Halobacterium yunchengense sp. nov., Natronomonas amylolytica sp. nov., Halorientalis halophila sp. nov., Halobellus salinisoli sp. nov., halophilic archaea isolated from a saline lake and inland saline soil.

Four halophilic archaeal strains YCN1T, YCN58T, LT38T, and LT62T were isolated from Yuncheng Salt Lake (Shanxi, China) and Tarim Basin (Xinjiang, China), respectively. Phylogenetic and phylogenomic analyses showed that these four strains tightly cluster with related species of Halobacterium, Natronomonas, Halorientalis, and Halobellus, respectively. The AAI, ANI, and dDDH values between these four strains and their related species of respective genera were lower than the proposed threshold values for species delineation. Strains YCN1T, YCN58T, LT38T, and LT62T could be differentiated from the current species of Halobacterium, Natronomonas, Halorientalis, and Halobellus, respectively, based on the comparison of diverse phenotypic characteristics. The polar lipid profiles of these four strains were closely similar to those of respective relatives within the genera Halobacterium, Natronomonas, Halorientalis, and Halobellus, respectively. The phenotypic, phylogenetic, and genome-based analyses indicated that strains YCN1T, YCN58T, LT38T, and LT62T represent respective novel species within the genera Halobacterium, Natronomonas, Halorentalis, and Halobellus, for which the names Halobacterium yunchengense sp. nov., Natronomonas amylolytica sp. nov., Halorientalis halophila sp. nov., and Halobellus salinisoli sp. nov. are proposed, respectively.

Genome comparison reveals that Halobacterium salinarum 63-R2 is the origin of the twin laboratory strains NRC-1 and R1.

The genome of Halobacterium strain 63-R2 was recently reported and provides the opportunity to resolve long-standing issues regarding the source of two widely used model strains of Halobacterium salinarum, NRC-1 and R1. Strain 63-R2 was isolated in 1934 from a salted buffalo hide (epithet "cutirubra"), along with another strain from a salted cow hide (91-R6T , epithet "salinaria," the type strain of Hbt. salinarum). Both strains belong to the same species according to genome-based taxonomy analysis (TYGS), with chromosome sequences showing 99.64% identity over 1.85 Mb. The chromosome of strain 63-R2 is 99.99% identical to the two laboratory strains NRC-1 and R1, with only five indels, excluding the mobilome. The two reported plasmids of strain 63-R2 share their architecture with plasmids of strain R1 (pHcu43/pHS4, 99.89% identity; pHcu235/pHS3, 100.0% identity). We detected and assembled additional plasmids using PacBio reads deposited at the SRA database, further corroborating that strain differences are minimal. One plasmid, pHcu190 (190,816 bp) corresponds to pHS1 (strain R1) but is even more similar in architecture to pNRC100 (strain NRC-1). Another plasmid, pHcu229, assembled partially and completed in silico (229,124 bp), shares most of its architecture with pHS2 (strain R1). In deviating regions, it corresponds to pNRC200 (strain NRC-1). Further architectural differences between the laboratory strain plasmids are not unique, but are present in strain 63-R2, which contains characteristics from both of them. Based on these observations, it is proposed that the early twentieth-century isolate 63-R2 is the immediate ancestor of the twin laboratory strains NRC-1 and R1.

Bioengineering of air-filled protein nanoparticles by genetic and chemical functionalization.

BACKGROUND: Various bacteria and archaea, including halophilic archaeon Halobacterium sp. NRC-1 produce gas vesicle nanoparticles (GVNPs), a unique class of stable, air-filled intracellular proteinaceous nanostructures. GVNPs are an attractive tool for biotechnological applications due to their readily production, purification, and unique physical properties. GVNPs are spindle- or cylinder-shaped, typically with a length of 100 nm to 1.5 µm and a width of 30-250 nm. Multiple monomeric subunits of GvpA and GvpC proteins form the GVNP shell, and several additional proteins are required as minor structural or assembly proteins. The haloarchaeal genetic system has been successfully used to produce and bioengineer GVNPs by fusing several foreign proteins with GvpC and has shown various applications, such as biocatalysis, diagnostics, bioimaging, drug delivery, and vaccine development. RESULTS: We demonstrated that native GvpC can be removed in a low salt buffer during the GVNP purification, leaving the GvpA-based GVNP's shell intact and stable under physiological conditions. Here, we report a genetic engineering and chemical modification approach for functionalizing the major GVNP protein, GvpA. This novel approach is based on combinatorial cysteine mutagenesis within GvpA and genetic expansion of the N-terminal and C-terminal regions. Consequently, we generated GvpA single, double, and triple cysteine variant libraries and investigated the impact of mutations on the structure and physical shape of the GVNPs formed. We used a thiol-maleimide chemistry strategy to introduce the biotechnological relevant activity by maleimide-activated streptavidin-biotin and maleimide-activated SpyTag003-SpyCatcher003 mediated functionalization of GVNPs. CONCLUSION: The merger of these genetic and chemical functionalization approaches significantly extends these novel protein nanomaterials' bioengineering and functionalization potential to assemble catalytically active proteins, biomaterials, and vaccines onto one nanoparticle in a modular fashion.

Bioengineering of Halobacterium sp. NRC-1 gas vesicle nanoparticles with GvpC fusion protein produced in E. coli.

Gas vesicle nanoparticles (GVNPs) are hollow, buoyant prokaryotic organelles used for cell flotation. GVNPs are encoded by a large gas vesicle protein (gvp) gene cluster in the haloarchaeon, Halobacterium sp. NRC-1, including one gene, gvpC, specifying a protein bound to the surface of the nanoparticles. Genetically engineered GVNPs in the Halobacterium sp. have been produced by fusion of foreign sequences to gvpC. To improve the versatility of the GVNP platform, we developed a method for displaying exogenously produced GvpC fusion proteins on the haloarchaeal nanoparticles. The streptococcal IgG-binding protein domain was fused at or near the C-terminus of GvpC, expressed and purified from E. coli, and shown to bind to wild-type GVNPs. The two fusion proteins, GvpC3GB and GvpC4GB, without or with a highly acidic GvpC C-terminal region, were found to be able to bind nanoparticles equally well. The GVNP-bound GvpC-IgG-binding fusion protein was also capable of binding to an enzyme-linked IgG-HRP complex which retained enzyme activity, demonstrating the hybrid system capability for display and delivery of protein complexes. This is the first report demonstrating functional binding of exogenously produced GvpC fusion proteins to wild-type haloarchaeal GVNPs which significantly expands the capability of the platform to produce bioengineered nanoparticles for biomedical applications. KEY POINTS: • Haloarchaeal gas vesicle nanoparticles (GVNPs) constitute a versatile display system. • GvpC-streptococcal IgG-binding fusion proteins expressed in E. coli bind to GVNPs. • IgG-binding proteins displayed on floating GVNPs bind and display IgG-HRP complex.

Divergent degeneration of creA antitoxin genes from minimal CRISPRs and the convergent strategy of tRNA-sequestering CreT toxins.

Aside from providing adaptive immunity, type I CRISPR-Cas was recently unearthed to employ a noncanonical RNA guide (CreA) to transcriptionally repress an RNA toxin (CreT). Here, we report that, for most archaeal and bacterial CreTA modules, the creA gene actually carries two flanking 'CRISPR repeats', which are, however, highly divergent and degenerated. By deep sequencing, we show that the two repeats give rise to an 8-nt 5' handle and a 22-nt 3' handle, respectively, i.e., the conserved elements of a canonical CRISPR RNA, indicating they both retained critical nucleotides for Cas6 processing during divergent degeneration. We also uncovered a minimal CreT toxin that sequesters the rare transfer RNA for isoleucine, tRNAIleCAU, with a six-codon open reading frame containing two consecutive AUA codons. To fully relieve its toxicity, both tRNAIleCAU overexpression and supply of extra agmatine (modifies the wobble base of tRNAIleCAU to decipher AUA codons) are required. By replacing AUA to AGA/AGG codons, we reprogrammed this toxin to sequester rare arginine tRNAs. These data provide essential information on CreTA origin and for future CreTA prediction, and enrich the knowledge of tRNA-sequestering small RNAs that are employed by CRISPR-Cas to get addictive to the host.

Microbial gas vesicles as nanotechnology tools: exploiting intracellular organelles for translational utility in biotechnology, medicine and the environment.

A range of bacteria and archaea produce gas vesicles as a means to facilitate flotation. These gas vesicles have been purified from a number of species and their applications in biotechnology and medicine are reviewed here. Halobacterium sp. NRC-1 gas vesicles have been engineered to display antigens from eukaryotic, bacterial and viral pathogens. The ability of these recombinant nanoparticles to generate an immune response has been quantified both in vitro and in vivo. These gas vesicles, along with those purified from Anabaena flos-aquae and Bacillus megaterium, have been developed as an acoustic reporter system. This system utilizes the ability of gas vesicles to retain gas within a stable, rigid structure to produce contrast upon exposure to ultrasound. The susceptibility of gas vesicles to collapse when exposed to excess pressure has also been proposed as a biocontrol mechanism to disperse cyanobacterial blooms, providing an environmental function for these structures.

Three ancient documents solve the jigsaw of the parchment purple spot deterioration and validate the microbial succession model.

The preservation of cultural heritage is one of the major challenges of today's society. Parchments, a semi-solid matrix of collagen produced from animal skin, are a significant part of the cultural heritage, being used as writing material since ancient times. Due to their animal origin, parchments easily undergo biodeterioration: the most common biological damage is characterized by isolated or coalescent purple spots, that often lead to the detachment of the superficial layer and the consequent loss of written content. Although many parchments with purple spot biodegradative features were studied, no common causative agent had been identified so far. In a previous study a successional model has been proposed, basing on the multidisciplinary analysis of damaged versus undamaged samples from a moderately damaged document. Although no specific sequences were observed, the results pointed to Halobacterium salinarum as the starting actor of the succession. In this study, to further investigate this topic, three dramatically damaged parchments were analysed; belonging to a collection archived as Faldone Patrizi A 19, and dated back XVI-XVII century A.D. With the same multidisciplinary approach, the Next Generation Sequencing (NGS, Illumina platform) revealed DNA sequences belonging to Halobacterium salinarum; the RAMAN spectroscopy identified the pigment within the purple spots as haloarchaeal bacterioruberin and bacteriorhodopsine, and the LTA technique quantified the extremely damaged collagen structures through the entire parchments, due to the biological attack to the parchment frame structures. These results allowed to propose a model of the progressive degradation pattern of the parchment collagen. Overall, these data validate a multi-phase microbial succession model. This demonstration is pivotal to possible new restoration strategies, important for a huge number of ancient documents.

Phyletic Distribution and Lineage-Specific Domain Architectures of Archaeal Two-Component Signal Transduction Systems.

The two-component signal transduction (TCS) machinery is a key mechanism of sensing environmental changes in the prokaryotic world. TCS systems have been characterized thoroughly in bacteria but to a much lesser extent in archaea. Here, we provide an updated census of more than 2,000 histidine kinases and response regulators encoded in 218 complete archaeal genomes, as well as unfinished genomes available from metagenomic data. We describe the domain architectures of the archaeal TCS components, including several novel output domains, and discuss the evolution of the archaeal TCS machinery. The distribution of TCS systems in archaea is strongly biased, with high levels of abundance in haloarchaea and thaumarchaea but none detected in the sequenced genomes from the phyla Crenarchaeota, Nanoarchaeota, and Korarchaeota The archaeal sensor histidine kinases are generally similar to their well-studied bacterial counterparts but are often located in the cytoplasm and carry multiple PAS and/or GAF domains. In contrast, archaeal response regulators differ dramatically from the bacterial ones. Most archaeal genomes do not encode any of the major classes of bacterial response regulators, such as the DNA-binding transcriptional regulators of the OmpR/PhoB, NarL/FixJ, NtrC, AgrA/LytR, and ActR/PrrA families and the response regulators with GGDEF and/or EAL output domains. Instead, archaea encode multiple copies of response regulators containing either the stand-alone receiver (REC) domain or combinations of REC with PAS and/or GAF domains. Therefore, the prevailing mechanism of archaeal TCS signaling appears to be via a variety of protein-protein interactions, rather than direct transcriptional regulation.IMPORTANCE Although the Archaea represent a separate domain of life, their signaling systems have been assumed to be closely similar to the bacterial ones. A study of the domain architectures of the archaeal two-component signal transduction (TCS) machinery revealed an overall similarity of archaeal and bacterial sensory modules but substantial differences in the signal output modules. The prevailing mechanism of archaeal TCS signaling appears to involve various protein-protein interactions rather than direct transcription regulation. The complete list of histidine kinases and response regulators encoded in the analyzed archaeal genomes is available online at http://www.ncbi.nlm.nih.gov/Complete_Genomes/TCSarchaea.html.

Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic ß-galactosidase.

The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic ß-galactosidase that functions in cold, hypersaline conditions. Six amino acid residues potentially important for cold activity were identified by comparative genomics and substituted with evolutionarily conserved residues (N251D, A263S, I299L, F387L, I476V, and V482L) in closely related homologs from mesophilic haloarchaea. Using a homology model, four residues (N251, A263, I299, and F387) were located in the TIM barrel around the active site in domain A, and two residues (I476 and V482) were within coiled or ß-sheet regions in domain B distant to the active site. Site-directed mutagenesis was performed by partial gene synthesis, and enzymes were overproduced from the cold-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-ß-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (one substitution each). The effects were correlated with perturbation of charge, hydrogen bonding, or packing, likely affecting the temperature-dependent flexibility and function of the enzyme. Our interdisciplinary approach, incorporating comparative genomics, mutagenesis, enzyme kinetics, and modeling, has shown that divergence of a very small number of amino acid residues can account for the cold temperature function of a polyextremophilic enzyme.

Halobacterium litoreum sp. nov., isolated from a marine solar saltern.

Halophilic archaeal strain ZS-54-S2T was isolated from Zhoushan marine solar saltern, China. Cells were rod-shaped, Gram-stain-negative and formed red-pigmented colonies on an agar plate. Strain ZS-54-S2T was able to grow at 20-50 °C (optimum 35 °C), at 1.7-4.8 M NaCl (optimum 3.9 M), at 0.005-1.0 M MgCl2 (optimum 0.05 M) and at pH 5.0-9.5 (optimum pH 7.0). The cells lysed in distilled water and the minimal NaCl concentration to prevent cell lysis was found to be 5 % (w/v). The major polar lipids of the strain were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, two glycolipids, which were chromatographically identical to sulfated galactosyl mannosyl galactofuranosyl glucosyl diether and galactosyl mannosyl glucosyl diether, and an unidentified glycolipid, which was chromatographically identical to one detected in Halobacterium salinarum ATCC 33171T. The 16S rRNA gene and rpoB' gene of strain ZS-54-S2T were phylogenetically related to the corresponding genes of Halobacterium noricense JCM 15102T (97.5 % and 90.6 % relatedness, respectively), Halobacterium jilantaiense CGMCC 1.5337T (96.9 and 91.2 %), Halobacterium rubrum CGMCC 1.12575T (96.8 and 90.3 %) and Halobacterium salinarum CGMCC 1.1958T (96.5 and 88.4 %). The DNA G+C content of strain ZS-54-S2T was 66.7 mol%. The phenotypic, chemotaxonomic and phylogenetic properties suggested that strain ZS-54-S2T (=CGMCC 1.12562T=JCM 30038T) represents a new species of Halobacterium, for which the name Halobacteriumlitoreum sp. nov. is proposed.

Genomic Analysis of the Extremely Halophilic Archaeon Halobacterium noricense CBA1132 Isolated from Solar Salt That Is an Essential Material for Fermented Foods.

The extremely halophilic archaeon Halobacterium noricense is a member of the genus Halobacterium. Strain CBA1132 (= KCCM 43183, JCM 31150) was isolated from solar salt. The genome of strain CBA1132 assembled with 4 contigs, including three rRNA genes, 44 tRNA genes, and 3,208 open reading frames. Strain CBA1132 had nine putative CRISPRs and the genome contained genes encoding metal resistance determinants: copper-translocating P-type ATPase (CtpA), arsenical pump-driving ATPase (ArsA), arsenate reductase (ArsC), and arsenical resistance operon repressor (ArsR). Strain CBA1132 was related to Halobacterium noricense, with 99.2% 16S rRNA gene sequence similarity. Based on the comparative genomic analysis, strain CBA1132 has distinctly evolved; moreover, essential genes related to nitrogen metabolism were only detected in the genome of strain CBA1132 among the reported genomes in the genus Halobacterium. This genome sequence of Halobacterium noricense CBA1132 may be of use in future molecular biological studies.

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.

The complete genome of a viable archaeum isolated from 123-million-year-old rock salt.

Live microbes have been isolated from rock salt up to Permian age. Only obligatory cellular functions can be performed in halite-buried cells. Consequently, their genomic sequences are likely to remain virtually unchanged. However, the available sequence information from these organisms is scarce and consists of mainly ribosomal 16S sequences. Here, live archaea were isolated from early Cretaceous (∼ 123 million years old) halite from the depth of 2000 m in Qianjiang Depression, Hubei Province, China. The sample was radiologically dated and subjected to rigorous surface sterilization before microbe isolation. The isolates represented a single novel species of Halobacterium, for which we suggest the name Halobacterium hubeiense, type strain Hbt. hubeiense JI20-1. The species was closely related to a Permian (225-280 million years old) isolate, Halobacterium noricense, originating from Alpine rock salt. This study is the first one to publish the complete genome of an organism originating from surface-sterilized ancient halite. In the future, genomic data from halite-buried microbes can become a key factor in understanding the mechanisms by which these organisms are able to survive in harsh conditions deep underground or possibly on other celestial bodies.

Immunogenicity and protective potential of a Plasmodium spp. enolase peptide displayed on archaeal gas vesicle nanoparticles.

BACKGROUND: Plasmodium falciparum enolase has been shown to localize on the surface of merozoites and ookinetes. Immunization of mice with recombinant Plasmodium enolase (rPfeno) showed partial protection against malaria. Anti-rPfeno antibodies inhibited growth of the parasite in in vitro cultures and blocked ookinete invasion of mosquito midgut epithelium. It is hypothesized that parasite specific moonlighting functions (e.g. host cell invasion) may map on to unique structural elements of Pfeno. Since enolases are highly conserved between the host and the parasite, a parasite-specific epitope of enolase was displayed on novel protein nanoparticles produced by a halophilic Archaeon Halobacterium sp. NRC-1 and tested their ability to protect mice against live challenge. METHODS: By genetic engineering, a Plasmodium-enolase specific peptide sequence (104)EWGWS(108) with protective antigenic potential was inserted into the Halobacterium gas vesicle protein GvpC, a protein localized on the surface of immunogenic gas vesicle nanoparticles (GVNPs). Two groups of mice were immunized with the wild type (WT) and the insert containing recombinant (Rec) GVNPs respectively. A third group of mice was kept as un-immunized control. Antibody titres were measured against three antigens (i.e. WT-GVNPs, Rec-GVNPs and rPfeno) using ELISA. The protective potential was determined by measuring percentage parasitaemia and survival after challenge with the lethal strain Plasmodium yoelii 17XL. RESULTS: Rec-GVNP-immunized mice showed higher antibody titres against rPfeno and Rec-GVNPs, indicating that the immunized mice had produced antibodies against the parasite enolase-specific insert sequence. Challenging the un-immunized, WT-GVNP and Rec-GVNP-immunized mice with a lethal strain of mice malarial parasite showed significantly lower parasitaemia and longer survival in the Rec-GVNP-immunized group as compared to control groups. The extent of survival advantage in the Rec-GVNP-group showed positive correlation with anti-rPfeno antibody titres while the parasitaemia showed a negative correlation. These results indicate that the parasite enolase peptide insert displayed on Halobacterium GVNPs is a good candidate as a protective antigenic epitope. CONCLUSION: The work reported here showed that the parasite-specific peptide sequence is a protective antigenic epitope. Although antibody response of B-cells to the guest sequence in Rec-GVNPs was mild, significant advantage in the control of parasitaemia and survival was observed. Future efforts are needed to display multiple antigens with protective properties to improve the performance of the GVNP-based approach.

Flagellar filament bio-templated inorganic oxide materials - towards an efficient lithium battery anode.

Designing a new generation of energy-intensive and sustainable electrode materials for batteries to power a variety of applications is an imperative task. The use of biomaterials as a nanosized structural template for these materials has the potential to produce hitherto unachievable structures. In this report, we have used genetically modified flagellar filaments of the extremely halophilic archaea species Halobacterium salinarum to synthesize nanostructured iron oxide composites for use as a lithium-ion battery anode. The electrode demonstrated a superior electrochemical performance compared to existing literature results, with good capacity retention of 1032 mAh g(-1) after 50 cycles and with high rate capability, delivering 770 mAh g(-1) at 5 A g(-1) (~5 C) discharge rate. This unique flagellar filament based template has the potential to provide access to other highly structured advanced energy materials in the future.

Characterization of hypothetical protein VNG0128C from Halobacterium NRC-1 reveals GALE like activity and its involvement in Leloir pathway of galactose metabolism.

VNG0128C, a hypothetical protein from Halobacterium NRC-1, was chosen for detailed insilico and experimental investigations. Computational exercises revealed that VNG0128C functions as NAD(+) binding protein. The phylogenetic analysis with the homolog sequences of VNG0128C suggested that it could act as UDP-galactose 4-epimerase. Hence, the VNG0128C sequence was modeled using a suitable template and docking studies were performed with NAD and UDP-galactose as ligands. The binding interactions strongly indicate that VNG0128C could plausibly act as UDP-galactose 4-epimerase. In order to validate these insilico results, VNG0128C was cloned in pUC57, subcloned in pET22b(+), expressed in BL21 cells and purified using nickel affinity chromatography. An assay using blue dextran was performed to confirm the presence of NAD binding domain. To corroborate the epimerase like enzymatic role of the hypothetical protein, i.e. the ability of the enzyme to convert UDP-galactose to UDP-glucose, the conversion of NAD to NADH was measured. The experimental assay significantly correlated with the insilico predictions, indicating that VNG0128C has a NAD(+) binding domain with epimerase activity. Consequently, its key role in nucleotide-sugar metabolism was thus established. Additionally, the work highlights the need for a methodical characterization of hypothetical proteins (less studied class of biopolymers) to exploit them for relevant applications in the field of biology.

Halophilic microorganisms are responsible for the rosy discolouration of saline environments in three historical buildings with mural paintings.

A number of mural paintings and building materials from monuments located in central and south Europe are characterized by the presence of an intriguing rosy discolouration phenomenon. Although some similarities were observed among the bacterial and archaeal microbiota detected in these monuments, their origin and nature is still unknown. In order to get a complete overview of this biodeterioration process, we investigated the microbial communities in saline environments causing the rosy discolouration of mural paintings in three Austrian historical buildings using a combination of culture-dependent and -independent techniques as well as microscopic techniques. The bacterial communities were dominated by halophilic members of Actinobacteria, mainly of the genus Rubrobacter. Representatives of the Archaea were also detected with the predominating genera Halobacterium, Halococcus and Halalkalicoccus. Furthermore, halophilic bacterial strains, mainly of the phylum Firmicutes, could be retrieved from two monuments using special culture media. Inoculation of building materials (limestone and gypsum plaster) with selected isolates reproduced the unaesthetic rosy effect and biodeterioration in the laboratory.

Halobacterium rubrum sp. nov., isolated from a marine solar saltern.

Halophilic archaeal strain TGN-42-S1(T) was isolated from the Tanggu marine solar saltern, China. Cells from strain TGN-42-S1(T) were observed to be pleomorphic rods, stained Gram-negative, and formed red-pigmented colonies on solid media. Strain TGN-42-S1(T) was found to be able to grow at 20-50 °C (optimum 35-37 °C), at 1.7-4.8 M NaCl (optimum 3.1 M), at 0-1.0 M MgCl2 (optimum 0.1 M), and at pH 5.0-9.0 (optimum pH 7.0-7.5). The cells lysed in distilled water, and the minimal NaCl concentration to prevent cell-lysis was found to be 10 % (w/v). The major polar lipids of the strain were phosphatidic acid, phosphatidylglycerol, phosphatidylglycerol phosphate methyl ester, phosphatidylglycerol sulfate, galactosyl mannosyl glucosyl diether (TGD-1), sulfated galactosyl mannosyl glucosyl diether (S-TGD-1), sulfated galactosyl mannosyl galactofuranosyl glucosyl diether (S-TeGD), and three unidentified glycolipids which were chromatographically identical to those of the Halobacterium species. The 16S rRNA gene and rpoB' gene of strain TGN-42-S1(T) were phylogenetically related to the corresponding genes of Halobacterium jilantaiense CGMCC 1.5337(T) (98.8 and 93.5 % nucleotide identity, respectively), Halobacterium salinarum CGMCC 1.1958(T) (98.4 and 91.9 %), and Halobacterium noricense JCM 15102(T) (96.9 and 91.1 %). The DNA G + C content of strain TGN-42-S1(T) was determined to be 69.2 mol %. Strain TGN-42-S1(T) showed low DNA-DNA relatedness with Hbt. jilantaiense CGMCC 1.5337(T) and Hbt. salinarum CGMCC 1.1958(T), the most closely related members of the genus Halobacterium. The phenotypic, chemotaxonomic, and phylogenetic properties suggested that strain TGN-42-S1(T) (=CGMCC 1.12575(T) =JCM 19908(T)) represents a new species of Halobacterium, for which the name Halobacterium rubrum sp. nov. is proposed.

Bioengineering radioresistance by overproduction of RPA, a mammalian-type single-stranded DNA-binding protein, in a halophilic archaeon.

Halobacterium sp. NRC-1 is a wild-type extremophilic microbe that is naturally tolerant to high levels of ionizing radiation. Mutants of strain NRC-1 with even higher levels of resistance to ionizing radiation, named RAD, were previously isolated after selecting survival to extremely high doses of ionizing radiation. These RAD mutants displayed higher transcription levels for the rfa3 operon, coding two subunits of the RPA-like putative single-stranded binding protein, rfa3 and rfa8, and a third downstream gene, ral. In order to bioengineer cells with increased tolerance to ionizing radiation and further explore the genetic basis of the RAD phenotype, we placed the rfa3 operon under control of the gvpA promoter in a Halobacterium expression plasmid, pDRK1. When pDRK1 was introduced into the wild-type NRC-1 strain, overproduction of the Rfa3 and Rfa8 proteins was observed by Western blotting and proteomic analysis. The Halobacterium strains expressing Rfa3 and Rfa8 also displayed improved survival after exposure to ionizing radiation, similar to the RAD mutants, when compared to wild-type strain NRC-1. The Rfa3 and Rfa8 proteins co-purified by affinity chromatography on single-stranded DNA cellulose columns, confirming the ability of the proteins to bind to single-stranded DNA as well as their relative abundance in the wild-type, RAD mutants, and rfa3 operon overexpression strains. These results clearly establish that overexpression of haloarchaeal RPA promotes ionizing radiation resistance in Halobacterium sp. NRC-1 and that the Rfa3 and Rfa8 subunits bind single-stranded DNA. Bioengineering cells with increased levels of ionizing radiation resistance may have potential value in medical and environmental applications.