Dataset Information

Role for outer membrane cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in reduction of manganese dioxide.

ABSTRACT: 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.

SUBMITTER: Myers JM

PROVIDER: S-EPMC92560 | biostudies-literature | 2001 Jan

REPOSITORIES: biostudies-literature

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Role for outer membrane cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in reduction of manganese dioxide.

Myers J M JM Myers C R CR

Applied and environmental microbiology 20010101 1

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 los ...[more]

PMID: 11133454

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Project description:Anaerobically grown cells of the metal-reducing bacterium Shewanella putrefaciens MR-1 contain multiple outer membrane (OM) cytochromes. A gene replacement mutant (strain OMCB1) lacking the OM cytochrome OmcB is markedly deficient in the reduction of MnO2 and exhibits reduced rates of Fe(III) reduction. The levels of other OM cytochromes are also decreased in OMCB1. Complementation of OMCB1 with wild-type omcB did not restore any of these defects. However, a 21-kb genomic fragment from MR-1, which included omcB and 19 kb of downstream DNA, fully restored MnO2 and Fe(III) reduction and the full complement of OM cytochromes to OMCB1. A 14.7-kb DNA fragment, including omcB and 12 kb of downstream DNA, provided only a modest increase in MnO2 reduction and OM cytochrome content, but it fully restored Fe(III) citrate reduction and partially restored FeOOH reduction. While omcB mRNA was readily detected in this complement, the OmcB protein was not detected in any cellular compartment. The restoration of Fe(III) reduction despite the absence of OmcB suggests that OmcB itself is not required for Fe(III) reduction. Another OM cytochrome, OmcA, was mislocalized to the cytoplasmic membrane of OMCB1. Only the 21-kb genomic fragment was able to restore proper localization of OmcA to the OM. This 21-kb fragment does not contain omcA, but it does contain several open reading frames (ORFs) downstream from omcB. The most downstream of these ORFs (altA) encodes a putative AraC-like transcriptional regulator. However, a gene replacement mutant of altA resembled the wild type with respect to MnO2 reduction, OM cytochrome content, and the localization of OmcA and OmcB to the OM. Since OMCB1 continues to express genes immediately downstream from omcB, the lack of expression of this downstream DNA does not explain its phenotype or the need for the large complementing fragment. The results suggest that the DNA downstream of omcB must be present in cis in order to restore Fe(III) reduction, MnO2 reduction, OM cytochrome content, and the localization of OmcA and OmcB to the OM.

Project description:The outer membrane c-type cytochromes (c-Cyts) OmcA and MtrC in Shewanella are key terminal reductases that bind and transfer electrons directly to iron (hydr)oxides. Although the amounts of OmcA and MtrC at the cell surface and their molecular structures are largely comparable, MtrC is known to play a more important role in dissimilatory iron reduction. To explore the roles of these outer membrane c-Cyts in the interaction of Shewanella oneidensis MR-1 with iron oxides, the processes of attachment of S. oneidensis MR-1 wild type and c-type cytochrome-deficient mutants (the ΔomcA, ΔmtrC, and ΔomcA ΔmtrC mutants) to goethite are compared via quartz crystal microbalance with dissipation monitoring (QCM-D). Strains with OmcA exhibit a rapid initial attachment. The quantitative model for QCM-D responses reveals that MtrC enhances the contact area and contact elasticity of cells with goethite by more than one and two times, respectively. In situ attenuated total reflectance Fourier transform infrared two-dimensional correlation spectroscopic (ATR-FTIR 2D-CoS) analysis shows that MtrC promotes the initial interfacial reaction via an inner-sphere coordination. Atomic force microscopy (AFM) analysis demonstrates that OmcA enhances the attractive force between cells and goethite by about 60%. As a result, OmcA contributes to a higher attractive force with goethite and induces a rapid short-term attachment, while MtrC is more important in the longer-term interaction through an enhanced contact area, which promotes interfacial reactions. These results reveal that c-Cyts OmcA and MtrC adopt different mechanisms for enhancing the attachment of S. oneidensis MR-1 cells to goethite. It improves our understanding of the function of outer membrane c-Cyts and the influence of cell surface macromolecules in cell-mineral interactions.IMPORTANCEShewanella species are one group of versatile and widespread dissimilatory iron-reducing bacteria, which are capable of respiring insoluble iron minerals via six multiheme c-type cytochromes. Outer membrane c-type cytochromes (c-Cyts) OmcA and MtrC are the terminal reductases in this pathway and have comparable protein structures. In this study, we elucidate the different roles of OmcA and MtrC in the interaction of S. oneidensis MR-1 with goethite at the whole-cell level. OmcA confers enhanced affinity toward goethite and results in rapid attachment. Meanwhile, MtrC significantly increases the contact area of bacterial cells with goethite and promotes the interfacial reaction, which may explain its central role in extracellular electron transfer. This study provides novel insights into the role of bacterial surface macromolecules in the interfacial interaction of bacteria with minerals, which is critical to the development of a comprehensive understanding of cell-mineral interactions.

Project description:The microbiota-the mixture of microorganisms in the intestinal tract of animals-plays an important role in host biology. Bacteriophages are a prominent, though often overlooked, component of the microbiota. The mechanisms that phage use to infect susceptible cells associated with animal hosts, and the broader role they could play in determining the substituents of the microbiota, are poorly understood. In this study, we isolated a zebrafish-associated bacteriophage, which we named Shewanella phage FishSpeaker. This phage infects Shewanella oneidensis strain MR-1, which cannot colonize zebrafish, but it is unable to infect Shewanella xiamenensis strain FH-1, a strain isolated from the zebrafish gut. Our data suggest that FishSpeaker uses the outer membrane decaheme cytochrome OmcA, which is an accessory component of the extracellular electron transfer (EET) pathway in S. oneidensis, as well as the flagellum to recognize and infect susceptible cells. In a zebrafish colony that lacks detectable FishSpeaker, we found that most Shewanella spp. are sensitive to infection and that some strains are resistant to infection. Our results suggest that phage could act as a selectivity filter for zebrafish-associated Shewanella and show that the EET machinery can be targeted by phage in the environment. IMPORTANCE Phage exert selective pressure on bacteria that influences and shapes the composition of microbial populations. However, there is a lack of native, experimentally tractable systems for studying how phage influence microbial population dynamics in complex communities. Here, we show that a zebrafish-associated phage requires both the outer membrane-associated extracellular electron transfer protein OmcA and the flagellum to infect Shewanella oneidensis strain MR-1. Our results suggest that the newly discovered phage-FishSpeaker-could exert selective pressure that restricts which Shewanella spp. colonize zebrafish. Moreover, the requirement of OmcA for infection by FishSpeaker suggests that the phage preferentially infects cells that are oxygen limited, a condition required for OmcA expression and an ecological feature of the zebrafish gut.

Dataset Information

Role for outer membrane cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in reduction of manganese dioxide.

Publications

Role for outer membrane cytochromes OmcA and OmcB of Shewanella putrefaciens MR-1 in reduction of manganese dioxide.

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