Temporal Dynamics of In-Field Bioreactor Populations Reflect the Groundwater System and Respond Predictably to Perturbation.

Temporal variability complicates testing the influences of environmental variability on microbial community structure and thus function. An in-field bioreactor system was developed to assess oxic versus anoxic manipulations on in situ groundwater communities. Each sample was sequenced (16S SSU rRNA genes, average 10,000 reads), and biogeochemical parameters are monitored by quantifying 53 metals, 12 organic acids, 14 anions, and 3 sugars. Changes in dissolved oxygen (DO), pH, and other variables were similar across bioreactors. Sequencing revealed a complex community that fluctuated in-step with the groundwater community and responded to DO. This also directly influenced the pH, and so the biotic impacts of DO and pH shifts are correlated. A null model demonstrated that bioreactor communities were driven in part not only by experimental conditions but also by stochastic variability and did not accurately capture alterations in diversity during perturbations. We identified two groups of abundant OTUs important to this system; one was abundant in high DO and pH and contained heterotrophs and oxidizers of iron, nitrite, and ammonium, whereas the other was abundant in low DO with the capability to reduce nitrate. In-field bioreactors are a powerful tool for capturing natural microbial community responses to alterations in geochemical factors beyond the bulk phase.

Mercury methylation by novel microorganisms from new environments.

Microbial mercury (Hg) methylation transforms a toxic trace metal into the highly bioaccumulated neurotoxin methylmercury (MeHg). The lack of a genetic marker for microbial MeHg production has prevented a clear understanding of Hg-methylating organism distribution in nature. Recently, a specific gene cluster (hgcAB) was linked to Hg methylation in two bacteria.1 Here we test if the presence of hgcAB orthologues is a reliable predictor of Hg methylation capability in microorganisms, a necessary confirmation for the development of molecular probes for Hg-methylation in nature. Although hgcAB orthologues are rare among all available microbial genomes, organisms are much more phylogenetically and environmentally diverse than previously thought. By directly measuring MeHg production in several bacterial and archaeal strains encoding hgcAB, we confirmed that possessing hgcAB predicts Hg methylation capability. For the first time, we demonstrated Hg methylation in a number of species other than sulfate- (SRB) and iron- (FeRB) reducing bacteria, including methanogens, and syntrophic, acetogenic, and fermentative Firmicutes. Several of these species occupy novel environmental niches for Hg methylation, including methanogenic habitats such as rice paddies, the animal gut, and extremes of pH and salinity. Identification of these organisms as Hg methylators now links methylation to discrete gene markers in microbial communities.

Association of the genus Acinetobacter with the decomposition of a swine carcass and the isolation and characterization of a novel strain of Acinetobacter sp. P4.

Using fluorescence in situ hybridization (FISH) and a selective and differential medium, Acinetobacter numbers were enumerated over the time course of decomposition, from fresh to putrid/dry, of a swine carcass. In addition, Acinetobacter diversity and succession were also characterized. Acinetobacter bacterial counts were observed to be the lowest before exposure (undetectable) and increased to their highest during active decay then decreased and leveled during advanced decay through putrid/dry. FISH analysis revealed Acinetobacter cells were mostly clustered together, which is consistent with growth in a non-mixed environment, such as soil. The abundance of Acinetobacter cells decreased from active decomposition to putrid/dry. BLAST analysis using the 16S rRNA-gene sequence identified the isolates as one of the following Acinetobacter spp: A. baumannii, A. haemolyticus, A. junii, A. johnsonii, and A. gerneri. Phenotypic description of the identified isolates closely matched those of known genomic species. One isolate, P4, was observed to be unique in its phenotypic and phylogenetic characteristics and was more closely related to A. sp E10. The isolates from this study displayed multi-antibiotic resistance. The results from the study revealed the association of Acinetobacter spp. with that of carrion which adds to our knowledge of the ecology of this genus along with the potential implications of infection for this opportunistic pathogen.

Growth inhibition and stimulation of Shewanella oneidensis MR-1 by surfactants and calcium polysulfide.

Foam delivery technology (FDT) uses surfactant based foam to immobilize subsurface contaminants in situ. Where traditional approaches are impractical, FDT has the potential to overcome many of the technical challenges facing the remediation of contaminated deep vadose zone environments. However, little is known about the effects these reactive chemicals may have on microorganisms inhabiting the contaminated subsurface. In addition, there are currently no standard assays to assess microbial responses to subsurface remedial treatments while these agents are under development. The objective of this study was to develop a rapid laboratory assay to assess the potential growth inhibition and/or stimulation of microorganisms following exposure to candidate FDT components. Calcium polysulfide (CPS) and several surfactants (i.e. sodium laureth sulfate (SLES), sodium dodecyl sulfate (SDS), cocamidopropyl betaine (CAPB) and NINOL40-CO) have diverse chemistries and are candidate components of FDT. Shewanella oneidensis MR-1 cultures were exposed to a range of concentrations of these chemicals to determine the minimum bactericidal concentration (MBC) and the growth and viability potential of these components. Concentrations of SDS higher than 700 µM were toxic to S. oneidensis MR-1 growth over the course of four days of exposure. The relative acute toxicity order for these compounds was SDS >> CPS >> NINOL 40-CO>SLES≥CAPB. Dose dependent growth decreases (20-100mM) were observed in the CAPB and SLES treated cultures and both CPS and NINOL 40-CO were toxic at all concentrations tested (1.45-7.25 mM CPS). Both SLES (20-100mM) and SDS at lower concentrations (20-500 µM) were stimulatory to S. oneidensis MR-1 indicating a capacity to be used as a carbon source. These studies also identified potentially key component characteristics, such as precipitate formation and oxygen availability, which may prove valuable in assessing the response of subsurface microorganisms. This benchtop system provides a capability to assess adverse microbial-remediation responses and contributes to the development of in situ remedial chemistries before they are deployed in the field.

High spatiotemporal variability of bacterial diversity over short time scales with unique hydrochemical associations within a shallow aquifer.

Understanding microbial community structure and function within the subsurface is critical to assessing overall quality and maintenance of groundwater; however, the factors that determine microbial community assembly, structure, and function in groundwater systems and their impact on water quality remains poorly understood. In this study, three shallow wells (FW301, FW303, FW305) in a non-contaminated shallow aquifer in the ENIGMA-Oak Ridge Field Research Center (Oak Ridge, TN) were sampled approximately 3 times a week over a period of three months to measure changes in groundwater geochemistry and microbial diversity. It was expected that the sampled microbial diversity from two historic field wells (FW301, FW303) would be relatively stable, while diversity from a newer well (FW305) would be less stable over time. The wells displayed some degree of hydrochemical variability over time unique to each well, with FW303 being overall the most stable well and FW301 being the most dynamic based upon dissolved oxygen, conductivity, and nitrate. Community analysis via ss-rRNA paired-end sequencing and distribution-based clustering revealed higher OTU richness, diversity, and variability in groundwater communities of FW301 than the other two wells for diversity binned over all time points. Microbial community composition of a given well was on average > 50% dissimilar to any other well at a given time (days), yet, functional gene diversity as measured with GeoChip remained relatively constant. Similarities in community structure across wells were observed with respect to the presence of 20 shared bacterial groups in all samples in all wells, although at varying levels over the tested time period. Similarity percentage (SIMPER) analysis revealed that variability in FW301 was largely attributed to low abundance, highly-transient populations, while variability in the most hydrochemically stable well (FW303) was due to fluctuations in more highly abundant and frequently present taxa. Additionally, the youngest well FW305 showed a dramatic shift in community composition towards the end of the sampling period that was not observed in the other wells, suggesting possible succession events over time. Time-series analysis using vector auto-regressive models and Granger causality showed unique relationships between richness and geochemistry over time in each well. These results indicate temporally dynamic microbial communities over short time scales, with day-to-day population shifts in local community structure influenced by available source community diversity and local groundwater hydrochemistry.

Natural bacterial communities serve as quantitative geochemical biosensors.

UNLABELLED: Biological sensors can be engineered to measure a wide range of environmental conditions. Here we show that statistical analysis of DNA from natural microbial communities can be used to accurately identify environmental contaminants, including uranium and nitrate at a nuclear waste site. In addition to contamination, sequence data from the 16S rRNA gene alone can quantitatively predict a rich catalogue of 26 geochemical features collected from 93 wells with highly differing geochemistry characteristics. We extend this approach to identify sites contaminated with hydrocarbons from the Deepwater Horizon oil spill, finding that altered bacterial communities encode a memory of prior contamination, even after the contaminants themselves have been fully degraded. We show that the bacterial strains that are most useful for detecting oil and uranium are known to interact with these substrates, indicating that this statistical approach uncovers ecologically meaningful interactions consistent with previous experimental observations. Future efforts should focus on evaluating the geographical generalizability of these associations. Taken as a whole, these results indicate that ubiquitous, natural bacterial communities can be used as in situ environmental sensors that respond to and capture perturbations caused by human impacts. These in situ biosensors rely on environmental selection rather than directed engineering, and so this approach could be rapidly deployed and scaled as sequencing technology continues to become faster, simpler, and less expensive. IMPORTANCE: Here we show that DNA from natural bacterial communities can be used as a quantitative biosensor to accurately distinguish unpolluted sites from those contaminated with uranium, nitrate, or oil. These results indicate that bacterial communities can be used as environmental sensors that respond to and capture perturbations caused by human impacts.

Granules harboring translationally active mRNAs provide a platform for P-body formation following stress.

The localization of mRNA to defined cytoplasmic sites in eukaryotic cells not only allows localized protein production but also determines the fate of mRNAs. For instance, translationally repressed mRNAs localize to P-bodies and stress granules where their decay and storage, respectively, are directed. Here, we find that several mRNAs are localized to granules in unstressed, actively growing cells. These granules play a key role in the stress-dependent formation of P-bodies. Specific glycolytic mRNAs are colocalized in multiple granules per cell, which aggregate during P-body formation. Such aggregation is still observed under conditions or in mutants where P-bodies do not form. In unstressed cells, the mRNA granules appear associated with active translation; this might enable a coregulation of protein expression from the same pathways or complexes. Parallels can be drawn between this coregulation and the advantage of operons in prokaryotic systems.

An immunohistochemical study of cases of sporadic and inherited frontotemporal lobar degeneration using 3R- and 4R-specific tau monoclonal antibodies.

The pathological distinctions between the various clinical and pathological manifestations of frontotemporal lobar degeneration (FTLD) remain unclear. Using monoclonal antibodies specific for 3- and 4-repeat isoforms of the microtubule associated protein, tau (3R- and 4R-tau), we have performed an immunohistochemical study of the tau pathology present in 14 cases of sporadic forms of FTLD, 12 cases with Pick bodies and two cases without and in 27 cases of familial FTLD associated with 12 different mutations in the tau gene (MAPT), five cases with Pick bodies and 22 cases without. In all 12 cases of sporadic FTLD where Pick bodies were present, these contained only 3R-tau isoforms. Clinically, ten of these cases had frontotemporal dementia and two had progressive apraxia. Only 3R-tau isoforms were present in Pick bodies in those patients with familial FTLD associated with L266V, Q336R, E342V, K369I or G389R MAPT mutations. Patients with familial FTLD associated with exon 10 N279K, N296H or +16 splice site mutations showed tau pathology characterised by neuronal neurofibrillary tangles (NFT) and glial cell tangles that contained only 4R-tau isoforms, as did the NFT in P301L MAPT mutation. With the R406W mutation, NFT contained both 3R- and 4R-tau isoforms. We also observed two patients with sporadic FTLD, but without Pick bodies, in whom the tau pathology comprised only of 4R-tau isoforms. We have therefore shown by immunohistochemistry that different specific tau isoform compositions underlie the various kinds of tau pathology present in sporadic and familial FTLD. The use of such tau isoform specific antibodies may refine pathological criteria underpinning FTLD.

Histopathological changes underlying frontotemporal lobar degeneration with clinicopathological correlation.

We have investigated the pathological correlates of dementia in the brains from a consecutive series of 70 patients dying with a clinical diagnosis of frontotemporal lobar degeneration (FTLD). Clinical misdiagnosis rate was low with only 3 patients (4%) failing to show pathological changes consistent with this diagnosis; 1 patient had Alzheimer's disease and 2 had cerebrovascular disease (CVD). In the remaining 67 patients, the most common underlying histological cause was ubiquitin pathology with 24 (36%) cases so affected. In these, ubiquitin-positive inclusions were present in the cerebral cortex as small, rounded or crescent-shaped structures within the cytoplasm of neurones of layer II, together with coiled or curvilinear bodies within neurites, and in the hippocampus as small, solid and more spherical-shaped inclusion bodies within the cytoplasm of dentate gyrus granule cells. In one patient, "cat's eye" or "lentiform" intranuclear ubiquitin inclusions were also present. The second most common histological type was dementia lacking distinctive histology (DLDH), in which neither tau nor ubiquitin inclusions were present, with 16 cases (24%) being affected. Pick-type histology was seen in 14 cases (21%) and tau histological changes associated with frontotemporal dementia (FTD) linked to chromosome 17 (FTDP-17) were present in 11 cases (16%). One case (1%) showed an unusual tau pathology that could not be allocated to any of the other tau groups. Only 1 case (1%) had neuronal intermediate filament inclusion dementia. No cases with ubiquitinated, valosin-containing protein-immunoreactive intranuclear inclusion bodies of the type seen in inclusion body myopathy with Paget's disease of bone and frontotemporal dementia were seen. Clinicopathological correlation showed that any of these histological subtypes can be associated with FTD. However, for FTD with motor neurone disease (FTD+MND), semantic dementia or primary progressive aphasia (PA), the histological profile was either ubiquitin type or DLDH type; Pick-type histology was seen in only 1 case of PA. None of these latter three clinical subtypes was associated with a mutation in tau gene and FTDP-17 type of tau pathology. All cases of progressive apraxia were associated with Pick-type histology. Present data therefore indicate that, although ubiquitin pathology is the most common histological form associated with FTLD, this pathology is not tightly linked with, nor is pathologically diagnostic for, any particular clinical form of the disease, including FTD+MND.