Driving apart and segregating genomes in Archaea

Genome segregation is a fundamental biological process in organisms from all domains of life. How this stage of the cell cycle unfolds in Eukarya has been clearly defined and considerable progress has been made to unravel chromosome partition in Bacteria. The picture is still elusive in Archaea. The lineages of this domain exhibit different cell-cycle lifestyles and wide-ranging chromosome copy numbers, fluctuating from 1 up to 55. This plurality of patterns suggests that a variety of mechanisms might underpin disentangling and delivery of DNA molecules to daughter cells. Here I describe recent developments in archaeal genome maintenance, including investigations of novel genome segregation machines that point to unforeseen bacterial and eukaryotic connections

Intrinsic properties of the two replicative DNA polymerases of Pyrococcus abyssi in replicating abasic sites: possible role in DNA damage tolerance?

The definitive version is available at ww3.interscience.wiley.com. En libre-accès sur Archimer : http://archimer.ifremer.fr/doc/2008/publication-6113.pdfInternational audienceSpontaneous and induced abasic sites in hyperthermophiles DNA have long been suspected to occur at high frequency. Here, Pyrococcus abyssi was used as an attractive model to analyse the impact of such lesions onto the maintenance of genome integrity. We demonstrated that endogenous AP sites persist at a slightly higher level in P. abyssi genome compared with Escherichia coli. Then, the two replicative DNA polymerases, PabpolB and PabpolD, were characterized in presence of DNA containing abasic sites. Both Pabpols had abortive DNA synthesis upon encountering AP sites. Under running start conditions, PabpolB could incorporate in front of the damage and even replicate to the full-length oligonucleotides containing a specific AP site, but only when present at a molar excess. Conversely, bypassing activity of PabpolD was strictly inhibited. The tight regulation of nucleotide incorporation opposite the AP site was assigned to the efficiency of the proof-reading function, because exonuclease-deficient enzymes exhibited effective TLS. Steady-state kinetics reinforced that Pabpols are high-fidelity DNA polymerases onto undamaged DNA. Moreover, Pabpols preferentially inserted dAMP opposite an AP site, albeit inefficiently. While the template sequence of the oligonucleotides did not influence the nucleotide insertion, the DNA topology could impact on the progression of Pabpols. Our results are interpreted in terms of DNA damage tolerance

Massive multiplication of genome and ribosomes in dormant cells (akinetes) of Aphanizomenon ovalisporum (Cyanobacteria)

Author Posting. © The Author(s), 2011. This is the author's version of the work. It is posted here by permission of Nature Publishing Group for personal use, not for redistribution. The definitive version was published in The ISME Journal 6 (2012): 670–679, doi:10.1038/ismej.2011.128.Akinetes are dormancy cells commonly found among filamentous cyanobacteria, many of which are toxic and/or nuisance, bloom-forming species. Development of akinetes from vegetative cells is a process that involves morphological and biochemical modifications. Here we applied a single cell approach to quantify genome and ribosome content of akinetes and vegetative cells in Aphanizomenon ovalisporum (Cyanobacteria). Vegetative cells of A. ovalisporum were naturally polyploid and contained on average 8 genome copies per cell. However, the chromosomal content of akinetes increased up to 450 copies, with an average value of 119 genome copies per akinete, 15 fold higher that in vegetative cells. Based on fluorescence in situ hybridization with a probe targeting 16S rRNA and detection with confocal laser scanning microscopy we conclude that ribosomes accumulated in akinetes to a higher level than that found in vegetative cells. We further present evidence that this massive accumulation of nucleic acids in akinetes is likely supported by phosphate supplied from inorganic polyphosphate bodies that were abundantly present in vegetative cells, but notably absent from akinetes. These results are interpreted in the context of cellular investments for proliferation following long term dormancy, as the high nucleic acid content would provide the basis for extended survival, rapid resumption of metabolic activity and cell division upon germination.Supported by the Gruss Lipper Foundation research award (AS). This study was part of the Joint German-Israeli-Project (FKZ 02WT0985, WR803) funded by the German Ministry of Research and Technology (BMBF) and Israel Ministry of Science and Technology (MOST)

Mre11-Rad50 Promotes Rapid Repair of DNA Damage in the Polyploid Archaeon Haloferax volcanii by Restraining Homologous Recombination

Polyploidy is frequent in nature and is a hallmark of cancer cells, but little is known about the strategy of DNA repair in polyploid organisms. We have studied DNA repair in the polyploid archaeon Haloferax volcanii, which contains up to 20 genome copies. We have focused on the role of Mre11 and Rad50 proteins, which are found in all domains of life and which form a complex that binds to and coordinates the repair of DNA double-strand breaks (DSBs). Surprisingly, mre11 rad50 mutants are more resistant to DNA damage than the wild-type. However, wild-type cells recover faster from DNA damage, and pulsed-field gel electrophoresis shows that DNA double-strand breaks are repaired more slowly in mre11 rad50 mutants. Using a plasmid repair assay, we show that wild-type and mre11 rad50 cells use different strategies of DSB repair. In the wild-type, Mre11-Rad50 appears to prevent the repair of DSBs by homologous recombination (HR), allowing microhomology-mediated end-joining to act as the primary repair pathway. However, genetic analysis of recombination-defective radA mutants suggests that DNA repair in wild-type cells ultimately requires HR, therefore Mre11-Rad50 merely delays this mode of repair. In polyploid organisms, DSB repair by HR is potentially hazardous, since each DNA end will have multiple partners. We show that in the polyploid archaeon H. volcanii the repair of DSBs by HR is restrained by Mre11-Rad50. The unrestrained use of HR in mre11 rad50 mutants enhances cell survival but leads to slow recovery from DNA damage, presumably due to difficulties in the resolution of DNA repair intermediates. Our results suggest that recombination might be similarly repressed in other polyploid organisms and at repetitive sequences in haploid and diploid species

The evolutionary significance of polyploidy

Polyploidy, or the duplication of entire genomes, has been observed in prokaryotic and eukaryotic organisms, and in somatic and germ cells. The consequences of polyploidization are complex and variable, and they differ greatly between systems (clonal or non-clonal) and species, but the process has often been considered to be an evolutionary 'dead end'. Here, we review the accumulating evidence that correlates polyploidization with environmental change or stress, and that has led to an increased recognition of its short-term adaptive potential. In addition, we discuss how, once polyploidy has been established, the unique retention profile of duplicated genes following whole-genome duplication might explain key longer-term evolutionary transitions and a general increase in biological complexity

Regulated polyploidy in halophilic archaea.

Polyploidy is common in higher eukaryotes, especially in plants, but it is generally assumed that most prokaryotes contain a single copy of a circular chromosome and are therefore monoploid. We have used two independent methods to determine the genome copy number in halophilic archaea, 1) cell lysis in agarose blocks and Southern blot analysis, and 2) Real-Time quantitative PCR. Fast growing H. salinarum cells contain on average about 25 copies of the chromosome in exponential phase, and their ploidy is downregulated to 15 copies in early stationary phase. The chromosome copy number is identical in cultures with a twofold lower growth rate, in contrast to the results reported for several other prokaryotic species. Of three additional replicons of H. salinarum, two have a low copy number that is not growth-phase regulated, while one replicon even shows a higher degree of growth phase-dependent regulation than the main replicon. The genome copy number of H. volcanii is similarly high during exponential phase (on average 18 copies/cell), and it is also downregulated (to 10 copies) as the cells enter stationary phase. The variation of genome copy numbers in the population was addressed by fluorescence microscopy and by FACS analysis. These methods allowed us to verify the growth phase-dependent regulation of ploidy in H. salinarum, and they revealed that there is a wide variation in genome copy numbers in individual cells that is much larger in exponential than in stationary phase. Our results indicate that polyploidy might be more widespread in archaea (or even prokaryotes in general) than previously assumed. Moreover, the presence of so many genome copies in a prokaryote raises questions about the evolutionary significance of this strategy

Figure 4

<p>Three independent cultures were used to determine the copy numbers of the chromosome and three additional replicons, i.e. pHS1 to pHS3, using the Real Time PCR method. One of the growth curves, the average replicon copy number per cell and the standard deviations are shown.</p

Figure 1

<p>A. Overview of the method. A culture of known cell density is embedded in low melting point agarose (step 1), agarose blocks with a defined number of cells are prepared, the cells are lysed and protein is digested (step 2). The blocks are melted and a restriction enzyme (step 3) as well as an internal standard (step 4) are added. After overnight digestion, DNA fragments are size fractionated by electrophoresis and a Southern blot is performed (step 5). A 1 kbp genomic fragment near the replication origin and the 0.9 kbp internal standard are both visualized with a single probe. Multiple aliquots containing different standard concentration are used for quantitation. B. Quantitation of the genome copy number of exponential cells. After gel electrophoresis and southern blotting, a genomic fragment (upper band) and different concentrations of an internal standard (lower band) were visualized with the same probe (step 5 in A). C. Quantitation of the genome copy number of stationary phase cells. After gel electrophoresis and Southern blotting, a genomic fragment (upper band) and different concentrations of an internal standard (lower band) were visualized with the same probe (step 5 in A). D. An example of a standard curve generated after quantitation of the bands shown in B. and C. The horizontal and vertical lines show the usage of the standard curve to determine the genome copy number in the biological replicate No. 9 (see E.). E. Summary of the results of the independent cultures that were used to determine the genome copy number with the “agarose block method”. In the first five experiments, the standard curve ranged from 0.5 molecules/cell to 10 molecules/cell (as in B.). The signal of exponential phase cells was much higher than the highest signal of the standard curve and could not be quantitated. Therefore the standard curve was chosen to range from 1 molecules/cell to 40 molecules/cell (as in C.) in subsequent experiments.</p

Figure 2

<p>A. Overview of the method. In short, a defined number of cells was harvested and lysed (steps 1 and 2). Serial dilutions of the cell lysate (step 3) were used as templates in quantitative Real Time PCR assays (step 4). Quantitation was performed by comparison with an external and an internal standard curve (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0000092#s4" target="_blank">Materials and Methods</a>). B. Selected real time PCR results. The fluorescence intensity curves from four standard dilutions (solid lines) and three sample dilutions (broken lines) are shown. In both cases serial tenfold dilutions of the templates were used. Note the identical slope of all curves and the equidistance of the curves of a dilution series, which is very close to the theoretical offset of 3.32 cycles per tenfold dilution. In addition to the selected reactions shown here, each experiment included more standards and more sample dilutions (filling the gap between tenfold dilutions) as well as a sample dilutions including a dilutions series of the standard added as internal control of PCR efficiency. C. A standard curve including nine standard concentrations distributed over three orders of magnitude. D. Average genome copy number values of three independent cultures and their standard deviation. E. Growth phase-dependent regulation of ploidy of H. salinarum. E is a graphical representation of the results tabulated in D.</p