Case Report: Shewanella Algae Pneumonia and Bacteremia in an Elderly Male Living at a Long-Term Care Facility.

Shewanella algae is a gram-negative, nonfermenting, oxidase-positive, motile bacillus that is ubiquitous in aquatic ecosystems. Human infections are rare and the immunocompromised are left most vulnerable. Risk factors for this infection include exposure to seawater, consumption of raw seafood, and underlying comorbid conditions such as hepatobiliary disease and chronic cutaneous ulcers. Previously documented cases of S. algae have involved near drownings, contaminated raw shellfish, or wound exposure to seawater, mud, sand, and sewage. This case study is unique in that it describes Shewanella bacteremia without any of these typical preceding exposures. We present a case of S. algae pneumonia and bacteremia in an elderly male patient living at a long-term care facility without any recent open water exposure.

Single-Cell Mass Spectroscopic Analysis for Quantifying Active Metabolic Pathway Heterogeneity in a Bacterial Population on an Electrode.

Microbial electrochemical catalysis based on respiratory reactions coupled with extracellular electron transport (EET), which is critical for bioenergy applications, strongly depends on the biocompatibility of the electrode material. However, the comparison of materials for such physiological responses has been difficult because of the lack of a quantitative assay for characterizing cellular metabolism at the electrode surface. Here, we developed a single-cell analysis method specific for the cells attached to the electrode to quantify active metabolic pathway heterogeneity as an index of physiological cell/electrode interaction, which generally increases with metabolic robustness in the microbial population. Nanoscale secondary ion mass spectrometry followed by microbial current production with model EET-capable bacteria, Shewanella oneidensis MR-1 and its mutant strains lacking carbon assimilation pathways, showed that different active metabolic pathways resulted in nearly identical 13C/15N assimilation ratios for individual cells in the presence of isotopically labeled nutrients, demonstrating a correlation between the 13C/15N ratio and the active metabolic pathway. Compared to the nonelectrode conditions, the heterogeneity of the assimilated 13C/15N ratio was highly enhanced on the electrode surface, suggesting that the metabolic robustness of the microbial population increased through the electrochemical interaction with the electrode. The present methodology enables us to quantitatively compare and screen electrode materials that increase the robustness of microbial electrocatalysis.

Probing Microbial Extracellular Respiration Ability Using Riboflavin.

Electrochemically active bacteria (EAB) are capable of extracellular electron transfer (EET) to insoluble metal oxides, and thus play a great role in the fields of environment, energy, and geosciences. However, rapid and accurate quantification of the EET ability of EAB is still challenging. In this work, we develop a riboflavin-based fluorescence method for facile, accurate, and in situ measurement of the EET ability of EAB. This method is successfully used to quantify the single-cellular EET ability of Geobacter sulfurreducens DL-1 (60.29 ± 13.02 fA) and Shewanella oneidensis MR-1 (2.11 ± 0.47 fA), the two widely present EAB in the environment. It also enables quantitative identification of EET-related c-type cytochromes in the outer membrane of S. oneidensis MR-1. This method provides a useful tool to rapidly identify EAB in diverse environments and elucidate their electron transfer mechanisms.

Influence of the Spatial Distribution of Cationic Functional Groups at Nanoparticle Surfaces on Bacterial Viability and Membrane Interactions.

While positively charged nanomaterials induce cytotoxicity in many organisms, much less is known about how the spatial distribution and presentation of molecular surface charge impact nanoparticle-biological interactions. We systematically functionalized diamond nanoparticle surfaces with five different cationic surface molecules having different molecular structures and conformations, including four small ligands and one polymer, and we then probed the molecular-level interaction between these nanoparticles and bacterial cells. Shewanella oneidensis MR-1 was used as a model bacterial cell system to investigate how the molecular length and conformation of cationic surface charges influence their interactions with the Gram-negative bacterial membranes. Nuclear magnetic resonance (NMR) and X-ray photoelectron spectroscopy (XPS) demonstrate the covalent modification of the nanoparticle surface with the desired cationic organic monolayers. Surprisingly, bacterial growth-based viability (GBV) and membrane damage assays both show only minimal biological impact by the NPs functionalized with short cationic ligands within the concentration range tested, yet NPs covalently linked to a cationic polymer induce strong cytotoxicity, including reduced cellular viability and significant membrane damage at the same concentration of cationic groups. Transmission electron microscopy (TEM) images of these NP-exposed bacterial cells show that NPs functionalized with cationic polymers induce significant membrane distortion and the production of outer membrane vesicle-like features, while NPs bearing short cationic ligands only exhibit weak membrane association. Our results demonstrate that the spatial distribution of molecular charge plays a key role in controlling the interaction of cationic nanoparticles with bacterial cell membranes and the subsequent biological impact. Nanoparticles functionalized with ligands having different lengths and conformations can have large differences in interactions even while having nearly identical zeta potentials. While the zeta potential is a convenient and commonly used measure of nanoparticle charge, it does not capture essential differences in molecular-level nanoparticle properties that control their biological impact.

Mesoscopic to Macroscopic Electron Transfer by Hopping in a Crystal Network of Cytochromes.

Rapid and directed electron transfer (ET) is essential for biological processes. While the rates of ET over 1-2 nm in proteins can largely be described by simplified nonadiabatic theory, it is not known how these processes scale to microscopic distances. We generated crystalline lattices of Small Tetraheme Cytochromes (STC) forming well-defined, three-dimensional networks of closely spaced redox centers that appear to be nearly ideal for multistep ET. Electrons were injected into specific locations in the STC crystals by direct photoreduction, and their redistribution was monitored by imaging. The results demonstrate ET over mesoscopic to microscopic (∼100 µm) distances through sequential hopping in a biologically based heme network. We estimate that a hypothetical "nanowire" composed of crystalline STC with a cross-section of about 100 cytochromes could support the anaerobic respiration of a Shewanella cell. The crystalline lattice insulates mobile electrons from oxidation by O2, as compared to those in cytochromes in solution, potentially allowing for efficient delivery of current without production of reactive oxygen species. The platform allows direct tests of whether the assumptions based on short-range ET hold for sequential ET over mesoscopic distances. We estimate that the interprotein ET across 6 Å between hemes in adjacent proteins was about 105 s-1, about 100-fold slower than expectations based on simplified theory. More detailed analyses implied that additional factors, possibly contributed by the crystal lattice, may strongly impact mesoscale ET mainly by increasing the reorganizational energy of interprotein ET, which suggests design strategies for engineering improved nanowires suitable for future bioelectronic materials.

Synergistic improvement of Shewanella loihica PV-4 extracellular electron transfer using a TiO2@TiN nanocomposite.

Extracellular electron transfer (EET) allows microorganisms to perform anaerobic respiration using insoluble electron acceptors, including minerals and electrodes. EET-based applications require efficient electron transfer between living and non-living systems. To improve EET efficiency, the TiO2@TiN nanocomposite was used to form hybrid biofilms with Shewanella loihica PV-4 (PV-4). Chronoamperometry showed that peak current was increased 4.6-fold via the addition of the TiO2@TiN nanocomposite. Different biofilms were further tested in a dual-chamber microbial fuel cell. The PV-4 biofilm resulted a maximum power density of 33.4 mW/m2, while the hybrid biofilm of the TiO2@TiN nanocomposite with PV-4 yielded a 92.8% increase of power density. Electrochemical impedance spectroscopy analyses showed a lower electron-transfer resistance in the hybrid biofilm. Biological measurements revealed that both flavin secretion and cytochrome c expression were increased when the TiO2@TiN nanocomposite presented. These results demonstrated that the TiO2@TiN nanocomposite could synergistically enhance the EET of PV-4 through altering its metabolism. Our findings provide a new strategy for optimizing biotic-abiotic interactions in bioelectrochemical systems.

PBP1a glycosyltransferase and transpeptidase activities are both required for maintaining cell morphology and envelope integrity in Shewanella oneidensis.

In rod-shaped Gram-negative bacteria, penicillin binding protein 1a (PBP1a) and 1b (PBP1b) form peptidoglycan-synthesizing complexes with the outer membrane lipoprotein LpoA and LpoB, respectively. Escherichia coli mutants lacking PBP1b/LpoB are sicker than those lacking PBP1a/LpoA. However, we previously found that mutants lacking PBP1a/LpoA but not PBP1b/LpoB are deleterious in Shewanella oneidensis. Here, we show that S. oneidensis PBP1a (SoPBP1a) contains conserved signature motifs with its E. coli counterpart, EcPBP1a. Although EcPBP1a play a less prominent role in E. coli, it is capable of substituting for the SoPBP1a in a manner dependent on SoLpoA. In S. oneidensis, expression of PBP1b is lower than PBP1a, and therefore the additional expression of SoPBP1b at low levels can functionally compensate for the absence of SoPBP1a. Importantly, S. oneidensis PBP1a variants lacking either glycosyltransferase (GTase) or transpeptidase (TPase) activity fail to maintain normal morphology and cell envelope integrity. Similarly, SoPBP1b variants also fail to compensate for the loss of SoPBP1a. Furthermore, overproduction of variants of SoPBP1a, but not SoPBP1b, has detrimental effects on cell morphology in S. oneidensis wild type cells. Overall, our results indicate that the combined enzymatic activities of SoPBP1a are essential for cell wall homeostasis.

Promoting bidirectional extracellular electron transfer of Shewanella oneidensis MR-1 for hexavalent chromium reduction via elevating intracellular cAMP level.

The bioreduction capacity of Cr(VI) by Shewanella is mainly governed by its bidirectional extracellular electron transfer (EET). However, the low bidirectional EET efficiency restricts its wider applications in remediation of the environments contaminated by Cr(VI). Cyclic adenosine 3',5'-monophosphate (cAMP) commonly exists in Shewanella strains and cAMP-cyclic adenosine 3',5'-monophosphate receptor protein (CRP) system regulates multiple bidirectional EET-related pathways. This inspires us to strengthen the bidirectional EET through elevating the intracellular cAMP level in Shewanella strains. In this study, an exogenous gene encoding adenylate cyclase from the soil bacterium Beggiatoa sp. PS is functionally expressed in Shewanella oneidensis MR-1 (the strain MR-1/pbPAC) and a MR-1 mutant lacking all endogenous adenylate cyclase encoding genes (the strain Δca/pbPAC). The engineered strains exhibit the enhanced bidirectional EET capacities in microbial electrochemical systems compared with their counterparts. Meanwhile, a three times more rapid reduction rate of Cr(VI) is achieved by the strain MR-1/pbPAC than the control in batch experiments. Furthermore, a higher Cr(VI) reduction efficiency is also achieved by the strain MR-1/pbPAC in the Cr(VI)-reducing biocathode experiments. Such a bidirectional enhancement is attributed to the improved production of cAMP-CRP complex, which upregulates the expression levels of the genes encoding the c-type cytochromes and flavins synthetic pathways. Specially, this strategy could be used as a broad-spectrum approach for the other Shewanella strains. Our results demonstrate that elevating the intracellular cAMP levels could be an efficient strategy to enhance the bidirectional EET of Shewanella strains and improve their pollutant transformation capacity.

Soluble electron acceptors affect bioluminescence from Shewanella woodyi.

Shewanella woodyi cultures were used to correlate bioluminescence intensity with changes in the electrochemical potential of a saltwater medium using soluble electron acceptors. A relationship between the concentration of NaNO3 or CoCl2 to bioluminescence intensity was confirmed using aerobic cultures of S. woodyi at 20°C with glucose as the sole carbon source. In general, increasing the concentration of nitrate or Co(II) reduced the bioluminescence per cell, with complete luminescence being repressed at ≥5 mM nitrate and ≥0.5 mM Co(II). Results from cell viability fluorescent staining concluded that increasing the concentration of Co(II) or nitrate did not affect the overall viability of the cells when compared with cultures lacking Co(II) or nitrate. These data show that potentials of <0.2 V vs Normal Hydrogen Electrode (NHE) repress the luminescence from the cells, but the exact mechanism is unclear. Our results indicated that the luminescence intensity from S. woodyi could be systematically reduced using these two soluble electron acceptors, making S. woodyi a potential model bacterium for whole-cell luminescence bioelectrochemical sensor applications.

Pellicle development of Shewanella oneidensis is an aerotaxis-piloted and energy-dependent process.

Pellicles are biofilms found at the air-liquid interface and are widely distributed in natural environments. In this study, a simple pellicle detection method was established, and using this new method, the pellicle formation activities of Shewanella oneidensis MR-1 and its 42 cytochrome c mutants were analysed. The results showed that the pellicle was initiated at very early stages of incubation. Aerotaxis was the major external factor, while energy acquirement was the main internal factor for pellicle initiation. Among the 42 cytochrome c mutants, 17 mutants, including those deficient in aerobic respiration, sulfur or sulfite/sulfate respiration, nitrite respiration, metal respiration, DMSO respiration and fumarate respiration, exhibited delayed pellicle initiation. The results suggest that S. oneidensis utilizes the electron acceptors simultaneously under anoxic conditions and that the disruption of any of these anaerobic respiration routes would retard pellicle initiation.

In situ imaging of the bacterial flagellar motor disassembly and assembly processes.

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo-tomography and sub-tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly in situ. We first show that motor disassembly results in stable outer membrane-embedded sub-complexes. These sub-complexes consist of the periplasmic embellished P- and L-rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner-membrane sub-complex consisting of the C-ring, MS-ring, and export apparatus. Finally, we show that the L-ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process.

In Vivo Water Dynamics in Shewanella oneidensis Bacteria at High Pressure.

Following observations of survival of microbes and other life forms in deep subsurface environments it is necessary to understand their biological functioning under high pressure conditions. Key aspects of biochemical reactions and transport processes within cells are determined by the intracellular water dynamics. We studied water diffusion and rotational relaxation in live Shewanella oneidensis bacteria at pressures up to 500 MPa using quasi-elastic neutron scattering (QENS). The intracellular diffusion exhibits a significantly greater slowdown (by -10-30%) and an increase in rotational relaxation times (+10-40%) compared with water dynamics in the aqueous solutions used to resuspend the bacterial samples. Those results indicate both a pressure-induced viscosity increase and slowdown in ionic/macromolecular transport properties within the cells affecting the rates of metabolic and other biological processes. Our new data support emerging models for intracellular organisation with nanoscale water channels threading between macromolecular regions within a dynamically organized structure rather than a homogenous gel-like cytoplasm.

The role of electron shuttle enhances Fe(III)-mediated reduction of Cr(VI) by Shewanella oneidensis MR-1.

Chromate is one of the hazardous toxic pollutants. Reduction of Cr(VI) to Cr(III) has shown to reduce the toxicity of chromate. This work examined the reduction of Cr(VI) using an anaerobic batch cultures of Shewanella oneidensis MR-1 containing Fe(III). To do so, 10 mg/L Cr(VI) was reduced to Cr(III) within 3 days along with the oxidization of Fe(II) to Fe(III). The removal rate of Cr(VI) increased with increasing the concentration of Fe(III). In the absence of Cr(VI), the Fe(II) concentration of the batch culture increased with the growth of S. oneidensis MR-1. These data showed that S. oneidensis MR-1 could reduce Fe(III) into Fe(II), resulting in reduction of Cr(VI) to Cr(III). During this process, the anthraquinone-2,6-disulfonate (AQDS) acted as an electron shuttle. Microscopic analysis showed that Cr(VI) had toxic effects on S. oneidensis MR-1 due to the appearance of Cr species on the bacterial surface. Cr2O3 or Cr(OH)3 precipitates formed during Cr(VI) reduction was identified using X-ray photoelectron spectroscopy. The AQDS as an electron shuttle enhanced the Cr(VI) reduction by S. oneidensis MR-1. Microbial reduction of Cr(VI) can be a useful technique for Cr detoxification.

Electrochemical Study on the Extracellular Electron Transfer Pathway from Shewanella Strain Hac319 to Electrodes.

Shewanella can transfer electrons to various extracellular electron acceptors. We electrochemically investigated the pathway of extracellular electron transfer from Shewanella strain Hac319 to electrodes. A resting cell suspension of Shewanella strain Hac319 containing lactate produced a steady-state sigmoidal wave in the presence of flavin mononucleotide (FMN) in cyclic voltammetry, but not in the absence of FMN. A harvested cell suspension without cell-washing also produced a similar catalytic wave without any external addition of free FMN. The midpoint potentials of the two sigmoidal waves were identical to the redox potential of free FMN. The data indicate that FMN secreted from the Shewanella strain Hac319 works as an electron-transfer mediator from the cell to electrodes. An addition of cyanide to a resting cell suspension of Shewanella strain Hac319 increased the rate of the FMN reduction in the presence of lactate, while it decreased the respiration rate. By considering the fact that cyanide is coordinated to the heme moiety of hemoproteins and shifts the redox potential to the negative potential side, the data indicate that the electron derived from lactate is predominantly transferred in a down-hill mode from an electron donor with a redox potential more negative than that of FMN without going through outer membrane cytochromes c molecules.

Deletion of PBP1a/LpoA complex compromises cell envelope integrity in Shewanella oneidensis.

High molecular weight penicillin-binding proteins (PBPs) are responsible for the biosynthesis of peptidoglycan. In Escherichia coli, PBP1a and PBP1b form multienzyme peptidoglycan-synthesizing complexes with outer membrane lipoproteins LpoA and LpoB, respectively. The two complexes appear to be largely redundant, although their distinct physiological roles remain unclear. PBP1a/LpoA and PBP1b/LpoB also exist in Shewanella oneidensis strain MR-1, but effects of the two complexes on aerobic growth and ß-lactam resistance are quite different. In this study, the phenotypes of strains lacking a certain complex in S. oneidensis were compared. Deletion of PBP1a/LpoA caused aberrant cell morphology (including branches and bulges), enhanced sensitivity to various envelope stresses and outer membrane permeability. On the contrary, strains lacking PBP1b/LpoB displayed phenotypes similar to the wild type.

Impedance spectroscopy of single bacterial nanofilament reveals water-mediated charge transfer.

For decades respiratory chain and photosystems were the main firing field of the studies devoted to mechanisms of electron transfer in proteins. The concept of conjugated lateral electron and transverse proton transport during cellular respiration and photosynthesis, which was formulated in the beginning of 1960-s, has been confirmed by thousands of experiments. However, charge transfer in recently discovered bacterial nanofilaments produced by various electrogenic bacteria is regarded currently outside of electron and proton conjugation concept. Here we report the new study of charge transfer within nanofilaments produced by Shewanella oneidensis MR-1 conducted in atmosphere of different relative humidity (RH). We utilize impedance spectroscopy and DC (direct current) transport measurements to find out the peculiarities of conductivity and Raman spectroscopy to analyze the nanofilaments' composition. Data analysis demonstrates that apparent conductivity of nanofilaments has crucial sensitivity to humidity and contains several components including one with unusual behavior which we assign to electron transport. We demonstrate that in the case of Shewanella oneidensis MR-1 charge transfer within these objects is strongly mediated by water. Basing on current data analysis of conductivity we conclude that the studied filaments of Shewanella oneidensis MR-1 are capable of hybrid (conjugated) electron and ion conductivity.

Effect of the anode potential on the physiology and proteome of Shewanella oneidensis MR-1.

Shewanella species respire using iron and manganese oxides as well as electrodes as solid terminal electron acceptors. Shewanella oneidenis MR-1 exploits mediated as well as direct extracellular electron transfer (EET) modes to transfer electrons at different formal potentials. These different EET modes at different potentials may utilise alternate electron transfer pathways. Therefore, we investigated how different anode potentials, providing different maximum microbial energy gains impacted S. oneidensis microbial physiology. Using quantitative proteomics, comparative analysis of the cellular variations to different anode potentials was performed. A label-free proteomic mass spectrometric analysis method, SWATH-MS, was used to gather quantitative information to determine physiological changes of Shewanella oneidensis MR-1 grown at different anodic potentials. S. oneidensis was cultured and grown in electrochemical cells at the set anode potentials of +0.71V, +0.21V & -0.19V versus SHE reference electrode, while the current production was monitored. At maximum current, electrodes were removed and whole-cell proteins extracted. Subsequent SWATH-MS analysis revealed information on 740 identified proteins across the three electrode potentials. For the first time, we show the abundance of S. oneidensis electron transfer proteins differs with electrode potential.

Living and Conducting: Coating Individual Bacterial Cells with In†Situ Formed Polypyrrole.

Coating individual bacterial cells with conjugated polymers to endow them with more functionalities is highly desirable. Here, we developed an in situ polymerization method to coat polypyrrole on the surface of individual Shewanella oneidensis MR-1, Escherichia coli, Ochrobacterium anthropic or Streptococcus thermophilus. All of these as-coated cells from different bacterial species displayed enhanced conductivities without affecting viability, suggesting the generality of our coating method. Because of their excellent conductivity, we employed polypyrrole-coated Shewanella oneidensis MR-1 as an anode in microbial fuel cells (MFCs) and found that not only direct contact-based extracellular electron transfer is dramatically enhanced, but also the viability of bacterial cells in MFCs is improved. Our results indicate that coating individual bacteria with conjugated polymers could be a promising strategy to enhance their performance or enrich them with more functionalities.

Optical Elemental Analysis of Metals Using Shewanella oneidensis.

A simple method for the detection of metal ions in solution is proposed, using Shewanella oneidensis, which has the ability to reduce metal ions into metal nanoparticles on the cell surface. The method can be used to identify metal ions in solution using the light-scattering characteristics of the metal nanoparticles formed on the cells.

Investigation Concerning the Formation Process of Gold Nanoparticles by Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a facultative anaerobic bacterium that is known to transfer electrons generated during metabolism to various metal ions and produce nanoparticles on the bacterial surface. In this study, we tracked the formation of gold nanoparticles (Au NPs) on the S. oneidensis cell surfaces and investigated the roles of membrane proteins and extracellular polysaccharides in this process by spectrometry, zeta potential analysis, and electron microscopy.