Quantification of ploidy in proteobacteria revealed the existence of monoploid, (mero-)oligoploid and polyploid species

Bacteria are generally assumed to be monoploid (haploid). This assumption is mainly based on generalization of the results obtained with the most intensely studied model bacterium, Escherichia coli (a gamma-proteobacterium), which is monoploid during very slow growth. However, several species of proteobacteria are oligo- or polyploid, respectively. To get a better overview of the distribution of ploidy levels, genome copy numbers were quantified in four species of three different groups of proteobacteria. A recently developed Real Time PCR approach, which had been used to determine the ploidy levels of halophilic archaea, was optimized for the quantification of genome copy numbers of bacteria. Slow-growing (doubling time 103 minutes) and fast-growing (doubling time 25 minutes) E. coli cultures were used as a positive control. The copy numbers of the origin and terminus region of the chromosome were determined and the results were in excellent agreement with published data. The approach was also used to determine the ploidy levels of Caulobacter crescentus (an alpha-proteobacterium) and Wolinella succinogenes (an epsilon-proteobacterium), both of which are monoploid. In contrast, Pseudomonas putida (a gamma-proteobacterium) contains 20 genome copies and is thus polyploid. A survey of the proteobacteria with experimentally-determined genome copy numbers revealed that only three to four of 11 species are monoploid and thus monoploidy is not typical for proteobacteria. The ploidy level is not conserved within the groups of proteobacteria, and there are no obvious correlations between the ploidy levels with other parameters like genome size, optimal growth temperature or mode of life

Halophilic archaea cultivated from surface sterilized middle-late eocene rock salt are polyploid

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DNA as a phosphate storage polymer and the alternative advantages of polyploidy for growth or survival

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

Effects of kasugamycin on the translatome of Escherichia coli

It is long known that Kasugamycin inhibits translation of canonical transcripts containing a 5’-UTR with a Shine Dalgarno (SD) motif, but not that of leaderless transcripts. To gain a global overview of the influence of Kasugamycin on translation efficiencies, the changes of the translatome of Escherichia coli induced by a 10 minutes Kasugamycin treatment were quantified. The effect of Kasugamycin differed widely, 102 transcripts were at least twofold more sensitive to Kasugamycin than average, and 137 transcripts were at least twofold more resistant, and there was a more than 100-fold difference between the most resistant and the most sensitive transcript. The 5’-ends of 19 transcripts were determined from treated and untreated cultures, but Kasugamycin resistance did neither correlate with the presence or absence of a SD motif, nor with differences in 5’-UTR lengths or GC content. RNA Structure Logos were generated for the 102 Kasugamycin-sensitive and for the 137 resistant transcripts. For both groups a short Shine Dalgarno (SD) motif was retrieved, but no specific motifs associated with resistance or sensitivity could be found. Notably, this was also true for the region -3 to -1 upstream of the start codon and the presence of an extended SD motif, which had been proposed to result in Kasugamycin resistance. Comparison of the translatome results with the database RegulonDB showed that the transcript with the highest resistance was leaderless, but no further leaderless transcripts were among the resistant transcripts. Unexpectedly, it was found that translational coupling might be a novel feature that is associated with Kasugamycin resistance. Taken together, Kasugamycin has a profound effect on translational efficiencies of E. coli transcripts, but the mechanism of action is different than previously described

<i>Hfx. volcanii</i> uses external DNA as a nutrient source and contains internal P and N storages.

<p><i>Hfx. volcanii</i> 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.</p

Desiccation resistances of cells of with different ploidy levels.

<p>Cultures were grown to mid-exponential phase in synthetic medium with casamino acids and 1 mM K₂HPO₄ to generate cells with 20 copies of the chromosome. For comparison, cultures were grown in synthetic medium in the absence of phosphate to generate cells with 2 copies of the chromosome. Both types of cells were exposed to a desiccation period of 12 days. Colony forming units (CFU) were quantified before and after desiccation, and the survival rates were calculated. Average results of three independent experiments and their standard deviations are shown. Left columns, prior to desiccation, right columns, after desiccation.</p

<i>Hfx. volcanii</i> consumes high molecular weight chromosomal DNA.

<p>Three <i>Hfx. volcanii</i> 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>B</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. <b>C</b>. 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.</p

Chromosome copy numbers after re-addition of phosphate to starved cells.

<p>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.</p

Phosphate content of the four <i>Hfx. volcanii</i> chromosomes before and after growth in the absence of phosphate.

<p>Phosphate content of the four <i>Hfx. volcanii</i> chromosomes before and after growth in the absence of phosphate.</p