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PubMed2006

Sulfide oxidation coupled to arsenate reduction by a diverse microbial community in a soda lake.

We characterized the arsenate-reducing, sulfide-oxidizing population of Mono Lake, California, by analyzing the distribution and diversity of rrnA, cbbL, and dissimilatory arsenate reductase (arrA) genes in environmental DNA, arsenate-plus sulfide-amended lake water, mixed cultures, and isolates. The arsenate-reducing community was diverse. An organism represented by an rrnA sequence previously retrieved from Mono Lake and affiliated with the Desulfobulbaceae (Deltaproteobacteria) appears to be an important member of the arsenate-reducing, sulfide-oxidizing community. Sulfide oxidation coupled with arsenate reduction appears to proceed via a two-electron transfer, resulting in the production of arsenite and an intermediate S compound that is subsequently disproportionated. A realgar-like As/S mineral was formed in some experiments.

Arsenates↗
PubMed2002

Molecular community analysis of microbial diversity.

New technologies that avoid the need for either gene amplification (e.g. microarrays) or nucleic acid extraction (e.g. in situ PCR) have recently been implemented in microbial ecology. Together with new approaches for culturing microorganisms and an increased understanding of the biases of molecular methods, these techniques form the most exciting advances in this field during the past year.

Ecosystem↗
PubMed1998

Microbial diversity in a hydrocarbon- and chlorinated-solvent-contaminated aquifer undergoing intrinsic bioremediation.

A culture-independent molecular phylogenetic approach was used to survey constituents of microbial communities associated with an aquifer contaminated with hydrocarbons (mainly jet fuel) and chlorinated solvents undergoing intrinsic bioremediation. Samples were obtained from three redox zones: methanogenic, methanogenic-sulfate reducing, and iron or sulfate reducing. Small-subunit rRNA genes were amplified directly from aquifer material DNA by PCR with universally conserved or Bacteria- or Archaea-specific primers and were cloned. A total of 812 clones were screened by restriction fragment length polymorphisms (RFLP), approximately 50% of which were unique. All RFLP types that occurred more than once in the libraries, as well as many of the unique types, were sequenced. A total of 104 (94 bacterial and 10 archaeal) sequence types were determined. Of the 94 bacterial sequence types, 10 have no phylogenetic association with known taxonomic divisions and are phylogenetically grouped in six novel division level groups (candidate divisions WS1 to WS6); 21 belong to four recently described candidate divisions with no cultivated representatives (OP5, OP8, OP10, and OP11); and 63 are phylogenetically associated with 10 well-recognized divisions. The physiology of two particularly abundant sequence types obtained from the methanogenic zone could be inferred from their phylogenetic association with groups of microorganisms with a consistent phenotype. One of these sequence types is associated with the genus Syntrophus; Syntrophus spp. produce energy from the anaerobic oxidation of organic acids, with the production of acetate and hydrogen. The organism represented by the other sequence type is closely related to Methanosaeta spp., which are known to be capable of energy generation only through aceticlastic methanogenesis. We hypothesize, therefore, that the terminal step of hydrocarbon degradation in the methanogenic zone of the aquifer is aceticlastic methanogenesis and that the microorganisms represented by these two sequence types occur in syntrophic association.

Bacteria↗
PubMed2003

A study on microbial diversity in different cultivars of Brassica napus in relation to its wilt pathogen, Verticillium longisporum.

Oilseed rape (Brassica napus) is one of the major oilseed crops in the world but is vulnerable to attack by many pathogens and insect pests. In addition to the host plant genotype, micro-organisms present in the rhizosphere and within plant tissues affect the susceptibility to plant pathogens. While rapid progress has been achieved concerning the concept of plant resistance genes, information on the role of the microbial community in plant protection is less apparent. We have studied the endophytic bacterial populations present in different tissues of oilseed rape and also analysed several cultivars (Express, Libraska, Maluka and Westar), which differ in their susceptibility to the wilt pathogen Verticillium longisporum. The population diversity was studied using agar plating assay, fatty acid methyl ester analysis and functional characterisation of isolated strains. Our work shows that already in the seeds there exists diversity in populations as well as in the total microbial load between two of the four tested cultivars. About 50% of the strains isolated from cultivars Express and Libraska showed moderate to strong direct inhibition of V. longisporum. The diversity of the endophytic flora isolated from oilseed rape and its implications in crop protection are discussed.

Alcaligenes↗
PubMed2004

Microbial diversity of extant stromatolites in the hypersaline marine environment of Shark Bay, Australia.

Stromatolites have been present on Earth, at various levels of distribution and diversity, for more than 3 billion years. Today, the best examples of stromatolites forming in hypersaline marine environments are in Hamelin Pool at Shark Bay, Western Australia. Despite their evolutionary significance, little is known about their associated microbial communities. Using a polyphasic approach of culture-dependent and culture-independent methods, we report the discovery of a wide range of microorganisms associated with these biosedimentary structures. There are no comparable reports combining these methodologies in the survey of cyanobacteria, bacteria, and archaea in marine stromatolites. The community was characterized by organisms of the cyanobacterial genera Synechococcus, Xenococcus, Microcoleus, Leptolyngbya, Plectonema, Symploca, Cyanothece, Pleurocapsa and Nostoc. We also report the discovery of potentially free-living Prochloron. The other eubacterial isolates and clones clustered into seven phylogenetic groups: OP9, OP10, Marine A group, Proteobacteria, Low G+C Gram-positive, Planctomycetes and Acidobacteria. We also demonstrate the presence of sequences corresponding to members of halophilic archaea of the divisions Euryarchaeota and Crenarchaeota and methanogenic archaea of the order Methanosarcinales. This is the first report of such archaeal diversity from this environment. This study provides a better understanding of the microbial community associated with these living rocks.

Archaea↗
PubMed2006

The microbial diversity of inland waters.

The conservation and sustainable use of freshwater resources is of global importance. Microorganisms are not only the most abundant organisms in natural freshwater systems, but are also key players in ecological processes controlling water quality. Detailed knowledge of the diversity and function of microorganisms dwelling in freshwater habitats is an essential prerequisite for the sustainable management of freshwater resources. Freshwater systems are inhabited by microbial communities that are indigenous to this habitat type and usually do not occur in marine systems, saline inland waters and terrestrial habitats. Despite recent advances in the characterization of the diversity of freshwater microorganisms, knowledge essential for a holistic understanding of their ecological roles is still lacking.

Adaptation, Physiological↗
PubMed2006

Microbial diversity and bioremediation of a hydrocarbon-contaminated aquifer (Vega Baja, Puerto Rico).

Hydrocarbon contamination of groundwater resources has become a major environmental and human health concern in many parts of the world. Our objectives were to employ both culture and culture-independent techniques to characterize the dynamics of microbial community structure within a fluidized bed reactor used to bioremediate a diesel-contaminated groundwater in a tropical environment. Under normal operating conditions, 97 to 99% of total hydrocarbons were removed with only 14 min hydraulic retention time. Over 25 different cultures were isolated from the treatment unit (96% which utilized diesel constituents as sole carbon source). Approximately 20% of the isolates were also capable of complete denitrification to nitrogen gas. Sequence analysis of 16S rDNA demonstrated ample diversity with most belonging to the infinity, beta and gamma subdivision of the Proteobacteria, Bacilli, and Actinobacteria groups. Moreover, the genetic constitution of the microbial community was examined at multiple time points with a Functional Gene Array (FGA) containing over 12,000 probes for genes involved in organic degradation and major biogeochemical cycles. Total community DNA was extracted and amplified using an isothermal phi29 polymerase-based technique, labeled with Cy5 dye, and hybridized to the arrays in 50% formimide overnight at 50 degrees C. Cluster analysis revealed comparable profiles over the course of treatment suggesting the early selection of a very stable microbial community. A total of 270 genes for organic contaminant degradation (including naphthalene, toluene [aerobic and anaerobic], octane, biphenyl, pyrene, xylene, phenanthrene, and benzene); and 333 genes involved in metabolic activities (nitrite and nitrous oxide reductases [nirS, nirK, and nosZ], dissimilatory sulfite reductases [dsrAB], potential metal reducing C-type cytochromes, and methane monooxygenase [pmoA]) were repeatedly detected. Genes for degradation of MTBE, nitroaromatics and chlorinated compounds were also present, indicating a broad catabolic potential of the treatment unit. FGA's demonstrated the early establishment of a diverse community with concurrent aerobic and anaerobic processes contributing to the bioremediation process.

Bacteria↗
PubMed2000

Molecular techniques for determining microbial diversity and community structure in natural environments.

The ability to quantify the number and kinds of microorganisms within a community is fundamental to the understanding of the structure and function of an ecosystem. The simple morphology of most microbes provides few clues for their identification and physiological traits are often ambiguous. In addition, many organisms resist cultivation, which is essential to their characterization. Recombinant DNA techniques have provided a means whereby many of the obstacles associated with cultivation and description can be overcome and subsequently has allowed many new insights into the complexity of natural microbial communities. Molecular approaches based on 16S ribosomal RNA (rRNA) sequence analysis allow direct investigation of the community structure, diversity, and phylogeny of microorganisms in almost any environment, while quantification of the individual types of microorganisms or entire microbial communities may be addressed by nucleic acid hybridization techniques. Furthermore, the use of fluorescently labeled population-specific rRNA probes allows microscopic examination of individual cells in complex microbial assemblages as well as their interactions in situ. In this review, we discuss strategies for characterizing microbial communities without the need for cultivation.

DNA, Ribosomal↗
PubMed2004

Microbial diversity of soil from two hot springs in Uttaranchal Himalaya.

Soil samples collected from two hot springs, Soldhar and Ringigad, both located in the Garhwal region of Uttaranchal Himalaya were analysed for their physical, chemical and microbial components. The alkaline pH, total absence of carbon and nitrogen, and high temperature were features common to soil samples from both sites. The Soldhar samples contained higher amounts of Cu, Fe and Mn. Ringigad soil was devoid of Cu, but had much higher phosphate. While the optimum incubation temperature for isolating the maximum microbial counts from soil samples from the two sites was 50 degrees C, microbial growth in broth was also observed when incubated at 80 degrees C. Microscopic examination revealed three types of microbial populations, i.e., bacteria, yeast and filamentous organisms. The soil samples were found to be dominated by spore forming rods. Out of 58 aerobic isolates, 53 were gram positive bacilli. Gram positive anaerobic oval rods were also observed up to 60 degrees C. Soil dilution plates revealed the presence of antagonistic and phosphate solubilizing populations.

Bacteria, Aerobic↗
PubMed2005

Identification of diverse microbial metabolites as potent inhibitors of HIV-1 Tat transactivation.

HIV-1 Tat is one of six regulatory proteins that are required for viral replication and is an attractive target for the development of new anti-HIV agents. Screening of microbial extracts using a whole cell Tat-dependent transactivation assay, which guided the separation of the active broths, led to the identification of five structurally diverse classes (M(R) range 232-1126) of natural products. These include i) three sesquiterpenoids, namely, sporogen-AO1, petasol, and 6-dehydropetasol, ii) two resorcylic 14-membered lactones, namely monorden and monocillin IV, iii) a ten-membered lactone, iv) a quinoline and quinoxiline bicyclic octadepsipeptides, namely echinomycin and UK-63598, and v) a cyclic heptapeptide, ternatin. These compounds displayed varying degrees of potencies with IC50 values ranging from 0.0002 to 100 microM. The most active compound was the quinoxiline bicyclic octadepsipeptides, UK-63598, which inhibited Tat-dependent transactivation with an IC50 value of 0.2 nM and exhibited a 100-fold therapeutic window with respect to toxicity. In a single-cycle antiviral assay, UK-6358 inhibited viral replication with an IC50 value of 0.5 nM; however, it appeared to be equally toxic at that concentration. Monocillin IV was significantly less active (Tat transactivation inhibitory IC50 of 5 microM) but was not toxic at 100 microM in an equivalent cytotoxicity assay. The compound exhibited antiviral activity with an IC50 value of 6.2 microM in the single-cycle antiviral assay and a sixfold therapeutic window. Details of the isolation, fermentation, and biological activities of these structurally diverse natural products are described.

Anti-HIV Agents↗
PubMed2001

Microbial diversity in hot synthetic compost as revealed by PCR-amplified rRNA sequences from cultivated isolates and extracted DNA.

High-temperature (>/=60 degrees C) synthetic food waste compost was examined by cultivation-dependent and -independent methods to determine predominant microbial populations. Fluorescent direct counts totaled 6.4 (+/-2.5)x10(10) cells gdw(-1) in a freeze-dried 74 degrees C compost sample, while plate counts for thermophilic heterotrophic aerobes averaged 2.6 (+/-1.0)x10(8) CFU gdw(-1). A pre-lysis cell fractionation method was developed to obtain community DNA and a suite of 16S and 18S rDNA-targeted PCR primers was used to examine the presence of Bacteria, Archaea and fungi. Bacterial 16S rDNA, including a domain-specific 1500-bp fragment and a 300-bp fragment specific for Actinobacteria, was amplified by PCR from all compost samples tested. Archaeal rDNA was not amplified in any sample. Fungal 18S rDNA was only amplified from a separate dairy manure compost that reached a peak temperature of 50 degrees C. Amplified rDNA restriction analysis (ARDRA) was used to screen isolated thermophilic bacteria and a clone library of full-length rDNA fragments. ARDRA screening revealed 14 unique patterns among 63 isolates, with one pattern accounting for 31 of the isolates. In the clone library, 52 unique patterns were detected among 70 clones, indicating high diversity of uncultivated bacteria in hot compost. Phylogenetic analysis revealed that the two most abundant isolates belonged in the genera Aneurinibacillus and Brevibacillus, which are not commonly associated with hot compost. With the exception of one Lactobacillus-type sequence, the clone library contained only sequences that clustered within the genus Bacillus. None of the isolates or cloned sequences could be assigned to the group of obligate thermophilic Bacillus spp. represented by B. stearothermophilus, commonly believed to dominate high-temperature compost. Amplified partial fragments from Actinobacteria, spanning the V3 variable region (Neefs et al. (1990) Nucleic Acids Res. 18, 2237-2242), included sequences related to the genera Saccharomonospora, Gordonia, Rhodococcus and Corynebacterium, although none of these organisms were detected among the isolates or full-length cloned rDNA sequences. All of the thermophilic isolates and sequenced rDNA fragments examined in this study were from Gram-positive organisms.

Journal Article↗
PubMed2006

Changes in microbial diversity in fed-batch reactor operation with wastewater containing nitroaromatic residues.

This study targeted the diversity of bacteria isolated from activated biomass of an ETP that treated wastewater from the dye industry. Phylogenetic analysis of 5'-partial 16S rDNA sequences revealed that the Proteobacteria (alpha, beta, and gamma) and Firmicutes were the major phyla in microbial community. Five unidentified culturable bacteria were also isolated. The activated biomass changed when acclimatized to wastewater with nitroaromatic residues. It was observed that the diversity of genera was reduced, but the degradative capacity of sludge was enhanced.

Base Sequence↗
PubMed2006

Phenotypic properties and microbial diversity of methanogenic granules from a full-scale upflow anaerobic sludge bed reactor treating brewery wastewater.

Methanogenic granules from an anaerobic bioreactor that treated wastewater of a beer brewery consisted of different morphological types of granules. In this study, the microbial compositions of the different granules were analyzed by molecular microbiological techniques: cloning, denaturing gradient gel electrophoresis and fluorescent in situ hybridization (FISH), and scanning and transmission electron microscopy. We propose here that the different types of granules reflect the different stages in the life cycle of granules. Young granules were small, black, and compact and harbored active cells. Gray granules were the most abundant granules. These granules have a multilayer structure with channels and void areas. The core was composed of dead or starving cells with low activity. The brown granules, which were the largest granules, showed a loose and amorphous structure with big channels that resulted in fractured zones and corresponded to the older granules. Firmicutes (as determined by FISH) and Nitrospira and Deferribacteres (as determined by cloning and sequencing) were the predominant Bacteria. Remarkably, Firmicutes could not be detected in the brown granules. The methanogenic Archaea identified were Methanosaeta concilii (70 to 90% by FISH and cloning), Methanosarcina mazei, and Methanospirillum spp. The phenotypic appearance of the granules reflected the physiological condition of the granules. This may be valuable to easily select appropriate seed sludges to start up other reactors.

Anaerobiosis↗
PubMed2006

Microbial diversity in natural environments: focusing on fundamental questions.

Interactions with Gijs Kuenen and other Dutch scientists have led my lab to fundamental insights into the composition, structure and function of a hot spring cyanobacterial mat community that should influence our thinking about all microbial communities. By focusing on the distribution of molecular sequence variants of predominant mat phototrophs, we have discovered that small-scale sequence variation can be ecologically meaningful. By applying novel cultivation approaches, we have been able to obtain genetically relevant community members and thus to test the hypothesis that closely related sequence variants arose via adaptive evolutionary radiation. By applying the analytical tools of organic geochemistry we have gained insight into the metabolisms of major phototrophic members of the mat community as well as interactions between phototrophic guilds. These observations challenge traditional paradigms about prokaryotic species and cause us to consider evolutionary ecology theory as we develop genome-based methods for high-resolution analysis of the species-like fundamental units comprising microbial communities, and for investigating how such units coordinate the physiological activities within guilds of the community.

Autotrophic Processes↗
PubMed2005

Vaginal microbial diversity among postmenopausal women with and without hormone replacement therapy.

Urogenital infections in postmenopausal women remain problematic. The use of estrogen replacement therapy has been shown to lower these infection rates, corresponding to increasing colonization by Lactobacillus species. Despite the gut's 500 microbial species and the proximity of the anus to the vagina, only a relatively few microbial strains appear to be able to colonize the urogenital area. In the present study, the sparsity of microbes in the vagina was confirmed by denaturing gradient gel electrophoresis analysis of swabs taken at time zero and monthly for 3 months from 40 postmenopausal subjects receiving Premarin (conjugated equine estrogen in combination with progesterone) hormone replacement therapy (HRT) and 20 who were not on HRT. Lactobacilli were recovered from the vagina of 95% or more women in both groups, but in the HRT group, Lactobacillus were more often the dominant and only colonizers and significantly fewer bacteria with pathogenic potential were found. The incidence of bacterial vaginosis was significantly lower in the HRT group than in the non-HRT-treated women (5.6% versus 31%). The use of HRTs has recently come under criticism. The ability of drugs such as Premarin to help recover the lactobacilli vaginal microbiota appears to be at least one benefit of HRT use. In women not using HRTs, use of probiotics may be the only way to restore a nonpathogen-dominated flora.

Adult↗
PubMed1998

Novel techniques for analysing microbial diversity in natural and perturbed environments.

Molecular techniques were applied for analysing the entire bacterial community, including both the cultivated and non-cultivated part of the community. DNA was extracted from samples of soils and sediments, and a combination of different molecular methods were used to investigate community structure and diversity in these environments. Reassociation of sheared and thermally denatured DNA in solution was used to measure the total genetical diversity. PCR-denaturing gradient gel electrophoresis (DGGE) analysis of rRNA genes gave information about changes in the numerically dominating bacterial populations. Hybridisation with phylogenetic group specific probes, and sequencing provided information about the affiliation of the bacterial populations. Using DNA reassociation analysis we demonstrated that bacterial communities in pristine soil and sediments may contain more than 10,000 different bacterial types. The diversity of the total soil community was at least 200 times higher than the diversity of bacterial isolates from the same soil. This indicates that the culturing conditions select for a distinct subpopulation of the bacteria present in the environment. Molecular methods were applied to monitor the effects of perturbations due to antropogenic activities and pollution on microbial communities. Our investigations show that agricultural management, fish farming and pollution may lead to profound changes in the community structure and a reduction in the bacterial diversity.

Bacteria↗
PubMed2005

New method to characterize microbial diversity using flow cytometry.

The majority of microorganisms have yet to be cultivated and represent a vast uncharacterized and untapped resource. Here, we report the utilization of a combination of flow cytometry, cultivation, and molecular genetics to develop new methodologies to access and characterize biodiversity in microbial samples. We demonstrate that fluorescent dyes and combinations of dyes can selectively stain portions of bacterial populations that can be isolated as sub-populations using fluorescence-activated cell sorting (FACS). Microbial sub-populations obtained by FACS differ substantially from the original microbial population, as demonstrated by denaturing gradient gel electrophoresis and determination of 16S rRNA gene sequences. These sub-populations can subsequently be used to inoculate microbial growth media, allowing the isolation of different microbial species from those that can be readily cultivated from the original sample using the same microbial growth media. When this technique was applied to the analysis of activated-sludge and Yellowstone Lake hydrothermal vent samples, comparative analysis of 16S rDNA sequences revealed that FACS allowed the detection of numerous bacterial species, including previously unknown species, not readily detectable in the original sample due to low relative abundance. This approach may result in a convenient methodology to more thoroughly characterize microbial biodiversity.

Bacteria↗