Heath J. Mills

Characterization of Microbial Community Structure in Gulf of Mexico Gas Hydrates: Comparative Analysis of DNA- and RNA-Derived Clone Libraries

ABSTRACT The characterization of microbial assemblages within solid gas hydrate, especially those that may be physiologically active under in situ hydrate conditions, is essential to gain a better understanding of the effects and contributions of microbial activities in Gulf of Mexico (GoM) hydrate ecosystems. In this study, the composition of the Bacteria and Archaea communities was determined by 16S rRNA phylogenetic analyses of clone libraries derived from RNA and DNA extracted from sediment-entrained hydrate (SEH) and interior hydrate (IH). The hydrate was recovered from an exposed mound located in the northern GoM continental slope with a hydrate chipper designed for use on the manned-submersible Johnson Sea Link (water depth, 550 m). Previous geochemical analyses indicated that there was increased metabolic activity in the SEH compared to the IH layer (B. N. Orcutt, A. Boetius, S. K. Lugo, I. R. Macdonald, V. A. Samarkin, and S. Joye, Chem. Geol. 205:239-251). Phylogenetic analysis of RNA- and DNA-derived clones indicated that there was greater diversity in the SEH libraries than in the IH libraries. A majority of the clones obtained from the metabolically active fraction of the microbial community were most closely related to putative sulfate-reducing bacteria and anaerobic methane-oxidizing archaea. Several novel bacterial and archaeal phylotypes for which there were no previously identified closely related cultured isolates were detected in the RNA- and DNA-derived clone libraries. This study was the first phylogenetic analysis of the metabolically active fraction of the microbial community extant in the distinct SEH and IH layers of GoM gas hydrate.

Identification of Members of the Metabolically Active Microbial Populations Associated with Beggiatoa Species Mat Communities from Gulf of Mexico Cold-Seep Sediments

ABSTRACT In this study, the composition of the metabolically active fraction of the microbial community occurring in Gulf of Mexico marine sediments (water depth, 550 to 575 m) with overlying filamentous bacterial mats was determined. The mats were mainly composed of either orange- or white-pigmented Beggiatoa spp. Complementary 16S ribosomal DNA (crDNA) was obtained from rRNA extracted from three different sediment depths (0 to 2, 6 to 8, and 10 to 12 cm) that had been subjected to reverse transcription-PCR amplification. Domain-specific 16S PCR primers were used to construct 12 different 16S crDNA libraries containing 333 Archaea and 329 Bacteria clones. Analysis of the Archaea clones indicated that all sediment depths associated with overlying orange- and white-pigmented microbial mats were almost exclusively dominated by ANME-2 (95% of total Archaea clones), a lineage related to the methanogenic order Methanosarcinales. In contrast, bacterial diversity was considerably higher, with the dominant phylotype varying by sediment depth. An equivalent number of clones detected at 0 to 2 cm, representing a total of 93%, were related to the γ and δ classes of Proteobacteria, whereas clones related to δ-Proteobacteria dominated the metabolically active fraction of the bacterial community occurring at 6 to 8 cm (79%) and 10 to 12 cm (85%). This is the first phylogenetics-based evaluation of the presumptive metabolically active fraction of the Bacteria and Archaea community structure investigated along a sediment depth profile in the northern Gulf of Mexico, a hydrocarbon-rich cold-seep region.

Microbial diversity in sediments associated with surface-breaching gas hydrate mounds in the Gulf of Mexico

Abstract A molecular phylogenetic approach was used to characterize the composition of microbial communities from two gas hydrate sedimentary systems in the Gulf of Mexico. Nucleic acids, extracted from sediments directly overlying surface-breaching gas hydrate mounds collected from a research submersible (water depth 550-575 m), were amplified with nine different 16S rDNA gene primer sets. The polymerase chain reaction primers targeted microorganisms at the domain-specific (Bacteria and Archaea) and group-specific (sulfate-reducing bacteria (SRB) and putative anaerobic methane-oxidizing (ANME) archaea) level. Amplicons were obtained with five of the nine primer sets including two of the six SRB Groups (SRB Group 5 and Group 6) and used to generate five different clone libraries. Analysis of 126 clones from the Archaea library revealed that the sediments associated with naturally occurring gas hydrate harbored a low diversity. Sequence analysis indicated the majority of archaeal clones were most closely related to Methanosarcinales, Methanomicrobiales and distinct phylogenetic lineages within the ANME groups. The most frequently recovered phylotypes in the ANME library were related to either ANME-2 or Methanomicrobiales. In contrast to the two archaeal libraries, bacterial diversity was higher with the majority of the 126 bacterial clones most closely related to uncultured clones dominated by the delta- and epsilon-Proteobacteria. Interestingly, while 82% of the clones in the SRB Group 5 library were affiliated with delta-Proteobacteria, the vast majority (83%) of clones in the SRB Group 6 library was affiliated with the Firmicutes. This is the first phylogenetic-based description of microbial communities extant in methane-rich hydrate-associated sediments from a hydrocarbon seep region in the Gulf of Mexico.

Microbial community diversity associated with carbon and nitrogen cycling in permeable shelf sediments.

ABSTRACT Though a large fraction of primary production and organic matter cycling in the oceans occurs on continental shelves dominated by sandy deposits, the microbial communities associated with permeable shelf sediments remain poorly characterized. Therefore, in this study, we provide the first detailed characterization of microbial diversity in marine sands of the South Atlantic Bight through parallel analyses of small-subunit (SSU) rRNA gene (Bacteria), nosZ (denitrifying bacteria), and amoA (ammonia-oxidizing bacteria) sequences. Communities were analyzed by parallel DNA extractions and clone library construction from both sediment core material and manipulated sediment within column experiments designed for geochemical rate determinations. Rapid organic-matter degradation and coupled nitrification-denitrification were observed in column experiments at flow rates resembling in situ conditions over a range of oxygen concentrations. Numerous SSU rRNA phylotypes were affiliated with the phyla Proteobacteria (classes Alpha-, Delta-, and Gammaproteobacteria), Planctomycetes, Cyanobacteria, Chloroflexi, and Bacteroidetes. Detectable sequence diversity of nosZ and SSU rRNA genes increased in stratified redox-stabilized columns compared to in situ sediments, with the Alphaproteobacteria comprising the most frequently detected group. Alternatively, nitrifier communities showed a relatively low and stable diversity that did not covary with the other gene targets. Our results elucidate predominant phylotypes that are likely to catalyze carbon and nitrogen cycling in marine sands. Although overall diversity increased in response to redox stabilization and stratification in column experiments, the major phylotypes remained the same in all of our libraries, indicating that the columns sufficiently mimic in situ conditions.

Chemically mediated competition between microbes and animals: microbes as consumers in food webs.

Microbes are known to affect ecosystems and communities as decomposers, pathogens, and mutualists. However, they also may function as classic consumers and competitors with animals if they chemically deter larger consumers from using rich food-falls such as carrion, fruits, and seeds that can represent critical windfalls to both microbes and animals. Microbes often use chemicals (i.e., antibiotics) to compete against other microbes. Thus using chemicals against larger competitors might be expected and could redirect significant energy subsidies from upper trophic levels to the detrital pathway. When we baited traps in a coastal marine ecosystem with fresh vs. microbe-laden fish carrion, fresh carrion attracted 2.6 times as many animals per trap as microbe-laden carrion. This resulted from fresh carrion being found more frequently and from attracting more animals when found. Microbe-laden carrion was four times more likely to be uncolonized by large consumers than was fresh carrion. In the lab, the most common animal found in our traps (the stone crab Menippe mercenaria) ate fresh carrion 2.4 times more frequently than microbe-laden carrion. Bacteria-removal experiments and feeding bioassays using organic extracts of microbe-laden carrion showed that bacteria produced noxious chemicals that deterred animal consumers. Thus bacteria compete with large animal scavengers by rendering carcasses chemically repugnant. Because food-fall resources such as carrion are major food subsidies in many ecosystems, chemically mediated competition between microbes and animals could be an important, common, but underappreciated interaction within many communities.

Functional Diversity and Electron Donor Dependence of Microbial Populations Capable of U(VI) Reduction in Radionuclide-Contaminated Subsurface Sediments

ABSTRACT In order to elucidate the potential mechanisms of U(VI) reduction for the optimization of bioremediation strategies, the structure-function relationships of microbial communities were investigated in microcosms of subsurface materials cocontaminated with radionuclides and nitrate. A polyphasic approach was used to assess the functional diversity of microbial populations likely to catalyze electron flow under conditions proposed for in situ uranium bioremediation. The addition of ethanol and glucose as supplemental electron donors stimulated microbial nitrate and Fe(III) reduction as the predominant terminal electron-accepting processes (TEAPs). U(VI), Fe(III), and sulfate reduction overlapped in the glucose treatment, whereas U(VI) reduction was concurrent with sulfate reduction but preceded Fe(III) reduction in the ethanol treatments. Phyllosilicate clays were shown to be the major source of Fe(III) for microbial respiration by using variable-temperature Mössbauer spectroscopy. Nitrate- and Fe(III)-reducing bacteria (FeRB) were abundant throughout the shifts in TEAPs observed in biostimulated microcosms and were affiliated with the genera Geobacter, Tolumonas, Clostridium, Arthrobacter, Dechloromonas, and Pseudomonas. Up to two orders of magnitude higher counts of FeRB and enhanced U(VI) removal were observed in ethanol-amended treatments compared to the results in glucose-amended treatments. Quantification of citrate synthase (gltA) levels demonstrated a stimulation of Geobacteraceae activity during metal reduction in carbon-amended microcosms, with the highest expression observed in the glucose treatment. Phylogenetic analysis indicated that the active FeRB share high sequence identity with Geobacteraceae members cultivated from contaminated subsurface environments. Our results show that the functional diversity of populations capable of U(VI) reduction is dependent upon the choice of electron donor.

Characterization of Nitrifying, Denitrifying, and Overall Bacterial Communities in Permeable Marine Sediments of the Northeastern Gulf of Mexico

ABSTRACT Sandy or permeable sediment deposits cover the majority of the shallow ocean seafloor, and yet the associated bacterial communities remain poorly described. The objective of this study was to expand the characterization of bacterial community diversity in permeable sediment impacted by advective pore water exchange and to assess effects of spatial, temporal, hydrodynamic, and geochemical gradients. Terminal restriction fragment length polymorphism (TRFLP) was used to analyze nearly 100 sediment samples collected from two northeastern Gulf of Mexico subtidal sites that primarily differed in their hydrodynamic conditions. Communities were described across multiple taxonomic levels using universal bacterial small subunit (SSU) rRNA targets (RNA- and DNA-based) and functional markers for nitrification (amoA) and denitrification (nosZ). Clonal analysis of SSU rRNA targets identified several taxa not previously detected in sandy sediments (i.e., Acidobacteria, Actinobacteria, Chloroflexi, Cyanobacteria, and Firmicutes). Sequence diversity was high among the overall bacterial and denitrifying communities, with members of the Alphaproteobacteria predominant in both. Diversity of bacterial nitrifiers (amoA) remained comparatively low and did not covary with the other gene targets. TRFLP fingerprinting revealed changes in sequence diversity from the family to species level across sediment depth and study site. The high diversity of facultative denitrifiers was consistent with the high permeability, deeper oxygen penetration, and high rates of aerobic respiration determined in these sediments. The high relative abundance of Gammaproteobacteria in RNA clone libraries suggests that this group may be poised to respond to short-term periodic pulses of growth substrates, and this observation warrants further investigation.

Introduction of a plasmid‐encoded phoA gene for constitutive overproduction of alkaline phosphatase in three subsurface Pseudomonas isolates

Abstract Three bacterial isolates, Pseudomonas fluorescens F1, Pseudomonas rhodesiae R1 and Pseudomonas veronii V1 were genetically modified by introduction of a plasmid, pJH123, with a phoA hybrid gene that directed constitutive overproduction of the enzyme alkaline phosphatase. The presence of the plasmid in the bacterial hosts elevated extracytoplasmic alkaline phosphatase production from 100- to 820-fold. The growth and survival of the plasmid-bearing hosts in sterilized soil slurries was comparable to parental control strains. In the absence of antibiotic selection, pJH123 was maintained in two of the three hosts (P. fluorescens F1 and P. veronii V1) during incubation in minimal medium. The effects of the genetically enhanced pseudomonads on the liberation of inorganic phosphate (PO(4) (3-)) were determined in sterilized soil slurries following the addition of an organophosphorus compound, glycerol-3-phosphate. A significant accumulation of PO(4) (3-) was measured in soil slurries amended with 10 mM glycerol-3-phosphate and any of the three phosphatase-enhanced pseudomonad isolates. In contrast, soil slurries containing unmodified parental strains did not exhibit significant PO(4) (3-) accumulation. Two of the three enhanced phosphate-liberating strains released sufficient PO(4) (3-) that cell-free supernatants from sterilized soil slurry incubations removed significant amounts of uranium (as much as 69%) from solution.