Abundance,Distribution, and Diversity of Viruses in Alkaline,Hypersaline Mono Lake,California

Mono Lake is a large (180 km²), alkaline (pH ~10), moderately hypersaline (70–85 g kg^–1) lake lying at the western edge of the Great Basin. An episode of persistent chemical stratification (meromixis) was initiated in 1995 and has resulted in depletion of oxygen and accumulation of ammonia and sulfide beneath the chemocline. Although previous studies have documented high bacterial abundances and marked seasonal changes in phytoplankton abundance and community composition, there have been no previous reports on the occurrence of viruses in this unique lake. Based on the high concentrations and diversity of microbial life in this lake, we hypothesized that planktonic viruses are also abundant and diverse. To examine the abundance and distribution of viruses and bacteria, water samples were collected from four stations along 5 to 15 vertical depths at each station. Viral abundance ranged from 1 × 10⁸ to 1 × 10⁹ mL^–1, among the highest observed in any natural aquatic system examined so far. Increases (p < 0.1) in viral densities were observed in the anoxic bottom water at multiple stations. However, regression analysis indicated that viral abundance could not be predicted by any single environmental parameter. Pulsed field gel electrophoresis revealed a diverse viral community in Mono Lake with genome sizes ranging from ~14 to >400 kb with most of the DNA in the 30 to 60 kb size range. Cluster analysis grouped the anoxic bottom-water viral community into a unique cluster differentiating it from surface and mid-water viral communities. A hybridization study using an indigenous viral isolate as a probe revealed an episodic pattern of temporal phage distribution with strong niche stratification between oxic and anoxic waters.     

Organic Osmolytes in Aerobic Bacteria from Mono Lake, an Alkaline, Moderately Hypersaline Environment

The identity and concentrations of intracellular organic solutes were determined by nuclear magnetic resonance spectroscopy for two strains of aerobic, gram-negative bacteria isolated from Mono Lake, Calif., an alkaline, moderately hypersaline lake. Ectoine (1,4,5,6-tetrahydro-2-methyl-4-pyrimidinecarboxylic acid) was the major endogenous solute in both organisms. Concentrations of ectoine varied with external NaCl levels in strain ML-D but not in strain ML-G, where the level was high but invariant from 1.5 to 3.0 M NaCl. Hydroxyectoine also occurred in strain ML-D, especially at elevated NaCl concentrations (2.5 and 3.0 M), but at levels lower than those of ectoine. Exogenous organic solutes that might occur in Mono Lake were examined for their effects on the de novo synthesis of ectoine. Dimethylsulfoniopropionate (DMSP) (0.1 or 1 mM) did not significantly lower ectoine levels in either isolate, and only strain ML-G showed any capacity for DMSP accumulation. With nitrogen limitation, however, DMSP (0.1 mM) substituted for ectoine in strain ML-G and became the main organic solute. Glycine betaine (GB) was more effective than DMSP in affecting ectoine levels, principally in strain ML-D. Strain ML-D accumulated GB to 50 or 67% of its organic solute pool at 2.5 M NaCl, at an external level of 0.1 or 1 mM GB, respectively. Strain ML-D also accumulated arsenobetaine. The methylated zwitterionic compounds, probably metabolic products of phytoplankton (DMSP and GB) or brine shrimps (arsenobetaine) in Mono Lake, may function as osmolytes for indigenous bacteria when present at high concentrations or under conditions of nitrogen limitation or salt stress.     

Contribution of Bacterial Cell Wall Components to DOM in Alkaline,Hypersaline Mono Lake,California

Soda lakes are often characterized by high densities of prokaryotes and high concentrations of dissolved organic carbon. Since bacterial cell walls are less degradable than most other cell constituents, accumulation of cell wall material may occur in these lakes and contribute to the DOM pool, but composition of DOM in soda lakes has rarely been examined. Here we report concentrations of DOM components likely originating from bacterial cell walls, including D amino acids, glucosamine (GluA) and muramic acid (MurA), in depth profiles of stratified, alkaline, hypersaline Mono Lake, CA. Concentrations of cell wall components were related to total pools of dissolved free and combined amino acids (DFAA and DCAA), and bacterial density and production. In the free pool, total DFAA ranged from 50 to 3250 nM and typically increased with depth, while GluA (5 to 140 nM) and MurA (< 0.5 nM and only detected in 2005) fluctuated with depth. In the combined pool, DCAA varied between 5000 and 15000 nM and did not show clear depth-related trends. GluA ranged from 1000 to 5000 nM and tended to increase in the hypolimnion, while MurA varied between 25 and 75 nM. Free D isomers in the DFAA pool either made up < 13% (Asp and Ser) or varied from 10 to 57% (Glu and Ala). In the combined pool, D isomers of Asp, Glu, Ser and Ala made up 24-48% of these DCAA and typically showed minor changes with depth. In 2005, lysozyme activity had highest rates in the surface and correlated negatively with most D isomers among the combined amino acids. Our observations demonstrate that the pool of dissolved combined amino compounds in the lake was about 5-fold higher than in other eutrophic lakes and that a substantial portion of these amino compounds originated from bacterial cell walls.     

Methanogenesis in Big Soda Lake, Nevada: an Alkaline, Moderately Hypersaline Desert Lake

Incubated sediment slurries from Big Soda Lake, Nevada, produced significant levels of CH₄, and production was inhibited by 2-bromoethanesulfonic acid and by autoclaving. Methane production was stimulated by methanol, trimethylamine, and, to a lesser extent, methionine. Surprisingly, hydrogen, acetate, and formate amendments provided only slight or no stimulation of methanogenesis. Methane production by sediment slurries had a pH optimum of 9.7. A methanol-grown enrichment culture containing a small, epifluorescent coccus as the predominant organism was recovered from sediments. The enrichment grew best when FeS or autoclaved sediment particles were included in the media, had a pH optimum of 9.7, and produced ¹⁴CH₄ from ¹⁴CH₃OH. The methane formed by methanolgrown enrichment cultures was depleted in ¹³C by 72 to 77‰ relative to the methanol.     

Contrasting Patterns of Community Assembly in the Stratified Water Column of Great Salt Lake, Utah

Phylogenetic examinations of communities sampled along geochemical gradients provide a framework for inferring the relative importance of niche-based ecological interactions (competition, environmental filtering) and neutral-based evolutionary interactions in structuring biodiversity. Great Salt Lake (GSL) in Utah exhibits strong spatial gradients due to both seasonal variation in freshwater input into the watershed and restricted fluid flow within North America’s largest saline terminal lake ecosystem. Here, we examine the phylogenetic structure and composition of archaeal, bacterial, and eukaryal small subunit (SSU) rRNA genes sampled along a stratified water column (DWR3) in the south arm of GSL in order to infer the underlying mechanism of community assembly. Communities sampled from the DWR3 epilimnion were phylogenetically clustered (i.e., coexistence of close relatives due to environmental filtering) whereas those sampled from the DWR3 hypolimnion were phylogenetically overdispersed (i.e., coexistence of distant relatives due to competitive interactions), with minimal evidence for a role for neutral processes in structuring any assemblage. The shift from phylogenetically clustered to overdispersed assemblages was associated with an increase in salinity and a decrease in dissolved O₂ (DO) concentration. Likewise, the phylogenetic diversity and phylogenetic similarity of assemblages was strongly associated with salinity or DO gradients. Thus, salinity and/or DO appeared to influence the mechanism of community assembly as well as the phylogenetic diversity and composition of communities. It is proposed that the observed patterns in the phylogenetic composition and structure of DWR3 assemblages are attributable to the meromictic nature of GSL, which prevents significant mixing between the epilimnion and the hypolimnion. This leads to strong physicochemical gradients at the halocline, which are capable of supporting a greater diversity. However, concomitant shifts in nutrient availability (e.g., DO) at and below the halocline drive competitive interactions leading to hypolimnion assemblages with minimal niche overlap.     

Analysis of Ammonia-Oxidizing Bacteria from Hypersaline Mono Lake, California, on the Basis of 16S rRNA Sequences

Ammonia-oxidizing bacteria were detected by PCR amplification of DNA extracted from filtered water samples throughout the water column of Mono Lake, California. Ammonia-oxidizing members of the β subdivision of the division Proteobacteria (β-subdivision Proteobacteria) were detected using previously characterized PCR primers; target sequences were detected by direct amplification in both surface water and below the chemocline. Denaturing gradient gel electrophoresis analysis indicated the presence of at least four different β-subdivision ammonia oxidizers in some samples. Subsequent sequencing of amplified 16S rDNA fragments verified the presence of sequences very similar to those of cultured Nitrosomonas strains. Two separate analyses, carried out under different conditions (different reagents, locations, PCR machines, sequencers, etc.), 2 years apart, detected similar ranges of sequence diversity in these samples. It seems likely that the physiological diversity of nitrifiers exceeds the diversity of their ribosomal sequences and that these sequences represent members of the Nitrosomonas europaea group that are acclimated to alkaline, high-salinity environments. Primers specific for Nitrosococcus oceanus, a marine ammonia-oxidizing bacterium in the γ subdivision of the Proteobacteria, did not amplify target from any samples.     

Distribution and Diversity of Archaea Corresponding to the Limnological Cycle of a Hypersaline Stratified Lake (Solar Lake, Sinai, Egypt)

The vertical and seasonal distribution and diversity of archaeal sequences was investigated in a hypersaline, stratified, monomictic lake, Solar Lake, Sinai, Egypt, during the limnological development of stratification and mixing. Archaeal sequences were studied via phylogenetic analysis of 16S rDNA sequences as well as denaturing gradient gel electrophoresis analysis. The 165 clones studied were grouped into four phylogenetically different clusters. Most of the clones isolated from both the aerobic epilimnion and the sulfide-rich hypolimnion were defined as cluster I, belonging to the Halobacteriaceae family. The three additional clusters were all isolated from the anaerobic hypolimnion. Cluster II is phylogenetically located between the genera Methanobacterium and Methanococcus. Clusters III and IV relate to two previously documented groups of uncultured euryarchaeota, remotely related to the genus Thermoplasma. No crenarchaeota were found in the water column of the Solar Lake. The archaeal community in the Solar Lake under both stratified and mixed conditions was dominated by halobacteria in salinities higher than 10%. During stratification, additional clusters, some of which may possibly relate to uncultured halophilic methanogens, were found in the sulfide- and methane-rich hypolimnion.     

Characterization of Microbial Arsenate Reduction in the Anoxic Bottom Waters of Mono Lake,California

Dissimilatory reduction of arsenate (DAsR) occurs in the arsenic-rich, anoxic water column of Mono Lake, California, yet the microorganisms responsible for this observed in situ activity have not been identified. To gain insight as to which microorganisms mediate this phenomenon, as well as to some of the biogeochemical constraints on this activity, we conducted incubations of arsenate-enriched bottom water coupled with inhibition/amendment studies and Denaturing Gradient Gel Electrophoresis (DGGE) characterization techniques. DAsR was totally inhibited by filter-sterilization and by nitrate, partially inhibited (~50%) by selenate, but only slightly (~25%) inhibited by oxyanions that block sulfate-reduction (molybdate and tungstate). The apparent inhibition by nitrate, however, was not due to action as a preferred electron acceptor to arsenate. Rather, nitrate addition caused a rapid, microbial re-oxidation of arsenite to arsenate, which gave the overall appearance of no arsenate loss. A similar microbial oxidation of As(III) was also found with Fe(III), a fact that has implications for the recycling of As(V) in Mono Lake's anoxic bottom waters. DAsR could be slightly (10%) stimulated by substrate amendments of lactate, succinate, malate, or glucose, but not by acetate, suggesting that the DAsR microflora is not electron donor limited. DGGE analysis of amplified 16S rDNA gene fragments from incubated arsenate-enriched bottom waters revealed the presence of two bands that were not present in controls without added arsenate. The resolved sequences of these excised bands indicated the presence of members of the epsilon ( Sulfurospirillum ) and delta ( Desulfovibrio ) subgroups of the Proteobacteria , both of which have representative species that are capable of anaerobic growth using arsenate as their electron acceptor.     

Novel and Unexpected Prokaryotic Diversity in Water and Sediments of the Alkaline,Hypersaline Lakes of the Wadi An Natrun,Egypt

The phylogenetic diversity of the bacterial and archaeal community in the water and sediments of three large lakes of the Wadi An Natrun was investigated using 16S rRNA clone libraries. The bacterial community was diverse: 769 clones formed 345 operational taxonomic units (OTUs) defined at 99% 16S rRNA sequence identity. The bacterial community in both the water and sediments of the lakes was dominated by clones affiliated with the low G + C Gram-type-positive group, alpha-proteobacteria, and Bacteroidetes, (11-39, 11-30, and 10-37% of OTUs observed, respectively), patterns that have been observed in previously described alkaline, athalassohaline systems. However, a relatively high proportion of Firmicutess-related clones in the water of the lakes and alpha-proteobacteria in the sediments was observed. The bacterial community composition of the water and sediment of the same lake and of different lakes was significantly different (p < 0.05). Operational taxonomic units related to the gamma-proteobacteria were more abundant in the sediment of Lake Fazda, whereas the sediment of Lake UmRisha was dominated by members of the delta-proteobacteria. The proportion of gamma-proteobacterial and Bacteroidetes-affiliated OTUs were predominant in the water of Lake UmRisha and differed significantly from other lake waters (chi-squared analysis, p < or = 0.01). The more oxygenated and dilute nature of Lake Hamra was reflected in its microbial community composition, with the abundance of Bacillales sequences in the water, the absence of Halanaerobiales, Clostridiales, and Archaea in the water, and the presence of representatives of more phyla such as the Actinobacteria, Spirochaetes, and Verrucomicrobia. The archaeal community composition appeared less diverse: 589 clones resulted in 198 OTUs defined at 99% 16S rRNA sequence identity, and all sequences fell into the phylum Euryarchaeota. Phylogenetic analysis showed that many of the sequences were distantly related (83-90% 16S rRNA sequence identity) to cultured and uncultured archaea, with many clones forming clusters that branched deeply within the Euryarchaeota. Forty-two and 53% of the bacterial and archaeal clones had less than 90% 16S rRNA sequence identity to previously described sequences. This indicates that the water and sediments of the Wadi An Natrun harbor a unique and novel prokaryotic diversity that is different from what has been described among other alkaline, athalassohaline lakes.     

Niche-Partitioning of Prochlorococcus Populations in a Stratified Water Column in the Eastern North Atlantic Ocean

The in situ community structure of Prochlorococcus populations in the eastern North Atlantic Ocean was examined by analysis of Prochlorococcus 16S rDNA sequences with three independent approaches: cloning and sequencing, hybridization to specific oligonucleotide probes, and denaturing gradient gel electrophoresis (DGGE). The hybridization of high-light (HL) and low-light (LL) Prochlorococcus genotype-specific probes to two depth profiles of PCR-amplified 16S rDNA sequences revealed that in these two stratified water columns, an obvious niche-partitioning of Prochlorococcus genotypes occurred. In each water column a shift from the HL to the LL genotype was observed, a transition correlating with the depth of the surface mixed layer (SML). Only the HL genotype was found in the SML in each water column, whereas the LL genotype was distributed below the SML. The range of in situ irradiance to which each genotype was subjected within these distinct niches was consistent with growth irradiance studies of cultured HL- and LL-adapted Prochlorococcus strains. DGGE analysis and the sequencing of Prochlorococcus 16S rDNA clones were in full agreement with the genotype-specific oligonucleotide probe hybridization data. These observations of a partitioning of Prochlorococcus genotypes in a stratified water column provide a genetic basis for the dim and bright Prochlorococcus populations observed in flow cytometric signatures in several oceanic provinces.     

Metagenomic Analysis of RNA Viruses in a Fresh Water Lake

Freshwater lakes and ponds present an ecological interface between humans and a variety of host organisms. They are a habitat for the larval stage of many insects and may serve as a medium for intraspecies and interspecies transmission of viruses such as avian influenza A virus. Furthermore, freshwater bodies are already known repositories for disease-causing viruses such as Norwalk Virus, Coxsackievirus, Echovirus, and Adenovirus. While RNA virus populations have been studied in marine environments, to this date there has been very limited analysis of the viral community in freshwater. Here we present a survey of RNA viruses in Lake Needwood, a freshwater lake in Maryland, USA. Our results indicate that just as in studies of other aquatic environments, the majority of nucleic acid sequences recovered did not show any significant similarity to known sequences. The remaining sequences are mainly from viral types with significant similarity to approximately 30 viral families. We speculate that these novel viruses may infect a variety of hosts including plants, insects, fish, domestic animals and humans. Among these viruses we have discovered a previously unknown dsRNA virus closely related to Banna Virus which is responsible for a febrile illness and is endemic to Southeast Asia. Moreover we found multiple viral sequences distantly related to Israeli Acute Paralysis virus which has been implicated in honeybee colony collapse disorder. Our data suggests that due to their direct contact with humans, domestic and wild animals, freshwater ecosystems might serve as repositories of a wide range of viruses (both pathogenic and non-pathogenic) and possibly be involved in the spread of emerging and pandemic diseases.     

Vertical Distribution of Nitrogen-Fixing Phylotypes in a Meromictic,Hypersaline Lake

We investigated the diversity of nitrogenase genes in the alkaline, moderately hypersaline Mono Lake, California to determine (1) whether nitrogen-fixing (diazotrophic) populations were similar to those in other aquatic environments and (2) if there was a pattern of distribution of phylotypes that reflected redox conditions, as well as (3) to identify populations that could be important in N dynamics in this nitrogen-limited lake. Mono Lake has been meromictic for almost a decade and has steep gradients in oxygen and reduced compounds that provide a wide range of aerobic and anaerobic habitats. We amplified a fragment of the nitrogenase gene (nifH) from planktonic DNA samples collected at three depths representing oxygenated surface waters, the oxycline, and anoxic, ammonium-rich deep waters. Forty-three percent of the 90 sequences grouped in nifH Cluster I. The majority of clones (57%) grouped in Cluster III, which contains many known anaerobic bacteria. Cluster I and Cluster III sequences were retrieved at every depth indicating little vertical zonation in sequence types related to the prominent gradients in oxygen and ammonia. One group in Cluster I was found most often at every depth and accounted for 29% of all the clones. These sequences formed a subcluster that contained other environmental clones, but no cultivated representatives. No significant nitrogen fixation was detected by the ¹⁵N₂ method after 48 h of incubation of surface, oxycline, or deep waters, suggesting that pelagic diazotrophs were contributing little to nitrogen fluxes in the lake. The failure to measure any significant nitrogen fixation, despite the detection of diverse and novel nitrogenase genes throughout the water column, raises interesting questions about the ecological controls on diazotrophy in Mono Lake and the distribution of functional genes in the environment.     

Century-Long Warming Trends in the Upper Water Column of Lake Tanganyika

Lake Tanganyika, the deepest and most voluminous lake in Africa, has warmed over the last century in response to climate change. Separate analyses of surface warming rates estimated from in situ instruments, satellites, and a paleolimnological temperature proxy (TEX₈₆) disagree, leaving uncertainty about the thermal sensitivity of Lake Tanganyika to climate change. Here, we use a comprehensive database of in situ temperature data from the top 100 meters of the water column that span the lake’s seasonal range and lateral extent to demonstrate that long-term temperature trends in Lake Tanganyika depend strongly on depth, season, and latitude. The observed spatiotemporal variation in surface warming rates accounts for small differences between warming rate estimates from in situ instruments and satellite data. However, after accounting for spatiotemporal variation in temperature and warming rates, the TEX₈₆ paleolimnological proxy yields lower surface temperatures (1.46 °C lower on average) and faster warming rates (by a factor of three) than in situ measurements. Based on the ecology of Thaumarchaeota (the microbes whose biomolecules are involved with generating the TEX₈₆ proxy), we offer a reinterpretation of the TEX₈₆ data from Lake Tanganyika as the temperature of the low-oxygen zone, rather than of the lake surface temperature as has been suggested previously. Our analyses provide a thorough accounting of spatiotemporal variation in warming rates, offering strong evidence that thermal and ecological shifts observed in this massive tropical lake over the last century are robust and in step with global climate change.     

Genome Sequence of Legionella tunisiensis Strain LegMT,a New Legionella Species Isolated from Hypersaline Lake Water

Legionella tunisiensis is a gammaproteobacterium from the class Legionellaceae, growing in amoebae. We sequenced the genome from strain LegM^T. It is composed of 3,508,121 bp and contains 4,747 protein-coding genes and 38 RNA genes, including 3 rRNA genes.     

Anaerobic Oxidation of Arsenite in Mono Lake Water and by a Facultative, Arsenite-Oxidizing Chemoautotroph, Strain MLHE-1

Arsenite [As(III)]-enriched anoxic bottom water from Mono Lake, California, produced arsenate [As(V)] during incubation with either nitrate or nitrite. No such oxidation occurred in killed controls or in live samples incubated without added nitrate or nitrite. A small amount of biological As(III) oxidation was observed in samples amended with Fe(III) chelated with nitrolotriacetic acid, although some chemical oxidation was also evident in killed controls. A pure culture, strain MLHE-1, that was capable of growth with As(III) as its electron donor and nitrate as its electron acceptor was isolated in a defined mineral salts medium. Cells were also able to grow in nitrate-mineral salts medium by using H₂ or sulfide as their electron donor in lieu of As(III). Arsenite-grown cells demonstrated dark ¹⁴CO₂ fixation, and PCR was used to indicate the presence of a gene encoding ribulose-1,5-biphosphate carboxylase/oxygenase. Strain MLHE-1 is a facultative chemoautotroph, able to grow with these inorganic electron donors and nitrate as its electron acceptor, but heterotrophic growth on acetate was also observed under both aerobic and anaerobic (nitrate) conditions. Phylogenetic analysis of its 16S ribosomal DNA sequence placed strain MLHE-1 within the haloalkaliphilic Ectothiorhodospira of the γ-Proteobacteria. Arsenite oxidation has never been reported for any members of this subgroup of the Proteobacteria.     

Osmoregulation in nestling California gulls at Mono Lake,California

• 1.1. Hematocrit (Hct), plasma sodium ([Na]_pl), chloride ([Cl]_pl) and osmotic concentration (Osm_pl); volume and concentration of 1.0MNaCl induced salt gland secretion (SGS); and weights of osmoregulatory organs: kidneys, adrenal glands, and salt glands and nonosmoregulatory organs (liver and heart) were determined in nestling California gulls, Larus californiens (CG), on Krakatoa Islet, Mono Lake, California.
• 2.2. The mean Hct was 40.0% + 1.0%, the mean [Na]_pl and [Cl]_pl were 153.9 ± 0.9 and 110.1 ± 0.5 mM (n = 22); and the mean Osm_pl was 323.6 ± 1.3 mOsm/kg (n = 18).
• 3.3. In CG nestlings with a mean age of 10 days (n = 7), the mean SGS [Na] was 719 ± 19 mM and the birds secreted 81 ± 18% of the injected fluid containing 59 ± 13% of the injected Na. By the mean age of 23 days (n = 7), mean SGS [Na] was slightly higher (790 ± 30 mM than in younger birds (P < 0.05), but the percentage of secreted fluid (54 ± 10%) and Na (42 ± 6%) tended to be less.
• 4.4. In 18 CG nestlings mean organ weights (% body weight) were: kidneys 1.52 ±0.06%; salt glands, 0.13 ±0.01%, and adrenal glands 0.07 ±0.01%.
• 5.5. Nestling CG had significantly greater Hct (P < 0.001), [Na]_pl and [ci]_pl (P < 0.001), Osm_pl, (P < 0.005), and adrenal gland weight (P < 0.01), compared to nestling glaucous-winged Gulls (GWG), L. glaucescens (Hughes, 1984), which nest under cooler, moister conditions. CG kidney weight was smaller (P < 0.001); salt gland weight and salt excretion were the same as GWG.

     

Microbiology Study of a Hypersaline Lake in French Somaliland

In a study of a lake having a higher concentration of salts than the Dead Sea, all of the heterotrophic bacteria isolated were aerobes; no strictly anaerobic strains were found. Ninety percent of the strains were euryhalines and ten percent were strict halophiles. The extreme halophiles belonged to the species Halobacterium trapanicum and Halococcus morrhuae.     

Analysis of fae and fhcD Genes in Mono Lake, California

Genes for two enzymes of the tetrahydromethanopterin-linked C₁ transfer pathway (fae and fhcD) were detected in hypersaline, hyperalkaline Mono Lake (California), via PCR amplification and analysis. Low diversity for fae and fhcD was noted, in contrast to the diversity previously detected in a freshwater lake, Lake Washington (Washington).