Low-Temperature Growth of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of ≈35°C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature (≈22°C) MR-1 grows with a doubling time of about 40 min, but when moved from 22°C to 3°C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of ≈67 h. In comparison to cells grown at 22°C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22°C.     

Hydrogen Metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of ΔhydA, ΔhyaB, and ΔhydA ΔhyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.     

Low-temperature growth of Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of approximately 35 degrees C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature ( approximately 22 degrees C) MR-1 grows with a doubling time of about 40 min, but when moved from 22 degrees C to 3 degrees C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of approximately 67 h. In comparison to cells grown at 22 degrees C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22 degrees C.     

Hydrogen metabolism in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative sediment microorganism which uses diverse compounds, such as oxygen and fumarate, as well as insoluble Fe(III) and Mn(IV) as electron acceptors. The electron donor spectrum is more limited and includes metabolic end products of primary fermenting bacteria, such as lactate, formate, and hydrogen. While the utilization of hydrogen as an electron donor has been described previously, we report here the formation of hydrogen from pyruvate under anaerobic, stationary-phase conditions in the absence of an external electron acceptor. Genes for the two S. oneidensis MR-1 hydrogenases, hydA, encoding a periplasmic [Fe-Fe] hydrogenase, and hyaB, encoding a periplasmic [Ni-Fe] hydrogenase, were found to be expressed only under anaerobic conditions during early exponential growth and into stationary-phase growth. Analyses of DeltahydA, DeltahyaB, and DeltahydA DeltahyaB in-frame-deletion mutants indicated that HydA functions primarily as a hydrogen-forming hydrogenase while HyaB has a bifunctional role and represents the dominant hydrogenase activity under the experimental conditions tested. Based on results from physiological and genetic experiments, we propose that hydrogen is formed from pyruvate by multiple parallel pathways, one pathway involving formate as an intermediate, pyruvate-formate lyase, and formate-hydrogen lyase, comprised of HydA hydrogenase and formate dehydrogenase, and a formate-independent pathway involving pyruvate dehydrogenase. A reverse electron transport chain is potentially involved in a formate-hydrogen lyase-independent pathway. While pyruvate does not support a fermentative mode of growth in this microorganism, pyruvate, in the absence of an electron acceptor, increased cell viability in anaerobic, stationary-phase cultures, suggesting a role in the survival of S. oneidensis MR-1 under stationary-phase conditions.     

Respiratory nitrate ammonification by Shewanella oneidensis MR-1

Anaerobic cultures of Shewanella oneidensis MR-1 grown with nitrate as the sole electron acceptor exhibited sequential reduction of nitrate to nitrite and then to ammonium. Little dinitrogen and nitrous oxide were detected, and no growth occurred on nitrous oxide. A mutant with the napA gene encoding periplasmic nitrate reductase deleted could not respire or assimilate nitrate and did not express nitrate reductase activity, confirming that the NapA enzyme is the sole nitrate reductase. Hence, S. oneidensis MR-1 conducts respiratory nitrate ammonification, also termed dissimilatory nitrate reduction to ammonium, but not respiratory denitrification.     

Transcriptome analysis of Shewanella oneidensis MR-1 in response to elevated salt conditions

Whole-genomic expression patterns were examined in Shewanella oneidensis cells exposed to elevated sodium chloride. Genes involved in Na(+) extrusion and glutamate biosynthesis were significantly up-regulated, and the majority of chemotaxis/motility-related genes were significantly down-regulated. The data also suggested an important role for metabolic adjustment in salt stress adaptation in S. oneidensis.     

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.     

Functional specificity of extracellular nucleases of Shewanella oneidensis MR-1

Bacterial species such as Shewanella oneidensis MR-1 require extracellular nucleolytic activity for the utilization of extracellular DNA (eDNA) as a source of nutrients and for the turnover of eDNA as a structural matrix component during biofilm formation. We have previously characterized two extracellular nucleases of S. oneidensis MR-1, ExeM and ExeS. Although both are involved in biofilm formation, they are not specifically required for the utilization of eDNA as a nutrient. Here we identified and characterized EndA, a third extracellular nuclease of Shewanella. The heterologously overproduced and purified protein was highly active and rapidly degraded linear and supercoiled DNAs of various origins. Divalent metal ions (Mg(2+) or Mn(2+)) were required for function. endA is cotranscribed with phoA, an extracellular phosphatase, and is not upregulated upon phosphostarvation. Deletion of endA abolished both extracellular degradation of DNA by S. oneidensis MR-1 and the ability to use eDNA as a sole source of phosphorus. PhoA is not strictly required for the exploitation of eDNA as a nutrient. The activity of EndA prevents the formation of large cell aggregates during planktonic growth. However, in contrast to the findings for ExeM, endA deletion had only minor effects on biofilm formation. The findings strongly suggest that the extracellular nucleases of S. oneidensis exert specific functions required under different conditions.     

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.     

Decolorization and detoxification of a sulfonated triphenylmethane dye aniline blue by Shewanella oneidensis MR-1 under anaerobic conditions

In this work, the extracellular decolorization of aniline blue, a sulfonated triphenylmethane dye, by Shewanella oneidensis MR-1 was confirmed. S. oneidensis MR-1 showed a high capacity for decolorizing aniline blue even at a concentration of up to 1,000 mg/l under anaerobic conditions. Maximum decolorization efficiency appeared at pH?7.0 and 30 °C. Lactate was a better candidate of electron donor for the decolorization of aniline blue. The addition of nitrate, hydrous ferric oxide, or trimethylamine N-oxide all could cause a significant decline of decolorization efficiency. The Mtr respiratory pathway was found to be involved into the decolorization of aniline blue by S. oneidensis MR-1. The toxicity evaluation through phytotoxicity and genotoxicity showed that S. oneidensis MR-1 could decrease the toxicity of aniline blue during the decolorization process. Thus, this work may facilitate a better understanding on the degradation mechanisms of the triphenylmethane dyes by Shewanella and is beneficial to their application in bioremediation.     

Butyrate-based n-butanol production from an engineered Shewanella oneidensis MR-1

Bioprocess and Biosystems Engineering - n-Butanol is considered as the next-generation biofuel, because its physiochemical properties are very similar to fossil fuels and it could be produced by...     

High frequency of glucose-utilizing mutants in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 has conventionally been considered unable to use glucose as a carbon substrate for growth. The genome sequence of S. oneidensis MR-1 however suggests the ability to use glucose. Here, we demonstrate that during initial glucose exposure, S. oneidensis MR-1 quickly and frequently gains the ability to utilize glucose as a sole carbon source, in contrast to wild-type S. oneidensis, which cannot immediately use glucose as a sole carbon substrate. High-performance liquid chromatography and (14)C glucose tracer studies confirm the disappearance in cultures and assimilation and respiration, respectively, of glucose. The relatively short time frame with which S. oneidensis MR-1 gained the ability to use glucose raises interesting ecological implications.     

Initial Phases of biofilm formation in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is a facultative Fe(III)- and Mn(IV)-reducing microorganism and serves as a model for studying microbially induced dissolution of Fe or Mn oxide minerals as well as biogeochemical cycles. In soil and sediment environments, S. oneidensis biofilms form on mineral surfaces and are critical for mediating the metabolic interaction between this microbe and insoluble metal oxide phases. In order to develop an understanding of the molecular basis of biofilm formation, we investigated S. oneidensis biofilms developing on glass surfaces in a hydrodynamic flow chamber system. After initial attachment, growth of microcolonies and lateral spreading of biofilm cells on the surface occurred simultaneously within the first 24 h. Once surface coverage was almost complete, biofilm development proceeded with extensive vertical growth, resulting in formation of towering structures giving rise to pronounced three-dimensional architecture. Biofilm development was found to be dependent on the nutrient conditions, suggesting a metabolic control. In global transposon mutagenesis, 173 insertion mutants out of 15,000 mutants screened were identified carrying defects in initial attachment and/or early stages in biofilm formation. Seventy-one of those mutants exhibited a nonswimming phenotype, suggesting a role of swimming motility or motility elements in biofilm formation. Disruption mutations in motility genes (flhB, fliK, and pomA), however, did not affect initial attachment but affected progression of biofilm development into pronounced three-dimensional architecture. In contrast, mutants defective in mannose-sensitive hemagglutinin type IV pilus biosynthesis and in pilus retraction (pilT) showed severe defects in adhesion to abiotic surfaces and biofilm formation, respectively. The results provide a basis for understanding microbe-mineral interactions in natural environments.     

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.     

Phage-induced lysis enhances biofilm formation in Shewanella oneidensis MR-1

Shewanella oneidensis MR-1 is capable of forming highly structured surface-attached communities. By DNase I treatment, we demonstrated that extracellular DNA (eDNA) serves as a structural component in all stages of biofilm formation under static and hydrodynamic conditions. We determined whether eDNA is released through cell lysis mediated by the three prophages LambdaSo, MuSo1 and MuSo2 that are harbored in the genome of S. oneidensis MR-1. Mutant analyses and infection studies revealed that all three prophages may individually lead to cell lysis. However, only LambdaSo and MuSo2 form infectious phage particles. Phage release and cell lysis already occur during early stages of static incubation. A mutant devoid of the prophages was significantly less prone to lysis in pure culture. In addition, the phage-less mutant was severely impaired in biofilm formation through all stages of development, and three-dimensional growth occurred independently of eDNA as a structural component. Thus, we suggest that in S. oneidensis MR-1 prophage-mediated lysis results in the release of crucial biofilm-promoting factors, in particular eDNA.     

The outer membrane cytochromes of Shewanella oneidensis MR-1 are lipoproteins

AIM: To determine if the outer membrane (OM) cytochromes OmcA and OmcB of the metal-reducing bacterium Shewanella oneidensis MR-1 are lipoproteins, and to assess cell surface exposure of the cytochromes by radioiodination. METHODS AND RESULTS: In anaerobic MR-1 cells grown with (3)H-palmitoleic acid, both OmcA and OmcB were radiolabelled. The identities of these bands were confirmed by the absence of each radiolabelled band in the respective mutants lacking individual OM cytochromes. Radioiodination of cell surface proteins in anaerobic cells resulted in (125)I-labelled OmcA. The identity of this band was confirmed by its absence in an OmcA-minus mutant. A ubiquitous radioiodinated band that migrates similarly to OmcB precluded the ability to determine the potential cell surface exposure of OmcB by this method. CONCLUSIONS: Both OmcA and OmcB are lipoproteins, and OmcA is cell surface exposed. SIGNIFICANCE: The lipoprotein modification of these OM cytochromes could be important for their localization or incorporation into the OM. The cell surface exposure of OmcA could allow it to directly transfer electrons to extracellular electron acceptors (e.g. manganese oxides) and is consistent with its in vivo role.