'All About' Extremophiles.

Despite common perception, most of Earth is what is often referred to as an 'extreme environment.' Yet to the organisms that call these places home, it is simply that (home). They have adapted to thrive in these environments and, in the process, have evolved many unique adaptations at the molecular- and 'omic-level. Scientists' interest in these organisms has typically been in how they and their products can be harnessed for biotechnological applications and the environments where they are found, while the general public's veers more toward a fascination with their deviation from the 'norm'. However, these organisms have so much more to tell us about Life and the myriad ways there are to perform 'simple' biological processes.

The information transfer system of halophilic archaea.

Information transfer is fundamental to all life forms. In the third domain of life, the archaea, many of the genes functioning in these processes are similar to their eukaryotic counterparts, including DNA replication and repair, basal transcription, and translation genes, while many transcriptional regulators and the overall genome structure are more bacterial-like. Among halophilic (salt-loving) archaea, the genomes commonly include extrachromosomal elements, many of which are large megaplasmids or minichromosomes. With the sequencing of genomes representing ten different genera of halophilic archaea and the availability of genetic systems in two diverse models, Halobacterium sp. NRC-1 and Haloferax volcanii, a large number of genes have now been annotated, classified, and studied. Here, we review the comparative genomic, genetic, and biochemical work primarily aimed at the information transfer system of halophilic archaea, highlighting gene conservation and differences in the chromosomes and the large extrachromosomal elements among these organisms.

Dissection of the regulatory mechanism of a heat-shock responsive promoter in Haloarchaea: a new paradigm for general transcription factor directed archaeal gene regulation.

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 (P(hsp5)) 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 P(hsp5) 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 P(hsp5) likely in a temperature-dependent manner. Taken together, the heat-responsiveness of P(hsp5) 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.

Multiple replication origins of Halobacterium sp. strain NRC-1: properties of the conserved orc7-dependent oriC1.

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.

Recent advances in understanding extremophiles.

Despite the typical human notion that the Earth is a habitable planet, over three quarters of our planet is uninhabitable by us without assistance. The organisms that live and thrive in these "inhospitable" environments are known by the name extremophiles and are found in all Domains of Life. Despite our general lack of knowledge about them, they have already assisted humans in many ways and still have much more to give. In this review, I describe how they have adapted to live/thrive/survive in their niches, helped scientists unlock major scientific discoveries, advance the field of biotechnology, and inform us about the boundaries of Life and where we might find it in the Universe.

Genetic and transcriptomic analysis of transcription factor genes in the model halophilic Archaeon: coordinate action of TbpD and TfbA.

BACKGROUND: 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. RESULTS: 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, Delta tbpD and DeltatfbA, 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 Delta tbpD and DeltatfbA 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 Delta tbpD and DeltatfbA mutant strains showed them to grow more slowly than the wild-type at an elevated temperature, 49 degrees C, and they showed reduced viability at 56 degrees 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. CONCLUSION: 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 Delta tbpD and DeltatfbA 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.

Extremophiles and biotechnology: current uses and prospects.

Biotechnology has almost unlimited potential to change our lives in very exciting ways. Many of the chemical reactions that produce these products can be fully optimized by performing them at extremes of temperature, pressure, salinity, and pH for efficient and cost-effective outcomes. Fortunately, there are many organisms (extremophiles) that thrive in extreme environments found in nature and offer an excellent source of replacement enzymes in lieu of mesophilic ones currently used in these processes. In this review, I discuss the current uses and some potential new applications of extremophiles and their products, including enzymes, in biotechnology.

The effects of extremes of pH on the growth and transcriptomic profiles of three haloarchaea.

The halophilic archaea (haloarchaea) live in saline environments which are found across the globe.  In addition to salinity, these niches can be quite dynamic and experience extreme conditions such as low oxygen content, radiation (gamma and UV), pH and temperature.  However, of all the naturally occurring stresses faced by the haloarchaea, only one, pH, has not been previously reported on. Therefore, we endeavored to determine the responses of the transcriptomes of three haloarchaea (Hla, Hvo, and NRC-1) to growth under acidic and alkaline pH. Our observations showed that the transcriptomes of Hvo and NRC-1 respond in a similar manner to each other as well as other prokaryotes when grown in an acidic environment, while the pattern for Hla was dissimilar. For alkaline stress, all three haloarchaea responded in a manner similar to well-studied archaea and bacteria and had four-times more significantly regulated transcripts in common, compared to acidic growth. Additionally, we performed an analysis on the changes in the transcriptomes of the three haloarchaea when shifting from one pH extreme to the other. The results showed that the transcriptomes of all three haloarchaea respond more similarly when moving from alkaline to acidic conditions compared to moving from an acidic to alkaline environment. Interestingly, our studies also showed that individual genes of multiple paralogous gene families ( tbp, tfb, orc/ cdc6, etc.) found in the haloarchaea were regulated under specific stresses thereby providing evidence that they modulate the response to various environmental stresses. The studies described here are the first to catalog the changes in the haloarchaeal transcriptomes under growth in extreme pH and help us understand how life is able to thrive under all conditions present on Earth and, if present, on extraterrestrial bodies as well.

Transcriptional profiling of the model Archaeon Halobacterium sp. NRC-1: responses to changes in salinity and temperature.

BACKGROUND: 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. RESULTS: 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 degrees 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. CONCLUSION: 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.

Protein engineering of a cold-active beta-galactosidase from Arthrobacter sp. SB to increase lactose hydrolysis reveals new sites affecting low temperature activity.

We examined variants of an especially cold-active beta-galactosidase (BgaS) to better understand features affecting enzyme activity at temperature extremes. We targeted locations corresponding to a region in the LacZ enzyme previously shown to increase activity and decrease thermostability. Changes in this region of BgaS consistently caused the elimination or reduction of activity. A gene (bgaS3) encoding a loss of function variant was subjected to random mutagenesis to restore activity and discover potential interactions important in cold activity. Gene sequences from the resulting library indicated that only two amino acid alterations, E229D and V405A, were required to restore activity. Genes with combinations of these mutations were constructed and their enzymes purified. Enzymes with the E229D/V405A/G803D alterations (BgaS6), or E229D/V405A (BgaS7) had similar thermal optima and thermostabilities as BgaS. BgaS7, however, showed a 2.5-fold increase in catalytic activity at 15 degrees C and hydrolyzed 80% of lactose in skim milk in less than half the time of BgaS at 2.5 degrees C. Computer-generated models predicted that the substitutions at positions 229 and 405 yielded fewer contacts at the enzyme's activating interface. Results from regional saturation mutagenesis supported this hypothesis and suggested that not easily predicted, subtle, cooperative intramolecular interactions contributed to thermal adaptation.

Post-genomics of the model haloarchaeon Halobacterium sp. NRC-1.

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.

Biochemical characterization of a beta-galactosidase with a low temperature optimum obtained from an Antarctic arthrobacter isolate.

A psychrophilic gram-positive isolate was obtained from Antarctic Dry Valley soil. It utilized lactose, had a rod-coccus cycle, and contained lysine as the diamino acid in its cell wall. Consistent with these physiological traits, the 16S ribosomal DNA sequence showed that it was phylogenetically related to other Arthrobacter species. A gene (bgaS) encoding a family 2 beta-galactosidase was cloned from this organism into an Escherichia coli host. Preliminary results showed that the enzyme was cold active (optimal activity at 18 degrees C and 50% activity remaining at 0 degrees C) and heat labile (inactivated within 10 min at 37 degrees C). To enable rapid purification, vectors were constructed adding histidine residues to the BgaS enzyme and its E. coli LacZ counterpart, which was purified for comparison. The His tag additions reduced the specific activities of both beta-galactosidases but did not alter the other characteristics of the enzymes. Kinetic studies using o-nitrophenyl-beta-D-galactopyranoside showed that BgaS with and without a His tag had greater catalytic activity at and below 20 degrees C than the comparable LacZ beta-galactosidases. The BgaS heat lability was investigated by ultracentrifugation, where the active enzyme was a homotetramer at 4 degrees C but dissociated into inactive monomers at 25 degrees C. Comparisons of family 2 beta-galactosidase amino acid compositions and modeling studies with the LacZ structure did not mimic suggested trends for conferring enzyme flexibility at low temperatures, consistent with the changes affecting thermal adaptation being localized and subtle. Mutation studies of the BgaS enzyme should aid our understanding of such specific, localized changes affecting enzyme thermal properties.