Halobacterium NRC-1 possesses the smallest genome to date among halophiles. It is 2,571,010 bp in size, and is composed of a large GC-rich chromosome (2,014,239 bp, 68 % G+C), and two smaller extrachromosomal replicons, pNRC100 (191,346 bp) and pNRC200 (365,425 bp), with 58–59 % G+C composition 91011. The two smaller replicons contain 145,428 bp of identical DNA and 33–39 kb inverted repeats catalyzing inversion isomers 27, and the majority of the 91 IS elements, representing 12 families, found in the genome 1011. As a result of the large number of repeated sequences, genome assembly required extensive genomic mapping and an ordered clone library of pNRC100 91027. Of the 2,630 likely protein-coding genes in the genome, 2,532 are unique. Halobacterium predicted proteins were found to be highly acidic 27 and a substantial number had bacterial homologs as their closest relatives, suggesting that they might have been acquired through lateral gene transfer 28. In addition, 52 RNA genes were also identified; however, the 16S rRNA sequence and other unique characteristics did not allow placement within a validly described Halobacterium species, and this point has been the subject of some controversy 293031. Interestingly, about 40 genes in pNRC100 and pNRC200 code for functions likely to be essential or important for cell viability (e.g. thioredoxin and thioredoxin reductase, a cytochrome oxidase, a DNA polymerase, multiple TATA-binding proteins (TBP) and transcription factor B (TFB) transcription factors, and the only arginyl-tRNA synthetase in the genome). As a result, these replicons were suggested to be essential "minichromosomes" rather than megaplasmids 9. Several reports on annotation of the Halobacterium sp. NRC-1 genome have been published 91011.

Halobacterium sp. NRC-1 provided the first opportunity for isolating autonomously replicating sequences from an archaeon and this was accomplished for both the chromosome and the large extrachromosomal replicon, pNRC100 3243. The replicating sequences, putative in vivo origins of replication, were isolated and cloned using their ability to endow autonomous replication ability on E. coli plasmids containing a selectable mevinolin resistance (Mev^r) marker. For the chromosome, a directed approach was taken to investigate whether chromosomal loci proximal to orc/cdc6 homologs possessed autonomous replication ability. A region of 2.3 kb containing the orc7 gene (one of ten eukaryotic-type origin recognition complex homologs), plus 750 bp upstream of the orc7 gene translational start, displayed replication ability 43. A nearly perfect inverted repeat of 33 bp flanking an extremely AT-rich stretch of 189 bp (56% GC) was found in the upstream region. This ori region corresponded to one of two chromosomal inflection points in cumulative GC-skew analysis (which has been used for bioinformatic prediction of replication origins) 2844. The ori region was also conserved in the genome sequences of other halophilic archaea, including Haloarcula marismortui and Haloferax volcanii 43. However, the region surrounding another orc gene near a second inflection point was not able to confer replication ability, suggesting the existence of only a single chromosomal origin in Halobacterium sp. NRC-1.

Previously, an autonomously replicating region of pNRC100, containing a 550 bp AT-rich region upstream of the repH gene, was isolated using similar methodology 32. However, in this case, the nature of the gene and the upstream region was unlike that found on any other replicon, except pHH, a closely related plasmid in another Halobacterium strain 45. For the pNRC100 replication region, autonomous replication was disrupted by linker scanning mutagenesis in repH, which was necessary for plasmid replication; however, the upstream region could be interrupted and partially deleted without knocking out replication ability. Genome sequencing showed that the repH gene region is present in both pNRC100 and pNRC200, and therefore may be involved in the replication of both replicons. Interestingly, the latter replicon also contains multiple orc gene homologs, the functions of which are unknown 91011. In addition to providing a genetic approach to DNA replication studies in archaea, the availability of Halobacterium replicating sequences has facilitated the development of vectors, including both shuttle plasmids and expression vectors 19.

The number of replication origins and their coordination in Halobacterium sp. NRC-1 has been of significant interest. The euryarchaeon, Pyrococcus abyssi, was shown to contain a single chromosomal replication origin composed of similar large inverted repeats 5' to the only orc/cdc6 gene of this archaeon 4647. However, the crenarchaea, Sulfolobus solfataricus and S. acidocaldarius, were reported to contain two or three chromosomal replication origins proximal to multiple orc/cdc6 genes in their genomes 4849. In Halobacterium sp. NRC-1, a euryarchaeon, the roles and relationships of multiple (10) orc/cdc6 and repH genes in replication require further experimentation for complete understanding.

Repair of DNA damage has been investigated in Halobacterium sp. NRC-1 because of the observed high levels of radiation resistance and the presence of homologs of both bacterial and eukaryotic-type repair genes 50. For UV resistance, biochemical and genetic work demonstrated the presence of cyclobutane pyrimidine dimer photolyase activity, corresponding to one of two phr gene homologs in the genome 51. Photolyase catalyzes the photoreversal of primary photoadducts from UV radiation, a process called photoreactivation, which is widely distributed in nature. In two DNA microarray studies, the transcriptional response of cells to UV irradiation was studied 2426; however, different results were obtained. In one study using 30–70 J/m², specific induction of radA1 and replication factor A genes (rfa3, rfa8, and ral) was observed 26. In the other study, using substantially higher doses, a large number of unexplained changes resulted, probably caused by an overwhelming of the cellular repair systems and other physiological perturbations that were introduced 24. In a biochemical study of the crenarchaeon, Sulfolobus solfataricus, replication factor A was found to bind to UV damaged DNA 52. In a study of responses to high energy radiation (γ) and desiccation, both of which lead to extensive double-strand DNA breaks, resistance to these conditions was correlated, and protective effects of salts and membrane pigments were observed 53.

The purple membrane of Halobacterium sp. NRC-1 contains the light-driven proton pump, bacteriorhodopsin, a complex of a protein, bacterio-opsin (bop gene product), and a retinal chromophore (Fig. 1). The purple membrane allows cells to grow phototrophically under high illumination, which may be important for survival under microaerobic and other stressful conditions 554. A combination of genetic, bioinformatic, and transcriptional analysis showed the involvement of the bop gene cluster 2354. The cluster includes, in addition to bop, crtB1 and brp, coding the first and last committed steps of retinal synthesis, blp, a gene of unknown function, and bat, the sensor-activator gene. The Bat sequence predicted a complex protein consisting of a GAF (cGMP-binding) domain, PAS/PAC (redox-sensing) domain, and C-terminal DNA-binding helix-turn-helix (HTH) motif 5455. Additional Bat-like putative regulatory genes have been found in the genome, and together are likely to be responsible for the complex response of this archaeon to light and oxygen. The role of brp in the oxidative cleavage of β-carotene to form retinal was shown using the ura3-based gene knockout system, which also led to the identification of an unlinked gene, blh, capable of performing the same function 21. Another unlinked gene involved in retinal biosynthesis, encoding lycopene cyclase (crtY), was also identified by a genetic knockout 56.

The bop gene promoter region was studied by a combination of saturation mutagenesis and bioinformatic analysis 57585960. The TATA-box sequence in the promoter deviates significantly from the archaeal consensus, suggesting the involvement of novel transcription factors in its recognition 58. In this context, the finding of 6 Tbp (TATA-binding protein) genes and 7 Tfb (transcription factor B) genes in the Halobacterium sp. NRC-1 genome suggested the use of alternative Tbp-Tfb pairs in promoter selection and transcriptional regulation, similar to that proposed for higher organisms 6061. Mutagenesis also revealed a regulatory site, UAS, 5' to bop 59. Additionally, UAS sites were found near two retinal synthesis genes, brp and crtB1, which are coordinately regulated with bop 54. Similarities of the UAS and Bat regulator to diverse organisms, including a plant and a γ-proteobacterium, suggested an ancient origin for this regulon 1154.

The metabolic capability of Halobacterium sp. NRC-1 includes anaerobic respiration using DMSO and TMAO as electron acceptors and fermentation with arginine via the arginine deiminase pathway (Fig. 1) 6263. The capability for anaerobic growth is likely advantageous for the organism in its natural habitat as high salt concentrations and elevated temperatures, together with high cell densities, reduce the availability of molecular oxygen. In a recent study of anaerobic respiration in Halobacterium sp. NRC-1 25, bioinformatic and transcriptional analyses, and gene knockouts showed it to harbor a bifunctional DMSO/TMAO reductase that is encoded by the dmsREABCD operon. This reductase is more closely related to NarG-type nitrate reductases than to bacterial DMSO/TMAO reductases, although phylogenetic analysis was inconclusive about its evolutionary origin. Whole-genome oligonucleotide microarray studies showed that the transcript level of the dms operon is strongly induced under anaerobic conditions 25. Gene knockouts showed that expression of the dms operon is under positive transcriptional control of the regulator, DmsR. The C-terminal region of DmsR contains an HTH DNA-binding motif similar to that of the bop gene activator, Bat 1925. DNA microarray analysis also indicated that Halobacterium stays primed for aerobic respiration even under anaerobic conditions. This regulatory scheme could result in a fast metabolic response to oxygen as electron acceptor, when available. It is complemented by the increased abundance of gas vesicles (see below) under anaerobic conditions, allowing the cells to float to more aerobic zones in the water column. In a different study where Halobacterium strains overproducing or lacking purple membrane (generated by extensive chemical mutagenesis) were compared, it was deduced that during phototrophy, genes required for arginine fermentation are repressed 23.

Gas vesicles are hollow, buoyant protein structures that allow Halobacterium sp. NRC-1 to float, increasing their access to light and oxygen. A large gene cluster on pNRC100 (gvpMLKJIHGFEDACNO) is necessary and sufficient to produce gas vesicles 646566676869. Transcript mapping established the presence of divergent promoters in the gvpD-A intergenic region 6667 and DNA microarrays confirmed that essentially all of the gvp genes are inducible under microaerobic, anaerobic, and other stressful conditions 25. Rightward transcription of gvpA and gvpC, encoding the two most abundant gas vesicle proteins, was induced in early exponential growth phase 69. Transcription was inhibited by aeration and by the addition of a DNA gyrase inhibitor, suggesting that increased DNA supercoiling is important for activation of gvpA transcription. A combination of genetic and bioinformatic analysis suggested that GvpE functions as an activator, with a leucine-zipper motif mediating dimer formation, and that GvpD, which contains an NTP-binding site, likely functions as a repressor by blocking GvpE-mediated activation 6770.

The protein composition of Halobacterium sp. NRC-1 gas vesicles was studied using immunoblotting 71. In addition to GvpA and GvpC, two small acidic polypeptides, GvpJ and GvpM, similar to GvpA were also identified as being present in the structure, and their function in determining vesicle membrane conformation was proposed 71. The GvpC protein, which contains a motif that is repeated 7–8 times, and is predicted to be involved in binding to GvpA, was shown to be present on the surface of vesicles. Recombinant GvpC fusion proteins were found to bind to the surface of gas vesicles and are being used in a biotechnology application as an antigen delivery system 7273. Three additional proteins, GvpF, G, and L, were also observed in Halobacterium NRC-1 gas vesicles, and coiled-coil domains were identified in GvpF and GvpL, suggestive of self-association 71. This was further confirmed by the observation of laddering of GvpL protein on gels. These genetic and biochemical results are consistent with the findings of comparative genomic studies, which showed that the corresponding gvp genes are present in all gas vesicle containing organisms examined 7174757677.

Halobacterium sp. NRC-1 is resistant to arsenic, a heavy metal which is frequently found in hypersaline environments 22. A gene cluster on pNRC100, arsMR2ADR1C, organized into three operons, was shown to be involved through construction and analysis of gene knockouts. Deletion of the arsADRC gene region increased sensitivity to arsenite. However, knockout of a putative arsB gene homolog on the chromosome showed no phenotypic effect, suggesting the existence of a novel arsenite pump in Halobacterium sp. NRC-1. Interestingly, knockout of the arsM gene also produced increased sensitivity to arsenite, indicating a second novel mechanism of arsenic resistance involving an arsenite methyltransferase. The arsenite resistance elements were shown to be regulated, with resistance to arsenic being inducible by exposure to a sublethal concentration of the metal 22. These results are consistent with Halobacterium sp. NRC-1 containing two arsenite detoxification systems.

Coenzyme B₁₂ metabolism has been explored in Halobacterium sp. NRC-1 using a combination of physiological and genetic approaches 78798081. Salvaging of coenzyme B₁₂ precursors from the environment was shown to require a previously unidentified amidohydrolase, the cbiZ gene product, which converts adenosylcobinamide to adenosylcobyric acid, an intermediate of the de novo coenzyme B₁₂ biosynthetic route 80. The salvaging of coenzyme B₁₂ precursors by the CbiZ enzyme appears to be a uniquely archaeal strategy, because all of the genomes of B₁₂-producing archaea have a cbiZ ortholog. Genetic studies showed that uptake of cobalamins for Halobacterium occurs via an ABC transporter similar to the bacterial Btu system 81.

Several additional experimental studies have been carried out on Halobacterium sp. NRC-1 genes, e.g. using Escherichia coli as host for complementation analysis and heterologous expression. An RNase H similar to the Type 1 bacterial enzyme, required for primer removal in DNA replication, was found to suppress the temperature-sensitive growth defect in E. coli, although the basic region present in the E. coli protein is absent in the haloarchaeal protein 82. The haloarchaeal enzyme was also shown to cleave an Okazaki fragment-like substrate, consistent with its function in DNA replication. In another study, the molecular recognition of a tRNA_Cys by the bacterial-type cysteinyl-tRNA synthetase of Halobacterium sp. NRC-1 was studied after expression in E. coli and showed similarities to its bacterial counterpart 83.

Isoelectric point analysis of the predicted proteome of Halobacterium sp. NRC-1 provided deep insight into the ability of this organism to survive under hypersaline conditions 9101128. A dramatically low average pI (~4.9) was found Halobacterium proteins and the generality of this observation was confirmed through analysis of subsequent haloarchaeal genome sequences. In contrast, the median pI of nearly all non-halophilic proteomes has been found to be close to neutral, usually with a bimodal distribution of basic (pI ~10) and acidic (pI ~5) proteins. Notable exceptions were for some methanogenic archaea and an extremely halophilic bacterium, Salinibacter ruber, which sometimes coexist with Halobacterium in hypersaline environments and also contain relatively high internal salt concentrations 848586. The acidity of halophilic proteins likely helps maintain their function through increased solvation in an intracellular milieu with markedly reduced water activity 87.

The acidic pI of Halobacterium proteins was correlated to a high concentration of surface negative charge in modeled structures 28. A transcription factor (TbpE) and a topoisomerase subunit (GyrA) both showed significantly higher surface negative charges when compared to their homologs in non-halophilic organisms using a Coulomb charge calculation. Although there is an overall reduction in deprotonation of acidic residues on the protein surface at 5 M NaCl as the result of a decrease in the dielectric constant 88, the surface charges of most proteins were still highly negative. For example, when the Tbp-Tfb-DNA complex was modeled using a dielectric constant reflecting intracellular conditions, a dramatic difference in surface charges, compared to the human complex, was still observed (Fig. 3). This pattern of increased negative charge and lowered pI was observed for the vast majority of haloarchaeal proteins when compared to their homologs.

In another study, de novo structure prediction was conducted on 1,185 proteins and protein domains of Halobacterium sp. NRC-1 89. Putative functions were predicted by searching the Protein DataBank. Three examples, chemotaxis proteins, possible prophage polypeptides and archaeal transcriptional regulators, were highlighted in the published report.

Initial whole-genome phylogenetic analysis of Halobacterium sp. NRC-1 confirmed the archaeal status of Halobacterium sp. NRC-1 10, but also noted interesting similarities to the Gram-positive spore-forming bacterium, Bacillus subtilis, and the radiation-resistant bacterium, Deinococcus radiodurans. Subsequent whole-genome analysis using a larger number of completed genomes produced phylogenetic trees with Halobacterium branching near the base of the archaeal branch, or even within the bacteria 9091. This was in contrast to phylogenetic analysis based on concatenated sequences of transcription and transcriptional machinery, which produced results similar to 16S rRNA analysis placing Halobacterium within the archaeal clade 92. One interpretation accounting for these incongruent results was that many LGTs have occurred between some bacteria and haloarchaea, although this conclusion appears to be open to some debate (Fig. 4) 93.

In spite of the initial uncertainties, several cases for LGT now appear to be convincing. Some components of the electron transport chain and biosynthetic proteins were found to have gene organization identical to homologous E. coli operons (nuo genes, coding subunits of NADH dehydrogenase; cox genes, coding subunits of cytochrome c oxidase; and men genes, coding for menaquinone biosynthesis) 28. GC composition analysis showed these genes to deviate significantly from nearby Halobacterium genes and a phylogenetic study indicated their grouping with bacterial genes. A further detailed study of the nuoI gene showed clear phylogenetic affinity with proteobacteria 94. These studies suggested that haloarchaea may have adapted to an oxidizing atmosphere by acquiring components of the electron transport chain through LGT events from bacteria 11.

A relatively recent case of LGT is the arginyl synthetase (argS) gene of Halobacterium sp. NRC-1, which is found on pNRC200 9. Although its GC composition did not differ significantly from the genomic average, maximum-likelihood phylogenetic analysis showed that ArgS from Halobacterium does not group with other archaeal (including other haloarchaeal) ArgS proteins, but is most closely related to a class of ArgS proteins from bacteria (Fig. 4). Given the essential nature of arginyl synthetases to protein synthesis, it is likely that a bacterial argS gene was captured and the archaeal argS was subsequently lost in Halobacterium sp. NRC-1. Another example of orthologous gene displacement has been found in studies of lipid biosynthesis genes 95.

One of the most interesting evolutionary questions concerning Halobacterium centers on the origin of the light-driven proton pump, bacteriorhodopsin 11. Such retinal chromoproteins (bacteriorhodopsin, halorhodopsin and sensory rhodopsins) originally discovered in haloarchaea have now been found in diverse bacteria and eukaryotes and therefore may have evolved before the divergence of the three domains of life 96. Alternatively, occurrence in relatively few and diverse clades, e.g. oceanic planktonic bacteria, some fungi, and haloarchaea, suggests dispersal by LGT. Although phylogenetic analysis has thus far been inconclusive, spectroscopic characteristics have suggested the possible co-evolution of retinal with chlorophyll-based pigments 1197.

An interesting study showed the importance of the Tat protein export pathway in Halobacterium sp. NRC-1 9899. This pathway allows transmembrane transport of proteins in a fully folded conformation and is used for export of a moderate number of proteins in bacteria, e.g. those binding cofactors such as iron-sulfur clusters and molybdopterin. However, in Halobacterium sp. NRC-1, the majority of exported proteins are predicted to use the Tat pathway. This has been explained as an adaptation to the highly saline conditions, which may require intracellular folding for protein stability 9899. In another study, several poorly conserved open reading frames (ORFans) of Halobacterium sp. NRC-1, with apparent paralogs in the genome, but with no clear homologs in other organisms, were shown to be transcribed. The results indicated that all of the studied paralogous ORFans corresponded to real genes, including those comprising relatively short proteins 100. Two comparative genomics studies have appeared, one comparing information transfer genes specifically among haloarchaeal species 101, and another comparing stress response genes in Halobacterium sp. NRC-1, E. coli, and Drosophila melanogaster 102.