The genome copy numbers of seven crenarchaeal species of four genera have been reported. All of them are monoploid and thus this seems to be a characteristic feature of Crenarchaeota. In stark contrast, none of six species representing six euryarchaeal genera is monoploid. Therefore Euryarchaea are typically oligoploid or polyploidy and their genome copy numbers are tightly regulated in response to growth phase and/or growth rate. A theoretical consideration called ‘Muller's ratchet’ predicts that asexually reproducing polyploid species should not be able to exist. An escape from Muller's ratchet would be a mechanism leading to the equalization of genome copies, such as gene conversion. Using two species of methanogenic and halophilic archaea, it was shown that heterozygous cells containing different genomes simultaneously can be selected, exemplifying gene redundancy as one possible evolutionary advantage of polyploidy. In both cases, the genomes were rapidly equalized in the absence of selection, showing that gene conversion operates at least in halophilic and methanogenic Euryarchaea.

Introduction

It is commonly assumed that prokaryotes have a single copy of a circular chromosome and are thus monoploid (haploid). This has been shown for, e.g., the widely used model species Bacillus subtilis and Escherichia coli, albeit in the latter species, it is only true for cells with a very long generation time and not for cultures grown under laboratory conditions in complex media. In addition to monoploid bacterial species, oligoploid and polyploid bacterial species have also been described. In fact more oligo-/poly-ploid than monoploid bacterial species are known, therefore it might be doubted that the common view that bacteria are typically monoploid is really true. The first quantification of the genome copy number in archaea was reported more than 10 years ago and revealed that two species of the genus Sulfolobus were monoploid [1]. However, it was reported previously that two species of halophilic archaea are polyploid [2], indicating that, similar to the domain of bacteria, in the domain of archaea, the ploidy level is also not uniform. To obtain an insight into typical genome copy numbers for archaeal species, an overview of the ploidy levels of all experimentally analysed archaeal species is given in the present paper. Furthermore, experimental evidence is reviewed which revealed that gene conversion occurs in polyploid archaeal species, leading to the equalization of genome copies.

Ploidy in Crenarchaeota

Currently the genome copy numbers of seven species of Crenarchaeota belonging to four different genera have been reported (see Table 1). All experiments were performed by the group of Rolf Bernander (University of Uppsala, Sweden) and in all cases the ploidy levels were determined using a FACS. The principle of the method is to stain the cells with a DNA-specific fluorescent dye and to quantify the fluorescence of single cells using a FACS. The genome copy number can be calculated by comparing the fluorescence with the fluorescence of another species with a known genome copy number and taking the genome sizes of both species into account. The advantages of the FACS method are (i) that single cells are analysed and thus the variance in the population can be addressed, and (ii) that cell-cycle characteristics can be derived. The disadvantages are (i) different replicons and different sites of the chromosome cannot be addressed separately, and (ii) the results rest on the comparison with another species and thus might be compromised if the efficiency of dye binding is not identical.

Table 1
Archaeal species and genome copy numbers in exponential and stationary growth phases
  Genome copy number   
Species Doubling time (h) Exponential phase Stationary phase Ploidy Reference 
Euryarchaeota      
Halobacterium cutirubrum 6.7 10.6 – Polyploid [18
Halobacterium cutirubrum 13.3 6.3 – Polyploid [18
Halobacterium salinarum 25 15 Polyploid [2
Halobacterium salinarum 25 15 Polyploid [2
Halobacterium salinarum (anaerobic) 25 15 Polyploid [2
Haloferax volcanii 17 10 Polyploid [2
Methanocaldococcus jannaschii 0.5 10–15 1–5 Polyploid [4
Methanococcus maripaludis 55 30 Polyploid [3
Methanosarcina acetivorans 18 16 Polyploid [3
Methanosarcina acetivorans 49 Oligoploid [3
Methanothermobacter thermoautotrophicus – 2* 1–2* Diploid [5
Crenarchaeota      
Acidianus hospitalis – 1–2 Monoploid [19
Aeropyrum pernix 3.3 1–2 Monoploid [19
Pyrobaculum aerophilum – 1–2 Monoploid [19
Pyrobaculum calidifontis 3.4 1–2 Monoploid [19
Sulfolobus acidocaldarius 3.5 1–2 Monoploid [1
Sulfolobus tokodai 1–2 Monoploid [19
Sulfolobus solfataricus 1–2 Monoploid [1
  Genome copy number   
Species Doubling time (h) Exponential phase Stationary phase Ploidy Reference 
Euryarchaeota      
Halobacterium cutirubrum 6.7 10.6 – Polyploid [18
Halobacterium cutirubrum 13.3 6.3 – Polyploid [18
Halobacterium salinarum 25 15 Polyploid [2
Halobacterium salinarum 25 15 Polyploid [2
Halobacterium salinarum (anaerobic) 25 15 Polyploid [2
Haloferax volcanii 17 10 Polyploid [2
Methanocaldococcus jannaschii 0.5 10–15 1–5 Polyploid [4
Methanococcus maripaludis 55 30 Polyploid [3
Methanosarcina acetivorans 18 16 Polyploid [3
Methanosarcina acetivorans 49 Oligoploid [3
Methanothermobacter thermoautotrophicus – 2* 1–2* Diploid [5
Crenarchaeota      
Acidianus hospitalis – 1–2 Monoploid [19
Aeropyrum pernix 3.3 1–2 Monoploid [19
Pyrobaculum aerophilum – 1–2 Monoploid [19
Pyrobaculum calidifontis 3.4 1–2 Monoploid [19
Sulfolobus acidocaldarius 3.5 1–2 Monoploid [1
Sulfolobus tokodai 1–2 Monoploid [19
Sulfolobus solfataricus 1–2 Monoploid [1
*

The cells grow in filaments; the numbers given are genome copies per cell, not per filament.

The results for the seven crenarchaeal species were very similar (Table 1): all species turned out to be monoploid and the cell cycles of all species were characterized by a long G2 and a short G1 phase. Therefore these seven crenarchaeal species at least harbour two chromosomes for the majority of the cell cycle. Furthermore, the cells of all species contained two copies of the chromosome in stationary phase. It is tempting to hypothesize that these results can be generalized to more crenarchaeal species of more genera and thus that monoploidy is a characteristic trait of Crenarchaeota.

Ploidy in Euryarchaeota

Currently, the genome copy numbers of six euryarchaeal species representing six genera are known. The majority of species have been analysed using a real-time PCR method that has been established with Halobacterium salinarum and Haloferax volcanii [2]. First, a standard PCR fragment of approx. 1 kb is generated and its concentration is determined. Next, serial dilutions of the standard fragment and a cytoplasmic extract of the species under investigation are analysed by real-time PCR, amplifying an ‘analysis fragment’ of approx. 200–300 nt. The genome copy number in the cytoplasmic extract can be calculated using a standard curve, and the ploidy level can be quantified by taking the cell density at the time of harvest into account. The advantages of the real-time PCR method are (i) that it is very sensitive, fast and precise, and (ii) that different replicons and different sites of the chromosome can be analysed separately (important, e.g., for mero-oligoploid species such as E. coli). The disadvantage of the method is that the results are average values for the population and single-cell analysis is not possible.

Both H. salinarum and H. volcanii turned out to be highly polyploid, harbouring approx. 25 and 15 genome copies per cell respectively in exponential phase. Both species moderately down-regulated the ploidy level in stationary phase to 15 and 12 copies per cell. For H. salinarum, it was shown that the copy numbers not only of chromosomes, but also of plasmids, are growth-phase-regulated, in contrast with the copy numbers of two other plasmids (which have a very low copy numbers of approx. five per cell). FACS analysis and fluorescence microscopy indicated that the copy number is not identical in all cells, but that there is a considerable variance in the population.

Four species of methanogenic archaea have been analysed. Methanosarcina acetivorans turned out to be polyploid during fast growth (15 genome copies; doubling time 6 h) and oligoploid during slow growth (about three genome copies; doubling time 40 h). The ploidy level was independent of growth phase during fast growth; in contrast, it was increased when cells entered stationary phase after slow growth, underscoring the fact that the genome copy number is precisely regulated in M. acetivorans [3]. Methanococcus maripaludis is the archaeal species with the highest level of polyploidy determined to date and contains approx. 55 genome copies per cell during mid-exponential growth [3]. The genome copy numbers of the remaining two species of methanogenic archaea were determined using the FACS method (see above). Methanothermococcus jannaschii has 10–15 genome copies during exponential phase, which are severely decreased to one to five genome copies in stationary phase [4]. The species is different from all other species discussed because it does not divide at mid-cell, but the septum forms such that mostly the two daughter cells differ in size and DNA content. The last species, Methanothermobacter thermoautotrophicum, is diploid and has thus the lowest genome copy number of all euryarchaeal species analysed to date [5].

In summary, none of six euryarchaeal species of six genera is monoploid. Therefore the ploidy level seems to be a fundamental difference between Euryarchaeota and Crenarchaeota and seems to indicate a severe genetic difference. It will be interesting to uncover the different molecular mechanisms of copy number control. There are various possible evolutionary advantages of polyploidy; however, in the present paper, it should only be noted that one is global gene dosage control, and the growth-phase- and/or growth-rate-dependent regulation of genome copy numbers observed in euryarchaeal species indicates that this control mechanism is indeed applied. Another possible advantage is gene redundancy, which is discussed below.

Problems with polyploidy in Euryarchaeota

Although several possible evolutionary advantages of polyploidy exist, problems with polyploid euryarchaeal species also exist. The most general theoretical counterargument was developed several decades ago and is called ‘Muller's ratchet’ [6]. In short, it states that asexually reproducing polyploid species simply cannot exist. The basic assumption is that if they did exist, this would lead to an accumulation of detrimental mutations in the diverse copies until not a single genome molecule with the wild-type information exists, leading unequivocally to extinction. There are two possible escapes from Muller's ratchet. The first one is that these species have found a mechanism acting in a similar way to recombination in sexually reproducing species. For several species, it has in fact described that lateral gene transfer followed by recombination happens with high frequency, but this will not be discussed in the present paper (see, e.g., [7]). A second escape from Muller's ratchet would be the existence of a mechanism that leads to an equalization of the genome copy in polyploid species, and it has been shown in two species of halophilic and methanogenic Archaea that such a mechanism called ‘gene conversion’ is indeed in operation in polyploid Euryarchaeota (see below).

A problem from the practical point of view is that mutants of halophilic and methanogenic archaea can easily be isolated after random mutagenesis and selection [810] and that protocols to introduce designed changes into the chromosome have been established [1114]. In both cases, the random or designed mutation is introduced into a single copy of the chromosome and, for statistical reasons, it seems impossible that homozygous polyploid mutants can arise from such a starting point, and yet they can be easily generated. However, also in this case, a mechanism such as gene conversion that leads to the equalization of genome copies would resolve the contradiction of the ease of mutant generation and polyploidy.

Gene conversion in Euryarchaeota

Gene conversion is defined as the non-reciprocal flow of information from one DNA molecule to another or from one part of a DNA molecule to another. For example, gene conversion is involved in mating-type switching in yeast, antigenic variation in bacterial pathogens and in the repair of the genomes of plastids, which are highly polyploid [15]. It is studied mostly in eukaryotes, somewhat in bacteria, but experimental studies using archaea are practically non-existent [16]. Two experiments have been performed to unravel whether gene conversion occurs in methanogenic and halophilic archaea and leads to the equalization of genome copies.

The first approach made use of a heterozygous strain of M. maripaludis that was described recently [17]. The aim had been to replace the selD gene encoding selenophosphate synthase with the puromycin-resistance gene pacN. However, it turned out that the selD gene is essential for M. maripaludis. Therefore selection in the presence of puromycin led to a heterozygous strain containing very few copies of the native selD-containing genome and more than 50 copies of the engineered pacN-containing genome. These cells were used for an experiment that is schematically summarized in Figure 1(A) [3]. Cultures were grown under four different conditions, i.e. in the absence of selection (puromycin), in the presence of the normal puromycin concentration (full selection) and in the presence of two intermediate concentrations. Quantification of the selD and pacN-containing genomes revealed that, in the absence of selection, the situation totally reversed within 14 generations: the cells nearly exclusively contained the native genome, whereas the number of pacN-containing genomes fell to about zero. The most likely explanation of this very fast genome replacement in the absence of any selection is gene conversion. In the presence of the two lower concentrations of puromycin, the replacement of the pacN genomes by the selD genomes also occurred, albeit with lower velocity. In the presence of the full selection, the situation remained unchanged, and after 14 generations, the cells still contained only one or two selD-containing genomes and a high number of pacN-containing genomes.

Schematic overview of gene conversion experiments with M. maripaludis (A) and H. volcanii (B)

Figure 1
Schematic overview of gene conversion experiments with M. maripaludis (A) and H. volcanii (B)

See the text for details.

Figure 1
Schematic overview of gene conversion experiments with M. maripaludis (A) and H. volcanii (B)

See the text for details.

The second approach aimed to verify that gene conversion also occurs in halophilic archaea (C. Lange, K. Zerulla, S. Breuert and J. Soppa, unpublished work). The experimental design is shown schematically in Figure 1(B). A strain with a deletion of the trpA gene was used to construct a heterozygous strain, which simultaneously contained genomes with the native leuB gene at the leuB locus, as well as genomes with the trpA gene at the leuB locus. In the absence of leucine and tryptophan, the strain must contain both types of genomes to enable growth, and this was verified experimentally by quantitative real-time PCR. Cells were then cultured under three different conditions: (i) in the presence of tryptophan selecting for the presence of the leuB gene, (ii) in the presence of leucine selecting for the presence of the trpA gene, and (iii) in the presence of leucine and tryptophan and thus in the absence of any selection. In scenario (i), the trpA-containing genomes were lost very rapidly within 3 days, and the culture contained solely the native leuB gene at the leuB locus. In scenario (ii), the leuB-containing genomes were lost, but it took much longer. The reason is that the experiment was not symmetrical, the conversion of trpA into leuB required the synthesis of 50 nt, whereas approx. 950 nt have to be synthesized for the conversion of leuB into trpA. In the absence of selection, the cells were free to keep both genome copies, but also in this case, the genomes were equalized and the trpA-containing genomes were lost within 5 days.

Conclusions and outlook

Quantification of the number of genome copies in 13 species of four crenarchaeal and six euryarchaeal genera revealed a sharp dichotomy between these two kingdoms: all Crenarchaea analysed until now are monoploid, whereas none of the Euryarchaea is monoploid. The ploidy level is very different in various euryarchaeal species and is precisely regulated in response to growth rate and/or growth phase. It will be interesting to unravel the molecular mechanisms of copy number control in several species of Euryarchaea. Two sets of experiments showed that gene conversion occurs in methanogenic and halophilic Archaea and is an efficient mechanism for the equalization of genomes in growing cells in the absence of any selection. These experiments also highlight one possible evolutionary advantage of polyploidy, i.e. that cells can become heterozygous under specific selective conditions. Novel genomes can, for example, be generated by mutations or by lateral gene transfer, and in polyploids, novel alleles can be ‘tested’ without losing the wild-type information. The isolation of ploidy mutants and the analysis of whether additional potential evolutionary advantages of polyploidy are used by Euryarchaea are currently under way.

Molecular Biology of Archaea II: A Biochemical Society Focused Meeting held at Robinson College, Cambridge, U.K., 16–18 August 2010. Organized and Edited by Stephen Bell (Oxford, U.K.) and Finn Werner (University College London, U.K.).

I thank Sebastian Breuert, Catherina Hildenbrand, Christian Lange and Gabriele Spohn for their excellent work on characterizing ploidy levels and gene conversion in archaea, and Thorsten Allers, Michael Rother and Tillman Stock for their very good co-operation.

Funding

This work was funded by the Deutsche Forschungsgemeinschaft [grant number So264/16-1].

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