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Abstract

Cyanobacteria are fascinating photosynthetic prokaryotes that are regarded as the ancestors of the plant chloroplast; the purveyors of oxygen and biomass for the food chain; and promising cell factories for an environmentally friendly production of chemicals. In colonizing most waters and soils of our planet, cyanobacteria are inevitably challenged by environmental stresses that generate DNA damages. Furthermore, many strains engineered for biotechnological purposes can use DNA recombination to stop synthesizing the biotechnological product. Hence, it is important to study DNA recombination and repair in cyanobacteria for both basic and applied research. This review reports what is known in a few widely studied model cyanobacteria and what can be inferred by mining the sequenced genomes of morphologically and physiologically diverse strains. We show that cyanobacteria possess manyE. coli-like DNA recombination and repair genes, and possibly other genes not yet identified.E. coli-homolog genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity. Many genes are extremely well conserved in cyanobacteria (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, anduvrABCD), even in small genomes, suggesting that they encode the core DNA repair process. In addition to these core genes, the marineProchlorococcus andSynechococcus strains harborrecBCD (DNA recombination),umuCD (mutational DNA replication), as well as the key SOS geneslexA (regulation of the SOS system) andsulA (postponing of cell division until completion of DNA reparation). Hence, these strains could possess anE. coli-type SOS system. In contrast, several cyanobacteria endowed with larger genomes lack typical SOS genes. For examples, the two studiedGloeobacter strains lackalkB, lexA, andsulA; andSynechococcus PCC7942 has neitherlexA norrecCD. Furthermore, theSynechocystis PCC6803lexA product does not regulate DNA repair genes. Collectively, these findings indicate that not all cyanobacteria have anE. coli-type SOS system. Also interestingly, several cyanobacteria possess multiple copies ofE. coli-like DNA repair genes, such asAcaryochloris marina MBIC11017 (2alkB, 3ogt, 7recA, 3recD, 2ssb, 3umuC, 4umuD, and 8xerC),Cyanothece ATCC51142 (2lexA and 4ruvC), andNostoc PCC7120 (2ssb and 3xerC).

Introduction

Cyanobacteria, the oldest and most diverse Gram-negative bacteria (Shih et al.,2013) are the only prokaryotes capable of oxygen-evolving photosynthesis (Hamilton et al.,2016). They are viewed as the ancestors of plant chloroplasts (Archibald,2009), and as major producers of (i) the Earth's oxygenic atmosphere (Schopf,2011) and (ii) the carbonates sedimentary deposits (Bosak et al.,2013; Benzerara et al.,2014).

Contemporary cyanobacteria produce a tremendous quantity of oxygen, and fix CO2 (Jansson and Northen,2010), NO₃ and N₂ (Zehr,2011) into an enormous biomass that supports a large part of the food chain. N₂-fixing cyanobacteria can be used to fertilize soils (Singh et al.,2016), in place of industrial N-fertilizers whose production consumes large amounts of fossil fuels (Grizeau et al.,2015). In colonizing a wealth of wastewater ecosystems that contain high levels of nitrate and phosphate (Abed et al.,2014) and/or heavy metals, cyanobacteria could be used for wastewater treatment (Abed et al.,2014; Singh et al.,2016).

Cyanobacteria produce a wealth of natural products that can influence human health (antioxidants, vitamins, antibacterial, toxins (Williams,2009; Dittmann et al.,2015; Kleigrewe et al.,2016; Narainsamy et al.,2016). Hence,Arthrospira has served as a human food since time immemorial (Gao,1998).

Cyanobacteria are also regarded as promising microbial factories for the production of chemicals from nature's most plentiful resources: solar light, water, CO₂ (Lai and Lan,2015; Savakis and Hellingwerf,2015; Zhou et al.,2016). To reach this objective, it is necessary to (i) introduce and express in cyanobacteria the (heterologous) chemicals-producing genes they lack; (ii) redirect the photosynthetically-fixed carbon toward the production of the intended chemicals; (iii) increase the tolerance of the engineered cyanobacteria to the intended products and (iv) maintain, or increase, the genomic stability of the producer strains. These biotechnological works are mainly performed with the unicellular modelsSynechocystis sp. strain PCC6803,Synechococcus sp. strain PCC7942 (formerlyAnacystis nidulans R2) andSynechococcus sp. strain PCC7002 (formerlyAgmenellum quadruplicatum PR6) that possess a small sequenced and manipulable genome (http://genome.microbedb.jp/cyanobase/). These cyanobacteria can take up and incorporate extracellular DNA into their chromosome to create insertion, deletion, or replacement mutations (Orkwiszewski and Kaney,1974; Stevens and Porter,1980; Grigorieva and Shestakov,1982). They can also be manipulated with replicative shuttle vectors derived from (i) their endogenous plasmids (Kuhlemeier et al.,1981; Buzby et al.,1983; Chauvat et al.,1986), or (ii) the non-cyanobacterial plasmid RSF1010 (Mermet-Bouvier et al.,1993). Interestingly, this promiscuous plasmid replicates also inThermosynechococcus elongatus (Mühlenhoff and Chauvat,1996),Prochlorococcus marinus sp. strain MIT9313 (Tolonen et al.,2006),Leptolyngbya sp. strain BL0902 andNostoc punctiforme sp. strain ATCC29133 (also registered as PCC73102) (Huang et al.,2010; Taton et al.,2014). Such RSF1010-derived plasmids proved useful tools forin vivo studies of (i) gene expression (Marraccini et al.,1993; Mermet-Bouvier and Chauvat,1994; Mazouni et al.,1998; Figge et al.,2000; Mazouni et al.,2003; Huang et al.,2010; Dutheil et al.,2012); (ii) cell division (Mazouni et al.,2004; Marbouty et al.,2009), DNA repair (Domain et al.,2004); (iii) hydrogen production (Dutheil et al.,2012; Sakr et al.,2013; Ortega-Ramos et al.,2014); (iv) insertion sequence (Cassier-Chauvat et al.,1997); and (v) redox metabolism and responses to heavy metals (Poncelet et al.,1998; Marteyn et al.,2009,2013).

Because of their photoautotrophic lifestyle, cyanobacteria are strongly challenged by DNA damages generated by solar UV rays and photosynthesis (for review see Cassier-Chauvat and Chauvat,2015), likely explaining their resistance to radiations. Furthermore, many cyanobacteria engineered for biotechnological purposes appeared to be genetically unstable in using DNA recombination to inactivate/eliminate the newly introduced genes of industrial interest. Hence, a better understanding of DNA recombination and repair in cyanobacteria could help increasing their robustness and the genetic stability of the engineered strains. This would represent an important contribution toward the development of an economically viable photo-biotechnology. In this perspective, we used a comparative genomic approach (Table1 and Supplemental Table1), to show that cyanobacteria possess a large number of genes homolog toEscherichia coli DNA recombination and repair genes, including the key SOS playerslexA andsulA. The presence/absence of these genes and information concerning their function and/or regulation indicate that some cyanobacteria may possess anE. coli-like SOS-type DNA repair system. These findings do not exclude the possible existence in cyanobacteria of other DNA repair genes, not yet identified.

Results and discussion

Genomic diversity of cyanobacteria

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 strainSynechocystis 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 forSynechococcus PCC7942 (Mann and Carr,1974; Griese et al.,2011; Watanabe et al.,2015); and 3.00 Mb, 50%, and likely 2–5 forSynechococcus 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); andSynechococcus 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 strainsProchlorococcus andSynechococcus 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).

Cyanobacteria can be resistant to radiations

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 andSynechococcus PCC7942 are found to be more resistant to UV than the (non-photosynthetic) bacteriumE. coli where DNA repair is best known (Baharoglu and Mazel,2014).Synechocystis PCC6803 is also more resistant to gamma rays thanSynechococcus PCC7942 andE. 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 bacteriumDeinococcus radiodurans [100% survival at 5kGy (Moseley and Mattingly,1971; Ito et al.,1983)]. These radiation-resistant cyanobacteria areChroococcidiopsis [10% survival to 4–5 kGy of gamma rays (Billi et al.,2000)], threeAnabaena strains [they can grow at 5 kGy (Singh et al.,2010)] andArthrospira 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).

Cyanobacteria can be naturally competent for genetic transformation mediated by DNA recombinations

The naturally transformable cyanobacteriaSynechococcus PCC7942,Synechococcus PCC7002, andSynechocystis 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 inBacillus subtilis andHelicobacter 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.

Thecom genes (Table1) are widely distributed in cyanobacteria (Supplemental Table1).Synechocystis PCC6803,Synechococcus PCC7942, andSynechococcus PCC7002 harbor thecomAEF genes (Supplemental Table1). TheSynechocystis PCC6803 genescomA andcomF truly operate in transformation (Yoshihara et al.,2001), andcomF is also involved in phototactic motility (Nakasugi et al.,2006). The role ofcomE could not be verified because thecomE-depleted mutant dies rapidly (Yoshihara et al.,2001). By contrast, theProchlorococcus cyanobacteria endowed with small genomes have nocomAEF genes, exceptedP. marinus MIT9303, andP. marinus MIT9313 that possesscomA, come, andComF (Supplemental Table1). These strains also have therecA, recG, andruvABC genes (Supplemental Table1). We have verified inSynechocystis PCC6803 thatruvB operates in genetic transformation (Domain et al.,2004). These finding suggest thatP. 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).

Cyanobacteria genetically engineered for biotechnological purposes can be genetically instable

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 ofSynechocystis 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 useSynechocystis PCC6803 for the production of a uniformly¹⁴C-labeled mouse urokinase (a serine protease). The urokinase producing plasmid, which replicated stably in therecA⁻ mutant ofE. coli, invariably lost part of the urokinase gene upon propagation inSynechocystis PCC6803 (Chauvat et al.,1988). AnotherSynechocystis PCC6803 strain harboringPseudomonas aeruginosa genes cloned its chromosome (at theslr0168 neutral docking site) for lactic acid production, happened to rescue its growth by introducing a duplication (~160 bp) that generated premature stop codons into thePseudomonas (NADPH/NADH) transhydrogenase gene (Angermayr et al.,2012).

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

InSynechococcus PCC7002, a recombinant strain managed to loose mannitol synthesis and recover healthy growth, in introducing a single-base deletion generating a stop codon in itsE. coli mannitol-1-phosphate dehydrogenasemtlD gene (Jacobsen and Frigaard,2014).

TheSynechocystis PCC6803 andSynechococcus PCC7002 recombinant strains producing theZymomonas mobilis pyruvate decarboxylase enzyme (PDC) for ethanol production, could introduce mutations, insertions, deletions or mobile genetic elements (insertion sequences) into thepdc 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 severalP. 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 ofMicrocystis aeruginosa NIES843, 1.5% of the 3.95 Mb genome ofSynechocystis PCC6803, and 1% of the 7.2 Mb genome ofNostoc (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 recombinantNostoc (Anabaena) PCC7120 strain harboring a plasmid encoding theB. subtilis SacB enzyme (levan sucrase), which kills cells incubated in the presence of sucrose, generated sucrose resistant mutants resulting from the disruption of thesacB gene by a mobile IS895 element (Alam et al.,1991).

Similarly, an IS5 element ofSynechocystis 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 threeSynechocystis 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. InCorynebacterium 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 cyanobacteriaSynechococcus PCC7942 andSynechococcus 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 forSynechocystis PCC6803 that possesses 128 transposase genes.

InE. coli, the stable propagation of recombinant DNA (usually cloned in plasmids) is achieved in strains whererecA, the key DNA-recombination gene (Baharoglu and Mazel,2014), has been inactivated to prevent unexpected DNA rearrangements. All cyanobacteria possess arecA gene (Acaryochoris marima MBIC11017 has 7recA genes, Supplemental Table1). TherecA gene appeared to be indispensable to cell life inSynechococcus PCC7002 (Murphy et al.,1990), whereas it could be deleted from all chromosome copies inSynechocystis PCC6803 (Minda et al.,2005). TheSynechocystis PCC6803recA 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 arecA⁻ 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 promoterscoaT, ziaA, etc (Berla et al.,2013; Zhou et al.,2016)], or (iv) the growth temperature [lambda phagepR promoter controlled by thecI857 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.

• “It should be inactive in absence of inducer”;

• “It should produce a predictable response to a given concentration of a regulator”;

• “The inducer should have no harmful effect on the host organism”;

• “The inducer should be cheap and stable under the growth conditions of the host”;

• “The inducible system should act orthogonally to the host cell's transcriptional program (ideal transcriptional repressors should not bind to native promoters.)”

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 cyanobacteriumSynechocystis PCC6803); intermediary expression at intermediate temperature 34–37°C; and strong expression at 39°C (whereSynechocystis 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).

Distribution of direct DNA-damages reversal genes in cyanobacteria

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 inE. 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 toE. coli DNA recombination and repair genes. Thephr, alkB andogt orthologs (Table1) are distributed unevenly in cyanobacteria (Supplemental Table1). Thephr 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 strainsP. marinus MIT9515 andP. marinus MED4 possessphr, whereas the low-light-adapted strainsP. marinus MIT9303 andP. marinus MIT9313 lackphr (Supplemental Table1), and are light sensitive (Biller et al.,2015). ThealkB andogt genes are less frequent thanphr. All three genesalkB, ogt, andphr are simultaneously present in several (twelve) studied cyanobacteria, such asNostoc (Anabaena) PCC7120 (filamentous), andCyanothece PCC7425 (unicellular) whereogt is duplicated. The other (evolutionary distant) unicellular modelsSynechocystis PCC6803,Synechococcus PCC7942, andSynechococcus PCC7002 possessphr (Supplemental Table1).Synechocystis PCC6803 hasalkB but notogt, Synechococcus PCC7942 hasogt (duplicated) but notalkB, andSynechococcus PCC7002 has neitheralkB norogt. Interestingly, the symbiotic (marine) cyanobacterium UCYN-A has nophr, alkB, andogt, in agreement with the fact that it possesses the smallest genome (1.44 Mb). The other symbiotic strainAcaryochloris marina MBIC11017 endowed with a larger genome (8.36 Mb) has twoalkB, threeogt (including one on a plasmid) but nophr (Supplemental Table1).

Distribution of nucleotide excision DNA repair genes in cyanobacteria

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). InE. 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 theuvrABCD single-copy genes (Supplemental Table1), whereuvrA anduvrB are not organized in operon (Supplemental Figure1), unlike what occurs inE. coli. In some cyanobacterial genomesuvrA, uvrB, uvrC, and/oruvrD are clustered with another DNA repair gene, such asphr orrecN (gene clusters a and c in Supplemental Table1 and Supplemental Figure1). In the radiation-resistant cyanobacteriumArthrospira PCC8005,uvrBCD were found to be upregulated by gamma rays (no information is provided foruvrA) (Badri et al.,2015).

Distribution of methyl-directed DNA mismatch repair genes in cyanobacteria

This pathway corrects the mispaired DNA bases generated by replication errors (Putnam,2016). InE. 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 havemutM (Supplemental Table1), which was shown inSynechococcus PCC7942 to operate in resistance to high light (Mühlenhoff,2000). All cyanobacteria possessmutS, which occurs in two copies, excepted inCrinalium epipsammum PCC 9333 (Supplemental Table1). By contrast,mutH is absent in all cyanobacteria. The genetic diversity of cyanobacteria is well illustrated with the presence/absence ofmutL, mutt, andmutY (Supplemental Table1), which lies in front ofrecR in a few cyanobacterial genomes (Table1 and Supplemental Figure1). SeveralP. marinus strains lackmutL, mutt, andmutY (Supplemental Table1). InArthrospira PCC8005 (radiation-resistant)mutST were upregulated by gamma rays (Badri et al.,2015).

The model strainsSynechocystis PCC6803,Synechococcus PCC7942,Synechococcus PCC7002 andNostoc (Anabaena) PCC7120 possessmutL, mutM, mutS (duplicated),mutT (exceptedSynechococcus PCC7002),mutY (exceptedSynechococcus PCC6803 andNostoc PCC7120) (Supplemental Table1). Thus,Synechococcus PCC7942 is best suited to study all these genes through deletion/over-expression in the otherwise same genetic context.

Distribution of recombinational DNA repair genes in cyanobacteria

This pathway repairs double-stranded breaks and cross-links. InE. 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 asH. pylori andB. subtilis the AddA and AddB proteins replace RecB and RecC, respectively (Dorer et al.,2011; Wigley,2013).

All cyanobacteria containrecA, which occurs as seven copies in the large genome (8.36 Mb) ofA. marina MBIC11017. Four of theserecA genes, possibly originating from gene duplication (Swingley et al.,2008), are located on four separate plasmids, while the otherrecA belong to the chromosome (Supplemental Table1).

LikerecA, radA andrecG are present in all cyanobacteria, andradA is duplicated inCyanothece PCC7425,M. aeruginosa NIES-843 and UCYNA (Supplemental Table1). It is the only duplicated gene in the very small UCYNA genome (1.44 Mb).

Many cyanobacteria have two copies ofrecJ andrecQ genes. They are noted asrecJ_ec orrecJ_cy, orrecQ_ec orrecQ_cy (ec forE. coli, cy for cyanobacteria), according to their high (recJ_ec,recQ_ec) or low (recJ_cy,recQ_cy) sequence similarity with theirE. coli counterparts (Table1 and Supplemental Table1). This is true forSynechococcus PCC7002 andNostoc PCC7120, where these duplicated genes can be studied and compared through deletion/over-expression. InArthrospira PCC8005 (radiation-resistant),recGJQ were found to be upregulated by gamma rays (Badri et al.,2015). In contrast a few cyanobacteria has neitherrecJ norrecQ, asP. marinus MIT9515 (Supplemental Table1). Also interestingly, the low-light-adaptedP. marinus MIT9313 andP. marinus MIT9303 possess therecQ genes (andogt and the competence genescomE andcomFC), which are not present in otherProchlorococcus (Supplemental Table1). In addition, bothP. marinus MIT9313 andP. marinus MIT9303 lack thephr gene, which occurs in otherProchlorococcus (Supplemental Table1), in agreement with their light-sensitivity (Biller et al.,2015). Collectively, these findings support the proposal thatP. marinus MIT9303 andP. marinus MIT9313 belong to the same clade, which diverged early from the otherProchloroccus clades (Sun and Blanchard,2014; Biller et al.,2015).

Almost all cyanobacteria have the single-copy genesrecF, recO andrecR, exceptedCyanobacterium aponinum PCC10605,C. epipsammum PCC9333 andCylindrospermum stagnale PCC7417 which lackrecR (Supplemental Table1)

TherecBCD genes are less conserved in cyanobacteria. For instance, the strain UCYN-A that possessesrecFOR has norecBCD genes (Supplemental Table1). MostP. marinus strains and several marineSynechococcus strains possessrecBCD. Most of these strains possess tworecB copies, notedrecB_ec (good similarity withE. coli recB) orrecB_cy (cy for cyanobacteria, low similarity withE. coli recB). In these strains,recB_ec belongs to the same genomic region thanrecC andrecD (cluster f in Supplemental Table1 and Supplemental Figure1). In a few other cyanobacteriarecD is duplicated (Microcoleus PCC7113) or triplicated (A. marina MBIC11017 andN. punctiforme PCC73102), irrespectively of the presence /absence orrecB_ec andrecB_cy (Supplemental Table1). The well-studied model cyanobacteria lackrecB, recC, orrecD. BothSynechocystis PCC6803 andNostoc (Anabaena) PCC7120 lackrecB_ec andrecC, while bothSynechococcus strains PCC7942 andSynechococcus PCC7002 lackrecCD.

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

In some cyanobacteria a fewrec genes are clustered together (recBCD see cluster f in Supplemental Table1 and Supplemental Figure1), or with other DNA repair genes, includinguvrA (cluster a) ormutY (cluster n; Supplemental Table1 and Supplemental Figure1).

All cyanobacteria have assb gene, which is repeated in a few strains. For instance,ssb is duplicated inNostoc (Anabaena) PCC7120 andA. marina MBIC11017), while it is triplicated inChroococcidiopsis thermalis PCC7203 and quadruplicated inCyanothece PCC7822 (Supplemental Table1). In these cyanobacteria (exceptedNostoc (Anabaena) PCC7120) onessb copy is propagated on a plasmid. One of the twoNostoc PCC7120ssb genes, (alr0088, but notalr7579) was shown to be involved in the tolerance to UV and mitomycin C which causes formation of DNA adducts (Kirti et al.,2013).

TheruvABC genes are present in all cyanobacteria, to the noticeable exception ofG. kilaueensis JS1 which lacksruvC (Supplemental Table1). TheruvA andruvB genes are not adjacent unlike their operonicE. coli counterparts. Furthermore,ruvA is duplicated inTrichodesmium erythraeum ISM101, whileruvC is quadruplicated inCyanothece ATCC51142 and quadruplicated inCyanothece PCC7822 (Supplemental Table1). InSynechocystis PCC6803ruvB 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).

UnlikerecAN andruvABC, xerC is a rare gene in cyanobacteria (Supplemental Table1). It occurs in a single copy in a few strains, as UCYN-A,Synechococcus PCC7942,Synechococcus PCC7002 andSynechocystis PCC6803, or in several copies inCyanothece 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. InE. coli, the Chi site used by RecBCD is 8 bases (GCTGGTGG), whereas inB. 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). InSynechocystis 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).

Distribution of mutagenic DNA repair genes in cyanobacteria

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 bydinB) and PolV (encoded byumuCD), which replicate damaged DNA in a mutagenic manner (Baharoglu and Mazel,2014).

TheumuCD genes (Table1) are unevenly distributed in cyanobacteria (Supplemental Table1). Many strains have noumuCD, like UCYN-A (small genome) andSynechococcus PCC7002. Others possessumuCD, such asNostoc (Anabaena) PCC7120,Synechococcus PCC7942,Synechocystis PCC6803, and theProchlorococcus strains. A few strains harbor a duplication ofumuC (Synechococcus PCC6312) and/orumuD (Cyanobium gracile PCC6307 andSynechococcus PCC6312).A. marina MBIC11017 possesses threeumuC and fourumuD (Swingley et al.,2008). In some cyanobacteriaumuDC are clustered together, nearbyruvA (cluster z in Supplemental Table1 and Supplemental Figure1).

The genedinB (Table1) is present in a very few cyanobacteria, such asA. marina MBIC11017,Anabaena PCC7120 andG. kilaueensis JS1 (Supplemental Table1).

Distribution of the keyE. coli-type SOS genesLexA andSulA in cyanobacteria

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. InE. 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.

ThelexA gene (Table1) is unevenly distributed in cyanobacteria. It is absent in bothArthrospira PCC8005 (Badri et al.,2015) and NIES39, and in several strains of the genusGloeobacter, Oscillatoria andSynechococcus (includingSynechococcus PCC7942, Supplemental Table1), similarly to what found in other bacteria asH. pylori (Dorer et al.,2011) andStreptococcus pneumoniae (Baharoglu and Mazel,2014). By contrast,lexA is present in the other tested cyanobacteria (it is duplicated inCyanothece ATCC51142). The marine cyanobacteria of the genusProchlorococcus andSynechococcus share a very similarlexA (clade C), while other strains possess a slightly differentlexA (clade B), such asA. marina MBIC11017, and bothNostoc PCC7120 andSynechocystis PCC6803 (Li et al.,2010). Interestingly, theSynechocystis PCC6803lexA 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, theNostoc PCC7120 LexA protein has a RecA-independent autoproteolytic cleavage (Kumar et al.,2015).

ThesulA homolog is present in almost all cyanobacteria, to the noticeable exception ofGloeobacter violaceus PCC7421,G. kilaueensis JS1,Anabaena sp. 90 and UCYN-A (Supplemental Table1). InSynechocystis PCC6803,sulA appeared to be indispensable to cell life and division (Raynaud et al.,2004).

The DNA repair genes present in all cyanobacteria likely encode the core process

Many genes are present in the 76 studied cyanobacteria (mutM, radA, recA, recFO, recG, recN, ruvABC, ssb, anduvrABCD; Table1 and Supplemental Table1), including the marine strain UCYN-A that possesses the smallest genome (1.44 Mb), and numerous marine strainsProchlorococcus andSynechococcus also endowed with a small genome (1.65–Mb). Similarly,mutS, recN, andruvC are present in almost all cyanobacteria (Supplemental Table1), namelyThermosynechococcus NK55a (absence ofmutS1),Cyanothece PCC51142 (absence ofrecN) and (G. kilaueensis JS1 absence ofruvC). Consequently, we propose that the genesmutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, anduvrABCD encode the core DNA repair system of cyanobacteria.

A few other genes are also very well conserved (Supplemental Table1), such asrecR (absent inC. stagnale PCC7417,Cyanobacterium aponinum PCC10605 andC. epipsammum PCC9333),phr (absent inA. marina MBIC11017, and fourProchlorococcus strains:SS120, MIT9211, MIT9303 and MIT9313), andsulA (absent in UCYN-A,Anabaena sp. 90, and the twoGloeobacter strainsG. violaceus PCC7421 andG. kilaueensis JS1).

By contrast, mutH is absent in all cyanobacteria (Supplemental Table1) whiledinB occurs in only five cyanobacteria (G. kilaueensis JS1,Nostoc (Anabaena) PCC7120,N. punctiforme PCC73102, Rivularia PCC7116 andA. marina MBIC11017), andrecC occurs mostly in the marineProchlorococcus andSynechococcus strains.

Acaryochloris marina MBIC11017 possesses the largest panel of DNA repair genes some of which occurring in multiple copies in the chromosome and/or plasmids

The cyanobacteriaA. 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 ofrecC. In addition to the core genes (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, anduvrABCD)A. marina MBIC11017 has the following genesalkB, dinB (rare in cyanobacteria),lexA, mutLTY, phr, ogt, mutLTY, recJQR, sulA, ssb, umuCD, andxerC (Supplemental Table1). 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), andxerC (eight copies, including six on plasmids).

The role of the DNA repair genes ofA. marina MBIC11017 cannot be studied in this host because it has no genetic system yet. However, these genes can be studied in the genetic modelsSynechocystis PCC6803,Synechococcus PCC7942,Synechococcus PCC7002 orNostoc (Anabaena) PCC7120, and their future DNA repair mutants. Hence, it would be interesting to study (and compare) the capability of each of the sevenA. marina MBIC11017recA genes to complement the detrimental absence of the endogenousrecA gene ofSynechococcus 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 theSynechococcus PCC7002 wild-type strain.

Together, the evolutionary-distant genetic modelsSynechocystis PCC6803,Synechococcus PCC7942,Synechococcus PCC7002 andNostoc (Anabaena) PCC7120 possess almost all DNA repair genes

The cyanobacterial core DNA repair genes (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, anduvrABCD) can be investigated in any genetic modelsSynechocystis PCC6803,Synechococcus PCC7942,Synechococcus PCC7002 and/orNostoc (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 investigatealkB, lexA, mutL, mutS (a second copy),mutT, phr, recBcy, recD, recQcy, sulA, umuC (two copies), andumuD (Supplemental Table1). The genes missing inSynechocystis PCC6803 (dinB, ogt, mutY, recBec, recC, recJcy, recJec, andrecQec) can be studied in the other models (Supplemental Table1):Synechococcus PCC7942 (mutY, recB_ec, and the two copies ofogt andrecJ),Synechococcus PCC7002 (mutY, recB_ec, the two copies ofrecJ andrecQ_ec) andNostoc PCC7120 (dinB, ogt, the two copies ofrecJ, andrecQ_ec). By contrast,recC in occurring only in the marine cyanobacteriaSynechococcus andProchlorococcus, 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 theruvB andlexA genes ofSynechocystis PCC6803 have been studiedin vivo. WhileruvB 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).

TheE.coli-like SOS model for DNA repair is possibly valid for the marineProchlorococcus andSynechococcus cyanobacteria, but not forGloeobacter, Synechocystis PCC6803, andSynechococcus PCC7942

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 cyanobacteriaProchlorococcus andSynechococcus possess several genes frequently absent in larger cyanobacterial genomes (recBCD andumuCD; Supplemental Table1).Prochlorococcus andSynechococcus also have homologs oflexA andsulA, which encode the keyE. 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 anduvrA are induced by UV inProchlorococcus andSynechococcus (no information is provided for the other genes), as occurs inE. coli (Mella-Flores et al.,2012). The distribution of DNA repair genes inProchlorococcus andSynechococcus marine strains suggest that they may possess anE.coli-like SOS system. This hypothesis is consistent with the fact that the mutation rate ofProchlorococcus is similar to that ofE. coli (Biller et al.,2015).

By contrast, several findings indicate that theE.coli-like SOS model for DNA repair is not valid for all cyanobacteria. The strongest evidence is that two cyanobacteriaG. violaceus PCC7421 andG. kilaueensis JS1 have none of the two key SOS geneslexA andsulA, and they also lackalkB, recBC andxerC (Supplemental Table1). Similarly,Synechococcus PCC7942 (and its sister strain PCC6301) has nolexA, alkB, dinB, andrecCD, whileAnabaena sp. 90 lackssulA, dinB, ogt, recBCD andumuCD.Synechocystis PCC6803 possesseslexA, but it does not regulate DNA repair genes; it controls carbon assimilation (Domain et al.,2004) and cell motility (Kizawa et al.,2016). Furthermore, theSynechocystis PCC6803lexA andrecA genes are not induced by UV-C as occur inE. 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, theSynechocystis PCC6803lexA andrecA promoters have neitherE. coli-like norB. subtilis-like SOS boxes (Domain et al.,2004). Similarly, no SOS box was found in the promoter region of theSynechococcus PCC7002recA gene (Murphy et al.,1990). Furthermore, thelexA gene ofAnabaena PCC7120 was neither induced by UV-B nor mitomycin C. In addition, theSynechocystis PCC6803 LexA protein has a RecA-independent autoproteolytic cleavage (Kumar et al.,2015).

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

Conclusion

From bacteria to higher eukaryotes, cells are equipped with various conserved systems to repair DNA damages generated by their own metabolism (Imlay,2013) or exogenous sources (solar UV, gamma radiations, chemicals, etc.). Inevitably, some DNA lesions are not correctly repaired leading to mutations that can influence cell fitness (Baharoglu and Mazel,2014).

For historical reasons, DNA recombination and repair in prokaryotes have been mostly studied in the (non-photosynthetic) bacteriumE. coli (Baharoglu and Mazel,2014). UnlikeE.coli, cyanobacteria are continuously exposed to DNA damages generated by solar UV rays and their own photosynthetic metabolism (Cassier-Chauvat and Chauvat,2015). As a likely consequence, all tested cyanobacteria were found to be more radiation resistant thanE. coli. It is also important to study DNA recombination and repair in cyanobacteria for biotechnological purposes, since many recombinant strains appeared to be genetically unstable. They somehow managed to inactivate the (newly-introduced) heterologous genes of industrial interest. Thus, a better understanding of DNA recombination and repair in cyanobacteria may lead to increasing the genetic stability of biotechnologically important strains, an important industrial goal.

Using a comparative genomic approach, we found that cyanobacteria possess many genes orthologous toE. coli DNA recombination and repair genes, notwithstanding the possibility that cyanobacteria have other, as yet unidentified, such genes.

TheseE. coli-like genes are unevenly distributed in cyanobacteria, in agreement with their wide genome diversity, in a way consistent with the size of their genomes, i.e., large genomes tend to possess more DNA repair genes than small genomes. Most of theseE. coli-like genes are scattered throughout cyanobacterial genomes, suggesting that there is a mechanism for their coordinate regulation or that they are mostly expressed constitutively. Many DNA repair genes (mutMS, radA, recA, recFO, recG, recN, ruvABC, ssb, anduvrABCD) are extremely well conserved in cyanobacteria, including in theProchlorococcus andSynechococcus marine strains which possess very small genomes (1.44–2.7 Mb). Consequently, we propose that these genes encode the core DNA repair system of cyanobacteria.

These marineProchlorococcus andSynechococcus cyanobacteria also have the genesrecBCD (DNA recombination),umuCD (mutational DNA replication), and the key SOS geneslexA (regulation of the SOS system) andsulA (postponing of cell division until completion of DNA reparation). These findings suggest that the marineProchlorococcus andSynechococcus cyanobacteria may possess anE. coli-type SOS system.

In contrast, other cyanobacteria endowed with larger genomes lack some of the SOS key genes (lexA, sulA, recBCD, orumuCD). For instance,G. violaceus PCC7421 andG. kilaueensis JS1 lacklexA, recBC, andsulA (they also lackalkB andxerC).Synechococcus PCC7942 has neitherlexA norrecCD. Furthermore, thelexA gene ofSynechocystis PCC6803 is not involved in the regulation of DNA repair genes (Domain et al.,2004). Collectively, these findings suggest that theE.coli-like SOS model for DNA repair is likely not valid for all cyanobacteria.

The cyanobacteriumA. marina MBIC11017 possesses the most complete, and complex, set of DNA repair genes:alkB (two copies),dinB (rare in cyanobacteria),lexA, mutL, mutM, mutS (two copies),mutT, mutY, ogt (three copies),phr, radA, recA (seven copies, four of them located on plasmids),recD (three copies, including two plasmidic copies),recF, recG, recJ (two copies),recN, recO, recQ (two copies),recR, ruvABC, ssb (two copies),sulA, umuC (three copies including two plasmid copies),umuD (four copies including two plasmid copies),uvrABCD andxerC (eight copies, including six on plasmids). However,A. marina MBIC11017 has not all DNA repair genes, since it lacksrecC. All cyanovacterial DNA repair genes naturally present (or not) in the few (evolutionary distant) genetic modelsSynechocystis PCC6803,Synechococcus PCC7002,Synechococcus PCC7942 andNostoc (Anabaena) PCC7120, can be studied through deletion and/or over-expression, and analysis of the corresponding mutants (e.g., resistance to DNA damaging agents). Such works would be most welcome since little is known about DNA recombination and repair in cyanobacteria. So far, only therecA, ruvB, andlexA genes have been studiedin vivo. TherecA gene appeared to be indispensable inSynechococcus PCC7002 (Murphy et al.,1990), and dispensable inSynechocystis PCC6803 (Minda et al.,2005). TheSynechocystis PCC6803recA-null mutant was sensitive to UV-C and white light. TheSynechocystis PCC6803ruvB gene was found to operate in DNA-recombination, whilelexA appeared to regulate carbon assimilation (Domain et al.,2004) and cell motility (Kizawa et al.,2016), but not DNA repair (Domain et al.,2004). We hope that this review will stimulate future studies of DNA recombination and repair in cyanobacteria so as to answer the following questions, among others. Do cyanobacteria possess DNA recombination and repair genes with no counterpart in a non-photosynthetic and radiation-sensitive bacterium such asE. coli? What is the specificity/redundancy of the various copies of the repeated genes of cyanobacteria (for example of the sevenrecA genes ofA. marina MBIC11017)? What are the molecular mechanisms responsible for the high radiation-resistance of some cyanobacteria (for instanceChroococcidiopsis). How to improve the genetic stability of cyanobacterial strains engineered for biotechnological puproses?

Author contribution

CC and FC conceived the study. CC, TV, and FC carried out the literature search and analyzed the data. CC, TV, and FC wrote the paper.

Conflict of interest statement

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.

Acknowledgments

TV was a recipient of PhD thesis fellowship from the CEA-Saclay France.

Conflict of interest

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.

Supplementary material

The Supplementary Material for this article can be found online at:http://journal.frontiersin.org/article/10.3389/fmicb.2016.01809/full#supplementary-material

Distribution of DNA repair genes in cyanobacteria. The presence (indicated by the number of copies) or absence (0) of the gene is indicated along with the letters referring to the conserved gene clusters depicted in Supplemental Figure1.

Conserved genomic organization around the DNA repair genes in cyanobacterial genomes. Genes are represented by boxes pointing in the direction of their transcription. DNA repair genes are colored in red. Genes encoding hypothetical proteins are indicated as “ho.”

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Summary

Keywords

cyanobacteria, photoproduction, DNA recombination, DNA repair, genetic instability, insertion sequences, natural transformation, radiation resistance

Citation

Cassier-Chauvat C, Veaudor T and Chauvat F (2016) Comparative Genomics of DNA Recombination and Repair in Cyanobacteria: Biotechnological Implications.Front. Microbiol. 7:1809. doi:10.3389/fmicb.2016.01809

Received

12 August 2016

Accepted

27 October 2016

Published

09 November 2016

Volume

7 - 2016

Edited by

Weiwen Zhang, Tianjin University, China

Reviewed by

Rajesh P. Rastogi, Ministry of Environment, Forests and Climate Change, India; Dmitry A. Los, Institute of Plant Physiology, Russia

Updates

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Copyright

*Correspondence: Franck Chauvatfranck.chauvat@cea.fr

Disclaimer

All claims expressed in this article are solely those of the authors and do not necessarily represent those of their affiliated organizations, or those of the publisher, the editors and the reviewers. Any product that may be evaluated in this article or claim that may be made by its manufacturer is not guaranteed or endorsed by the publisher.