Mechanism study of photo-induced gold nanoparticles formation by Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1, a bioelectricity generating bacterium, is broadly used in bioremediation, microbial fuel cell and dissimilatory reduction and recovery of precious metals. Herein, we report for the first time that photo induction as a trigger to stimulate gold nanoparticles (Au@NPs) formation by MR-1, with wavelength and light intensity as two key variables. Results indicated that sigmoidal model is the best fit for Au@NPs formation at various wavelengths (with R2 > 0.97). Light intensity in terms of photosynthetic photon flux density (PPFD) critically influences the rate constant in the low-light intensity region (PPFD < 20), while wavelength controls the maximum rate constant in the high-light region (PPFD > 20). By deletion of Mtr pathway genes in MR-1, we proposed the mechanism for light induced Au@NP formation is the excitation effect of light on certain active groups and extracellular polymeric substances (EPS) on the cell surface. Also, the release of electrons from proteins and co-enzyme complexes enhance electron generation. To the best of our knowledge, this is the first-attempt to explore the effect of photo-induction on Au@NPs production by MR-1, which provides an alternative cost-effective and eco-friendly process in green chemical industry.

Transcriptome analysis reveals a stress response of Shewanella oneidensis deprived of background levels of ionizing radiation.

Natural ionizing background radiation has exerted a constant pressure on organisms since the first forms of life appeared on Earth, so that cells have developed molecular mechanisms to avoid or repair damages caused directly by radiation or indirectly by radiation-induced reactive oxygen species (ROS). In the present study, we investigated the transcriptional effect of depriving Shewanella oneidensis cultures of background levels of radiation by growing the cells in a mine 655 m underground, thus reducing the dose rate from 72.1 to 0.9 nGy h-1 from control to treatment, respectively. RNASeq transcriptome analysis showed the differential expression of 4.6 and 7.6% of the S. oneidensis genome during early- and late-exponential phases of growth, respectively. The greatest change observed in the treatment was the downregulation of ribosomal proteins (21% of all annotated ribosomal protein genes during early- and 14% during late-exponential) and tRNA genes (14% of all annotated tRNA genes in early-exponential), indicating a marked decrease in protein translation. Other significant changes were the upregulation of membrane transporters, implying an increase in the traffic of substrates across the cell membrane, as well as the up and downregulation of genes related to respiration, which could be interpreted as a response to insufficient oxidants in the cells. In other reports, there is evidence in multiple species that some ROS not just lead to oxidative stress, but act as signaling molecules to control cellular metabolism at the transcriptional level. Consistent with these reports, several genes involved in the metabolism of carbon and biosynthesis of amino acids were also regulated, lending support to the idea of a wide metabolic response. Our results indicate that S. oneidensis is sensitive to the withdrawal of background levels of ionizing radiation and suggest that a transcriptional response is required to maintain homeostasis and retain normal growth.

DNA Backbone Sulfur-Modification Expands Microbial Growth Range under Multiple Stresses by its anti-oxidation function.

DNA phosphorothioate (PT) modification is a sulfur modification on the backbone of DNA introduced by the proteins DndA-E. It has been detected within many bacteria isolates and metagenomic datasets, including human pathogens, and is considered to be widely distributed in nature. However, little is known about the physiological function of this modification, and thus its evolutionary significance and application potential remains largely a mystery. In this study, we focused on the advantages of DNA PT modification to bacterial cells coping with environmental stresses. We show that the mesophile Escherichia coli and the extremophile Shewanella piezotolerans both expanded their growth ranges following exposure to extreme temperature, salinity, pH, pressure, UV, X-ray and heavy metals as a result of DNA phophorothioation. The phophorothioated DNA reacted to both H2O2 and hydroxyl radicals in vivo, and protected genomic DNA as well as sensitive enzymes from intracellular oxidative damage. We further demonstrate that this process has evolved separate from its associated role in DNA restriction and modification. These findings provide a physiological role for a covalent modification widespread in nature and suggest possible applications in biotechnology and biomedicine.

Filamentous phage SW1 is active and influences the transcriptome of the host at high-pressure and low-temperature.

As the most abundant biological entities on the planet, viruses are involved in global biogeochemical cycles, and they have been shown to play an important role in the overall functioning of the deep-sea ecosystem. Nevertheless, little is known about whether and how deep-sea viruses affect the physiology of their bacterial hosts. Previously, the filamentous phage SW1 was identified in the bathypelagic bacterium Shewanella piezotolerans WP3, which was isolated from the upper sediment of West Pacific ocean. In this study, phage SW1 was shown to be active under in situ environmental conditions (20 MPa and 4°C) by transmission electron microscopy and reverse-transcription quantitative polymerase chain reaction. Further comparative analysis showed that SW1 had a significant influence on the growth and transcriptome of its host. The transcription of genes responsible for basic cellular activities, including the transcriptional/translational apparatus, arginine synthesis, purine metabolism and the flagellar motor, were down-regulated by the phage. Our results present the first characterization of a phage-host interaction under high-pressure and low-temperature conditions, which indicated that the phage adjusted the energy utilization strategy of the host for improved survival in deep-sea environments.

Stress induction in the bacteria Shewanella oneidensis and Deinococcus radiodurans in response to below-background ionizing radiation.

PURPOSE: The 'Linear no-threshold' (LNT) model predicts that any amount of radiation increases the risk of organisms to accumulate negative effects. Several studies at below background radiation levels (4.5-11.4 nGy h(-1)) show decreased growth rates and an increased susceptibility to oxidative stress. The purpose of our study is to obtain molecular evidence of a stress response in Shewanella oneidensis and Deinococcus radiodurans grown at a gamma dose rate of 0.16 nGy h(-1), about 400 times less than normal background radiation. MATERIALS AND METHODS: Bacteria cultures were grown at a dose rate of 0.16 or 71.3 nGy h(-1) gamma irradiation. Total RNA was extracted from samples at early-exponential and stationary phases for the rt-PCR relative quantification (radiation-deprived treatment/background radiation control) of the stress-related genes katB (catalase), recA (recombinase), oxyR (oxidative stress transcriptional regulator), lexA (SOS regulon transcriptional repressor), dnaK (heat shock protein 70) and SOA0154 (putative heavy metal efflux pump). RESULTS: Deprivation of normal levels of radiation caused a reduction in growth of both bacterial species, accompanied by the upregulation of katB, recA, SOA0154 genes in S. oneidensis and the upregulation of dnaK in D. radiodurans. When cells were returned to background radiation levels, growth rates recovered and the stress response dissipated. CONCLUSIONS: Our results indicate that below-background levels of radiation inhibited growth and elicited a stress response in two species of bacteria, contrary to the LNT model prediction.

Phenotypic Characterisation of Shewanella oneidensis MR-1 Exposed to X-Radiation.

Biogeochemical processes mediated by Fe(III)-reducing bacteria such as Shewanella oneidensis have the potential to influence the post-closure evolution of a geological disposal facility for radioactive wastes and to affect the solubility of some radionuclides. Furthermore, their potential to reduce both Fe(III) and radionuclides can be harnessed for the bioremediation of radionuclide-contaminated land. As some such sites are likely to have significant radiation fluxes, there is a need to characterise the impact of radiation stress on such microorganisms. There have, however, been few global cell analyses on the impact of ionizing radiation on subsurface bacteria, so here we address the metabolic response of S. oneidensis MR-1 to acute doses of X-radiation. UV/Vis spectroscopy and CFU counts showed that although X-radiation decreased initial viability and extended the lag phase of batch cultures, final biomass yields remained unchanged. FT-IR spectroscopy of whole cells indicated an increase in lipid associated vibrations and decreases in vibrations tentatively assigned to nucleic acids, phosphate, saccharides and amines. MALDI-TOF-MS detected an increase in total protein expression in cultures exposed to 12 Gy. At 95 Gy, a decrease in total protein levels was generally observed, although an increase in a putative cold shock protein was observed, which may be related to the radiation stress response of this organism. Multivariate statistical analyses applied to these FT-IR and MALDI-TOF-MS spectral data suggested that an irradiated phenotype developed throughout subsequent generations. This study suggests that significant alteration to the metabolism of S. oneidensis MR-1 is incurred as a result of X-irradiation and that dose dependent changes to specific biomolecules characterise this response. Irradiated S. oneidensis also displayed enhanced levels of poorly crystalline Fe(III) oxide reduction, though the mechanism underpinning this phenomenon is unclear.

Dynamic of bacterial communities attached to lightened phytodetritus.

The effects of singlet oxygen ((1)O2) transfer to bacteria attached on phytodetritus were investigated under laboratory-controlled conditions. For this purpose, a nonaxenic culture of Emiliania huxleyi in late stationary phase was studied for bacterial viability. Our results indicated that only 9 ± 3% of attached bacteria were alive compared to 46 ± 23% for free bacteria in the E. huxleyi culture. Apparently, under conditions of low irradiance (36 W m(-2)), during the culture, the cumulative dose received (22,000 kJ m(-2)) was sufficiently important to induce an efficient (1)O2 transfer to attached bacteria during the senescence of E. huxleyi cells. At this stage, attached bacteria appeared to be dominated by pigmented bacteria (Maribacter, Roseobacter, Roseovarius), which should resist to (1)O2 stress probably due to their high contents of carotenoids. After subsequent irradiation of the culture until fully photodegradation of chlorophyll, DGGE analyses showed that the diversity of bacteria attached to E. huxleyi cells is modified by light. Photooxidative alterations of bacteria were confirmed by the increasing amounts of cis-vaccenic photoproducts (bacterial marker) per bacteria observed during irradiation time. Interestingly, preliminary chemotaxis experiments showed that Shewanella oneidensis considered here as a model of motile bacteria was attracted by phytodetritus producing or not (1)O2. This lack of repulsive effects could explain the high mortality rate of bacteria measured on E. huxleyi cells.

The impact of γ radiation on the bioavailability of Fe(III) minerals for microbial respiration.

Conservation of energy by Fe(III)-reducing species such as Shewanella oneidensis could potentially control the redox potential of environments relevant to the geological disposal of radioactive waste and radionuclide contaminated land. Such environments will be exposed to ionizing radiation so characterization of radiation alteration to the mineralogy and the resultant impact upon microbial respiration of iron is essential. Radiation induced changes to the iron mineralogy may impact upon microbial respiration and, subsequently, influence the oxidation state of redox-sensitive radionuclides. In the present work, Mössbauer spectroscopy and electron microscopy indicate that irradiation (1 MGy gamma) of 2-line ferrihydrite can lead to conversion to a more crystalline phase, one similar to akaganeite. The room temperature Mössbauer spectrum of irradiated hematite shows the emergence of a paramagnetic Fe(III) phase. Spectrophotometric determination of Fe(II) reveals a radiation-induced increase in the rate and extent of ferrihydrite and hematite reduction by S. oneidensis in the presence of an electron shuttle (riboflavin). Characterization of bioreduced solids via XRD indicate that this additional Fe(II) is incorporated into siderite and ferrous hydroxy carbonate, along with magnetite, in ferrihydrite systems, and siderite in hematite systems. This study suggests that mineralogical changes to ferrihydrite and hematite induced by radiation may lead to an increase in bioavailability of Fe(III) for respiration by Fe(III)-reducing bacteria.

On the influence of the culture conditions in bacterial antifouling bioassays and biofilm properties: Shewanella algae, a case study.

BACKGROUND: A variety of conditions (culture media, inocula, incubation temperatures) are employed in antifouling tests with marine bacteria. Shewanella algae was selected as model organism to evaluate the effect of these parameters on: bacterial growth, biofilm formation, the activity of model antifoulants, and the development and nanomechanical properties of the biofilms.The main objectives were: 1) To highlight and quantify the effect of these conditions on relevant parameters for antifouling studies: biofilm morphology, thickness, roughness, surface coverage, elasticity and adhesion forces. 2) To establish and characterise in detail a biofilm model with a relevant marine strain. RESULTS: Both the medium and the temperature significantly influenced the total cell densities and biofilm biomasses in 24-hour cultures. Likewise, the IC50 of three antifouling standards (TBTO, tralopyril and zinc pyrithione) was significantly affected by the medium and the initial cell density. Four media (Marine Broth, MB; 2% NaCl Mueller-Hinton Broth, MH2; Luria Marine Broth, LMB; and Supplemented Artificial Seawater, SASW) were selected to explore their effect on the morphological and nanomechanical properties of 24-h biofilms. Two biofilm growth patterns were observed: a clear trend to vertical development, with varying thickness and surface coverage in MB, LMB and SASW, and a horizontal, relatively thin film in MH2. The Atomic Force Microscopy analysis showed the lowest Young modulii for MB (0.16 ± 0.10 MPa), followed by SASW (0.19 ± 0.09 MPa), LMB (0.22 ± 0.13 MPa) and MH2 (0.34 ± 0.16 MPa). Adhesion forces followed an inverted trend, being higher in MB (1.33 ± 0.38 nN) and lower in MH2 (0.73 ± 0.29 nN). CONCLUSIONS: All the parameters significantly affected the ability of S. algae to grow and form biofilms, as well as the activity of antifouling molecules. A detailed study has been carried out in order to establish a biofilm model for further assays. The morphology and nanomechanics of S. algae biofilms were markedly influenced by the nutritional environments in which they were developed. As strategies for biofilm formation inhibition and biofilm detachment are of particular interest in antifouling research, the present findings also highlight the need for a careful selection of the assay conditions.

Cu-doped TiO(2) nanoparticles enhance survival of Shewanella oneidensis MR-1 under ultraviolet light (UV) exposure.

It has been shown that photocatalytic TiO(2) nanoparticles (NPs) can be used as an efficient anti-microbial agent under UV light due to generation of reactive oxygen species (ROS), while Shewanella oneidensis MR-1 is a metal-reducing bacterium highly susceptible to UV radiation. Interestingly, we found that the presence of Cu-doped TiO(2) NPs in the cultural medium dramatically increased the survival rates (based on colony-forming unit) of strain MR-1 by over 10,000-fold (incubation without shaking) and ~200 fold (incubation with shaking) after a 2-h exposure to UV light. Gene expression results (via qPCR measurement) indicated that the DNA repair gene recA in MR-1 was significantly induced by UV exposure (indicating cellular damage under UV stress), but the influence of NPs on recA expression was not statistically evident. Plausible explanations to NP attenuation of UV stresses are: 1. TiO(2) based NPs are capable of scattering and absorbing UV light and thus create a shading effect to protect MR-1 from UV radiation; 2. more importantly, Cu-doped TiO(2) NPs can co-agglomerate with MR-1 to form large flocs that improves cells' survival against the environmental stresses. This study improves our understanding of NP ecological impacts under natural solar radiation and provides useful insights to application of photocatalytic-NPs for bacterial disinfection.

A novel filamentous phage from the deep-sea bacterium Shewanella piezotolerans WP3 is induced at low temperature.

Active filamentous phage particles were isolated from the deep-sea bacterium Shewanella piezotolerans WP3. A putative single-stranded DNA binding protein of the phage was found to be overexpressed at 4 degrees C compared to its expression at 25 degrees C by two-dimensional polyacrylamide gel electrophoresis. Reverse transcription quantitative PCR further revealed that the key genes of the SW1 phage were significantly induced at low temperature.

Transcriptome analysis applied to survival of Shewanella oneidensis MR-1 exposed to ionizing radiation.

The ionizing radiation (IR) dose that yields 20% survival (D20) of Shewanella oneidensis MR-1 is lower by factors of 20 and 200 than those for Escherichia coli and Deinococcus radiodurans, respectively. Transcriptome analysis was used to identify the genes of MR-1 responding to 40 Gy (D20). We observed the induction of 170 genes and repression of 87 genes in MR-1 during a 1-h recovery period after irradiation. The genomic response of MR-1 to IR is very similar to its response to UV radiation (254 nm), which included induction of systems involved in DNA repair and prophage synthesis and the absence of differential regulation of tricarboxylic acid cycle activity, which occurs in IR-irradiated D. radiodurans. Furthermore, strong induction of genes encoding antioxidant enzymes in MR-1 was observed. DNA damage may not be the principal cause of high sensitivity to IR, considering that MR-1 carries genes encoding a complex set of DNA repair systems and 40 Gy IR induces less than one double-strand break in its genome. Instead, a combination of oxidative stress, protein damage, and prophage-mediated cell lysis during irradiation and recovery might underlie this organism's great sensitivity to IR.

Genome-wide examination of the natural solar radiation response in Shewanella oneidensis MR-1.

We previously reported that Shewanella oneidensis MR-1 is extremely sensitive to natural solar radiation (NSR). Here we analyzed the global transcriptional profile of MR-1 during a 1-h recovering period after exposure to ambient solar light at a dose that yields about 20% survival rate on a Luria-Bertani (LB) plate. We observed the induction of DNA damage-repair genes, the SOS response as well as detoxification strategies that we previously observed in MR-1 following artificial UV-A irradiation. Few prophage-related genes were induced by natural solar UV radiation, however, in contrast to what was observed following artificial UV-B irradiation. Overall, the cellular response to NSR in MR-1 was more similar to that of UV-A than that of UV-B, but additional genes involved in detoxification were induced compared with induction by either UV-B or UV-A or their sum. Thus, oxidative stress appeared to contribute greatly to the NSR-induced cytotoxic effects in MR-1. A total of 29.1% of genome showed differential expression following NSR exposure, which is much greater than following exposure by UV-B (4.0%), UV-A (8.2%) or their sum (10.7%). Our data suggest that NSR may impact biological processes in a much more complex manner than previously thought.

Effects of a strong static magnetic field on bacterium Shewanella oneidensis: an assessment by using whole genome microarray.

The effect of a strong static 14.1 T magnetic field on log phase cells of bacterial strain Shewanella oneidensis MR-1 was evaluated by using whole genome microarray of this bacterium. Although differences were not observed between the treatment and control by measuring the optical density (OD), colony forming unit (CFU), as well as post-exposure growth of cells, transcriptional expression levels of 65 genes were altered according to our microarray data. Among these genes, 21 were upregulated while other 44 were downregulated, compared with control.

Comparative analysis of differentially expressed genes in Shewanella oneidensis MR-1 following exposure to UVC, UVB, and UVA radiation.

We previously reported that Shewanella oneidensis MR-1 is highly sensitive to UVC (254 nm), UVB (290 to 320 nm), and UVA (320 to 400 nm). Here we delineated the cellular response of MR-1 to UV radiation damage by analyzing the transcriptional profile during a 1-h recovering period after UVC, UVB, and UVA exposure at a dose that yields about a 20% survival rate. Although the SOS response was observed with all three treatments, the induction was more robust in response to short-wavelength UV radiation (UVB and UVC). Similarly, more prophage-related genes were induced by short-wavelength UV radiation. MR-1 showed an active detoxification mechanism in response to UVA, which included the induction of antioxidant enzymes and iron-sequestering proteins to scavenge reactive oxygen species. In addition, a great number of genes encoding multidrug and heavy metal efflux pumps were induced following UVA irradiation. Our data suggested that activation of prophages appears the major lethal factor in MR-1 following UVC or UVB irradiation, whereas oxidative damage contributes greatly to the high UVA sensitivity in MR-1.

How radiation kills cells: survival of Deinococcus radiodurans and Shewanella oneidensis under oxidative stress.

We have recently shown that Deinococcus radiodurans and other radiation resistant bacteria accumulate exceptionally high intracellular manganese and low iron levels. In comparison, the dissimilatory metal-reducing bacterium Shewanella oneidensis accumulates Fe but not Mn and is extremely sensitive to radiation. We have proposed that for Fe-rich, Mn-poor cells killed at radiation doses which cause very little DNA damage, cell death might be induced by the release of Fe(II) from proteins during irradiation, leading to additional cellular damage by Fe(II)-dependent oxidative stress. In contrast, Mn(II) ions concentrated in D. radiodurans might serve as antioxidants that reinforce enzymic systems which defend against oxidative stress during recovery. We extend our hypothesis here to include consideration of respiration, tricarboxylic acid cycle activity, peptide transport and metal reduction, which together with Mn(II) transport represent potential new targets to control recovery from radiation injury.

Global profiling of Shewanella oneidensis MR-1: expression of hypothetical genes and improved functional annotations.

The gamma-proteobacterium Shewanella oneidensis strain MR-1 is a metabolically versatile organism that can reduce a wide range of organic compounds, metal ions, and radionuclides. Similar to most other sequenced organisms, approximately 40% of the predicted ORFs in the S. oneidensis genome were annotated as uncharacterized "hypothetical" genes. We implemented an integrative approach by using experimental and computational analyses to provide more detailed insight into gene function. Global expression profiles were determined for cells after UV irradiation and under aerobic and suboxic growth conditions. Transcriptomic and proteomic analyses confidently identified 538 hypothetical genes as expressed in S. oneidensis cells both as mRNAs and proteins (33% of all predicted hypothetical proteins). Publicly available analysis tools and databases and the expression data were applied to improve the annotation of these genes. The annotation results were scored by using a seven-category schema that ranked both confidence and precision of the functional assignment. We were able to identify homologs for nearly all of these hypothetical proteins (97%), but could confidently assign exact biochemical functions for only 16 proteins (category 1; 3%). Altogether, computational and experimental evidence provided functional assignments or insights for 240 more genes (categories 2-5; 45%). These functional annotations advance our understanding of genes involved in vital cellular processes, including energy conversion, ion transport, secondary metabolism, and signal transduction. We propose that this integrative approach offers a valuable means to undertake the enormous challenge of characterizing the rapidly growing number of hypothetical proteins with each newly sequenced genome.

Survival of Shewanella oneidensis MR-1 after UV radiation exposure.

We systematically investigated the physiological response as well as DNA damage repair and damage tolerance in Shewanella oneidensis MR-1 following UVC, UVB, UVA, and solar light exposure. MR-1 showed the highest UVC sensitivity among Shewanella strains examined, with D37 and D10 values of 5.6 and 16.5% of Escherichia coli K-12 values. Stationary cells did not show an increased UVA resistance compared to exponential-phase cells; instead, they were more sensitive at high UVA dose. UVA-irradiated MR-1 survived better on tryptic soy agar than Luria-Bertani plates regardless of the growth stage. A 20% survival rate of MR-1 was observed following doses of 3.3 J of UVC m(-2), 568 J of UVB m(-2), 25 kJ of UVA m(-2), and 558 J of solar UVB m(-2), respectively. Photoreactivation conferred an increased survival rate to MR-1 of as much as 177- to 365-fold, 11- to 23-fold, and 3- to 10-fold following UVC, UVB, and solar light irradiation, respectively. A significant UV mutability to rifampin resistance was detected in both UVC- and UVB-treated samples, with the mutation frequency in the range of 10(-5) to 10(-6). Unlike in E. coli, the expression levels of the nucleotide excision repair (NER) component genes uvrA, uvrB, and uvrD were not damage inducible in MR-1. Complementation of Pseudomonas aeruginosa UA11079 (uvrA deficient) with uvrA of MR-1 increased the UVC survival of this strain by more than 3 orders of magnitude. Loss of damage inducibility of the NER system appears to contribute to the high sensitivity of this bacterium to UVR as well as to other DNA-damaging agents.