What's New Is Old: Resolving the Identity of Leptothrix ochracea Using Single Cell Genomics, Pyrosequencing and FISH

Leptothrix ochracea is a common inhabitant of freshwater iron seeps and iron-rich wetlands. Its defining characteristic is copious production of extracellular sheaths encrusted with iron oxyhydroxides. Surprisingly, over 90% of these sheaths are empty, hence, what appears to be an abundant population of iron-oxidizing bacteria, consists of relatively few cells. Because L. ochracea has proven difficult to cultivate, its identification is based solely on habitat preference and morphology. We utilized cultivation-independent techniques to resolve this long-standing enigma. By selecting the actively growing edge of a Leptothrix-containing iron mat, a conventional SSU rRNA gene clone library was obtained that had 29 clones (42% of the total library) related to the Leptothrix/Sphaerotilus group (≤96% identical to cultured representatives). A pyrotagged library of the V4 hypervariable region constructed from the bulk mat showed that 7.2% of the total sequences also belonged to the Leptothrix/Sphaerotilus group. Sorting of individual L. ochracea sheaths, followed by whole genome amplification (WGA) and PCR identified a SSU rRNA sequence that clustered closely with the putative Leptothrix clones and pyrotags. Using these data, a fluorescence in-situ hybridization (FISH) probe, Lepto175, was designed that bound to ensheathed cells. Quantitative use of this probe demonstrated that up to 35% of microbial cells in an actively accreting iron mat were L. ochracea. The SSU rRNA gene of L. ochracea shares 96% homology with its closet cultivated relative, L. cholodnii, This establishes that L. ochracea is indeed related to this group of morphologically similar, filamentous, sheathed microorganisms

Two naphthalene degrading bacteria belonging to the genera Paenibacillus and Pseudomonas isolated from a highly polluted lagoon perform different sensitivities to the organic and heavy metal contaminants

Two bacterial strains were isolated in the presence of naphthalene as the sole carbon and energy source from sediments of the Orbetello Lagoon, Italy, which is highly contaminated with both organic compounds and metals. 16S rRNA gene sequence analysis of the two isolates assigned the strains to the genera Paenibacillus and Pseudomonas. The effect of different contaminants on the growth behaviors of the two strains was investigated. Pseudomonas sp. ORNaP2 showed a higher tolerance to benzene, toluene, and ethylbenzene than Paenibacillus sp. ORNaP1. In addition, the toxicity of heavy metals potentially present as co-pollutants in the investigated site was tested. Here, strain Paenibacillus sp. ORNaP1 showed a higher tolerance towards arsenic, cadmium, and lead, whereas it was far more sensitive towards mercury than strain Pseudomonas sp. ORNaP2. These differences between the Gram-negative Pseudomonas and the Gram-positive Paenibacillus strain can be explained by different general adaptive response systems present in the two bacteria

Microbial life in the deep terrestrial subsurface

The distribution and function of microorganisms is a vital issue in microbial ecology. The US Department of Energy`s Program, ``Microbiology of the Deep Subsurface,`` concentrates on establishing fundamental scientific information about organisms at depth, and the use of these organisms for remediation of contaminants in deep vadose zone and groundwater environments. This investigation effectively extends the Biosphere hundreds of meters into the Geosphere and has implications to a variety of subsurface activities

Nutrient availability as a control of the abundance and activities of deep subsurface microorganisms. Final report, June 30, 1991--June 30, 1992

The focus of our research is to determine whether the availability of major nutrients (C, N, P) control the abundance, distribution, and activity of heterotrophic microorganisms in subsurface sediments. Specifically we have developed and are testing a microcosm system using subsurface sediment samples from a DOE site. We are using this system to conduct a preliminary nutrient enrichment experiments. The flow-through microcosm system simulates conditions in natural aquifers and can be regulated with respect to water flow rate, temperature, oxygen concentration, and nutrient concentration. Furthermore, the system cam be run aseptically, excluding exogenous bacteria from test cores. In our preliminary nutrient enrichment experiment, we collected deep vadose sediment from the Savannah River Site and pumped sterilized well water from sampling area through microcosms that were filled with this sediment. Experimental treatments included the flow-through of this water amen with different combinations of nutrients (+N+P, +C, +N+P+C) or no additional nutrients. We found that asepsis was maintained for the entire 30 day experiment. We also found that microbial abundance, biomass, and activity increased in several microcosms in response to the flow through of the well water, which was rich in nitrate. This response, however, was variable among both microcosms containing intact sediments and microcosms containing intact sediment core sections. No significant treatment responses were observed, but each microcosm that had an increase in microbial growth also immobilized N from the well water. We infer that water and N availability, and water table fluctuations strongly influence microbial growth and activity in the deep vadose