Mark A. Schneegurt

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.

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.

Bacterial Growth at the High Concentrations of Magnesium Sulfate Found in Martian Soils

The martian surface environment exhibits extremes of salinity, temperature, desiccation, and radiation that would make it difficult for terrestrial microbes to survive. Recent evidence suggests that martian soils contain high concentrations of MgSO₄ minerals. Through warming of the soils, meltwater derived from subterranean ice-rich regolith may exist for an extended period of time and thus allow the propagation of terrestrial microbes and create significant bioburden at the near surface of Mars. The current report demonstrates that halotolerant bacteria from the Great Salt Plains (GSP) of Oklahoma are capable of growing at high concentrations of MgSO₄ in the form of 2 M solutions of epsomite. The epsotolerance of isolates in the GSP bacterial collection was determined, with 35% growing at 2 M MgSO₄. There was a complex physiological response to mixtures of MgSO₄ and NaCl coupled with other environmental stressors. Growth also was measured at 1 M concentrations of other magnesium and sulfate salts. The complex responses may be partially explained by the pattern of chaotropicity observed for high-salt solutions as measured by agar gelation temperature. Select isolates could grow at the high salt concentrations and low temperatures found on Mars. Survival during repetitive freeze-thaw or drying-rewetting cycles was used as other measures of potential success on the martian surface. Our results indicate that terrestrial microbes might survive under the high-salt, low-temperature, anaerobic conditions on Mars and present significant potential for forward contamination. Stringent planetary protection requirements are needed for future life-detection missions to Mars.

Media and Conditions for the Growth of Halophilic and Halotolerant Bacteria and Archaea

An awareness of haloarchaea has existed since ancient times, with published descriptions of “red waters” associated with salt mining, the “red heat” of salted hides, and the “reddening” of salted fish (Bass-Becking 1931; Kurlansky 2002). For a society without refrigeration, the economic impact of codfish deterioration garnered particular attention, with Farlow (1878) oft cited as the first to publish on what were presumably haloarchaea . The early growth media of Eddington (1887) and Le Dantec (1891) reflected natural high-protein substrates, using beef peptone, gelatins, and fish broths, solidified with agar, flour, or bread paste. While some early studies used pieces of fish soaked in various brines (Hoye 1908; Klebahn 1919; Harrison and Kennedy 1922), many included ground cod or a cod broth, or media based on beef bouillon or beef gelatin (Beckwith 1911; Bitting 1911; Becker 1912; Kellerman 1915; Clayton and Gibbs 1927; Velu 1929). Milk was introduced as a preferred organic constituent by Bitting (1911) and Kellerman (1915), but was popularized by Lockhead (1934). Rice flour, wheat flour or whole rice grains often were used as gelling agents (Clayton and Gibbs 1927; Robertson 1931; Boury 1934; Gibbons 1937). Silica gel was suggested to reduce organic content of solidified media (Hanks and Weintraub 1936; Moore 1940 1941). It was recognized that alkaline culture conditions were useful for growing certain halophilic microbes (Stather and Liebscher 1929) and that halophilic obligate anaerobes could be grown on a cooked meat medium (Baumgartner 1937). The seminal paper of Harrison and Kennedy (1922) focused on the difficulties of growing the organisms responsible for red discolorations on salted fish, trying many media recipes including those based on cider, milk, broths, sugars, and potatoes. While the red organisms proved difficult to isolate, as an aside, the paper discusses a broad diversity of non-red halophilic organisms that were more easily isolated on these media.

Carbon substrate utilization, antibiotic sensitivity, and numerical taxonomy of bacterial isolates from the Great Salt Plains of Oklahoma

The current work extends the phenotypic characterization of a bacterial culture collection from the Great Salt Plains of Oklahoma. This barren expanse of mud flats is typically crusted with thalassohaline salt evaporites. The initial account of the aerobic heterotrophic bacteria from the Great Salt Plains described 105 halotolerant isolates that represented 47 phylotypes. Extensive phenotypic analyses were performed on 76 isolates representing 37 unique phylotypes. The current report extends these observations for 60 of the isolates by measuring a wider set of phenotypic characteristics. Utilization patterns for 45 carbon substrates were used to assign the isolates into seven coherent phenons, along with several singletons and a group of isolates that did not grow on single carbon substrates. Most of the isolates were able to utilize nearly all of the nitrogen sources tested, with nitrate being the least utilized. Little antibiotic resistance was seen in the collection as a whole; however, certain phenons were enriched for antibiotic-resistant organisms. A total of 81 phenotypic characteristics were used to generate dendrograms. The numerical taxonomy trees essentially agreed with those generated using 16S rRNA gene sequences. The pattern of carbon substrate utilization showed substantial changes at different salinities that may have relevance to the variable salinities microbes experience at the Salt Plains over time.

Molecular and phenetic characterization of the bacterial assemblage of Hot Lake, WA, an environment with high concentrations of magnesium sulphate, and its relevance to Mars

Hot Lake (Oroville, WA) is an athalassohaline epsomite lake that can have precipitating concentrations of MgSO4 salts, mainly epsomite. Little biotic study has been done on epsomite lakes and it was unclear whether microbes isolated from epsomite lakes and their margins would fall within recognized halotolerant genera, common soil genera, or novel phyla. Our initial study cultivated and characterized epsotolerant bacteria from the lake and its margins. Approximately 100 aerobic heterotrophic microbial isolates were obtained by repetitive streak-plating in high-salt media including either 10% NaCl or 2 M MgSO4. The collected isolates were all bacteria, nearly evenly divided between Gram-positive and Gram-negative clades, the most abundant genera being Halomonas, Idiomarina, Marinobacter, Marinococcus, Nesterenkonia, Nocardiopsis, and Planococcus. Bacillus, Corynebacterium, Exiguobacterium, Kocuria, and Staphylococcus also were cultured. This initial study included culture-independent community analysis of direct DNA extracts of lake margin soil using PCR-based clone libraries and 16S rRNA gene phylogeny. Clones assigned Gram-positive bacterial clades (70% of total clones) were dominated by sequences related to uncultured actinobacteria. There were abundant Deltaproteobacteria clones related to bacterial sulfur metabolisms and clones of Legionella and Coxiella. These epsomite lake microbial communities seem to be divided between bacteria primarily associated with hyperhaline environments rich in NaCl and salinotolerant relatives of common soil organisms. Archaea appear to be in low abundance and none were isolated, despite near-saturated salinities. Growth of microbes at very high concentrations of magnesium and other sulfates has relevance to planetary protection and life-detection missions to Mars, where scant liquid water may form as deliquescent brines and appear as eutectic liquids.

Comparative molecular analysis of the prokaryotic diversity of two salt mine soils in southwest China

While much is known about the microbial diversity in some hypersaline environments, little is known about those of salt mine tunnel soils. The objective of this study was to conduct a comprehensive phylogenetic comparison of the archaeal and bacterial communities present in Yipinglang salt mine (YPL) and Qiaohou salt mine (QH) tunnels differing in salinity and salt composition using 16S rRNA gene clone libraries. Two hundred twenty‐eight sequences for QH and 182 sequences for YPL were analyzed by amplified ribosomal DNA‐restriction analysis. Libraries revealed 44 bacterial and 57 archaeal different operational taxonomic units belonging to at least 8 bacterial and 3 archaeal divisions, but not all divisions were observed in both salt mines. The bacterial community affiliated with the Bacteroidetes was the most abundant (60% of clones) in QH, while the community in YPL was dominated by δ‐Proteobacteria (45% of clones). All archaeal clones from QH were affiliated with Halobacteriaceae. In contrast, in the YPL library, 49% of clones belonged to Halobacteriaceae, 31% of clones related to unclassified archaea, and 21% of clones belonged to Crenarchaeota. Bioinformatic analysis and comparisons showed that the clone libraries were significantly different between two salt mines.

Isolation and characterization of bacteria from the feathers of wild Dark-eyed Juncos (Junco hyemalis)

ABSTRACT We dislodged microbes from samples of composites of ventral feathers from different birds of overwintering Dark-eyed Juncos (Junco hyemalis) after mist-net capture in south-central Kansas, USA. Bacterial loads were measured by standard plate counts and >300 isolates were purified by repetitive streak-plating on R2A medium (+ cycloheximide). Biochemical and physiological characterization included identification by 16S rRNA gene phylogeny. Nearly half of the isolates grew on keratin and 80% exhibited lipase activity, suggesting that these isolates can degrade feathers and thus may affect survival and reproduction. Individual bacterial loads from 8 juncos varied within a 3-fold range, 105–106 colony-forming units g−1 feather. At 97% DNA sequence identity (species-level), 63 operational taxonomic units were detected among 202 sequences; the Chao1 estimate was 123. The Shannon diversity index (H; 97% identity) was 3.75, Simpsons diversity index (1/D) was 16.1, and Goods coverage was 82.4. Gram-positive bacteria dominated the culture collection, balanced between low and high G+C clades. Bacillus spp. were abundant, including B. asahii, B. cereus, B. megaterium, and B. pumilus. Lysinibacillus, Paenibacillus, and Staphylococcus also were isolated. Remarkably, substantial numbers of Actinomycetes were isolated, including representatives of Clavibacter, Curtobacterium, Microbacterium, and Rathayibacter, genera recognized as being populated by xylem-filling crop plant pathogens. Apposed to these were feather isolates implicated as beneficial to host plants, Frigoribacterium and Kitasatospora, being antagonists to plant pathogens or acting as plant growth promoters. High G+C Gram-positive bacterial isolates included Blastococcus, Cellulomonas, Humicoccus, Nocardioides, Promicromonospora, and Rhodococcus. Proteobacteria dominated the Gram-negative bacteria, with Alphaproteobacteria most abundant, including the potential plant pathogens Agrobacterium and Sphingomonas, and the oligotrophs Aurantimonas, Brevundimonas, Methylobacterium, Rhizobium, and Rhodobacter. Gammaproteobacteria included Pantoea, Pseudomonas, and Stenotrophomonas. Ours is the first report of abundant helpful and harmful phyllosphere bacteria on wild bird feathers. The clear implication is that free-living migratory birds may carry bacteria throughout their geographic ranges and may transmit pathogens and beneficial bacteria to plants.

Culture-independent analysis of the soil bacterial assemblage at the Great Salt Plains of Oklahoma.

The Great Salt Plains (GSP) of Oklahoma is a natural inland terrestrial hypersaline environment that forms evaporite crusts of mainly NaCl. Previous work described GSP bacterial assemblages through the phylogenetic and phenetic characterization of 105 isolates from 46 phylotypes. The current report describes the same bacterial assemblages through culture‐independent 16S rRNA gene clone libraries. Although from similar hypersaline mud flats, the bacterial libraries from two sites, WP3 and WP6, were quite different. The WP3 library was dominated by cyanobacteria, mainly Cyanothece and Euhalothece. The WP6 library was rich in anaerobic sulfur‐cycle organisms, including abundant Desulfuromonas. This pattern likely reflects differences in abiotic factors, such as frequency of flooding and hydrologic push. While more than 100 OTUs were identified, the assemblages were not as diverse, based on Shannon indexes, as bacterial communities from oligohaline soils. Since natural inland hypersaline soils are relatively unstudied, it was not clear what kind of bacteria would be present. The bacterial assemblage is predominantly genera typically found in hypersaline systems, although some were relatives of microbes common in oligohaline and marine environments. The bacterial clones did not reflect wide functional diversity, beyond phototrophs, sulfur metabolizers, and numerous heterotrophs. (© 2012 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)