Characterization of Microbial Diversity in Hypersaline Environments by Melting Profiles and Reassociation Kinetics in Combination with Terminal Restriction Fragment Length Polymorphism (T-RFLP)

The diversity of prokaryotes inhabiting solar saltern ponds was determined by thermal melting and reassociation of community DNA. These measurements were compared with fingerprinting techniques such as terminal restriction fragment length polymorphisms (T-RFLP) analysis, denaturant gradient gel electrophoresis (DGGE), and cloning and sequencing approaches. Three ponds with salinities of 22, 32, and 37% (NaCl saturation) were studied. The combination of independent molecular techniques to estimate the total genetic diversity provided a realistic assessment to reveal the microbial diversity in these environments. The changes in the prokaryotic communities at different salinity (22, 32, and 37% salt) were significant and revealed that the total genetic diversity increased from 22% to 32% salinity. At 37% salinity the diversity was reduced again to nearly half that at 22% salinity. Our results revealed that the community genome had a DNA complexity that was 7 (in 22% salinity pond), 13 (in 32% salinity pond), and 4 (in 37% salinity pond) times the complexity of an Escherichia coli genome. The base composition profiles showed two abundant populations, which changed in relative amount between the three ponds. They indicated an uneven taxon distribution at 22% and 37% salinity and a more even distribution at 32% salinity. The results indicated a large predominating population at 37% salinity, which might correspond to the abundance of square archaea (SPhT) observed by transmission electron microscopy (TEM) and also indicated by the same T-RFLP fragment as the SPhT. The SPhT phylotype has also been reported to be the most frequently retrieved phylotype from this environment by culture independent techniques. In addition, two different operational taxonomic units (OTU) were detected at 37% salinity based on PCR with bacterial specific primers and T-RFLP. One of these predominant phylotypes is the extreme halophilic bacterium belonging to the bacteroidetes group, Salinibacter ruber.     

Changes in archaeal,bacterial and eukaryal assemblages along a salinity gradient by comparison of genetic fingerprinting methods in a multipond solar saltern

Microbial communities inhabiting a multipond solar saltern were analysed and compared using SSU rRNA polymerase chain reaction (PCR)-based fingerprintings carried out in parallel by four laboratories. A salinity gradient from seawater (3.7%) to NaCl precipitation (37%) was studied for Bacteria, Archaea and Eukarya, and laboratories applied their own techniques and protocols on the same set of samples. Members of all three domains were retrieved from all salt concentrations. Three fingerprinting techniques were used: denaturing gradient gel electrophoresis (DGGE), ribosomal internal spacer analysis (RISA), and terminal-restriction fragments length polymorphism (T-RFLP). In addition, each laboratory used its own biomass collection method and DNA extraction protocols. Prokaryotes were addressed using DGGE and RISA with different 'domain-specific' primers sets. Eukaryotes were analysed by one laboratory using DGGE and T-RFLP, but targeting the same 18S rDNA site. Fingerprints were compared through cluster analysis and non-metric multidimensional scaling plots. This exercise allowed fast comparison of microbial assemblages and determined to what extent the picture provided by each laboratory was similar to those of others. Formation of two main, salinity-based groups of samples in prokaryotes (4-15% and 22-37% salinity) was consistent for all the laboratories. When other clusters appeared, this was a result of the particular technique and the protocol used in each case, but more affected by the primers set used. Eukaryotic microorganisms changed more from pond to pond; 4-5% and 8-37% salinity were but the two main groups detected. Archaea showed the lowest number of bands whereas Eukarya showed the highest number of operational taxonomic units (OTUs) in the initial ponds. Artefacts appeared in the DGGE from ponds with extremely low microbial richness. On the other hand, different 16S rDNA fragments with the same restriction or internal transcribed spacer (ITS) length were the main limitations for T-RFLP and RISA analyses, respectively, in ponds with the highest OTUs richness. However, although the particular taxonomic composition could vary among protocols, the general structure of the microbial assemblages was maintained.     

Evaluation of terminal-restriction fragment length polymorphism analysis in contrasting marine environments

Terminal-restriction fragment length polymorphism (T-RFLP) analysis is widely used in microbial ecology studies. In the present study, T-RFLP analysis of PCR products digested by five restriction enzymes (AluI, HaeIII, MspI, Sau3AI and TaqI) was applied for 20 samples from three contrasting coastal environments to assess the biases associated with the choice of enzyme digestion and T-RF analysis. The five enzyme digestions produced highly variable species richness (in terms of number of T-RFs). Analysis of peak areas with a threshold of 0.5% of the total peak area, which recovered 92-96% of the total peak area, revealed different diversity indexes from the five enzyme digestions. Multidimensional scaling, based on matrices that were generated by scoring peak presence/absence and area, revealed similar bacterial community structure patterns among the 20 samples, regardless of the choice of restriction enzymes. Our results strongly argue that the choice of different digestion enzymes in the T-RFLP technique generated valid and consistent bacterial community structures but highly variable species richness and diversity indices. The biases associated with the choice of digestion enzymes needs to be evaluated carefully or at least to be addressed when using T-RFLP analysis.     

Comparative diversity analysis of halophiles at two polar saltern systems in Indramayu,West Java,Indonesia

Successive microbes in solar salt ponds are essential since it is well correlated with the quality of salt produced. This research aimed to analyse the microbial diversity of the solar salt ponds in Indonesia, which use high-density polyethylene in the ponds. There are two systems, that is, an integrated open system (In-system) and a closed system (Tt-system). The In-system uses seawater while the Tt-system uses seawater from the saline artesian well. Results showed that the In-system had richer microbes than the Tt-system. Both systems shared similar halophilic microbes profile. Ponds with low salinity (3–4 Be) had very low archaea, that is, 0·2 and 0·7% for the In-system and Tt-system respectively and were dominantly inhabited by phylum Proteobacteria. In the pond with high salinity, that is, 25 Be, both systems were dominated by the phylum Euryarchaeota, family Halobacteriaceae, and genera Halorubrum was dominantly found in In25 ponds and Tt25 ponds. Even though the two systems use the same parent seawater, that is, the Java Sea and share similar microbial composition at the phylum level, we found the dominance identified microbes in both systems were different.     

Complexity in natural microbial ecosystems: the Guerrero Negro experience

The goal of this project is to describe and understand the organismal composition, structure, and physiology of microbial ecosystems from hypersaline environments. One collection of such ecosystems occurs at North America's largest saltworks, the Exportadora de Sal, in Guerrero Negro, Baja California Sur. There, seawater flows through a series of evaporative basins with an increase in salinity until saturation is reached and halite crystallization begins. Several of these ponds are lined with thick (10 cm) microbial mats that have received some biological study. To determine the nature and extent of diversity of the microbial organisms that constitute these ecosystems, we are conducting a phylogenetic analysis using molecular approaches, based on cloning and sequencing of small subunit (SSU) rRNA genes (16S for Bacteria and Archaea, 18S for Eukarya). In addition, we report preliminary results on the microbial composition of a laminated community that occurs in a crystallized gypsum-halite matrix in near-saturated salt water. Exposure of the interior of these large (kilogram) wet, endoevaporite crystals reveals a multitude of colors: layers of yellow, green, pink, and purple microbiota. To date, analyses of these two environments indicate the ubiquitous dominance of uncultured organisms of phylogenetic kinds not generally thought to be associated with hypersaline environments.     

Characterization of protistan plankton diversity in ancient salt evaporation ponds located in a volcanic crater on the island Sal,Cape Verde

Salinity is an important factor when exploring the limits known for life. Therefore, hypersaline systems have attracted much attention in recent years. In this study, we investigated the protistan diversity and community composition in two natural salt evaporation ponds (27–30% salinity) located in an ancient volcanic crater on the Cape Verde island Sal using high-throughput DNA sequencing. Our study revealed a broad range of protistan taxa and a high taxonomic diversity within the Ciliophora, Dinophyceae, and Chlorophyta. We detected a total of 23 Dinophyceae families, although Dinophyceae were generally considered to be only this diverse in aquatic environments of less than 10% salinity. Moreover, we uncovered a high degree of genetic novelty in this habitat. The mean similarity of all detected OTUs to previously described sequences was only 93.6%. These findings strongly dispute the traditional view that extreme hypersaline environments generally maintain low protistan diversity. A meta-analysis covering our and previously published data from other inland and coastal salt ponds clearly showed that our samples clustered according to salinity and not biogeography. This result further supports the claim that salinity is a major transition boundary for protistan communities, regardless of their biogeographic origin.

     

Contrasting patterns of quantitative and neutral genetic variation in locally adapted populations of the natterjack toad, Bufo calamita

The relative importance of natural selection and genetic drift in determining patterns of phenotypic diversity observed in nature is still unclear. The natterjack toad (Bufo calamita) is one of a few amphibian species capable of breeding in saline ponds, even though water salinity represents a considerable stress for them. Results from two common-garden experiments showed a pattern of geographic variation in embryonic salinity tolerance among populations from either fresh or brackish environments, consistent with the hypothesis of local adaptation. Full-sib analysis showed increased variation in survival among sibships within population for all populations as osmotic stress was increased (broad-sense heritability increased as salinity raised). Nevertheless, toads native to the brackish water environment had the highest overall survival under brackish conditions. Levels of population genetic differentiation for salinity tolerance were higher than those of neutral genetic differentiation, the latter obtained through the analysis of eight microsatellite loci. Microsatellite markers also revealed little population differentiation, lack of an isolation-by-distance pattern, and moderate gene flow connecting the populations. Therefore, environmental stress tolerance appears to have evolved in absence of geographic isolation, and consequently we reject the null hypothesis of neutral differentiation.     

The Santa Pola saltern as a model for studying the microbiota of hypersaline environments

Multi-pond salterns constitute an excellent model for the study of the microbial diversity and ecology of hypersaline environments, showing a wide range of salt concentrations, from seawater to salt saturation. Accumulated studies on the Santa Pola (Alicante, Spain) multi-pond solar saltern during the last 35 years include culture-dependent and culture-independent molecular methods and metagenomics more recently. These approaches have permitted to determine in depth the microbial diversity of the ponds with intermediate salinities (from 10 % salts) up to salt saturation, with haloarchaea and bacteria as the two main dominant groups. In this review, we describe the main results obtained using the different methodologies, the most relevant contributions for understanding the ecology of these extreme environments and the future perspectives for such studies.     

From community approaches to single-cell genomics: the discovery of ubiquitous hyperhalophilic Bacteroidetes generalists

The microbiota of multi-pond solar salterns around the world has been analyzed using a variety of culture-dependent and molecular techniques. However, studies addressing the dynamic nature of these systems are very scarce. Here we have characterized the temporal variation during 1 year of the microbiota of five ponds with increasing salinity (from 18% to >40%), by means of CARD-FISH and DGGE. Microbial community structure was statistically correlated with several environmental parameters, including ionic composition and meteorological factors, indicating that the microbial community was dynamic as specific phylotypes appeared only at certain times of the year. In addition to total salinity, microbial composition was strongly influenced by temperature and specific ionic composition. Remarkably, DGGE analyses unveiled the presence of most phylotypes previously detected in hypersaline systems using metagenomics and other molecular techniques, such as the very abundant Haloquadratum and Salinibacter representatives or the recently described low GC Actinobacteria and Nanohaloarchaeota. In addition, an uncultured group of Bacteroidetes was present along the whole range of salinity. Database searches indicated a previously unrecognized widespread distribution of this phylotype. Single-cell genome analysis of five members of this group suggested a set of metabolic characteristics that could provide competitive advantages in hypersaline environments, such as polymer degradation capabilities, the presence of retinal-binding light-activated proton pumps and arsenate reduction potential. In addition, the fairly high metagenomic fragment recruitment obtained for these single cells in both the intermediate and hypersaline ponds further confirm the DGGE data and point to the generalist lifestyle of this new Bacteroidetes group.     

一种从活性污泥中提取微生物总DNA的方法

对活性污泥的微生物群落进行研究的首要前提是获得大量的高纯度微生物基因组DNA。本文建立了一种高效、简便的提取活性污泥总DNA方法。从提取的核酸总量、纯度、基因组完整性等多方面对所得到的DNA质量进行了评价,结果表明,本法从单位活性污泥中提取的DNA得率为105-823μg/g,结构完整,纯度很高,无需进一步的纯化,可直接进行微生物群落分析及构建文库等后续分子生物学操作。现在实验室使用的提取活性污泥中DNA的方法,纯度普遍都无法达到PCR反应和建立文库的要求,本文建立的活性污泥DNA提取方法则可以克服这一难题。     

Comparisons of the polar lipid and pigment profiles of two solar salterns located in Newark, California, U.S.A., and Eilat, Israel

The whole community pigments and lipids have been examined during a 5-year period in two commercial solar salterns located in the United States and in Israel. There were significant differences in the complexity of the lipid and pigment patterns within the California saltern system, and these differences were not consistent over the sampling period despite examination of ponds with the same salinity. The solar saltern system in Eilat, Israel, showed greater consistency during this sampling period and compared directly with previous studies. The complexity of the saltern in Newark, California, could be explained on the basis of the prevailing weather conditions (cooler and more rainfall) and the nutrient-enriched source water. The Eilat saltern, however, has an oligotrophic water source and has a considerably warmer and drier climate. This difference resulted in more diverse and more complex pigment and lipid patterns and presumably microbial populations in the Newark, California, plant than in the saltern in Eilat, Israel. Received: December 10, 1999 / Accepted: April 6, 2000     

Characterization of morphospecies and strains of the genus Microcystis (Cyanobacteria) for a reconsideration of species classification

For characterization of Microcystis species and strains, cell size, growth temperature optimum, salinity tolerance, dark chemoheterotrophy, photoheterotrophy, guanine + cytosine content in DNA, total fatty acid composition and restriction fragment length polymorphism of a polymerase chain reaction product (PCR-RFLP) of the cpcBA intergenic spacer and flanking region were examined using 24 strains of Microcystis isolated from various lakes and ponds in Japan. From the results obtained it was observed that Microcystis spp. displayed low phenotypic diversity. Cell diameters of these strains were overlapping and there was no clear correlation with morphospecies. Slight differences in growth temperature optimum and salinity tolerance were observed among all strains. No strains showed either chemoheterotrophy or photoheterotrophy. The fatty acids present were the same in different strains although the amounts were different. All the strains had a similar G + C content ranging from 39 to 43 mol%. The phonogram constructed from the PCR-RFLP analysis showed that the species assignment for Microcystis species by morphology did not correspond with the genetic background.     

Prokaryotic genetic diversity throughout the salinity gradient of a coastal solar saltern

Bacterial and archaeal assemblages have been studied in a multipond solar saltern using a range of microbial ecology techniques by four laboratories simultaneously. These include 16S rDNA sequencing from both denaturing gradient gel electrophoresis (DGGE) and clone libraries, and culturing methods. Water samples from eight ponds were analysed, covering a salinity range from near sea water (4% salt) to saturated sodium chloride (37% salt; ponds called crystallizers). Clone libraries focused on ponds with salinity of 8%, 22% and 32%. Although different cloning strategies were able to retrieve the same type of dominant sequences, there were differing degrees of success with less abundant sequences. Thus, the use of two sets of primers recovered a higher number of phylotypes. Bacterial and archaeal isolates were, however, different from any of the retrieved environmental sequences. For Bacteria, most sequences in the 8% salt pond were related to organisms of marine origin. Thus, representatives of the alpha-, beta-, gamma- and epsilon-subdivisions of Proteobacteria, the Cytophaga-Flavobacterium-Bacteroides group (CFB), high-G+C Gram-positive bacteria and cyanobacteria were found. In the 22% salt pond, alpha- and gamma-Proteobacteria, cyanobacteria and CFB were the only groups found, and most of them were related to specialized halophilic bacteria. From the 32% salt pond, only CFB were found, and most of the sequences retrieved clustered with Salinibacter ruber, an extremely halophilic bacterium. A decrease in the richness of bacterial genera was therefore apparent along the gradient. Archaea behaved quite similarly. In the lowest salinity ponds, sequences were related to environmental clones of Marine Archaea Group III (Thermoplasmales relatives) and to unclassified branches of Euryarchaeaota. In the 8%, 22% and 32% ponds, most of the clones were related to different cultured strains of Halobacteriaceae. Finally, most sequences from the crystallizers clustered with the uncultured square archaeon SPhT. Crenarchaeaota were not detected. Despite the fact that higher prokaryotic richness was apparent in the lower salinity ponds than in the crystallizers, the diversity index from clone libraries calculated according to Shannon and Weaver did not show this trend. This was because diversity in the crystallizers can be considered as 'microdiversity', the co-existence of several closely related clones of Bacteria (the S. ruber cluster) and Archaea (the SPhT cluster). Regardless of the changes in abundance, both Bacteria and Archaea showed the same pattern; as salinity increased, the number of different clusters decreased, and only one cluster became dominant. Both clusters, however, showed a considerable degree of microdiversity. The meaning of such microdiversity remains to be determined.     

Comparative metabolic diversity in two solar salterns

The purpose of this study was to compare the metabolic diversity of the whole microbial community in an oligotrophic saltern (Eilat, Israel) and in a saltern with a more enriched source water (Newark, California). Between 1993 and 1998 water samples were taken from selected locations within the solar salterns of the Cargill Solar Salt Plant, Newark, California, and the Israel Salt Co. in Eilat, Israel. To examine the whole community metabolic diversity, we used the 96-well BIOLOG GN^{{ TM}} plates which contain 95 different carbon sources and a control well. Plates from samples containing greater than 15% salt were excluded from the final analyses because of a lack of reproducibility when multiple plates were inoculated with the same sample. The data were analyzed by simple matching coefficient and principal component analysis. Both methods gave similar results. Two major clusters were formed. These could be subdivided into 10 sub-clusters with only three samples from the Newark saltern in December 1997 joining at the 95% similarity level. Most of the inlet and lower salinity samples from the Cargill samples comprised one large subcluster. Several carbon sources were used by 85% of the microbial community from the California samples, while 85% of the Eilat samples had no commonly used carbon sources. These results suggest that ponds in different geographic locations may have communities with different microbial populations despite the similarities in salt content and processing for salt production.     

Comparison of Metagenomic DNA Extraction Methods for Soil Sediments of High Elevation Puga Hot Spring in Ladakh,India to Explore Bacterial Diversity

Extraction of good-quality metagenomic DNA from extreme environments is quite challenging, particularly from high elevation hot spring sediments. Low microbial load, high humic acid content and other contaminants complicate the process of extraction of metagenomic DNA from hot spring sediments. In the present study, efficacy of five manual DNA extraction protocols with modifications has been evaluated for metagenomic DNA extraction from boron–sulfur rich high elevation Puga hot spring sediments. Best suited protocol was identified based on the cell lysis efficiency, DNA yield, humic acid content, PCR reproducibility and representation of bacterial diversity. Quantity as well as quality of crude metagenomic DNA differed remarkably between various protocols used and were not pure enough to give PCR amplification using 16S rRNA bacterial and archaeal primers. Crude metagenomic DNA extracted using five different DNA extraction protocols was purified using spin column based purification method. Even after purification, only three protocols C, D and E yielded metagenomic DNA that could be amplified using both archaeal and bacterial primers. To evaluate the degree of microbial diversity represented by protocols C, D and E, phylogenetic genes amplified were subjected to amplified ribosomal DNA restriction analysis (ARDRA) and denaturing gradient gel electrophoresis analysis (DGGE) analysis. ARDRA banding pattern of amplicons generated for all the three extraction protocols, i.e., C, D and E were found to be similar. DGGE of protocol E derived amplicons resulted in the similar number of dominant bands but a greater number of non-dominant bands, i.e., the highest microbial diversity in comparison to protocols C and D, respectively. In the present study, protocol E developed from Yeates et al. protocol has been found to be best in terms of DNA yield, DNA purity and bacterial diversity depiction associated with boron–sulfur rich sediment of high elevation hot springs.     

Estimation of the contribution of halobacterial to the bacterial biomass and activity in solar salterns by the use of bile salts

Abstract Bile salts (deoxycholate, taurocholate) were used to estimate the contribution of bacteria of the Halobacterium group to bacterial community size and activity at different salinities as found in a multi-pond saltern. Low concentrations of bile salts cause lysis of halophilic archaebacteria of the Halobacterium group, while halophilic eubacteria and halococci remain microscopically intact. Upon addition of bile salts, total bacterial numbers (as estimated microscopically) in saltern ponds at salinities below 250 g/l did not decrease, while above this salinity bacterial numbers decreased by 30–50%. To estimate the contribution of halobacteria to overall heterotrophic activity, the effect of bile salt addition was tested on the incorporation of labelled amino acids. In saltern ponds of a salinity below 250 g/l activity was not greatly inhibited by taurocholate, while at salinity above 300 g/l taurocholate completely abolished incorporation of amino acids.     

Pulsed-field gel electrophoresis analysis of virus assemblages present in a hypersaline environment.

A method for analyzing virus assemblages in aquatic environments was developed and used for studying the highest-salinity ponds (from 13.4 to 35% salinity) from a multi-pond solar saltern in Alicante, Spain. The protocol consisted of a series of concentration and purification steps including tangential flow filtration and ultracentrifugation, followed by the preparation of total viral nucleic acids that were subsequently separated by pulsed-field gel electrophoresis. For every sample analyzed, a characteristic DNA pattern was obtained, whose complexity was related to viral diversity. The comparison of our results with a similar analysis carried out with marine virus assemblages shows that, as expected, the viral diversity corresponding to the analyzed hypersaline environment is considerably lower than that of a marine environment.     

The use of protein synthesis inhibitors in the estimation of the contribution of halophilic archaebacteria to bacterial activity in hypersaline environments

Abstract Antibiotics affecting protein synthesis were used to differentiate between the activity of different groups of organisms (halophilic archaebacteria, eubacteria and eukaryotes) in water samples from hypersaline ecosystems. Anisomycin (inhibiting both archaebacterial halophiles and eukaryotes) can be used to quantitate the contribution of the archaebacterial halophiles to amino acid incorporation by the microbial community, when cycloheximide (inhibiting eukaryotic protein synthesis, but not affecting halobacteria) is used as a control. Both in saltern ponds at salinities above 300 g/1 and in Dead Sea surface water more than 95% of the amino acid incorporation activity was abolished by anisomycin, but not by cycloheximide. Inhibition by anisomycin was well correlated with inhibition by low concentrations of bile salts, which specifically affect bacteria of the Halobacterium group. Chloramphenicol (an inhibitor of eubacterial protein synthesis) quantitatively inhibited amino acid incorporation in saltern brines of relatively low salinity, but also caused significant (28–42%) inhibition at high salinities. Erythromycin was also found valuable in the estimation of activities of the different bacterial groups.     

Genetic Diversity of Picocyanobacteria in Tibetan Lakes: Assessing the Endemic and Universal Distributions

The phylogenetic diversity of picocyanobacteria in seven alkaline lakes on the Tibetan Plateau was analyzed using the molecular marker 16S-23S rRNA internal transcribed spacer sequence. A total of 1,077 environmental sequences retrieved from the seven lakes were grouped into seven picocyanobacterial clusters, with two clusters newly described here. Each of the lakes was dominated by only one or two clusters, while different lakes could have disparate communities, suggesting low alpha diversity but high beta diversity of picocyanobacteria in these high-altitude freshwater and saline lakes. Several globally distributed clusters were found in these Tibetan lakes, such as subalpine cluster I and the Cyanobium gracile cluster. Although other clusters likely exhibit geographic restriction to the plateau temporally, reflecting endemicity, they can indeed be distributed widely on the plateau. Lakes with similar salinities may have similar genetic populations despite a large geographic distance. Canonical correspondence analysis identified salinity as the only environmental factor that may in part explain the diversity variations among lakes. Mantel tests suggested that the community similarities among lakes are independent of geographic distance. A portion of the picocyanobacterial clusters appear to be restricted to a narrow salinity range, while others are likely adapted to a broad range. A seasonal survey of Lake Namucuo across 3 years did not show season-related variations in diversity, and depth-related population partitioning was observed along a vertical profile of the lake. Our study emphasizes the high dispersive potential of picocyanobacteria and suggests that the regional distribution may result from adaptation to specified environments.