Microbial community biofabrics in a geothermal mine adit

Speleothems such as stalactites and stalagmites are usually considered to be mineralogical in composition and origin; however, microorganisms have been implicated in the development of some speleothems. We have identified and characterized the biological and mineralogical composition of mat-like biofabrics in two novel kinds of speleothems from a 50 degrees C geothermal mine adit near Glenwood Springs, CO. One type of structure consists of 2- to 3-cm-long, 3- to 4-mm-wide, leather-like, hollow, soda straw stalactites. Light and electron microscopy indicated that the stalactites are composed of a mineralized biofabric with several cell morphotypes in a laminated form, with gypsum and sulfur as the dominant mineral components. A small-subunit rRNA gene phylogenetic community analysis along the stalactite length yielded a diverse gradient of organisms, with a relatively simple suite of main constituents: Thermus spp., crenarchaeotes, Chloroflexi, and Gammaproteobacteria. PCR analysis also detected putative crenarchaeal ammonia monooxygenase subunit A (amoA) genes in this community, the majority related to sequences from other geothermal systems. The second type of speleothem, dumpling-like rafts floating on a 50 degrees C pool on the floor of the adit, showed a mat-like fabric of evidently living organisms on the outside of the dumpling, with a multimineral, amorphous, gypsum-based internal composition. These two novel types of biofabrics are examples of the complex roles that microbes can play in mineralization, weathering, and deposition processes in karst environments.     

Molecular and Cellular Fossils of a Mat-Like Microbial Community in Geothermal Boratic Sinters

Silica and travertine deposits, the two most common surface manifestations of terrestrial hot springs, have so far been the only markers universally helpful in locating and interpreting geothermal systems and past lifeforms potentially associated with them. In the current study, we for the first time report microbial fossils from a third type of geothermal sinter, viz. the boron mineral deposits, which are characteristic of the relatively uncommon silicate-poor hot springs. Organic biomarker analyses of the boratic sinters framing the hot springs of Puga valley, Ladakh, identified molecular fossils (viz. respiratory and photosynthetic isoprenoid quinones and photosynthetic pigments) of a mat-like microbial community putatively comprised of algae, fungi, cyanobacteria and other photosynthetic bacteria, some of which may be proteobacteria. So far as microfossil preservation is concerned, mineralized mat-like biofabrics with diverse cellular morphotypes could be identified in scanning electron microscopy of sub-recent sediments as well as hard and consolidated old sinters. Though this putative past community is quite unusual in volcanic niches, its ubiquitous and enduring presence in the Puga geothermal area is highlighted by the discovery of its biosignatures along all of the three dimensions of the explored boratic deposits. These findings usher a new paradigm of looking at past or present geothermal life in as well as outside the Earth.     

Isolation and Distribution of a Novel Iron-Oxidizing Crenarchaeon from Acidic Geothermal Springs in Yellowstone National Park

Novel thermophilic crenarchaea have been observed in Fe(III) oxide microbial mats of Yellowstone National Park (YNP); however, no definitive work has identified specific microorganisms responsible for the oxidation of Fe(II). The objectives of the current study were to isolate and characterize an Fe(II)-oxidizing member of the Sulfolobales observed in previous 16S rRNA gene surveys and to determine the abundance and distribution of close relatives of this organism in acidic geothermal springs containing high concentrations of dissolved Fe(II). Here we report the isolation and characterization of the novel, Fe(II)-oxidizing, thermophilic, acidophilic organism Metallosphaera sp. strain MK1 obtained from a well-characterized acid-sulfate-chloride geothermal spring in Norris Geyser Basin, YNP. Full-length 16S rRNA gene sequence analysis revealed that strain MK1 exhibits only 94.9 to 96.1% sequence similarity to other known Metallosphaera spp. and less than 89.1% similarity to known Sulfolobus spp. Strain MK1 is a facultative chemolithoautotroph with an optimum pH range of 2.0 to 3.0 and an optimum temperature range of 65 to 75°C. Strain MK1 grows optimally on pyrite or Fe(II) sorbed onto ferrihydrite, exhibiting doubling times between 10 and 11 h under aerobic conditions (65°C). The distribution and relative abundance of MK1-like 16S rRNA gene sequences in 14 acidic geothermal springs containing Fe(III) oxide microbial mats were evaluated. Highly related MK1-like 16S rRNA gene sequences (>99% sequence similarity) were consistently observed in Fe(III) oxide mats at temperatures ranging from 55 to 80°C. Quantitative PCR using Metallosphaera-specific primers confirmed that organisms highly similar to strain MK1 comprised up to 40% of the total archaeal community at selected sites. The broad distribution of highly related MK1-like 16S rRNA gene sequences in acidic Fe(III) oxide microbial mats is consistent with the observed characteristics and growth optima of Metallosphaera-like strain MK1 and emphasizes the importance of this newly described taxon in Fe(II) chemolithotrophy in acidic high-temperature environments of YNP.     

Phylogenetic Diversity Analysis of Subterranean Hot Springs in Iceland

Geothermal energy has been harnessed and used for domestic heating in Iceland. In wells that are typically drilled to a depth of 1,500 to 2,000 m, the temperature of the source water is 50 to 130°C. The bottoms of the boreholes can therefore be regarded as subterranean hot springs and provide a unique opportunity to study the subterranean biosphere. Large volumes of geothermal fluid from five wells and a mixture of geothermal water from 50 geothermal wells (hot tap water) were sampled and concentrated through a 0.2-μm-pore-size filter. Cells were observed in wells RG-39 (91.4°C) and MG-18 (71.8°C) and in hot tap water (76°C), but no cells were detected in wells SN-4, SN-5 (95 to 117°C), and RV-5 (130°C). Archaea and Bacteria were detected by whole-cell fluorescent in situ hybridization. DNAs were extracted from the biomass, and small-subunit rRNA genes (16S rDNAs) were amplified by PCR using primers specific for the Archaea and Bacteria domains. The PCR products were cloned and sequenced. The sequence analysis showed 11 new operational taxonomic units (OTUs) out of 14, 3 of which were affiliated with known surface OTUs. Samples from RG-39 and hot tap water were inoculated into enrichment media and incubated at 65 and 85°C. Growth was observed only in media based on geothermal water. 16S rDNA analysis showed enrichments dominated with Desulfurococcales relatives. Two strains belonging to Desulfurococcus mobilis and to the Thermus/Deinococcus group were isolated from borehole RG-39. The results indicate that subsurface volcanic zones are an environment that provides a rich subsurface for novel thermophiles.     

Phylogenetic and Comparative Sequence Analysis of Thermostable Alpha Amylases of kingdom Archea,Prokaryotes and Eukaryotes

Alpha amylase family is generally defined as a group of enzymes that can hydrolyse and transglycosylase α-(1, 4) or α-(1, 6) glycosidic bonds along with the preservation of anomeric configuration. For the comparative analysis of alpha amylase family, nucleotide sequences of seven thermo stable organisms of Kingdom Archea i.e. Pyrococcus furiosus (100-105°C), Kingdom Prokaryotes i.e. Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C), Bacillus amyloliquefaciens (72°C), Bacillus subtilis (70°C) and Bacillus KSM K38 (55°C) and Eukaryotes i.e. Aspergillus oryzae (60°C) were selected from NCBI. Primary structure composition analysis and Conserved sequence analysis were conducted through Bio Edit tools. Results from BioEdit shown only three conserved regions of base pairs and least similarity in MSA of the above mentioned alpha amylases. In Mega 5.1 Phylogeny of thermo stable alpha amylases of Kingdom Archea, Prokaryotes and Eukaryote was handled by Neighbor-Joining (NJ) algorithm. Mega 5.1 phylogenetic results suggested that alpha amylases of thermo stable organisms i.e. Pyrococcus furiosus (100-105°C), Bacillus licheniformis (90-95°C), Geobacillus stearothermophilus (75°C) and Bacillus amyloliquefaciens (72°C) are more distantly related as compared to less thermo stable organisms. By keeping in mind the characteristics of most thermo stable alpha amylases novel and improved features can be introduced in less thermo stable alpha amylases so that they become more thermo tolerant and productive for industry.     

Involvement of Microorganisms in the Formation of Carbonate Speleothems in the Cervo Cave (L'Aquila-Italy)

Much is known about the bacterial precipitation of carbonate rocks, but comparatively little is known about the involvement of microbes in the formation of secondary mineral structures in caves. We hypothesized that bacteria isolated from calcareous stalactites, which are able to mediate CaCO₃ precipitation in vitro, play a role in the formation of carbonate speleothems. We collected numerous cultivable calcifying bacteria from calcareous speleothems from Cervo cave, implying that their presence was not occasional. The relative abundance of calcifying bacteria among total cultivable microflora was found to be related to the calcifying activity in the stalactites. We also determined the δ ¹³C and δ ¹⁸ O values of the Cervo cave speleothems from which bacteria were isolated and of the carbonates obtained in vitro to determine whether bacteria were indeed involved in the formation of secondary mineral structures. We identified three groups of biological carbonates produced in vitro at 11°C on the basis of their carbon isotopic composition: carbonates with δ ¹³C values (a) slightly more positive, (b) more negative, and (c) much more negative than those of the stalactite carbonates. The carbonates belonging to the first group, characterized by the most similar δ ¹³C values to stalactites, were produced by the most abundant strains. Most of calcifying isolates belonged to the genus Kocuria. Scanning electron microscopy showed that dominant morphologies of the bioliths were sherulithic with fibrous radiated interiors. We suggest a mechanism of carbonate crystal formation by bacteria.     

Legionella Species Diversity in an Acidic Biofilm Community in Yellowstone National Park

Legionella species are frequently detected in aquatic environments, but their occurrence in extreme, acidic, geothermal habitats has not been explored with cultivation-independent methods. We investigated a predominately eukaryotic algal mat community in a pH 2.7 geothermal stream in Yellowstone National Park for the presence of Legionella and potential host amoebae. Our analyses, using PCR amplification with Legionella-specific primers targeting 16S rRNA genes, detected four known Legionella species, as well as Legionella sequences from species that are not represented in sequence databases, in mat samples and cultivated isolates. The nonrandom occurrence of sequences detected at lower (30°C) and higher (35 to 38°C) temperatures suggests that natural thermal gradients in the stream influence Legionella species distributions in this mat community. We detected only one sequence, Legionella micdadei, from cultivated isolates. We cultured and sequenced partial 18S rRNA gene regions from two potential hosts, Acanthamoeba and Euglena species.     

Isolation, Characterization, and Ecology of Sulfur-Respiring Crenarchaea Inhabiting Acid-Sulfate-Chloride-Containing Geothermal Springs in Yellowstone National Park

Elemental sulfur (S⁰) is associated with many geochemically diverse hot springs, yet little is known about the phylogeny, physiology, and ecology of the organisms involved in its cycling. Here we report the isolation, characterization, and ecology of two novel, S⁰-reducing Crenarchaea from an acid geothermal spring referred to as Dragon Spring. Isolate 18U65 grows optimally at 70 to 72°C and at pH 2.5 to 3.0, while isolate 18D70 grows optimally at 81°C and pH 3.0. Both isolates are chemoorganotrophs, dependent on complex peptide-containing carbon sources, S⁰, and anaerobic conditions for respiration-dependent growth. Glycerol dialkyl glycerol tetraethers (GDGTs) containing four to six cyclopentyl rings were present in the lipid fraction of isolates 18U65 and 18D70. Physiological characterization suggests that the isolates are adapted to the physicochemical conditions of Dragon Spring and can utilize the natural organic matter in the spring as a carbon and energy source. Quantitative PCR analysis of 16S rRNA genes associated with the S⁰ flocs recovered from several acid geothermal springs using isolate-specific primers indicates that these two populations together represent 17 to 37% of the floc-associated DNA. The physiological characteristics of isolates 18U65 and 18D70 are consistent with their potential widespread distribution and putative role in the cycling of sulfur in acid geothermal springs throughout the Yellowstone National Park geothermal complex. Based on phenotypic and genetic characterization, the designations Caldisphaera draconis sp. nov. and Acidilobus sulfurireducens sp. nov. are proposed for isolates 18U65 and 18D70, respectively.     

Metagenomes from High-Temperature Chemotrophic Systems Reveal Geochemical Controls on Microbial Community Structure and Function

The Yellowstone caldera contains the most numerous and diverse geothermal systems on Earth, yielding an extensive array of unique high-temperature environments that host a variety of deeply-rooted and understudied Archaea, Bacteria and Eukarya. The combination of extreme temperature and chemical conditions encountered in geothermal environments often results in considerably less microbial diversity than other terrestrial habitats and offers a tremendous opportunity for studying the structure and function of indigenous microbial communities and for establishing linkages between putative metabolisms and element cycling. Metagenome sequence (14–15,000 Sanger reads per site) was obtained for five high-temperature (>65°C) chemotrophic microbial communities sampled from geothermal springs (or pools) in Yellowstone National Park (YNP) that exhibit a wide range in geochemistry including pH, dissolved sulfide, dissolved oxygen and ferrous iron. Metagenome data revealed significant differences in the predominant phyla associated with each of these geochemical environments. Novel members of the Sulfolobales are dominant in low pH environments, while other Crenarchaeota including distantly-related Thermoproteales and Desulfurococcales populations dominate in suboxic sulfidic sediments. Several novel archaeal groups are well represented in an acidic (pH 3) Fe-oxyhydroxide mat, where a higher O₂ influx is accompanied with an increase in archaeal diversity. The presence or absence of genes and pathways important in S oxidation-reduction, H₂-oxidation, and aerobic respiration (terminal oxidation) provide insight regarding the metabolic strategies of indigenous organisms present in geothermal systems. Multiple-pathway and protein-specific functional analysis of metagenome sequence data corroborated results from phylogenetic analyses and clearly demonstrate major differences in metabolic potential across sites. The distribution of functional genes involved in electron transport is consistent with the hypothesis that geochemical parameters (e.g., pH, sulfide, Fe, O₂) control microbial community structure and function in YNP geothermal springs.     

Algal Species and Light Microenvironment in a Low-pH, Geothermal Microbial Mat Community

Unicellular algae are the predominant microbial mat-forming phototrophs in the extreme environments of acidic geothermal springs. The ecology of these algae is not well known because concepts of species composition are inferred from cultivated isolates and microscopic observations, methods known to provide incomplete and inaccurate assessments of species in situ. We used sequence analysis of 18S rRNA genes PCR amplified from mat samples from different seasons and different temperatures along a thermal gradient to identify algae in an often-studied acidic (pH 2.7) geothermal creek in Yellowstone National Park. Fiber-optic microprobes were used to show that light for algal photosynthesis is attenuated to <1% over the 1-mm surface interval of the mat. Three algal sequences were detected, and each was present year-round. A Cyanidioschyzon merolae sequence was predominant at temperatures of ≥49°C. A Chlorella protothecoides var. acidicola sequence and a Paradoxia multisita-like sequence were predominant at temperatures of ≤39°C.     

A Leech Capable of Surviving Exposure to Extremely Low Temperatures

It is widely considered that most organisms cannot survive prolonged exposure to temperatures below 0°C, primarily because of the damage caused by the water in cells as it freezes. However, some organisms are capable of surviving extreme variations in environmental conditions. In the case of temperature, the ability to survive subzero temperatures is referred to as cryobiosis. We show that the ozobranchid leech, Ozobranchus jantseanus, a parasite of freshwater turtles, has a surprisingly high tolerance to freezing and thawing. This finding is particularly interesting because the leach can survive these temperatures without any acclimation period or pretreatment. Specifically, the leech survived exposure to super-low temperatures by storage in liquid nitrogen (−196°C) for 24 hours, as well as long-term storage at temperatures as low as −90°C for up to 32 months. The leech was also capable of enduring repeated freeze-thaw cycles in the temperature range 20°C to −100°C and then back to 20°C. The results demonstrated that the novel cryotolerance mechanisms employed by O. jantseanus enable the leech to withstand a wider range of temperatures than those reported previously for cryobiotic organisms. We anticipate that the mechanism for the observed tolerance to freezing and thawing in O. jantseanus will prove useful for future studies of cryopreservation.     

Effect of Temperature on Structure and Function of the Methanogenic Archaeal Community in an Anoxic Rice Field Soil

Soil temperatures in Italian rice fields typically range between about 15 and 30°C. A change in the incubation temperature of anoxic methanogenic soil slurry from 30°C to 15°C typically resulted in a decrease in the CH₄ production rate, a decrease in the steady-state H₂ partial pressure, and a transient accumulation of acetate. Previous experiments have shown that these changes were due to an alteration of the carbon and electron flow in the methanogenic degradation pathway of organic matter caused by the temperature shift (K. J. Chin and R. Conrad, FEMS Microbiol. Ecol. 18:85–102, 1995). To investigate how temperature affects the structure of the methanogenic archaeal community, total DNA was extracted from soil slurries incubated at 30 and 15°C. The archaeal small-subunit (SSU) rRNA-encoding genes (rDNA) of these environmental DNA samples were amplified by PCR with an archaeal-specific primer system and used for the generation of clone libraries. Representative rDNA clones (n = 90) were characterized by terminal restriction fragment length polymorphism (T-RFLP) and sequence analysis. T-RFLP analysis produced for the clones terminally labeled fragments with a characteristic length of mostly 185, 284, or 392 bp. Sequence analysis allowed determination of the phylogenetic affiliation of the individual clones with their characteristic T-RFLP fragment lengths and showed that the archaeal community of the anoxic rice soil slurry was dominated by members of the families Methanosarcinaceae (185 bp) and Methanosaetaceae (284 bp), the kingdom Crenarchaeota (185 or 284 bp), and a novel, deeply branching lineage of the (probably methanogenic) kingdom Euryarchaeota (392 bp) that has recently been detected on rice roots (R. Großkopf, S. Stubner, and W. Liesack, Appl. Environ. Microbiol. 64:4983–4989, 1998). The structure of the archaeal community changed when the temperature was shifted from 30°C to 15°C. Before the temperature shift, the clones (n = 30) retrieved from the community were dominated by Crenarchaeota (70%), “novel Euryarchaeota” (23%), and Methanosarcinacaeae (7%). Further incubation at 30°C (n = 30 clones) resulted in a relative increase in members of the Methanosarcinaceae (77%), whereas further incubation at 15°C (n = 30 clones) resulted in a much more diverse community consisting of 33% Methanosarcinaceae, 23% Crenarchaeota, 20% Methanosaetaceae, and 17% novel Euryarchaeota. The appearance of Methanosaetaceae at 15°C was conspicuous. These results demonstrate that the structure of the archaeal community in anoxic rice field soil changed with time and incubation temperature.     

Diatoms and Other Epibionts Associated with Olive Ridley (Lepidochelys olivacea) Sea Turtles from the Pacific Coast of Costa Rica

Although the sea turtles have long been familiar and even iconic to marine biologists, many aspects of their ecology remain unaddressed. The present study is the first of the epizoic diatom community covering the olive ridley turtle’s (Lepidochelys olivacea) carapace and the first describing diatoms living on sea turtles in general, with the primary objective of providing detailed information on turtle epibiotic associations. Samples of turtle carapace including the associated diatom biofilm and epizoic macro-fauna were collected from Ostional beach (9° 59´ 23.7´´ N 85° 41´ 52.6´´ W), Costa Rica, during the arribada event in October 2013. A complex diatom community was present in every sample. In total, 11 macro-faunal and 21 diatom taxa were recorded. Amongst diatoms, the most numerous were erect (Achnanthes spp., Tripterion spp.) and motile (Haslea sp., Navicula spp., Nitzschia spp., Proschkinia sp.) forms, followed by adnate Amphora spp., while the most common macro-faunal species was Stomatolepas elegans (Cirripedia). Diatom densities ranged from 8179 ± 750 to 27685 ± 4885 cells mm^-2. Epizoic microalgae were either partly immersed or entirely encapsulated within an exopolymeric coat. The relatively low diatom species number, stable species composition and low inter-sample dissimilarities (14.4% on average) may indicate a mutualistic relationship between the epibiont and the basibiont. Dispersal of sea turtle diatoms is probably highly restricted and similar studies will help to understand both diatom diversity, evolution and biogeography, and sea turtle ecology and foraging strategies.     

THE RATE OF OXYGEN UTILIZATION BY YEAST AS RELATED TO TEMPERATURE

Suspensions of the yeast Saccharomyces cerevisiae gave reproducible rates of O₂ uptake over a period of 6 months. The relation of rate of consumption of O₂ to temperature was tested over a wide range of temperatures, and the constant in the formulation of the relationship is found to be reproducible. The values of this constant (µ) have been obtained for five separate series of experiments by three methods of estimation. The variability of µ has the following magnitudes: the average deviation of a single determination expressed as per cent of the mean is ±2 per cent in the range 30–15°, and ±0.8 per cent in the range 15–3°C. This constancy of metabolic activity measured as a function of temperature can then be utilized for more precise investigations of processes controlling the velocity of oxidations of substrates, and of respiratory systems controlled by intracellular respiratory pigments. The data plotted according to the Arrhemus equation give average values of the constant µ as follows: for the range 35–30°, µ = 8,290; 30–15°, µ = 12,440 ±290; 15–3°, µ = 19,530 ±154. The critical temperatures are at 29.0° and 15.7°C. A close similarity exists between these temperature characteristics (µ) and values in the series usually obtained for respiratory activities in other organisms. This fact supports the view that a common system of processes controls the velocities of physiological activities in yeast and in other organisms.     

Novel Clostridium populations involved in the anaerobic degradation of Microcystis blooms

Understanding the microbial degradation of Microcystis biomass is crucial for determining the ecological consequences of Microcystis blooms in freshwater lakes. The purpose of this study was to identify bacteria involved in the anaerobic degradation of Microcystis blooms. Microcystis scum was anaerobically incubated for 90 days at three temperatures (15 °C, 25 °C and 35 °C). We used terminal restriction fragment length polymorphism (T-RFLP) analysis of bacterial 16S rRNA genes, followed by cloning and sequencing of selected samples, to reveal the community composition of bacteria and their dynamics during decomposition. Clostridium spp. were found to be the most dominant bacteria in the incubations, accounting for 72% of the sequenced clones. Eight new clusters or subclusters (designated CLOS.1–8) were identified in the Clostridium phylogenetic tree. The bacterial populations displayed distinct successions during Microcystis decomposition. Temperature had a strong effect on the dynamics of the bacterial populations. At 15 °C, the initial dominance of a 207-bp T-RF (Betaproteobacteria) was largely substituted by a 227-bp T-RF (Clostridium, new cluster CLOS.2) at 30 days. In contrast, at 25 °C and 35 °C, we observed an alternating succession of the 227-bp T-RF and a 231-bp T-RF (Clostridium, new cluster CLOS.1) that occurred more than four times; no one species dominated the flora for the entire experiment. Our study shows that novel Clostridium clusters and their diverse consortiums dominate the bacterial communities during anaerobic degradation of Microcystis, suggesting that these microbes'' function in the degradation process.     

Few Highly Abundant Operational Taxonomic Units Dominate within Rumen Methanogenic Archaeal Species in New Zealand Sheep and Cattle

Sequencing and analyses of 16S rRNA gene amplicons were performed to estimate the composition of the rumen methanogen community in 252 samples from eight cohorts of sheep and cattle, separated into 16 different sample groups by diet, and to determine which methanogens are most prominent in the rumens of farmed New Zealand ruminants. Methanobacteriales (relative abundance ± standard deviation, 89.6% ± 9.8%) and Methanomassiliicoccales (10.4% ± 9.8%) were the two major orders and contributed 99.98% (±0.1%) to the rumen methanogen communities in the samples. Sequences from Methanobacteriales were almost entirely from only four different species (or clades of very closely related species). Each was detectable in at least 89% of the samples. These four species or clades were the Methanobrevibacter gottschalkii clade and Methanobrevibacter ruminantium clade with a mean abundance of 42.4% (±19.5% standard deviation) and 32.9% (±18.8%), respectively, and Methanosphaera sp. ISO3-F5 (8.2% ± 6.7%) and Methanosphaera sp. group5 (5.6% ± 5.7%). These four species or clades appeared to be primarily represented by only one or, in one case, two dominant sequence types per species or clade when the sequences were grouped into operational taxonomic units (OTUs) at 99% sequence identity. The mean relative abundance of Methanomassiliicoccales in the samples was relatively low but exceeded 40% in some of the treatment groups. Animal feed affected the apparent methanogen community structure of both orders, as evident from differences in relative abundances of the major OTUs in animals under different feeding regimens.     

Production of Novel Antibiotics Zeamines through Optimizing Dickeya zeae Fermentation Conditions

Dickeya zeae strain EC1 was recently shown to produce a new type of phytotoxins designated as zeamine and zeamine II, which are potent wide-spectrum antibiotics against Gram-positive and Gram-negative bacterial pathogens, suggesting their promising potential as clinical medicines. In this study, the optimized medium composition and culture conditions for biosynthesis of novel antibiotics zeamines have been established by using response surface methodology, largely increasing the yield of zeamines from original about 7.35 µg·mL⁻¹ in minimal medium to about 150 µg·mL⁻¹ in LS5 medium. The study identified the major factors contributing to zeamines production, which include nitrate, sucrose, asparaginate, mineral elements Mg²⁺ and K⁺, and optimized amount of phosphate. In addition, the results showed that overexpression of zmsK in D. zeae strain EC1 could further increase zeamines yield to about 180 µg·mL⁻¹ in LS5 medium. The findings from this study could facilitate further characterization and utilization of these two novel antibiotics, and also provide useful clues for understanding the regulatory mechanisms that govern D. zeae virulence.     

A Comprehensive Census of Microbial Diversity in Hot Springs of Tengchong,Yunnan Province China Using 16S rRNA Gene Pyrosequencing

The Rehai and Ruidian geothermal fields, located in Tengchong County, Yunnan Province, China, host a variety of geochemically distinct hot springs. In this study, we report a comprehensive, cultivation-independent census of microbial communities in 37 samples collected from these geothermal fields, encompassing sites ranging in temperature from 55.1 to 93.6°C, in pH from 2.5 to 9.4, and in mineralogy from silicates in Rehai to carbonates in Ruidian. Richness was low in all samples, with 21–123 species-level OTUs detected. The bacterial phylum Aquificae or archaeal phylum Crenarchaeota were dominant in Rehai samples, yet the dominant taxa within those phyla depended on temperature, pH, and geochemistry. Rehai springs with low pH (2.5–2.6), high temperature (85.1–89.1°C), and high sulfur contents favored the crenarchaeal order Sulfolobales, whereas those with low pH (2.6–4.8) and cooler temperature (55.1–64.5°C) favored the Aquificae genus Hydrogenobaculum. Rehai springs with neutral-alkaline pH (7.2–9.4) and high temperature (>80°C) with high concentrations of silica and salt ions (Na, K, and Cl) favored the Aquificae genus Hydrogenobacter and crenarchaeal orders Desulfurococcales and Thermoproteales. Desulfurococcales and Thermoproteales became predominant in springs with pH much higher than the optimum and even the maximum pH known for these orders. Ruidian water samples harbored a single Aquificae genus Hydrogenobacter, whereas microbial communities in Ruidian sediment samples were more diverse at the phylum level and distinctly different from those in Rehai and Ruidian water samples, with a higher abundance of uncultivated lineages, close relatives of the ammonia-oxidizing archaeon “Candidatus Nitrosocaldus yellowstonii”, and candidate division O1aA90 and OP1. These differences between Ruidian sediments and Rehai samples were likely caused by temperature, pH, and sediment mineralogy. The results of this study significantly expand the current understanding of the microbiology in Tengchong hot springs and provide a basis for comparison with other geothermal systems around the world.     

PCR Analysis of the Distribution of Unicellular Cyanobacterial Diazotrophs in the Arabian Sea

An oligonucleotide primer, NITRO821R, targeting the 16S rRNA gene of unicellular cyanobacterial N₂ fixers was developed based on newly derived sequences from Crocosphaera sp. strain WH 8501 and Cyanothece sp. strains WH 8902 and WH 8904 as well as several previously described sequences of Cyanothece sp. and sequences of intracellular cyanobacterial symbionts of the marine diatom Climacodium frauenfeldianum. This oligonucleotide is specific for the targeted organisms, which represent a well-defined phylogenetic lineage, and can detect as few as 50 cells in a standard PCR when it is used as a reverse primer together with the cyanobacterium- and plastid-specific forward primer CYA359F (U. Nübel, F. Garcia-Pichel, and G. Muyzer, Appl. Environ. Microbiol. 63:3327-3332, 1997). Use of this primer pair in the PCR allowed analysis of the distribution of marine unicellular cyanobacterial diazotrophs along a transect following the 67°E meridian from Victoria, Seychelles, to Muscat, Oman (0.5°S to 26°N) in the Arabian Sea. These organisms were found to be preferentially located in warm (>29°C) oligotrophic subsurface waters between 0 and 7°N, but they were also found at a station north of Oman at 26°N, 56°35′E, where similar water column conditions prevailed. Slightly cooler oligotrophic waters (<29°C) did not contain these organisms or the numbers were considerably reduced, suggesting that temperature is a key factor in dictating the abundance of this unicellular cyanobacterial diazotroph lineage in marine environments.