Stephanie A. Connon

Bacterial diversity in hyperarid Atacama Desert soils

[1] Surface and subsurface soil samples analyzed for this investigation were collected from the hyperarid Yungay region in the Atacama Desert, Chile. This report details the bacterial diversity derived from DNA and PLFA extracted directly from these extremely desiccated soils. Actinobacteria, Proteobacteria, Firmicutes and TM7 division bacteria were detected. Ninety-four percent of the 16S rRNA genes cloned from these soils belong to the Actinobacteria phylum, and the majority of these were most closely related to the genus Frankia. A 24-hour water activity (a w ) time course showed a diurnal cycle that peaked at 0.52 in the early predawn hours, and ranged from 0.01-0.08 during the day. All measured water activity values were below the levels required for microbial growth or enzyme activity. Total organic carbon (TOC) concentrations were above the limit of detection and below the limit of quantification (i.e., 200 μg/g < TOC < 1000 μg/g), and phospholipid fatty acid (PLFA) concentrations ranged from 2 x 105 to 7 x 10 cell equivalents per gram of soil. Soil extracts analyzed for culturable biomass yielded mostly no growth on R2A media; the highest single extract yielded 47 colony forming units (CFU) per gram of soil.

Visualizing in situ translational activity for identifying and sorting slow-growing archaeal−bacterial consortia

Significance One of the biggest challenges in environmental microbiology is to determine the activity of uncultured cells directly in their habitat. We report on the application of bioorthogonal noncanonical amino acid tagging (BONCAT), a high-throughput approach to detecting protein synthesis in individual cells by fluorescence staining, on deep-sea methane seep sediments. By combining BONCAT with fluorescence in situ hybridization, we visualized active archaeal−bacterial consortia catalyzing the anaerobic oxidation of methane. We further developed a novel approach that combines BONCAT with fluorescence-activated cell sorting (FACS) to separate translationally active cells from complex samples. BONCAT-FACS enabled us to directly link the identities of anaerobic methane-oxidizing archaea with their partner bacteria for individual active consortia, uncovering previously unknown interactions between these archaea and Verrucomicrobia. To understand the biogeochemical roles of microorganisms in the environment, it is important to determine when and under which conditions they are metabolically active. Bioorthogonal noncanonical amino acid tagging (BONCAT) can reveal active cells by tracking the incorporation of synthetic amino acids into newly synthesized proteins. The phylogenetic identity of translationally active cells can be determined by combining BONCAT with rRNA-targeted fluorescence in situ hybridization (BONCAT-FISH). In theory, BONCAT-labeled cells could be isolated with fluorescence-activated cell sorting (BONCAT-FACS) for subsequent genetic analyses. Here, in the first application, to our knowledge, of BONCAT-FISH and BONCAT-FACS within an environmental context, we probe the translational activity of microbial consortia catalyzing the anaerobic oxidation of methane (AOM), a dominant sink of methane in the ocean. These consortia, which typically are composed of anaerobic methane-oxidizing archaea (ANME) and sulfate-reducing bacteria, have been difficult to study due to their slow in situ growth rates, and fundamental questions remain about their ecology and diversity of interactions occurring between ANME and associated partners. Our activity-correlated analyses of >16,400 microbial aggregates provide the first evidence, to our knowledge, that AOM consortia affiliated with all five major ANME clades are concurrently active under controlled conditions. Surprisingly, sorting of individual BONCAT-labeled consortia followed by whole-genome amplification and 16S rRNA gene sequencing revealed previously unrecognized interactions of ANME with members of the poorly understood phylum Verrucomicrobia. This finding, together with our observation that ANME-associated Verrucomicrobia are found in a variety of geographically distinct methane seep environments, suggests a broader range of symbiotic relationships within AOM consortia than previously thought.

Microbial abundance and diversity patterns associated with sediments and carbonates from the methane seep environments of Hydrate Ridge, OR

Methane seeps are among the most productive habitats along continental margins, as anaerobic methane-oxidizing euryarchaeaota and sulfur-metabolizing deltaproteobacteria form the biological base of a dynamic deep-sea ecosystem. The degree of methane seepage therefore represents one important variable in ecosystem dynamics, and the recent discovery of carbonate-hosted endolithic methanotrophy exposes another potentially discriminating factor: physical substrate type. Methanotrophic microbial communities have been detected within diverse seep-associated habitats, including unlithified sediments, protolithic carbonate nodules, and lithified carbonate slabs and chemoherms of distinct mineralogies. However, a systematic assessment of the diversity and community structure associated with these different habitats has been lacking. In this study, microbial aggregate analysis, microbial abundance quantification, mineralogical identification, and archaeal and bacterial 16S rRNA gene clone libraries were used to deconvolve the relationships between seepage activity, substrate type, and microbial community structure. We report prevalent methane-oxidizing archaeal lineages in both active and low-activity seep settings, and a strong community dependence on both seepage activity and substrate type. Statistical treatments of relative taxa abundances indicate that archaeal community structure is more dependent on the degree of methane seepage than physical substrate type; bacterial assemblages appear to be more strongly influenced by the type of colonization substrate than seepage activity. These findings provide a window into the determinants of community structure and function, improving our understanding of potential elemental cycling at seep sites.

Activity and interactions of methane seep microorganisms assessed by parallel transcription and FISH-NanoSIMS analyses

To characterize the activity and interactions of methanotrophic archaea (ANME) and Deltaproteobacteria at a methane-seeping mud volcano, we used two complimentary measures of microbial activity: a community-level analysis of the transcription of four genes (16S rRNA, methyl coenzyme M reductase A (mcrA), adenosine-5′-phosphosulfate reductase α-subunit (aprA), dinitrogenase reductase (nifH)), and a single-cell-level analysis of anabolic activity using fluorescence in situ hybridization coupled to nanoscale secondary ion mass spectrometry (FISH-NanoSIMS). Transcript analysis revealed that members of the deltaproteobacterial groups Desulfosarcina/Desulfococcus (DSS) and Desulfobulbaceae (DSB) exhibit increased rRNA expression in incubations with methane, suggestive of ANME-coupled activity. Direct analysis of anabolic activity in DSS cells in consortia with ANME by FISH-NanoSIMS confirmed their dependence on methanotrophy, with no 15NH4+ assimilation detected without methane. In contrast, DSS and DSB cells found physically independent of ANME (i.e., single cells) were anabolically active in incubations both with and without methane. These single cells therefore comprise an active ‘free-living’ population, and are not dependent on methane or ANME activity. We investigated the possibility of N2 fixation by seep Deltaproteobacteria and detected nifH transcripts closely related to those of cultured diazotrophic Deltaproteobacteria. However, nifH expression was methane-dependent. 15N2 incorporation was not observed in single DSS cells, but was detected in single DSB cells. Interestingly, 15N2 incorporation in single DSB cells was methane-dependent, raising the possibility that DSB cells acquired reduced 15N products from diazotrophic ANME while spatially coupled, and then subsequently dissociated. With this combined data set we address several outstanding questions in methane seep microbial ecosystems and highlight the benefit of measuring microbial activity in the context of spatial associations.

Intragenomic heterogeneity of the 16S rRNA gene in strain UFO1 caused by a 100‐bp insertion in helix 6

Two different versions of the 16S rRNA gene, one of which contained an unusual 100-bp insertion in helix 6, were detected in isolate UFO1 acquired from the Oak Ridge Integrated Field-Research Challenge (ORIFRC) site in Tennessee. rRNA was extracted from UFO1 and analyzed by reverse transcriptase-quantitative PCR with insert- and non-insert-specific primers; only the noninsert 16S rRNA gene sequence was detected. Similarly, PCR-based screening of a cDNA library (190 clones) constructed from reverse-transcribed rRNA from UFO1 did not detect any clones containing the 100-bp insert. Examination of cDNA with primers specific to the insert-bearing 16S rRNA gene, but downstream of the insert, suggests that the insert was excised from rRNA. Inspection of other 16S rRNA genes in the GenBank database revealed that a homologous insert sequence, also found in helix 6, has been reported in other environmental clones, including those acquired from ORIFRC enrichments. These findings demonstrate the existence of widely divergent copies of the 16S rRNA gene within the same organism, which may confound 16S rRNA gene-based methods of estimating microbial diversity in environmental samples.

Detection and Viability Assessment of Endospore-Forming Pathogens

In this chapter, we explore technology developments for the rapid detection, identification, and viability assessment of endospore-forming pathogens with a focus on Bacillus anthracis. First, we introduce various toxin-producing species and their role as bioinsecticides, probiotics, and bioweapons. We also review the role of endospores as biological indicators (i.e., dosimeters) for evaluating sterilization regimens, such as autoclaving and wastewater remediation. Monitoring the effectiveness of cleaning and sterilization regimens to maintain good hygiene is required in several major industries, including health care, food, and pharmaceutical industries. In the next section, we review recent developments in DNA-, immuno-, and dipicolinic acid assays, and their applications for detection and monitoring of Bacillus anthracis and other endospore-forming pathogens. Finally, we review viability assays capable of rapid validation of endospore inactivation after sterilization, including assays based on ATP synthesis during stage II germination, and DPA release during stage I germination.

Metal Transformation by a Novel Pelosinus Isolate From a Subsurface Environment

The capability of microorganisms to alter metal speciation offers potential for the development of new strategies for immobilization of toxic metals in the environment. A metal-reducing microbe, “Pelosinus lilae” strain UFO1, was isolated under strictly anaerobic conditions from an Fe(III)-reducing enrichment established with uncontaminated soil from the Department of Energy Oak Ridge Field Research Center, Tennessee. “P. lilae” UFO1 is a rod-shaped, spore-forming, and Gram-variable anaerobe with a fermentative metabolism. It is capable of reducing the humic acid analog anthraquinone-2,6-disulfonate (AQDS) using a variety of fermentable substrates and H2. Reduction of Fe(III)-nitrilotriacetic acid occurred in the presence of lactate as carbon and electron donor. Ferrihydrite was not reduced in the absence of AQDS. Nearly complete reduction of 1, 3, and 5 ppm Cr(VI) occurred within 24 h in suspensions containing 108 cells mL−1 when provided with 10 mM lactate; when 1 mM AQDS was added, 3 and 5 ppm Cr(VI) were reduced to 0.1 ppm within 2 h. Strain UFO1 is a novel species within the bacterial genus Pelosinus, having 98.16% 16S rRNA gene sequence similarity with the most closely related described species, Pelosinus fermentans R7T. The G+C content of the genomic DNA was 38 mol%, and DNA-DNA hybridization of “P. lilae” UFO1 against P. fermentans R7T indicated an average 16.8% DNA-DNA similarity. The unique phylogenetic, physiologic, and metal-transforming characteristics of “P. lilae” UFO1 reveal it is a novel isolate of the described genus Pelosinus.

Widespread nitrogen fixation in sediments from diverse deep‐sea sites of elevated carbon loading

Nitrogen fixation, the biological conversion of N2 to NH3 , is critical to alleviating nitrogen limitation in many marine ecosystems. To date, few measurements exist of N2 fixation in deep-sea sediments. Here, we conducted > 400 bottle incubations with sediments from methane seeps, whale falls and background sites off the western coast of the United States from 600 to 2893 m water depth to investigate the potential rates, spatial distribution and biological mediators of benthic N2 fixation. We found that N2 fixation was widespread, yet heterogeneously distributed with sediment depth at all sites. In some locations, rates exceeded previous measurements by > 10×, and provided up to 30% of the community anabolic growth requirement for nitrogen. Diazotrophic activity appeared to be inhibited by pore water ammonium: N2 fixation was only observed if incubation ammonium concentrations were ≤ 25 μM, and experimental additions of ammonium reduced diazotrophy. In seep sediments, N2 fixation was dependent on CH4 and coincident with sulphate reduction, consistent with previous work showing diazotrophy by microorganisms mediating sulphate-coupled methane oxidation. However, the pattern of diazotrophy was different in whale-fall and associated reference sediments, where it was largely unaffected by CH4 , suggesting catabolically different diazotrophs at these sites.