Charles R. Myers

Bacterial Manganese Reduction and Growth with Manganese Oxide as the Sole Electron Acceptor

Microbes that couple growth to the reduction of manganese could play an important role in the biogeochemistry of certain anaerobic environments. Such a bacterium, Alteromonas putrefaciens MR-1, couples its growth to the reduction of manganese oxides only under anaerobic conditions. The characteristics of this reduction are consistent with a biological, and not an indirect chemical, reduction of manganese, which suggest that this bacterium uses manganic oxide as a terminal electron acceptor. It can also utilize a large number of other compounds as terminal electron acceptors; this versatility could provide a distinct advantage in environments where electron-acceptor concentrations may vary.

Microbial reduction of manganese oxides: Interactions with iron and sulfur

Alteromonas putrefaciens (strain MR-1) is capable of rapid Mn(IV) reduction under conditions of neutral pH and temperatures characteristic of the Oneida Lake, New York, sediments from which it was isolated. MR-1 also reduces Fe3+ to Fe2+, and disproportionates thiosulfate to sulfide and sulfite; independently, the Fe2+ and sulfide act as rapid reductants of Mn. The addition of Fe3+ or thiosulfate to cultures of MR-1 in the presence of oxidized Mn increases the rate and extent of Mn reduction relative to that observed in the absence of Fe3+ or thiosulfate. Furthermore, when Fe3+ and Mn oxides are present conjointly, Fe2+ does not appear until reduction of the oxidized Mn is complete. These results demonstrate that observed rates of Fe2+ and sulfide production may underestimate the total rates of Fe and sulfate reduction in those environments containing oxidized Mn. These results also demonstrate the potential impact that a single microbe can exert on sediment geochemistry, and provide the basis for preliminary models of the complexity of microbial and geochemical interactions that occur.

Chromium(VI) reductase activity is associated with the cytoplasmic membrane of anaerobically grown Shewanella putrefaciens MR-1.

C.R. MYERS, B.P. CARSTENS, W.E. ANTHOLINE and J.M. MYERS.2000. Shewanella putrefaciens MR‐1 can reduce a diverse array of compounds under anaerobic conditions, including manganese and iron oxides, fumarate, nitrate, and many other compounds. These reductive processes are apparently linked to a complex electron transport system. Chromium (Cr) is a toxic and mutagenic metal and bacteria could potentially be utilized to immobilize Cr by reducing the soluble and bioavailable state, Cr(VI), to the insoluble and less bioavailable state, Cr(III). Formate‐dependent Cr(VI) reductase activity was detected in anaerobically grown cells of S. putrefaciens MR‐1, with highest specific activity in the cytoplasmic membrane. Both formate and NADH served as electron donors for Cr(VI) reductase, whereas l‐lactate or NADPH did not support any activity. The addition of 10 μmol l−1 FMN markedly stimulated formate‐dependent Cr(VI) reductase, and the activity was almost completely inhibited by diphenyliodonium chloride, an inhibitor of flavoproteins. Cr(VI) reductase activity was also inhibited by p‐chloromercuriphenylsulphonate, azide, 2‐heptyl‐4‐hydroxyquinolone‐N‐oxide, and antimycin A, suggesting involvement of a multi‐component electron transport chain which could include cytochromes and quinones. Cr(V) was detected by electron paramagnetic resonance (EPR) spectroscopy, suggesting a one‐electron reduction as the first step.

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 omcBmutant (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 MnO2. 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.

Role of the Tetraheme Cytochrome CymA in Anaerobic Electron Transport in Cells of Shewanella putrefaciens MR-1 with Normal Levels of Menaquinone

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.

Outer membrane cytochromes of Shewanella putrefaciens MR-1: spectral analysis, and purification of the 83-kDa c-type cytochrome

The metal-reducing bacterium Shewanella putrefaciens MR-1 is known to localize a majority of its membrane-bound cytochromes to its outer membrane when grown under anaerobic conditions. In this study, pyridine hemochrome spectra confirmed that these outer membrane cytochromes are c-type, and electrophoretic data demonstrated the presence of four distinct outer membrane cytochromes, with apparent molecular masses of 150, 83, 65, and 53 kDa. Fourth-order derivative analysis of 77 K spectra of the outer membrane revealed four spectrally distinct c-type hemes, with peaks at 545.4, 548.0, 550.6, and 552.6 nm. Outer membrane cytochromes in the reduced state were rapidly re-oxidized by oxidized iron and manganese, which have previously been shown to serve as electron acceptors for anaerobic respiration in this bacterium. The 83-kDa outer membrane cytochrome was purified and a specific polyclonal antibody was generated against this protein. Western blot analysis demonstrated that the vast majority of this protein was localized to the outer membrane and an intermediate density membrane fraction of similar composition. Its levels, but not its subcellular distribution, were somewhat influenced by the electron acceptor used to support anaerobic growth, with levels higher in fumarate-grown cells relative to iron(III)- or trimethylamine N-oxide-grown cells. Its specific content in cells grown under aerobic conditions was only 14% of that of fumarate-grown cells, suggesting that a switch to anaerobic conditions significantly increases the de novo synthesis of this outer membrane cytochrome.

Isolation and sequence of omcA, a gene encoding a decaheme outer membrane cytochrome c of Shewanella putrefaciens MR-1, and detection of omcA homologs in other strains of S. putrefaciens.

The sequence of the omcA gene, which encodes a decaheme cytochrome c that is localized to the outer membrane (OM) of Shewanella putrefaciens MR-1, was determined. The 2202 bp nucleotide sequence of omcA encodes for 734 amino acids with a predicted molecular protein mass of 78.6 kDa. Comparison with the amino-terminal sequence of the mature protein suggests the presence of a hydrophobic leader sequence which is cleaved during translocation of the protein to the OM. This leader sequence has a lipoprotein consensus sequence for signal peptidase II at the cleavage site. The predicted mature protein is comprised of 708 amino acids with a predicted molecular mass of 75.8 kDa, but the addition of ten covalently attached heme c groups and covalent lipid modification to the amino-terminal cysteine increases the predicted mass to 82.7 kDa. This is consistent with its apparent mass of 83 kDa in SDS-PAGE gels. The predicted amino acid sequence for the OmcA protein shows no significant homology to known proteins. A RNA of approx. 2300 bases that hybridizes to the omcA gene was detected in anaerobically grown MR-1 cells. The size of this transcript is similar to the coding region of the omcA gene, suggesting that it is not part of a multicistronic operon. Similar to MR-1, four other strains of S. putrefaciens were all found to localize a majority of their membrane-bound cytochromes to the OM when grown under anaerobic conditions, and all contained an OM cytochrome of similar size to OmcA. In two of these strains, MR-4 and MR-8, a homolog of omcA was identified by RT-PCR and Southern blotting using primers and probes specific for omcA of MR-1. Western blot analysis using a polyclonal antibody to OmcA was similarly positive in strains MR-4 and MR-8. Partial nucleotide sequence analysis of these homologs demonstrated 74-77% predicted amino acid homology with OmcA of MR-1. In contrast, strains MR-30 and MR-42 tested negative for omcA homologs by Southern and Northern blots, RT-PCR, and Western blots.

MtrB Is Required for Proper Incorporation of the Cytochromes OmcA and OmcB into the Outer Membrane of Shewanella putrefaciens MR-1

ABSTRACT When grown under anaerobic conditions, Shewanella putrefaciens MR-1 synthesizes multiple outer membrane (OM) cytochromes, some of which have a role in the use of insoluble electron acceptors (e.g., MnO2) for anaerobic respiration. The cytochromes OmcA and OmcB are localized to the OM and the OM-like intermediate-density membrane (IM) in MR-1. The components necessary for proper localization of these cytochromes to the OM have not been identified. A gene replacement mutant (strain MTRB1) lacking the putative OM protein MtrB was isolated and characterized. The specific cytochrome content of the OM of MTRB1 was only 36% that of MR-1. This was not the result of a general decline in cytochrome content, however, because the cytoplasmic membrane (CM) and soluble fractions were not cytochrome deficient. While OmcA and OmcB were detected in the OM and IM fractions of MTRB1, significant amounts were mislocalized to the CM. OmcA was also detected in the soluble fraction of MTRB1. While OmcA and OmcB in MR-1 fractions were resistant to solubilization with Triton X-100 in the presence of Mg2+, Triton X-100 readily solubilized these proteins from all subcellular fractions of MTRB1. Together, these data suggest that MtrB is required for the proper localization and insertion of OmcA and OmcB into the OM of MR-1. The inability of MTRB1 to properly insert these, and possibly other, proteins into its OM likely contributes to its marked deficiency in manganese(IV) and iron(III) reduction. While the localization of another putative OM cytochrome (MtrF) could not be directly determined, an mtrF gene replacement mutant exhibited wild-types rates of Mn(IV) and Fe(III) reduction. Therefore, even if MtrF were mislocalized in MTRB1, it would not contribute to the loss of metal reduction activity in this strain.

Isolation and identification of manganese-reducing bacteria and estimates of microbial Mn(IV)-reducing potential in the Black Sea

During the summer of 1988, profiles of major and minor nutrients in the western and central basins of the Black Sea showed a pronounced suboxic zone in which could be seen distinct zones of nitrate, manganese and iron reduction. Enrichment culture techniques, used to determine relative levels of microbial Mn(IV) and Fe(III) reduction potential, indicated very high levels of iron-reducing potential throughout the suboxic zone, and high levels of Mn(IV)-reducing potential in both the nitrate reduction (55 m) and the Mn(IV) reduction (85 m) zones. Bacteria were also directly isolated from various depths in the water column and tested for their ability to reduce Mn(IV). Using these direct techniques, the major group of organisms isolated from the 80–90 m (Mn reduction) zone were in the genus Shewanella, accounting for up to 20–50% of the total bacterial viable counts, and up to 1–5 × 105 bacteria per ml. At other depths, there were fewer Mn(IV)-reducing bacteria, and those that were found were in different taxonomic groups, including Pseudomonas spp., Bacillus spp., and some unidentified Gram-negative rods and coccobacilli. All of these bacteria were respiratory bacteria, and many were capable of both Fe(III) and Mn(IV) reduction.

Nitric oxide inhibits aldosterone synthesis by a guanylyl cyclase-independent effect.

To investigate the mechanism of nitric oxide (NO) inhibition of aldosterone release, this study compared the effects of type A natriuretic peptide and heat-stable enterotoxin to a nitric oxide donor, deta nonoate, on cGMP production and angiotensin II-stimulated aldosterone synthesis in primary cultures of bovine adrenal zona glomerulosa cells. Type A natriuretic peptide (10 210 -10 26 M) and deta nonoate (10 26 -10 23 M) stimulated concentration-related increases in cGMP production. Heat-stable enterotoxin (10 26 M) failed to stimulate cGMP synthesis in zona glomerulosa cells. Type A natriuretic peptide and deta nonoate attenuated angiotensin II-stimulated aldosterone production over the same concentration range that stimulated cGMP production. Heat-stable enterotoxin (10 26 M) was without effect on aldosterone release. To further test the hypothesis that cGMP mediated the inhibition of aldosterone synthesis, the selective inhibitor of soluble guanylyl cyclase, 1H-(1,2,4)oxadiazolo[4,3-a]quinoxalin-1one (ODQ) was used. ODQ pretreatment (10 25 M) completely prevented deta nonoate-stimulated cGMP production without altering the inhibitory effect of deta nonoate on angiotensin II-stimulated steroidogenesis. Consistent with its selectivity for inhibiting soluble guanylyl cyclase, ODQ did not block type A natriuretic peptide-stimulated cGMP synthesis or type A natriuretic peptide inhibition of steroidogenesis. Deta nonoate completely blocked 25-hydroxycholesterol- and progesterone-stimulated aldosterone synthesis in zona glomerulosa cells and inhibited the conversion of 25-hydroxycholesterol to pregnenolone in mitochondrial fractions from bovine adrenal cortex. Deta nonoate-derived NO gave an absorbance maximum of the mitochondrial cytochrome P450 of 453 nm and inhibited the absorbance at 450 nm caused by carbon monoxide binding to the enzyme. These results suggest that deta nonoate reduces steroidogenesis independent of guanylyl cyclase activation and that NO has a direct effect to inhibit the activity of cytochrome P450, probably by binding to the heme groups of the cytochrome. (Endocrinology 139: 4053‐ 4060, 1998)