Archaeal diversity at the great salt plains of Oklahoma described by cultivation and molecular analyses.

The Great Salt Plains of Oklahoma is a natural inland terrestrial hypersaline environment that forms evaporite crusts of mainly NaCl. Previous work described the bacterial community through the characterization of 105 isolates from 46 phylotypes. The current report describes the archaeal community through both microbial isolation and culture-independent techniques. Nineteen distinct archaea were isolated, and ten were characterized phenetically. Included were isolates phylogenetically related to Haloarcula, Haloferax, Halorubrum, Haloterrigena, and Natrinema. The isolates were aerobic, non-motile, Gram-negative organisms and exhibited little capacity for fermentation. All of the isolates were halophilic, with most requiring at least 15% salinity for growth, and all grew at 30% salinity. The isolates were mainly mesothermic and could grow at alkaline pH (8.5). A 16S rRNA gene library was generated by polymerase chain reaction amplification of direct soil DNA extracts, and 200 clones were sequenced and analyzed. At 99% and 94% sequence identity, 36 and 19 operational taxonomic units (OTUs) were detected, respectively, while 53 and 22 OTUs were estimated by Chao1, respectively. Coverage was relatively high (100% and 59% at 89% and 99% sequence identity, respectively), and the Shannon Index was 3.01 at 99% sequence identity, comparable to or somewhat lower than hypersaline habitats previously studied. Only sequences from Euryarchaeota in the Halobacteriales were detected, and the strength of matches to known sequences was generally low, most near 90% sequence identity. Large clusters were observed that are related to Haloarcula and Halorubrum. More than two-thirds of the sequences were in clusters that did not have close relatives reported in public databases.

Saccharin aversions in hamsters as a result of nicotine injections.

Golden Syrian hamsters (males, N = 70) showed dose-related conditioned taste aversion (CTA) when saccharin drinking was followed by delayed nicotine injections. Baseline consisted of measuring amounts consumed after 20 minutes of daily access to tap water. Measures were taken for five days. The hamsters were then conditioned by offering them saccharin solution (0.1%, w/v) for 20 minutes; afterwhich a 30 minute delay was imposed. Subsequent to the delay, groups of 10 animals were treated as follows: nicotine injection (1.0, 3.0, or 9.0 mg/kg, IP), saline injection, lithium chloride injection (2% body weight of a 0.15 M solution), sham injection, or left in their cages as handling/stress controls. Following two recovery days with plain water available for 20 minutes, all animals were tested for CTA by offering them saccharin solution. Dose-related CTA was demonstrated in the nicotine animals as measured by a decrease in saccharin consumption compared to drinking measures obtained from animals injected with saline. Lithium chloride produced the same degree of CTA as 9 mg/kg of nicotine, and the aversions had extinguished in all groups by the third test day.

Conditioned saccharin aversions in rats as a result of cutaneous nicotine or intraperitoneal nicotine administered in divided doses.

Nicotine base was used in a conditioned taste aversion (CTA) paradigm to avert male Sprague-Dawley rats to saccharin solution (0.1%, w/v). Experiments investigated different dose routes of nicotine administration and duration of action as determinants in nicotine-induced CTA. In Experiment 1 nicotine was injected intraperitoneally (IP) at doses of 0.5, 1.0, or 3.0 mg/kg 30 min after drinking saccharin solution. Using a two-bottle choice test, no CTA was observed, although all nicotine animals showed obvious symptoms of malaise including seizures in the highest dose group. Experiment 2 showed dose-related CTA when nicotine (10.0, 30.0, or 50.0 mg/kg) was cutaneously applied 30 min following saccharin drinking. Experiment 2B showed that the aversions were due to associative rather than nonassociative factors such as sensitization or enhanced neophobia. In Experiment 3, the following group treatments were begun 30 min after saccharin drinking to distribute identical total nicotine doses over an extended period of time: One IP injection of 2.0 mg/kg nicotine (in a saline vehicle) and four injections of saline solution, three injections of 0.67 mg/kg nicotine and two injections of saline, five injections of 0.40 mg/kg nicotine, or five injections of saline. All injections were spaced 30 min apart. Compared with saline-injected controls, CTA occurred in the rats receiving either three or five injections of nicotine but the group receiving one injection did not differ from the control group. There was no difference in CTA between the groups receiving three or five injections.

An improved method for calculating labeling indices of lung epithelial and interstitial cells.

The sampling method was developed as an improvement in calculating labeling indices (LIs) of epithelial and interstitial cells in the airways and alveolar region of the lungs of rats. This method is more accurate and requires less time than the traditional cytokinetic methods, which involve counting all labeled and unlabeled cells. In the sampling method, all labeled cells are counted, but the total number of cells is estimated based on a representative sample count of airway and alveolar cells in each animal. The LIs as calculated by the two methods correlated very well (r greater than 0.97). The small amount of error introduced by estimating the total number of cells was more than compensated for by the increased accuracy associated with a larger sample size.

Halotolerant aerobic heterotrophic bacteria from the Great Salt Plains of Oklahoma.

The Salt Plains National Wildlife Refuge (SPNWR) near Cherokee, Oklahoma, contains a barren salt flat where Permian brine rises to the surface and evaporates under dry conditions to leave a crust of white salt. Rainfall events dissolve the salt crust and create ephemeral streams and ponds. The rapidly changing salinity and high surface temperatures, salinity, and UV exposure make this an extreme environment. The Salt Plains Microbial Observatory (SPMO) examined the soil microbial community of this habitat using classic enrichment and isolation techniques and phylogenetic rDNA studies. Rich growth media have been emphasized that differ in total salt concentration and composition. Aerobic heterotrophic enrichments were performed under a variety of conditions. Heterotrophic enrichments and dilution plates have generated 105 bacterial isolates, representing 46 phylotypes. The bacterial isolates have been characterized phenotypically and subjected to rDNA sequencing and phylogenetic analyses. Fast-growing isolates obtained from enrichments with 10% salt are predominantly from the gamma subgroup of the Proteobacteria and from the low GC Gram-positive cluster. Several different areas on the salt flats have yielded a variety of isolates from the Gram-negative genera Halomonas, Idiomarina, Salinivibrio, and Bacteroidetes. Gram-positive bacteria are well represented in the culture collection including members of the Bacillus, Salibacillus, Oceanobacillus, and Halobacillus.

DNA-repair potential of Halomonas spp. from the Salt Plains Microbial Observatory of Oklahoma.

The Great Salt Plains (GSP), an unvegetated, barren salt flat that is part of the Salt Plains National Wildlife Refuge near Cherokee, Oklahoma, is the site of the Salt Plains Microbial Observatory. At the GSP the briny remains of an ancient sea rise to the surface, evaporate under dry conditions, and leave crusts of white salt. Adaptation to this environment requires development of coping mechanisms providing tolerance to desiccating conditions due to the high salinity, extreme temperatures, alkaline pH, unrelenting exposure to solar UV radiation, and prevailing winds. Several lines of evidence suggest that the same DNA repair mechanisms that are usually associated with UV light or chemically induced DNA damage are also important in protecting microbes from desiccation. Because little is known about the DNA repair capacity of microorganisms from hypersaline terrestrial environments, we explored the DNA repair capacity of microbial isolates from the GSP. We used survival following exposure to UV light as a convenient tool to assess DNA repair capacity. Two species of Halomonas (H. salina and H. venusta) that have been isolated repeatedly from the GSP were chosen for analysis. The survival profiles were compared to those of Escherichia coli, Pseudomonas aeruginosa, and Halomonas spp. from aquatic saline environments. Survival of GSP organisms exceeded that of the freshwater organism P. aeruginosa, although they survived no better than E. coli. The GSP isolates were much more resistance to killing by UV than were the aquatic species of Halomonas reported in the literature [Martin et al. (2000) Can J Microbiol 46:180-187]. Unlike E. coli, the GSP isolates did not appear to have an inducible, error-prone repair mechanism. However, they demonstrated high levels of spontaneous mutation.