Redox Sensing within the Genus Shewanella.

A novel bacterial behavior called congregation was recently described in Shewanella oneidensis MR-1 as the accumulation of cells around insoluble electron acceptors (IEA). It is the result of a series of "run-and-reversal" events enabled by modulation of swimming speed and direction. The model proposed that the swimming cells constantly sense their surroundings with specialized outer membrane cytochromes capable of extracellular electron transport (EET). Up to this point, neither the congregation nor attachment behavior have been studied in any other strains. In this study, the wild type of S. oneidensis MR-1 and several deletion mutants as well as eight other Shewanella strains (Shewanella putrefaciens CN32, S. sp. ANA-3, S. sp. W3-18-1, Shewanella amazonensis SB2B, Shewanella loihica PV-4, Shewanella denitrificans OS217, Shewanella baltica OS155, and Shewanella frigidimarina NCIMB400) were screened for the ability to congregate. To monitor congregation and attachment, specialized cell-tracking techniques, as well as a novel cell accumulation after photo-bleaching (CAAP) confocal microscopy technique were utilized in this study. We found a strong correlation between the ability of strain MR-1 to accumulate on mineral surface and the presence of key EET genes such as mtrBC/omcA (SO_1778, SO_1776, and SO_1779) and gene coding for methyl-accepting protein (MCPs) with Ca+ channel chemotaxis receptor (Cache) domain (SO_2240). These EET and taxis genes were previously identified as essential for characteristic run and reversal swimming around IEA surfaces. CN32, ANA-3, and PV-4 congregated around both Fe(OH)3 and MnO2. Two other Shewanella spp. showed preferences for one oxide over the other: preferences that correlated with the metal content of the environments from which the strains were isolated: e.g., W3-18-1, which was isolated from an iron-rich habitat congregated and attached preferentially to Fe(OH)3, while SB2B, which was isolated from a MnO2-rich environment, preferred MnO2.

In situ Detection of Microbial Life in the Deep Biosphere in Igneous Ocean Crust.

The deep biosphere is a major frontier to science. Recent studies have shown the presence and activity of cells in deep marine sediments and in the continental deep biosphere. Volcanic lavas in the deep ocean subsurface, through which substantial fluid flow occurs, present another potentially massive deep biosphere. We present results from the deployment of a novel in situ logging tool designed to detect microbial life harbored in a deep, native, borehole environment within igneous oceanic crust, using deep ultraviolet native fluorescence spectroscopy. Results demonstrate the predominance of microbial-like signatures within the borehole environment, with densities in the range of 10(5) cells/mL. Based on transport and flux models, we estimate that such a concentration of microbial cells could not be supported by transport through the crust, suggesting in situ growth of these communities.

Label-free bacterial imaging with deep-UV-laser-induced native fluorescence.

We introduce a near-real-time optical imaging method that works via the detection of the intrinsic fluorescence of life forms upon excitation by deep-UV (DUV) illumination. A DUV (<250-nm) source enables the detection of microbes in their native state on natural materials, avoiding background autofluorescence and without the need for fluorescent dyes or tags. We demonstrate that DUV-laser-induced native fluorescence can detect bacteria on opaque surfaces at spatial scales ranging from tens of centimeters to micrometers and from communities to single cells. Given exposure times of 100 µs and low excitation intensities, this technique enables rapid imaging of bacterial communities and cells without irreversible sample alteration or destruction. We also demonstrate the first noninvasive detection of bacteria on in situ-incubated environmental experimental samples from the deep ocean (Lo'ihi Seamount), showing the use of DUV native fluorescence for in situ detection in the deep biosphere and other nutrient-limited environments.

Reduction of graphene oxide via bacterial respiration.

Here we present that graphene oxide (GO) can act as a terminal electron acceptor for heterotrophic, metal-reducing, and environmental bacteria. The conductance and physical characteristics of bacterially converted graphene (BCG) are comparable to other forms of chemically converted graphene (CCG). Electron transfer to GO is mediated by cytochromes MtrA, MtrB, and MtrC/OmcA, while mutants lacking CymA, another cytochrome associated with extracellular electron transfer, retain the ability to reduce GO. Our results demonstrate that biodegradation of GO can occur under ambient conditions and at rapid time scales. The capacity of microbes to degrade GO, restoring it to the naturally occurring ubiquitous graphite mineral form, presents a positive prospect for its bioremediation. This capability also provides an opportunity for further investigation into the application of environmental bacteria in the area of green nanochemistries.

The Impact of Bacterial Strain on the Products of Dissimilatory Iron Reduction.

Three bacterial strains from the genus Shewanella were used to examine the influence of specific bacteria on the products of dissimilatory iron reduction. Strains CN32, MR-4 and W3-18-1 were incubated with HFO (hydrous ferric oxide) as the terminal electron acceptor and lactate as the organic carbon and energy source. Mineral products of iron reduction were analyzed using X-ray powder diffraction, electron microscopy, coulometry and susceptometry. Under identical nutrient loadings, iron reduction rates for strains CN32 and W3-18-1 were similar, and about twice as fast as MR-4. Qualitative and quantitative assessment of mineralized end products (secondary minerals) indicated that different products were formed during experiments with similar reduction rates but different strains (CN32 and W3-18-1), and similar products were formed during experiments with different iron reduction rates and different strains (CN32 and MR-4). The major product of iron reduction by strains CN32 and MR-4 was magnetite, while for W3-18-1 it was a mixture of magnetite and iron carbonate hydroxide hydrate (green rust), a precursor to fougerite. Another notable difference was that strains CN32 and MR-4 converted all of the starting ferric iron material into magnetite, while W3-18-1 did not convert most of the Fe(3+) into a recognizable crystalline material. Biofilm formation is more robust in W3-18-1 than in the other two strains used in this study. The differences in mineralization may be an indicator that EPS (or another cellular product from W3-18-1) may interfere with the crystallization of magnetite or facilitate formation of green rust. These results suggest that the relative abundance of mineral end products and the relative distribution of these products are strongly dependent on the bacterial species or strain catalyzing iron reduction.

Classification of organic and biological materials with deep ultraviolet excitation.

We show that native fluorescence can be used to differentiate classes or groups of organic molecules and biological materials when excitation occurs at specific excitation wavelengths in the deep ultraviolet (UV) region. Native fluorescence excitation-emission maps (EEMs) of pure organic materials, microbiological samples, and environmental background materials were compared using excitation wavelengths between 200-400 nm with emission wavelengths from 270 to 500 nm. These samples included polycyclic aromatic hydrocarbons (PAHs), nitrogen- and sulfur-bearing organic heterocycles, bacterial spores, and bacterial vegetative whole cells (both Gram positive and Gram negative). Each sample was categorized into ten distinct groups based on fluorescence properties. Emission spectra at each of 40 excitation wavelengths were analyzed using principal component analysis (PCA). Optimum excitation wavelengths for differentiating groups were determined using two metrics. We show that deep UV excitation at 235 (+/-2) nm optimally separates all organic and biological groups within our dataset with >90% confidence. For the specific case of separation of bacterial spores from all other samples in the database, excitation at wavelengths less than 250 nm provides maximum separation with >6sigma confidence.