• Comparative Genomic Analysis of Halophilic Archaea

      Capes, Melinda Dyanne; DasSarma, Shiladitya (2012)
      Halophilic Archaea (Haloarchaea) form a clade in the Domain Archaea in phylogenetic studies. The genome for a model organism, Halobacterium sp. NRC-1 was available in 2000, with 14 additional complete haloarchaeal genomes representing 13 different genera available by 2011. In this thesis, comparative genomic and bioinformatic analyses of complete haloarchaeal genomes have advanced the understanding of their characteristics that allow for survival in harsh environments, an inventory of genes and molecular machineries that Haloarchaea possess compared to other Archaea, Bacteria and Eukarya, and the genome dynamics and evolution, particularly in the megaplasmids of the Halobacterium genus. Best reciprocal hits were used to identify haloarchaeal core and unique conserved proteins. 4,455 haloarchaeal orthologous groups (HOGs), of which 784, named the core haloarchaeal proteome (cHOGs), were identified in 13 haloarchaeal genomes. Of the cHOGs, 55 were identified as truly unique (tucHOGs) to Haloarchaea. Among the cHOG clusters, 33 % was predicted to function in metabolism, 25 % in information transfer and storage, and 10 % in cell processes and signaling. The conservation of information transfer genes were analyzed in further detail, and the vast majority was conserved in all of the haloarchaeal genomes. Several expanded gene families were identified and many of the paralogs were coded on large extrachromosomal elements. Detailed comparative genomic analysis of two sequenced genomes of Halobacterium strains showed that the chromosomes were very similar, with less than fifty divergent regions over 2 Mbp, while the extrachromosomal elements (two in strain NRC-1 and four in strain R-1) were more highly diverged, with, a mosaic of over 300 divergent regions. PCR amplification of extrachromosomal genes in strains NRC-1, R-1, and 14 other Halobacterium strains showed that only 14 % of the genes were conserved in all the strains, and clustered into six subgroups. Utilizing the 619 non-paralogous cHOGs for 13 Haloarchaea, amino acid substitutions unique to a cold-adapted species isolated in Antarctica, Halorubrum lacusprofundi, were determined using position specific scoring matrices (PSSM) and deviations from mesophilic identity sequence (MIS). Seven amino acid substitutions were identified that likely increase flexibility, improve interactions with the solvent, and counteract weakened hydrophobic effects.
    • Survival of a Polyextremophilic Archaeon and Function of Its Enzyme in Potentially Astrobiological Conditions

      Laye, Victoria Janet; DasSarma, Shiladitya (2018)
      Haloarchaea are extremophilic microorganisms used as models for life in niche environments on Earth, Mars, and other planets. We compared growth of Halorubrum lacusprofundi, a cold-adapted isolate from Vestfold Hills, Antarctica, with Halobacterium sp. NRC-1, a mesophilic laboratory strain. Both tolerated concentrations of magnesium and perchlorate higher than expected on Mars. These Haloarchaea were exposed to and tolerated stratospheric conditions. Psychrophilic Halorubrum has a better survival rate than Halobacterium after freeze-thaw. Cold-active β-galactosidase from Halorubrum was used as a model to establish effects of inhibitory conditions. The enzyme was more inhibited by magnesium. Cold-activity of the enzyme was investigated through bioinformatics, genomics, and mutagenesis by identifying divergent amino acids and creating single amino acid mutations. Importance of the residues for cold activity was tested and confirmed using steady-state kinetics. Based on this work, Halorubrum and its enzyme are ideal models for studying adaptation in polyextreme environments on Earth and elsewhere.