agrobacterium tumefaciens str. C58 (NC_003062)

Bacillus subtilis subsp. subtilis str. 168 (NC_000964)

Bordetella parapertussis 12822 (NC_002928)

Escherichia coli APEC O1 (NC_008563)

Enterobacter sp. 638 (NC_009436)

Janthinobacterium sp. Marseille (NC_009659)

Klebsiella pneumoniae subsp. pneumoniae MGH 78578 (CP000647)

Listeria monocytogenes EGD-e (NC_003210)

Methylococcus capsulatus str. Bath (NC_002977)

Ochrobactrum anthropi ATCC 49188 chromosome 1 (NC_009667)

Polaromonas naphthalenivorans CJ2 (NC_008781)

Pseudomonas aeruginosa PA7 (NC_009656)

Pseudomonas fluorescens PfO-1 (NC_007492)

Rhizobium etli CFN 42 (NC_007761)

Rhizobium leguminosarum bv. viciae 3841 (NC_008380)

Rhodobacter sphaeroides ATCC 17025 (NC_009428)

Rhodoferax ferrireducens T118 (NC_007908)

Shewanella loihica PV-4 (NC_009092)

Shewanella oneidensis MR-1 (NC_004347)

Shewanella sp. W3-18-1 (NC_008750)

Shigella boydii Sb227 (NC_007613)

Silicibacter sp. TM1040 (NC_008044)

Yersinia enterocolitica subsp. enterocolitica 8081 (NC_008800)

Yersinia pestis CO92 (NC_003143)

Photorhabdus luminescens subsp. laumondii TTO1 (NC_005126)