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1.  Haloarchaeal gas vesicle nanoparticles displaying Salmonella SopB antigen reduce bacterial burden when administered with live attenuated bacteria 
Vaccine  2014;32(35):4543-4549.
Innovative vaccines against typhoid and other Salmonella diseases that are safe, effective, and inexpensive are urgently needed. In order to address this need, buoyant, self-adjuvating gas vesicle nanoparticles (GVNPs) from the halophilic archaeon Halobacterium sp. NRC-1 were bioengineered to display the highly conserved Salmonella enterica antigen SopB, a secreted inosine phosphate effector protein injected by pathogenic bacteria during infection into the host cell. Two highly conserved sopB gene segments near the 3’-coding region, named sopB4 and B5, were each fused to the C-terminal coding region of the gvpC gene, and resulting GVNPs were purified by centrifugally accelerated flotation. Display of SopB4 and B5 antigenic epitopes on GVNPs was established by Western blotting analysis using antisera raised against short synthetic peptides of SopB. Immunostimulatory activities of the SopB4 and B5 nanoparticles were tested by intraperitoneal administration of recombinant GVNPs to BALB/c mice which had been immunized with S. enterica serovar Typhimurium 14028 ΔpmrG-HM-D (DV-STM-07), a live attenuated vaccine strain. Proinflammatory cytokines IFN-γ, IL-2, and IL-9 were significantly induced in mice boosted with SopB5-GVNPs, consistent with a robust Th1 response. After challenge with virulent S. enterica serovar Typhimurium 14028, bacterial burden was found to be diminished in spleen of mice boosted with SopB4-GVNPs and absent or significantly diminished in liver, mesenteric lymph node, and spleen of mice boosted with SopB5-GVNPs, indicating that the C-terminal portions of SopB displayed on GVNPs elicit a protective response to Salmonella infection in mice. SopB antigen-GVNPs were found to be stable at elevated temperatures for extended periods without refrigeration in Halobacterium cells. The results all together show that bioengineered GVNPs are likely to represent a valuable platform for the development of improved vaccines against Salmonella diseases.
PMCID: PMC4729386  PMID: 24950351
antigen display; nanoparticle; Salmonella; Haloarchaea; enteric disease
2.  Immunogenicity and protective potential of a Plasmodium spp. enolase peptide displayed on archaeal gas vesicle nanoparticles 
Malaria Journal  2015;14:406.
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.
By genetic engineering, a Plasmodium-enolase specific peptide sequence 104EWGWS108 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.
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.
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.
PMCID: PMC4605222  PMID: 26463341
Plasmodium falciparum enolase; Protective epitope; Halobacterium gas vesicles; Nanoparticles
3.  Gas Vesicle Nanoparticles for Antigen Display 
Vaccines  2015;3(3):686-702.
Microorganisms like the halophilic archaeon Halobacterium sp. NRC-1 produce gas-filled buoyant organelles, which are easily purified as protein nanoparticles (called gas vesicles or GVNPs). GVNPs are non-toxic, exceptionally stable, bioengineerable, and self-adjuvanting. A large gene cluster encoding more than a dozen proteins has been implicated in their biogenesis. One protein, GvpC, found on the exterior surface of the nanoparticles, can accommodate insertions near the C-terminal region and results in GVNPs displaying the inserted sequences on the surface of the nanoparticles. Here, we review the current state of knowledge on GVNP structure and biogenesis as well as available studies on immunogenicity of pathogenic viral, bacterial, and eukaryotic proteins and peptides displayed on the nanoparticles. Recent improvements in genetic tools for bioengineering of GVNPs are discussed, along with future opportunities and challenges for development of vaccines and other applications.
PMCID: PMC4586473  PMID: 26350601
protein nanoparticle; adjuvant; carrier; SIV; Chlamydia; Salmonella; typhoid; Plasmodium; malaria; luciferase
4.  Aquatic Biosystems reviewer acknowledgement 2013 
Aquatic Biosystems  2014;10:1.
Contributing reviewers
The Aquatic Biosystems editorial team would like to thank the following colleagues who contributed to peer review for the journal in 2013.
PMCID: PMC3896783  PMID: 24433368
5.  An improved genetic system for bioengineering buoyant gas vesicle nanoparticles from Haloarchaea 
BMC Biotechnology  2013;13:112.
Gas vesicles are hollow, buoyant organelles bounded by a thin and extremely stable protein membrane. They are coded by a cluster of gvp genes in the halophilic archaeon, Halobacterium sp. NRC-1. Using an expression vector containing the entire gvp gene cluster, gas vesicle nanoparticles (GVNPs) have been successfully bioengineered for antigen display by constructing gene fusions between the gvpC gene and coding sequences from bacterial and viral pathogens.
To improve and streamline the genetic system for bioengineering of GVNPs, we first constructed a strain of Halobacterium sp. NRC-1 deleted solely for the gvpC gene. The deleted strain contained smaller, more spindle-shaped nanoparticles observable by transmission electron microscopy, confirming a shape-determining role for GvpC in gas vesicle biogenesis. Next, we constructed expression plasmids containing N-terminal coding portions or the complete gvpC gene. After introducing the expression plasmids into the Halobacterium sp. NRC-1 ΔgvpC strain, GvpC protein and variants were localized to the GVNPs by Western blotting analysis and their effects on increasing the size and shape of nanoparticles established by electron microscopy. Finally, a synthetic gene coding for Gaussia princeps luciferase was fused to the gvpC gene fragments on expression plasmids, resulting in an enzymatically active GvpC-luciferase fusion protein bound to the buoyant nanoparticles from Halobacterium.
GvpC protein and its N-terminal fragments expressed from plasmid constructs complemented a Halobacterium sp. NRC-1 ΔgvpC strain and bound to buoyant GVNPs. Fusion of the luciferase reporter gene from Gaussia princeps to the gvpC gene derivatives in expression plasmids produced GVNPs with enzymatically active luciferase bound. These results establish a significantly improved genetic system for displaying foreign proteins on Halobacterium gas vesicles and extend the bioengineering potential of these novel nanoparticles to catalytically active enzymes.
PMCID: PMC3878110  PMID: 24359319
Vaccine; Halophiles; Archaea; Luciferase
6.  Genome-Wide Responses of the Model Archaeon Halobacterium sp. Strain NRC-1 to Oxygen Limitation 
Journal of Bacteriology  2012;194(20):5530-5537.
As part of a comprehensive postgenomic investigation of the model archaeon Halobacterium sp. strain NRC-1, we used whole-genome DNA microarrays to compare transcriptional profiles of cells grown under anaerobic or aerobic conditions. When anaerobic growth supported by arginine fermentation was compared to aerobic growth, genes for arginine fermentation (arc) and anaerobic respiration (dms), using trimethylamine N-oxide (TMAO) as the terminal electron acceptor, were highly upregulated, as was the bop gene, required for phototrophic growth. When arginine fermentation was compared to anaerobic respiration with TMAO, the arc and dms genes were both induced with arginine, while TMAO induced the bop gene and major gas vesicle protein (gvpAC) genes specifying buoyant gas vesicles. Anaerobic conditions with either TMAO or arginine also upregulated the cba genes, encoding one of three cytochrome oxidases. In-frame deletion of two COG3413 family regulatory genes, bat and dmsR, showed downregulation of the bop gene cluster and loss of purple membrane synthesis and downregulation of the dms operon and loss of anaerobic respiration capability, respectively. Bioinformatic analysis identified additional regulatory and sensor genes that are likely involved in the full range of cellular responses to oxygen limitation. Our results show that the Halobacterium sp. has evolved a carefully orchestrated set of responses to oxygen limitation. As conditions become more reducing, cells progressively increase buoyancy, as well as capabilities for phototrophy, scavenging of molecular oxygen, anaerobic respiration, and fermentation.
PMCID: PMC3458682  PMID: 22865851
7.  Amino Acid Substitutions in Cold-Adapted Proteins from Halorubrum lacusprofundi, an Extremely Halophilic Microbe from Antarctica 
PLoS ONE  2013;8(3):e58587.
The halophilic Archaeon Halorubrum lacusprofundi, isolated from the perennially cold and hypersaline Deep Lake in Antarctica, was recently sequenced and compared to 12 Haloarchaea from temperate climates by comparative genomics. Amino acid substitutions for 604 H. lacusprofundi proteins belonging to conserved haloarchaeal orthologous groups (cHOGs) were determined and found to occur at 7.85% of positions invariant in proteins from mesophilic Haloarchaea. The following substitutions were observed most frequently: (a) glutamic acid with aspartic acid or alanine; (b) small polar residues with other small polar or non-polar amino acids; (c) small non-polar residues with other small non-polar residues; (d) aromatic residues, especially tryptophan, with other aromatic residues; and (e) some larger polar residues with other similar residues. Amino acid substitutions for a cold-active H. lacusprofundi β-galactosidase were then examined in the context of a homology modeled structure at residues invariant in homologous enzymes from mesophilic Haloarchaea. Similar substitutions were observed as in the genome-wide approach, with the surface accessible regions of β-galactosidase displaying reduced acidity and increased hydrophobicity, and internal regions displaying mainly subtle changes among smaller non-polar and polar residues. These findings are consistent with H. lacusprofundi proteins displaying amino acid substitutions that increase structural flexibility and protein function at low temperature. We discuss the likely mechanisms of protein adaptation to a cold, hypersaline environment on Earth, with possible relevance to life elsewhere.
PMCID: PMC3594186  PMID: 23536799
8.  Cloning, overexpression, purification, and characterization of a polyextremophilic β-galactosidase from the Antarctic haloarchaeon Halorubrum lacusprofundi 
BMC Biotechnology  2013;13:3.
Halorubrum lacusprofundi is a cold-adapted halophilic archaeon isolated from Deep Lake, a perennially cold and hypersaline lake in Antarctica. Its genome sequencing project was recently completed, providing access to many genes predicted to encode polyextremophilic enzymes active in both extremely high salinity and cold temperatures.
Analysis of the genome sequence of H. lacusprofundi showed a gene cluster for carbohydrate utilization containing a glycoside hydrolase family 42 β-galactosidase gene, named bga. In order to study the biochemical properties of the β-galactosidase enzyme, the bga gene was PCR amplified, cloned, and expressed in the genetically tractable haloarchaeon Halobacterium sp. NRC-1 under the control of a cold shock protein (cspD2) gene promoter. The recombinant β-galactosidase protein was produced at 20-fold higher levels compared to H. lacusprofundi, purified using gel filtration and hydrophobic interaction chromatography, and identified by SDS-PAGE, LC-MS/MS, and ONPG hydrolysis activity. The purified enzyme was found to be active over a wide temperature range (−5 to 60°C) with an optimum of 50°C, and 10% of its maximum activity at 4°C. The enzyme also exhibited extremely halophilic character, with maximal activity in either 4 M NaCl or KCl. The polyextremophilic β-galactosidase was also stable and active in 10–20% alcohol-aqueous solutions, containing methanol, ethanol, n-butanol, or isoamyl alcohol.
The H. lacusprofundi β-galactosidase is a polyextremophilic enzyme active in high salt concentrations and low and high temperature. The enzyme is also active in aqueous-organic mixed solvents, with potential applications in synthetic chemistry. H. lacuprofundi proteins represent a significant biotechnology resource and for developing insights into enzyme catalysis under water limiting conditions. This study provides a system for better understanding how H. lacusprofundi is successful in a perennially cold, hypersaline environment, with relevance to astrobiology.
PMCID: PMC3556326  PMID: 23320757
Polyextremophiles; Extremozymes; Protein stability; Halophiles; Psychrophiles; Biofuels
9.  Function and biotechnology of extremophilic enzymes in low water activity 
Aquatic Biosystems  2012;8:4.
Enzymes from extremophilic microorganisms usually catalyze chemical reactions in non-standard conditions. Such conditions promote aggregation, precipitation, and denaturation, reducing the activity of most non-extremophilic enzymes, frequently due to the absence of sufficient hydration. Some extremophilic enzymes maintain a tight hydration shell and remain active in solution even when liquid water is limiting, e.g. in the presence of high ionic concentrations, or at cold temperature when water is close to the freezing point. Extremophilic enzymes are able to compete for hydration via alterations especially to their surface through greater surface charges and increased molecular motion. These properties have enabled some extremophilic enzymes to function in the presence of non-aqueous organic solvents, with potential for design of useful catalysts. In this review, we summarize the current state of knowledge of extremophilic enzymes functioning in high salinity and cold temperatures, focusing on their strategy for function at low water activity. We discuss how the understanding of extremophilic enzyme function is leading to the design of a new generation of enzyme catalysts and their applications to biotechnology.
PMCID: PMC3310334  PMID: 22480329
Extremophile; Extremozymes; Protein stability; Halophiles; Psychrophile; Cold activity; Organic solvent; Low temperature; High salinity; Biofuel; Bioenergy
10.  Aquatic biosystems: reactions and actions 
Aquatic Biosystems  2012;8:1.
Aquatic biological systems are a critical part of the structure and function of earth's biosphere. While attention of the scientific community is often focused on the reaction of biological systems to changes in the environment, these systems also have profound effects, or actions, on the environment. Throughout the evolutionary history of earth, the rise and/or fall of different aquatic biosystems has impacted the character of the biosphere. At no time have environmental changes been more important to all life on earth than in the modern era, which underscores the need for the new journal, Aquatic Biosystems. We welcome submission of original research manuscripts, reviews, and commentaries to the journal.
PMCID: PMC3310335  PMID: 22520865
11.  The core and unique proteins of haloarchaea 
BMC Genomics  2012;13:39.
Since the first genome of a halophilic archaeon was sequenced in 2000, biologists have been advancing the understanding of genomic characteristics that allow for survival in the harsh natural environments of these organisms. An increase in protein acidity and GC-bias in the genome have been implicated as factors in tolerance to extreme salinity, desiccation, and high solar radiation. However, few previous attempts have been made to identify novel genes that would permit survival in such extreme conditions.
With the recent release of several new complete haloarchaeal genome sequences, we have conducted a comprehensive comparative genomic analysis focusing on the identification of unique haloarchaeal conserved proteins that likely play key roles in environmental adaptation. Using bioinformatic methods, we have clustered 31,312 predicted proteins from nine haloarchaeal genomes into 4,455 haloarchaeal orthologous groups (HOGs). We assigned likely functions by association with established COG and KOG databases in NCBI. After identifying homologs in four additional haloarchaeal genomes, we determined that there were 784 core haloarchaeal protein clusters (cHOGs), of which 83 clusters were found primarily in haloarchaea. Further analysis found that 55 clusters were truly unique (tucHOGs) to haloarchaea and qualify as signature proteins while 28 were nearly unique (nucHOGs), the vast majority of which were coded for on the haloarchaeal chromosomes. Of the signature proteins, only one example with any predicted function, Ral, involved in desiccation/radiation tolerance in Halobacterium sp. NRC-1, was identified. Among the core clusters, 33% was predicted to function in metabolism, 25% in information transfer and storage, 10% in cell processes and signaling, and 22% belong to poorly characterized or general function groups.
Our studies have established conserved groups of nearly 800 protein clusters present in all haloarchaea, with a subset of 55 which are predicted to be accessory proteins that may be critical or essential for success in an extreme environment. These studies support core and signature genes and proteins as valuable concepts for understanding phylogenetic and phenotypic characteristics of coherent groups of organisms.
PMCID: PMC3287961  PMID: 22272718
12.  HaloWeb: the haloarchaeal genomes database 
Saline Systems  2010;6:12.
Complete genome sequencing together with post-genomic studies provide the opportunity for a comprehensive 'systems biology' understanding of model organisms. For maximum effectiveness, an integrated database containing genomic, transcriptomic, and proteomic data is necessary.
To improve data access and facilitate functional genomic studies on haloarchaea in our laboratory, a dedicated database and website, named HaloWeb, was developed. It incorporates all finished and publicly released haloarchaeal genomes, including gene, protein and RNA sequences and annotation data, as well as other features such as insertion element sequences. The HaloWeb database was designed for easy data access and mining, and includes tools for tasks such as genome map generation, sequence extraction, and sequence editing. Popular resources at other sites, e.g., NCBI PubMed and BLAST, COG and KOG protein clusters, KEGG pathways, and GTOP structures were dynamically linked. The HaloWeb site is located at, and at a mirror site,, with all public genomic data and NCBI, KEGG, and GTOP links available for use by the academic community. The database is curated and updated on a regular basis.
The HaloWeb site includes all completely sequenced haloarchaeal genomes from public databases. It is currently being used as a tool for comparative genomics, including analysis of gene and genome structure, organization, and function. The database and website are up-to-date resources for researchers worldwide.
PMCID: PMC3023673  PMID: 21192823
13.  Multiple Replication Origins of Halobacterium sp. Strain NRC-1: Properties of the Conserved orc7-Dependent oriC1▿  
Journal of Bacteriology  2009;191(16):5253-5261.
The eukaryote-like DNA replication system of the model haloarchaeon Halobacterium NRC-1 is encoded within a circular chromosome and two large megaplasmids or minichromosomes, pNRC100 and pNRC200. We previously showed by genetic analysis that 2 (orc2 and orc10) of the 10 genes coding for Orc-Cdc6 replication initiator proteins were essential, while a third (orc7), located near a highly conserved autonomously replicating sequence, oriC1, was nonessential for cell viability. Here we used whole-genome marker frequency analysis (MFA) and found multiple peaks, indicative of multiple replication origins. The largest chromosomal peaks were located proximal to orc7 (oriC1) and orc10 (oriC2), and the largest peaks on the extrachromosomal elements were near orc9 (oriP1) in both pNRC100 and -200 and near orc4 (oriP2) in pNRC200. MFA of deletion strains containing different combinations of chromosomal orc genes showed that replication initiation at oriC1 requires orc7 but not orc6 and orc8. The initiation sites at oriC1 were determined by replication initiation point analysis and found to map divergently within and near an AT-rich element flanked by likely Orc binding sites. The oriC1 region, Orc binding sites, and orc7 gene orthologs were conserved in all sequenced haloarchaea. Serial deletion of orc genes resulted in the construction of a minimal strain containing not only orc2 and orc10 but also orc9. Our results suggest that replication in this model system is intriguing and more complex than previously thought. We discuss these results from the perspective of the replication strategy and evolution of haloarchaeal genomes.
PMCID: PMC2725591  PMID: 19502403
14.  Transcriptional responses to biologically relevant doses of UV-B radiation in the model archaeon, Halobacterium sp. NRC-1 
Saline Systems  2008;4:13.
Most studies of the transcriptional response to UV radiation in living cells have used UV doses that are much higher than those encountered in the natural environment, and most focus on short-wave UV (UV-C) at 254 nm, a wavelength that never reaches the Earth's surface. We have studied the transcriptional response of the sunlight-tolerant model archaeon, Halobacterium sp. NRC-1, to low doses of mid-wave UV (UV-B) to assess its response to UV radiation that is likely to be more biologically relevant.
Halobacterium NRC-1 cells were irradiated with UV-B at doses equivalent to 30 J/m2 and 5 J/m2 of UV-C. Transcriptional profiling showed that only 11 genes were up-regulated 1.5-fold or more by both UV-B doses. The most strongly up-regulated gene was radA1 (vng2473), the archaeal homologue of RAD51/recA recombinase. The others included arj1 (vng779) (recJ-like exonuclease), top6A (vng884) and top6B (vng885) (coding for Topoisomerase VI subunits), and nrdJ (vng1644) (which encodes a subunit of ribonucleotide reductase). We have found that four of the consistently UV-B up-regulated genes, radA1 (vng2473), vng17, top6B (vng885) and vng280, share a common 11-base pair motif in their promoter region, TTTCACTTTCA. Similar sequences were found in radA promoters in other halophilic archaea, as well as in the radA promoter of Methanospirillum hungatei. We analysed the transcriptional response of a repair-deficient ΔuvrA (vng2636) ΔuvrC (vng2381) double-deletion mutant and found common themes between it and the response in repair proficient cells.
Our results show a core set of genes is consistently up-regulated after exposure to UV-B light at low, biologically relevant doses. Eleven genes were up-regulated, in wild-type cells, after two UV-B doses (comparable to UV-C doses of 30 J/m2 and 5 J/m2), and only four genes were up-regulated by all doses of UV-B and UV-C that we have used in this work and previously. These results suggest that high doses of UV-C radiation do not necessarily provide a good model for the natural response to environmental UV. We have found an 11-base pair motif upstream of the TATA box in four of the UV-B up-regulated genes and suggest that this motif is the binding site for a transcriptional regulator involved in their response to UV damage in this model archaeon.
PMCID: PMC2556686  PMID: 18759987
15.  On the origin of prokaryotic "species": the taxonomy of halophilic Archaea 
Saline Systems  2008;4:5.
The consistent use of the taxonomic system of binomial nomenclature (genus and species) was first popularized by Linnaeus nearly three-hundred years ago to classify mainly plants and animals. His main goal was to give labels that would ensure that biologists could agree on which organism was under investigation. One-hundred fifty years later, Darwin considered the term species as one of convenience and not essentially different from variety. In the modern era, exploration of the world's niches together with advances in genomics have expanded the number of named species to over 1.8 million, including many microorganisms. However, even this large number excludes over 90% of microorganisms that have yet to be cultured or classified. In naming new isolates in the microbial world, the challenge remains the lack of a universally held and evenly applied standard for a species. The definition of species based on the capacity to form fertile offspring is not applicable to microorganisms and 70% DNA-DNA hybridization appears rather crude in light of the many completed genome sequences. The popular phylogenetic marker, 16S rRNA, is tricky for classification since it does not provide multiple characteristics or phenotypes used classically for this purpose. Using most criteria, agreement may usually be found at the genus level, but species level distinctions are problematic. These observations lend credence to the proposal that the species concept is flawed when applied to prokaryotes. In order to address this topic, we have examined the taxonomy of extremely halophilic Archaea, where the order, family, and even a genus designation have become obsolete, and the naming and renaming of certain species has led to much confusion in the scientific community.
PMCID: PMC2397426  PMID: 18485204
16.  Dissection of the regulatory mechanism of a heat-shock responsive promoter in Haloarchaea: a new paradigm for general transcription factor directed archaeal gene regulation 
Nucleic Acids Research  2008;36(9):3031-3042.
Multiple general transcription factors (GTFs), TBP and TFB, are present in many haloarchaea, and are deemed to accomplish global gene regulation. However, details and the role of GTF-directed transcriptional regulation in stress response are still not clear. Here, we report a comprehensive investigation of the regulatory mechanism of a heat-induced gene (hsp5) from Halobacterium salinarum. We demonstrated by mutation analysis that the sequences 5′ and 3′ to the core elements (TATA box and BRE) of the hsp5 promoter (Phsp5) did not significantly affect the basal and heat-induced gene expression, as long as the transcription initiation site was not altered. Moreover, the BRE and TATA box of Phsp5 were sufficient to render a nonheat-responsive promoter heat-inducible, in both Haloferax volcanii and Halobacterium sp. NRC-1. DNA–protein interactions revealed that two heat-inducible GTFs, TFB2 from H. volcanii and TFBb from Halobacterium sp. NRC-1, could specifically bind to Phsp5 likely in a temperature-dependent manner. Taken together, the heat-responsiveness of Phsp5 was mainly ascribed to the core promoter elements that were efficiently recognized by specific heat-induced GTFs at elevated temperature, thus providing a new paradigm for GTF-directed gene regulation in the domain of Archaea.
PMCID: PMC2396416  PMID: 18390887
17.  Structure and Function of Cold Shock Proteins in Archaea▿  
Journal of Bacteriology  2007;189(15):5738-5748.
Archaea are abundant and drive critical microbial processes in the Earth's cold biosphere. Despite this, not enough is known about the molecular mechanisms of cold adaptation and no biochemical studies have been performed on stenopsychrophilic archaea (e.g., Methanogenium frigidum). This study examined the structural and functional properties of cold shock proteins (Csps) from archaea, including biochemical analysis of the Csp from M. frigidum. csp genes are present in most bacteria and some eucarya but absent from most archaeal genome sequences, most notably, those of all archaeal thermophiles and hyperthermophiles. In bacteria, Csps are small, nucleic acid binding proteins involved in a variety of cellular processes, such as transcription. In this study, archaeal Csp function was assessed by examining the ability of csp genes from psychrophilic and mesophilic Euryarchaeota and Crenarchaeota to complement a cold-sensitive growth defect in Escherichia coli. In addition, an archaeal gene with a cold shock domain (CSD) fold but little sequence identity to Csps was also examined. Genes encoding Csps or a CSD structural analog from three psychrophilic archaea rescued the E. coli growth defect. The three proteins were predicted to have a higher content of solvent-exposed basic residues than the noncomplementing proteins, and the basic residues were located on the nucleic acid binding surface, similar to their arrangement in E. coli CspA. The M. frigidum Csp was purified and found to be a single-domain protein that folds by a reversible two-state mechanism and to exhibit a low conformational stability typical of cold-adapted proteins. Moreover, M. frigidum Csp was characterized as binding E. coli single-stranded RNA, consistent with its ability to complement function in E. coli. The studies show that some Csp and CSD fold proteins have retained sufficient similarity throughout evolution in the Archaea to be able to function effectively in the Bacteria and that the function of the archaeal proteins relates to cold adaptation. The initial biochemical analysis of M. frigidum Csp has developed a platform for further characterization and demonstrates the potential for expanding molecular studies of proteins from this important archaeal stenopsychrophile.
PMCID: PMC1951829  PMID: 17545280
18.  Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA 
BMC Genetics  2007;8:61.
Archaea are prokaryotic organisms with simplified versions of eukaryotic transcription systems. Genes coding for the general transcription factors TBP and TFB are present in multiple copies in several Archaea, including Halobacterium sp. NRC-1. Multiple TBP and TFBs have been proposed to participate in transcription of genes via recognition and recruitment of RNA polymerase to different classes of promoters.
We attempted to knock out all six TBP and seven TFB genes in Halobacterium sp. NRC-1 using the ura3-based gene deletion system. Knockouts were obtained for six out of thirteen genes, tbpCDF and tfbACG, indicating that they are not essential for cell viability under standard conditions. Screening of a population of 1,000 candidate mutants showed that genes which did not yield mutants contained less that 0.1% knockouts, strongly suggesting that they are essential. The transcriptomes of two mutants, ΔtbpD and ΔtfbA, were compared to the parental strain and showed coordinate down regulation of many genes. Over 500 out of 2,677 total genes were regulated in the ΔtbpD and ΔtfbA mutants with 363 regulated in both, indicating that over 10% of genes in both strains require the action of both TbpD and TfbA for normal transcription. Culturing studies on the ΔtbpD and ΔtfbA mutant strains showed them to grow more slowly than the wild-type at an elevated temperature, 49°C, and they showed reduced viability at 56°C, suggesting TbpD and TfbA are involved in the heat shock response. Alignment of TBP and TFB protein sequences suggested the expansion of the TBP gene family, especially in Halobacterium sp. NRC-1, and TFB gene family in representatives of five different genera of haloarchaea in which genome sequences are available.
Six of thirteen TBP and TFB genes of Halobacterium sp. NRC-1 are non-essential under standard growth conditions. TbpD and TfbA coordinate the expression of over 10% of the genes in the NRC-1 genome. The ΔtbpD and ΔtfbA mutant strains are temperature sensitive, possibly as a result of down regulation of heat shock genes. Sequence alignments suggest the existence of several families of TBP and TFB transcription factors in Halobacterium which may function in transcription of different classes of genes.
PMCID: PMC2121645  PMID: 17892563
19.  Transcriptional profiling of the model Archaeon Halobacterium sp. NRC-1: responses to changes in salinity and temperature 
Saline Systems  2007;3:6.
The model halophile Halobacterium sp. NRC-1 was among the first Archaea to be completely sequenced and many post-genomic tools, including whole genome DNA microarrays are now being applied to its analysis. This extremophile displays tolerance to multiple stresses, including high salinity, extreme (non-mesophilic) temperatures, lack of oxygen, and ultraviolet and ionizing radiation.
In order to study the response of Halobacterium sp. NRC-1 to two common stressors, salinity and temperature, we used whole genome DNA microarrays to assay for changes in gene expression under differential growth conditions. Cultures grown aerobically in rich medium at 42°C were compared to cultures grown at elevated or reduced temperature and high or low salinity. The results obtained were analyzed using a custom database and microarray analysis tools. Growth under salt stress conditions resulted in the modulation of genes coding for many ion transporters, including potassium, phosphate, and iron transporters, as well as some peptide transporters and stress proteins. Growth at cold temperature altered the expression of genes involved in lipid metabolism, buoyant gas vesicles, and cold shock proteins. Heat shock showed induction of several known chaperone genes. The results showed that Halobacterium sp. NRC-1 cells are highly responsive to environmental changes at the level of gene expression.
Transcriptional profiling showed that Halobacterium sp. NRC-1 is highly responsive to its environment and provided insights into some of the specific responses at the level of gene expression. Responses to changes in salt conditions appear to be designed to minimize the loss of essential ionic species and abate possible toxic effects of others, while exposure to temperature extremes elicit responses to promote protein folding and limit factors responsible for growth inhibition. This work lays the foundation for further bioinformatic and genetic studies which will lead to a more comprehensive understanding of the biology of a model halophilic Archaeon.
PMCID: PMC1971269  PMID: 17651475
20.  Essential and non-essential DNA replication genes in the model halophilic Archaeon, Halobacterium sp. NRC-1 
BMC Genetics  2007;8:31.
Information transfer systems in Archaea, including many components of the DNA replication machinery, are similar to those found in eukaryotes. Functional assignments of archaeal DNA replication genes have been primarily based upon sequence homology and biochemical studies of replisome components, but few genetic studies have been conducted thus far. We have developed a tractable genetic system for knockout analysis of genes in the model halophilic archaeon, Halobacterium sp. NRC-1, and used it to determine which DNA replication genes are essential.
Using a directed in-frame gene knockout method in Halobacterium sp. NRC-1, we examined nineteen genes predicted to be involved in DNA replication. Preliminary bioinformatic analysis of the large haloarchaeal Orc/Cdc6 family, related to eukaryotic Orc1 and Cdc6, showed five distinct clades of Orc/Cdc6 proteins conserved in all sequenced haloarchaea. Of ten orc/cdc6 genes in Halobacterium sp. NRC-1, only two were found to be essential, orc10, on the large chromosome, and orc2, on the minichromosome, pNRC200. Of the three replicative-type DNA polymerase genes, two were essential: the chromosomally encoded B family, polB1, and the chromosomally encoded euryarchaeal-specific D family, polD1/D2 (formerly called polA1/polA2 in the Halobacterium sp. NRC-1 genome sequence). The pNRC200-encoded B family polymerase, polB2, was non-essential. Accessory genes for DNA replication initiation and elongation factors, including the putative replicative helicase, mcm, the eukaryotic-type DNA primase, pri1/pri2, the DNA polymerase sliding clamp, pcn, and the flap endonuclease, rad2, were all essential. Targeted genes were classified as non-essential if knockouts were obtained and essential based on statistical analysis and/or by demonstrating the inability to isolate chromosomal knockouts except in the presence of a complementing plasmid copy of the gene.
The results showed that ten out of nineteen eukaryotic-type DNA replication genes are essential for Halobacterium sp. NRC-1, consistent with their requirement for DNA replication. The essential genes code for two of ten Orc/Cdc6 proteins, two out of three DNA polymerases, the MCM helicase, two DNA primase subunits, the DNA polymerase sliding clamp, and the flap endonuclease.
PMCID: PMC1906834  PMID: 17559652
21.  Saline Systems highlights for 2006 
Saline Systems  2007;3:1.
Saline Systems is a journal devoted to both basic and applied studies of saline and hypersaline environments and their biodiversity. Here, I review the reports and commentaries published in the journal in 2006, including some exploring the geochemistry of saline estuaries, lakes, and ponds, others on the ecology and molecular biology of the indigenous halophilic organisms, and still others addressing the environmental challenges facing saline environments. Several studies are relevant to applications in biotechnology and aquaculture.
PMCID: PMC1784102  PMID: 17244355
22.  The uvrA, uvrB and uvrC genes are required for repair of ultraviolet light induced DNA photoproducts in Halobacterium sp. NRC-1 
Saline Systems  2006;2:11.
Sequenced archaeal genomes contain a variety of bacterial and eukaryotic DNA repair gene homologs, but relatively little is known about how these microorganisms actually perform DNA repair. At least some archaea, including the extreme halophile Halobacterium sp. NRC-1, are able to repair ultraviolet light (UV) induced DNA damage in the absence of light-dependent photoreactivation but this 'dark' repair capacity remains largely uncharacterized. Halobacterium sp. NRC-1 possesses homologs of the bacterial uvrA, uvrB, and uvrC nucleotide excision repair genes as well as several eukaryotic repair genes and it has been thought that multiple DNA repair pathways may account for the high UV resistance and dark repair capacity of this model halophilic archaeon. We have carried out a functional analysis, measuring repair capability in uvrA, uvrB and uvrC deletion mutants.
Deletion mutants lacking functional uvrA, uvrB or uvrC genes, including a uvrA uvrC double mutant, are hypersensitive to UV and are unable to remove cyclobutane pyrimidine dimers or 6–4 photoproducts from their DNA after irradiation with 150 J/m2 of 254 nm UV-C. The UV sensitivity of the uvr mutants is greatly attenuated following incubation under visible light, emphasizing that photoreactivation is highly efficient in this organism. Phylogenetic analysis of the Halobacterium uvr genes indicates a complex ancestry.
Our results demonstrate that homologs of the bacterial nucleotide excision repair genes uvrA, uvrB, and uvrC are required for the removal of UV damage in the absence of photoreactivating light in Halobacterium sp. NRC-1. Deletion of these genes renders cells hypersensitive to UV and abolishes their ability to remove cyclobutane pyrimidine dimers and 6–4 photoproducts in the absence of photoreactivating light. In spite of this inability to repair UV damaged DNA, uvrA, uvrB and uvrC deletion mutants are substantially less UV sensitive than excision repair mutants of E. coli or yeast. This may be due to efficient damage tolerance mechanisms such as recombinational lesion bypass, bypass DNA polymerase(s) and the existence of multiple genomes in Halobacterium. Phylogenetic analysis provides no clear evidence for lateral transfer of these genes from bacteria to archaea.
PMCID: PMC1590041  PMID: 16970815
23.  Post-genomics of the model haloarchaeon Halobacterium sp. NRC-1 
Saline Systems  2006;2:3.
Halobacteriumsp. NRC-1 is an extremely halophilic archaeon that is easily cultured and genetically tractable. Since its genome sequence was completed in 2000, a combination of genetic, transcriptomic, proteomic, and bioinformatic approaches have provided insights into both its extremophilic lifestyle as well as fundamental cellular processes common to all life forms. Here, we review post-genomic research on this archaeon, including investigations of DNA replication and repair systems, phototrophic, anaerobic, and other physiological capabilities, acidity of the proteome for function at high salinity, and role of lateral gene transfer in its evolution.
PMCID: PMC1447603  PMID: 16542428
24.  Saline Systems highlights for 2005 
Saline Systems  2006;2:1.
On the 4th of July, 2005, the Saline Systems editorial group launched the new online open access journal, Saline Systems, with BioMed Central as the publisher. The scope of the journal includes both basic and applied research on halophilic organisms and saline environments, from gene systems to ecosystems. The stated goal of the journal is to meet publication needs for researchers working in coastal and inland saline environments and provide an interdisciplinary and readily accessible forum for scientists worldwide. The inaugural volume of the journal contains a significant number of high quality original research papers and reviews on a wide range of relevant topics. At the end of the launch period, from January 1, 2006 onwards, the journal will be introducing article-processing charges to cover the cost of publication. Charges will be partly or completely waived for authors from BioMed Central institutional subscribers and in cases of financial hardship.
PMCID: PMC1360670  PMID: 16417635
25.  UV irradiation induces homologous recombination genes in the model archaeon, Halobacterium sp. NRC-1 
Saline Systems  2005;1:3.
A variety of strategies for survival of UV irradiation are used by cells, ranging from repair of UV-damaged DNA, cell cycle arrest, tolerance of unrepaired UV photoproducts, and shielding from UV light. Some of these responses involve UV-inducible genes, including the SOS response in bacteria and an array of genes in eukaryotes. To address the mechanisms used in the third branch of life, we have studied the model archaeon, Halobacterium sp. strain NRC-1, which tolerates high levels of solar radiation in its natural hypersaline environment.
Cells were irradiated with 30–70 J/m2 UV-C and an immunoassay showed that the resulting DNA damage was largely repaired within 3 hours in the dark. Under such conditions, transcriptional profiling showed the most strongly up-regulated gene was radA1, the archaeal homolog of rad51/recA, which was induced 7-fold. Additional genes involved in homologous recombination, such as arj1 (recJ-like exonuclease), dbp (eukaryote-like DNA binding protein of the superfamily I DNA and RNA helicases), and rfa3 (replication protein A complex), as well as nrdJ, encoding for cobalamin-dependent ribonucleotide reductase involved in DNA metabolism, were also significantly induced in one or more of our experimental conditions. Neither prokaryotic nor eukaryotic excision repair gene homologs were induced and there was no evidence of an SOS-like response.
These results show that homologous recombination plays an important role in the cellular response of Halobacterium sp. NRC-1 to UV damage. Homologous recombination may permit rescue of stalled replication forks, and/or facilitate recombinational repair. In either case, this provides a mechanism for the observed high-frequency recombination among natural populations of halophilic archaea.
PMCID: PMC1224876  PMID: 16176594

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