U (VI) tolerance affects Shewanella sp. RCRI7 biological responses: growth, morphology and bioreduction ability.

Native Shewanella sp. RCRI7 is recently counted as an operative bacterium in the uranium bio-reduction. The aim of this study was to investigate the effects of uranium tolerance on the morphology and population of RCRI7, following its potential removal capacity in different time intervals. In this research, the bacterial growth and uranium removal kinetic were evaluated in aerobic TSB medium, uranium-reducing condition (URC), aerobic uranium-containing (AUC) and anaerobic uranium-free (AUF) solution, following evaluations of omcAB gene expressions. In addition, spectrophotometry analyses were performed in URC confirming the bio-reduction mechanism. It was found that the bacteria can grow efficiently in the presence of 0.5 mM uranium anaerobically, unlike AUC and AUF solutions. Since the bacterium's adsorption capacity is quickly saturated, it can be deduced that uranium reduction should be dominant as incubation times proceed up to 84 h in URC. In 92 h incubation, the adsorbed uranium containing unreduced and reduced (U (IV) monomeric), was released to the solution due to either increased pH or bacterial death. In AUC and AUF, improper conditions lead to the reduced bacterial size (coccus-shape formation) and increased bacterial aggregations; however, membrane vesicles produced by the bacteria avoid the uranium incrustation in AUC. In overall, this study implies that Shewanella sp. RCRI7 are well tolerated by uranium under anaerobic conditions and the amount of regenerated uranium increases over time in the reduced form.

Biogenic synthesis of selenium nanoparticles by Shewanella sp. HN-41 using a modified bioelectrochemical system

BACKGROUND Synthesis of selenium nanoparticles from selenite by Shewanella sp. HN-41 demonstrated that particle size depended on the reaction time and biomass of cells. The slow reaction and low biomass tended to form small particles. In this study, Shewanella sp. HN-41 was introduced into the anode of a nonexternal circuit bioelectrochemical system (nec_BES) to convert chemical energy from lactate to low electron current to the cathode, where selenite was reduced. RESULTS Our experiment with two systems, one bioelectrochemical system with a cathode flushed with nitrogen and the other with a no-nitrogen-flushing cathode, showed that the former could not produce Se nanoparticles after 21 d, but the latter formed them with an average size of 37.7 nm. The SEM and TEM images demonstrated that the particle size of 10 nm occupied over 10% and most of the particles were in the range of 30­60 nm. The XRD result and SAED image demonstrated no clear peaks of crystal and proved that the Se nanoparticles are amorphous. CONCLUSIONS : The clean Se nanoparticles were synthesized and completely separated from bacterial cells in the bioelectrochemical system. This study opened a new approach for the biological synthesis of metal nanoparticles. Finally, the Se products in the range of 30­60 nm can be tested for antimicrobial activities in medical applications

Enhanced Bioreduction of Radionuclides by Driving Microbial Extracellular Electron Pumping with an Engineered CRISPR Platform.

Dissimilatory metal-reducing bacteria (DMRB) with extracellular electron transfer (EET) capability show great potential in bioremediating the subsurface environments contaminated by uranium through bioreduction and precipitation of hexavalent uranium [U(VI)]. However, the low EET efficiency of DMRB remains a bottleneck for their applications. Herein, we develop an engineered CRISPR platform to drive the extracellular electron pumping of Shewanella oneidensis, a representative DMRB species widely present in aquatic environments. The CRISPR platform allows for highly efficient and multiplex genome editing and rapid platform elimination post-editing in S. oneidensis. Enabled by such a platform, a genomic promoter engineering strategy (GPS) for genome-widely engineering the EET-encoding gene network was established. The production of electron conductive Mtr complex, synthesis of electron shuttle flavin, and generation of NADH as intracellular electron carrier are globally optimized and promoted, leading to a significantly enhanced EET ability. Applied to U(VI) bioreduction, the edited strains achieve up to 3.62-fold higher reduction capacity over the control. Our work endows DMRB with an enhanced ability to remediate the radionuclides-contaminated environments and provides a gene editing approach to handle the growing environmental challenges of radionuclide contaminations.

Evaluation of mtr cluster expression in Shewanella RCRI7 during uranium removal.

In recent years, bioremediation is considered as an efficient method to remove the pollutants from the industrial wastewater. In this study, quantitative gene expressions (Real-time RT-PCR) of mtr gene cluster (mtrA, mtrB, mtrC, mtrD, mtrE, mtrF and omcA) in five different uranium concentrations (0.1, 0.25, 0.5, 1 and 2 mM) were performed with ICP and microscopic live cell counting analysis under anaerobic condition, by Shewanella RCRI7 as a native bacterium. The results indicated that the amount of uranium removal and live-cell counting were decreased in the higher uranium concentrations (1 and 2 mM), due to the uranium toxicity, suggesting 0.5 mM as the optimum uranium concentration for Shewanella RCRI7 resistance. The expression of mtrCED and omcA genes presented increasing trend in the lower uranium concentrations (0.1, 0.25 and 0.5 mM) and a decreasing trend in 1 and 2 mM, while mtrABF, presented an inverse pattern, proving the alternative role of mtrF for mtrC and omcA, as the substantial multiheme cytochromes in Extracellular Electron Transfer (EET) pathway. These data are a proof of these gene vital roles in the EET pathway, proposing them for genetic engineering toward EET optimization, as the certain pathway in heavy metal bioremediation process.

Shewanella algae Relatives Capable of Generating Electricity from Acetate Contribute to Coastal-Sediment Microbial Fuel Cells Treating Complex Organic Matter.

To identify exoelectrogens involved in the generation of electricity from complex organic matter in coastal sediment (CS) microbial fuel cells (MFCs), MFCs were inoculated with CS obtained from tidal flats and estuaries in the Tokyo bay and supplemented with starch, peptone, and fish extract as substrates. Power output was dependent on the CS used as inocula and ranged between 100 and 600 mW m-2 (based on the projected area of the anode). Analyses of anode microbiomes using 16S rRNA gene amplicons revealed that the read abundance of some bacteria, including those related to Shewanella algae, positively correlated with power outputs from MFCs. Some fermentative bacteria were also detected as major populations in anode microbiomes. A bacterial strain related to S. algae was isolated from MFC using an electrode plate-culture device, and pure-culture experiments demonstrated that this strain exhibited the ability to generate electricity from organic acids, including acetate. These results suggest that acetate-oxidizing S. algae relatives generate electricity from fermentation products in CS-MFCs that decompose complex organic matter.

Differential gene content and gene expression for bacterial evolution and speciation of Shewanella in terms of biosynthesis of heme and heme-requiring proteins.

BACKGROUND: Most species of Shewanella harbor two ferrochelatase paralogues for the biosynthesis of c-type cytochromes, which are crucial for their respiratory versatility. In our previous study of the Shewanella loihica PV-4 strain, we found that the disruption of hemH1 but not hemH2 resulted in a significant accumulation of extracellular protoporphyrin IX (PPIX), but it is different in Shewanella oneidensis MR-1. Hence, the function and transcriptional regulation of two ferrochelatase genes, hemH1 and hemH2, are investigated in S. oneidensis MR-1. RESULT: In the present study, deletion of either hemH1 or hemH2 in S. oneidensis MR-1 did not lead to overproduction of extracellular protoporphyrin IX (PPIX) as previously described in the hemH1 mutants of S. loihica PV-4. Moreover, supplement of exogenous hemins made it possible to generate the hemH1 and hemH2 double mutant in MR-1, but not in PV-4. Under aerobic condition, exogenous hemins were required for the growth of MR-1ΔhemH1ΔhemH2, which also overproduced extracellular PPIX. These results suggest that heme is essential for aerobic growth of Shewanella species and MR-1 could also uptake hemin for biosynthesis of essential cytochrome(s) and respiration. Besides, the exogenous hemin mediated CymA cytochrome maturation and the cellular KatB catalase activity. Both hemH paralogues were transcribed in wild-type MR-1, and the hemH2 transcription was remarkably up-regulated in MR-1ΔhemH1 mutant to compensate for the loss of hemH1. The periplasmic glutathione peroxidase gene pgpD, located in the same operon with hemH2, and a large gene cluster coding for iron, heme (hemin) uptake systems are absent in the PV-4 genome. CONCLUSION: Our results indicate that the genetic divergence in gene content and gene expression between these Shewanella species, accounting for the phenotypic difference described here, might be due to their speciation and adaptation to the specific habitats (iron-rich deep-sea vent versus iron-poor freshwater) in which they evolved and the generated mutants could potentially be utilized for commercial production of PPIX.

Secreted Flavin Cofactors for Anaerobic Respiration of Fumarate and Urocanate by Shewanella oneidensis: Cost and Role.

Shewanella oneidensis strain MR-1, a facultative anaerobe and model organism for dissimilatory metal reduction, uses a periplasmic flavocytochrome, FccA, both as a terminal fumarate reductase and as a periplasmic electron transfer hub for extracellular respiration of a variety of substrates. It is currently unclear how maturation of FccA and other periplasmic flavoproteins is achieved, specifically in the context of flavin cofactor loading, and the fitness cost of flavin secretion has not been quantified. We demonstrate that deletion of the inner membrane flavin adenine dinucleotide (FAD) exporter Bfe results in a 23% slower growth rate than that of the wild type during fumarate respiration and an 80 to 90% loss in fumarate reductase activity. Exogenous flavin supplementation does not restore FccA activity in a Δbfe mutant unless the gene encoding the periplasmic FAD hydrolase UshA is also deleted. We demonstrate that the small Bfe-independent pool of FccA is sufficient for anaerobic growth with fumarate. Strains lacking Bfe were unable to grow using urocanate as the sole electron acceptor, which relies on the periplasmic flavoprotein UrdA. We show that periplasmic flavoprotein maturation occurs in careful balance with periplasmic FAD hydrolysis, and that the current model for periplasmic flavin cofactor loading must account for a Bfe-independent mechanism for flavin transport. Finally, we determine that the metabolic burden of flavin secretion is not significant during growth with flavin-independent anaerobic electron acceptors. Our work helps frame the physiological motivations that drove evolution of flavin secretion by ShewanellaIMPORTANCEShewanella species are prevalent in marine and aquatic environments, throughout stratified water columns, in mineral-rich sediments, and in association with multicellular marine and aquatic organisms. The diversity of niches shewanellae can occupy are due largely to their respiratory versatility. Shewanella oneidensis is a model organism for dissimilatory metal reduction and can respire a diverse array of organic and inorganic compounds, including dissolved and solid metal oxides. The fumarate reductase FccA is a highly abundant multifunctional periplasmic protein that acts to bridge the periplasm and temporarily store electrons in a variety of respiratory nodes, including metal, nitrate, and dimethyl sulfoxide respiration. However, maturation of this central protein, particularly flavin cofactor acquisition, is poorly understood. Here, we quantify the fitness cost of flavin secretion and describe how free flavins are acquired by FccA and a homologous periplasmic flavoprotein, UrdA.

Genomic Analysis of Shewanella sp. O23S-The Natural Host of the pSheB Plasmid Carrying Genes for Arsenic Resistance and Dissimilatory Reduction.

Shewanella sp. O23S is a dissimilatory arsenate reducing bacterial strain involved in arsenic transformations within the abandoned gold mine in Zloty Stok (SW Poland). Previous physiological studies revealed that O23S may not only release arsenic from minerals, but also facilitate its immobilization through co-precipitation with reduced sulfur species. Given these uncommon, complementary characteristics and the application potential of the strain in arsenic-removal technologies, its genome (~5.3 Mbp), consisting of a single chromosome, two large plasmids (pSheA and pSheB) and three small plasmid-like phages (pSheC-E) was sequenced and annotated. Genes encoding putative proteins involved in heavy metal transformations, antibiotic resistance and other phenotypic traits were identified. An in-depth comparative analysis of arsenic respiration (arr) and resistance (ars) genes and their genetic context was also performed, revealing that pSheB carries the only copy of the arr genes, and a complete ars operon. The plasmid pSheB is therefore a unique natural vector of these genes, providing the host cells arsenic respiration and resistance abilities. The functionality of the identified genes was determined based on the results of the previous and additional physiological studies, including: the assessment of heavy metal and antibiotic resistance under various conditions, adhesion-biofilm formation assay and BiologTM metabolic preferences test. This combined genetic and physiological approach shed a new light on the capabilities of O23S and their molecular basis, and helped to confirm the biosafety of the strain in relation to its application in bioremediation technologies.

Evaluation of anionic surfactant removal in anaerobic reactor with Fe(III) supplementation.

The objective of this study was to evaluate the removal of linear alkylbenzene sulfonate (LAS) associated with Fe(III) supplementation using an expanded granular sludge bed (EGSB) reactor. The reactor was inoculated with a granular sludge and fed with synthetic wastewater containing a specific LAS load rate (SLLR) of 1.5 mg gVS-1 d-1 (∼16.4 mgLAS L-1 influent) and supplied with 7276 µMol L-1 of Fe(III). The biomasses from the inoculum and at the end of the EGSB-Fe operation (127 days) were characterized using 16S rRNA Ion Tag sequencing. An increase of 20% in the removal efficiency was observed compared to reactors without Fe(III) supplementation that was reported in the literature, and the LAS removal was approximately 84%. The Fe(III) reduction was dissimilatory (the total iron concentration in the influent and effluent were similar) and reached approximately 64%. The higher Fe(III) reduction and LAS removal were corroborated by the enrichment of genera, such as Shewanella (only EGSB-Fe - 0.5%) and Geobacter (1% - inoculum; 18% - EGSB-Fe). Furthermore, the enrichment of genera that degrade LAS and/or aromatic compounds (3.8% - inoculum; 29.6% - EGSB-Fe of relative abundance) was observed for a total of 20 different genera.

Inhibition of quorum sensing, biofilm, and spoilage potential in Shewanella baltica by green tea polyphenols.

We investigated the quorum sensing (QS) system of Shewanella baltica and the anti-QS related activities of green tea polyphenols (TP) against spoilage bacteria in refrigerated large yellow croaker. Autoinducer-2 (AI-2) and the diketopiperazines (DKPs) cyclo-(L-Pro-L-Leu) and cyclo-(L-Pro-L-Phe) were detected in the culture extract of S. baltica XH2, however, no N-acylhomoserine lactones (AHLs) activity was observed. Green TP at sub-inhibitory concentrations interfered with AI-2 and DKPs activities of S. baltica without inhibiting cell growth and promoted degradation of AI-2. The green TP treatment inhibited biofilm development, exopolysaccharide production and swimming motility of S. baltica in a concentration- dependent manner. In addition, green TP decreased extracellular protease activities and trimethylamine production in S. baltica. A transcriptional analysis showed that green TP repressed the luxS and torA genes in S. baltica, which agreed with the observed reductions in QS activity and the spoilage phenotype. Epigallocatechin gallate (EGCG)-enriched in green TP significantly inhibited AI-2 activity of S. baltica. These findings strongly suggest that green TP could be developed as a new QS inhibitor for seafood preservation to enhance shelf life.

[Biosynthesis of selemium nanobars by Shewallena oneidensis MR-1].

OBJECTIVE: We used Shewallena oneidensis MR-1 to produce selemium (Se) nanobars and studied the influence of Se(IV) concentrations and incubation time on nanobars production. METHODS: We incubated Shewallena oneidensis MR-1 under anaerobic condition with Luria-Bertani (LB) liquid medium containing 0.1, 1.0, 10.0 or 100.0 mmol/L Se (IV) in Na2SeO3, to determine the optimal Se (IV) concentration for bacterial growth. Then, we incubated Shewallena oneidensis MR-1 with the optimal Se (IV) concentration and collected deposits 24 and 72 h after anearobic incubation. We used scanning electron microscopy, energy-dispersive X-ray and X-ray diffraction to analyse the deposits. RESULTS: The cross sectional diameter and length of deposits that were produced by Shewallena oneidensis MR-1 after 24 h incubation with 1 mmol/L Se(IV) was around 80 nm and 2-3 µm, respectively. However, the deposits after 72 h incubation exceeded the size limit of nano material. Furthermore, the energy-dispersive X-ray and the X-ray diffraction spectroscopy confirmed that the deposits were elemental Se. CONCLUSION: This study provides a viable method for the biosynthesis of Se nanoban Shewallena oneidensis MR-1 can produce a large number of Se nanobars at exponential phase under 0.1 mmol/L Se (IV).

Engineering of EPA/DHA omega-3 fatty acid production by Lactococcus lactis subsp. cremoris MG1363.

Eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) have been shown to be of major importance in human health. Therefore, these essential polyunsaturated fatty acids have received considerable attention in both human and farm animal nutrition. Currently, fish and fish oils are the main dietary sources of EPA/DHA. To generate sustainable novel sources for EPA and DHA, the 35-kb EPA/DHA synthesis gene cluster was isolated from a marine bacterium, Shewanella baltica MAC1. To streamline the introduction of the genes into food-grade microorganisms such as lactic acid bacteria, unnecessary genes located upstream and downstream of the EPA/DHA gene cluster were deleted. Recombinant Escherichia coli harboring the 20-kb gene cluster produced 3.5- to 6.1-fold more EPA than those carrying the 35-kb DNA fragment coding for EPA/DHA synthesis. The 20-kb EPA/DHA gene cluster was cloned into a modified broad-host-range low copy number vector, pIL252m (4.7 kb, Ery) and expressed in Lactococcus lactis subsp. cremoris MG1363. Recombinant L. lactis produced DHA (1.35 ± 0.5 mg g(-1) cell dry weight) and EPA (0.12 ± 0.04 mg g(-1) cell dry weight). This is believed to be the first successful cloning and expression of EPA/DHA synthesis gene cluster in lactic acid bacteria. Our findings advance the future use of EPA/DHA-producing lactic acid bacteria in such applications as dairy starters, silage adjuncts, and animal feed supplements.

Effects of the anaerobic respiration of Shewanella oneidensis MR-1 on the stability of extracellular U(VI) nanofibers.

Uranium (VI) is considered to be one of the most widely dispersed and problematic environmental contaminants, due in large part to its high solubility and great mobility in natural aquatic systems. We previously reported that under anaerobic conditions, Shewanella oneidensis MR-1 grown in medium containing uranyl acetate rapidly accumulated long, extracellular, ultrafine U(VI) nanofibers composed of polycrystalline chains of discrete meta-schoepite (UO(3)·2H2O) nanocrystallites. Wild-type MR-1 finally transformed the uranium (VI) nanofibers to uranium (IV) nanoparticles via further reduction. In order to investigate the influence of the respiratory chain in the uranium transformation process, a series of mutant strains lacking a periplasmic cytochrome MtrA, outer membrane (OM) cytochrome MtrC and OmcA, a tetraheme cytochrome CymA anchored to the cytoplasmic membrane, and a trans-OM protein MtrB, were tested in this study. Although all the mutants produced U(VI) nanofibers like the wild type, the transformation rates from U(VI) nanofibers to U(IV) nanoparticles varied; in particular, the mutant with deletion in tetraheme cytochrome CymA stably maintained the uranium (VI) nanofibers, suggesting that the respiratory chain of S. oneidensis MR-1 is probably involved in the stability of extracellular U(VI) nanofibers, which might be easily treated via the physical processes of filtration or flocculation for the remediation of uranium contamination in sediments and aquifers, as well as the recovery of uranium in manufacturing processes.

Linking bacterial diversity and geochemistry of uranium-contaminated groundwater.

To understand the link between bacterial diversity and geochemistry in uranium-contaminated groundwater, microbial communities were assessed based on clone libraries of 16S rDNA genes from the USDOE Oak Ridge Field Research Centre (FRC) site. Four groundwater wells (GW835, GW836, FW113-47 and FW215-49) with a wide range of pH (3 to 7), nitrate (44 to 23,400 mg L(-1)), uranium (0.73 to 60.36 mg L(-1)) and other metal contamination, were investigated. Results indicated that bacterial diversity correlated with the geochemistry of the groundwater. Microbial diversity decreased in relation to the contamination levels of the wells. The highly contaminated well (FW113-47) had lower gene diversity than less contaminated wells (FW215-49, GW835 and GW836). The high concentrations of contaminants present in well FW113-47 stimulated the growth of organisms capable of reducing uranium (Shewanella and Pseudomonas), nitrate (Pseudomonas, Rhodanobacter and Xanthomonas) and iron (Stenotrophomonas), and which were unique to this well. The clone libraries consisted primarily of sequences closely related to the phylum Proteobacteria, with FW-113-47 almost exclusively containing this phylum. Metal-reducing bacteria were present in all four wells, which may suggest that there is potential for successful bioremediation of the groundwater at the Oak Ridge FRC. The microbial community information gained from this study and previous studies at the site can be used to develop predictive multivariate and geographical information system (GIS) based models for microbial populations at the Oak Ridge FRC. This will allow for a better understanding of what organisms are likely to occur where and when, based on geochemistry, and how these organisms relate to bioremediation processes at the site.

Roles of siderophore in manganese-oxide reduction by Shewanella oneidensis MR-1.

Dissimilatory metal-reducing bacteria (DMRB), such as Shewanella oneidensis MR-1, are of great interest for their importance in the biogeochemical cycling of metals and utility in biotechnological processes, such as bioremediation and microbial fuel cells. To identify genes necessary for metal reduction, this study constructed a random transposon-insertion mutant library of MR-1 and screened it for isolating mutants that were deficient in metal reduction. Examination of approximately 5000 mutants on lactate minimal-medium plates containing MnO(2) resulted in the isolation of one mutant, strain N22-7, that showed a decreased MnO(2)-reduction activity. Determination of a transposon-insertion site in N22-7 followed by deletion and complementation experiments revealed that the disruption of SO3030, a siderophore biosynthesis gene, was responsible for the decreased MnO(2)-reduction activity. In ΔSO3030 cells, iron and cytochrome contents were decreased to approximately 50% of those in the wild-type cells, when they were incubated under MnO(2)-reduction conditions. In addition, the transcription of genes encoding outer-membrane cytochromes necessary for metal reduction was repressed in ΔSO3030 under MnO(2)-reduction conditions, while their transcription was upregulated after supplementation of culture media with ferrous iron. These results suggest that siderophore is important for S. oneidensis MR-1 to respire MnO(2), because iron availability influences the expression of cytochromes necessary for metal reduction.

Large-scale comparative phenotypic and genomic analyses reveal ecological preferences of shewanella species and identify metabolic pathways conserved at the genus level.

The use of comparative genomics for the study of different microbiological species has increased substantially as sequence technologies become more affordable. However, efforts to fully link a genotype to its phenotype remain limited to the development of one mutant at a time. In this study, we provided a high-throughput alternative to this limiting step by coupling comparative genomics to the use of phenotype arrays for five sequenced Shewanella strains. Positive phenotypes were obtained for 441 nutrients (C, N, P, and S sources), with N-based compounds being the most utilized for all strains. Many genes and pathways predicted by genome analyses were confirmed with the comparative phenotype assay, and three degradation pathways believed to be missing in Shewanella were confirmed as missing. A number of previously unknown gene products were predicted to be parts of pathways or to have a function, expanding the number of gene targets for future genetic analyses. Ecologically, the comparative high-throughput phenotype analysis provided insights into niche specialization among the five different strains. For example, Shewanella amazonensis strain SB2B, isolated from the Amazon River delta, was capable of utilizing 60 C compounds, whereas Shewanella sp. strain W3-18-1, isolated from deep marine sediment, utilized only 25 of them. In spite of the large number of nutrient sources yielding positive results, our study indicated that except for the N sources, they were not sufficiently informative to predict growth phenotypes from increasing evolutionary distances. Our results indicate the importance of phenotypic evaluation for confirming genome predictions. This strategy will accelerate the functional discovery of genes and provide an ecological framework for microbial genome sequencing projects.

Identification and characterization of UndAHRCR-6, an outer membrane endecaheme c-type cytochrome of Shewanella sp. strain HRCR-6.

UndA(HRCR-6) was identified from the metal-reducing bacterium Shewanella sp. strain HRCR-6. Both in vivo and in vitro characterization results indicate that UndA(HRCR-6) is an outer membrane endecaheme c-type cytochrome and probably has a key functional role in the extracellular reduction of iron [Fe(III)] oxides and uranium [U(VI)] by Shewanella sp. HRCR-6.

Roles of two Shewanella oneidensis MR-1 extracellular endonucleases.

The dissimilatory iron-reducing bacterium Shewanella oneidensis MR-1 is capable of using extracellular DNA (eDNA) as the sole source of carbon, phosphorus, and nitrogen. In addition, we recently demonstrated that S. oneidensis MR-1 requires eDNA as a structural component during all stages of biofilm formation. In this study, we characterize the roles of two Shewanella extracellular endonucleases, ExeS and ExeM. While ExeS is likely secreted into the medium, ExeM is predicted to remain associated with the cell envelope. Both exeM and exeS are highly expressed under phosphate-limited conditions. Mutants lacking exeS and/or exeM exhibit decreased eDNA degradation; however, the capability of S. oneidensis MR-1 to use DNA as the sole source of phosphorus is only affected in mutants lacking exeM. Neither of the two endonucleases alleviates toxic effects of increased eDNA concentrations. The deletion of exeM and/or exeS significantly affects biofilm formation of S. oneidensis MR-1 under static conditions, and expression of exeM and exeS drastically increases during static biofilm formation. Under hydrodynamic conditions, a deletion of exeM leads to altered biofilms that consist of densely packed structures which are covered by a thick layer of eDNA. Based on these results, we hypothesize that a major role of ExeS and, in particular, ExeM of S. oneidensis MR-1, is to degrade eDNA as a matrix component during biofilm formation to improve nutrient supply and to enable detachment.

Favourable effects of eicosapentaenoic acid on the late step of the cell division in a piezophilic bacterium, Shewanella violacea DSS12, at high-hydrostatic pressures.

Shewanella violacea DSS12, a deep-sea bacterium, produces eicosapentaenoic acid (EPA) as a component of membrane phospholipids. Although various isolates from the deep sea, such as Photobacterium profundum SS9, Colwellia psychrerythraea 34H and various Shewanella strains, produce EPA- or docosahexaenoic acid-containing phospholipids, the physiological role of these polyunsaturated fatty acids remains unclear. In this article, we illustrate the physiological importance of EPA for high-pressure adaptation in strain DSS12 with the help of an EPA-deficient mutant (DSS12(pfaA)). DSS12(pfaA) showed significant growth retardation at 30 MPa, but not at 0.1 MPa. We also found that DSS12(pfaA) grown at 30 MPa forms filamentous cells. When an EPA-containing phospholipid (sn-1-oleoly-sn-2-eicosapentaenoyl phosphatidylethanolamine) was supplemented, the growth retardation and the morphological defect of DSS12(pfaA) were suppressed, indicating that the externally added EPA-containing phospholipid compensated for the loss of endogenous EPA. In contrast, the addition of an oleic acid-containing phospholipid (sn-1,2-dioleoyl phosphatidylethanolamine) did not affect the growth and the morphology of the cells. Immunofluorescent microscopic analysis with anti-FtsZ antibody revealed a number of Z-rings and separated nucleoids in DSS12(pfaA) grown at 30 MPa. These results demonstrate the physiological importance of EPA for the later step of Z-ring formation of S. violacea DSS12 under high-pressure conditions.

Biodegradation of crystal violet by a Shewanella sp. NTOU1.

A bacterial isolate, strain NTOU1, originally isolated from the cooling system in an oil refinery could decolorize and detoxify crystal violet under anaerobic conditions. The strain was characterized and identified as a member of Shewanella decolorationis based on Gram staining, morphology characters, biochemical tests, the 16S rRNA gene and the gyrase subunit beta gene (gyrB). The optimum pH value and temperature for decolorization of crystal violet by this strain under anaerobic conditions were pH 8-9 and 30-40 degrees C, respectively. Formate (20 mM) was the best electron donor. Addition of ferric citrate did not inhibit decolorization of crystal violet, the addition of thiosulfate, ferric oxide, or manganese oxide slightly decreased decolorization, while addition of nitrite (20 mM) inhibited the decolorization of crystal violet. By supplementing the medium with formate and ferric citrate and cultivating it under optimum pH and temperature, this strain could remove crystal violet, at a concentration of 1500 mg l(-1), at the rate of 298 mg l(-1) h(-1) (during decolorization the OD(600) of the cell culture increased from approximately 0.6 to approximately 1.2). GC/MS analysis of the degradation products of crystal violet detected the presence of N,N'-bis(dimethylamino) benzophenone (Michler's Ketone), [N,N-dimethylaminophenyl] [N-methylaminophenyl] benzophenone, N,N-dimethylaminobenzaldehyde, N,N-dimethylaminophenol, and 4-methylaminophenol. These results suggest that crystal violet was biotransformed into N,N-dimethylaminophenol and Michler's Ketone prior to further degradation of these intermediates. This paper proposes a probable pathway for the degradation of crystal violet by this Shewanella sp. Cytotoxicity and antimicrobial tests showed that the process of decolorization also detoxify crystal violet.