Characterization of Non-selected Intermolecular Gene Conversion in the Polyploid Haloarchaeon Haloferax volcanii.

Gene conversion is defined as the non-reciprocal transfer of genetic information from one site to a homologous, but not identical site of the genome. In prokaryotes, gene conversion can increase the variance of sequences, like in antigenic variation, but can also lead to a homogenization of sequences, like in the concerted evolution of multigene families. In contrast to these intramolecular mechanisms, the intermolecular gene conversion in polyploid prokaryotes, which leads to the equalization of the multiple genome copies, has hardly been studied. We have previously shown the intermolecular gene conversion in halophilic and methanogenic archaea is so efficient that it can be studied without selecting for conversion events. Here, we have established an approach to characterize unselected intermolecular gene conversion in Haloferax volcanii making use of two genes that encode enzymes involved in carotenoid biosynthesis. Heterozygous strains were generated by protoplast fusion, and gene conversion was quantified by phenotype analysis or/and PCR. It was verified that unselected gene conversion is extremely efficient and it was shown that gene conversion tracts are much longer than in antigenic variation or concerted evolution in bacteria. Two sites were nearly always co-converted when they were 600 bp apart, and more than 30% co-conversion even occurred when two sites were 5 kbp apart. The gene conversion frequency was independent from the extent of genome differences, and even a one nucleotide difference triggered conversion.

Polyploidy in halophilic archaea: regulation, evolutionary advantages, and gene conversion.

All analyzed haloarachea are polyploid. In addition, haloarchaea contain more than one type of chromosome, and thus the gene dosage can be regulated independently on different replicons. Haloarchaea and several additional archaea have more than one replication origin on their major chromosome, in stark contrast with bacteria, which have a single replication origin. Two of these replication origins of Haloferax volcanii have been studied in detail and turned out to have very different properties. The chromosome copy number appears to be regulated in response to growth phases and environmental factors. Archaea typically contain about two Origin Recognition Complex (ORC) proteins, which are homologous to eukaryotic ORC proteins. However, haloarchaea are the only archaeal group that contains a multitude of ORC proteins. All 16 ORC protein paralogs from H. volcanii are involved in chromosome copy number regulation. Polyploidy has many evolutionary advantages for haloarchaea, e.g. a high resistance to desiccation, survival over geological times, and the relaxation of cell cycle-specific replication control. A further advantage is the ability to grow in the absence of external phosphate while using the many genome copies as internal phosphate storage polymers. Very efficient gene conversion operates in haloarchaea and results in the unification of genome copies. Taken together, haloarchaea are excellent models to study many aspects of genome biology in prokaryotes, exhibiting properties that have not been found in bacteria.

Influence of Origin Recognition Complex Proteins on the Copy Numbers of Three Chromosomes in Haloferax volcanii.

Replication initiation in archaea involves a protein named ORC, Cdc6, or ORC1/Cdc6, which is homologous to the eukaryotic origin recognition complex (ORC) proteins and to the eukaryotic Cdc6. Archaeal replication origins are comprised of origin repeat regions and adjacent orc genes. Some archaea contain a single replication origin and a single orc gene, while others have more than one of each. Haloferax volcanii is exceptional because it contains, in total, six replication origins on three chromosomes and 16 orc genes. Phylogenetic trees were constructed that showed that orc gene duplications occurred at very different times in evolution. To unravel the influence of the ORC proteins on chromosome copy number and cellular fitness, it was attempted to generate deletion mutants of all 16 genes. A total of 12 single-gene deletion mutants could be generated, and only three orc gene turned out to be essential. For one gene, the deletion analysis failed. Growth analyses revealed that no deletion mutant had a growth defect, but some had a slight growth advantage compared to the wild type. Quantification of the chromosome copy numbers in the deletion mutants showed that all 12 ORC proteins influenced the copy numbers of one, two, or all three chromosomes. The lack of an ORC led to an increase or decrease of chromosome copy number. Therefore, chromosome copy numbers in Hfxvolcanii are regulated by an intricate network of ORC proteins. This is in contrast to other archaea, in which ORC proteins typically bind specifically to the adjacent origin.IMPORTANCE The core origins of archaea are comprised of a repeat region and an adjacent gene for an origin recognition complex (ORC) protein, which is homologous to eukaryotic ORC proteins. Haloferax volcanii is exceptional because it contains six replication origins on three chromosomes and an additional 10 orc genes that are not adjacent to an origin. This unique ORC protein repertoire was used to unravel the importance of core origin orc genes and of origin-remote orc genes. Remarkably, all ORC proteins influenced the copy number of at least one chromosome. Some of them influenced those of all three chromosomes, showing that cross-regulation in trans exists in Hfx. volcanii Furthermore, the evolution of the archaeal ORC protein family was analyzed.

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

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.

Characterization of Copy Number Control of Two Haloferax volcanii Replication Origins Using Deletion Mutants and Haloarchaeal Artificial Chromosomes.

Haloferax volcanii is polyploid and contains about 20 genome copies under optimal conditions. However, the chromosome copy number is highly regulated and ranges from two during phosphate starvation to more than 40 under conditions of phosphate surplus. The aim of this study was the characterization of the influence of two replication origins on the genome copy number. The origin repeats and the genes encoding origin recognition complex (ORC) proteins were deleted. The core origin oriC1-orc1 (ori1) deletion mutant had a lower genome copy number and a higher level of fitness than the wild type, in stark contrast to the oriC2-orc5 (ori2) deletion mutant. The genes adjacent to ori1 could not be deleted, and thus, at least two of them are probably essential, while deletion of the genes adjacent to ori2 was possible. Various fragments of and around the origins were cloned into a suicide plasmid to generate haloarchaeal artificial chromosomes (HACs). The copy number of the oriC1-orc1 HAC was much higher than that of the oriC2-orc5 HAC. The addition of adjacent genes influenced both the HAC copy number and the chromosome copy number. The results indicate that the origins of H. volcanii are not independent but that the copy number is regulated via a network of genes around the origins.IMPORTANCE Several species of archaea have more than one origin of replication on their major chromosome and are thus the only known prokaryotic species that allow the analysis of the evolution of multiorigin replication. The widely studied Haloferax volcanii H26 strain has a major chromosome with four origins of replication. Two origins, ori1 and ori2, were chosen for an in-depth analysis using deletion mutants and haloarchaeal artificial chromosomes. The analysis was not restricted to the core origin regions; origin-adjacent genes were also included. Because H. volcanii is polyploid, the effects on the chromosome copy number were of specific importance. The results revealed extreme differences between the two origins.

The ploidy level of Synechocystis sp. PCC 6803 is highly variable and is influenced by growth phase and by chemical and physical external parameters.

Synechocystis sp. PCC 6803 is a cyanobacterial model strain widely used to study many biological processes and is also applied for the production of biopolymers. Recently, it was reported that two of its substrains are highly polyploid. To test whether this can be generalized to the whole strain, six substrains were selected and their ploidy levels quantified. The ploidy levels of all substrains were highly growth phase regulated and the copy number was on average about 20 at an OD750 of 0.1 and about 4 at an OD750 of 2.5. In addition to growth phase, external conditions were found to influence the ploidy level, i.e. the copy number was elevated at lower light intensity and at higher phosphate concentrations (53 and 35 copies, respectively). In the absence of external phosphate, considerable growth was observed, although growth rate and growth yield were much lower than in the presence of either orthophosphate or genomic DNA as external source of phosphate. A rapid reduction in genome copy number was observed during growth in the absence of phosphate, indicating that replication ceased and genomes were distributed to the daughter cells. During prolonged incubation of stationary-phase cultures in the absence of phosphate, the cells eventually became monoploid. Taking the data together, the ploidy level of Synechocystis sp. PCC 6803 is extremely variable and is influenced by both growth phase and physical and chemical environmental parameters.