Regulated ploidy of Bacillus subtilis and three new isolates of Bacillus and Paenibacillus

Regulated ploidy of Bacillus subtilis and three new isolates of Bacillus and Paenibacillus Abstract Bacteria were long assumed to be monoploid, maintaining one copy of a circular chromosome. In recent years it became obvious that the majority of species in several phylogenetic groups of prokaryotes are oligoploid or polyploid. The present study aimed at investigating the ploidy in Gram-positive aerobic endospore-forming bacteria. First, the numbers of origins and termini of the widely used laboratory strain Bacillus subtilis 168 were quantified. The strain was found to be mero-oligoploid in exponential phase (5.9 origins, 1.2 termini) and to down-regulate the number of origins in stationary phase. After inoculation of fresh medium with stationary-phase cells the onset of replication preceded the onset of mass increase. For the analysis of the ploidy in fresh isolates, three strains were isolated from soil, which were found to belong to the genera of Bacillus and Paenibacillus. All three strains were found to be mero-oligoploid in exponential phase and exhibit a growth phase-dependent down-regulation of the ploidy level in stationary phase. Taken together, these results indicate that mero-oligoploidy as well as growth phase-dependent copy number regulation might be widespread in and typical for Bacillus and related genera. Bacillus subtilis, Paenibacillus, chromosome copy number, mero-oligoploidy, Firmicutes, real-time PCR INTRODUCTION Polyploidy, the presence of multiple copies of the genome, is common in eukaryotes such as ciliates, fish, amphibians and plants (Soltis et al.2015; Schmid, Evans and Bogart 2015). In contrast to eukaryotes, prokaryotes have long been assumed to be monoploid and contain one copy of a circular chromosome. This assumption originated from the best-studied Gram-negative bacterium, Escherichia coli, which contains one copy of the chromosome when its generation time is longer than the time required for chromosomal replication and segregation (Skarstad, Steen and Boye 1983). However, under optimal laboratory conditions the generation time of E. coli is smaller than the replication time, and a new round of replication is initiated before termination of the previous round. The number of replication origins per cell is then larger than the number of termini and the cell becomes mero-oligoploid (Bremer and Dennis 1996; Pecoraro et al.2011). Therefore, E. coli is not a truly monoploid species. However, several truly monoploid prokaryotic species exist that contain one copy of the chromosome irrespective of the growth rate, e.g. Caulobacter crescentus and Wolinella succinogenes (Pecoraro et al.2011). In contrast, many prokaryotic species in several phylogenetic groups have been shown to be oligoploid or polyploid (Soppa 2015). The fraction of oligo-/polyploid species is high in halophilic archaea, methanogenic archaea, cyanobacteria and proteobacteria (Breuert et al.2006; Soppa 2011; Hildenbrand et al.2011; Griese, Lange and Soppa 2011; Pecoraro et al.2011). An intensely studied example is the extreme radiation-resistant bacterium Deinococcus radiodurans, which is extremely resistant to DNA-shattering treatments such as ionizing radiation or desiccation and can regenerate a functional genome from hundreds of chromosomal fragments (Hansen 1978; Bentchikou et al.2010). In contrast to these groups, information about the ploidy distribution in Gram-positive bacteria has been sparse. Genome copy numbers have been experimentally determined for Lactococcus lactis and for Bacillus subtilis. Lactococcus lactis was found to have two copies of the chromosome when it was grown very slowly, which were replicated into four chromosomes during the C period of the cell cycle. Therefore, this species is diploid without overlapping chromosomal replication cycles (Michelsen et al.2010). For B. subtilis several experimental approaches have shown that the ploidy level depends on the growth rate and that it is monoploid during very slow growth and mero-oligoploid during fast growth, similar to Escherichia coli. For example, tagging the replication origin with the green fluorescent protein yielded one fluorescent spot before and two spots after replication during slow growth, but two or four spots during fast growth (Webb et al.1998). However, recently it was reported that newborn cells contain two origins regardless of growth rate, even if the cells had a doubling time of 98 min (Wang, Llopis and Rudner 2014). This challenged the view that B. subtilis becomes monoploid during slow growth. Quantifying the number of replication origins per cell by blocking initiation, incubation to allow run-off of replication, and subsequent analysis of the DNA content of individual cells by flow cytometry revealed that the cells had two origins and four origins (Kadoya et al.2002) or four origins and eight origins (Moriya et al.2009). Quantification of the average genome content by fluorescence microscopy and by a chemical method revealed that cells growing with a doubling time of 73 min contained about 1.5 genomes, while cells growing with a doubling time of 30 min contained 3.2 genomes (Sharpe et al.1998). These results motivated us to choose B. subtilis as the first Gram-positive species for the application of another method for the quantification of genome copy numbers, i.e. real-time PCR. The numbers of origins and termini of B. subtilis were quantified, and the effect of growth phase on the ploidy level was analyzed. In addition, stationary phase cells were used to inoculate fresh medium, and the onset of replication and growth was analyzed. Furthermore, three new spore-forming aerobic strains were freshly isolated from soil, and they were integrated into 16S rRNA trees. Their ploidy levels were also determined in exponential and in stationary phase. MATERIALS AND METHODS Bacterial species, media and growth conditions Bacillus subtilis 168 (DSM strain No. 23778) was obtained from Prof. Dr Karl-Dieter Entian (Goethe-University, Frankfurt, Germany). It was grown in a complex medium that is recommended by the German Culture Collection (DSMZ; www.dsmz.de), i.e. medium No. 1: 0.5% (w/v) peptone, 0.3% (w/v) meat extract and 0.5% (w/v) NaCl. Thirty-milliliter cultures were grown in 100 ml Erlenmeyer flasks at 37°C with a rotating frequency of 200 rpm. Isolation and characterization of aerobic spore-forming bacteria For the isolation of new strains a soil sample was taken near the Biocentre of Goethe-University, Frankfurt, Germany. One cubic centimeter of soil was transferred to a 15 ml Falcon tube and thoroughly mixed with 10 ml of sterile water. A 1 ml volume of the suspension was transferred to a 1.5 ml Eppendorf cup and was heated for 10 min to 80°C to kill all vegetative cells. Serial dilutions in sterile water were prepared, plated on complex medium agar plates (1.2% (w/v) agar) and incubated at 37°C overnight. Several colonies were re-streaked to guarantee that colonies represented pure clones. Individual colonies were used to inoculate complex medium cultures. Exponentially growing and stationary-phase cells were analyzed microscopically. Three clones were chosen arbitrarily that seemed to represent different species based on colony and cell morphology. For sequencing part of the 16S rRNA gene 1 ml aliquots of the cultures were removed and cells were harvested by centrifugation. They were resuspended in 1 ml lysis buffer (10 mM Tris/HCl pH 7.2, 1 mM EDTA, 10 mg ml−1 lysozyme) and incubated for 30 min at 37°C. Silica beads A3B (Analytik Jena, Jena, Germany) weighing 1.15 g were added, and the cells were lysed by shaking three times for 40 s in a FastPrep (MP Biomedicals, Solon, OH, USA). The beads and cell debris were removed by centrifugation, and aliquots of the supernatants were used as templates in PCR reactions to amplify part of the respective 16S rRNA genes using the primers ‘16S1kin’ and ‘16S2kin’ (Table S1 in the online supplementary material). The resulting PCR fragments were sequenced from both ends using the above-mentioned primers, and the sequences were combined using Clone Manager (Scientific and Educational, Cary, NC, USA). Multiple sequence alignments of the new sequences and the 16S rRNA sequences of selected species of Bacillus and Paenibacillus, respectively, were generated using ClustalW (www.ebi.ac.uk). Phylogenetic trees were constructed using the program MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 (Kumar, Stecher and Tamura 2016). The maximum parsimony approach was used and 1000 bootstrap replications were performed. Sequencing part of a single-copy gene of the new isolates The real-time PCR method for ploidy determination requires the presence of sequence information. Of course, no sequence information apart from the 16S rRNA sequences was available for the new isolates. However, the 16S sequence could not be used because the copy numbers of the ribosomal RNA operons of the isolates were unknown, and many bacterial species contain multiple copies of the rRNA operon. Therefore, the aim was to generate sequence information of a single-copy protein-encoding gene. The sigL gene encoding the sigma factor 54 was chosen because sigL is universally conserved in Bacillus and is a single-copy gene in all analyzed species. The sigL sequences of 10 species of the genus Bacillus and 2 species of the genus Paenibacillus were retrieved from the database and a multiple sequence alignment was generated using ClustalW. Two highly conserved regions were chosen and degenerated oligonucleotides were designed (for sequences see Table S1 in the online supplementary material). The oligonucleotides were used for the amplification and sequencing of a sigL fragment of about 1 kb using standard PCRs, using the respective genomic DNAs of the three isolates as templates. Based on the sequences of the three sigL genes species-specific oligonucleotides were designed for the amplification of standard fragments and analysis fragments for the three new isolates (Table S1). Quantification of the ploidy levels was performed as described below. Growth curves and quantification of cell densities For the generation of growth curves cultures were grown in 30 ml of medium in 100 ml Klett flasks (37°C, 200 rpm). Growth was recorded using a Klett Colorimeter (diameter 14.25 mm). In each case, three biological replicates were performed. Average values of the optical densities and their standard deviations were calculated. The doubling time was determined by fitting a straight line to the half-logarithmic representation of the optical densities in exponential phase. All growth curves are shown in the online Supplementary material. Cell densities were determined microscopically using a Neubauer counting chamber to enable the calculation of the genome copy numbers per cell. Preparation of cell extracts Aliquots of about 3 × 108 cells were withdrawn from cultures in exponential or stationary phase, and cells were harvested by centrifugation (5 min, 13 000 rpm). Cell pellets were suspended in 190 μl lysis buffer (1.2% (v/v) Triton X-100, 20 mg ml−1 lysozyme, 2 mM EDTA and 20 mM Tris/HCl, pH 8.0). In the case of the new isolate I2, 50 U or 100 U of mutanolysin (M9901, Sigme-Aldrich, St. Louis, MO, USA) were added to suspensions of exponential-phase and stationary-phase cells, respectively. The cells were incubated for 30 min at 37°C and subsequently 10 μl Proteinase K (20 mg ml−1; Applichem, Darmstadt, Germany) and 300 μl lysis buffer (Buffer AL, Qiagen, Venlo, Netherlands) were added to the suspension, followed by a second incubation for 20 min at 65°C. Subsequently the samples were heated to 96°C for 5 min. Quantification of the cell densities before and after this treatment revealed that more than 98% of the cells had been lysed. Alternatively, cells were lysed using osmotic shock after protoplast preparation (Biedendieck et al.2010). Cell debris was pelleted by centrifugation (10 min, 13 000 rpm) and the integrity of the genomic DNA was verified by analytical agarose gel electrophoresis. Aliquots of the cell extracts were dialyzed on membrane filters against distilled water. Serial dilutions of cell extracts were generated and 5 μl aliquots were included as templates in real-time PCR analyses for quantification of genome copy numbers (see below). Quantification of ploidy levels using a real-time PCR method To determine genome copy numbers, a real-time PCR approach was applied (Breuert et al.2006). At first, fragments of ∼1 kb were amplified using standard PCRs with genomic DNA of B. subtilis and the three isolates I1, I2 and I3 as templates. The sequences of the oligonucleotides are included in Table S1 in the online supplementary material. The amplified genomic regions are summarized in Table S2 in the online supplementary material. The PCR fragments were purified by using preparative agarose gel electrophoresis and an AxyPrepDNA gel extraction kit (Axygen Biosciences, Union City, CA, USA). DNA concentrations were determined photometrically and the numbers of DNA molecules per volume were calculated with ‘oligo calc’ (www.basic.northwestern.edu/biotools) and the Avogadro number. For each standard fragment, a dilution series was generated and used for real-time PCR analysis in parallel with a dilution series of the respective cell extracts. The ‘analysis fragments’ were about 200–300 bp and exact sizes and genomic localizations (when possible) are summarized in Table S2. The real-time PCR analyses were performed as previously described (Breuert et al.2006; Pecoraro et al.2011). A standard curve was generated and used to calculate the genome copy numbers present in the dilutions of the cell extracts. In each case three biological replicates were performed. For each biological replicate four dilutions of the cytoplasmic extracts were analyzed in duplicates, therefore, the calculated average ploidy levels rest on 24 technical replicates. In combination with the cell densities of the three biological replicates, the numbers of genome copies per cell were calculated. RESULTS AND DISCUSSION Ploidy of Bacillus subtilis Bacillus subtilis was isolated more than 100 years ago and has been cultivated in the laboratory ever since. The strain B. subtilis 168 (DSM strain No. 23778) is widely used. For B. subtilis 168 as well as for the other species described in this study the method of cell lysis was optimized prior to the genome copy number quantification. The method had to fulfill the following three criteria: (i) more than 95% of all cells were lysed, (ii) the genomic DNA remained mainly intact and no fragments smaller than 20 kb were visible in analytical agarose gels, and (iii) the resulting cell extract did not inhibit exponential amplification during real-time PCR, which was verified by a ΔCt value of about 3.32 of serial 10-fold dilutions. Bacillus subtilis cultures were grown in complex medium, and the average growth curve of three independent cultures is shown in Fig. 1A. During exponential growth, the cultures had a doubling time of 24 min. The copy numbers of two genomic regions were quantified, which represent the intracellular concentration of the replication origin and the terminus, respectively (see Table 1). As expected, the average number of origins (5.9 ± 0.6) was found to be considerably higher than the average number of termini (1.2 ± 0.2), and thus B. subtilis is mero-oligoploid. The average value of 5.9 indicated that most cells contained 4 or 8 origins, respectively, in congruence with an earlier report based on a different method (Moriya et al.2009). The small average number of termini indicated that B. subtilis divides soon after replication is complete and has a very short D period (or G2 phase). These results are very similar to fast-growing E. coli cultures, which have been reported to contain an average number of 6.8 origins and 1.7 termini (Bremer and Dennis 1996; Pecoraro et al.2011). Therefore, both B. subtilis and E. coli are mero-oligoploid during fast growth. Figure 1. View largeDownload slide Growth curves of B. subtilis in complex medium. (A) Growth from exponential to stationary phase. (B) Growth curve of B. subtilis after inoculation of fresh medium with late-stationary-phase cells. Three biological replicates were grown, and average values and standard deviations are shown. Arrows: time points at which the samples were taken. Filled triangles: time period used to calculate the generation time. Figure 1. View largeDownload slide Growth curves of B. subtilis in complex medium. (A) Growth from exponential to stationary phase. (B) Growth curve of B. subtilis after inoculation of fresh medium with late-stationary-phase cells. Three biological replicates were grown, and average values and standard deviations are shown. Arrows: time points at which the samples were taken. Filled triangles: time period used to calculate the generation time. Table 1. Origin and termini copy numbers in B. subtilis 168. Growth condition  Doubling time [min]  Average cell density [cells ml−1]  No. origins per cell  Standard deviation (origins)  No. termini per cell  Standard deviation (termini)  Complex medium  24  2.8 × 108  5.9  0.6  1.2  0.1    Stationary  1.6 × 109  2.8  0.7  1.3  0.4  After inoculation  –  4.1 × 107  1.4  0.4  0.9  0.4    –  6.5 × 107  3.0  0.4  1.7  0.3    27  4.0 × 108  4.5  0.6  1.9  0.3  Growth condition  Doubling time [min]  Average cell density [cells ml−1]  No. origins per cell  Standard deviation (origins)  No. termini per cell  Standard deviation (termini)  Complex medium  24  2.8 × 108  5.9  0.6  1.2  0.1    Stationary  1.6 × 109  2.8  0.7  1.3  0.4  After inoculation  –  4.1 × 107  1.4  0.4  0.9  0.4    –  6.5 × 107  3.0  0.4  1.7  0.3    27  4.0 × 108  4.5  0.6  1.9  0.3  View Large In stationary-phase cells of B. subtilis the average number of origins per cell was found to be considerably lower (2.8 ± 0.7) than in exponentially growing cultures (5.9 ± 0.6), whereas the numbers of termini per cell were nearly the same in exponential and stationary phase (1.2 ± 0.2 and 1.3 ± 0.4). Therefore, the origin copy number is growth phase regulated. These results are in accordance with earlier reports that showed that the DNA content of B. subtilis correlates with growth rate and faster growing cells contain more DNA than slower growing cells (Sharpe et al.1998; Webb et al.1998; Kadoya et al.2002; Moriya et al.2009). Notably, the number of origins was higher than two, in accordance with a recent study that showed that under normal growth conditions the cells never contain a single unreplicated chromosome (Wang, Llopis and Rudner 2014). Many of the earlier studies used fluorescence microscopy or flow cytometry to quantify the bulk DNA content, and thus the number of origins and termini were indirectly calculated and not quantified directly, as reported here. To our knowledge only one earlier study also used real-time PCR for direct quantification of origins and termini in B. subtilis (Defeu Soufo et al.2008). It was found that the origin/terminus ratio is growth rate dependent and is 4.1 during growth in complex medium at 37°C and 2.1 during growth in synthetic medium at 25°C. Not only in B. subtilis, but also in E. coli the genome copy number is growth rate regulated. Slowly growing E. coli cells with generation times of about 100 min contain average numbers of 2.5 origins and 1.2 termini (ratio 2.1), while cells growing with a generation time of about 25 min contain 6.8 origins and 1.7 termini (ratio 4.0) (Bremer and Dennis 1996; Pecoraro et al.2011). Onset of replication and growth in freshly inoculated cultures Because the origin copy number in stationary-phase cells is much lower than in exponentially growing cells, we aimed at characterizing when and how fast the number of origins increases during the onset of growth. To this end, fresh medium was inoculated with late-stationary-phase cells, and the onset of growth was monitored. Figure 1B shows that there was a long lag phase of about 2.5 h before the onset of exponential growth. The numbers of origins and termini per cell were quantified after 1.0, 2.0 and 3.5 h following inoculation (Table 1). One hour after inoculation the numbers of origins and termini were still very low (1.4 ± 0.4 and 0.9 ± 0.4). Remarkably, the number of origins was smaller than two, indicating that a fraction of the late-stationary-phase culture that was used for inoculation had become truly monoploid, in contrast to the culture described above that had only spent 10 h in stationary phase (Fig. 1A). It has also been reported for the cyanobacterium Synechocystis PCC 6803 that the copy number in stationary phase was not constant, but decreased during prolonged incubation (Zerulla, Ludt and Soppa 2016). While the number of origins was only 1.4 at 1 h after inoculation, only 1 h later the average number of origins had increased to 3.0 (±0.4), indicating that replication had started. At that time point growth had not yet started, revealing that in B. subtilis the onset of replication precedes the onset of mass increase. At 3.5 h after inoculation the cells were actively growing, and the average number of origins had further increased to 4.5 (±0.6), but it was still somewhat lower than in cells that had been exponentially growing for a long time (5.9 ± 0.6). To our knowledge such an experiment has been performed as yet only for one additional species, i.e. Synechococcus elongatus PCC 7942 (Watanabe et al.2015). The cells were pre-incubated in the dark for 18 h and then transferred into light conditions to enable the onset of photosynthetic growth. After the transfer, a lag phase of 18 h was observed before photosynthetic growth started. At the beginning of the light incubation the cells contained two to three genome copies. However, already during the lag phase the value increased to 4–10 genome copies (median: 6). Therefore, also in this Gram-negative cyanobacterium the onset of replication preceded the onset of mass increase, similar to our observation in the Gram-positive B. subtilis. Isolation and characterization of three new species of aerobic spore-forming bacteria Bacillus subtilis has been cultured in the laboratory for decades under optimal conditions, which might have led to mutations that influence the genome copy number. Therefore, we aimed at quantification of the ploidy levels of several freshly isolated species of Bacillus or related genera. The isolation of aerobic spore-forming bacteria from soil is straightforward, i.e. a soil sample is suspended in sterile water and heated to 80°C to simultaneously kill all vegetative cells and induce germination of spores. After isolation of pure clones and an initial morphological analysis of colonies and cells, three examples, most probably representing three different species, were chosen arbitrarily and further characterized. Table 2 summarizes some selected features of the three new isolates. A large part of the 16S rRNA gene of the three isolates was amplified and sequenced. A phylogenetic tree was constructed with the sequences of the three new isolates and 16S rRNA genes from 17 Gram-positive bacteria of four genera (data not shown). The tree revealed that isolates I1 and I2 belonged to the genus Bacillus and isolate I3 belonged to the genus Paenibacillus. To obtain a higher phylogenetic resolution, two additional trees were generated with species of these genera. Figure 2 shows a tree based on the 16S rRNA sequences of isolates I1 and I2, 45 species of the genus Bacillus, and three species of the genus Lactobacillus as an outgroup. Isolate I1 groups with B. simplex and B. megaterium, which nicely fits to its large cell size of up to 10 μm. Isolate I2 forms a group with B. thuringiensis, B. mycoides and B. wheihenstephanensis. Figure S1 in the online supplementary material shows a tree based on the 16S rRNA sequences of isolate I3, 68 species of Paenibacillus, and three species of the genus Lactobacillus as an outgroup. Isolate I3 forms a group with P. tautus, P. glucanolyticus and P. vortex. Thus the new isolates represent two diverse positions within the genus Bacillus and one position within the genus Paenibacillus and are excellently suited to analyze the ploidy levels of Gram-positive spore formers newly isolated from soil. Table 2. Cell characteristics of the three new isolates. Species  Growth phase  Cell shape  Length  Filamentous  Motility  Bacillus sp. I1  Exponential  Rods  2–5 μm  Short filaments  Yes  Bacillus sp. I1  Stationary  Rods  2–5 μm  Short filaments  Yes  Bacillus sp. I2  Exponential  Rods  5–10 μm  Filaments  Yes  Bacillus sp. I2  Stationary  Rods  2.5–5 μm  Short filaments  Yes  Paenibacillus sp. I3  Exponential  Rods  5 μm  Short filaments  Yes  Paenibacillus sp. I3  Stationary  Rods  2 μm  Short filaments  Yes  Species  Growth phase  Cell shape  Length  Filamentous  Motility  Bacillus sp. I1  Exponential  Rods  2–5 μm  Short filaments  Yes  Bacillus sp. I1  Stationary  Rods  2–5 μm  Short filaments  Yes  Bacillus sp. I2  Exponential  Rods  5–10 μm  Filaments  Yes  Bacillus sp. I2  Stationary  Rods  2.5–5 μm  Short filaments  Yes  Paenibacillus sp. I3  Exponential  Rods  5 μm  Short filaments  Yes  Paenibacillus sp. I3  Stationary  Rods  2 μm  Short filaments  Yes  View Large Figure 2. View largeDownload slide Phylogenetic tree of isolates I1 and I2, and 45 selected Bacillus species. The tree is based on 16S rRNA sequences. A maximum parsimony algorithm was used. Three species of Lactobacillus were used as an outgroup. One thousand bootstrap repetitions were performed, and the results are included as percent values. Figure 2. View largeDownload slide Phylogenetic tree of isolates I1 and I2, and 45 selected Bacillus species. The tree is based on 16S rRNA sequences. A maximum parsimony algorithm was used. Three species of Lactobacillus were used as an outgroup. One thousand bootstrap repetitions were performed, and the results are included as percent values. Ploidy levels of the three new isolates Obviously, the genome sequences of the three new isolates were unknown. However, for the application of the real-time PCR method for ploidy quantification sequence information is a prerequisite. The sequence of the 16S rRNA gene could not be used because many bacterial species contain more than one copy of the gene, and the copy number in the three new isolates was unknown. Therefore, a large part of the single-copy gene sigL, which encodes the sigma factor 54, was amplified and sequenced for all three isolates (see Materials and Methods). The sigL gene is highly conserved in Bacillus and ubiquitously present as a single-copy gene (Schmidt, Scott and Dyer 2011). The sigL sequences of the three new isolates enabled quantification of their chromosome copy. For each isolate three independent cultures were grown in complex medium. They had doubling times of 26 min (isolate I1, growth curve: Supplementary Fig. S2, available online), 24 min (isolate I2, growth curve: Supplementary Fig. S3, available online) and 48 min (isolate I3, growth curve: Supplementary Fig. S4, available online). The genome copy numbers were quantified for exponentially growing and stationary-phase cultures (compare arrows in growth curves). The results are summarized in Table 3. Isolate I1 had average genome copy numbers of 4.7 (±1.1) during exponential phase and 2.3 (±0.4) during stationary phase. The genome copy number of isolate I2 was also found to be growth phase regulated, the average genome copy numbers were 6.4 (±1.4) during exponential phase and 2.4 (±0.3) during stationary phase. Thus, the values are very similar although the two isolates are only distantly related within the genus Bacillus. The average values of the genome copy numbers of isolate I3 were 3.4 (±0.5) during exponential phase and 2.5 (±0.5) during stationary phase. Table 3. Ploidy levels of the three new isolates. Culture No.  Doubling time [min]  Cell density [cells ml−1]  No. of genomes per cell  Standard deviation  Isolate I1  26  1.4 × 108  4.7  1.1    Stationary  1.0 × 109  2.3  0.4  Isolate I2  24  2.9 × 108  6.4  1.4    Stationary  7.7 × 108  2.4  0.3  Isolate I3  48  4.8 × 108  3.4  0.5    Stationary  7.1 × 108  2.5  0.5  Culture No.  Doubling time [min]  Cell density [cells ml−1]  No. of genomes per cell  Standard deviation  Isolate I1  26  1.4 × 108  4.7  1.1    Stationary  1.0 × 109  2.3  0.4  Isolate I2  24  2.9 × 108  6.4  1.4    Stationary  7.7 × 108  2.4  0.3  Isolate I3  48  4.8 × 108  3.4  0.5    Stationary  7.1 × 108  2.5  0.5  View Large The doubling times of all three isolates (24–48 min) were smaller than the time for replication of chromosomes in B. subtilis and E. coli. Reported replication times for B. subtilis growing at 37°C are 73 min for cells with a generation time of 30 min, and 84 min for cells with a generation time of 40 min (Sharpe et al.1998). For E. coli growing at 37°C, replication times of 65 and 70 min, respectively, have been reported for cells with generation times of 24 and 40 min (Bremer and Dennis 1996). Therefore, the fast growth of the new isolates already indicated that they could not be monoploid, which is in accordance with the determined sigL copy numbers in exponentially growing cultures. The values of 3.4–6.4 could indicate that the three new isolates are oligoploid. However, the most parsimonious explanation is that the three new isolates are mero-oligoploid, like B. subtilis and E. coli. In B. subtilis, the sigL gene is located in a distance of 0.7 Mb from the origin and 1.4 Mb from the terminus. If the genomic localization of the sigL gene was conserved in the three new isolates and it was closer to the origin than to the terminus, the determined sigL copy numbers would be in full agreement with mero-oligoploidy. In any case, we could show that none of three new isolates of the genera Bacillus and Paenibacillus is monoploid. In addition, all three new isolates exhibited a growth phase-dependent down-regulation of the copy number in stationary phase, similar to B. subtilis and several other bacterial and archaeal species (Breuert et al.2006; Zerulla, Ludt and Soppa 2016). The three additional examples considerably increase the number of experimentally characterized species with this regulatory pattern. Overview of ploidy levels in different species of Gram-positive bacteria An overview of Gram-positive bacteria with experimentally determined ploidy levels is given in Table 4. Among seven species investigated thus far, only four strains of one species are truly monoploid. In contrast, most species are mero-oligoploid, and one species is hyperpolyploid. Therefore, it seems that mero-oligoploidy and polyploidy might be more widespread in Bacillus and related genera and that monoploidy is not typical. A similar large variance of ploidy levels and a low fraction of monoploid species has also been observed for other phylogenetic groups of bacteria, e.g. the cyanobacteria (Griese, Lange and Soppa 2011) and the proteobacteria (Pecoraro et al.2011). Table 4. Overview of ploidy levels in different species of Gram-positive bacteria. Species  Number of genomes per cell  Ploidy  References  Lactococcus lactis        Five strains  2–4  Diploid  Michelsen et al.2010  Four strains  1–2  Monoploid  Michelsen et al.2010  Corynebacterium glutamicum  2  Diploid  Böhm et al.2017  Bacillus subtilis  4–8  Mero-oligoploida  Webb et al.1998  Bacillus subtilis  4–8  Mero-oligoploida  Moriya et al.2009  Bacillus subtilis  6/3b  Mero-oligoploida  This study  Wild-type isolate I1 Bacillus sp.  5/2c  (Mero-)Oligoploid  This study  Wild-type isolate I2 Bacillus sp.  6/2c  (Mero-)Oligoploid  This study  Wild-type isolate I3 Paenibacillus sp.  3/3c  (Mero-)Oligoploid  This study  Epulopiscium spp.  10 000–100 000  Hyperpolyploid  Mendell et al.2008  Species  Number of genomes per cell  Ploidy  References  Lactococcus lactis        Five strains  2–4  Diploid  Michelsen et al.2010  Four strains  1–2  Monoploid  Michelsen et al.2010  Corynebacterium glutamicum  2  Diploid  Böhm et al.2017  Bacillus subtilis  4–8  Mero-oligoploida  Webb et al.1998  Bacillus subtilis  4–8  Mero-oligoploida  Moriya et al.2009  Bacillus subtilis  6/3b  Mero-oligoploida  This study  Wild-type isolate I1 Bacillus sp.  5/2c  (Mero-)Oligoploid  This study  Wild-type isolate I2 Bacillus sp.  6/2c  (Mero-)Oligoploid  This study  Wild-type isolate I3 Paenibacillus sp.  3/3c  (Mero-)Oligoploid  This study  Epulopiscium spp.  10 000–100 000  Hyperpolyploid  Mendell et al.2008  aDuring fast growth. bNumber of origins per cell in exponential and stationary phase in complex medium. cNumber of genomes per cell in exponential and stationary phase. View Large SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. Acknowledgements We thank Prof. Dr Karl-Dieter Entian for supplying the species Bacillus subtilis 168. FUNDING This work was supported by the German Research Council (Deutsche Forschungsgemeinschaft) through grant So264/24. Conflict of interest. None declared. REFERENCES Bentchikou E, Servant P, Coste G et al.   A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans. PLoS Genet  2010; 6: e1000774. Google Scholar CrossRef Search ADS PubMed  Biedendieck R, Bunk B, Fürch T et al.   Systems biology of recombinant protein production in Bacillus megaterium. In: Wittman C, Krull R (eds). Biosystems Engineering: Creating Superior Biocatalysts . Berlin, Heidelberg: Springer, 2010, 133– 61. Google Scholar CrossRef Search ADS   Böhm K, Meyer F, Rhomberg A et al.   Novel chromosome organization pattern in Actinomycetales—overlapping replication cycles combined with diploidy. MBio  2017; 8: e00511– 17. Google Scholar CrossRef Search ADS PubMed  Bremer H, Dennis PP. Modulation of chemical composition and other parameters of the cell by growth rate. In: Neidhardt FC (ed.). Escherichia coli and Salmonella . Washington: ASM Press, 1996, 1553– 69. Breuert S, Allers T, Spohn G et al.   Regulated polyploidy in halophilic archaea. PLoS One  2006; 1: e92. Google Scholar CrossRef Search ADS PubMed  Defeu Soufo C, Defeu Soufo HJ, Noirot-Gros M-F et al.   Cell-cycle-dependent spatial sequestration of the DnaA replicator initator protein in Bacillus subtilis. Dev Cell  2008; 15: 935– 41. Google Scholar CrossRef Search ADS PubMed  Griese M, Lange C, Soppa J. Ploidy in cyanobacteria. FEMS Microbiol Lett  2011; 323: 124– 31. Google Scholar CrossRef Search ADS PubMed  Hansen MT. Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans. J Bacteriol  1978; 134: 71– 5. Google Scholar PubMed  Hildenbrand C, Stock T, Lange C et al.   Genome copy numbers and gene conversion in methanogenic archaea. J Bacteriol  2011; 193: 734– 43. Google Scholar CrossRef Search ADS PubMed  Kadoya R, Hassan AK, Kasahara Y et al.   Two separate DNA sequences within oriC participate in accurate chromosome segregation in Bacillus subtilis. Mol Microbiol  2002; 45: 73– 87. Google Scholar CrossRef Search ADS PubMed  Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol  2016; 33: 1870– 4. Google Scholar CrossRef Search ADS PubMed  Mendell JE, Clements KD, Choat JH et al.   Extreme polyploidy in a large bacterium. Proc Natl Acad Sci USA  2008; 105: 6730– 4. Google Scholar CrossRef Search ADS PubMed  Michelsen O, Hansen FG, Albrechtsen B et al.   The MG1363 and IL1403 laboratory strains of Lactococcus lactis and several dairy strains are diploid. J Bacteriol  2010; 192: 1058– 65. Google Scholar CrossRef Search ADS PubMed  Moriya S, Kawai Y, Kaji S et al.   Effects of oriC relocation on control of replication initiation in Bacillus subtilis. Microbiology  2009; 155: 3070– 82. Google Scholar CrossRef Search ADS PubMed  Pecoraro V, Zerulla K, Lange C et al.   Quantification of ploidy in proteobacteria revealed the existence of monoploid, (mero-)oligoploid and polyploid species. PLoS One  2011; 6: e16392. Google Scholar CrossRef Search ADS PubMed  Schmid M, Evans BJ, Bogart JP. Polyploidy in amphibia. Cytogenet Genome Res  2015; 145: 315– 330. Google Scholar CrossRef Search ADS PubMed  Schmidt TR, Scott EJ, Dyer DW. Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group. BMC Genomics  2011; 12: 430. Google Scholar CrossRef Search ADS PubMed  Sharpe ME, Hauser PM, Sharpe RG et al. Bacillus subtilis cell cycle as studied by fluorescence microscopy: constancy of cell length at initiation of DNA replication and evidence for active nucleoid partitioning. J Bacteriol  1998; 180: 547– 55. Google Scholar PubMed  Skarstad K, Steen HB, Boye E. Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry. J Bacteriol  1983; 154: 656– 62. Google Scholar PubMed  Soltis PS, Marchant DG, Van de Peer Y et al.   Ploidy and genome evolution in plants. Curr Opin Genet Dev  2015; 35: 119– 25. Google Scholar CrossRef Search ADS PubMed  Soppa J. Ploidy and gene conversion in Archaea. Biochem Soc Trans  2011; 39: 150– 4. Google Scholar CrossRef Search ADS PubMed  Soppa J. Polyploidy in archaea and bacteria: about desiccation resistance, giant cell size, long-term survival, enforcement by a eukaryotic host and additional aspects. J Mol Microbiol Biotech  2015; 24: 409– 19. Google Scholar CrossRef Search ADS   Wang X, Llopis PM, Rudner DZ. Bacillus subtilis chromosome organization oscillates between two distinct patterns. Proc Natl Acad Sci U S A  2014; 111: 12877– 82. Google Scholar CrossRef Search ADS PubMed  Watanabe S, Ohbayashi R, Kanesaki Y et al.   Intensive replication and metabolism during the lag phase in cyanobacteria. PLoS One  2015; 10: e0136800. Google Scholar CrossRef Search ADS PubMed  Webb CD, Graumann PL, Kahana JA et al.   Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis. Mol Microbiol  1998; 28: 883– 92. Google Scholar CrossRef Search ADS PubMed  Zerulla K, Ludt K, Soppa J. The ploidy level of Synechocystis sp. PCC6803 is highly variable and is influenced by growth phase and by chemical and physical external parameters. Microbiology  2016; 162: 730– 9. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png FEMS Microbiology Letters Oxford University Press

Regulated ploidy of Bacillus subtilis and three new isolates of Bacillus and Paenibacillus

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Abstract

Abstract Bacteria were long assumed to be monoploid, maintaining one copy of a circular chromosome. In recent years it became obvious that the majority of species in several phylogenetic groups of prokaryotes are oligoploid or polyploid. The present study aimed at investigating the ploidy in Gram-positive aerobic endospore-forming bacteria. First, the numbers of origins and termini of the widely used laboratory strain Bacillus subtilis 168 were quantified. The strain was found to be mero-oligoploid in exponential phase (5.9 origins, 1.2 termini) and to down-regulate the number of origins in stationary phase. After inoculation of fresh medium with stationary-phase cells the onset of replication preceded the onset of mass increase. For the analysis of the ploidy in fresh isolates, three strains were isolated from soil, which were found to belong to the genera of Bacillus and Paenibacillus. All three strains were found to be mero-oligoploid in exponential phase and exhibit a growth phase-dependent down-regulation of the ploidy level in stationary phase. Taken together, these results indicate that mero-oligoploidy as well as growth phase-dependent copy number regulation might be widespread in and typical for Bacillus and related genera. Bacillus subtilis, Paenibacillus, chromosome copy number, mero-oligoploidy, Firmicutes, real-time PCR INTRODUCTION Polyploidy, the presence of multiple copies of the genome, is common in eukaryotes such as ciliates, fish, amphibians and plants (Soltis et al.2015; Schmid, Evans and Bogart 2015). In contrast to eukaryotes, prokaryotes have long been assumed to be monoploid and contain one copy of a circular chromosome. This assumption originated from the best-studied Gram-negative bacterium, Escherichia coli, which contains one copy of the chromosome when its generation time is longer than the time required for chromosomal replication and segregation (Skarstad, Steen and Boye 1983). However, under optimal laboratory conditions the generation time of E. coli is smaller than the replication time, and a new round of replication is initiated before termination of the previous round. The number of replication origins per cell is then larger than the number of termini and the cell becomes mero-oligoploid (Bremer and Dennis 1996; Pecoraro et al.2011). Therefore, E. coli is not a truly monoploid species. However, several truly monoploid prokaryotic species exist that contain one copy of the chromosome irrespective of the growth rate, e.g. Caulobacter crescentus and Wolinella succinogenes (Pecoraro et al.2011). In contrast, many prokaryotic species in several phylogenetic groups have been shown to be oligoploid or polyploid (Soppa 2015). The fraction of oligo-/polyploid species is high in halophilic archaea, methanogenic archaea, cyanobacteria and proteobacteria (Breuert et al.2006; Soppa 2011; Hildenbrand et al.2011; Griese, Lange and Soppa 2011; Pecoraro et al.2011). An intensely studied example is the extreme radiation-resistant bacterium Deinococcus radiodurans, which is extremely resistant to DNA-shattering treatments such as ionizing radiation or desiccation and can regenerate a functional genome from hundreds of chromosomal fragments (Hansen 1978; Bentchikou et al.2010). In contrast to these groups, information about the ploidy distribution in Gram-positive bacteria has been sparse. Genome copy numbers have been experimentally determined for Lactococcus lactis and for Bacillus subtilis. Lactococcus lactis was found to have two copies of the chromosome when it was grown very slowly, which were replicated into four chromosomes during the C period of the cell cycle. Therefore, this species is diploid without overlapping chromosomal replication cycles (Michelsen et al.2010). For B. subtilis several experimental approaches have shown that the ploidy level depends on the growth rate and that it is monoploid during very slow growth and mero-oligoploid during fast growth, similar to Escherichia coli. For example, tagging the replication origin with the green fluorescent protein yielded one fluorescent spot before and two spots after replication during slow growth, but two or four spots during fast growth (Webb et al.1998). However, recently it was reported that newborn cells contain two origins regardless of growth rate, even if the cells had a doubling time of 98 min (Wang, Llopis and Rudner 2014). This challenged the view that B. subtilis becomes monoploid during slow growth. Quantifying the number of replication origins per cell by blocking initiation, incubation to allow run-off of replication, and subsequent analysis of the DNA content of individual cells by flow cytometry revealed that the cells had two origins and four origins (Kadoya et al.2002) or four origins and eight origins (Moriya et al.2009). Quantification of the average genome content by fluorescence microscopy and by a chemical method revealed that cells growing with a doubling time of 73 min contained about 1.5 genomes, while cells growing with a doubling time of 30 min contained 3.2 genomes (Sharpe et al.1998). These results motivated us to choose B. subtilis as the first Gram-positive species for the application of another method for the quantification of genome copy numbers, i.e. real-time PCR. The numbers of origins and termini of B. subtilis were quantified, and the effect of growth phase on the ploidy level was analyzed. In addition, stationary phase cells were used to inoculate fresh medium, and the onset of replication and growth was analyzed. Furthermore, three new spore-forming aerobic strains were freshly isolated from soil, and they were integrated into 16S rRNA trees. Their ploidy levels were also determined in exponential and in stationary phase. MATERIALS AND METHODS Bacterial species, media and growth conditions Bacillus subtilis 168 (DSM strain No. 23778) was obtained from Prof. Dr Karl-Dieter Entian (Goethe-University, Frankfurt, Germany). It was grown in a complex medium that is recommended by the German Culture Collection (DSMZ; www.dsmz.de), i.e. medium No. 1: 0.5% (w/v) peptone, 0.3% (w/v) meat extract and 0.5% (w/v) NaCl. Thirty-milliliter cultures were grown in 100 ml Erlenmeyer flasks at 37°C with a rotating frequency of 200 rpm. Isolation and characterization of aerobic spore-forming bacteria For the isolation of new strains a soil sample was taken near the Biocentre of Goethe-University, Frankfurt, Germany. One cubic centimeter of soil was transferred to a 15 ml Falcon tube and thoroughly mixed with 10 ml of sterile water. A 1 ml volume of the suspension was transferred to a 1.5 ml Eppendorf cup and was heated for 10 min to 80°C to kill all vegetative cells. Serial dilutions in sterile water were prepared, plated on complex medium agar plates (1.2% (w/v) agar) and incubated at 37°C overnight. Several colonies were re-streaked to guarantee that colonies represented pure clones. Individual colonies were used to inoculate complex medium cultures. Exponentially growing and stationary-phase cells were analyzed microscopically. Three clones were chosen arbitrarily that seemed to represent different species based on colony and cell morphology. For sequencing part of the 16S rRNA gene 1 ml aliquots of the cultures were removed and cells were harvested by centrifugation. They were resuspended in 1 ml lysis buffer (10 mM Tris/HCl pH 7.2, 1 mM EDTA, 10 mg ml−1 lysozyme) and incubated for 30 min at 37°C. Silica beads A3B (Analytik Jena, Jena, Germany) weighing 1.15 g were added, and the cells were lysed by shaking three times for 40 s in a FastPrep (MP Biomedicals, Solon, OH, USA). The beads and cell debris were removed by centrifugation, and aliquots of the supernatants were used as templates in PCR reactions to amplify part of the respective 16S rRNA genes using the primers ‘16S1kin’ and ‘16S2kin’ (Table S1 in the online supplementary material). The resulting PCR fragments were sequenced from both ends using the above-mentioned primers, and the sequences were combined using Clone Manager (Scientific and Educational, Cary, NC, USA). Multiple sequence alignments of the new sequences and the 16S rRNA sequences of selected species of Bacillus and Paenibacillus, respectively, were generated using ClustalW (www.ebi.ac.uk). Phylogenetic trees were constructed using the program MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 (Kumar, Stecher and Tamura 2016). The maximum parsimony approach was used and 1000 bootstrap replications were performed. Sequencing part of a single-copy gene of the new isolates The real-time PCR method for ploidy determination requires the presence of sequence information. Of course, no sequence information apart from the 16S rRNA sequences was available for the new isolates. However, the 16S sequence could not be used because the copy numbers of the ribosomal RNA operons of the isolates were unknown, and many bacterial species contain multiple copies of the rRNA operon. Therefore, the aim was to generate sequence information of a single-copy protein-encoding gene. The sigL gene encoding the sigma factor 54 was chosen because sigL is universally conserved in Bacillus and is a single-copy gene in all analyzed species. The sigL sequences of 10 species of the genus Bacillus and 2 species of the genus Paenibacillus were retrieved from the database and a multiple sequence alignment was generated using ClustalW. Two highly conserved regions were chosen and degenerated oligonucleotides were designed (for sequences see Table S1 in the online supplementary material). The oligonucleotides were used for the amplification and sequencing of a sigL fragment of about 1 kb using standard PCRs, using the respective genomic DNAs of the three isolates as templates. Based on the sequences of the three sigL genes species-specific oligonucleotides were designed for the amplification of standard fragments and analysis fragments for the three new isolates (Table S1). Quantification of the ploidy levels was performed as described below. Growth curves and quantification of cell densities For the generation of growth curves cultures were grown in 30 ml of medium in 100 ml Klett flasks (37°C, 200 rpm). Growth was recorded using a Klett Colorimeter (diameter 14.25 mm). In each case, three biological replicates were performed. Average values of the optical densities and their standard deviations were calculated. The doubling time was determined by fitting a straight line to the half-logarithmic representation of the optical densities in exponential phase. All growth curves are shown in the online Supplementary material. Cell densities were determined microscopically using a Neubauer counting chamber to enable the calculation of the genome copy numbers per cell. Preparation of cell extracts Aliquots of about 3 × 108 cells were withdrawn from cultures in exponential or stationary phase, and cells were harvested by centrifugation (5 min, 13 000 rpm). Cell pellets were suspended in 190 μl lysis buffer (1.2% (v/v) Triton X-100, 20 mg ml−1 lysozyme, 2 mM EDTA and 20 mM Tris/HCl, pH 8.0). In the case of the new isolate I2, 50 U or 100 U of mutanolysin (M9901, Sigme-Aldrich, St. Louis, MO, USA) were added to suspensions of exponential-phase and stationary-phase cells, respectively. The cells were incubated for 30 min at 37°C and subsequently 10 μl Proteinase K (20 mg ml−1; Applichem, Darmstadt, Germany) and 300 μl lysis buffer (Buffer AL, Qiagen, Venlo, Netherlands) were added to the suspension, followed by a second incubation for 20 min at 65°C. Subsequently the samples were heated to 96°C for 5 min. Quantification of the cell densities before and after this treatment revealed that more than 98% of the cells had been lysed. Alternatively, cells were lysed using osmotic shock after protoplast preparation (Biedendieck et al.2010). Cell debris was pelleted by centrifugation (10 min, 13 000 rpm) and the integrity of the genomic DNA was verified by analytical agarose gel electrophoresis. Aliquots of the cell extracts were dialyzed on membrane filters against distilled water. Serial dilutions of cell extracts were generated and 5 μl aliquots were included as templates in real-time PCR analyses for quantification of genome copy numbers (see below). Quantification of ploidy levels using a real-time PCR method To determine genome copy numbers, a real-time PCR approach was applied (Breuert et al.2006). At first, fragments of ∼1 kb were amplified using standard PCRs with genomic DNA of B. subtilis and the three isolates I1, I2 and I3 as templates. The sequences of the oligonucleotides are included in Table S1 in the online supplementary material. The amplified genomic regions are summarized in Table S2 in the online supplementary material. The PCR fragments were purified by using preparative agarose gel electrophoresis and an AxyPrepDNA gel extraction kit (Axygen Biosciences, Union City, CA, USA). DNA concentrations were determined photometrically and the numbers of DNA molecules per volume were calculated with ‘oligo calc’ (www.basic.northwestern.edu/biotools) and the Avogadro number. For each standard fragment, a dilution series was generated and used for real-time PCR analysis in parallel with a dilution series of the respective cell extracts. The ‘analysis fragments’ were about 200–300 bp and exact sizes and genomic localizations (when possible) are summarized in Table S2. The real-time PCR analyses were performed as previously described (Breuert et al.2006; Pecoraro et al.2011). A standard curve was generated and used to calculate the genome copy numbers present in the dilutions of the cell extracts. In each case three biological replicates were performed. For each biological replicate four dilutions of the cytoplasmic extracts were analyzed in duplicates, therefore, the calculated average ploidy levels rest on 24 technical replicates. In combination with the cell densities of the three biological replicates, the numbers of genome copies per cell were calculated. RESULTS AND DISCUSSION Ploidy of Bacillus subtilis Bacillus subtilis was isolated more than 100 years ago and has been cultivated in the laboratory ever since. The strain B. subtilis 168 (DSM strain No. 23778) is widely used. For B. subtilis 168 as well as for the other species described in this study the method of cell lysis was optimized prior to the genome copy number quantification. The method had to fulfill the following three criteria: (i) more than 95% of all cells were lysed, (ii) the genomic DNA remained mainly intact and no fragments smaller than 20 kb were visible in analytical agarose gels, and (iii) the resulting cell extract did not inhibit exponential amplification during real-time PCR, which was verified by a ΔCt value of about 3.32 of serial 10-fold dilutions. Bacillus subtilis cultures were grown in complex medium, and the average growth curve of three independent cultures is shown in Fig. 1A. During exponential growth, the cultures had a doubling time of 24 min. The copy numbers of two genomic regions were quantified, which represent the intracellular concentration of the replication origin and the terminus, respectively (see Table 1). As expected, the average number of origins (5.9 ± 0.6) was found to be considerably higher than the average number of termini (1.2 ± 0.2), and thus B. subtilis is mero-oligoploid. The average value of 5.9 indicated that most cells contained 4 or 8 origins, respectively, in congruence with an earlier report based on a different method (Moriya et al.2009). The small average number of termini indicated that B. subtilis divides soon after replication is complete and has a very short D period (or G2 phase). These results are very similar to fast-growing E. coli cultures, which have been reported to contain an average number of 6.8 origins and 1.7 termini (Bremer and Dennis 1996; Pecoraro et al.2011). Therefore, both B. subtilis and E. coli are mero-oligoploid during fast growth. Figure 1. View largeDownload slide Growth curves of B. subtilis in complex medium. (A) Growth from exponential to stationary phase. (B) Growth curve of B. subtilis after inoculation of fresh medium with late-stationary-phase cells. Three biological replicates were grown, and average values and standard deviations are shown. Arrows: time points at which the samples were taken. Filled triangles: time period used to calculate the generation time. Figure 1. View largeDownload slide Growth curves of B. subtilis in complex medium. (A) Growth from exponential to stationary phase. (B) Growth curve of B. subtilis after inoculation of fresh medium with late-stationary-phase cells. Three biological replicates were grown, and average values and standard deviations are shown. Arrows: time points at which the samples were taken. Filled triangles: time period used to calculate the generation time. Table 1. Origin and termini copy numbers in B. subtilis 168. Growth condition  Doubling time [min]  Average cell density [cells ml−1]  No. origins per cell  Standard deviation (origins)  No. termini per cell  Standard deviation (termini)  Complex medium  24  2.8 × 108  5.9  0.6  1.2  0.1    Stationary  1.6 × 109  2.8  0.7  1.3  0.4  After inoculation  –  4.1 × 107  1.4  0.4  0.9  0.4    –  6.5 × 107  3.0  0.4  1.7  0.3    27  4.0 × 108  4.5  0.6  1.9  0.3  Growth condition  Doubling time [min]  Average cell density [cells ml−1]  No. origins per cell  Standard deviation (origins)  No. termini per cell  Standard deviation (termini)  Complex medium  24  2.8 × 108  5.9  0.6  1.2  0.1    Stationary  1.6 × 109  2.8  0.7  1.3  0.4  After inoculation  –  4.1 × 107  1.4  0.4  0.9  0.4    –  6.5 × 107  3.0  0.4  1.7  0.3    27  4.0 × 108  4.5  0.6  1.9  0.3  View Large In stationary-phase cells of B. subtilis the average number of origins per cell was found to be considerably lower (2.8 ± 0.7) than in exponentially growing cultures (5.9 ± 0.6), whereas the numbers of termini per cell were nearly the same in exponential and stationary phase (1.2 ± 0.2 and 1.3 ± 0.4). Therefore, the origin copy number is growth phase regulated. These results are in accordance with earlier reports that showed that the DNA content of B. subtilis correlates with growth rate and faster growing cells contain more DNA than slower growing cells (Sharpe et al.1998; Webb et al.1998; Kadoya et al.2002; Moriya et al.2009). Notably, the number of origins was higher than two, in accordance with a recent study that showed that under normal growth conditions the cells never contain a single unreplicated chromosome (Wang, Llopis and Rudner 2014). Many of the earlier studies used fluorescence microscopy or flow cytometry to quantify the bulk DNA content, and thus the number of origins and termini were indirectly calculated and not quantified directly, as reported here. To our knowledge only one earlier study also used real-time PCR for direct quantification of origins and termini in B. subtilis (Defeu Soufo et al.2008). It was found that the origin/terminus ratio is growth rate dependent and is 4.1 during growth in complex medium at 37°C and 2.1 during growth in synthetic medium at 25°C. Not only in B. subtilis, but also in E. coli the genome copy number is growth rate regulated. Slowly growing E. coli cells with generation times of about 100 min contain average numbers of 2.5 origins and 1.2 termini (ratio 2.1), while cells growing with a generation time of about 25 min contain 6.8 origins and 1.7 termini (ratio 4.0) (Bremer and Dennis 1996; Pecoraro et al.2011). Onset of replication and growth in freshly inoculated cultures Because the origin copy number in stationary-phase cells is much lower than in exponentially growing cells, we aimed at characterizing when and how fast the number of origins increases during the onset of growth. To this end, fresh medium was inoculated with late-stationary-phase cells, and the onset of growth was monitored. Figure 1B shows that there was a long lag phase of about 2.5 h before the onset of exponential growth. The numbers of origins and termini per cell were quantified after 1.0, 2.0 and 3.5 h following inoculation (Table 1). One hour after inoculation the numbers of origins and termini were still very low (1.4 ± 0.4 and 0.9 ± 0.4). Remarkably, the number of origins was smaller than two, indicating that a fraction of the late-stationary-phase culture that was used for inoculation had become truly monoploid, in contrast to the culture described above that had only spent 10 h in stationary phase (Fig. 1A). It has also been reported for the cyanobacterium Synechocystis PCC 6803 that the copy number in stationary phase was not constant, but decreased during prolonged incubation (Zerulla, Ludt and Soppa 2016). While the number of origins was only 1.4 at 1 h after inoculation, only 1 h later the average number of origins had increased to 3.0 (±0.4), indicating that replication had started. At that time point growth had not yet started, revealing that in B. subtilis the onset of replication precedes the onset of mass increase. At 3.5 h after inoculation the cells were actively growing, and the average number of origins had further increased to 4.5 (±0.6), but it was still somewhat lower than in cells that had been exponentially growing for a long time (5.9 ± 0.6). To our knowledge such an experiment has been performed as yet only for one additional species, i.e. Synechococcus elongatus PCC 7942 (Watanabe et al.2015). The cells were pre-incubated in the dark for 18 h and then transferred into light conditions to enable the onset of photosynthetic growth. After the transfer, a lag phase of 18 h was observed before photosynthetic growth started. At the beginning of the light incubation the cells contained two to three genome copies. However, already during the lag phase the value increased to 4–10 genome copies (median: 6). Therefore, also in this Gram-negative cyanobacterium the onset of replication preceded the onset of mass increase, similar to our observation in the Gram-positive B. subtilis. Isolation and characterization of three new species of aerobic spore-forming bacteria Bacillus subtilis has been cultured in the laboratory for decades under optimal conditions, which might have led to mutations that influence the genome copy number. Therefore, we aimed at quantification of the ploidy levels of several freshly isolated species of Bacillus or related genera. The isolation of aerobic spore-forming bacteria from soil is straightforward, i.e. a soil sample is suspended in sterile water and heated to 80°C to simultaneously kill all vegetative cells and induce germination of spores. After isolation of pure clones and an initial morphological analysis of colonies and cells, three examples, most probably representing three different species, were chosen arbitrarily and further characterized. Table 2 summarizes some selected features of the three new isolates. A large part of the 16S rRNA gene of the three isolates was amplified and sequenced. A phylogenetic tree was constructed with the sequences of the three new isolates and 16S rRNA genes from 17 Gram-positive bacteria of four genera (data not shown). The tree revealed that isolates I1 and I2 belonged to the genus Bacillus and isolate I3 belonged to the genus Paenibacillus. To obtain a higher phylogenetic resolution, two additional trees were generated with species of these genera. Figure 2 shows a tree based on the 16S rRNA sequences of isolates I1 and I2, 45 species of the genus Bacillus, and three species of the genus Lactobacillus as an outgroup. Isolate I1 groups with B. simplex and B. megaterium, which nicely fits to its large cell size of up to 10 μm. Isolate I2 forms a group with B. thuringiensis, B. mycoides and B. wheihenstephanensis. Figure S1 in the online supplementary material shows a tree based on the 16S rRNA sequences of isolate I3, 68 species of Paenibacillus, and three species of the genus Lactobacillus as an outgroup. Isolate I3 forms a group with P. tautus, P. glucanolyticus and P. vortex. Thus the new isolates represent two diverse positions within the genus Bacillus and one position within the genus Paenibacillus and are excellently suited to analyze the ploidy levels of Gram-positive spore formers newly isolated from soil. Table 2. Cell characteristics of the three new isolates. Species  Growth phase  Cell shape  Length  Filamentous  Motility  Bacillus sp. I1  Exponential  Rods  2–5 μm  Short filaments  Yes  Bacillus sp. I1  Stationary  Rods  2–5 μm  Short filaments  Yes  Bacillus sp. I2  Exponential  Rods  5–10 μm  Filaments  Yes  Bacillus sp. I2  Stationary  Rods  2.5–5 μm  Short filaments  Yes  Paenibacillus sp. I3  Exponential  Rods  5 μm  Short filaments  Yes  Paenibacillus sp. I3  Stationary  Rods  2 μm  Short filaments  Yes  Species  Growth phase  Cell shape  Length  Filamentous  Motility  Bacillus sp. I1  Exponential  Rods  2–5 μm  Short filaments  Yes  Bacillus sp. I1  Stationary  Rods  2–5 μm  Short filaments  Yes  Bacillus sp. I2  Exponential  Rods  5–10 μm  Filaments  Yes  Bacillus sp. I2  Stationary  Rods  2.5–5 μm  Short filaments  Yes  Paenibacillus sp. I3  Exponential  Rods  5 μm  Short filaments  Yes  Paenibacillus sp. I3  Stationary  Rods  2 μm  Short filaments  Yes  View Large Figure 2. View largeDownload slide Phylogenetic tree of isolates I1 and I2, and 45 selected Bacillus species. The tree is based on 16S rRNA sequences. A maximum parsimony algorithm was used. Three species of Lactobacillus were used as an outgroup. One thousand bootstrap repetitions were performed, and the results are included as percent values. Figure 2. View largeDownload slide Phylogenetic tree of isolates I1 and I2, and 45 selected Bacillus species. The tree is based on 16S rRNA sequences. A maximum parsimony algorithm was used. Three species of Lactobacillus were used as an outgroup. One thousand bootstrap repetitions were performed, and the results are included as percent values. Ploidy levels of the three new isolates Obviously, the genome sequences of the three new isolates were unknown. However, for the application of the real-time PCR method for ploidy quantification sequence information is a prerequisite. The sequence of the 16S rRNA gene could not be used because many bacterial species contain more than one copy of the gene, and the copy number in the three new isolates was unknown. Therefore, a large part of the single-copy gene sigL, which encodes the sigma factor 54, was amplified and sequenced for all three isolates (see Materials and Methods). The sigL gene is highly conserved in Bacillus and ubiquitously present as a single-copy gene (Schmidt, Scott and Dyer 2011). The sigL sequences of the three new isolates enabled quantification of their chromosome copy. For each isolate three independent cultures were grown in complex medium. They had doubling times of 26 min (isolate I1, growth curve: Supplementary Fig. S2, available online), 24 min (isolate I2, growth curve: Supplementary Fig. S3, available online) and 48 min (isolate I3, growth curve: Supplementary Fig. S4, available online). The genome copy numbers were quantified for exponentially growing and stationary-phase cultures (compare arrows in growth curves). The results are summarized in Table 3. Isolate I1 had average genome copy numbers of 4.7 (±1.1) during exponential phase and 2.3 (±0.4) during stationary phase. The genome copy number of isolate I2 was also found to be growth phase regulated, the average genome copy numbers were 6.4 (±1.4) during exponential phase and 2.4 (±0.3) during stationary phase. Thus, the values are very similar although the two isolates are only distantly related within the genus Bacillus. The average values of the genome copy numbers of isolate I3 were 3.4 (±0.5) during exponential phase and 2.5 (±0.5) during stationary phase. Table 3. Ploidy levels of the three new isolates. Culture No.  Doubling time [min]  Cell density [cells ml−1]  No. of genomes per cell  Standard deviation  Isolate I1  26  1.4 × 108  4.7  1.1    Stationary  1.0 × 109  2.3  0.4  Isolate I2  24  2.9 × 108  6.4  1.4    Stationary  7.7 × 108  2.4  0.3  Isolate I3  48  4.8 × 108  3.4  0.5    Stationary  7.1 × 108  2.5  0.5  Culture No.  Doubling time [min]  Cell density [cells ml−1]  No. of genomes per cell  Standard deviation  Isolate I1  26  1.4 × 108  4.7  1.1    Stationary  1.0 × 109  2.3  0.4  Isolate I2  24  2.9 × 108  6.4  1.4    Stationary  7.7 × 108  2.4  0.3  Isolate I3  48  4.8 × 108  3.4  0.5    Stationary  7.1 × 108  2.5  0.5  View Large The doubling times of all three isolates (24–48 min) were smaller than the time for replication of chromosomes in B. subtilis and E. coli. Reported replication times for B. subtilis growing at 37°C are 73 min for cells with a generation time of 30 min, and 84 min for cells with a generation time of 40 min (Sharpe et al.1998). For E. coli growing at 37°C, replication times of 65 and 70 min, respectively, have been reported for cells with generation times of 24 and 40 min (Bremer and Dennis 1996). Therefore, the fast growth of the new isolates already indicated that they could not be monoploid, which is in accordance with the determined sigL copy numbers in exponentially growing cultures. The values of 3.4–6.4 could indicate that the three new isolates are oligoploid. However, the most parsimonious explanation is that the three new isolates are mero-oligoploid, like B. subtilis and E. coli. In B. subtilis, the sigL gene is located in a distance of 0.7 Mb from the origin and 1.4 Mb from the terminus. If the genomic localization of the sigL gene was conserved in the three new isolates and it was closer to the origin than to the terminus, the determined sigL copy numbers would be in full agreement with mero-oligoploidy. In any case, we could show that none of three new isolates of the genera Bacillus and Paenibacillus is monoploid. In addition, all three new isolates exhibited a growth phase-dependent down-regulation of the copy number in stationary phase, similar to B. subtilis and several other bacterial and archaeal species (Breuert et al.2006; Zerulla, Ludt and Soppa 2016). The three additional examples considerably increase the number of experimentally characterized species with this regulatory pattern. Overview of ploidy levels in different species of Gram-positive bacteria An overview of Gram-positive bacteria with experimentally determined ploidy levels is given in Table 4. Among seven species investigated thus far, only four strains of one species are truly monoploid. In contrast, most species are mero-oligoploid, and one species is hyperpolyploid. Therefore, it seems that mero-oligoploidy and polyploidy might be more widespread in Bacillus and related genera and that monoploidy is not typical. A similar large variance of ploidy levels and a low fraction of monoploid species has also been observed for other phylogenetic groups of bacteria, e.g. the cyanobacteria (Griese, Lange and Soppa 2011) and the proteobacteria (Pecoraro et al.2011). Table 4. Overview of ploidy levels in different species of Gram-positive bacteria. Species  Number of genomes per cell  Ploidy  References  Lactococcus lactis        Five strains  2–4  Diploid  Michelsen et al.2010  Four strains  1–2  Monoploid  Michelsen et al.2010  Corynebacterium glutamicum  2  Diploid  Böhm et al.2017  Bacillus subtilis  4–8  Mero-oligoploida  Webb et al.1998  Bacillus subtilis  4–8  Mero-oligoploida  Moriya et al.2009  Bacillus subtilis  6/3b  Mero-oligoploida  This study  Wild-type isolate I1 Bacillus sp.  5/2c  (Mero-)Oligoploid  This study  Wild-type isolate I2 Bacillus sp.  6/2c  (Mero-)Oligoploid  This study  Wild-type isolate I3 Paenibacillus sp.  3/3c  (Mero-)Oligoploid  This study  Epulopiscium spp.  10 000–100 000  Hyperpolyploid  Mendell et al.2008  Species  Number of genomes per cell  Ploidy  References  Lactococcus lactis        Five strains  2–4  Diploid  Michelsen et al.2010  Four strains  1–2  Monoploid  Michelsen et al.2010  Corynebacterium glutamicum  2  Diploid  Böhm et al.2017  Bacillus subtilis  4–8  Mero-oligoploida  Webb et al.1998  Bacillus subtilis  4–8  Mero-oligoploida  Moriya et al.2009  Bacillus subtilis  6/3b  Mero-oligoploida  This study  Wild-type isolate I1 Bacillus sp.  5/2c  (Mero-)Oligoploid  This study  Wild-type isolate I2 Bacillus sp.  6/2c  (Mero-)Oligoploid  This study  Wild-type isolate I3 Paenibacillus sp.  3/3c  (Mero-)Oligoploid  This study  Epulopiscium spp.  10 000–100 000  Hyperpolyploid  Mendell et al.2008  aDuring fast growth. bNumber of origins per cell in exponential and stationary phase in complex medium. cNumber of genomes per cell in exponential and stationary phase. View Large SUPPLEMENTARY DATA Supplementary data are available at FEMSLE online. Acknowledgements We thank Prof. Dr Karl-Dieter Entian for supplying the species Bacillus subtilis 168. FUNDING This work was supported by the German Research Council (Deutsche Forschungsgemeinschaft) through grant So264/24. Conflict of interest. None declared. REFERENCES Bentchikou E, Servant P, Coste G et al.   A major role of the RecFOR pathway in DNA double-strand-break repair through ESDSA in Deinococcus radiodurans. PLoS Genet  2010; 6: e1000774. Google Scholar CrossRef Search ADS PubMed  Biedendieck R, Bunk B, Fürch T et al.   Systems biology of recombinant protein production in Bacillus megaterium. In: Wittman C, Krull R (eds). Biosystems Engineering: Creating Superior Biocatalysts . Berlin, Heidelberg: Springer, 2010, 133– 61. Google Scholar CrossRef Search ADS   Böhm K, Meyer F, Rhomberg A et al.   Novel chromosome organization pattern in Actinomycetales—overlapping replication cycles combined with diploidy. MBio  2017; 8: e00511– 17. Google Scholar CrossRef Search ADS PubMed  Bremer H, Dennis PP. Modulation of chemical composition and other parameters of the cell by growth rate. In: Neidhardt FC (ed.). Escherichia coli and Salmonella . Washington: ASM Press, 1996, 1553– 69. Breuert S, Allers T, Spohn G et al.   Regulated polyploidy in halophilic archaea. PLoS One  2006; 1: e92. Google Scholar CrossRef Search ADS PubMed  Defeu Soufo C, Defeu Soufo HJ, Noirot-Gros M-F et al.   Cell-cycle-dependent spatial sequestration of the DnaA replicator initator protein in Bacillus subtilis. Dev Cell  2008; 15: 935– 41. Google Scholar CrossRef Search ADS PubMed  Griese M, Lange C, Soppa J. Ploidy in cyanobacteria. FEMS Microbiol Lett  2011; 323: 124– 31. Google Scholar CrossRef Search ADS PubMed  Hansen MT. Multiplicity of genome equivalents in the radiation-resistant bacterium Micrococcus radiodurans. J Bacteriol  1978; 134: 71– 5. Google Scholar PubMed  Hildenbrand C, Stock T, Lange C et al.   Genome copy numbers and gene conversion in methanogenic archaea. J Bacteriol  2011; 193: 734– 43. Google Scholar CrossRef Search ADS PubMed  Kadoya R, Hassan AK, Kasahara Y et al.   Two separate DNA sequences within oriC participate in accurate chromosome segregation in Bacillus subtilis. Mol Microbiol  2002; 45: 73– 87. Google Scholar CrossRef Search ADS PubMed  Kumar S, Stecher G, Tamura K. MEGA7: Molecular Evolutionary Genetics Analysis version 7.0 for bigger datasets. Mol Biol Evol  2016; 33: 1870– 4. Google Scholar CrossRef Search ADS PubMed  Mendell JE, Clements KD, Choat JH et al.   Extreme polyploidy in a large bacterium. Proc Natl Acad Sci USA  2008; 105: 6730– 4. Google Scholar CrossRef Search ADS PubMed  Michelsen O, Hansen FG, Albrechtsen B et al.   The MG1363 and IL1403 laboratory strains of Lactococcus lactis and several dairy strains are diploid. J Bacteriol  2010; 192: 1058– 65. Google Scholar CrossRef Search ADS PubMed  Moriya S, Kawai Y, Kaji S et al.   Effects of oriC relocation on control of replication initiation in Bacillus subtilis. Microbiology  2009; 155: 3070– 82. Google Scholar CrossRef Search ADS PubMed  Pecoraro V, Zerulla K, Lange C et al.   Quantification of ploidy in proteobacteria revealed the existence of monoploid, (mero-)oligoploid and polyploid species. PLoS One  2011; 6: e16392. Google Scholar CrossRef Search ADS PubMed  Schmid M, Evans BJ, Bogart JP. Polyploidy in amphibia. Cytogenet Genome Res  2015; 145: 315– 330. Google Scholar CrossRef Search ADS PubMed  Schmidt TR, Scott EJ, Dyer DW. Whole-genome phylogenies of the family Bacillaceae and expansion of the sigma factor gene family in the Bacillus cereus species-group. BMC Genomics  2011; 12: 430. Google Scholar CrossRef Search ADS PubMed  Sharpe ME, Hauser PM, Sharpe RG et al. Bacillus subtilis cell cycle as studied by fluorescence microscopy: constancy of cell length at initiation of DNA replication and evidence for active nucleoid partitioning. J Bacteriol  1998; 180: 547– 55. Google Scholar PubMed  Skarstad K, Steen HB, Boye E. Cell cycle parameters of slowly growing Escherichia coli B/r studied by flow cytometry. J Bacteriol  1983; 154: 656– 62. Google Scholar PubMed  Soltis PS, Marchant DG, Van de Peer Y et al.   Ploidy and genome evolution in plants. Curr Opin Genet Dev  2015; 35: 119– 25. Google Scholar CrossRef Search ADS PubMed  Soppa J. Ploidy and gene conversion in Archaea. Biochem Soc Trans  2011; 39: 150– 4. Google Scholar CrossRef Search ADS PubMed  Soppa J. Polyploidy in archaea and bacteria: about desiccation resistance, giant cell size, long-term survival, enforcement by a eukaryotic host and additional aspects. J Mol Microbiol Biotech  2015; 24: 409– 19. Google Scholar CrossRef Search ADS   Wang X, Llopis PM, Rudner DZ. Bacillus subtilis chromosome organization oscillates between two distinct patterns. Proc Natl Acad Sci U S A  2014; 111: 12877– 82. Google Scholar CrossRef Search ADS PubMed  Watanabe S, Ohbayashi R, Kanesaki Y et al.   Intensive replication and metabolism during the lag phase in cyanobacteria. PLoS One  2015; 10: e0136800. Google Scholar CrossRef Search ADS PubMed  Webb CD, Graumann PL, Kahana JA et al.   Use of time-lapse microscopy to visualize rapid movement of the replication origin region of the chromosome during the cell cycle in Bacillus subtilis. Mol Microbiol  1998; 28: 883– 92. Google Scholar CrossRef Search ADS PubMed  Zerulla K, Ludt K, Soppa J. The ploidy level of Synechocystis sp. PCC6803 is highly variable and is influenced by growth phase and by chemical and physical external parameters. Microbiology  2016; 162: 730– 9. Google Scholar CrossRef Search ADS PubMed  © FEMS 2018. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com

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FEMS Microbiology LettersOxford University Press

Published: Feb 1, 2018

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