Role of the tetraheme cytochrome CymA in anaerobic electron transport in cells of Shewanella putrefaciens MR-1 with normal levels of menaquinone.
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Shewanella putrefaciens MR-1 possesses a complex electron transport system which facilitates its ability to use a diverse array of compounds as terminal electron acceptors for anaerobic respiration. A previous report described a mutant strain (CMTn-1) deficient in CymA, a tetraheme cytochrome c. However, the interpretation of the electron transport role of CymA was complicated by the fact that CMTn-1 was also markedly deficient in menaquinones. This report demonstrates that the depressed menaquinone levels were the result of the rifampin resistance phenotype of the parent of CMTn-1 and not the interruption of the cymA gene. This is the first report of rifampin resistance leading to decreased menaquinone levels, indicating that rifampin-resistant strains should be used with caution when analyzing electron transport processes. A site-directed gene replacement approach was used to isolate a cymA knockout strain (MR1-CYMA) directly from MR-1. While MR1-CYMA retained menaquinone levels comparable to those of MR-1, it lost the ability to reduce iron(III), manganese(IV), and nitrate and to grow by using fumarate as an electron acceptor. All of these functions were restored to wild-type efficacy, and the presence of the cymA transcript and CymA protein was also restored, by complementation of MR1-CYMA with the cymA gene. The requirement for CymA in anaerobic electron transport to iron(III), fumarate, nitrate, and manganese(IV) is therefore not dependent on the levels of menaquinone in these cells. This represents the first successful use of a suicide vector for directed gene replacement in MR-1. (+info)
Effect of electron donor and solution chemistry on products of dissimilatory reduction of technetium by Shewanella putrefaciens.
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To help provide a fundamental basis for use of microbial dissimilatory reduction processes in separating or immobilizing (99)Tc in waste or groundwaters, the effects of electron donor and the presence of the bicarbonate ion on the rate and extent of pertechnetate ion [Tc(VII)O(4)(-)] enzymatic reduction by the subsurface metal-reducing bacterium Shewanella putrefaciens CN32 were determined, and the forms of aqueous and solid-phase reduction products were evaluated through a combination of high-resolution transmission electron microscopy, X-ray absorption spectroscopy, and thermodynamic calculations. When H(2) served as the electron donor, dissolved Tc(VII) was rapidly reduced to amorphous Tc(IV) hydrous oxide, which was largely associated with the cell in unbuffered 0. 85% NaCl and with extracellular particulates (0.2 to 0.001 microm) in bicarbonate buffer. Cell-associated Tc was present principally in the periplasm and outside the outer membrane. The reduction rate was much lower when lactate was the electron donor, with extracellular Tc(IV) hydrous oxide the dominant solid-phase reduction product, but in bicarbonate systems much less Tc(IV) was associated directly with the cell and solid-phase Tc(IV) carbonate may have been present. In the presence of carbonate, soluble (<0.001 microm) electronegative, Tc(IV) carbonate complexes were also formed that exceeded Tc(VII)O(4)(-) in electrophoretic mobility. Thermodynamic calculations indicate that the dominant reduced Tc species identified in the experiments would be stable over a range of E(h) and pH conditions typical of natural waters. Thus, carbonate complexes may represent an important pathway for Tc transport in anaerobic subsurface environments, where it has generally been assumed that Tc mobility is controlled by low-solubility Tc(IV) hydrous oxide and adsorptive, aqueous Tc(IV) hydrolysis products. (+info)
A new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon and its comparison with white spot syndrome (WSS) caused by virus.
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This paper describes a new bacterial white spot syndrome (BWSS) in cultured tiger shrimp Penaeus monodon. The affected shrimp showed white spots similar to those caused by white spot syndrome virus (WSSV), but the shrimp remained active and grew normally without significant mortalities. The study revealed no evidence of WSSV infection using electron microscopy, histopathology and nested polymerase chain reaction. Electron microscopy indicated bacteria associated with white spot formation, and with degeneration and discoloration of the cuticle as a result of erosion of the epicuticle and underlying cuticular layers. Grossly the white spots in BWSS and WSS look similar but showed different profiles under wet mount microscopy. The bacterial white spots were lichen-like, having perforated centers unlike the melanized dots in WSSV-induced white spots. Bacteriological examination showed that the dominant isolate in the lesions was Bacillus subtilis. The occurrence of BWSS may be associated with the regular use of probiotics containing B. subtilis in shrimp ponds. The externally induced white spot lesions were localized at the integumental tissues, i.e., cuticle and epidermis, and connective tissues. Damage to the deeper tissues was limited. The BWS lesions are non-fatal in the absence of other complications and are usually shed through molting. (+info)
Role for outer membrane cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in reduction of manganese dioxide.
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Shewanella putrefaciens MR-1 can use a wide variety of terminal electron acceptors for anaerobic respiration, including certain insoluble manganese and iron oxides. To examine whether the outer membrane (OM) cytochromes of MR-1 play a role in Mn(IV) and Fe(III) reduction, mutants lacking the OM cytochrome OmcA or OmcB were isolated by gene replacement. Southern blotting and PCR confirmed replacement of the omcA and omcB genes, respectively, and reverse transcription-PCR analysis demonstrated loss of the respective mRNAs, whereas mRNAs for upstream and downstream genes were retained. The omcA mutant (OMCA1) resembled MR-1 in its growth on trimethylamine N-oxide (TMAO), dimethyl sulfoxide, nitrate, fumarate, thiosulfate, and tetrathionate and its reduction of nitrate, nitrite, ferric citrate, FeOOH, and anthraquinone-2,6-disulfonic acid. Similarly, the omcB mutant (OMCB1) grew on fumarate, nitrate, TMAO, and thiosulfate and reduced ferric citrate and FeOOH. However, OMCA1 and OMCB1 were 45 and 75% slower than MR-1, respectively, at reducing MnO(2). OMCA1 lacked only OmcA. While OMCB1 lacked OmcB, other OM cytochromes were also missing or markedly depressed. The total cytochrome content of the OM of OMCB1 was less than 15% of that of MR-1. Western blots demonstrated that OMCB1 still synthesized OmcA, but most of it was localized in the cytoplasmic membrane and soluble fractions rather than in the OM. OMCB1 had therefore lost the ability to properly localize multiple OM cytochromes to the OM. Together, the results suggest that the OM cytochromes of MR-1 participate in the reduction of Mn(IV) but are not required for the reduction of Fe(III) or other electron acceptors. (+info)
Prevalence of nonfermenters in clinical specimens.
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One hundred and thirty three non fermenting gram negative bacilli isolated out of 625 different clinical specimens were identified and characterised. Samples were exudate from chronic suppractive otits media (341), diabetic foot (117) wound (116) and blood (51). Of these isolates Pseudomonas aeruginosa 105(78.94%) predominated followed by Acinetobacter sp 8 [6.1%], Pseudomonas putrifaciens 6(4.5%), Flavobacterium sp 6(4.5%), Xanthomonas maltophilia 5(3.75%), Alkaligenes sp 3 (2.25). 31 (23.30%) were resistant to commonly used antibiotics. Amikacin 85 (63.90%) was found to be more effective than fluoroquinolones (27.8-48.12%). (+info)
Shewanella putrefaciens adhesion and biofilm formation on food processing surfaces.
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Laboratory model systems were developed for studying Shewanella putrefaciens adhesion and biofilm formation under batch and flow conditions. S. putrefaciens plays a major role in food spoilage and may cause microbially induced corrosion on steel surfaces. S. putrefaciens bacteria suspended in buffer adhered readily to stainless steel surfaces. Maximum numbers of adherent bacteria per square centimeter were reached in 8 h at 25 degrees C and reflected the cell density in suspension. Numbers of adhering bacteria from a suspension containing 10(8) CFU/ml were much lower in a laminar flow system (modified Robbins device) (reaching 10(2) CFU/cm(2)) than in a batch system (reaching 10(7) CFU/cm(2)), and maximum numbers were reached after 24 h. When nutrients were supplied, S. putrefaciens grew in biofilms with layers of bacteria. The rate of biofilm formation and the thickness of the film were not dependent on the availability of carbohydrate (lactate or glucose) or on iron starvation. The number of S. putrefaciens bacteria on the surface was partly influenced by the presence of other bacteria (Pseudomonas fluorescens) which reduced the numbers of S. putrefaciens bacteria in the biofilm. Numbers of bacteria on the surface must be quantified to evaluate the influence of environmental factors on adhesion and biofilm formation. We used a combination of fluorescence microscopy (4',6'-diamidino-2-phenylindole staining and in situ hybridization, for mixed-culture studies), ultrasonic removal of bacteria from surfaces, and indirect conductometry and found this combination sufficient to quantify bacteria on surfaces. (+info)
Isolation and characterization of a Shewanella putrefaciens MR-1 electron transport regulator etrA mutant: reassessment of the role of EtrA.
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Shewanella putrefaciens MR-1 has emerged as a good model to study anaerobic respiration and electron transport-linked metal reduction. Its remarkable respiratory plasticity suggests the potential for a complex regulatory system to coordinate electron acceptor use in the absence of O(2). It had previously been suggested that EtrA (electron transport regulator A), an analog of Fnr (fumarate nitrate regulator) from Escherichia coli, may regulate gene expression for anaerobic electron transport. An etrA knockout strain (ETRA-153) was isolated from MR-1 using a gene replacement strategy. Reverse transcription-PCR analysis of total RNA demonstrated the loss of the etrA mRNA in ETRA-153. ETRA-153 cells retained the ability to grow on all electron acceptors tested, including fumarate, trimethylamine N-oxide (TMAO), thiosulfate, dimethyl sulfoxide, ferric citrate, nitrate, and O(2), as well as the ability to reduce ferric citrate, manganese(IV), nitrate, and nitrite. EtrA is therefore not necessary for growth on, or the reduction of, these electron acceptors. However, ETRA-153 had reduced initial growth rates on fumarate and nitrate but not on TMAO. The activities for fumarate and nitrate reductase were lower in ETRA-153, as were the levels of fumarate reductase protein and transcript. ETRA-153 was also deficient in one type of ubiquinone. These results are in contrast to those previously reported for the putative etrA mutant METR-1. Molecular analysis of METR-1 indicated that its etrA gene is not interrupted; its reported phenotype was likely due to the use of inappropriate anaerobic growth conditions. (+info)
Sorption of Fe (hydr)oxides to the surface of Shewanella putrefaciens: cell-bound fine-grained minerals are not always formed de novo.
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Shewanella putrefaciens, a gram-negative, facultative anaerobe, is active in the cycling of iron through its interaction with Fe (hydr)oxides in natural environments. Fine-grained Fe precipitates that are attached to the outer membranes of many gram-negative bacteria have most often been attributed to precipitation and growth of the mineral at the cell surface. Our study of the sorption of nonbiogenic Fe (hydr)oxides revealed, however, that large quantities of nanometer-scale ferrihydrite (hydrous ferric oxide), goethite (alpha-FeOOH), and hematite (alpha-Fe(2)O(3)) adhered to the cell surface. Attempts to separate suspensions of cells and minerals with an 80% glycerin cushion proved that the sorbed minerals were tightly attached to the bacteria. The interaction between minerals and cells resulted in the formation of mineral-cell aggregates, which increased biomass density and provided better sedimentation of mineral Fe compared to suspensions of minerals alone. Transmission electron microscopy observations of cells prepared by whole-mount, conventional embedding, and freeze-substitution methods confirmed the close association between cells and minerals and suggested that in some instances, the mineral crystals had even penetrated the outer membrane and peptidoglycan layers. Given the abundance of these mineral types in natural environments, the data suggest that not all naturally occurring cell surface-associated minerals are necessarily formed de novo on the cell wall. (+info)