Frequent Recombination in a Saltern Population of Halorubrum

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Science  10 Dec 2004:
Vol. 306, Issue 5703, pp. 1928-1929
DOI: 10.1126/science.1103289


Sex and recombination are driving forces in the evolution of eukaryotes. Homologous recombination is known to be the dominant process in the divergence of many bacterial species. For Archaea, the only direct evidence bearing on the importance or natural occurrence of homologous recombination is anecdotal reports of mosaicism from comparative genomic studies. Genetic studies, however, reveal that recombination may play a significant role in generating diversity among members of at least one archaeal group, the haloarchaea. We used multi-locus sequence typing to demonstrate that haloarchaea exchange genetic information promiscuously, exhibiting a degree of linkage equilibrium approaching that of a sexual population.

The evidence that haloarchaea are potentially sexual organisms comes from laboratory studies. Prototrophic recombinants of the genus Haloferax can be obtained when auxotrophic strains are filtered together and plated on growth medium (1, 2), a process that may involve the formation of intercellular bridges. Transformation can be artificially induced in several species, and recombination occurs readily between chromosomes and introduced homologous DNA fragments (35). In nature, haloarchaea, which prefer elevated salt concentrations, are characteristically prominent in multipond solar salterns, where seawater is concentrated in stages from 4 to 37% sodium chloride (NaCl). Using ribosomal RNA (rRNA) genes (6, 7), we have identified multiple clusters of related (conspecific) ribotypes in the salterns at Santa Pola, near Alicante, Spain. In these salterns, apparent species diversity decreased with increasing salinity but microdiversity increased. Diversity at a scale finer than that accessible to rRNA-based methods can be revealed by multi-locus sequence typing (MLST), which also offers a window into genetic exchange processes (8, 9).

We undertook an MLST survey of haloarchaea isolated from different salinity ponds at this site (10). Polymerase chain reaction (PCR) was used to amplify a 325–base pair (bp) stretch of small subunit (SSU) rRNA genes. Sequenced PCR products showed few ambiguities, suggesting that the strains did not harbor multiple SSU rRNA genes of substantially different sequences, as some species of haloarchaea clearly do (11). In total, 122 strains were characterized and could be assigned to five clusters, corresponding to four named haloarchaeal genera and one novel haloarchaeal assemblage. Sixty-nine of the strains assignable to the Halorubrum cluster had SSU rRNA sequences identical to each other and to an Australian isolate [Halobacterium sp. AUS1 (JCM9573)] (12). Thirty-six of these isoribotypes were selected for MLST analysis. Primers were designed with reference to the sequenced genome of Halobacterium NRC-1 and individual GenBank entries (13) to generate products of approximately 500 bp from atpB, ef-2, radA (10), and secY genes. For each gene, each different sequence obtained was considered an allele and assigned a number (Table 1). Alleles at each locus were also analyzed phylogenetically and assigned to one of two to four clades (fig. S1).

Table 1.

Members of different allele clades differ by 6 to 43 nucleotides at any locus and are represented by different colors for heuristic value (fig. S1). Column P shows recombination detected by phylogenetic analysis. Column S shows recombination detected by SLV analysis.

Among the isolates, allelic profiles were highly mosaic; all loci (except for SSU rRNA) were polymorphic with 8 to 15 alleles and 30 to 61 polymorphic nucleotide sites per locus. We estimated the genetic diversity as Math, where x is the relative frequency of the ith allele, and we averaged the values to obtain the average genetic diversity in our sample set (14). The estimated value for the entire data set was H = 0.69. For the 36 and 23% salinity ponds, separately, we estimated the average genetic diversity to be 0.83 and 0.57, respectively. All values were higher than the average genetic diversity of 0.47 reported for Escherichia coli, which itself is approximately five times higher than that of typical eukaryotic species (14).

Strain phylogenies based on the different genes were incongruent, which was expected if recombination had occurred during the divergence of these organisms (fig. S1). Comparison of isolate-specific allelic profiles (Table 1) using linkage disequilibrium freeware (15, 16) also demonstrated recombination. This freeware measures the variance in allelic profiles in all pairwise combinations and contrasts it to the value obtained from 1000 randomizations (P = 0.001) of the data set (simulating a freely recombining population). If the observed variance is greater than the maximum variance obtained from randomizing the data set, then the association of alleles is not random: There is linkage disequilibrium, and recombination may be rare. However, if the measured variance is less than the maximum value obtained from randomization, then alleles are arbitrarily associated: the population is more likely to be freely recombining and in linkage equilibrium (15, 16). Linkage analysis of our entire data set revealed that the observed variance of 0.92 was lower than the maximum variance of 1.26 and close to the mean variance of 0.83 obtained in 1000 randomized data. When isolates cultured from ponds of different salinities were analyzed as separate data sets, even wider differences in variance were measured. From the 36% salinity pond, the observed variance was 0.510 and the maximum and mean variances for 1000 trials were 1.30 and 0.69, respectively. From the 23% salinity pond, the observed variance was 0.707 and the maximum and mean variances for 1000 randomizations were 1.31 and 0.77, respectively. This analysis suggests that the Halorubrum population is near linkage equilibrium and that random mating and recombination occur both within and (possibly at a somewhat reduced rate) between ponds of different salinities.

It is also possible to quantify the relative contributions to diversity of mutation and recombination using rules of procedure based on single-locus variation (SLV) developed in MLST studies on bacteria (17). SLV, a situation in which two strains are identical at all alleles except for one, can arise by either mutation or recombination. Mutational alterations are single-nucleotide changes and are unique in a data set, whereas recombination events can have single or multiple nucleotide changes and are encountered several times, independently. In either case, uniqueness, direction of change, and independence of occurrence can be mapped against a clonal complex model (fig. S2). We noted 87 nucleotide changes in 10 alleles that likely arose from recombination and two nucleotide changes in 2 alleles that likely arose from mutation.

For certain pathogenic bacterial species, comparable excesses of recombination over mutation as source of variation have been reported (17, 18), leading to a radical rethinking of mechanisms of bacterial adaptation and diversification. Adaptive sweeps do occur, reducing variation within a population. But such variants arise within populations that are interrelated in complex weblike patterns through recombination, and may come to dominate because they bear favorable combinations of preexisting alleles, rather than because they carry novel mutations (9). Much recombination may occur within so-called “species boundaries,” but there may be no absolute barrier to recombination between species. Some of the recombination events we documented involved sequences differing by more than 7% and may have transgressed such boundaries.

Recombination, our results show, can be an important force in the generation of diversity and adaptive novelty in Archaea. Although the underlying cellular mechanisms are as yet poorly known, cytoplasmic connections (1) may represent an evolved gene exchange system within the Euryarchaeota. There is also evidence for natural conjugative mechanisms for gene exchange and recombination in species of Sulfolobus, a member of the second major division of Archaea, the Crenarchaeota (1921).

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