16S rRNA Gene Diversity in Ancient Gray and Pink Salt from San Simón Salt Mines in Tarija, Bolivia.

DNA from 250 million-year-old pink and gray salts from mines in Tarija, Bolivia, subjected to 16S rRNA gene amplicon sequencing and analysis provided evidence for similar but distinct prokaryotic communities. The results constitute a snapshot of archaeal and bacterial microorganisms in these remote and ancient salt deposits.

16S rRNA Gene Diversity in the Salt Crust of Salar de Uyuni, Bolivia, the World's Largest Salt Flat.

Salar de Uyuni is a vast, high-altitude salt flat in Bolivia with extreme physico-geochemical properties approaching multiple limits of life. Evidence for diverse halophilic bacteria and archaea was found in its surface and near-surface salt crust using 16S amplicon analysis, providing a snapshot of prokaryotic life.

Effects of road salt on microbial communities: Halophiles as biomarkers of road salt pollution.

Increased use of salting to de-ice roadways, especially in urban areas, is leading to elevated salinity levels in soil as well as surface- and ground water. This salt pollution may cause long-term ecological changes to soil and aquatic microbial communities. In this study, we examined the impact on microbial communities in soils exposed to urban road salt runoff using both culturing and 16S amplicon sequencing. Both methods showed an increase in halophilic Bacteria and Archaea in samples from road salt-exposed areas and suggested that halophiles are becoming persistent members of microbial communities in urban, road salt-impacted soils. Since salt is a pollutant that can accumulate in soils over time, it is critical to begin assessing its impact on the environment immediately. Toward this goal, we have developed a facile semi-quantitative assay utilizing halophilic microbes as biomarkers to evaluate on-going salt pollution of soils.

Key amino acid residues conferring enhanced enzyme activity at cold temperatures in an Antarctic polyextremophilic ß-galactosidase.

The Antarctic microorganism Halorubrum lacusprofundi harbors a model polyextremophilic ß-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 ß-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-inducible cspD2 promoter in the genetically tractable Haloarchaeon, Halobacterium sp. NRC-1. Purified enzymes were characterized by steady-state kinetic analysis at temperatures from 0 to 25 °C using the chromogenic substrate o-nitrophenyl-ß-galactoside. All substitutions resulted in altered temperature activity profiles compared with wild type, with five of the six clearly exhibiting reduced catalytic efficiency (kcat/Km) at colder temperatures and/or higher efficiency at warmer temperatures. These results could be accounted for by temperature-dependent changes in both Km and kcat (three substitutions) or either Km or kcat (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.

Microneedle-Assisted Skin Permeation by Nontoxic Bioengineerable Gas Vesicle Nanoparticles.

Gas vesicle nanoparticles (GVNPs) are hollow, buoyant protein organelles produced by the extremophilic microbe Halobacterium sp. NRC-1 and are being developed as bioengineerable and biocompatible antigen and drug-delivery systems (DDS). Dynamic light scattering measurements of purified GVNP suspensions showed a mean diameter of 245 nm. In vitro diffusion studies using Yucatan miniature pig skin showed GVNP permeation to be enhanced after MN-treatment compared to untreated skin. GVNPs were found to be nontoxic to mammalian cells (human kidney and rat mycocardial myoblasts). These findings support the use of GVNPs as DDS for intradermal/transdermal permeation of protein- and peptide-based drugs.

Assessment of the northern distribution range of selected Perkinsus species in eastern oysters (Crassostrea virginica) and hard clams (Mercenaria mercenaria) with the use of PCR-based detection assays.

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.

Identification of a second rRNA gene unit in the Perkinsus andrewsi genome.

Perkinsus species are parasitic protozoa of mollusks, currently classified within the Perkinsozoa, a recently established phylum that is basal to the Apicomplexa and Dinozoa. Ribosomal RNA (rRNA) genes and their intergenic spacers have been used to support the taxonomy of Perkinsus species, the description of new species, and to develop molecular probes for their detection and identification. We previously described ultrastructure, behavior in culture, and partial sequence of the rRNA locus of a Perkinsus species isolated from the baltic clam Macoma balthica. The rRNA genes and intergenic spacers of this Perkinsus isolate differed from those described in the currently accepted species to a degree that led to its designation as a new species, Perkinsus andrewsi. In this study, we identify an additional rRNA gene unit (rRNA-B) in the P. andrewsi holotype, and report the complete sequences of both rRNA gene units. Except for the 5.8S, all regions of the rRNA-B gene unit exhibited sequence differences from that initially described (rRNA-A). Each rRNA gene unit is arranged in a "head-to-tail" tandem repeat. This is the first report demonstrating two distinct rRNA units in a Perkinsus species.

The protistan parasite Perkinsus marinus is resistant to selected reactive oxygen species.

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.