Fumarate reductase is a soluble enzyme in anaerobically grown Shewanella putrefaciens MR-1

Abstract The expression and distribution of fumarate reductase activity was examined in Shewanella putrefaciens MR-1. Fumarate reductase was expressed at very low levels in aerobically grown cell and was markedly induced by growth under anaerobic conditions. Cells were fractionated into soluble and purified membrane components by four different methods. For all four methods used, and in marked contrast to the membrane-bound fumarate reductases of other bacteria, ≧ 98% of the fumarate reductase activity was localized in the soluble fraction. In cells subjected to osmotic shock or treated with lysozyme and EDTA to form spheroplasts, the specific activity of fumarate reductase was highest in the periplasmic fraction, while the majority of total fumarate reductase activity was in the cytoplasmic fraction.     

CpdA is involved in amino acid metabolism in Shewanella oneidensis MR-1

Cyclic 3′,5′-adenosine monophosphate (cAMP) phosphodiesterase (CPD) is an enzyme that catalyzes the hydrolysis of cAMP, a signaling molecule affecting diverse cellular and metabolic processes in bacteria. Some CPDs are also known to function in cAMP-independent manners, while their physiological roles remain largely unknown. Here, we investigated physiological roles of CPD in Shewanella oneidensis MR-1, a model environmental bacterium, and report that CPD is involved in amino-acid metabolism. We found that a CPD-deficient mutant of MR-1 (ΔcpdA) showed decreased expression of genes for the synthesis of methionine, S-adenosylmethionine, and histidine and required these three compounds to grow in minimal media. Interestingly, deletion of adenylate cyclases in ΔcpdA did not restore the ability to grow in minimal media, indicating that the amino acid requirements were not due to the accumulation of cAMP. These results suggest that CPD is involved in the regulation of amino acid metabolism in MR-1 in a cAMP-independent manner.     

Over-expression,characterization, and modification of highly active alkaline phosphatase from a Shewanella genus bacterium

We isolated a Shewanella sp. T3-3 bacterium that yielded highly active alkaline phosphatase (APase). We then cloned the APase gene from Shewanella sp. T3-3 (T3-3AP), and expressed and purified the enzyme from Escherichia coli. Recombinant T3-3AP showed high comparative reactivity on colorimetric (pNPP) and luminescent substrates (PPD and ASP-5). Subsequently, we improved the residual activity after maleimide activation by introducing amino acid substitutions of two Lys residues that were located near the active site. The double mutant enzyme (K161S + K184S) showed much higher residual specific activity after maleimide activation than the wild type enzyme, and had approximately twofold increased sensitivity on sandwich enzyme linked immunosorbent assays (ELISA) compared with calf intestinal APase (CIAP), which is routinely used as a labeling enzyme for ELISA.     

Changes in Shewanella putrefaciens CN32 Membrane Stability upon Growth in the Presence of Soluble Mn(II), V(IV), and U(VI)

In natural reducing environments, such as anoxic sediments and soils, bacteria may be exposed to high concentrations of soluble transition metals. The aim of this study was to identify physiological and biochemical adaptations of Shewanella putrefaciens CN32 membranes to soluble Mn(II), V(IV), and U(VI). We assessed responses of CN32 to these metals, in aerobic and anaerobic cultures, by means of membrane fluidity and fatty acid composition assays. During aerobic growth, all metals had a stabilizing effect on fluidity, while under anoxic conditions this was only observed for bacteria treated with U(VI). Membrane gel-to-fluid phase transition temperatures were higher under anaerobic conditions and were not affected by the metal treatments. Fatty acid desaturation demonstrated linear correlation with significant increases in membrane fluidity, despite metal treatments that did not significantly alter fatty acid chemistry. Scanning transmission X-ray microscopy (STXM) and near-edge X-ray absorption fine structure spectroscopy (NEXAFS) at Mn 2p- and V 2p-edges revealed that both Mn(II) and V(IV) were associated with CN32 membranes, with V(IV) associating as VO²⁺ under anoxic conditions only. The results of this study indicate that the bacterial growth environment greatly impacts membrane chemistry and stability, with overall implications for in vitro as well as in situ studies. Supplemental materials are available for this article. Please go to the publisher's online edition of Geomicrobiology Journal to view the supplemental file.     

Piezoresponse of the cyo-Operon Coding for Quinol Oxidase Subunits in a Deep-sea Piezophilic Bacterium,Shewanella violacea

We have isolated the genes for quinol oxidase from a deep-sea piezophilic bacterium, Shewanella violacea. Analysis of the deduced amino acid sequences of the cyo subunits showed that this oxidase has high similarity to Escherichia coli bo-type quinol oxidase. Northern blot analysis showed that these genes are expressed at a high level when the bacterium is grown at elevated pressure. Upstream in the cyo-operon, a σ⁵⁴-binding motif and an octamer sequence unit were found, suggesting that these elements may play a role in regulation of expression of the cyo-operon in response to changes in pressure.     

Early detection of oxidized surfaces using Shewanella oneidensis MR-1 as a tool

Corrosion is a natural global problem of immense importance. Oxidation of iron and steel not only compromises the structural stability of a widely used and versatile material but it also creates an abrasive compound (iron oxide) that can score the surfaces of metals, rendering them useless for the purpose for which they were designed. Clearly, the identification of corrosion in its nascent stages is a high priority for reasons that range from aesthetics to economics. Many bacteria in the facultatively aerobic genus Shewanella have the capacity to respire some metal oxides, such as iron oxide, by way of a variety of oxide-binding proteins lodged in their outer membrane. In this study, a rapid, cost-effective system for the specific early detection of a variety of oxidized steel surfaces is described, taking advantage of bacteria with natural affinities for iron oxides, to identify the sites of nascent corrosion.     

Purification and Identification of Uracil and 5-Methylcytosine from Chick Embryo DNA

It was found that minor components were approximately 1% of the total deoxyribosides in the analysis for base composition of chick embryo DNA. In order to identify the minor components, this work was done. The separation of the minor components from the major components in a DNA hydrolysate was carried out by celite column chromatography and the final purification was accomplished by paper chromatography. For detection of minor components at the deoxyriboside level, the microbial assay method with Lactobacillus leichmannii ATCC 7830 was applied. Thus, two minor deoxyribosides of the DNA were identical with deoxyuridine and 5-methyldeoxycytidine in spectral, electrophoretic and chromatographic properties, respectively. Furthermore, uracil and 5-methylcytosine were purified and identified from perchloric acid hydrolysate of the DNA.     

Anaerobic production of polyunsaturated fatty acids by Shewanella putrefaciens strain ACAM 342

Abstract The fatty acid composition of cultures of Shewanella putrefaciens strain ACAM 342 grown aero-bically and anaerobically at 15°C and 25°C were analysed by capillary gas chromatography. The bacterium was found to produce the polyunsaturated fatty acids (PUFA) 18:2ω3, 18:3ω3 and 20:5ω3 under aerobic and anaerobic conditions at both growth temperatures. This result suggests that the bacterium possesses both the aerobic and anaerobic pathways for unsaturated fatty acid synthesis, where an alternate terminal electron acceptor(s) is utilised in the absence of oxygen.     

In situ effective diffusion coefficient profiles in live biofilms using pulsed‐field gradient nuclear magnetic resonance

Diffusive mass transfer in biofilms is characterized by the effective diffusion coefficient. It is well documented that the effective diffusion coefficient can vary by location in a biofilm. The current literature is dominated by effective diffusion coefficient measurements for distinct cell clusters and stratified biofilms showing this spatial variation. Regardless of whether distinct cell clusters or surface‐averaging methods are used, position‐dependent measurements of the effective diffusion coefficient are currently: (1) invasive to the biofilm, (2) performed under unnatural conditions, (3) lethal to cells, and/or (4) spatially restricted to only certain regions of the biofilm. Invasive measurements can lead to inaccurate results and prohibit further (time‐dependent) measurements which are important for the mathematical modeling of biofilms. In this study our goals were to: (1) measure the effective diffusion coefficient for water in live biofilms, (2) monitor how the effective diffusion coefficient changes over time under growth conditions, and (3) correlate the effective diffusion coefficient with depth in the biofilm. We measured in situ two‐dimensional effective diffusion coefficient maps within Shewanella oneidensis MR‐1 biofilms using pulsed‐field gradient nuclear magnetic resonance methods, and used them to calculate surface‐averaged relative effective diffusion coefficient (D_rs) profiles. We found that (1) D_rs decreased from the top of the biofilm to the bottom, (2) D_rs profiles differed for biofilms of different ages, (3) D_rs profiles changed over time and generally decreased with time, (4) all the biofilms showed very similar D_rs profiles near the top of the biofilm, and (5) the D_rs profile near the bottom of the biofilm was different for each biofilm. Practically, our results demonstrate that advanced biofilm models should use a variable effective diffusivity which changes with time and location in the biofilm. Biotechnol. Bioeng. 2010;106: 928–937. © 2010 Wiley Periodicals, Inc.