In colonizing most waters (fresh, brackish and marine) and soils, where they face various challenges (Cassier-Chauvat and Chauvat, 2015), cyanobacteria have developed as widely diverse organisms (Narainsamy et al., 2013). Their genomes differ in size (from 1.44 to 12.07 Mb), ploidy (from two to more than 20 chromosome copies per cell) or GC content (30–60%), probably as a result from gains and losses of genes transferred by plasmids, insertion sequences (Alam et al., 1991; Cassier-Chauvat et al., 1997) and/or cyanophages (Hess, 2011; Shih et al., 2013). Most cyanobacteria possess a single circular chromosome, ranging from 1.44 Mb in size (the marine symbiotic strain UCYN-A) to 12.07 Mb (Scytonema hofmanni PCC7110) (Dagan et al., 2013). The well-studied strain Synechocystis PCC6803 has a 3.57 Mb chromosome, with a 48% GC content (http://genome.microbedb.jp/cyanobase/) and a copy number of 10–50 (Labarre et al., 1989; Griese et al., 2011). For the other models the values are 2.69 Mb, 55% and 2–5 for Synechococcus PCC7942 (Mann and Carr, 1974; Griese et al., 2011; Watanabe et al., 2015); and 3.00 Mb, 50%, and likely 2–5 for Synechococcus PCC7002 (Griese et al., 2011; Watanabe et al., 2015). Synechocystis PCC6803 also has seven plasmids, ranging from 2.3 Kb (Chauvat et al., 1986) to 119vKb (http://genome.microbedb.jp/cyanobase/); Synechococcus PCC7942 has one plasmid (46 Kb); and Synechococcus PCC7002 has seven plasmids (4.8–186 Kb). Interestingly, Cyanothece ATCC51142 possesses two chromosomes (one circular, 4.39 Mb; and one linear, 0.4 Mb) and four plasmids (10–39 Kb), whereas the marine strains Prochlorococcus and Synechococcus have a small chromosome (1.6–2.7 Mb), and no plasmids (Scanlan et al., 2009).

As a consequence of their genomic diversity, cyanobacteria produce a wealth of metabolites (Dittmann et al., 2015; Kleigrewe et al., 2016), display different cell morphologies (Cassier-Chauvat and Chauvat, 2014) and can differentiate cells, akinetes and/or heterocysts, respectively dedicated to cell survival in adverse conditions (Chauvat et al., 1982) or the fixation of atmospheric nitrogen (Flores and Herrero, 2010).

Because of their photoautotrophic lifestyle, cyanobacteria are strongly challenged by solar UV rays and reactive oxygen species generated by photosynthesis (Cassier-Chauvat and Chauvat, 2015). Consequently, Synechocystis PCC6803 and Synechococcus PCC7942 are found to be more resistant to UV than the (non-photosynthetic) bacterium E. coli where DNA repair is best known (Baharoglu and Mazel, 2014). Synechocystis PCC6803 is also more resistant to gamma rays than Synechococcus PCC7942 and E. coli in that order (the doses yielding 10% survival are 660, 230, and 130 Gy, respectively (Domain et al., 2004). Other cyanobacteria are even more radioresistant, almost as the champion bacterium Deinococcus radiodurans [100% survival at 5kGy (Moseley and Mattingly, 1971; Ito et al., 1983)]. These radiation-resistant cyanobacteria are Chroococcidiopsis [10% survival to 4–5 kGy of gamma rays (Billi et al., 2000)], three Anabaena strains [they can grow at 5 kGy (Singh et al., 2010)] and Arthrospira PCC8005 [it grows at 800 Gy (Badri et al., 2015)]. Thus, cyanobacteria might be used in the future for leaching (and/or sequestration) of radionuclides (Acharya and Apte, 2013).

The naturally transformable cyanobacteria Synechococcus PCC7942, Synechococcus PCC7002, and Synechocystis PCC6803 can take up extracellular DNA and to recombine it into their own genome (Orkwiszewski and Kaney, 1974; Stevens and Porter, 1980; Grigorieva and Shestakov, 1982). This capability served to create a wealth of insertions, deletions or replacement mutations (Lai and Lan, 2015; Savakis and Hellingwerf, 2015; Zhou et al., 2016).

Natural transformation is best studied in Bacillus subtilis and Helicobacter pylori (Dorer et al., 2011). DNA transported into the cytosol by the Com proteins (com for competence) is integrated into the recipient genome by the RecA, RecG, and RuvABC recombination proteins.

The com genes (Table 1) are widely distributed in cyanobacteria (Supplemental Table 1). Synechocystis PCC6803, Synechococcus PCC7942, and Synechococcus PCC7002 harbor the comAEF genes (Supplemental Table 1). The Synechocystis PCC6803 genes comA and comF truly operate in transformation (Yoshihara et al., 2001), and comF is also involved in phototactic motility (Nakasugi et al., 2006). The role of comE could not be verified because the comE-depleted mutant dies rapidly (Yoshihara et al., 2001). By contrast, the Prochlorococcus cyanobacteria endowed with small genomes have no comAEF genes, excepted P. marinus MIT9303, and P. marinus MIT9313 that possess comA, come, and ComF (Supplemental Table 1). These strains also have the recA, recG, and ruvABC genes (Supplemental Table 1). We have verified in Synechocystis PCC6803 that ruvB operates in genetic transformation (Domain et al., 2004). These finding suggest that P. marinus MIT9313 may be transformable in appropriate conditions.

Recently, the CRISPR/Cas9 genome editing system, which enhances the recombination efficiency and accelerates the process for chromosome segregation, was used for efficient genome editing in cyanobacteria (Li et al., 2016; Wendt et al., 2016).

Microbial organisms can genetically adapt themselves to their “laboratory” environment. This phenomenon explains the phenotypic differences observed between various sub-strains of the same organism cultivated in diverse laboratories. Hence, the four laboratory sub-strains of Synechocystis PCC6803 with different cell motility and/or ability to feed from glucose, harbor mutations, insertion or deletion, as compared to each others (Okamoto et al., 1999; Kanesaki et al., 2012; Trautmann et al., 2012).

Genetic instability can also be observed in strains genetically engineered for the synthesis of chemicals, where it can decrease the amplitude and/or durability of production. Genetic instability correlates with the toxicity of the products, and homologous recombination between repeated DNA motifs (Gellert and Nash, 1987; Holder et al., 2015), which are frequent in cyanobacteria (Elhai, 2015).

In the 61 articles reporting the genetic engineering of a model cyanobacterium for the synthesis of a biotechnological product, the level of production were analyzed only during short periods of times (usually not more than 30 days after the generation of the producer strains; Lai and Lan, 2015). Consequently, we know very little regarding genome (in)stability in engineered cyanobacteria growing under laboratory conditions. This genome (in)stability is an important issue in large industrial cultures that require many cell divisions of the engineered cyanobacteria. The longer the cultivation, the higher the probability of selecting spontaneous mutations decreasing the synthesis of the product to increase cell fitness.

A few studies reported the genetic instability of engineered cyanobacteria. We observed this phenomenon while attempting to use Synechocystis PCC6803 for the production of a uniformly ¹⁴C-labeled mouse urokinase (a serine protease). The urokinase producing plasmid, which replicated stably in the recA⁻ mutant of E. coli, invariably lost part of the urokinase gene upon propagation in Synechocystis PCC6803 (Chauvat et al., 1988). Another Synechocystis PCC6803 strain harboring Pseudomonas aeruginosa genes cloned its chromosome (at the slr0168 neutral docking site) for lactic acid production, happened to rescue its growth by introducing a duplication (~160 bp) that generated premature stop codons into the Pseudomonas (NADPH/NADH) transhydrogenase gene (Angermayr et al., 2012).

Similarly, the Synechococcus PCC7942 strain harboring the Pseudomonas syringae gene (efe) encoding the ethylene-forming enzyme (Fukuda et al., 1992; Sakai et al., 1997), managed to introduce short nucleotide insertions in efe to stop ethylene production and recover a healthy growth (Takahama et al., 2003). Another recombinant Synechococcus PCC7942 strain could introduce a missense mutation in the E. coli atoD gene (acetoacetyl-CoA transferase) to decrease isopropanol production (Kusakabe et al., 2013).

In Synechococcus PCC7002, a recombinant strain managed to loose mannitol synthesis and recover healthy growth, in introducing a single-base deletion generating a stop codon in its E. coli mannitol-1-phosphate dehydrogenase mtlD gene (Jacobsen and Frigaard, 2014).

The Synechocystis PCC6803 and Synechococcus PCC7002 recombinant strains producing the Zymomonas mobilis pyruvate decarboxylase enzyme (PDC) for ethanol production, could introduce mutations, insertions, deletions or mobile genetic elements (insertion sequences) into the pdc gene to stop ethanol production (Schulze et al., 2015).

Insertion sequences (ISs) are approximately 1 kbp long DNA segments found in the genome of most living organisms, where they can interrupt genes (Bennett, 2004). Generally, an IS comprises an inverted repeat DNA sequence flanking one or two genes encoding the mobilization protein (transposase), which drives the excision and reinsertion of IS in genomes.

Many cyanobacterial chromosomes and/or plasmids harbor a few or numerous copies of ISs, as the widely distributed IS families IS4, IS5, IS630 and IS200-605, which are regarded as ancestral (Lin et al., 2011). Though several P. marinus strains harboring a small genome have no IS, the frequencies of IS do not systematically increase with the genome size. Indeed, IS represent 10% of the 5.8 Mb genome of Microcystis aeruginosa NIES843, 1.5% of the 3.95 Mb genome of Synechocystis PCC6803, and 1% of the 7.2 Mb genome of Nostoc (Anabaena) PCC7120 (Lin et al., 2011). Consistent with the findings that transposase genes can be induced by stresses (Hernández-Prieto et al., 2016), several studies employing a positive selection procedure showed that ISs can be truly mobile in cyanobacteria. First, a recombinant Nostoc (Anabaena) PCC7120 strain harboring a plasmid encoding the B. subtilis SacB enzyme (levan sucrase), which kills cells incubated in the presence of sucrose, generated sucrose resistant mutants resulting from the disruption of the sacB gene by a mobile IS895 element (Alam et al., 1991).

Similarly, an IS5 element of Synechocystis PCC6803 was shown to be mobile in rescuing the growth of a conditionally lethal mutant by disrupting the repressor gene that normally blocks the transcription of an essential ferredoxin-encoding gene (Cassier-Chauvat et al., 1997; Poncelet et al., 1998). Other recently transposed IS4 elements were identified through Southern blotting and DNA sequencing analysis of three Synechocystis PCC6803 sub-strains (Okamoto et al., 1999).

In addition, the presence of multiple copies of an IS in a genome can promote homologous recombination, leading to genome rearrangements (inversions or deletions; Gellert and Nash, 1987) that can modify cell fitness. Moreover, ISs can be transferred between genomes by horizontal gene transfer mechanisms. Thus, ISs are an important force in genome evolution (Bennett, 2004).

So far very few studies attempted to decrease or eliminate the negative influence of IS on biotechnological production. In Corynebacterium glutamicum, the deletion of two major IS elements generated a cell chassis with an increased ability to stably produce recombinant proteins (Choi et al., 2015). A similar strategy could be tested in the genetically manipulable cyanobacteria Synechococcus PCC7942 and Synechococcus PCC7002 because they possess only one and ten transposase genes, respectively (http://genome.microbedb.jp/cyanobase/). In contrast, an IS-deletion strategy is not an appealing for Synechocystis PCC6803 that possesses 128 transposase genes.

In E. coli, the stable propagation of recombinant DNA (usually cloned in plasmids) is achieved in strains where recA, the key DNA-recombination gene (Baharoglu and Mazel, 2014), has been inactivated to prevent unexpected DNA rearrangements. All cyanobacteria possess a recA gene (Acaryochoris marima MBIC11017 has 7 recA genes, Supplemental Table 1). The recA gene appeared to be indispensable to cell life in Synechococcus PCC7002 (Murphy et al., 1990), whereas it could be deleted from all chromosome copies in Synechocystis PCC6803 (Minda et al., 2005). The Synechocystis PCC6803 recA null mutant is bound to be of limited biotechnological interest because it is not only sensitive to UV-C, but also to standard fluence of white light required for cell growth. Furthermore, in being defective in DNA recombination a recA⁻ mutant is not appropriate for genetic manipulation of the cyanobacterial chromosome (cloning of heterologous genes encoding the synthesis of biotechnological products and/or deletion of endogenous genes limiting the intended production).

An interesting way to limit genetic instability of engineered bacteria is to clone the product-synthesizing genes under the control of regulatable expression signals to afford a user-controlled synthesis of the potentially harmful product. Using such regulatory signals, one can grow the engineered strain up to a large biomass, before triggering the synthesis of the intended product, which, otherwise, could have impaired the fitness and/or the genetic stability of the producer.

In cyanobacteria gene expression can be regulated by (i) light (psbA2 promoter), (ii) the IPTG metabolite (lac promoter/repressor system), (iii) metals [cyanobacterial promoters coaT, ziaA, etc (Berla et al., 2013; Zhou et al., 2016)], or (iv) the growth temperature [lambda phage pR promoter controlled by the cI857 temperature-sensitive repressor (Ferino and Chauvat, 1989; Mermet-Bouvier and Chauvat, 1994)]. As put forward by other workers (Berla et al., 2013) an ideal system should combine the following properties.

In our laboratory, we often used the temperature-controlled system that appeared to combine most of these advantageous properties (Dutheil et al., 2012; Marteyn et al., 2013; Ortega-Ramos et al., 2014) and references therein. This system tightly controls gene expression proportionally to growth temperatures i.e., absence of expression at temperature ≤30°C (the standard growth temperature of our favorite cyanobacterium Synechocystis PCC6803); intermediary expression at intermediate temperature 34–37°C; and strong expression at 39°C (where Synechocystis PCC6803 keep growing well). For instance, when this system was used to control the production of the heterologous enzymes chloramphenicol-acetyl-transferase and beta-galactosidase, which possess an easily quantified activity, the values were respectively ≤3 units (30°C); 700–1000 units (34–37°C) and 2000–4000 units (39°C) (Ferino and Chauvat, 1989; Mermet-Bouvier and Chauvat, 1994). Hence this system can be also used for basic research that requires the construction of conditionally-lethal mutants (Poncelet et al., 1998; Sakr et al., 2013).

From bacteria to higher eukaryotes, cells are continuously exposed to DNA damages generated by their own metabolism (Imlay, 2013) and/or exogenous sources (radiations, chemicals, etc). DNA lesions are repaired by conserved pathways that have been extensively studied in E. coli (Baharoglu and Mazel, 2014). The simplest system, the direct damage reversal pathway, removes only the base-modifying agent in one single step (Resende et al., 2011) catalyzed by the AlkB demethylase, the Ogt alkyltranferase, and the Phr (photorepairs of pyrimidine) photolyase.

Using a comparative genomic approach, we found that the 76 cyanobacterial genome sequences in the MBGD data base (http://mbgd.genome.ad.jp/) possess many genes orthologous to E. coli DNA recombination and repair genes. The phr, alkB and ogt orthologs (Table 1) are distributed unevenly in cyanobacteria (Supplemental Table 1). The phr gene is present in almost all cyanobacteria including some, but not all, P. marinus strains endowed with a small genome (1.6–2.7 Mb). In agreement with the light fluence they receive in their oceanic biotopes (Biller et al., 2015), the high-light-adapted strains P. marinus MIT9515 and P. marinus MED4 possess phr, whereas the low-light-adapted strains P. marinus MIT9303 and P. marinus MIT9313 lack phr (Supplemental Table 1), and are light sensitive (Biller et al., 2015). The alkB and ogt genes are less frequent than phr. All three genes alkB, ogt, and phr are simultaneously present in several (twelve) studied cyanobacteria, such as Nostoc (Anabaena) PCC7120 (filamentous), and Cyanothece PCC7425 (unicellular) where ogt is duplicated. The other (evolutionary distant) unicellular models Synechocystis PCC6803, Synechococcus PCC7942, and Synechococcus PCC7002 possess phr (Supplemental Table 1). Synechocystis PCC6803 has alkB but not ogt, Synechococcus PCC7942 has ogt (duplicated) but not alkB, and Synechococcus PCC7002 has neither alkB nor ogt. Interestingly, the symbiotic (marine) cyanobacterium UCYN-A has no phr, alkB, and ogt, in agreement with the fact that it possesses the smallest genome (1.44 Mb). The other symbiotic strain Acaryochloris marina MBIC11017 endowed with a larger genome (8.36 Mb) has two alkB, three ogt (including one on a plasmid) but no phr (Supplemental Table 1).

This pathway removes distortions of the double helix of DNA (pyrimidine dimers or DNA intra-strand cross-links), by excising a small group of bases (Baharoglu and Mazel, 2014). In E. coli the two-proteins complex UvrAB recognizes the DNA lesion; UvrC generates a double incision on both sides of the lesion and the UvrD helicase removes the single-strand DNA carrying the lesion. The missing DNA is re-synthesized by the DNA polymerase I (Pol I), and subsequently sealed by a ligase.

All tested cyanobacterial genomes possess the uvrABCD single-copy genes (Supplemental Table 1), where uvrA and uvrB are not organized in operon (Supplemental Figure 1), unlike what occurs in E. coli. In some cyanobacterial genomes uvrA, uvrB, uvrC, and/or uvrD are clustered with another DNA repair gene, such as phr or recN (gene clusters a and c in Supplemental Table 1 and Supplemental Figure 1). In the radiation-resistant cyanobacterium Arthrospira PCC8005, uvrBCD were found to be upregulated by gamma rays (no information is provided for uvrA) (Badri et al., 2015).

This pathway corrects the mispaired DNA bases generated by replication errors (Putnam, 2016). In E. coli, MutS recognizes mispaired DNA bases and coordinates with MutH and MutL (nucleases), MutM, MutT and MutY (DNA glycosylases) and UvrD (helicase) to direct excision of the newly synthesized DNA strand (not yet methylated at GATC sites by the Dam methylase) up to the mismatch. The resulting gap is filled up by a DNA polymerase (likely PolIII) and a ligase (Putnam, 2016).

All tested cyanobacteria have mutM (Supplemental Table 1), which was shown in Synechococcus PCC7942 to operate in resistance to high light (Mühlenhoff, 2000). All cyanobacteria possess mutS, which occurs in two copies, excepted in Crinalium epipsammum PCC 9333 (Supplemental Table 1). By contrast, mutH is absent in all cyanobacteria. The genetic diversity of cyanobacteria is well illustrated with the presence/absence of mutL, mutt, and mutY (Supplemental Table 1), which lies in front of recR in a few cyanobacterial genomes (Table 1 and Supplemental Figure 1). Several P. marinus strains lack mutL, mutt, and mutY (Supplemental Table 1). In Arthrospira PCC8005 (radiation-resistant) mutST were upregulated by gamma rays (Badri et al., 2015).

The model strains Synechocystis PCC6803, Synechococcus PCC7942, Synechococcus PCC7002 and Nostoc (Anabaena) PCC7120 possess mutL, mutM, mutS (duplicated), mutT (excepted Synechococcus PCC7002), mutY (excepted Synechococcus PCC6803 and Nostoc PCC7120) (Supplemental Table 1). Thus, Synechococcus PCC7942 is best suited to study all these genes through deletion/over-expression in the otherwise same genetic context.

This pathway repairs double-stranded breaks and cross-links. In E. coli, single-strand DNA nicks are enlarged by the RecQ helicase and RecJ exonuclease, into gaps that are recognized by the proteins RecFOR. The double-strand DNA breaks (DSB) are recognized by the RecBCD proteins that form an exonuclease/helicase complex. Subsequently, the RecFOR/RecBCD complexes (and RecN) load RecA to initiate homologous recombination and DNA repair. RecA mediates synapsis, forming a Holliday junction. Replication fills gaps. RecG, Ssb (single-stranded DNA binding protein) and RuvAB mediate branch migration (stimulated by RadA), and RuvC resolves the junctions (Baharoglu and Mazel, 2014).

DNA recombination also involves the XerC-XerD complex. It converts dimers of the chromosome into monomers to permit their segregation during cell division, and it contributes to the segregational stability of plasmids (Resende et al., 2011; Buljubašic et al., 2013).

In many bacteria, such as H. pylori and B. subtilis the AddA and AddB proteins replace RecB and RecC, respectively (Dorer et al., 2011; Wigley, 2013).

All cyanobacteria contain recA, which occurs as seven copies in the large genome (8.36 Mb) of A. marina MBIC11017. Four of these recA genes, possibly originating from gene duplication (Swingley et al., 2008), are located on four separate plasmids, while the other recA belong to the chromosome (Supplemental Table 1).

Like recA, radA and recG are present in all cyanobacteria, and radA is duplicated in Cyanothece PCC7425, M. aeruginosa NIES-843 and UCYNA (Supplemental Table 1). It is the only duplicated gene in the very small UCYNA genome (1.44 Mb).

Many cyanobacteria have two copies of recJ and recQ genes. They are noted as recJ_ec or recJ_cy, or recQ_ec or recQ_cy (ec for E. coli, cy for cyanobacteria), according to their high (recJ_ec, recQ_ec) or low (recJ_cy, recQ_cy) sequence similarity with their E. coli counterparts (Table 1 and Supplemental Table 1). This is true for Synechococcus PCC7002 and Nostoc PCC7120, where these duplicated genes can be studied and compared through deletion/over-expression. In Arthrospira PCC8005 (radiation-resistant), recGJQ were found to be upregulated by gamma rays (Badri et al., 2015). In contrast a few cyanobacteria has neither recJ nor recQ, as P. marinus MIT9515 (Supplemental Table 1). Also interestingly, the low-light-adapted P. marinus MIT9313 and P. marinus MIT9303 possess the recQ genes (and ogt and the competence genes comE and comFC), which are not present in other Prochlorococcus (Supplemental Table 1). In addition, both P. marinus MIT9313 and P. marinus MIT9303 lack the phr gene, which occurs in other Prochlorococcus (Supplemental Table 1), in agreement with their light-sensitivity (Biller et al., 2015). Collectively, these findings support the proposal that P. marinus MIT9303 and P. marinus MIT9313 belong to the same clade, which diverged early from the other Prochloroccus clades (Sun and Blanchard, 2014; Biller et al., 2015).

Almost all cyanobacteria have the single-copy genes recF, recO and recR, excepted Cyanobacterium aponinum PCC10605, C. epipsammum PCC9333 and Cylindrospermum stagnale PCC7417 which lack recR (Supplemental Table 1)

The recBCD genes are less conserved in cyanobacteria. For instance, the strain UCYN-A that possesses recFOR has no recBCD genes (Supplemental Table 1). Most P. marinus strains and several marine Synechococcus strains possess recBCD. Most of these strains possess two recB copies, noted recB_ec (good similarity with E. coli recB) or recB_cy (cy for cyanobacteria, low similarity with E. coli recB). In these strains, recB_ec belongs to the same genomic region than recC and recD (cluster f in Supplemental Table 1 and Supplemental Figure 1). In a few other cyanobacteria recD is duplicated (Microcoleus PCC7113) or triplicated (A. marina MBIC11017 and N. punctiforme PCC73102), irrespectively of the presence /absence or recB_ec and recB_cy (Supplemental Table 1). The well-studied model cyanobacteria lack recB, recC, or recD. Both Synechocystis PCC6803 and Nostoc (Anabaena) PCC7120 lack recB_ec and recC, while both Synechococcus strains PCC7942 and Synechococcus PCC7002 lack recCD.

The recN gene is present in all cyanobacteria to the noticeable exception of Chamaesiphon minutus PCC6605. Interestingly the RecN protein was absent in mature heterocysts of Anabaena PCC7120, the differentiated nitrogen-fixing cells that have lost the ability to divide (Hu et al., 2015).

In some cyanobacteria a few rec genes are clustered together (recBCD see cluster f in Supplemental Table 1 and Supplemental Figure 1), or with other DNA repair genes, including uvrA (cluster a) or mutY (cluster n; Supplemental Table 1 and Supplemental Figure 1).

All cyanobacteria have a ssb gene, which is repeated in a few strains. For instance, ssb is duplicated in Nostoc (Anabaena) PCC7120 and A. marina MBIC11017), while it is triplicated in Chroococcidiopsis thermalis PCC7203 and quadruplicated in Cyanothece PCC7822 (Supplemental Table 1). In these cyanobacteria (excepted Nostoc (Anabaena) PCC7120) one ssb copy is propagated on a plasmid. One of the two Nostoc PCC7120 ssb genes, (alr0088, but not alr7579) was shown to be involved in the tolerance to UV and mitomycin C which causes formation of DNA adducts (Kirti et al., 2013).

The ruvABC genes are present in all cyanobacteria, to the noticeable exception of G. kilaueensis JS1 which lacks ruvC (Supplemental Table 1). The ruvA and ruvB genes are not adjacent unlike their operonic E. coli counterparts. Furthermore, ruvA is duplicated in Trichodesmium erythraeum ISM101, while ruvC is quadruplicated in Cyanothece ATCC51142 and quadruplicated in Cyanothece PCC7822 (Supplemental Table 1). In Synechocystis PCC6803 ruvB was shown to be dispensable to cell growth in standard laboratory conditions, and to operate in the resistance to UV and H₂O₂ (Domain et al., 2004).

Unlike recAN and ruvABC, xerC is a rare gene in cyanobacteria (Supplemental Table 1). It occurs in a single copy in a few strains, as UCYN-A, Synechococcus PCC7942, Synechococcus PCC7002 and Synechocystis PCC6803, or in several copies in Cyanothece PCC7424, Cyanothece PCC7822 (two copies), Nostoc (Anabaena) PCC7120 (three copies), A. marina MBIC11017 (eight copies).

In bacteria, homologous recombination preferentially initiates at highly repeated, oligomeric DNA sequences designated as Chi (crossover hotspot instigator) sites. In E. coli, the Chi site used by RecBCD is 8 bases (GCTGGTGG), whereas in B. subtilis Chi used by AddAB is just 5 bases (AGCGG) (Wigley, 2013). Similarly, the GCGATCGC sequence is overrepresented in many cyanobacteria where one or more methylases recognize some portion of the sequence (Elhai, 2015). In Synechocystis PCC6803 the repeated sequence HIP1 (Highly Iterated Palindrome) is associated to a CGATCG-specific methylase (M.Ssp6803I) that is required for rapid growth (Elhai, 2015).

The above-mentioned repair systems usually remove the initial DNA lesions and restore the genetic material back to its original state. When facing many DNA injuries cells start synthesizing several proteins (endonucleases, polymerases and ligases) to accelerate DNA repair, even though there may be some incorporated errors. In this case, the replicative DNA polymerase PolIII, which cannot replicate damaged DNA, is replaced by other polymerases PolIV (encoded by dinB) and PolV (encoded by umuCD), which replicate damaged DNA in a mutagenic manner (Baharoglu and Mazel, 2014).

The umuCD genes (Table 1) are unevenly distributed in cyanobacteria (Supplemental Table 1). Many strains have no umuCD, like UCYN-A (small genome) and Synechococcus PCC7002. Others possess umuCD, such as Nostoc (Anabaena) PCC7120, Synechococcus PCC7942, Synechocystis PCC6803, and the Prochlorococcus strains. A few strains harbor a duplication of umuC (Synechococcus PCC6312) and/or umuD (Cyanobium gracile PCC6307 and Synechococcus PCC6312). A. marina MBIC11017 possesses three umuC and four umuD (Swingley et al., 2008). In some cyanobacteria umuDC are clustered together, nearby ruvA (cluster z in Supplemental Table 1 and Supplemental Figure 1).

The gene dinB (Table 1) is present in a very few cyanobacteria, such as A. marina MBIC11017, Anabaena PCC7120 and G. kilaueensis JS1 (Supplemental Table 1).

In many bacteria, the so-called “SOS” regulatory system is the main transcriptional circuit that detects DNA damages and regulates the repair systems according to cells needs (Baharoglu and Mazel, 2014). The SOS response is activated when RecA binds single-stranded DNA and generates a nucleofilament triggering the auto-proteolysis of the LexA regulator. In E. coli, LexA normally represses about 40 SOS genes (recABCD, ruvABC, etc.) by binding to its cognate LexA-box sequence on their promoters (5′-taCTGTatatatatACAGta-3′; the upper cases indicate the conserved nucleotides), thereby precluding their transcription (Baharoglu and Mazel, 2014). One of the SOS-controlled gene codes for the key SulA protein that delays cell division until DNA damages are repaired.

The lexA gene (Table 1) is unevenly distributed in cyanobacteria. It is absent in both Arthrospira PCC8005 (Badri et al., 2015) and NIES39, and in several strains of the genus Gloeobacter, Oscillatoria and Synechococcus (including Synechococcus PCC7942, Supplemental Table 1), similarly to what found in other bacteria as H. pylori (Dorer et al., 2011) and Streptococcus pneumoniae (Baharoglu and Mazel, 2014). By contrast, lexA is present in the other tested cyanobacteria (it is duplicated in Cyanothece ATCC51142). The marine cyanobacteria of the genus Prochlorococcus and Synechococcus share a very similar lexA (clade C), while other strains possess a slightly different lexA (clade B), such as A. marina MBIC11017, and both Nostoc PCC7120 and Synechocystis PCC6803 (Li et al., 2010). Interestingly, the Synechocystis PCC6803 lexA gene appeared to regulate carbon assimilation (Domain et al., 2004) and cell motility (Kizawa et al., 2016), but not DNA recombination and repair (Domain et al., 2004). Furthermore, the Nostoc PCC7120 LexA protein has a RecA-independent autoproteolytic cleavage (Kumar et al., 2015).

The sulA homolog is present in almost all cyanobacteria, to the noticeable exception of Gloeobacter violaceus PCC7421, G. kilaueensis JS1, Anabaena sp. 90 and UCYN-A (Supplemental Table 1). In Synechocystis PCC6803, sulA appeared to be indispensable to cell life and division (Raynaud et al., 2004).

Many genes are present in the 76 studied cyanobacteria (mutM, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD; Table 1 and Supplemental Table 1), including the marine strain UCYN-A that possesses the smallest genome (1.44 Mb), and numerous marine strains Prochlorococcus and Synechococcus also endowed with a small genome (1.65–Mb). Similarly, mutS, recN, and ruvC are present in almost all cyanobacteria (Supplemental Table 1), namely Thermosynechococcus NK55a (absence of mutS1), Cyanothece PCC51142 (absence of recN) and (G. kilaueensis JS1 absence of ruvC). Consequently, we propose that the genes mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD encode the core DNA repair system of cyanobacteria.

A few other genes are also very well conserved (Supplemental Table 1), such as recR (absent in C. stagnale PCC7417, Cyanobacterium aponinum PCC10605 and C. epipsammum PCC9333), phr (absent in A. marina MBIC11017, and four Prochlorococcus strains: SS120, MIT9211, MIT9303 and MIT9313), and sulA (absent in UCYN-A, Anabaena sp. 90, and the two Gloeobacter strains G. violaceus PCC7421 and G. kilaueensis JS1).

By contrast, mutH is absent in all cyanobacteria (Supplemental Table 1) while dinB occurs in only five cyanobacteria (G. kilaueensis JS1, Nostoc (Anabaena) PCC7120, N. punctiforme PCC73102, Rivularia PCC7116 and A. marina MBIC11017), and recC occurs mostly in the marine Prochlorococcus and Synechococcus strains.

The cyanobacteria A. marina are unique in that they use chlorophyll d to absorb far-red light for photosynthesis. A. marina MBIC11017 possesses a large genome (836 Mb) comprising a circular chromosome (6.5 Mb) and nine plasmids [2.13–374 Kb, (Swingley et al., 2008)]. Consistent with its large genome size, A. marina MBIC11017 possesses almost all DNA repair genes observed in cyanobacteria, to the noticeable exception of recC. In addition to the core genes (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD) A. marina MBIC11017 has the following genes alkB, dinB (rare in cyanobacteria), lexA, mutLTY, phr, ogt, mutLTY, recJQR, sulA, ssb, umuCD, and xerC (Supplemental Table 1). Several of these genes occur in multiple copies (some located on plasmids): alkB (two copies), mutS (two copies), ogt (three copies), recA (seven copies, four of them located on four distinct plasmids), recD (three copies, two of them propagated on plasmid), recJ (two copies), recQ (two copies), ssb (two copies), umuC (three copies including two plasmid copies), umuD (four copies including two plasmid copies), and xerC (eight copies, including six on plasmids).

The role of the DNA repair genes of A. marina MBIC11017 cannot be studied in this host because it has no genetic system yet. However, these genes can be studied in the genetic models Synechocystis PCC6803, Synechococcus PCC7942, Synechococcus PCC7002 or Nostoc (Anabaena) PCC7120, and their future DNA repair mutants. Hence, it would be interesting to study (and compare) the capability of each of the seven A. marina MBIC11017 recA genes to complement the detrimental absence of the endogenous recA gene of Synechococcus PCC7002 (Murphy et al., 1990). If so, the responses of the resulting mutants to DNA damaging agents could be further studied and compared to those of the Synechococcus PCC7002 wild-type strain.

The cyanobacterial core DNA repair genes (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD) can be investigated in any genetic models Synechocystis PCC6803, Synechococcus PCC7942, Synechococcus PCC7002 and/or Nostoc (Anabaena) PCC7120, through deletion and/or over-expression, and phenotypic analysis of the resulting mutants (resistance to DNA damaging agents, etc).

Besides the core DNA repair genes, Synechocystis PCC6803, the best-studied model, can be used to investigate alkB, lexA, mutL, mutS (a second copy), mutT, phr, recBcy, recD, recQcy, sulA, umuC (two copies), and umuD (Supplemental Table 1). The genes missing in Synechocystis PCC6803 (dinB, ogt, mutY, recBec, recC, recJcy, recJec, and recQec) can be studied in the other models (Supplemental Table 1): Synechococcus PCC7942 (mutY, recB_ec, and the two copies of ogt and recJ), Synechococcus PCC7002 (mutY, recB_ec, the two copies of recJ and recQ_ec) and Nostoc PCC7120 (dinB, ogt, the two copies of recJ, and recQ_ec). By contrast, recC in occurring only in the marine cyanobacteria Synechococcus and Prochlorococcus, with no genetics, cannot be studied in its truly natural genetic context. Nevertheless, recC can be investigated in any model cyanobacteria mentioned above.

So far only the ruvB and lexA genes of Synechocystis PCC6803 have been studied in vivo. While ruvB was found to operate in DNA-recombination, lexA appeared to regulate carbon assimilation (Domain et al., 2004) and cell motility (Kizawa et al., 2016) but not DNA repair (Domain et al., 2004).

In addition to the core DNA repair genes (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, and uvrABCD) the small genomes (1.6–2.7 Mb) of the marine cyanobacteria Prochlorococcus and Synechococcus possess several genes frequently absent in larger cyanobacterial genomes (recBCD and umuCD; Supplemental Table 1). Prochlorococcus and Synechococcus also have homologs of lexA and sulA, which encode the key E. coli SOS proteins LexA (regulation of the SOS system) and SulA (postponing of cell division until completion of DNA reparation) (Baharoglu and Mazel, 2014). Furthermore, recA and uvrA are induced by UV in Prochlorococcus and Synechococcus (no information is provided for the other genes), as occurs in E. coli (Mella-Flores et al., 2012). The distribution of DNA repair genes in Prochlorococcus and Synechococcus marine strains suggest that they may possess an E.coli-like SOS system. This hypothesis is consistent with the fact that the mutation rate of Prochlorococcus is similar to that of E. coli (Biller et al., 2015).

By contrast, several findings indicate that the E.coli-like SOS model for DNA repair is not valid for all cyanobacteria. The strongest evidence is that two cyanobacteria G. violaceus PCC7421 and G. kilaueensis JS1 have none of the two key SOS genes lexA and sulA, and they also lack alkB, recBC and xerC (Supplemental Table 1). Similarly, Synechococcus PCC7942 (and its sister strain PCC6301) has no lexA, alkB, dinB, and recCD, while Anabaena sp. 90 lacks sulA, dinB, ogt, recBCD and umuCD. Synechocystis PCC6803 possesses lexA, but it does not regulate DNA repair genes; it controls carbon assimilation (Domain et al., 2004) and cell motility (Kizawa et al., 2016). Furthermore, the Synechocystis PCC6803 lexA and recA genes are not induced by UV-C as occur in E. coli, actually they are downregulated by UV-C (Domain et al., 2004) [lexA is also negatively regulated by UV-B (Huang et al., 2002)]. In addition, the Synechocystis PCC6803 lexA and recA promoters have neither E. coli-like nor B. subtilis-like SOS boxes (Domain et al., 2004). Similarly, no SOS box was found in the promoter region of the Synechococcus PCC7002 recA gene (Murphy et al., 1990). Furthermore, the lexA gene of Anabaena PCC7120 was neither induced by UV-B nor mitomycin C. In addition, the Synechocystis PCC6803 LexA protein has a RecA-independent autoproteolytic cleavage (Kumar et al., 2015).

In Synechococcus PCC7942, the Weigle-reactivation of irradiated phage (As-1) was neither induced by mitomycin-C nor nalidixic acid, unlike what was found in E.coli (Lanham and Houghton, 1988).

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.