Development of a genetic system for Haloferax gibbonsii LR2-5, model host for haloarchaeal viruses.

Archaeal viruses are among the most enigmatic members of the virosphere, and their diverse morphologies raise many questions about their infection mechanisms. The study of molecular mechanisms underlying virus-host interactions hinges upon robust model organisms with a system for gene expression and deletion. Currently, there are only a limited number of archaea that have associated viruses and have a well-developed genetic system. Here, we report the development of a genetic system for the euryarchaeon Haloferax gibbonsii LR2-5. This strain can be infected by multiple viruses and is a model for the study of virus-host interactions. We created a Hfx. gibbonsii LR2-5 ∆pyrE strain, resulting in uracil auxotrophy, which could be used as a selection marker. An expression plasmid carrying a pyrE gene from the well-established Haloferax volcanii system was tested for functionality. Expression of a GFP-MinD fusion under a tryptophan inducible promoter was fully functional and showed similar cellular localization as in Hfx. volcanii. Thus, the plasmids of the Hfx. volcanii system can be used directly for the Hfx. gibbonsii LR2-5 genetic system, facilitating the transfer of tools between the two. Finally, we tested for the functionality of gene deletions by knocking out two genes of the archaeal motility structure, the archaellum. These deletion mutants were as expected non-motile and the phenotype of one deletion could be rescued by the expression of the deleted archaellum gene from a plasmid. Thus, we developed a functional genetic toolbox for the euryarchaeal virus host Hfx. gibbonsii LR2-5, which will propel future studies on archaeal viruses. IMPORTANCE: Species from all domains of life are infected by viruses. In some environments, viruses outnumber their microbial hosts by a factor of 10, and viruses are the most important predators of microorganisms. While much has been discovered about the infection mechanisms of bacterial and eukaryotic viruses, archaeal viruses remain understudied. Good model systems are needed to study their virus-host interactions in detail. The salt-loving archaeon Haloferax gibbonsii LR2-5 has been shown to be infected by a variety of different viruses and, thus, is an excellent model to study archaeal viruses. By establishing a genetic system, we have significantly expanded the toolbox for this model organism, which will fuel our understanding of infection strategies of the underexplored archaeal viruses.

Genome-wide transcriptional response to silver stress in extremely halophilic archaeon Haloferax alexandrinus DSM 27206 T.

BACKGROUND: The extremely halophilic archaeon Haloferax (Hfx.) alexandrinus DSM 27206 T was previously documented for the ability to biosynthesize silver nanoparticles while mechanisms underlying its silver tolerance were overlooked. In the current study, we aimed to assess the transcriptional response of this haloarchaeon to varying concentrations of silver, seeking a comprehensive understanding of the molecular determinants underpinning its heavy metal tolerance. RESULTS: The growth curves confirmed the capacity of Hfx. alexandrinus to surmount silver stress, while the SEM-EDS analysis illustrated the presence of silver nanoparticles in cultures exposed to 0.5 mM silver nitrate. The RNA-Seq based transcriptomic analysis of Hfx. alexandrinus cells exposed to 0.1, 0.25, and 0.5 mM silver nitrate revealed the differential expression of multiple sets of genes potentially employed in heavy-metal stress response, genes mostly related to metal transporters, basic metabolism, oxidative stress response and cellular motility. The RT-qPCR analysis of selected transcripts was conducted to verify and validate the generated RNA-Seq data. CONCLUSIONS: Our results indicated that copA, encoding the copper ATPase, is essential for the survival of Hfx. alexandrinus cells in silver-containing saline media. The silver-exposed cultures underwent several metabolic adjustments that enabled the activation of enzymes involved in the oxidative stress response and impairment of the cellular movement capacity. To our knowledge, this study represents the first comprehensive analysis of gene expression in halophillic archaea facing increased levels of heavy metals.

Archaeal Host Cell Recognition and Viral Binding of HFTV1 to Its Haloferax Host.

Viruses are highly abundant and the main predator of microorganisms. Microorganisms of each domain of life are infected by dedicated viruses. Viruses infecting archaea are genomically and structurally highly diverse. Archaea are undersampled for viruses in comparison with bacteria and eukaryotes. Consequently, the infection mechanisms of archaeal viruses are largely unknown, and most available knowledge stems from viruses infecting a select group of archaea, such as crenarchaea. We employed Haloferax tailed virus 1 (HFTV1) and its host, Haloferax gibbonsii LR2-5, to study viral infection in euryarchaea. We found that HFTV1, which has a siphovirus morphology, is virulent, and interestingly, viral particles adsorb to their host several orders of magnitude faster than most studied haloarchaeal viruses. As the binding site for infection, HFTV1 uses the cell wall component surface (S)-layer protein. Electron microscopy of infected cells revealed that viral particles often made direct contact with their heads to the cell surface, whereby the virion tails were perpendicular to the surface. This seemingly unfavorable orientation for genome delivery might represent a first reversible contact between virus and cell and could enhance viral adsorption rates. In a next irreversible step, the virion tail is orientated toward the cell surface for genome delivery. With these findings, we uncover parallels between entry mechanisms of archaeal viruses and those of bacterial jumbo phages and bacterial gene transfer agents. IMPORTANCE Archaeal viruses are the most enigmatic members of the virosphere. These viruses infect ubiquitous archaea and display an unusually high structural and genetic diversity. Unraveling their mechanisms of infection will shed light on the question if entry and egress mechanisms are highly conserved between viruses infecting a single domain of life or if these mechanisms are dependent on the morphology of the virus and the growth conditions of the host. We studied the entry mechanism of the tailed archaeal virus HFTV1. This showed that despite "typical" siphovirus morphology, the infection mechanism is different from standard laboratory models of tailed phages. We observed that particles bound first with their head to the host cell envelope, and, as such, we discovered parallels between archaeal viruses and nonmodel bacteriophages. This work contributes to a better understanding of entry mechanisms of archaeal viruses and a more complete view of microbial viruses in general.

The Viral Susceptibility of the Haloferax Species.

Viruses can infect members of all three domains of life. However, little is known about viruses infecting archaea and the mechanisms that determine their host interactions are poorly understood. Investigations of molecular mechanisms of viral infection rely on genetically accessible virus-host model systems. Euryarchaea belonging to the genus Haloferax are interesting models, as a reliable genetic system and versatile microscopy methods are available. However, only one virus infecting the Haloferax species is currently available. In this study, we tested ~100 haloarchaeal virus isolates for their infectivity on 14 Haloferax strains. From this, we identified 10 virus isolates in total capable of infecting Haloferax strains, which represented myovirus or siphovirus morphotypes. Surprisingly, the only susceptible strain of all 14 tested was Haloferax gibbonsii LR2-5, which serves as an auspicious host for all of these 10 viruses. By applying comparative genomics, we shed light on factors determining the host range of haloarchaeal viruses on Haloferax. We anticipate our study to be a starting point in the study of haloarchaeal virus-host interactions.

Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., low salt-tolerant haloarchaea isolated from seawater and sediment.

Three novel halophilic archaea were isolated from seawater and sediment near Yeoungheungdo Island, Republic of Korea. The genome size and G + C content of the isolates MBLA0076T, MBLA0077T, and MBLA0078T were 3.56, 3.48, and 3.48 Mb and 61.7, 60.8, and 61.1 mol%, respectively. The three strains shared 98.5-99.5 % sequence similarity of the 16 S rRNA gene, whereas their sequence similarity to the 16 S rRNA gene of type strains was below 98.5 %. Phylogenetic analysis based on sequences of the 16 S rRNA and RNA polymerase subunit beta genes indicated that the isolates belonged to the genus Haloferax. The orthologous average nucleotide identity, average amino-acid identity, and in silico DNA-DNA hybridization values were below species delineation thresholds. Pan-genomic analysis indicated that the three novel strains and 11 reference strains had 8981 pan-orthologous groups in total. Fourteen Haloferax strains shared 1766 core pan-genome orthologous groups, which were mainly related to amino acid transport and metabolism. Cells of the three isolates were gram-negative, motile, red-pink pigmented, and pleomorphic. The strains grew optimally at 30 °C (MBLA0076T) and 40 °C (MBLA0077T, MBLA0078T) in the presence of 1.28 M (MBLA0077T) and 1.7 M (MBLA0076T, MBLA0078T) NaCl and 0.1 M (MBLA0077T), 0.2 M (MBLA0076T), and 0.3 M (MBLA0078T) MgCl2·6H2O at pH 7.0-8.0. Cells of all isolates lysed in distilled water; the minimum NaCl concentration necessary to prevent lysis was 0.43 M. The major polar lipids of the three strains were phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, and sulphated diglycosyl archaeol-1. Based on their phenotypic and genotypic properties, MBLA0076T, MBLA0077T, and MBLA0078T were described as novel species of Haloferax, for which we propose the names Haloferax litoreum sp. nov., Haloferax marinisediminis sp. nov., and Haloferax marinum sp. nov., respectively. The respective type strains of these species are MBLA0076T (= KCTC 4288T = JCM 34,169T), MBLA0077T (= KCTC 4289T = JCM 34,170T), and MBLA0078T (= KCTC 4290T = JCM 34,171T).

Ubiquitousness of Haloferax and Carotenoid Producing Genes in Arabian Sea Coastal Biosystems of India.

This study presents a comparative analysis of halophiles from the global open sea and coastal biosystems through shotgun metagenomes (n = 209) retrieved from public repositories. The open sea was significantly enriched with Prochlorococcus and Candidatus pelagibacter. Meanwhile, coastal biosystems were dominated by Marinobacter and Alcanivorax. Halophilic archaea Haloarcula and Haloquandratum, predominant in the coastal biosystem, were significantly (p < 0.05) enriched in coastal biosystems compared to the open sea. Analysis of whole genomes (n = 23,540), retrieved from EzBioCloud, detected crtI in 64.66% of genomes, while cruF was observed in 1.69% Bacteria and 40.75% Archaea. We further confirmed the viability and carotenoid pigment production by pure culture isolation (n = 1351) of extreme halophiles from sediments (n = 410 × 3) sampling at the Arabian coastline of India. All red-pigmented isolates were represented exclusively by Haloferax, resistant to saturated NaCl (6 M), and had >60% G + C content. Multidrug resistance to tetracycline, gentamicin, ampicillin, and chloramphenicol were also observed. Our study showed that coastal biosystems could be more suited for bioprospection of halophiles rather than the open sea.

Production and characterization of a bioactive extracellular homopolysaccharide produced by Haloferax sp. BKW301.

The production of extracellular polysaccharides (EPS) by haloarchaeal members, with novel and unusual physicochemical properties, is of special importance and has the potential for extensive biotechnological exploitation. An extremely halophilic archaeon, Haloferax sp. BKW301 (GenBank Accession No. KT240044) isolated from a solar saltern of Baksal, West Bengal, India has been optimized for the production of EPS under batch culture. It produced a considerable amount (5.95 g/L) of EPS in the medium for halophiles with 15% NaCl, 3% glucose, 0.5% yeast extract, and 6% inoculum under shake flask culture at 120 rpm. The purified EPS, a homopolymer of galactose as revealed by chromatographic methods and Fourier-transform infrared spectroscopy, is noncrystalline (CIxrd , 0.82), amorphous, and could emulsify hydrocarbons like kerosene, petrol, xylene, and so forth. Moreover, the polymer is highly thermostable (up to 420°C) and displayed pseudoplastic rheology. Biologically, the EPS was able to scavenge DPPH (2,2-diphenyl-1-picrylhydrazyl) radical efficiently and inhibit the proliferation of the Huh-7 cell line at an IC50 value of 6.25 µg/ml with a Hill coefficient of 0.844. Large-scale production of this thermostable, pseudoplastic homopolysaccharide, therefore, could find suitable applications in industry and biotechnology.

Specificity of protein-DNA interactions in hypersaline environment: structural studies on complexes of Halobacterium salinarum oxidative stress-dependent protein hsRosR.

Interactions between proteins and DNA are crucial for all biological systems. Many studies have shown the dependence of protein-DNA interactions on the surrounding salt concentration. How these interactions are maintained in the hypersaline environments that halophiles inhabit remains puzzling. Towards solving this enigma, we identified the DNA motif recognized by the Halobactrium salinarum ROS-dependent transcription factor (hsRosR), determined the structure of several hsRosR-DNA complexes and investigated the DNA-binding process under extreme high-salt conditions. The picture that emerges from this work contributes to our understanding of the principles underlying the interplay between electrostatic interactions and salt-mediated protein-DNA interactions in an ionic environment characterized by molar salt concentrations.

Division plane placement in pleomorphic archaea is dynamically coupled to cell shape.

One mechanism for achieving accurate placement of the cell division machinery is via Turing patterns, where nonlinear molecular interactions spontaneously produce spatiotemporal concentration gradients. The resulting patterns are dictated by cell shape. For example, the Min system of Escherichia coli shows spatiotemporal oscillation between cell poles, leaving a mid-cell zone for division. The universality of pattern-forming mechanisms in divisome placement is currently unclear. We examined the location of the division plane in two pleomorphic archaea, Haloferax volcanii and Haloarcula japonica, and showed that it correlates with the predictions of Turing patterning. Time-lapse analysis of H. volcanii shows that divisome locations after successive rounds of division are dynamically determined by daughter cell shape. For H. volcanii, we show that the location of DNA does not influence division plane location, ruling out nucleoid occlusion. Triangular cells provide a stringent test for Turing patterning, where there is a bifurcation in division plane orientation. For the two archaea examined, most triangular cells divide as predicted by a Turing mechanism; however, in some cases multiple division planes are observed resulting in cells dividing into three viable progeny. Our results suggest that the division site placement is consistent with a Turing patterning system in these archaea.

Discovery of bacteriorhodopsins in Haloarchaeal species isolated from Indian solar salterns: deciphering the role of the N-terminal residues in protein folding and functional expression.

Interesting optical and photochemical properties make microbial rhodopsin a promising biological material suitable for various applications, but the cost-prohibitive nature of production has limited its commercialization. The aim of this study was to explore the natural biodiversity of Indian solar salterns to isolate natural bacteriorhodopsin (BR) variants that can be functionally expressed in Escherichia coli. In this study, we report the isolation, functional expression and purification of BRs from three pigmented haloarchaea, wsp3 (water sample Pondicherry), wsp5 and K1T isolated from two Indian solar salterns. The results of the 16S rRNA data analysis suggest that wsp3, wsp5 and K1T are novel strains belonging to the genera Halogeometricum, Haloferax and Haloarcula respectively. Overall, the results of our study suggest that 17 N-terminal residues, that were not included in the gene annotation of the close sequence homologues, are essential for functional expression of BRs. The primary sequence, secondary structural content, thermal stability and absorbance spectral properties of these recombinant BRs are similar to those of the previously reported Haloarcula marismortui HmBRI. This study demonstrates the cost-effective, functional expression of BRs isolated from haloarchaeal species using E. coli as an expression host and paves the way for feasibility studies for future applications.

The nuts and bolts of the Haloferax CRISPR-Cas system I-B.

Invading genetic elements pose a constant threat to prokaryotic survival, requiring an effective defence. Eleven years ago, the arsenal of known defence mechanisms was expanded by the discovery of the CRISPR-Cas system. Although CRISPR-Cas is present in the majority of archaea, research often focuses on bacterial models. Here, we provide a perspective based on insights gained studying CRISPR-Cas system I-B of the archaeon Haloferax volcanii. The system relies on more than 50 different crRNAs, whose stability and maintenance critically depend on the proteins Cas5 and Cas7, which bind the crRNA and form the Cascade complex. The interference machinery requires a seed sequence and can interact with multiple PAM sequences. H. volcanii stands out as the first example of an organism that can tolerate autoimmunity via the CRISPR-Cas system while maintaining a constitutively active system. In addition, the H. volcanii system was successfully developed into a tool for gene regulation.

A Cobalamin Activity-Based Probe Enables Microbial Cell Growth and Finds New Cobalamin-Protein Interactions across Domains.

Understanding the factors that regulate microbe function and microbial community assembly, function, and fitness is a grand challenge. A critical factor and an important enzyme cofactor and regulator of gene expression is cobalamin (vitamin B12). Our knowledge of the roles of vitamin B12 is limited, because technologies that enable in situ characterization of microbial metabolism and gene regulation with minimal impact on cell physiology are needed. To meet this need, we show that a synthetic probe mimic of B12 supports the growth of B12-auxotrophic bacteria and archaea. We demonstrate that a B12 activity-based probe (B12-ABP) is actively transported into Escherichia coli cells and converted to adenosyl-B12-ABP akin to native B12 Identification of the proteins that bind the B12-ABP in vivo in E. coli, a Rhodobacteraceae sp. and Haloferax volcanii, demonstrate the specificity for known and novel B12 protein targets. The B12-ABP also regulates the B12 dependent RNA riboswitch btuB and the transcription factor EutR. Our results demonstrate a new approach to gain knowledge about the role of B12 in microbe functions. Our approach provides a powerful nondisruptive tool to analyze B12 interactions in living cells and can be used to discover the role of B12 in diverse microbial systems.IMPORTANCE We demonstrate that a cobalamin chemical probe can be used to investigate in vivo roles of vitamin B12 in microbial growth and regulation by supporting the growth of B12 auxotrophic bacteria and archaea, enabling biological activity with three different cell macromolecules (RNA, DNA, and proteins), and facilitating functional proteomics to characterize B12-protein interactions. The B12-ABP is both transcriptionally and translationally able to regulate gene expression analogous to natural vitamin B12 The application of the B12-ABP at biologically relevant concentrations facilitates a unique way to measure B12 microbial dynamics and identify new B12 protein targets in bacteria and archaea. We demonstrate that the B12-ABP can be used to identify in vivo protein interactions across diverse microbes, from E. coli to microbes isolated from naturally occurring phototrophic biofilms to the salt-tolerant archaea Haloferax volcanii.

Genome sequence and description of Haloferax massiliense sp. nov., a new halophilic archaeon isolated from the human gut.

By applying the culturomics concept and using culture conditions containing a high salt concentration, we herein isolated the first known halophilic archaeon colonizing the human gut. Here we described its phenotypic and biochemical characterization as well as its genome annotation. Strain Arc-HrT (= CSUR P0974 = CECT 9307) was mesophile and grew optimally at 37 °C and pH 7. Strain Arc-HrT was also extremely halophilic with an optimal growth observed at 15% NaCl. It showed gram-negative cocci, was strictly aerobic, non-motile and non-spore-forming, and exhibited catalase and oxidase activities. The 4,015,175 bp long genome exhibits a G + C% content of 65.36% and contains 3911 protein-coding and 64 predicted RNA genes. PCR-amplified 16S rRNA gene of strain Arc-HrT yielded a 99.2% sequence similarity with Haloferax prahovense, the phylogenetically closest validly published species in the Haloferax genus. The DDH was of 50.70 ± 5.2% with H. prahovense, 53.70 ± 2.69% with H. volcanii, 50.90 ± 2.64% with H. alexandrinus, 52.90 ± 2.67% with H. gibbonsii and 54.30 ± 2.70% with H. lucentense. The data herein represented confirm strain Arc-HrT as a unique species and consequently we propose its classification as representative of a novel species belonging to the genus Haloferax, as Haloferax massiliense sp. nov.

Transcription-coupled repair of UV damage in the halophilic archaea.

Transcription-coupled repair (TCR) is a subpathway of nucleotide excision repair (NER) in which excision repair proteins are targeted to RNA polymerase-arresting lesions located in the transcribed strand of active genes. TCR has been documented in a variety of bacterial and eukaryotic organisms but has yet to be observed in the Archaea. We used Halobacterium sp. NRC-1 and Haloferax volcanii to determine if TCR occurs in the halophilic archaea. Following UV irradiation of exponentially growing cultures, we quantified the rate of repair of cyclobutane pyrimidine dimers in the two strands of the rpoB2B1A1A2 and the trpDFEG operons of Halobacterium sp. NRC-1 and the pts operon of H. volcanii through the use of a Southern blot assay and strand-specific probes. TCR was observed in all three operons and was dependent on the NER gene uvrA in Halobacterium sp. NRC-1, but not in H. volcanii. The halophilic archaea likely employ a novel mechanism for TCR in which an as yet unknown coupling factor recognizes the arrested archaeal RNA polymerase complex and recruits certain NER proteins to complete the process.

Isolation and characterization of extremely halophilic CO-oxidizing Euryarchaeota from hypersaline cinders, sediments and soils and description of a novel CO oxidizer, Haloferax namakaokahaiae Mke2.3T, sp. nov.

The phylogenetic affiliations of organisms responsible for aerobic CO oxidation in hypersaline soils and sediments were assessed using media containing 3.8 M NaCl. CO-oxidizing strains of the euryarchaeotes, Haloarcula, Halorubrum, Haloterrigena and Natronorubrum, were isolated from the Bonneville Salt Flats (UT) and Atacama Desert salterns (Chile). A halophilic euryarchaeote, Haloferax strain Mke2.3(T), was isolated from Hawai'i Island saline cinders. Haloferax strain Mke2.3(T) was most closely related to Haloferax larsenii JCM 13917(T) (97.0% 16S rRNA sequence identity). It grew with a limited range of substrates, and oxidized CO at a headspace concentration of 0.1%. However, it did not grow with CO as a sole carbon and energy source. Its ability to oxidize CO, its polar lipid composition, substrate utilization and numerous other traits distinguished it from H. larsenii JCM 13917(T), and supported designation of the novel isolate as Haloferax namakaokahaiae Mke2.3(T), sp. nov (= DSM 29988, = LMG 29162). CO oxidation was also documented for 'Natronorubrum thiooxidans' HG1 (Sorokin, Tourova and Muyzer 2005), N. bangense (Xu, Zhou and Tian 1999) and N. sulfidifaciens AD2(T) (Cui et al. 2007). Collectively, these results established a previously unsuspected capacity for extremely halophilic aerobic CO oxidation, and indicated that the trait might be widespread among the Halobacteriaceae, and occur in a wide range of hypersaline habitats.

Sequence analysis and minimal replicon determination of a new haloarchaeal plasmid pHF2 isolated from Haloferax sp. strain Q22.

A new cryptic plasmid, pHF2 (2520 bp), was isolated from Haloferax sp. strain Q22 (CGMCC 1.15317), a haloarchaeal strain living in a subterranean halite deposit. Sequence analysis revealed that it is the smallest plasmid in the genus Haloferax so far, and three syntropic open reading frames (ORF1, ORF2, and ORF3) were identified on the same strand. ORF1 encodes a putative replication initiation protein (Rep). Three typical motifs (I, II, and III) were presented in the Rep proteins of rolling-circle replicating (RCR) plasmids. The amino acid sequence of the Rep protein is very similar to that of another haloarchaeal plasmid pNB101 in Natronobacterium sp. AS-7091 (coverage 97%, identity 56%). The minimal replicon (~1000 bp) of pHF2 was determined through the construction of a series of truncated plasmids. Interestingly, we also found that the incomplete rep gene still can drive plasmid replication. This plasmid has provided another valuable extra-chromosomal genetic resource, and deepened our knowledge in DNA replication.

Complete genome sequence of Haloferax gibbonsii strain ARA6, a potential producer of polyhydroxyalkanoates and halocins isolated from Araruama, Rio de Janeiro, Brasil.

Haloferax gibbonsii strain ARA6 is a haloarchaea isolated from saline saltern samples from Vermelha lake, located in Araruama region, Rio de Janeiro, Brazil. Its genome displays 66,2% G+C content and is composed by one circular chromosome of 2,945,391 bp and four circular plasmids comprising 993,063 bp. This genomic information shows H. gibbonsii's potential for biotechnological applications and can also contribute to assign evolutionary traits in the genus Haloferax.

Identification and codon reading properties of 5-cyanomethyl uridine, a new modified nucleoside found in the anticodon wobble position of mutant haloarchaeal isoleucine tRNAs.

Most archaea and bacteria use a modified C in the anticodon wobble position of isoleucine tRNA to base pair with A but not with G of the mRNA. This allows the tRNA to read the isoleucine codon AUA without also reading the methionine codon AUG. To understand why a modified C, and not U or modified U, is used to base pair with A, we mutated the C34 in the anticodon of Haloarcula marismortui isoleucine tRNA (tRNA2(Ile)) to U, expressed the mutant tRNA in Haloferax volcanii, and purified and analyzed the tRNA. Ribosome binding experiments show that although the wild-type tRNA2(Ile) binds exclusively to the isoleucine codon AUA, the mutant tRNA binds not only to AUA but also to AUU, another isoleucine codon, and to AUG, a methionine codon. The G34 to U mutant in the anticodon of another H. marismortui isoleucine tRNA species showed similar codon binding properties. Binding of the mutant tRNA to AUG could lead to misreading of the AUG codon and insertion of isoleucine in place of methionine. This result would explain why most archaea and bacteria do not normally use U or a modified U in the anticodon wobble position of isoleucine tRNA for reading the codon AUA. Biochemical and mass spectrometric analyses of the mutant tRNAs have led to the discovery of a new modified nucleoside, 5-cyanomethyl U in the anticodon wobble position of the mutant tRNAs. 5-Cyanomethyl U is present in total tRNAs from euryarchaea but not in crenarchaea, eubacteria, or eukaryotes.

Mapping of active replication origins in vivo in thaum- and euryarchaeal replicons.

We report mapping of active replication origins in thaum- and euryarchaeal replicons using high-throughput sequencing-based marker frequency analysis. The chromosome of the thaumarchaeon Nitrosopumilus maritimus is shown to contain a single origin of replication, whereas the main chromosome in the halophilic euryarchaea Haloferax mediterranei and Haloferax volcanii each contains two origins. All replication origins specified bidirectional replication, and the two origins in the halophiles were initiated in synchrony. The pHM500 plasmid of H. mediterranei is shown to contain a single origin, and the copy numbers of five plasmid replicons in the two halophiles were inferred to be close to that of the main chromosome. Origin recognition boxes (ORBs) that provide binding sites for Orc1/Cdc6 replication initiator proteins are identified at all chromosomal origins, as well as in a range of additional thaumarchaeal species. An annotation update is provided for all three species.

Accumulation of polyhydroxyalkanoates by halophilic archaea isolated from traditional solar salterns of India.

Extremely halophilic archaeal isolates obtained from brine and sediment samples of solar salterns of Goa and Tamil Nadu, India were screened for accumulation of polyhydroxyalkanoates (PHA). Seven polymer accumulating haloarchaeal strains (TN4, TN5, TN6, TN7, TN9, TN10 and BBK2) were selected based on their growth and intensity of fluorescence when grown on 20 % NaCl synthetic medium supplemented with 2 % glucose and incorporated with Nile red dye. The polymer was quantified by conversion of PHA to crotonic acid which gave a characteristic absorption maxima at 235 nm. On the basis of phenotypic and genotypic characterization the cultures TN4, TN5, TN6, TN7, TN10 and BBK2 were grouped under genus Haloferax whereas isolate TN9 was grouped under the genus Halogeometricum. Growth kinetics and polymer accumulation studies revealed that the culture Halogeometricum borinquense strain TN9 accumulates PHA maximally at the mid-log phase, i.e. 5th day of growth (approx. 14 wt% PHA of CDW). Analysis of the polymer by IR, (1)H NMR and (13)C NMR confirmed it to be a homopolymer of 3-hydroxybutyrate.