Halophilic microorganisms are found in all domains of life and thrive in hypersaline (high salt content) environments. These unusual microbes have been a subject of study for many years due to their interesting properties and physiology. Study of the genetics of halophilic microorganisms (from gene expression and regulation to genomics) has provided understanding into mechanisms of how life can occur at high salinity levels. Here we highlight recent studies that advance knowledge of biological function through study of the genetics of halophilic microorganisms and their viruses. Full article

One of the most commonly encountered species in the small basidiomycetous sub-phylum Wallemiomycotina is Wallemia mellicola, a xerotolerant fungus with a widespread distribution. To investigate the population characteristics of the species, whole genomes of twenty-five strains were sequenced. Apart from identification of four strains of clonal origin, the distances between the genomes failed to reflect either the isolation habitat of the strains or their geographical origin. Strains from different parts of the world appeared to represent a relatively homogenous and widespread population. The lack of concordance between individual gene phylogenies and the decay of linkage disequilibrium indicated that W. mellicola is at least occasionally recombining. Two versions of a putative mating-type locus have been found in all sequenced genomes, each present in approximately half of the strains. W. mellicola thus appears to be capable of (sexual) recombination and shows no signs of allopatric speciation or specialization to specific habitats. Full article

The Cabo Rojo solar salterns are a hypersaline environment located in a tropical climate, where conditions remain stable throughout the year. These conditions can favor the establishment of steady microbial communities. Little is known about the microbial composition that thrives in hypersaline environments in the tropics. The main goal of this study was to assess the microbial diversity present in the crystallizer ponds of Cabo Rojo, in terms of structure and metabolic processes across time using metagenomic techniques. Three samplings (December 2014, March and July 2016) were carried out, where water samples (50 L each) were filtered through a Millipore pressurized filtering system. DNA was subsequently extracted using physical–chemical methods and sequenced using paired end Illumina technologies. The sequencing effort produced three paired end libraries with a total of 111,816,040 reads, that were subsequently assembled into three metagenomes. Out of the phyla detected, the microbial diversity was dominated in all three samples by Euryarchaeota, followed by Bacteroidetes and Proteobacteria. However, sample MFF1 (for Muestreo Final Fraternidad) exhibited a higher diversity, with 12 prokaryotic phyla detected at 34% NaCl (w/v), when compared to samples MFF2 and MFF3, which only exhibited three phyla. Precipitation events might be one of the contributing factors to the change in the microbial community composition through time. Diversity at genus level revealed a more stable community structure, with an overwhelming dominance of the square archaeon Haloquadratum in the three metagenomes. Furthermore, functional annotation was carried out in order to detect genes related to metabolic processes, such as carbon, nitrogen, and sulfur cycles. The presence of gene sequences related to nitrogen fixation, ammonia oxidation, sulfate reduction, sulfur oxidation, and phosphate solubilization were detected. Through binning methods, four putative novel genomes were obtained, including a possible novel genus belonging to the Bacteroidetes and possible new species for the genera Natronomonas, Halomicrobium, and Haloquadratum. Using a metagenomic approach, a 3-year study has been performed in a Caribbean hypersaline environment. When compared to other salterns around the world, the Cabo Rojo salterns harbor a similar community composition, which is stable through time. Moreover, an analysis of gene composition highlights the importance of the microbial community in the biogeochemical cycles at hypersaline environments. Full article

Salt mines are among the most extreme environments as they combine darkness, low nutrient availability, and hypersaline conditions. Based on comparative genomics and transcriptomics, we describe in this work the adaptive strategies of the true halophilic fungus Aspergillus salisburgensis, found in a salt mine in Austria, and compare this strain to the ex-type halotolerant fungal strain Aspergillus sclerotialis. On a genomic level, A. salisburgensis exhibits a reduced genome size compared to A. sclerotialis, as well as a contraction of genes involved in transport processes. The proteome of A. sclerotialis exhibits an increased proportion of alanine, glycine, and proline compared to the proteome of non-halophilic species. Transcriptome analyses of both strains growing at 5% and 20% NaCl show that A. salisburgensis regulates three-times fewer genes than A. sclerotialis in order to adapt to the higher salt concentration. In A. sclerotialis, the increased osmotic stress impacted processes related to translation, transcription, transport, and energy. In contrast, membrane-related and lignolytic proteins were significantly affected in A. salisburgensis. Full article

Zinc finger domains are highly structured and can mediate interactions to DNA, RNA, proteins, lipids, and small molecules. Accordingly, zinc finger proteins are very versatile and involved in many biological functions. Eukaryotes contain a wealth of zinc finger proteins, but zinc finger proteins have also been found in archaea and bacteria. Large zinc finger proteins have been well studied, however, in stark contrast, single domain zinc finger µ-proteins of less than 70 amino acids have not been studied at all, with one single exception. Therefore, 16 zinc finger µ-proteins of the haloarchaeon Haloferax volcanii were chosen and in frame deletion mutants of the cognate genes were generated. The phenotypes of mutants and wild-type were compared under eight different conditions, which were chosen to represent various pathways and involve many genes. None of the mutants differed from the wild-type under optimal or near-optimal conditions. However, 12 of the 16 mutants exhibited a phenotypic difference under at least one of the four following conditions: Growth in synthetic medium with glycerol, growth in the presence of bile acids, biofilm formation, and swarming. In total, 16 loss of function and 11 gain of function phenotypes were observed. Five mutants indicated counter-regulation of a sessile versus a motile life style in H. volcanii. In conclusion, the generation and analysis of a set of deletion mutants demonstrated the high importance of zinc finger µ-proteins for various biological functions, and it will be the basis for future mechanistic insight. Full article

Antisense RNAs (asRNAs) are present in diverse organisms and play important roles in gene regulation. In this work, we mapped the primary antisense transcriptome in the halophilic archaeon Halobacterium salinarum NRC-1. By reanalyzing publicly available data, we mapped antisense transcription start sites (aTSSs) and inferred the probable 3′ ends of these transcripts. We analyzed the resulting asRNAs according to the size, location, function of genes on the opposite strand, expression levels and conservation. We show that at least 21% of the genes contain asRNAs in H. salinarum. Most of these asRNAs are expressed at low levels. They are located antisense to genes related to distinctive characteristics of H. salinarum, such as bacteriorhodopsin, gas vesicles, transposases and other important biological processes such as translation. We provide evidence to support asRNAs in type II toxin–antitoxin systems in archaea. We also analyzed public Ribosome profiling (Ribo-seq) data and found that ~10% of the asRNAs are ribosome-associated non-coding RNAs (rancRNAs), with asRNAs from transposases overrepresented. Using a comparative transcriptomics approach, we found that ~19% of the asRNAs annotated in H. salinarum belong to genes with an ortholog in Haloferax volcanii, in which an aTSS could be identified with positional equivalence. This shows that most asRNAs are not conserved between these halophilic archaea. Full article

Restriction–modification (RM) systems in bacteria are implicated in multiple biological roles ranging from defense against parasitic genetic elements, to selfish addiction cassettes, and barriers to gene transfer and lineage homogenization. In bacteria, DNA-methylation without cognate restriction also plays important roles in DNA replication, mismatch repair, protein expression, and in biasing DNA uptake. Little is known about archaeal RM systems and DNA methylation. To elucidate further understanding for the role of RM systems and DNA methylation in Archaea, we undertook a survey of the presence of RM system genes and related genes, including orphan DNA methylases, in the halophilic archaeal class Halobacteria. Our results reveal that some orphan DNA methyltransferase genes were highly conserved among lineages indicating an important functional constraint, whereas RM systems demonstrated patchy patterns of presence and absence. This irregular distribution is due to frequent horizontal gene transfer and gene loss, a finding suggesting that the evolution and life cycle of RM systems may be best described as that of a selfish genetic element. A putative target motif (CTAG) of one of the orphan methylases was underrepresented in all of the analyzed genomes, whereas another motif (GATC) was overrepresented in most of the haloarchaeal genomes, particularly in those that encoded the cognate orphan methylase. Full article

The unexpected lysis of a large culture of Halobacterium salinarum strain S9 was found to be caused by a novel myovirus, designated ChaoS9. Virus purification from the culture lysate revealed a homogeneous population of caudovirus-like particles. The viral genome is linear, dsDNA that is partially redundant and circularly permuted, has a unit length of 55,145 nt, a G + C% of 65.3, and has 85 predicted coding sequences (CDS) and one tRNA (Arg) gene. The left arm of the genome (0–28 kbp) encodes proteins similar in sequence to those from known caudoviruses and was most similar to myohaloviruses phiCh1 (host: Natrialba magadii) and phiH1 (host: Hbt. salinarum). It carries a tail-fiber gene module similar to the invertible modules present in phiH1 and phiCh1. However, while the tail genes of ChaoS9 were similar to those of phiCh1 and phiH1, the Mcp of ChaoS9 was most similar (36% aa identity) to that of Haloarcula hispanica tailed virus 1 (HHTV-1). Provirus elements related to ChaoS9 showed most similarity to tail/assembly proteins but varied in their similarity with head/assembly proteins. The right arm (29–55 kbp) of ChaoS9 encoded proteins involved in DNA replication (ParA, RepH, and Orc1) but the other proteins showed little similarity to those from phiH1, phiCh1, or provirus elements, and most of them could not be assigned a function. ChaoS9 is probably best classified within the genus Myohalovirus, as it shares many characteristics with phiH1 (and phiCh1), including many similar proteins. However, the head/assembly gene region appears to have undergone a recombination event, and the inferred proteins are different to those of phiH1 and phiCh1, including the major capsid protein. This makes the taxonomic classification of ChaoS9 more ambiguous. We also report a revised genome sequence and annotation of Natrialba virus phiCh1. Full article

The obligately anaerobic haloalkaliphilic bacterium Alkalitalea saponilacus can use xylan as the sole carbon source and produce propionate as the main fermentation product. Using mixed carbon sources of 0.4% (w/v) sucrose and 0.1% (w/v) birch xylan, xylanase production from A. saponilacus was 3.2-fold greater than that of individual carbon sources of 0.5% (w/v) sucrose or 0.5% (w/v) birch xylan. The xylanse is halostable and exhibits optimal activity over a broad salt concentration (2–6% NaCl). Its activity increased approximately 1.16-fold by adding 0.2% (v/v) Tween 20. To understand the potential genetic mechanisms of xylan degradation and molecular adaptation to saline-alkali extremes, the complete genome sequence of A. saponilacus was performed with the pacBio single-molecule real-time (SMRT) and Illumina Misseq platforms. The genome contained one chromosome with a total size of 4,775,573 bps, and a G+C genomic content of 39.27%. Ten genes relating to the pathway for complete xylan degradation were systematically identified. Furthermore, various genes were predicted to be involved in isosmotic cytoplasm via the “compatible-solutes strategy” and cytoplasmic pH homeostasis though the “influx of hydrogen ions”. The halostable xylanase from A. saponilacus and its genomic sequence information provide some insight for potential applications in industry under double extreme conditions. Full article

Halophilic archaea thrive in hypersaline conditions associated with desiccation, ultraviolet (UV) irradiation and redox active compounds, and thus are naturally tolerant to a variety of stresses. Here, we identified mutations that promote enhanced tolerance of halophilic archaea to redox-active compounds using Haloferax volcanii as a model organism. The strains were isolated from a library of random transposon mutants for growth on high doses of sodium hypochlorite (NaOCl), an agent that forms hypochlorous acid (HOCl) and other redox acid compounds common to aqueous environments of high concentrations of chloride. The transposon insertion site in each of twenty isolated clones was mapped using the following: (i) inverse nested two-step PCR (INT-PCR) and (ii) semi-random two-step PCR (ST-PCR). Genes that were found to be disrupted in hypertolerant strains were associated with lysine deacetylation, proteasomes, transporters, polyamine biosynthesis, electron transfer, and other cellular processes. Further analysis revealed a ΔpsmA1 (α1) markerless deletion strain that produces only the α2 and β proteins of 20S proteasomes was hypertolerant to hypochlorite stress compared with wild type, which produces α1, α2, and β proteins. The results of this study provide new insights into archaeal tolerance of redox active compounds such as hypochlorite. Full article

The halophilic myohalovirus Halobacterium virus phiH (ΦH) was first described in 1982 and was isolated from a spontaneously lysed culture of Halobacterium salinarum strain R1. Until 1994, it was used extensively as a model to study the molecular genetics of haloarchaea, but only parts of the viral genome were sequenced during this period. Using Sanger sequencing combined with high-coverage Illumina sequencing, the full genome sequence of the major variant (phiH1) of this halovirus has been determined. The dsDNA genome is 58,072 bp in length and carries 97 protein-coding genes. We have integrated this information with the previously described transcription mapping data. PhiH could be classified into Myoviridae Type1, Cluster 4 based on capsid assembly and structural proteins (VIRFAM). The closest relative was Natrialba virus phiCh1 (φCh1), which shared 63% nucleotide identity and displayed a high level of gene synteny. This close relationship was supported by phylogenetic tree reconstructions. The complete sequence of this historically important virus will allow its inclusion in studies of comparative genomics and virus diversity. Full article

In the past decades, the study of microbial life through shotgun metagenomic sequencing has rapidly expanded our understanding of environmental, synthetic, and clinical microbial communities. Here, we review how shotgun metagenomics has affected the field of halophilic microbial ecology, including functional potential reconstruction, virus–host interactions, pathway selection, strain dispersal, and novel genome discoveries. However, there still remain pitfalls and limitations from conventional metagenomic analysis being applied to halophilic microbial communities. Deconvolution of halophilic metagenomes has been difficult due to the high G + C content of these microbiomes and their high intraspecific diversity, which has made both metagenomic assembly and binning a challenge. Halophiles are also underrepresented in public genome databases, which in turn slows progress. With this in mind, this review proposes experimental and analytical strategies to overcome the challenges specific to the halophilic microbiome, from experimental designs to data acquisition and the computational analysis of metagenomic sequences. Finally, we speculate about the potential applications of other next-generation sequencing technologies in halophilic communities. RNA sequencing, long-read technologies, and chromosome conformation assays, not initially intended for microbiomes, are becoming available in the study of microbial communities. Together with recent analytical advancements, these new methods and technologies have the potential to rapidly advance the field of halophile research. Full article