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Keywords:

  • ploidy;
  • polyploidy;
  • genome copy number;
  • Synechococcus;
  • Synechocystis;
  • cyanobacteria

Abstract

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

A recently developed real-time PCR method for the determination of genome copy numbers was optimized for the application to cyanobacteria. Three species were chosen to represent a fresh water species, a salt water species, and two strains of a widely used laboratory species. SynechococcusPCC 7942 and SynechococcusWH7803 were found to contain 3–4 genome copies per cell and are thus oligoploid, confirming earlier publications. In contrast, SynechocystisPCC 6803 is highly polyploid. The motile wild-type strain contains 218 genome copies in exponential phase and 58 genome copies in linear and in stationary growth phase. The GT wild-type strain contains 142 genome copies in exponential phase and 42 genome copies in linear and stationary growth phase. These are the highest numbers found for any cyanobacterial species. Notably these values are much higher than the value of 12 genome copies published for the ‘Kazusa’ strain more than 20 years ago. The results reveal that for SynechocystisPCC 6803 strain differences exist and that the ploidy level is highly growth phase-regulated. A compilation of the ploidy levels of all investigated cyanobacterial species gives an overview of the genome copy number distribution and shows that monoploid, oligoploid, and polyploid cyanobacteria exist.


Introduction

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Many eukaryotic species including ciliates, fish, flowering plants, and even some cell types of humans are polyploid, and advantages as well as disadvantages of polyploidy have been discussed in various reviews (e.g. Wendel, 2000; Osborn et al., 2003; Comai, 2005; Thorpe et al., 2007; Hegarty & Hiscock, 2008). In contrast, it is generally believed that prokaryotes typically contain a single copy of their chromosome. This is usually called ‘haploidy’, but as the term ‘haploid’ does not seem to make much sense in species without a diploid stage, the term ‘monoploid’ will be used throughout this contribution. The idea that prokaryotes are typically monoploid is a generalization from the results obtained with Escherichia coli, the best studied bacterium. Escherichia coli is monoploid when it is grown very slowly, e.g. with a doubling time of 16 h (Skarstad et al., 1983). When the doubling time becomes shorter than the time to replicate and segregate the chromosome, E. coli starts a new round of replication before the previous round had been terminated, and thus the gene dosage of regions near the replication origin becomes higher than of regions near the terminus. This unequal gene dosage is called merodiploidy or mero-oligoploidy. Under optimal conditions, E. coli grows with a doubling time of 20 min and contains on average 6.8 origins and nearly two termini (Bremer & Dennis, 1996; Pecoraro et al., 2011). The dependence of DNA content and growth rate shows that E. coli ‘tries’ to grow as monoploid as possible. Several other species of bacteria are truely monoploid, e.g. Bacillus subtilis, Caulobacter crescentus, and Wolinella succinogenes (Webb et al., 1998; Pecoraro et al., 2011).

However, several species of prokaryotes also have been described to be oligoploid or polyploid. A prominent example is Deinococcus radiodurans, which contains 5–8 genome copies (Hansen, 1978). It is long known that D. radiodurans can restore intact chromosomes from heavily fragmented chromosomes, which is not possible in monoploid species. Recently, it has been shown that this is a two-stage mechanism involving a high induction of DNA repair synthesis followed by recombination (Slade et al., 2009). The efficient repair of a high number of double strand breaks (induced by irradiation or, more naturally, desiccation) is one evolutionary advantage of polyploidy for prokaryotes. Additional advantages include gene redundancy, a low rate of spontaneous mutations, global regulation of gene expression, cell size increase, or the possibility of rapid cell divisions of pathogens after entering a host. Therefore, it might not be surprising that the number of known oligoploid and polyploid prokaryotic species outnumbers the monoploid species and it seems that monoploidy is not typical for prokaryotes, in contrast to the general belief. Polyploid, oligoploid, and monoploid species can co-exist within one group of prokaryotes, an example is the gamma-proteobacteria (Pecoraro et al., 2011), whereas other groups like the euryarchaeota seem to be devoid of monoploid species (Hildenbrand et al., 2011).

Therefore, we found it interesting to clarify the situation for another group of prokaryotes, the cyanobacteria. It has been described more than 30 years ago that Anabaena cylindrica and Anabaena variabilis are polyploid and contain 25 and 8–9 genome copies, respectively (Simon, 1977, 1980). In contrast, other species like Synechococcus WH8101 were shown to be monoploid (Armbrust et al., 1989). Three species of cyanobacteria, representing a salt water species, a fresh water species, and a widely used laboratory species, were selected, and their genome copy numbers were quantified. A fast, sensitive, and precise real time PCR method was used that had originally been established for genome copy number quantification of haloarchaea (Breuert et al., 2006), but has recently been applied successfuly to proteobacteria (Pecoraro et al., 2011). In addition, a literature survey was performed and an overview of all cyanobacterial species with experimentally determined ploidy levels is given.

Materials and methods

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Cyanobacterial strains, media and growth conditions

Two Synechocystis PCC 6803 wild-type strains were obtained from Annegret Wilde (University of Giessen, Germany). Synechocystis PCC6803 was isolated in 1968 by R. Kunisawa from a freshwater lake in California (Stanier et al., 1971) and deposited at the Pasteur Culture Collection (PCC6803) and the American Type Culture Collection (ATCC 27184). Several variants arose and are currently under investigation. One strain will be called ‘motile strain’ (originally obtained from the lab of Sergey Shestakov, Moscow State University, in the cyanobacteria community known as ‘Moscow strain’), the other will be called ‘GT strain’ (glusose tolerant; originally obtained from the lab of Martin Tichy, Trebon; in the cyanobacteria community known as ‘Vermaas strain’). The genome sequence was determined from a third strain not used in this study. It was derived from the GT strain, and is known in the cyanobacteria community as the ‘Kazusa strain’ (Okamoto et al., 1999).

Synechococcus elongatus PCC 7942 and Synechococcus sp. WH7803 were obtained from Wolfgang R. Hess (University of Freiburg, Germany). Synechocystis and S. elongatus were grown in BG11 medium (Rippka et al., 1979). The marine Synechococcus sp. WH7803 was grown in artificial sea water (ASW; Waterbury & Wiley, 1988). All strains were grown at a temperature of 28 °C on a rotary shaker (120 r.p.m.) to prevent aggregation. They were grown phototrophically at a fluence rate of 10–60 photons m−2 s−1 (Osram daylight lamp LUMILUX de Lux L18W/954; Osram, Munich, Germany) with constant illumination. Growth was monitored spectroscopically at 750 nm.

Cell harvest, cell disruption and control of DNA integrity

Cultures for genome copy number determination were inoculated from precultures in the linear growth phase and grown to the respective optical densities (see text and tables). At the times of harvest, the cultures were checked microscopically to detect possible aggregation, which was not observed, and to determine cell densities using a Neubauer counting chamber. The cells of 40 mL culture were harvested by centrifugation (3200 g, 30 min, room temperature). The supernatant was checked microscopically to verify that it was free of cells. The pellet was suspended in 2 mL distilled water. The cell density was determined microscopically using a Neubauer counting chamber. 0.5 mL of the cell suspension was mixed with either 0.75 g (Synechocystis PCC 6803) or 1 g (S. elongatus PCC 7942 and Synechococcus sp. WH7803) zirconia/silica beads (0.1 mm; Roth, Karlsruhe, Germany) in a 2 mL screw cup (Sarstedt, Nümbrecht, Germany). Cells were disrupted by shaking for 1.5 min (Synechocystis PCC 6803) or 2 min (S. elongatus PCC 7942 and Synechococcus sp. WH7803) in a Speedmill P12 (Analytik Jena, Jena, Germany). The cell density was determined again, and the values before and after cell disruption were used to calculate the efficiency of cell disruption. Cell debris was removed by centrifugation (15 000 g, 20 min room temperature). 0.3 mL of the supernatant was used as cytoplasmic extract for further analysis. The integrity of genomic DNA was checked using analytical agarose electrophoresis. The extract was dialyzed against distilled water, and volumes prior and after dialysis were used to calculate the dilution.

Real time PCR method for ploidy determination

To determine genome copy numbers, a real time PCR approach was applied (Breuert et al., 2006; an overview is given in Figure 1 of Pecoraro et al., 2011). For each species, a fragment of about 1 kbp was amplified using standard PCRs with isolated genomic DNA as template. The primers are summarized in Table S1 (Supporting Information). The fragments were purified using preparative agarose gel electrophoresis and the AxyPrepDNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA). The DNA mass concentrations were determined photometrically, and the concentrations of DNA molecules were calculated using the molecular weights computed with ‘oligo calc’ (www.basic.northwestern.edu/biotools). For each standard fragment, a dilution series was generated and used for real time PCR analysis in parallel with the dilution series of the respective cell extract. The ‘analysis fragments’ were 300–400 bp, and exact sizes and primers are summarized in Table S1. The real time PCR analyses were performed as previously described (Breuert et al., 2006), but without glycerol addition. A negative control (no template control) was also included. By comparison of the Ct differences of the different dilutions, it was verified that the PCR was exponential at least up to the threshold DNA concentration used for the analysis (i.e. a 10-fold dilution corresponds to a Ct difference of about 3.32). The size of the analysis product and the absence of other products were verified using analytical agarose gel electrophoresis. A standard curve was generated and used to calculate the genome copy numbers present in the dilutions of the cell extract. Together with the known cell densities (see above), this number was used to calculate the genome copy number per cell. At least three independent experiments (biologic replicates) were performed for each species, and average values and standard deviations were calculated.

Spectroscopic method for ploidy determination

Dialyzed cytoplasmic extracts of Synechocystis PCC6803 (see above) were used to record spectra from 220 to 340 nm. The spectra had the typical shapes of nucleic acids spectra and E260/E280 quotients typical for pure nucleic acids. The cell densities (see above) and the absorption at 260 nm were used to calculate the genome copy numbers per cell using the following parameters: absorption of one equals a DNA concentration of 50 μg mL−1, the average molecular mass of one base pair is 660 g mol−1, and the Avogadro number. The best value for the genome size is less clear, the chromosome size is 3.57 Mbp, and the genome size including plasmids is 3.96 Mbp. The plasmid copy number is unknown and e.g. in Halobacterium salinarum, two plasmids have a copy number of five, whereas the genome has a copy number of 25 (Breuert et al., 2006). To take the unknown plasmid copy numbers into account, genome sizes of 3.96 Mbp (high plasmid copy number) and 3.65 Mbp (low plasmid copy number) were used to calculate the ploidy level of the chromosome.

It should be noted that in highly polyploid species, the absorbance of RNA is much lower than that of genomic DNA and can be neglected. A short calculation should demonstrate this point: E. coli cells growing with a doubling time of 100 min. contain about 7000 ribosomes per cell (Bremer & Dennis, 1996). If the same number is assumed for Synechocystis with a much longer doubling time, the cells would contain 3.2 × 107 nt ribosomal RNA, which makes up nearly 90% of cellular RNA. Fifty copies of a genome of 3.6 Mbp are equal to 3.6 × 108 nt. Therefore, under these conditions, DNA outnumbers RNA by more than a factor of 10.

Results and discussion

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Optimization of the real time PCR method

The real time PCR method for the quantification of genome copy numbers had been established for haloarchaea (Breuert et al., 2006), but, in the meantime, was also applied to methanogenic archaea and proteobacteria (Hildenbrand et al., 2011; Pecoraro et al., 2011). It has been validated against several independent methods, i.e. quantitative Southern blotting (Breuert et al., 2006), DNA isolation, and spectroscopic quantification (Hildenbrand et al., 2011), and the wealth of results published for E. coli, which was obtained by radioactive labeling and Fluorescence Activated Cell Sorter (FACS) analyses (Pecoraro et al., 2011). In each case, the results of the real time PCR method were in excellent agreement with the respective independent method.

To give a short overview, genomic DNA was used as a template in a conventional PCR reaction to amplify a fragment of about 1 kbp. A dilution series of this fragment was prepared and used for real time PCR analysis. A fragment of about 300 bp, internal to the standard fragment, was amplified. The results were used to generate a standard curve. To determine the genome copy number, cells were lysed and a dilution series of the resulting cell extract was analyzed using real time PCR in parallel to the standards. The results allowed calculating the number of genome copies in the cell extract and, in combination with the cell density of the culture, the ploidy level.

The following points have to be optimized for every new species under investigation and were optimized for the three species of cyanobacteria used in this study: (1) the cell density has to be quantified with a very low variance, (2) it has to be verified that culture growth is highly reproducible, (3) the method of cell disruption has to be about 100% effective yet leaving the genomic DNA intact, and (4) the real time PCR has to be truly exponential.

For cyanobacteria, the method for cell disruption turned out to be the most critical point. Several standard methods (sonification, enzymatic murein digestion, ‘normal shaking’ with glass beads) could not be used, either because the efficiency of cell lysis was too low or because damage of the genomic DNA was too high. Shaking the cells in a Speedmill with 0.1 mm glass beads led to satisfactory results, lysis efficiency was higher than 90%, and the genomic DNA was only slightly damaged (fragment sizes from 4 kbp to >20 kbp, data not shown). The amount of beads and shaking time were optimized for every species. To exemplify the results, Fig. S1 (Supporting Information) shows one typical example of a real time PCR analysis (Fig. S1a), a standard curve (Fig. S1b), a melting point analysis, and an analytical agarose gel of the analysis fragments (Fig. S1c, d). At least three independent cultures were analyzed (and each culture was analyzed at least in triplicates), and average values and standard deviations (SD) were calculated.

Synechococcus elongatusPCC 7942 and SynechococcusWH7803 are oligoploid

Synechococcus elongatus PCC 7942 grew with a doubling time of 24 h. An average growth curve of three cultures is shown in Fig. S2. The results of genome quantification of three independent cultures are summarized in Table 1. At an OD750 nm of 0.6, S. elongatus contained about four genome copies per cell and thus the species is oligoploid. This is termed ‘exponential phase’, although growth of the cultures was not truly exponential, but the OD750 nm of 0.6 was prior to the onset of the linear growth phase (compare Fig. S2). This value is in accordance with the previously published value of 3–5 genome copies that was obtained using FACS analysis (Mori et al., 1996). Stationary phase cells also contained 3–4 genomes per cell. Therefore, in S. elongatus, the ploidy level is not growth phase-regulated, in contrast to many other species.

Table 1. Genome copy numbers of Synechococcus elongatusPCC 7942 and Synechococcus sp. WH 7803
CultureOD750 nmExponential phaseStationary phase
Genome copiesAverage ± SDGenome copiesAverage ± SD
S. elongatus PCC 7942
10.64.2 3.1 
20.63.64.0 ± 0.33.54.0 ± 1.3
30.64.2 5.5 
Synechococcus sp. WH 7803
10.62.6   
20.64.33.6 ± 0.9  
30.63.8   

The results of genome quantification for Synechococcus WH7803 are also summarized in Table 1. This species also contained between three and four genome copies at an OD750 nm of 0.6 and during stationary phase, and is thus oligoploid. Again, this is in accordance with an earlier study that applied FACS analysis for genome copy number determination and found 2–4 copies per cell (Binder & Chisholm, 1990). Taken together, the freshwater as well as the salt water species were found to be oligoploid, irrespective of the applied method for quantification (based either on one specific site of the genome (this study) or the average DNA content), growth in continuous light (this study) or growth in light–dark cycles (Mori et al., 1996), and the growth phase.

SynchocystisPCC 6803 is highly polyploid

First, the motile Synechocystic PCC 6803 wild-type strain was analyzed. An average growth curve of three independent cultures is shown in Fig. S3. The results of genome copy number determination are summarized in Table 2. The doubling time at the cell harvest in linear growth phase (OD750 nm = 0.6) was around 20 h. Synechocystis PCC 6803 turned out to be highly polyploid, and it contained nearly 60 genomes per cell, both in linear and in stationary growth phase. As this value is very high and in fact higher than any value published until now for any cyanobacterial species, the genome copy number in stationary phase cells was also determined using an independent method, namely spectroscopic determination of the DNA concentration. The average values of 57.9 (if the plasmid copy number would be low) and 53.3 (if the plasmid copy number would be high) genomes per cell were in excellent agreement with the real time PCR result, and thus underscored that Synechocystis PCC 6803 is highly polyploid.

Table 2. Genome copy numbers of two SynechocystisPCC 6803 strains
No.Exponential phaseLinear phaseStationary phase
OD750 nmGenome copiesAverage ± SDOD750 nmGenome copiesAverage ± SDGenome copiesAverage ± SD
Motile wild-type strain
10.1210.3 0.666.1 52.9 
20.1218.3218.0 ± 7.60.646.758.0 ± 10.161.057.6 ± 4.2
30.1225.5 0.661.2 58.9 
GT wild-type strain
10.1144.5 0.641.1 42.1 
20.1136.1142.2 ± 5.30.646.347.2 ± 6.645.243.3 ± 2.8
30.1146.0 0.654.1 39.7 

An earlier study had also shown that this species is polyploid, but the reported value of 12 genome copies per cell for the ‘Kazusa’ wild-type of Synechocystis PCC 6803 (Labarre et al., 1989) is much lower than the value determined in this study. The reason for the discrepancy is not obvious, as in the previous study also the lysis efficiency was quantified, genome size was underestimated by only 32%, and the colorimetric assay for DNA quantification probably cannot be that wrong. The same medium was used, and a similar doubling time of 15–20 h was reported. Therefore, it might be that both reports are correct and that the ploidy level of various strains of the species Synechocystis PCC 6803 are different.

To test this hypothesis, another wild-type strain of Synechocystis PCC 6803 was used, i.e. the so-called GT wild-type. This strain was chosen because genotypes segregate much faster in the GT strain than in the motile strain in the course of chromosomal mutant construction (Annegret Wilde, personal communication). Average growth curves of three independent cultures are shown in Fig. S4, and again, cells in linear growth phase and in stationary phase were analyzed. The results are also shown in Table 2. The GT wild-type was also highly polyploid; however, the genome copy number was with 42 genome copies nearly 30% lower than that of the motile wild-type, verifying that different strains of PCC 6803 vary in their ploidy level.

Notably, the 12 genome copies reported for the ‘Kazusa’ strain (Labarre et al., 1989) are much lower compared with the 42 and 58 genome copies of the two other wild-type strains analyzed in this study. Three explanations appear possible: (1) the ‘Kazusa’ strain highly deviates from the other two strains, (2) the genome copy number changed during the last 20 years of cultivation in the laboratory and today the ploidy level of the ‘Kazusa’ strain is higher than in 1989, (3) strains cultivated for long times under identical names in different laboratories accumulated different mutations, including mutations that affect the ploidy level, and thus ‘identical’ strains have different ploidy levels in different laboratories.

The species Synechocystis PCC 6803 was isolated from freshwater in California more than 40 years ago (Stanier et al., 1971). Several mutations are known that occurred during its further ‘evolution in the laboratory’. The sequenced ‘Kazusa’ strain contains insertion elements at four places of the genome that were devoid of an insertion element in the original isolate (Okamoto et al., 1999). In addition, the sequenced ‘Kazusa’ strain contains a frameshift mutation in the gene encoding a protein kinase that is not present in other strains (Kamei et al., 2001). It will be interesting to unravel how different strains differ in their ploidy level. An in-depth analysis including several samples of each of the three wild-type strains obtained from different laboratories around the world will be needed to clarify the situation. In any case, all Synechocystis PCC 6803 strains analyzed until now are polyploid, and we could show that the ploidy levels of different strains vary. For experiments that are sensitive to the ploidy level, this should be taken into account and the ploidy level of the strain under investigation should be quantified.

Anonymous reviewers of the first version of this article pointed out that we only analyzed the linear and the stationary growth phase, and that an analysis of exponentially growing cells would also be desirable. Therefore, again three independent cultures of both strains were grown and were harvested during exponential growth at an OD750 nm of 0.1. The results are included in Table 3. Surprisingly, it turned out that the GT wild-type contained 142 genome copies per cells and the motile wild-type contained 218 genome copies per cell, much higher values than in linear and stationary growth phase. Several bacterial and archaeal species exhibit growth phase-dependent regulation of their ploidy levels and have higher genome copy numbers in exponential phase than in stationary phase (Hildenbrand et al., 2011; Pecoraro et al., 2011). Synechocystis PCC 6803 adds to this list, but is extraordinary in that the genome copy number is already down-regulated in linear growth phase.

Table 3. Overview of cyanobacterial species with experimentally determined ploidy levels and selected parameters
SpeciesGrowth temperature (°C)Doubling time (h)Genome size (Mbp)Average genome copy No.PloidyReferences
  1. stat.ph., stationary phase; LDC, light–dark cycles.

  2. a

    Exponential phase/linear phase/stationary phase.

  3. b

    Longer doubling times correspond to lower genome copy numbers.

  4. c

    Calculated from relative fluorescence and genome size of WH7803.

Anabaena cylindrica3018.525PolyploidSimon (1977)
Anabaena variabilis307.15–8OligoploidSimon (1980)
10 Microcystis strains20stat.ph.1–10OligoploidKurmayer & Kutzenberger (2003)
Anabaena sp. PCC 7120287.28.2OligoploidHu et al. (2007)
Prochlorococcus1.7MonoploidVaulot et al. (1995)
Synechococcus elongatus PCC 794228242.83.9/3.3bOligoploidThis study
S. elongatus PCC 79423011, LDC2.83–5OligoploidMori et al. (1996)
Synechococcus sp. PCC 6301385 to >502.72–6 to >1–2cOligoploidBinder & Chisholm (1990)
Synechococcus sp. WH 7803282.43.6OligoploidThis study
Synechococcus sp. WH 7803252.42–4OligoploidBinder & Chisholm (1995)
Synechococcus sp. WH 780525152.6a1MonoploidBinder & Chisholm (1995)
Synechococcus sp. WH 810125173.2a1MonoploidArmbrust et al. (1989)
Synechococcus sp. WH810325222.7a1–2MonoploidBinder & Chisholm (1995)
Synechocystis sp. PCC 6803 (motile)28203.6218/58/58bPolyploidThis study
Synechocystis sp. PCC 6803 (GT)28203.6142/47/43bPolyploidThis study
Synechocystis sp. PCC 6803 (‘Kazusa’)3015–203.612PolyploidLabarre et al. (1989)

The genome copy numbers of 218 and 142 in exponentially growing cells of the two Synechocystis strains are considerably higher than the 120 genome copies per cell that have been reported for Buchnera, a symbiotic bacterium with a reduced genome size (Komaki & Ishikawa, 1999). To our knowledge, a higher value has been reported only for Epulopiscium sp. that contains tens of thousands of genome copies (Mendell et al., 2008). However, Epulopiscium sp. is a giant bacterium exhibiting cell lengths in excess of 600 μm. Therefore, Synechocystis PCC 6803 has the highest ploidy level of any ‘normal’ sized prokaryote. However, it is unclear whether such high ploidy levels also exist in natural habitats, or whether this is an artifact of decades of cultivation in the laboratory. Synechocystis PCC 6803 was isolated 40 years ago and has been cultivated in the laboratory since then (Stanier et al., 1971). Therefore, an in-depth analysis (see above) should also include fresh isolates of Synechocystis PCC6803 as well as samples from different culture collections that had been kept frozen since their submission.

Ploidy in cyanobacteria

A literature search was performed to identify (hopefully) all cyanobacterial species with experimentally determined ploidy levels. Table 3 summarizes the results together with selected features. Three species are polyploid and contain at least 10 genome copies. They belong to different genera and grow either as single cells or as filaments. More than ten species are oligoploid and contain between three and nine genome copies. Again, among them are unicellular and filamentous species of several genera. Four species are monoploid, and thus monoploidy is not the rule, but an exception in cyanobacteria. The ploidy level is highly variable in cyanobacteria similar to the proteobacteria (Pecoraro et al., 2011). One genus can harbor monoploid and oligoploid species (Synechococcus) or oligoploid and polyploid species (Anabaena). There is no obvious correlation between the number of genome copies and any of the listed features, i.e. genome size, growth temperature, and doubling time. Various evolutionary advantages of oligo- and polyploidy for prokaryotes exist. As has been extensively studied with D. radiodurans, one of the advantages is resistance against double strand breaks that can be induced by X-ray irradiation (an artificial situation) and desiccation (regularly occurring in natural habitats). In fact, it could be shown that the resistance of polyploid Synechocystis PCC 6803 against X-ray irradiation is much higher than that of the oligoploid Synechococcus PCC 7942 (Domain et al., 2004). Another advantage is that the cells can live without a stringent control of equal chromosome segregation, and for Synechocystis PCC 6803 and Nostoc sp. PCC 7120, it has indeed been shown that the amount of DNA in the two newborn daughter cells after cell division is not always identical, but can vary (Hu et al., 2007; Schneider et al., 2007). An additional advantage is gene redundancy, which opens the possibility that under unfavorable conditions, mutations are induced in some genome copies, whereas the wildtype information is retained in others. It has indeed been shown that heterozygous cells of S. elongatus PCC 7942 and of Synechocystis PCC 6803 can be selected, at least under laboratory conditions (Labarre et al., 1989; Spence et al., 2004; Takahama et al., 2004; Nodop et al., 2008). Heterozygous strains have also been selected of two halophilic and methanogenic archaea, Haloferax volcanii and Methanococcus maripaludis. In both cases, it was shown that in the absence of selection gene conversion leads to the equalization of genomes and reappearance of homozygous cells (Hildenbrand et al., 2011; Lange et al., 2011). By analogy, we predict that gene conversion also operates in oligo- and polyploid species of cyanobacteria. The higher efficiency of gene replacement with linear DNA compared with circular DNA in Synechocystis PCC 6803 indicates that this is really the case (Labarre et al., 1989).

Acknowledgements

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

This work was supported by grant So264/16-1 of the German Research Council (Deutsche Forschungsgemeinschaft). We thank Annegret Wilde (University of Giessen, Germany) for the motile and the GT Synechocystis PCC 6803 strains, Wolfgang R. Hess for S. elongatus PCC 7942 and Synechococcus sp. WH7803, and both for very valuable advice concerning growth of cyanobacteria. We thank Enrico Schleiff for the possibility to grow cyanobacterial cultures in his light incubator. We are grateful to two reviewers who were patient with us as non-experts of cyanobacteria, and gave us very good suggestions and literature references.

References

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information

Supporting Information

  1. Top of page
  2. Abstract
  3. Introduction
  4. Materials and methods
  5. Results and discussion
  6. Acknowledgements
  7. References
  8. Supporting Information
FilenameFormatSizeDescription
fml2368-sup-0001-FigureS1.epsimage/eps413KFig. S1. Selected results to exemplify the real time PCR method for genome copy number quantification.
fml2368-sup-0002-FigureS2.epsimage/eps80KFig. S2. Average growth curve of Synechococcus PCC 7942.
fml2368-sup-0003-FigureS3.epsimage/eps80KFig. S3. Average growth curve of motile Synechocystis PCC 6803.
fml2368-sup-0004-FigureS4.epsimage/eps79KFig. S4. Average growth curve of the GT strain of Synechocystis PCC 6803.
fml2368-sup-0005-TableS1.docWord document21KTable S1. Oligonucleotides and PCR fragments.

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