Microbial diversity in acid mine drainage of Xiang Mountain sulfide mine, Anhui Province, China

To understand the composition and structure of microbial communities in acid (pH 3.0) mine drainage (AMD) associated with pyrite mine tailings in Anhui Province, China, molecular diversities of 16S rRNA and 18S rRNA genes were examined using a PCR-based cloning approach. Bacterial, archaeal and microeukaryotic clone libraries were constructed. In contrast to typical dominance of autotrophic acidophiles, genus Acidiphilium, which consists of mixotrophic acidophiles capable of chemoorganotrophic and photosynthetic metabolisms, was the largest group in the bacterial clone library. These mixotrophic organisms may be advantageous in the oligotrophic AMD environment of the study site (certain amounts of dissolved organic carbon and light) by switching between two modes of metabolisms. Unexpectedly, a large fraction of bacterial clones (12.7%) were related to the neutrophilic genus Legionella, which can cause Legionnaires’ disease, a potentially lethal pneumonia. The eukaryotic 18S rRNA gene sequences were mostly related to Oxytricha, Nuclearia, and Penicillium. In the archaeal clone library, all the sequences were affiliated to the phylum Crenarchaeota, while the Euryarchaeota was not present.     

Comparison of Acid Mine Drainage Microbial Communities in Physically and Geochemically Distinct Ecosystems

This study presents population analyses of microbial communities inhabiting a site of extreme acid mine drainage (AMD) production. The site is the inactive underground Richmond mine at Iron Mountain, Calif., where the weathering of a massive sulfide ore body (mostly pyrite) produces solutions with pHs of ~0.5 to ~1.0. Here we used a suite of oligonucleotide probes, designed from molecular data recently acquired from the site, to analyze a number of microbial environments by fluorescent in situ hybridization. Microbial-community analyses were correlated with geochemical and mineralogical data from those environments. The environments investigated were within the ore body and thus at the site of pyrite dissolution, as opposed to environments that occur downstream of the dissolution. Few organism types, as defined by the specificities of the oligonucleotide probes, dominated the microbial communities. The majority of the dominant organisms detected were newly discovered or organisms only recently associated with acid-leaching environments. “Ferroplasma” spp. were detected in many of the communities and were particularly dominant in environments of lowest pH and highest ionic strength. Leptospirillum spp. were also detected in many slime and pyrite-dominated environments. In samples of an unusual subaerial slime, a new uncultured Leptospirillum sp. dominated. Sulfobacillus spp. were detected as a prominent inhabitant in warmer (~43°C) environments. The information gathered here is critical for determining organisms important to AMD production at Iron Mountain and for directing future studies of this process. The findings presented here also have relevance to the microbiology of industrial bioleaching and to the understanding of geochemical iron and sulfur cycles.     

Detection of sphingomonads and in situ identification in activated sludge using 16S rRNA-targeted oligonucleotide probes

The increasing significance of members of the genus Sphingomonas in biotechnological applications has led to an increased interest in the diversity, abundance and ecophysiological potential of this group of Gram-negative bacteria. This general focus provides a challenge to improve means for identification of sphingomonads; eg molecular genetic methods for rapid and specific detection could facilitate screening of new isolates. Here, fluorescently labeled oligonucleotide probes targeted against 16S rRNA were used to typify strains previously assigned to the genus. All 46 sphingomonads tested including type strains of 21 Sphingomonasspecies could be detected with a probe originally designed for the genus and all but one with a probe designed for the alpha-4 subgroup of the Proteobacteria. The two probes are suitable for direct detection of sphingomonads in pure and mixed cultures as well as in environmental samples of unknown composition. The probes were used to identify sphingomonads in situ in activated sludge samples. Sphingomonads were rather abundant accounting for about 5–10% of the total cells in municipal sludges. Distinct patterns in aggregation of the cells suggest that these organisms could be involved in the formation process of sludge flocs. Received 27 May 1999/ Accepted in revised form 22 August 1999     

Oligonucleotide Probes for the Detection of Representatives of the Genus Thermoanaerobacter

Based on the analysis of 16S rRNA nucleotide sequences, oligonucleotide probes were designed for the detection of representatives of the genus Thermoanaerobacter. To increase the specificity of detection, the genus Thermoanaerobacter was divided into three groups. The probe Tab 827 (5"-GCTTCCGCDYCCCACACCTA-3") detected all known representatives of the genus Thermoanaerobacter; the probe Tab_1 844 (5"-TTAACTACGGCACGRAATGCTTC-3") was specific for the first group of species of the genus (T. wiegelii, T. siderophilus, T. sulfurophilus, T. brockii, T. kivui, T. ethanolicus, T. acetoethylicus, and T. thermohydrosulfuricus); the probe Tab_2 424 (5"-CACTAMYGGGGTTTACAACC-3") targeted the second group (T. thermocopriae, T. mathranii, and T. italicus); and the probe Tab_3 184 (5"-TCCTCCATCAGGATGCCCTA-3") was specific for the third group (T. tengcongensis, T. yonseiensis, T. subterraneus, and Carboxydibrachium pacificum, an organism related to the genus Thermoanaerobacter according to its 16S rRNA sequence). The oligonucleotide probes were labeled with Dig-11-dUTP. Hybridization with the probes showed the affiliation with Thermoanaerobacter of several pure cultures that were morphologically similar to representatives of this genus but possessed metabolic features unusual for it (capacity for agarose hydrolysis, anaerobic oxidation of CO, growth at low pH values) or were isolated from habitats previously unknown for Thermoanaerobacter (deep-sea hydrothermal vents).     

Varied resonses in gene expression of culturable heterotrophic bacteria isolated from the environment

Summary Gene expression in heterotrophic bacteria isolated from environmental samples was studied using a combination of non-selective and selective plating techniques and gene probe methodology. The gene probes and their respective phenotypes were nahAB, for naphthalene degradation, merA, for narrowlspectrum mercury resistance, and merB, for broad-spectrum mercury resistance. Gene-probe-positive organisms could be placed into one of three categories: (1) organisms that could express their genetic information immediately upon isolation from the environment; (2) organisms that expressed their genotype only after cultivation before selection for the genotype; and (3) organisms that did not express their genotype at all in our hands. For all three probes it was found that most organisms fell into category 2. This phenomenon was also observed with bacteria isolated from lake water that probed positive with the nitrogenase (nifHDK) gene probe. The data suggest that the numbers of isolates identified by gene probes merely reflect the genetic potential of a community whereas various expression data suggest that differences in the actual activity of those genotypes exist in the natural environment. Correspondence to: B. H. Olson     

Effect of water flow velocity on growth and morphology of cultured <Emphasis Type="Italic">Undaria pinnatifida</Emphasis> sporophytes (Laminariales,Phaeophyceae) in Okirai Bay on the Sanriku coast,Northeast Japan

Undaria pinnatifida sporophytes, originating from the same strain, were cultured at the commercial cultivation site exposed to wave action and the uncultivated site protected from water action of Okirai Bay, Northeast Japan, from January to April 2007; simultaneously, water flow velocity, water temperature, salinity, NO₃ + NO₂, and chlorophyll a were monitored to investigate the effect of water environment on their growth and morphology. Water temperature and salinity fluctuated within the optimal range for their growth whereas water flow velocity at the cultivation site was greatly fast compared with that at the uncultivated site. Successive chlorophyll a increases synchronized with NO₃ + NO₂ decreases were observed only at the uncultivated site for over a month; indicating developments of phytoplankton blooms and their nutrient consumption under the low-flow condition. Meanwhile, blade growth rate of cultured sporophytes was higher at the cultivation site than at the uncultivated site. Their thallus size expressed by six morphological characters (blade length, stipe length, blade wet weight, stipe wet weight, blade width, and undivided blade width) at the cultivation site became large in comparison with that at the uncultivated site. Their three morphological correlations (correlations between blade length and thallus length; blade wet weight and thallus wet weight; and undivided blade width and blade width) differed between the sites. They produced a thick and flat blade at the cultivation site but formed a thin and wrinkled blade at the uncultivated site. These results show the significant impact of water flow velocity on their growth and morphology.     

Comparison of acid mine drainage microbial communities in physically and geochemically distinct ecosystems

This study presents population analyses of microbial communities inhabiting a site of extreme acid mine drainage (AMD) production. The site is the inactive underground Richmond mine at Iron Mountain, Calif., where the weathering of a massive sulfide ore body (mostly pyrite) produces solutions with pHs of approximately 0.5 to approximately 1.0. Here we used a suite of oligonucleotide probes, designed from molecular data recently acquired from the site, to analyze a number of microbial environments by fluorescent in situ hybridization. Microbial-community analyses were correlated with geochemical and mineralogical data from those environments. The environments investigated were within the ore body and thus at the site of pyrite dissolution, as opposed to environments that occur downstream of the dissolution. Few organism types, as defined by the specificities of the oligonucleotide probes, dominated the microbial communities. The majority of the dominant organisms detected were newly discovered or organisms only recently associated with acid-leaching environments. "Ferroplasma" spp. were detected in many of the communities and were particularly dominant in environments of lowest pH and highest ionic strength. Leptospirillum spp. were also detected in many slime and pyrite-dominated environments. In samples of an unusual subaerial slime, a new uncultured Leptospirillum sp. dominated. Sulfobacillus spp. were detected as a prominent inhabitant in warmer ( approximately 43 degrees C) environments. The information gathered here is critical for determining organisms important to AMD production at Iron Mountain and for directing future studies of this process. The findings presented here also have relevance to the microbiology of industrial bioleaching and to the understanding of geochemical iron and sulfur cycles.     

Validation of the H<Subscript>2</Subscript>S method to detect bacteria of fecal origin by cultured and molecular methods

Using biochemical and molecular methods, this research determined whether or not the H₂S test did correctly identify sewage-contaminated waters by being the first to use culturing and molecular methods to identify the types and numbers of fecal indicator organisms, pathogens, and other microbes present in sewage samples with positive H₂S test results. For the culture-based method, samples were analyzed for the presence of fecal bacteria by spread plating the sewage sample onto differential and selective media for Aeromonas spp., Escherichia coli, sulfite-reducing clostridia, H₂S-producing bacteria, and Salmonella/Shigella spp. The isolates were then: (1) tested to determine whether they were H₂S-producing organisms and (2) identified to the genus and species level using biochemical methods. The molecular method used to characterize the microbial populations of select samples was terminal restriction fragment length polymorphisms. These experiments on sewage provided evidence that positive H₂S tests consistently contained fecal bacteria and pathogens. There were strong relationships of agreement between the organisms identified by both methods tested. This study is an important advance in microbial water quality detection since it is focused on the evaluation of a novel, low-cost, water microbiology test that has the potential to provide millions of people worldwide access to water quality detection technology. Of prime consideration in evaluating water quality tests is the determination of the test’s accuracy and specificity, and this article is a fundamental step in providing that information.     

A survey of culturable aerobic and anaerobic marine bacteria in de novo biofilm formation on natural substrates in St. Andrews Bay,Scotland

This study reports a novel study of marine biofilm formation comprising aerobic and anaerobic bacteria. Samples of quartz and feldspar, minerals commonly found on the earth, were suspended 5 m deep in the North Sea off the east coast of St. Andrews, Scotland for 5 weeks. The assemblage of organisms attached to these stones was cultivated under aerobic and anaerobic conditions in the laboratory. Bacteria isolated on Marine Agar 2216 were all Gram-negative and identified to genus level by sequencing the gene encoding 16S rRNA. Colwellia, Maribacter, Pseudoaltermonas and Shewanella were observed in aerobically-grown cultures while Vibrio was found to be present in both aerobic and anaerobic cultures. The obligate anaerobic bacterium Psychrilyobacter atlanticus, a recently defined genus, was identified as a close relative of isolates grown anaerobically. The results provide valuable information as to the main players that attach and form de novo biofilms on common minerals in sea water.     

Molecular diversity of microbial community in acid mine drainages of Yunfu sulfide mine

Two acid mine drainage (AMD) samples were studied by a PCR-based cloning approach, which were from Yunfu sulfide mine in Guangdong province, China. A total of 15 operational taxonomic units (OTUs) were obtained from the two AMD samples. The percentage of overlapped OTUs in two AMD samples was 42.1%. Phylogenetic analysis revealed that the bacterium in the two samples fell into four putative divisions, which were Nitrospira, α-Proteobacteria, β-Proteobacteria, and γ-Proteobacteria four families. Organisms of genuses Acidithiobacillus and Gallionella, which were in γ-Proteobacteria family and β-Proteobacteria family, respectively, were dominant in two samples. The proportions of clones affiliated with Gallionella in each sample were 47.2% (G2) and 16.9% (G1). The result suggested that organisms of Gallionella were a very important composition in microbial communities of the two AMD samples we studied. In addition, the PCR amplification of archaeal 16S rDNA genes form these two AMD samples have been performed with two sets of archaea-specific primers, but no PCR product found. Zhiguo He, Shengmu Xiao, Xuehui Xie, and Hui Zhong equally contributed to this work.     

Selective enrichment,isolation and molecular detection of <Emphasis Type="Italic">Salinibacter</Emphasis> and related extremely halophilic <Emphasis Type="Italic">Bacteria</Emphasis> from hypersaline environments

Salinibacter is a genus of red, extremely halophilic Bacteria. Thus far the genus is represented by a single species, Salinibacter ruber, strains of which have been isolated from saltern crystallizer ponds in Spain and on the Balearic Islands. Both with respect to its growth conditions and its physiology, Salinibacter resembles the halophilic Archaea of the order Halobacteriales. We have designed selective enrichment and isolation techniques to obtain Salinibacter and related red extremely halophilic Bacteria from different hypersaline environments, based on their resistance to anisomycin and bacitracin, two antibiotics that are potent inhibitors of the halophilic Archaea. Using direct plating on media containing bacitracin, we found Salinibacter-like organisms in numbers between 1.4×10³ and 1.4×10⁶ml⁻¹ in brines collected from the crystallizer ponds of the salterns in Eilat, Israel, being equivalent to 1.8–18% of the total colony counts obtained on identical media without bacitracin. A number of strains from Eilat were subjected to a preliminary characterization, and they proved similar to the type strain of S. ruber. We also report here the isolation and molecular detection of Salinibacter-like organisms from an evaporite crust on the bottom of salt pools at the Badwater site in Death Valley, CA. These isolates and environmental 16S rRNA gene sequences differ in a number of properties from S. ruber, and they may represent a new species of Salinibacter or a new related genus. Guest Editor: John M. Melack Saline Waters and their Biota     

On the Organization and Affinities of the Amoeba,Pompholyxophrys punicea Archer,Based on Ultrastructural Examination of Individual Cells from Wild Material1

The genus Pompholyxophrys includes amoebae which have a spherical body, fine radiating pseudopodia, and a layer of adhering siliceous “perles.” These organisms are normally regarded as a type of heliozoon. Ultrastructural examination of P. punicea reveals that those characters associated with well characterized heliozoa, such as microtubular axonemes and extrusomes, are lacking. The species has much in common with nucleariid filose amoebae with which it, and the related genus Pinaciophora, are regarded as having affinities. The species P. punicea is rare, and this study was made possible by the application of techniques developed for the ultrastructural examination of single cells. The assessment of protistan diversity and interrelationships relies heavily on the use of ultrastructural characters. Although techniques that are based on the examination of a small number of individual cells have limitations, they do allow rare organisms to be included in the re-evaluation of protistan systematics.     

Recent developments in the thermophilic microbiology of deep-sea hydrothermal vents

The diversity of thermophilic prokaryotes inhabiting deep-sea hot vents was actively studied over the last two decades. The ever growing interest is reflected in the exponentially increasing number of novel thermophilic genera described. The goal of this paper is to survey the progress in this field made in the years 2000–2005. In this period, representatives of several new taxa of hyperthermophilic archaea were obtained from deep-sea environments. Two of these isolates had phenotypic features new for this group of organisms: the presence of an outer cell membrane (the genus Ignicoccus) and the ability to grow anaerobically with acetate and ferric iron (the genus Geoglobus). Also, our knowledge on the diversity of thermophilic bacteria from deep-sea thermal environments extended significantly. The new bacterial isolates represented diverse bacterial divisions: the phylum Aquificae, the subclass Epsilonproteobacteria, the order Thermotogales, the families Thermodesulfobacteriaceae, Deferribacteraceae, and Thermaceae, and a novel bacterial phylum represented by the genus Caldithrix. Most of these isolates are obligate or facultative lithotrophs, oxidizing molecular hydrogen in the course of different types of anaerobic respiration or microaerobic growth. The existence and significant ecological role of some of new bacterial thermophilic isolates was initially established by molecular methods.     

The <Emphasis Type="Italic">Fibrobacteres</Emphasis>: an Important Phylum of Cellulose-Degrading Bacteria

The phylum Fibrobacteres currently comprises one formal genus, Fibrobacter, and two cultured species, Fibrobacter succinogenes and Fibrobacter intestinalis, that are recognised as major bacterial degraders of lignocellulosic material in the herbivore gut. Historically, members of the genus Fibrobacter were thought to only occupy mammalian intestinal tracts. However, recent 16S rRNA gene-targeted molecular approaches have demonstrated that novel centres of variation within the genus Fibrobacter are present in landfill sites and freshwater lakes, and their relative abundance suggests a potential role for fibrobacters in cellulose degradation beyond the herbivore gut. Furthermore, a novel subphylum within the Fibrobacteres has been detected in the gut of wood-feeding termites, and proteomic analyses have confirmed their involvement in cellulose hydrolysis. The genome sequence of F. succinogenes rumen strain S85 has recently suggested that within this group of organisms a “third” way of attacking the most abundant form of organic carbon in the biosphere, cellulose, has evolved. This observation not only has evolutionary significance, but the superior efficiency of anaerobic cellulose hydrolysis by Fibrobacter spp., in comparison to other cellulolytic rumen bacteria that typically utilise membrane-bound enzyme complexes (cellulosomes), may be explained by this novel cellulase system. There are few bacterial phyla with potential functional importance for which there is such a paucity of phenotypic and functional data. In this review, we highlight current knowledge of the Fibrobacteres phylum, its taxonomy, phylogeny, ecology and potential as a source of novel glycosyl hydrolases of biotechnological importance.     

Yeast and mammalian replication intermediates migrate similarly in two-dimensional gels

In the budding yeast, Saccharomyces cerevisiae, DNA replication initiates at specific, discrete chromosomal locations. At each initiation site, a single small replication bubble is generated, which subsequently expands at Y-like replication forks. We wanted to know whether other eukaryotic organisms utilize similar initiation mechanisms. For this purpose, replication intermediates (RIs) from three different organisms (Schizosaccharomyces pombe, Chinese hamster and human) were mixed individually with RIs from S. cerevisiae and then subjected to two-dimensional (2D) gel electrophoresis under conditions known to resolve molecules having different structures. All of the RIs detected by the hybridization probes we used for each organism migrated nearly identically to specific RIs of similar size from S. cerevisiae, implying that the detected RIs from all the studied organisms have very similar structures and may therefore employ the same basic initiation mechanism.     

In situ DNA‐hybridization chain reaction (HCR): a facilitated in situ HCR system for the detection of environmental microorganisms

In situ detection of microorganisms by fluorescence in situ hybridization (FISH) is a powerful tool for environmental microbiology, but analyses can be hampered by low rRNA content in target organisms, especially in oligotrophic environments. Here, we present a non‐enzymatic, hybridization chain reaction (HCR)‐based signal amplified in situ whole‐cell detection technique (in situ DNA‐HCR). The components of the amplification buffer were optimized to polymerize DNA amplifier probes for in situ DNA‐HCR. In situ hybridization of initiator probes followed by signal amplification via HCR produced bright signals with high specificity and probe permeation into cells. The detection rates for Bacteria in a seawater sample and Archaea in anaerobic sludge samples were comparable with or greater than those obtained by catalyzed reporter deposition (CARD)‐FISH or standard FISH. Detection of multiple organisms (Bacteria, Archaea and Methanosaetaceae) in an anaerobic sludge sample was achieved by simultaneous in situ DNA‐HCR. In summary, in situ DNA‐HCR is a simple and easy technique for detecting single microbial cells and enhancing understanding of the ecology and behaviour of environmental microorganisms in situ.     

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.     

Prevalence,complete genome,and metabolic potentials of a phylogenetically novel cyanobacterial symbiont in the coral-killing sponge,Terpios hoshinota

Terpios hoshinota is an aggressive, space-competing sponge that kills various stony corals. Outbreaks of this species have led to intense damage to coral reefs in many locations. Here, the first large-scale 16S rRNA gene survey across three oceans revealed that bacteria related to the taxa Prochloron, Endozoicomonas, SAR116, Ruegeria, and unclassified Proteobacteria were prevalent in T. hoshinota. A Prochloron-related bacterium was the most dominant and prevalent cyanobacterium in T. hoshinota. The complete genome of this uncultivated cyanobacterium and pigment analysis demonstrated that it has phycobiliproteins and lacks chlorophyll b, which is inconsistent with the definition of Prochloron. Furthermore, the cyanobacterium was phylogenetically distinct from Prochloron, strongly suggesting that it should be a sister taxon to Prochloron. Therefore, we proposed this symbiotic cyanobacterium as a novel species under the new genus Candidatus Paraprochloron terpiosi. Comparative genomic analyses revealed that ‘Paraprochloron’ and Prochloron exhibit distinct genomic features and DNA replication machinery. We also characterized the metabolic potentials of ‘Paraprochloron terpiosi’ in carbon and nitrogen cycling and propose a model for interactions between it and T. hoshinota. This study builds a foundation for the study of the T. hoshinota microbiome and paves the way for better understanding of ecosystems involving this coral-killing sponge.     

Development and Application of Random DIG-Labelled Probes Based on Total Genomic Lactobacillus DNA

Summary Nine Lactobacillus-specific and non-isotopically (digoxygenin) labelled probes were developed on the basis of Lactobacillus total chromosomal DNA. Their specificity and applicability for Lactobacillus discrimination was proven by DNA–DNA hybridization to reference strains from the American Type Culture Collection (ATCC). The DNA probes were divided into three groups depending on the ability to hybridize to DNA from the same and/or from a group of related Lactobacillus strains. They were assayed in the species-specific detection of vaginal strains from the genus Lactobacillus. Six DNA probes were successfully applied for characterization of 21 newly isolated vaginal Lactobacilli. The species affliation of some isolates was determined. The developed DNA probes were evaluated for usage as a qualitative hybridization test for detection of Lactobacillus species in mixed cultures, obtained directly from vaginal samples without strain isolation.