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1.  Population and genomic analysis of the genus Halorubrum 
The Halobacteria are known to engage in frequent gene transfer and homologous recombination. For stably diverged lineages to persist some checks on the rate of between lineage recombination must exist. We surveyed a group of isolates from the Aran-Bidgol endorheic lake in Iran and sequenced a selection of them. Multilocus Sequence Analysis (MLSA) and Average Nucleotide Identity (ANI) revealed multiple clusters (phylogroups) of organisms present in the lake. Patterns of intein and Clustered Regularly Interspaced Short Palindromic Repeats (CRISPRs) presence/absence and their sequence similarity, GC usage along with the ANI and the identities of the genes used in the MLSA revealed that two of these clusters share an exchange bias toward others in their phylogroup while showing reduced rates of exchange with other organisms in the environment. However, a third cluster, composed in part of named species from other areas of central Asia, displayed many indications of variability in exchange partners, from within the lake as well as outside the lake. We conclude that barriers to gene exchange exist between the two purely Aran-Bidgol phylogroups, and that the third cluster with members from other regions is not a single population and likely reflects an amalgamation of several populations.
doi:10.3389/fmicb.2014.00140
PMCID: PMC3990103  PMID: 24782836
Halobacteria; Multilocus Sequence Analysis (MLSA); Average Nucleotide Identity (ANI); intein; CRISPR
2.  Evidence from phylogenetic and genome fingerprinting analyses suggests rapidly changing variation in Halorubrum and Haloarcula populations 
Halobacteria require high NaCl concentrations for growth and are the dominant inhabitants of hypersaline environments above 15% NaCl. They are well-documented to be highly recombinogenic, both in frequency and in the range of exchange partners. In this study, we examine the genetic and genomic variation of cultured, naturally co-occurring environmental populations of Halobacteria. Sequence data from multiple loci (~2500 bp) identified many closely and more distantly related strains belonging to the genera Halorubrum and Haloarcula. Genome fingerprinting using a random priming PCR amplification method to analyze these isolates revealed diverse banding patterns across each of the genera and surprisingly even for isolates that are identical at the nucleotide level for five protein coding sequenced loci. This variance in genome structure even between identical multilocus sequence analysis (MLSA) haplotypes indicates that accumulation of genomic variation is rapid: faster than the rate of third codon substitutions.
doi:10.3389/fmicb.2014.00143
PMCID: PMC3988388  PMID: 24782838
Halobacteria; MLSA; genome fingerprinting; Aran-Bidgol lake; environmental population
3.  Extracellular DNA metabolism in Haloferax volcanii 
Extracellular DNA is found in all environments and is a dynamic component of the microbial ecosystem. Microbial cells produce and interact with extracellular DNA through many endogenous mechanisms. Extracellular DNA is processed and internalized for use as genetic information and as a major source of macronutrients, and plays several key roles within prokaryotic biofilms. Hypersaline sites contain some of the highest extracellular DNA concentrations measured in nature–a potential rich source of carbon, nitrogen, and phosphorus for halophilic microorganisms. We conducted DNA growth studies for the halophilic archaeon Haloferax volcanii DS2 and show that this model Halobacteriales strain is capable of using exogenous double-stranded DNA as a nutrient. Further experiments with varying medium composition, DNA concentration, and DNA types revealed that DNA is utilized primarily as a phosphorus source, that growth on DNA is concentration-dependent, and that DNA isolated from different sources is metabolized selectively, with a bias against highly divergent methylated DNA. Additionally, fluorescence microscopy showed that labeled DNA co-localized with H. volcanii cells. The gene Hvo_1477 was also identified using a comparative genomic approach as a factor likely to be involved in DNA processing at the cell surface, and deletion of Hvo_1477 created a strain deficient in the ability to grow on extracellular DNA. Widespread distribution of Hvo_1477 homologs in archaea suggests metabolism of extracellular DNA may be of broad ecological and physiological relevance in this domain of life.
doi:10.3389/fmicb.2014.00057
PMCID: PMC3929857  PMID: 24600440
extracellular DNA; Haloferax volcanii; DNA metabolism; Halobacteria; halophiles; archaea; natural competence; archaeal genetics
4.  Dihydroxyacetone metabolism in Haloferax volcanii 
Dihydroxyacetone (DHA) is a ketose sugar that can be produced by oxidizing glycerol. DHA in the environment is taken up and phosphorylated to DHA-phosphate by glycerol kinase or DHA kinase. In hypersaline environments, it is hypothesized that DHA is produced as an overflow product from glycerol utilization by organisms such as Salinibacter ruber. Previous research has demonstrated that the halobacterial species Haloquadratum walsbyi can use DHA as a carbon source, and putative DHA kinase genes were hypothesized to be involved in this process. However, DHA metabolism has not been demonstrated in other halobacterial species, and the role of the DHA kinase genes was not confirmed. In this study, we examined the metabolism of DHA in Haloferax volcanii because putative DHA kinase genes were annotated in its genome, and it has an established genetic system to assay growth of mutant knockouts. Experiments in which Hfx. volcanii was grown on DHA as the sole carbon source demonstrated growth, and that it is concentration dependent. Three annotated DHA kinase genes (HVO_1544, HVO_1545, and HVO_1546), which are homologous to the putative DHA kinase genes present in Hqm. walsbyi, as well as the glycerol kinase gene (HVO_1541), were deleted to examine the effect of these genes on the growth of Hfx. volcanii on DHA. Experiments demonstrated that the DHA kinase deletion mutant exhibited diminished, but not absence of growth on DHA compared to the parent strain. Deletion of the glycerol kinase gene also reduced growth on DHA, and did so more than deletion of the DHA kinase. The results indicate that Hfx. volcanii can metabolize DHA and that DHA kinase plays a role in this metabolism. However, the glycerol kinase appears to be the primary enzyme involved in this process. BLASTp analyses demonstrate that the DHA kinase genes are patchily distributed among the Halobacteria, whereas the glycerol kinase gene is widely distributed, suggesting a widespread capability for DHA metabolism.
doi:10.3389/fmicb.2013.00376
PMCID: PMC3863723  PMID: 24379808
dihydroxyacetone metabolism; dihydroxyacetone kinase; glycerol kinase; archaea; Halobacteria; Haloarchaea
5.  Comparison of prokaryotic community structure from Mediterranean and Atlantic saltern concentrator ponds by a metagenomic approach 
We analyzed the prokaryotic community structure of a saltern pond with 21% total salts located in Isla Cristina, Huelva, Southwest Spain, close to the Atlantic ocean coast. For this purpose, we constructed a metagenome (designated as IC21) obtained by pyrosequencing consisting of 486 Mb with an average read length of 397 bp and compared it with other metagenomic datasets obtained from ponds with 19, 33, and 37% total salts acquired from Santa Pola marine saltern, located in Alicante, East Spain, on the Mediterranean coast. Although the salinity in IC21 is closer to the pond with 19% total salts from Santa Pola saltern (designated as SS19), IC21 is more similar at higher taxonomic levels to the pond with 33% total salts from Santa Pola saltern (designated as SS33), since both are predominated by the phylum Euryarchaeota. However, there are significant differences at lower taxonomic levels where most sequences were related to the genus Halorubrum in IC21 and to Haloquadratum in SS33. Within the Bacteroidetes, the genus Psychroflexus is the most abundant in IC21 while Salinibacter dominates in SS33. Sequences related to bacteriorhodopsins and halorhodopsins correlate with the abundance of Haloquadratum in Santa Pola SS19 to SS33 and of Halorubrum in Isla Cristina IC21 dataset, respectively. Differences in composition might be attributed to local ecological conditions since IC21 showed a decrease in the number of sequences related to the synthesis of compatible solutes and in the utilization of phosphonate.
doi:10.3389/fmicb.2014.00196
PMCID: PMC4021199  PMID: 24847316
metagenomics; haloarchaea; halophilic bacteria; saltern; prokaryotic diversity
6.  DNA as a Phosphate Storage Polymer and the Alternative Advantages of Polyploidy for Growth or Survival 
PLoS ONE  2014;9(4):e94819.
Haloferax volcanii uses extracellular DNA as a source for carbon, nitrogen, and phosphorous. However, it can also grow to a limited extend in the absence of added phosphorous, indicating that it contains an intracellular phosphate storage molecule. As Hfx. volcanii is polyploid, it was investigated whether DNA might be used as storage polymer, in addition to its role as genetic material. It could be verified that during phosphate starvation cells multiply by distributing as well as by degrading their chromosomes. In contrast, the number of ribosomes stayed constant, revealing that ribosomes are distributed to descendant cells, but not degraded. These results suggest that the phosphate of phosphate-containing biomolecules (other than DNA and RNA) originates from that stored in DNA, not in rRNA. Adding phosphate to chromosome depleted cells rapidly restores polyploidy. Quantification of desiccation survival of cells with different ploidy levels showed that under phosphate starvation Hfx. volcanii diminishes genetic advantages of polyploidy in favor of cell multiplication. The consequences of the usage of genomic DNA as phosphate storage polymer are discussed as well as the hypothesis that DNA might have initially evolved in evolution as a storage polymer, and the various genetic benefits evolved later.
doi:10.1371/journal.pone.0094819
PMCID: PMC3986227  PMID: 24733558
7.  Draft Genome Sequence of the Moderately Halophilic Bacterium Marinobacter lipolyticus Strain SM19 
Genome Announcements  2013;1(4):e00379-13.
Marinobacter lipolyticus strain SM19, isolated from saline soil in Spain, is a moderately halophilic bacterium belonging to the class Gammaproteobacteria. Here, we report the draft genome sequence of this strain, which consists of a 4.0-Mb chromosome and which is able to produce the halophilic enzyme lipase LipBL.
doi:10.1128/genomeA.00379-13
PMCID: PMC3695434  PMID: 23814106
8.  Draft Genome Sequence of the Moderately Halophilic Bacterium Pseudoalteromonas ruthenica Strain CP76 
Genome Announcements  2013;1(3):e00268-13.
Pseudoalteromonas ruthenica strain CP76, isolated from a saltern in Spain, is a moderately halophilic bacterium belonging to the Gammaproteobacteria. Here we report the draft genome sequence, which consists of a 4.0-Mb chromosome, of this strain, which is able to produce the extracellular enzyme haloprotease CPI.
doi:10.1128/genomeA.00268-13
PMCID: PMC3662824  PMID: 23704184
9.  Quantifying Homologous Replacement of Loci between Haloarchaeal Species 
Genome Biology and Evolution  2012;4(12):1223-1244.
In vitro studies of the haloarchaeal genus Haloferax have demonstrated their ability to frequently exchange DNA between species, whereas rates of homologous recombination estimated from natural populations in the genus Halorubrum are high enough to maintain random association of alleles between five loci. To quantify the effects of gene transfer and recombination of commonly held (relaxed core) genes during the evolution of the class Halobacteria (haloarchaea), we reconstructed the history of 21 genomes representing all major groups. Using a novel algorithm and a concatenated ribosomal protein phylogeny as a reference, we created a directed horizontal genetic transfer (HGT) network of contemporary and ancestral genomes. Gene order analysis revealed that 90% of testable HGTs were by direct homologous replacement, rather than nonhomologous integration followed by a loss. Network analysis revealed an inverse log-linear relationship between HGT frequency and ribosomal protein evolutionary distance that is maintained across the deepest divergences in Halobacteria. We use this mathematical relationship to estimate the total transfers and amino acid substitutions delivered by HGTs in each genome, providing a measure of chimerism. For the relaxed core genes of each genome, we conservatively estimate that 11–20% of their evolution occurred in other haloarchaea. Our findings are unexpected, because the transfer and homologous recombination of relaxed core genes between members of the class Halobacteria disrupts the coevolution of genes; however, the generation of new combinations of divergent but functionally related genes may lead to adaptive phenotypes not available through cumulative mutations and recombination within a single population.
doi:10.1093/gbe/evs098
PMCID: PMC3542582  PMID: 23160063
homologous recombination; horizontal gene transfer; lateral gene transfer; fitness landscape; populations; microbial evolution
10.  Ancient origin of the divergent forms of leucyl-tRNA synthetases in the Halobacteriales 
Background
Horizontal gene transfer (HGT) has greatly impacted the genealogical history of many lineages, particularly for prokaryotes, with genes frequently moving in and out of a line of descent. Many genes that were acquired by a lineage in the past likely originated from ancestral relatives that have since gone extinct. During the course of evolution, HGT has played an essential role in the origin and dissemination of genetic and metabolic novelty.
Results
Three divergent forms of leucyl-tRNA synthetase (LeuRS) exist in the archaeal order Halobacteriales, commonly known as haloarchaea. Few haloarchaeal genomes have the typical archaeal form of this enzyme and phylogenetic analysis indicates it clusters within the Euryarchaeota as expected. The majority of sequenced halobacterial genomes possess a bacterial form of LeuRS. Phylogenetic reconstruction puts this larger group of haloarchaea at the base of the bacterial domain. The most parsimonious explanation is that an ancient transfer of LeuRS took place from an organism related to the ancestor of the bacterial domain to the haloarchaea. The bacterial form of LeuRS further underwent gene duplications and/or gene transfers within the haloarchaea, with some genomes possessing two distinct types of bacterial LeuRS. The cognate tRNALeu also reveals two distinct clusters for the haloarchaea; however, these tRNALeu clusters do not coincide with the groupings found in the LeuRS tree, revealing that LeuRS evolved independently of its cognate tRNA.
Conclusions
The study of leucyl-tRNA synthetase in haloarchaea illustrates the importance of gene transfer originating in lineages that went extinct since the transfer occurred. The haloarchaeal LeuRS and tRNALeu did not co-evolve.
doi:10.1186/1471-2148-12-85
PMCID: PMC3436685  PMID: 22694720
11.  In Vivo Characterization of the Homing Endonuclease within the polB Gene in the Halophilic Archaeon Haloferax volcanii 
PLoS ONE  2011;6(1):e15833.
Inteins are parasitic genetic elements, analogous to introns that excise themselves at the protein level by self-splicing, allowing the formation of functional non-disrupted proteins. Many inteins contain a homing endonuclease (HEN) gene, and rely on its activity for horizontal propagation. In the halophilic archaeon, Haloferax volcanii, the gene encoding DNA polymerase B (polB) contains an intein with an annotated but uncharacterized HEN. Here we examine the activity of the polB HEN in vivo, within its natural archaeal host. We show that this HEN is highly active, and able to insert the intein into both a chromosomal target and an extra-chromosomal plasmid target, by gene conversion. We also demonstrate that the frequency of its incorporation depends on the length of the flanking homologous sequences around the target site, reflecting its dependence on the homologous recombination machinery. Although several evolutionary models predict that the presence of an intein involves a change in the fitness of the host organism, our results show that a strain deleted for the intein sequence shows no significant changes in growth rate compared to the wild type.
doi:10.1371/journal.pone.0015833
PMCID: PMC3024317  PMID: 21283796
12.  Intertwined Evolutionary Histories of Marine Synechococcus and Prochlorococcus marinus 
Prochlorococcus is a genus of marine cyanobacteria characterized by small cell and genome size, an evolutionary trend toward low GC content, the possession of chlorophyll b, and the absence of phycobilisomes. Whereas many shared derived characters define Prochlorococcus as a clade, many genome-based analyses recover them as paraphyletic, with some low-light adapted Prochlorococcus spp. grouping with marine Synechococcus. Here, we use 18 Prochlorococcus and marine Synechococcus genomes to analyze gene flow within and between these taxa. We introduce embedded quartet scatter plots as a tool to screen for genes whose phylogeny agrees or conflicts with the plurality phylogenetic signal, with accepted taxonomy and naming, with GC content, and with the ecological adaptation to high and low light intensities. We find that most gene families support high-light adapted Prochlorococcus spp. as a monophyletic clade and low-light adapted Prochlorococcus sp. as a paraphyletic group. But we also detect 16 gene families that were transferred between high-light adapted and low-light adapted Prochlorococcus sp. and 495 gene families, including 19 ribosomal proteins, that do not cluster designated Prochlorococcus and Synechococcus strains in the expected manner. To explain the observed data, we propose that frequent gene transfer between marine Synechococcus spp. and low-light adapted Prochlorococcus spp. has created a “highway of gene sharing” (Beiko RG, Harlow TJ, Ragan MA. 2005. Highways of gene sharing in prokaryotes. Proc Natl Acad Sci USA. 102:14332–14337) that tends to erode genus boundaries without erasing the Prochlorococcus-specific ecological adaptations.
doi:10.1093/gbe/evp032
PMCID: PMC2817427  PMID: 20333202
marine cyanobacteria; horizontal gene transfer; introgression; quartet decomposition; supertree; genome evolution
13.  Evolution of rhodopsin ion pumps in haloarchaea 
Background
The type 1 (microbial) rhodopsins are a diverse group of photochemically reactive proteins that display a broad yet patchy distribution among the three domains of life. Recent work indicates that this pattern is likely the result of lateral gene transfer (LGT) of rhodopsin genes between major lineages, and even across domain boundaries. Within the lineage in which the microbial rhodopsins were initially discovered, the haloarchaea, a similar patchy distribution is observed. In this initial study, we assess the roles of LGT and gene loss in the evolution of haloarchaeal rhodopsin ion pump genes, using phylogenetics and comparative genomics approaches.
Results
Mapping presence/absence of rhodopsins onto the phylogeny of the RNA polymerase B' subunit (RpoB') of the haloarchaea supports previous notions that rhodopsins are patchily distributed. The phylogeny for the bacteriorhodopsin (BR) protein revealed two discrepancies in comparison to the RpoB' marker, while the halorhodopsin (HR) tree showed incongruence to both markers. Comparative analyses of bacteriorhodopsin-linked regions of five haloarchaeal genomes supported relationships observed in the BR tree, and also identified two open reading frames (ORFs) that were more frequently linked to the bacteriorhodopsin gene than those genes previously shown to be important to the function and expression of BR.
Conclusion
The evidence presented here reveals a complex evolutionary history for the haloarchaeal rhodopsins, with both LGT and gene loss contributing to the patchy distribution of rhodopsins within this group. Similarities between the BR and RpoB' phylogenies provide supportive evidence for the presence of bacteriorhodopsin in the last common ancestor of haloarchaea. Furthermore, two loci that we have designated bacterio-opsin associated chaperone (bac) and bacterio-opsin associated protein (bap) are inferred to have important roles in BR biogenesis based on frequent linkage and co-transfer with bacteriorhodopsin genes.
doi:10.1186/1471-2148-7-79
PMCID: PMC1885257  PMID: 17511874
14.  Genomics and the bacterial species problem 
Genome Biology  2006;7(9):116.
Do we need to describe bacteria as species, and if so, can we?
Whether or not bacteria have species is a perennially vexatious question. Given what we now know about variation among bacterial genomes, we argue that there is no intrinsic reason why the processes driving diversification and adaptation must produce groups of individuals sufficiently coherent in their genetic and phenotypic properties to merit the designation 'species' - although sometimes they might.
doi:10.1186/gb-2006-7-9-116
PMCID: PMC1794555  PMID: 17020593
15.  Environmental genomics of "Haloquadratum walsbyi" in a saltern crystallizer indicates a large pool of accessory genes in an otherwise coherent species 
BMC Genomics  2006;7:171.
Background
Mature saturated brine (crystallizers) communities are largely dominated (>80% of cells) by the square halophilic archaeon "Haloquadratum walsbyi". The recent cultivation of the strain HBSQ001 and thesequencing of its genome allows comparison with the metagenome of this taxonomically simplified environment. Similar studies carried out in other extreme environments have revealed very little diversity in gene content among the cell lineages present.
Results
The metagenome of the microbial community of a crystallizer pond has been analyzed by end sequencing a 2000 clone fosmid library and comparing the sequences obtained with the genome sequence of "Haloquadratum walsbyi". The genome of the sequenced strain was retrieved nearly complete within this environmental DNA library. However, many ORF's that could be ascribed to the "Haloquadratum" metapopulation by common genome characteristics or scaffolding to the strain genome were not present in the specific sequenced isolate. Particularly, three regions of the sequenced genome were associated with multiple rearrangements and the presence of different genes from the metapopulation. Many transposition and phage related genes were found within this pool which, together with the associated atypical GC content in these areas, supports lateral gene transfer mediated by these elements as the most probable genetic cause of this variability. Additionally, these sequences were highly enriched in putative regulatory and signal transduction functions.
Conclusion
These results point to a large pan-genome (total gene repertoire of the genus/species) even in this highly specialized extremophile and at a single geographic location. The extensive gene repertoire is what might be expected of a population that exploits a diverse nutrient pool, resulting from the degradation of biomass produced at lower salinities.
doi:10.1186/1471-2164-7-171
PMCID: PMC1560387  PMID: 16820057
16.  Ancient origins determine global biogeography of hot and cold desert cyanobacteria 
Nature Communications  2011;2:163-.
Factors governing large-scale spatio-temporal distribution of microorganisms remain unresolved, yet are pivotal to understanding ecosystem value and function. Molecular genetic analyses have focused on the influence of niche and neutral processes in determining spatial patterns without considering the temporal scale. Here, we use temporal phylogenetic analysis calibrated using microfossil data for a globally sampled desert cyanobacterium, Chroococcidiopsis, to investigate spatio-temporal patterns in microbial biogeography and evolution. Multilocus phylogenetic associations were dependent on contemporary climate with no evidence for distance-related patterns. Massively parallel pyrosequencing of environmental samples confirmed that Chroococcidiopsis variants were specific to either hot or cold deserts. Temporally scaled phylogenetic analyses showed no evidence of recent inter-regional gene flow, indicating populations have not shared common ancestry since before the formation of modern continents. These results indicate that global distribution of desert cyanobacteria has not resulted from widespread contemporary dispersal but is an ancient evolutionary legacy. This highlights the importance of considering temporal scales in microbial biogeography.
Microorganisms are abundant in many environments and understanding their dispersal between ecosystems is important for ecology and conservation. These authors demonstrate that cyanobacterial populations are specific to hot or cold deserts and that gene flow between different populations does not occur.
doi:10.1038/ncomms1167
PMCID: PMC3105302  PMID: 21266963

Results 1-16 (16)