Haloferax volcanii uses extracellular DNA as a source for carbon, nitrogen, and phosphorous. However, it can also grow to a limited extend in the absence of added phosphorous, indicating that it contains an intracellular phosphate storage molecule. As Hfx. volcanii is polyploid, it was investigated whether DNA might be used as storage polymer, in addition to its role as genetic material. It could be verified that during phosphate starvation cells multiply by distributing as well as by degrading their chromosomes. In contrast, the number of ribosomes stayed constant, revealing that ribosomes are distributed to descendant cells, but not degraded. These results suggest that the phosphate of phosphate-containing biomolecules (other than DNA and RNA) originates from that stored in DNA, not in rRNA. Adding phosphate to chromosome depleted cells rapidly restores polyploidy. Quantification of desiccation survival of cells with different ploidy levels showed that under phosphate starvation Hfx. volcanii diminishes genetic advantages of polyploidy in favor of cell multiplication. The consequences of the usage of genomic DNA as phosphate storage polymer are discussed as well as the hypothesis that DNA might have initially evolved in evolution as a storage polymer, and the various genetic benefits evolved later.

Funding: This project has been supported by the German Research Council (DFG grant So264/16), the National Science Foundation, USA (0919290 and 0830024), the US-Israel Binational Science Foundation (award No. 2007043), and the NASA Astrobiology Program (Grant NNX12AD70G). The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2014 Zerulla et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Halophilic Archaea of the family Halobacteriaceae are polyploids with phenotypic traits consistent with polyploidy. Both species Hfx. volcanii and Halobacterium salinarum are demonstrated to contain more than 20 chromosome copies during exponential phase and 10 during stationary phase [4] . Hbt. salinarum has been shown to be very resistant to gamma radiation [11] , and Halorubrum chaoviator strain Halo-G survived the conditions of outer space for two weeks [12] , which would be unlikely if these species were monoploids. Furthermore, halobacteria in general experience homologous recombination and gene transfer from distant species [13] , [14] and Halorubrum populations exist in genetic equilibrium [15] . Haloarchaea produce heterozygous cells after fusion of membranes and cell walls [16] . This is even true for different species thus displaying an unusually low species barrier to homologous recombination [17] and thus can account for their genetic exchange partner promiscuity. These phenotypic characteristics of haloarchaea show that they make intensive use of various genetic advantages of polyploidy. However, here we show that nutrient availability determines ploidy level and that extracellular and intracellular genomic DNA is used as a storage polymer. Notably, it is also shown that Hfx. volcanii diminishes genetic advantages of polyploidy under conditions of phosphate starvation.

A few polyploid prokaryotic species and their probable selective advantage of polyploidy have been well characterized. For example, cells from the unusually large bacterium Epulospicium type B, whose dimensions make it visible to the naked eye, are estimated to contain 50,000–120,000 chromosome copies per cell, which are positively correlated with cytoplasmic volume [8] . Because of diffusion limitations, the extreme polyploidy of Epulospicium is thought to be necessary for efficient gene expression. Though interesting and biological relevant, this polyploidy system probably has evolved rather late in evolution because a giant cell size requires a cytoskeleton and advanced intracellular transport. Another example is the bacterium Deinococcus radiodurans, which survives high doses of ionizing radiation that generate hundreds of double strand breaks. Its survival strategy relies on polyploidy for performing interchromosomal recombination, which is necessary for repairing its fragmented DNA [9] , [10] . While X-ray irradiation is used to induce double strand breaks in the laboratory, the cause of double strand breaks and chromosome fragmentation in nature is desiccation. Polyploidy as a basis for the repair of scattered chromosomes probably evolved early, nevertheless, it requires the pre-existence of a sophisticated DNA repair system. In summary, nearly 10 putative evolutionary advantages that led to the development of polyploidy at different times in different prokaryotic lineages have been discussed [4] – [7] , most of which require the pre-existence of homologous recombination. Here we add an additional evolutionary advantage of polyploidy that does not require the pre-existence of homologous recombination, namely the usage of genomic DNA as a storage polymer. The experiments revealing that a prokaryotic species uses DNA as a storage polymer were performed with Haloferax volcanii, a halophilic archaeon.

The advantages of polyploidy that led to its development in evolution has long been discussed in the framework of eukaryotes, because prokaryotes were long thought to be typically monoploid (a single copy of the chromosome before replication), which is often erroneously termed “haploid”. Evolutionary explanations for organisms with homologous sets of chromosomes have long been linked to the invention of sexual reproduction [1] , and have been developed from mathematical modeling using population genetics principles and assumptions. Those analyses indicate that ploidy levels ≥2 n could be selectively advantageous by preventing the expression of deleterious recessive alleles [2] . Additional hypotheses are interconnected with high recombination rates [2] or cell size and r vs. K selection [3] . However, in recent years polyploidy has been demonstrated to be widespread in bacteria and archaea as well [4] – [7] , indicating that it is an ancient trait preceding eukaryotes, and that any explanation for the origin and maintenance of higher ploidy levels must address asexually reproducing prokaryotes.

Results

Intracellular storage capacities and growth on external genomic DNA The first aim of this study was to clarify whether Hfx. volcanii can use external (environmental) genomic DNA as a source of carbon (C), nitrogen (N), and/or phosphorous (P). Control cultures supplemented with all three nutrients in the form of glucose, ammonium chloride and potassium phosphate were compared to cultures in which each one of the substances, respectively, was omitted. In each case three independent cultures were grown, and average growth curves and their standard deviations are shown in Fig. 1. In the absence of externally added genomic DNA no growth occurred when C was omitted, indicating that Hfx. volcanii has no intracellular carbon storage (Fig. 1, curve –C). In contrast, considerable growth occurred when P was omitted, showing that Hfx. volcanii contains an intracellular phosphate storage pool. The growth yield was about 40% of the control culture grown in the presence of all three nutrients. Also the omission of ammonium chloride resulted in considerable growth with a growth yield of about 80% of the control culture. However, in preparation of future genetic experiments Hfx. volcanii strain H26 was used, which is auxotrophic for uracil. Therefore, uracil had to be supplemented, which might have been used as nitrogen source, and thus the experiment is uninformative about the absence or presence of an internal nitrogen storage pool. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 1. Hfx. volcanii uses external DNA as a nutrient source and contains internal P and N storages. Hfx. volcanii was grown in microtiter plates in synthetic medium with added carbon (C), nitrogen (N), and phosphate (P) as positive control (diaments). In additional cultures each one of the three nutrients was replaced with genomic DNA (dotted lines), i.e. C was replaced (squares), N was replaced (circles), and P was replaced (triangles). In further cultures each one of the respective nutrients was omitted without replacement (solid lines), i.e. C was omitted (squares), N was omitted (circle), and P was omitted (triangles). To verify that spill over did not occur, for each medium also non-inoculated controls (sterile controls) were performed (open symbols). In each case average values of three independent cultures and their standard deviations are shown. https://doi.org/10.1371/journal.pone.0094819.g001 The addition of external genomic DNA to cultures lacking any of the three nutrients in all three cases enhanced the growth yield, revealing that genomic DNA can be a source for C, N, and P for Hfx. volcanii (filled symbols and dotted lines in Fig. 1). The addition of genomic DNA to cultures lacking phosphate or ammonium resulted even in faster growth compared to the control culture grown in the presence of all three nutrients. Only the culture with genomic DNA instead of glucose as a C-source had a substantially lower growth rate, showing that genomic DNA is metabolized more slowly than glucose as a carbon source. These results revealed on the one hand that external (environmental) DNA can be used as a source for C, N, and P, and on the other hand showed that Hfx. volcanii must have intracellular storage capacities for P, but not for C. In the following experiments we concentrated on the usage of external genomic DNA as a source of P and the identity of the intracellular P storage polymer. To further confirm that high molecular weight genomic DNA was indeed the source of the phosphorous, and not potential impurities or contaminations, Hfx. volcanii was grown in the presence of DNA and the absence of any other supplemented P. As a control, non-inoculated cultures were incubated under identical conditions. Fig. 2A shows average OD 600 values of three independent cultures and their standard deviations. Notably, the OD 600 values of Fig. 2A cannot be compared to that of Fig. 1, because in this experiment cultures were grown in Erlenmeyer flasks and not in microtiter plates and thus path length, photometer, and +/−dilution prior to measurements differ in the two experiments. At the eight time points indicated in Fig. 2 aliquots were removed and the cells were pelleted by centrifugation. The DNA content of the supernatant was analyzed by analytical agarose gel electrophoresis (after dialysis to remove the high salt concentration of the medium). Fig. 2B shows one representative gel of the mock treated culture. It can be seen that the high molecular weight input genomic DNA is broken into small fragments, either by chemical hydrolysis or, more probable, by mechanical shearing forces due to the shaking with 250 rpm. The amount of DNA was quantified using the program ImageJ and the result is included in Fig. 2A (filled squares, dotted line). Within 21 hours the values dropped to 80% and then stayed constant throughout the remaining 120 hours of the experiment. Most probably the initial drop of 20% in integrated signal intensity is not due to a real loss of DNA, but to a broader distribution of the fragments in the gel compared to the full-size genomic DNA. Fig. 2C shows one representative gel of the DNA content in the supernatants of the inoculated culture. In contrast to the mock treated culture the amount of DNA steadily decreased and less than 10% of the input DNA was left after 142 hours. Taken together, these results clearly show that Hfx. volcanii can use external (environmental) genomic DNA as a source of phosphorous (Fig. 1 and 2) and also as a source of carbon and nitrogen (Fig. 1). PPT PowerPoint slide

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larger image TIFF original image Download: Figure 2. Hfx. volcanii consumes high molecular weight chromosomal DNA. Three Hfx. volcanii cultures were grown in synthetic medium with chromosomal DNA as sole source of phosphorous and a growth curve was recorded (solid line, squares). As negative controls three non-inoculated cultures were incubated under identical conditions (solid line, circles). At the indicated times the optical densities were recorded and aliquots were removed for the quantification of the DNA content. Average optical densities and their standard deviations are shown (solid lines). The cells were pelleted by centrifugation and the DNA content of the supernatants was analyzed by analytical agarose gel electrophoresis (compare B and C). The DNA concentration was quantified using ImageJ, and average values and their standard deviations are shown (dotted lines, circles for the mock-treated non-inoculated control, squares for the inoculated culture). B. The supernatants of the aliquots of non-inoculated negative control cultures were dialyzed to remove salts and analyzed by analytical agarose gel electrophoresis. One representative gel is shown. For comparison the input DNA (gDNA) and a size marker (1 kb plus) were included. C. The supernatants of the aliquots of cultures grown with genomic DNA as phosphate source were dialyzed to remove salts and analyzed by analytical agarose gel electrophoresis. One representative gel is shown. For comparison the input DNA (gDNA) and a size marker (1 kb plus) were included. https://doi.org/10.1371/journal.pone.0094819.g002

Genomic DNA is the intracellular storage polymer of phosphate For a further characterization of the growth of Hfx. volcanii in the absence of any externally added P source cultures were grown in the presence of two different phosphate concentrations (1 mM, and 10 mM) and in the absence of added P. The results are shown in Fig. 3. Growth with 1 mM and with 10 mM phosphate was identical, indicating that phosphate is not the limiting nutrient under these conditions. Again, considerable growth was observed in the absence of added P, indicating that the liberation of phosphate from the intracellular phosphate storage polymer is growth rate-limiting. The OD 600 at the start of the experiment was about 0.05. The sterile controls showed that the microtiter plates had and OD 600 of about 0.03 and thus the inoculum had an OD 600 of about 0.02. After 140 h growth in the absence of added P the cells had an OD 600 of about 0.17 (measured OD 600 of 0.2 minus the OD 600 of the sterile control, 0.03). This is an 8.5-fold increase in OD 600 , which would be equivalent to about three doublings in the absence of added phosphate if the light scatter of the cells would not change. Microscopic observation of the cells indicated that they had normal morphology and were of similar size. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 3. Comparison of growth with different phosphate concentrations and with DNA. Hfx. volcanii was grown in microtiter plates in synthetic medium in the absence of any added phosphate source (triangles) and in the presence of, respectively, 1 mM (standard concentration, solid circles) and 10 mM phosphate (diamonds). Non-inoculated sterile controls were also incubated (dotted lines). Growth was followed by measuring the optical density at 600 nm. Average values of three independent cultures and their standard deviations are shown. https://doi.org/10.1371/journal.pone.0094819.g003 These observations that Hfx. volcanii maintains an intracellular phosphorous storage and our previous results that Hfx. volcanii is highly polyploid and contains about 25–30 copies of the chromosome in exponential phase [4] led to the hypothesis that genomic DNA might be the intracellular phosphate storage polymer. To test our hypothesis we used Hfx. volcanii cells grown to exponential phase in complex medium as an inoculum for assessing growth in synthetic media supplemented with two different phosphate concentrations (1 mM, and 10 mM) and no added phosphate. Using quantitative PCR (qPCR), chromosome copy numbers were estimated for the inoculum (an estimate of the pre-growth condition) as well as cells grown to exponential phase and stationary phase without added phosphate (exponential phase: 9.4×107 cells/ml, stationary phase: 2.7×108 cells/ml) and with 1 mM and 10 mM phosphate supplementation (for both: exponential phase: 5.2×108 cells/ml, stationary phase: 1.3×109 cells/ml). During exponential growth, the phosphate concentration was found to influence the ploidy level, with 24 copies on average in cells grown with 10 mM phosphate, 19 copies in cells grown with 1 mM phosphate, and 14 in cells grown in the absence of an added source of phosphorous (Fig. 4). Stationary phase cells that were grown in the presence of added phosphate (10 and 1 mM) maintained approximately 13 chromosomal copies of their genome. However, in the absence of phosphate supplementation, stationary cells had on average reduced their genome copy number to two. This result showed that Hfx. volcanii indeed uses genomic DNA as a phosphate storage polymer and indicated that it diminishes the putative genetic and long-term advantages of polyploidy (e.g. DNA repair, desiccation resistance, long term survival) to enable short-term reproductive gains. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 4. Chromosomal copy numbers during and after growth with and without added phosphate. Hfx. volcanii was grown in synthetic medium in the presence of 10 mM and 1 mM phosphate and in the absence of phosphate, respectively. Aliquots were removed during mid-exponential growth phase and at stationary phase (compare text). An aliquot from the pre-culture used for inoculation was also included. Cells were harvested by centrifugation and the chromosome copy number was quantified using Real Time PCR. Three biological replicates were performed and average values and standard deviations are shown, from left to right 10 mM phosphate, 1 mM phosphate, and no externally added phosphate. https://doi.org/10.1371/journal.pone.0094819.g004 Polyploidy dependence upon nutrient availability was further substantiated when cells from P-starved stationary phase cultures that were depleted of extra chromosomes were amended with phosphate: within three hours the chromosome copy number more than tripled and within 24 hours they increased by greater than 10-fold to more than 40 copies per cell (Fig. 5). Thus phosphate-starved Hfx. volcanii cells take up phosphate very fast after re-addition and use it to re-establish the polyploid state, even with an overshoot phase with more than 40 chromosomal copies per cell. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 5. Chromosome copy numbers after re-addition of phosphate to starved cells. Stationary phase, phosphate-starved, chromosome-depleted cells were resuspended in medium containing 1 mM phosphate. At various times, as indicated, aliquots were removed and the chromosome copy number was determined using Real Time PCR. Three biological replicates were performed and average values and standard deviations are shown. https://doi.org/10.1371/journal.pone.0094819.g005 The genome sequence of Hfx. volcanii contains five genes that are annotated to encode polyphosphate kinases (HVO_0074, HVO_0837, HVO_1650, HVO_2363, HVO_2598), opening the possibility that Hfx. volcanii might also use polyphosphate as a phosphate-storage polymer, in addition to genomic DNA. To investigate this possibility, Hfx. volcanii was grown in the presence of added phosphate and a culture aliquot was removed during exponential growth. The cells were fixed and stained with DAPI to simultaneously detect genomic DNA as well as polyphosphate based on the differential wavelengths of fluorescence emission for these biopolymers [18]. Chromosomal DNA was readily observed in cells using this approach, in contrast to polyphosphate, which was not detected (data not shown). Therefore, at least under the conditions of the experiments of this study, Hfx. volcanii appears to use only genomic DNA as polymer for the storage of phosphate and not polyphosphate. However, it should be noted that even the detection of polyphosphate would not have disproven our observation that Hfx. volcanii uses genomic DNA as a phosphate storage polymer.

During phosphate starvation other phosphate containing biomolecules are produced from genomic DNA, not from rRNA The results showed that during growth under phosphate starvation Hfx. volcanii dramatically decreased its chromosome copy number from about 30 to only 2, suggesting that it uses genomic DNA as a phosphate storage polymer. Another possible source of phosphate might be ribosomal RNA. The numbers of ribosomes per cell are influenced by parameters like growth rate and it can vary widely, both in E. coli [19] and in Hfx. volcanii [20]. Therefore, for a better understanding of the phosphate balance of cells during phosphate starvation, also the number of ribosomes was quantified. Hfx. volcanii cultures were again grown in the absence of added P. The cell density was quantified and increased from 3.22×107 cells ml−1 to 2.70×108 cells ml−1. This is an 8.4-fold increase in cell number, which is in excellent agreement with the 8.5-fold increase in OD 600 observed in previous experiments (compare Fig. 3). The number of ribosomes prior to and after phosphate starvation was quantified using a previously described approach [20]. Cells of the preculture grown in complex medium contained 29250 ribosomes (SD 1290, n = 3), a number similar to the number of 26000 ribosomes per cell determined earlier [20]. Stationary phase cells after phosphate starvation contained on average 3290 ribosomes (SD 97, n = 3). This is an 8.8-fold reduction of the number of ribosomes per cell during phosphate starvation, a value that is very similar to the 8.4-fold increase in cell number during phosphate starvation. Together these results revealed that ribosomal RNA is neither source nor sink of phosphate during phosphate starvation, but that ribosomes are distributed among the daughter cells and that the phosphate content bound in rRNA is self-sufficient during phosphate starvation. Hfx. volcanii harbors not only the major chromosome, but also three additional small chromosomes and a very small plasmid. To enable a comprehensive comparison of the total amount of phosphate bound in rRNA and in DNA, three independent cultures were again grown in the absence of added phosphate and the numbers of four replicons were quantified prior to and after phosphate starvation (the replicon pHV2 is a very small plasmid that is not present in strain H26). The results are summarized in Table 1. As expected, the numbers of all replicons were severely reduced after phosphate starvation. It was revealed that a polyploid Haloferax cell growing exponentially in complex medium contains about 2.2×108 molecules of phosphate in its DNA. With approximately 4600 nucleotides per ribosome and 29250 ribosomes per cell, the estimated total amount of phosphate in rRNA is 1.2×108 molecules per cell. Thus in the polyploid Hfx. volcanii the amount of phosphate bound in DNA is about twice that bound in ribosomes. This is contrast to monoploid species, which contain more phosphate in rRNA than in genomic DNA. PPT PowerPoint slide

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larger image TIFF original image Download: Table 1. Phosphate content of the four Hfx. volcanii chromosomes before and after growth in the absence of phosphate. https://doi.org/10.1371/journal.pone.0094819.t001 Fig. 6 summarizes the balancing of phosphate during growth of Hfx. volcanii under phosphate starvation, which is based on the quantification of the numbers of cells, chromosomes and ribosomes. Taken together, the results revealed that the numbers of rRNA-bound phosphate molecules are identical prior to and after phosphate starvation, and thus ribosomal RNA is neither source nor sink of phosphate. In contrast, only about 2/3 of the phosphate that was DNA-bound prior to phosphate starvation was still found in chromosomes after starvation. This indicates that 1/3 of the chromosomes had been degraded and suggests that they were the source of intracellular phosphate for the production of other phosphate-containing biomolecules, e.g. phospholipids, phosphoproteins, phosphosugars, ATP, NADP+, etc. Thus it seems that the polyploid Hfx. volcanii uses chromosomal DNA as a phosphate storage polymer in two different ways: 1) cell division in the absence of replication is enabled by distribution of preexisting chromosomes to the daughter cells, and 2) chromosomal DNA is degraded to liberate phosphate needed for other biomolecules that do not have a storage pool in the cell. PPT PowerPoint slide

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larger image TIFF original image Download: Figure 6. Phosphate balance in cells prior to and after growth in the absence of external phosphate. A preculture in complex medium was grown to mid-exponential phase. Aliquots were harvested, washed, and used to inoculate synthetic medium lacking any added phosphate source. Aliquots were removed at the beginning of the experiment and after growth in the absence of phosphate ceased. The cell densities were quantified using a counting chamber, the genome copy numbers were quantified by Real Time PCR, and the numbers of ribosomes were quantified after RNA isolation and two DNase treatments as described in the text. The figure gives a schematic overview of the phosphate balance prior to and after growth during phosphate starvation. https://doi.org/10.1371/journal.pone.0094819.g006