Perkinsus species are protistan parasites of molluscs. In Chesapeake Bay, Perkinsus marinus, Perkinsus chesapeaki, and Perkinsus andrewsi are sympatric, infecting oysters and clams. Although P. marinus is a pathogen for Crassostrea virginica, it remains unknown whether P. andrewsi and P. chesapeaki are equally pathogenic. Perkinsus species have been reported in C. virginica as far north as Maine, sometimes associated with high prevalence, but low mortality. Thus, we hypothesized that, in addition to P. marinus, Perkinsus species with little or no pathogenicity for C. virginica may be present. Accordingly, we investigated the distribution of Perkinsus species in C. virginica and Mercenaria mercenaria, collected from Maine to Virginia, by applying PCR-based assays specific for P. marinus, P. andrewsi, and a Perkinsus sp. isolated from M. mercenaria. DNA samples of M. mercenaria possessed potent PCR inhibitory activity, which was overcome by the addition of 1 mg/ml BSA and 5% (v/v) DMSO to the PCR reaction mixture. All 3 Perkinsus species were found in both host species throughout the study area. Interestingly, the prevalence of P. marinus in M. mercenaria was significantly lower than in C. virginica, suggesting that M. mercenaria is not an optimal host for P. marinus.

The Antarctic microorganism harbors a model polyextremophilic β-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 β-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-inducibleD2 promoter in the genetically tractable Haloarchaeon, sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate-nitrophenyl-β-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (/) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both and (three substitutions) or either or (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.

The parasite Perkinsus marinus has devastated natural and farmed oyster populations along the Atlantic and Gulf coasts of North America. When viable P. marinus trophozoites are engulfed by oyster hemocytes, the typical accumulation of reactive oxygen species (ROS) normally associated with phagocyte activity is not observed. One hypothesis to explain this is that the parasite rapidly removes ROS. A manifestation of efficient ROS removal should be a high level of resistance to exogenous ROS. We investigated the in vitro susceptibility of P. marinus to ROS as compared to the estuarine bacterium Vibrio splendidus. We find that P. marinus is markedly less susceptible than V. splendidus to superoxide and hydrogen peroxide (H(2)O(2)), but equally sensitive to hypochlorite. Viable P. marinus trophozoites degrade H(2)O(2) in vitro, but lack detectable catalase activity. However, extracts contain an ascorbate dependent peroxidase activity that may contribute to H(2)O(2) removal in vitro and in vivo.

Hypersaline regions are terrestrial analogs of the Earth's primitive ecosystem and extraterrestrial environment. The salt range in Pakistan is considered among a few of the ancient salt deposits in the subcontinent. Karak salt mine is situated at the Northwest end in Pakistan. Despite the fact that halophiles initiated the formation of terrestrial ecosystems, their products and identities remain hidden. Some preliminary studies limited to culture-dependent isolations have been reported. Characterizing the microbiome that spans over centuries of ecosystem development is crucial, given their role in shaping landscape succession and biogeochemical cycles. Here, we used metagenomics techniques to explore the microbial diversity of the Karak salt mine. We used 16S rRNA Illumina amplicon sequencing to characterize the halophilic communities entrapped in Karak mine. The results were interpreted using Illumina Basespace, QIIME, and Cytoscape. Cultures were isolated at 16-25% salinity. Metagenomics data was consistent with our preliminary culturing data, indicating remarkable species to strainlevel diversity of unique halophiles. A total of 107,099 (brine) and 122,679 (salt) reads were obtained. 16S rRNA based sequencing revealed a microbiome with bacteria (66% brine and 72% salt) dominated by Bacteroidetes and Proteobacteria with a strikingly high abundance of Archaea (18% brine and 13% salt). Alpha diversity has higher values in salt than in the brine. The study of the halophiles in the Karak salt mine provides clues for species contributing to the maintenance of biogeochemical cycles of the ecosystem. This is the first report of a metagenomic study of any hypersaline region of Pakistan.