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1.  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
2.  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
3.  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
4.  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
5.  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

Results 1-5 (5)