Diversity of phototrophic bacteria in microbial mats from Arctic hot springs (Greenland)

We investigated the genotypic diversity of oxygenic and anoxygenic phototrophic microorganisms in microbial mat samples collected from three hot spring localities on the east coast of Greenland. These hot springs harbour unique Arctic microbial ecosystems that have never been studied in detail before. Specific oligonucleotide primers for cyanobacteria, purple sulfur bacteria, green sulfur bacteria and Choroflexus/Roseiflexus-like green non-sulfur bacteria were used for the selective amplification of 16S rRNA gene fragments. Amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced. In addition, several cyanobacteria were isolated from the mat samples, and classified morphologically and by 16S rRNA-based methods. The cyanobacterial 16S rRNA sequences obtained from DGGE represented a diverse, polyphyletic collection of cyanobacteria. The microbial mat communities were dominated by heterocystous and non-heterocystous filamentous cyanobacteria. Our results indicate that the cyanobacterial community composition in the samples were different for each sampling site. Different layers of the same heterogeneous mat often contained distinct and different communities of cyanobacteria. We observed a relationship between the cyanobacterial community composition and the in situ temperatures of different mat parts. The Greenland mats exhibited a low diversity of anoxygenic phototrophs as compared with other hot spring mats which is possibly related to the photochemical conditions within the mats resulting from the Arctic light regime.     

Matching molecular diversity and ecophysiology of benthic cyanobacteria and diatoms in communities along a salinity gradient

The phylogenetic diversity of oxygenic phototrophic microorganisms in hypersaline microbial mats and their distribution along a salinity gradient were investigated and compared with the halotolerances of closely related cultivated strains. Segments of 16S rRNA genes from cyanobacteria and diatom plastids were retrieved from mat samples by DNA extraction and polymerase chain reaction (PCR), and subsequently analysed by denaturing gradient gel electrophoresis (DGGE). Sequence analyses of DNA from individual DGGE bands suggested that the majority of these organisms was related to cultivated strains at levels that had previously been demonstrated to correlate with characteristic salinity responses. Proportional abundances of amplified 16S rRNA gene segments from phylogenetic groupings of cyanobacteria and diatoms were estimated by image analysis of DGGE gels and were generally found to correspond to abundances of the respective morphotypes determined by microscopic analyses. The results indicated that diatoms accounted for low proportions of cells throughout, that the cyanobacterium Microcoleus chthonoplastes and close relatives dominated the communities up to a salinity of 11% and that, at a salinity of 14%, the most abundant cyanobacteria were related to highly halotolerant cultivated cyanobacteria, such as the recently established phylogenetic clusters of Euhalothece and Halospirulina . Although these organisms in cultures had previously demonstrated their ability to grow with close to optimal rates over a wide range of salinities, their occurrence in the field was restricted to the highest salinities investigated.     

Community composition of a hypersaline endoevaporitic microbial mat

A hypersaline, endoevaporitic microbial community in Eilat, Israel, was studied by microscopy and by PCR amplification of genes for 16S rRNA from different layers. In terms of biomass, the oxygenic layers of the community were dominated by Cyanobacteria of the Halothece, Spirulina, and Phormidium types, but cell counts (based on 4',6'-diamidino-2-phenylindole staining) and molecular surveys (clone libraries of PCR-amplified genes for 16S rRNA) showed that oxygenic phototrophs were outnumbered by the other constituents of the community, including chemotrophs and anoxygenic phototrophs. Bacterial clone libraries were dominated by phylotypes affiliated with the Bacteroidetes group and both photo- and chemotrophic groups of alpha-proteobacteria. Green filaments related to the Chloroflexi were less abundant than reported from hypersaline microbial mats growing at lower salinities and were only detected in the deepest part of the anoxygenic phototrophic zone. Also detected were nonphototrophic gamma- and delta-proteobacteria, Planctomycetes, the TM6 group, Firmicutes, and Spirochetes. Several of the phylotypes showed a distinct vertical distribution in the crust, suggesting specific adaptations to the presence or absence of oxygen and light. Archaea were less abundant than Bacteria, their diversity was lower, and the community was less stratified. Detected archaeal groups included organisms affiliated with the Methanosarcinales, the Halobacteriales, and uncultured groups of Euryarchaeota.     

Diversity and Distribution in Hypersaline Microbial Mats of Bacteria Related to Chloroflexus spp.

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of “green nonsulfur bacteria.” PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.     

Benthic phototrophic community from Kiran soda lake,south-eastern Siberia

Phototrophic bacterial mats from Kiran soda lake (south-eastern Siberia) were studied using integrated approach including analysis of the ion composition of water, pigments composition, bacterial diversity and the vertical distribution of phototrophic microorganisms in the mats. Bacterial diversity was investigated using microscopic examination, 16S rRNA gene Illumina sequencing and culturing methods. The mats were formed as a result of decomposition of sedimented planktonic microorganisms, among which cyanobacteria of the genus Arthrospira predominated. Cyanobacteria were the largest part of phototrophs in the mats, but anoxygenic phototrophs were significant fraction. The prevailing species of the anoxygenic phototrophic bacteria are typical for soda lakes. The mats harbored aerobic anoxygenic phototrophic bacteria, purple sulfur and non-sulfur bacteria, as well as new filamentous phototrophic Chloroflexi. New strains of Thiocapsa sp. Kir-1, Ectothiorhodospira sp. Kir-2 and Kir-4, Thiorhodospira sp. Kir-3 and novel phototrophic Chloroflexi bacterium Kir15-3F were isolated and identified.

     

Community Composition of a Hypersaline Endoevaporitic Microbial Mat

A hypersaline, endoevaporitic microbial community in Eilat, Israel, was studied by microscopy and by PCR amplification of genes for 16S rRNA from different layers. In terms of biomass, the oxygenic layers of the community were dominated by Cyanobacteria of the Halothece, Spirulina, and Phormidium types, but cell counts (based on 4′,6′-diamidino-2-phenylindole staining) and molecular surveys (clone libraries of PCR-amplified genes for 16S rRNA) showed that oxygenic phototrophs were outnumbered by the other constituents of the community, including chemotrophs and anoxygenic phototrophs. Bacterial clone libraries were dominated by phylotypes affiliated with the Bacteroidetes group and both photo- and chemotrophic groups of α-proteobacteria. Green filaments related to the Chloroflexi were less abundant than reported from hypersaline microbial mats growing at lower salinities and were only detected in the deepest part of the anoxygenic phototrophic zone. Also detected were nonphototrophic γ- and δ-proteobacteria, Planctomycetes, the TM6 group, Firmicutes, and Spirochetes. Several of the phylotypes showed a distinct vertical distribution in the crust, suggesting specific adaptations to the presence or absence of oxygen and light. Archaea were less abundant than Bacteria, their diversity was lower, and the community was less stratified. Detected archaeal groups included organisms affiliated with the Methanosarcinales, the Halobacteriales, and uncultured groups of Euryarchaeota.     

Diversity and distribution in hypersaline microbial mats of bacteria related to Chloroflexus spp

Filamentous bacteria containing bacteriochlorophylls c and a were enriched from hypersaline microbial mats. Based on phylogenetic analyses of 16S rRNA gene sequences, these organisms form a previously undescribed lineage distantly related to Chloroflexus spp. We developed and tested a set of PCR primers for the specific amplification of 16S rRNA genes from filamentous phototrophic bacteria within the kingdom of "green nonsulfur bacteria." PCR products recovered from microbial mats in a saltern in Guerrero Negro, Mexico, were subjected to cloning or denaturing gradient gel electrophoresis and then sequenced. We found evidence of a high diversity of bacteria related to Chloroflexus which exhibit different distributions along a gradient of salinity from 5.5 to 16%.     

PCR primers to amplify 16S rRNA genes from cyanobacteria.

We developed and tested a set of oligonucleotide primers for the specific amplification of 16S rRNA gene segments from cyanobacteria and plastids by PCR. PCR products were recovered from all cultures of cyanobacteria and diatoms that were checked but not from other bacteria and archaea. Gene segments selectively retrieved from cyanobacteria and diatoms in unialgal but nonaxenic cultures and from cyanobionts in lichens could be directly sequenced. In the context of growing sequence databases, this procedure allows rapid and phylogenetically meaningful identification without pure cultures or molecular cloning. We demonstrate the use of this specific PCR in combination with denaturing gradient gel electrophoresis to probe the diversity of oxygenic phototrophic microorganisms in cultures, lichens, and complex microbial communities.     

Diversity and Function of Chloroflexus-Like Bacteria in a Hypersaline Microbial Mat: Phylogenetic Characterization and Impact on Aerobic Respiration

We studied the diversity of Chloroflexus-like bacteria (CLB) in a hypersaline phototrophic microbial mat and assayed their near-infrared (NIR) light-dependent oxygen respiration rates. PCR with primers that were reported to specifically target the 16S rRNA gene from members of the phylum Chloroflexi resulted in the recovery of 49 sequences and 16 phylotypes (sequences of the same phylotype share more than 96% similarity), and 10 of the sequences (four phylotypes) appeared to be related to filamentous anoxygenic phototrophic members of the family Chloroflexaceae. Photopigment analysis revealed the presence of bacteriochlorophyll c (BChlc), BChld, and γ-carotene, pigments known to be produced by phototrophic CLB. Oxygen microsensor measurements for intact mats revealed a NIR (710 to 770 nm) light-dependent decrease in aerobic respiration, a phenomenon that we also observed in an axenic culture of Chloroflexus aurantiacus. The metabolic ability of phototrophic CLB to switch from anoxygenic photosynthesis under NIR illumination to aerobic respiration under non-NIR illumination was further used to estimate the contribution of these organisms to mat community respiration. Steady-state oxygen profiles under dark conditions and in the presence of visible (VIS) light (400 to 700 nm), NIR light (710 to 770 nm), and VIS light plus NIR light were compared. NIR light illumination led to a substantial increase in the oxygen concentration in the mat. The observed impact on oxygen dynamics shows that CLB play a significant role in the cycling of carbon in this hypersaline microbial mat ecosystem. This study further demonstrates that the method applied, a combination of microsensor techniques and VIS and NIR illumination, allows rapid establishment of the presence and significance of CLB in environmental samples.     

Detection and Capturing of <Superscript>14</Superscript>C Radioactively-Labeled Small Subunit rRNA from Mixed Microbial Communities of a Microbial Mat Using Magnetic Beads

Carbon cycling in the hypersaline microbial mats from Chiprana Lake, Spain is primarily dependent on phototrophic microorganisms with the ability to fix CO₂ into organics that can be further utilized by aerobic as well as anaerobic heterotrophic bacteria. Here, mat pieces were incubated in seawater amended with ¹⁴C sodium bicarbonate and the incorporation of the radiocarbon in the small subunit ribosomal RNA (SSU rRNA) of mat organisms was followed using scintillation counter and autoradiography. Different domains of SSU rRNA were separated from the total RNA by means of streptavidin-coated magnetic beads and biotin-labeled oligonucleotide probes. The ¹⁴C label was detected in isolated RNA by both scintillation counter and autoradiography, however the latter technique was less sensitive. Using scintillation counter, the radiolabel incorporation increased with time with a maximum rate of 0.18 Bq ng⁻¹ detected after 25 days. The bacterial SSU rRNA could be captured using the magnetic beads, however the hybridization efficiency was around 20%. The captured RNA was radioactively labeled, which could be mainly due to the fixation of radiocarbon by phototrophic organisms. In conclusion, the incubation of microbial mats in the presence of radiolabeled bicarbonate leads to the incorporation of the ¹⁴C label into RNA molecules through photosynthesis and this label can be detected using scintillation counter. The used approach could be useful in studying the fate of fixed carbon and its uptake by other microorganisms in complex microbial mats, particularly when species-specific probes are used and the hybridization efficiency and RNA yield are further optimized.     

Microbial communities in iron-silica-rich microbial mats at deep-sea hydrothermal fields of the Southern Mariana Trough

The abundance, diversity and composition of bacterial and archaeal communities in the microbial mats at deep-sea hydrothermal fields were investigated, using culture-independent 16S rRNA and functional gene analyses combined with mineralogical analysis. Microbial mats were collected at two hydrothermal areas on the ridge of the back-arc spreading centre in the Southern Mariana Trough. Scanning electron microscope and energy dispersive X-ray spectroscopic (SEM-EDS) analyses revealed that the mats were mainly composed of amorphous silica and contained numerous filamentous structures of iron hydroxides. Direct cell counting with SYBR Green I staining showed that the prokaryotic cell densities were more than 10⁸ cells g⁻¹. Quantitative polymerase chain reaction (Q-PCR) analysis revealed that Bacteria are more abundant than Archaea in the microbial communities. Furthermore, zetaproteobacterial cells accounted for 6% and 22% of the prokaryotic cells in each mat estimated by Q-PCR with newly designed primers and TaqMan probe. Phylotypes related to iron-oxidizers, methanotrophs/methylotrophs, ammonia-oxidizers and sulfate-reducers were found in the 16S rRNA gene clone libraries constructed from each mat sample. A variety of unique archaeal 16S rRNA gene phylotypes, several pmoA , dsrAB and archaeal amoA gene phylotypes were also recovered from the microbial mats. Our results provide insights into the diversity and abundance of microbial communities within microbial mats in deep-sea hydrothermal fields.     

Diversity and function of Chloroflexus-like bacteria in a hypersaline microbial mat: phylogenetic characterization and impact on aerobic respiration

We studied the diversity of Chloroflexus-like bacteria (CLB) in a hypersaline phototrophic microbial mat and assayed their near-infrared (NIR) light-dependent oxygen respiration rates. PCR with primers that were reported to specifically target the 16S rRNA gene from members of the phylum Chloroflexi resulted in the recovery of 49 sequences and 16 phylotypes (sequences of the same phylotype share more than 96% similarity), and 10 of the sequences (four phylotypes) appeared to be related to filamentous anoxygenic phototrophic members of the family Chloroflexaceae. Photopigment analysis revealed the presence of bacteriochlorophyll c (BChlc), BChld, and gamma-carotene, pigments known to be produced by phototrophic CLB. Oxygen microsensor measurements for intact mats revealed a NIR (710 to 770 nm) light-dependent decrease in aerobic respiration, a phenomenon that we also observed in an axenic culture of Chloroflexus aurantiacus. The metabolic ability of phototrophic CLB to switch from anoxygenic photosynthesis under NIR illumination to aerobic respiration under non-NIR illumination was further used to estimate the contribution of these organisms to mat community respiration. Steady-state oxygen profiles under dark conditions and in the presence of visible (VIS) light (400 to 700 nm), NIR light (710 to 770 nm), and VIS light plus NIR light were compared. NIR light illumination led to a substantial increase in the oxygen concentration in the mat. The observed impact on oxygen dynamics shows that CLB play a significant role in the cycling of carbon in this hypersaline microbial mat ecosystem. This study further demonstrates that the method applied, a combination of microsensor techniques and VIS and NIR illumination, allows rapid establishment of the presence and significance of CLB in environmental samples.     

Unexpected Diversity and Complexity of the Guerrero Negro Hypersaline Microbial Mat

We applied nucleic acid-based molecular methods, combined with estimates of biomass (ATP), pigments, and microelectrode measurements of chemical gradients, to map microbial diversity vertically on a millimeter scale in a hypersaline microbial mat from Guerrero Negro, Baja California Sur, Mexico. To identify the constituents of the mat, small-subunit rRNA genes were amplified by PCR from community genomic DNA extracted from layers, cloned, and sequenced. Bacteria dominated the mat and displayed unexpected and unprecedented diversity. The majority (1,336) of the 1,586 bacterial 16S rRNA sequences generated were unique, representing 752 species (≥97% rRNA sequence identity) in 42 of the main bacterial phyla, including 15 novel candidate phyla. The diversity of the mat samples differentiated according to the chemical milieu defined by concentrations of O₂ and H₂S. Bacteria of the phylum Chloroflexi formed the majority of the biomass by percentage of bulk rRNA and of clones in rRNA gene libraries. This result contradicts the general belief that cyanobacteria dominate these communities. Although cyanobacteria constituted a large fraction of the biomass in the upper few millimeters (>80% of the total rRNA and photosynthetic pigments), Chloroflexi sequences were conspicuous throughout the mat. Filamentous Chloroflexi bacteria were identified by fluorescence in situ hybridization within the polysaccharide sheaths of the prominent cyanobacterium Microcoleus chthonoplastes, in addition to free living in the mat. The biological complexity of the mat far exceeds that observed in other polysaccharide-rich microbial ecosystems, such as the human and mouse distal guts, and suggests that positive feedbacks exist between chemical complexity and biological diversity.     

Microscopic Examination of Distribution and Phenotypic Properties of Phylogenetically Diverse Chloroflexaceae-Related Bacteria in Hot Spring Microbial Mats†

We investigated the diversity, distribution, and phenotypes of uncultivated Chloroflexaceae-related bacteria in photosynthetic microbial mats of an alkaline hot spring (Mushroom Spring, Yellowstone National Park). By applying a directed PCR approach, molecular cloning, and sequence analysis of 16S rRNA genes, an unexpectedly large phylogenetic diversity among these bacteria was detected. Oligonucleotide probes were designed to target 16S rRNAs from organisms affiliated with the genus Chloroflexus or with the type C cluster, a group of previously discovered Chloroflexaceae relatives of this mat community. The application of peroxidase-labeled probes in conjunction with tyramide signal amplification enabled the identification of these organisms within the microbial mats by fluorescence in situ hybridization (FISH) and the investigation of their morphology, abundance, and small-scale distribution. FISH was combined with oxygen microelectrode measurements, microscope spectrometry, and microautoradiography to examine their microenvironment, pigmentation, and carbon source usage. Abundant type C-related, filamentous bacteria were found to flourish within the cyanobacterium-dominated, highly oxygenated top layers and to predominate numerically in deeper orange-colored zones of the investigated microbial mats, correlating with the distribution of bacteriochlorophyll a. Chloroflexus sp. filaments were rare at 60°C but were more abundant at 70°C, where they were confined to the upper millimeter of the mat. Both type C organisms and Chloroflexus spp. were observed to assimilate radiolabeled acetate under in situ conditions.     

Influence of molecular resolution on sequence-based discovery of ecological diversity among Synechococcus populations in an alkaline siliceous hot spring microbial mat

Previous research has shown that sequences of 16S rRNA genes and 16S-23S rRNA internal transcribed spacer regions may not have enough genetic resolution to define all ecologically distinct Synechococcus populations (ecotypes) inhabiting alkaline, siliceous hot spring microbial mats. To achieve higher molecular resolution, we studied sequence variation in three protein-encoding loci sampled by PCR from 60°C and 65°C sites in the Mushroom Spring mat (Yellowstone National Park, WY). Sequences were analyzed using the ecotype simulation (ES) and AdaptML algorithms to identify putative ecotypes. Between 4 and 14 times more putative ecotypes were predicted from variation in protein-encoding locus sequences than from variation in 16S rRNA and 16S-23S rRNA internal transcribed spacer sequences. The number of putative ecotypes predicted depended on the number of sequences sampled and the molecular resolution of the locus. Chao estimates of diversity indicated that few rare ecotypes were missed. Many ecotypes hypothesized by sequence analyses were different in their habitat specificities, suggesting different adaptations to temperature or other parameters that vary along the flow channel.     

Extraction of high molecular weight DNA from microbial mats

Successful and accurate analysis and interpretation of metagenomic data is dependent upon the efficient extraction of high-quality, high molecular weight (HMW) community DNA. However, environmental mat samples often pose difficulties to obtaining large concentrations of high-quality, HMW DNA. Hypersaline microbial mats contain high amounts of extracellular polymeric substances (EPS)1 and salts that may inhibit downstream applications of extracted DNA. Direct and harsh methods are often used in DNA extraction from refractory samples. These methods are typically used because the EPS in mats, an adhesive matrix, binds DNA during direct lysis. As a result of harsher extraction methods, DNA becomes fragmented into small sizes. The DNA thus becomes inappropriate for large-insert vector cloning. In order to circumvent these limitations, we report an improved methodology to extract HMW DNA of good quality and quantity from hypersaline microbial mats. We employed an indirect method involving the separation of microbial cells from the background mat matrix through blending and differential centrifugation. A combination of mechanical and chemical procedures was used to extract and purify DNA from the extracted microbial cells. Our protocol yields approximately 2 μg of HMW DNA (35-50 kb) per gram of mat sample, with an A(260/280) ratio of 1.6. Furthermore, amplification of 16S rRNA genes suggests that the protocol is able to minimize or eliminate any inhibitory effects of contaminants. Our results provide an appropriate methodology for the extraction of HMW DNA from microbial mats for functional metagenomic studies and may be applicable to other environmental samples from which DNA extraction is challenging.     

Investigation of the Microbial Ecology of Intertidal Hot Springs by Using Diversity Analysis of 16S rRNA and Chitinase Genes

The microbial diversity of intertidal hot springs on the seashore of northwest Iceland was examined by combining directed in situ enrichments, artificial support colonization, and mat sampling. Analysis of 16S rRNA genes revealed the presence of clones related to both marine and terrestrial, thermophilic, mesophilic, and psychrophilic microorganisms scattered among 11 bacterial divisions. No archaea were found. The species composition of the enrichments was affected by the length of the hot periods experienced at low tide and was very different from those found in the biomass. A total of 36 chitinase genes were detected by molecular screening of the samples with degenerate primers for glycoside hydrolase family 18. The chitinase gene diversity was at least twofold higher in the enrichment samples than in the controls, indicating that a much higher diversity of hydrolytic genes can be accessed with this approach.