Exploring Patient Decision Making Regarding Discontinuation of Tyrosine Kinase Inhibitors for Chronic Myeloid Leukemia.

BACKGROUND: The standard treatment for chronic phase chronic myeloid leukemia (CML) is lifelong oral tyrosine kinase inhibitor (TKI) therapy. Multiple clinical trials have demonstrated that some patients with a sustained deep molecular response to TKI therapy can safely stop therapy and remain in a treatment-free remission. TKI discontinuation is now offered to select patients in routine clinical care. In order to better support patient decision making, we explored patients' views on TKI discontinuation and the factors patients consider when making this decision. MATERIALS AND METHODS: Patients were recruited from three U.S. academic cancer centers. Qualitative interviews were recorded, transcribed, and content analyzed. RESULTS: We interviewed 22 patients, half of whom wanted to try TKI discontinuation. Eleven factors relevant to the decision emerged, and patients weighed these factors differently. Commonly mentioned factors included perceived risk of relapse, TKI side effects, financial considerations, polypharmacy, and willingness to change something that was working (status quo). There were notable differences in patients' understanding of the likelihood of achieving a treatment-free remission, with patients who did not want to stop TKIs more accurately reporting the risk of relapse than patients who wanted to stop. CONCLUSION: This is a novel decision that will become more common as the prevalence of patients with well-controlled CML continues to increase. These results highlight the need for patient education and decision support so that patients and providers can make shared decisions that are informed and values based. IMPLICATIONS FOR PRACTICE: The standard treatment for chronic phase chronic myeloid leukemia (CML) is lifelong oral tyrosine kinase inhibitor (TKI) therapy. Clinical trials have shown that some patients with a sustained deep molecular response to TKI therapy can safely stop therapy and remain in a treatment-free remission. TKI discontinuation is now being offered to patients outside of clinical trials. This study explored factors that patients who are eligible to try TKI discontinuation considered when making this decision. Factors relevant to the decision included risk of relapse, side effects, financial considerations, polypharmacy, and willingness to change something that was working. This is a novel decision that will become more common as the prevalence of patients with well-controlled CML continues to increase. These results highlight the need for decision support and outline the factors that should be included so that patients and providers can make shared decisions that are informed and values based.

The selenium-independent inherent pro-oxidant NADPH oxidase activity of mammalian thioredoxin reductase and its selenium-dependent direct peroxidase activities.

Mammalian thioredoxin reductase (TrxR) is an NADPH-dependent homodimer with three redox-active centers per subunit: a FAD, an N-terminal domain dithiol (Cys(59)/Cys(64)), and a C-terminal cysteine/selenocysteine motif (Cys(497)/Sec(498)). TrxR has multiple roles in antioxidant defense. Opposing these functions, it may also assume a pro-oxidant role under some conditions. In the absence of its main electron-accepting substrates (e.g. thioredoxin), wild-type TrxR generates superoxide (O ), which was here detected and quantified by ESR spin trapping with 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO). The peroxidase activity of wild-type TrxR efficiently converted the O adduct (DEPMPO/HOO(*)) to the hydroxyl radical adduct (DEPMPO/HO(*)). This peroxidase activity was Sec-dependent, although multiple mutants lacking Sec could still generate O . Variants of TrxR with C59S and/or C64S mutations displayed markedly reduced inherent NADPH oxidase activity, suggesting that the Cys(59)/Cys(64) dithiol is required for O generation and that O is not derived directly from the FAD. Mutations in the Cys(59)/Cys(64) dithiol also blocked the peroxidase and disulfide reductase activities presumably because of an inability to reduce the Cys(497)/Sec(498) active site. Although the bulk of the DEPMPO/HO(*) signal generated by wild-type TrxR was due to its combined NADPH oxidase and Sec-dependent peroxidase activities, additional experiments showed that some free HO(*) could be generated by the enzyme in an H(2)O(2)-dependent and Sec-independent manner. The direct NADPH oxidase and peroxidase activities of TrxR characterized here give insights into the full catalytic potential of this enzyme and may have biological consequences beyond those solely related to its reduction of thioredoxin.

The effects of acrolein on peroxiredoxins, thioredoxins, and thioredoxin reductase in human bronchial epithelial cells.

Inhalation is a common form of exposure to acrolein, a toxic reactive volatile aldehyde that is a ubiquitous environmental pollutant. Bronchial epithelial cells would be directly exposed to inhaled acrolein. The thioredoxin (Trx) system is essential for the maintenance of cellular thiol redox balance, and is critical for cell survival. Normally, thioredoxin reductase (TrxR) maintains the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins in the reduced state, and the thioredoxins keep the peroxiredoxins (Prx) reduced, thereby supporting their peroxidase function. The effects of acrolein on TrxR, Trx and Prx in human bronchial epithelial (BEAS-2B) cells were determined. A 30-min exposure to 5 microM acrolein oxidized both Trx1 and Trx2, although significant effects were noted for Trx1 at even lower acrolein concentrations. The effects on Trx1 and Trx2 could not be reversed by treatment with disulfide reductants. TrxR activity was inhibited 60% and >85% by 2.5 and 5 microM acrolein, respectively. The endogenous electron donor for TrxR, NADPH, could not restore its activity, and activity did not recover in cells during a 4-h acrolein-free period in complete medium. The effects of acrolein on TrxR and Trx therefore extend beyond the duration of exposure. While there was a strong correlation between TrxR inhibition and Trx1 oxidation, the irreversible effects on Trx1 suggest direct effects of acrolein rather than loss of reducing equivalents from TrxR. Trx2 did not become oxidized until > or = 90% of TrxR was inhibited, but irreversible effects on Trx2 also suggest direct effects of acrolein. Prx1 (cytosolic) and Prx3 (mitochondrial) shifted to a largely oxidized state only when >90 and 100% of their respective Trxs were oxidized. Prx oxidation was readily reversed with a disulfide reductant, suggesting that Prx oxidation resulted from lack of reducing equivalents from Trx and not direct reaction with acrolein. The effects of acrolein on the thioredoxin system and peroxiredoxins could have important implications for cell survival, redox-sensitive cell signaling, and tolerance to other oxidant insults.

Hexavalent chromium causes the oxidation of thioredoxin in human bronchial epithelial cells.

Hexavalent chromium [Cr(VI)] species such as chromates are cytotoxic. Inhalational exposure is a primary concern in many Cr-related industries and their immediate environments, and bronchial epithelial cells are directly exposed to inhaled Cr(VI). Chromates are readily taken up by cells and are reduced to reactive Cr species which may also result in the generation of reactive oxygen species (ROS). The thioredoxin (Trx) system has a key role in the maintenance of cellular thiol redox balance and is essential for cell survival. Cells normally maintain the cytosolic (Trx1) and mitochondrial (Trx2) thioredoxins largely in the reduced state. Redox Western blots were used to assess the redox status of the thioredoxins in normal human bronchial epithelial cells (BEAS-2B) incubated with soluble Na2CrO4 or insoluble ZnCrO4 for different periods of time. Both chromates caused a dose- and time-dependent oxidation of Trx2 and Trx1. Trx2 was more susceptible in that it could all be converted to the oxidized form, whereas a small amount of reduced Trx1 remained even after prolonged treatment with higher Cr concentrations. Only one of the dithiols, presumably the active site, of Trx1 was oxidized by Cr(VI). Cr(VI) did not cause significant GSH depletion or oxidation indicating that Trx oxidation does not result from a general oxidation of cellular thiols. With purified Trx and thioredoxin reductase (TrxR) in vitro, Cr(VI) also resulted in Trx oxidation. It was determined that purified TrxR has pronounced Cr(VI) reducing activity, so competition for electron flow from TrxR might impair its ability to reduce Trx. The in vitro data also suggested some direct redox interaction between Cr(VI) and Trx. The ability of Cr(VI) to cause Trx oxidation in cells could contribute to its cytotoxic effects, and could have important implications for cell survival, redox-sensitive cell signaling, and the cells' tolerance of other oxidant insults.

Addition of DNA to Cr(VI) and cytochrome b5 containing proteoliposomes leads to generation of DNA strand breaks and Cr(III) complexes.

Chromium (Cr) is a cytotoxic metal that can be associated with a variety of types of DNA damage, including Cr-DNA adducts and strand breaks. Prior studies with purified human cytochrome b(5) and NADPH:P450 reductase in reconstituted proteoliposomes (PLs) demonstrated rapid reduction of Cr(VI) (hexavalent chromium, as CrO(4)(2-), and the generation of Cr(V), superoxide (O(2)(*-)), and hydroxyl radical (HO(*)). Studies reported here examined the potential for the species produced by this system to interact with DNA. Strand breaks of purified plasmid DNA increased over time aerobically, but were not observed in the absence of O(2). Cr(V) is formed under both conditions, so the breaks are not mediated directly by Cr(V). The aerobic strand breaks were significantly prevented by catalase and EtOH, but not by the metal chelator diethylenetriaminepentaacetic acid (DTPA), suggesting that they are largely due to HO(*) from Cr-mediated redox cycling. EPR was used to assess the formation of Cr-DNA complexes. Following a 10-min incubation of PLs, CrO(4)(2-), and plasmid DNA, intense EPR signals at g=5.7 and g=5.0 were observed. These signals are attributed to specific Cr(III) complexes with large zero field splitting (ZFS). Without DNA, the signals in the g=5 region were weak. The large ZFS signals were not seen, when Cr(III)Cl(3) was incubated with DNA, suggesting that the Cr(III)-DNA interactions are different when generated by the PLs. After 24 h, a broad signal at g=2 is attributed to Cr(III) complexes with a small ZFS. This g=2 signal was observed without DNA, but it was different from that seen with plasmid. It is concluded that EPR can detect specific Cr(III) complexes that depend on the presence of plasmid DNA and the manner in which the Cr(III) is formed.

Reduction of hexavalent chromium by human cytochrome b5: generation of hydroxyl radical and superoxide.

The reduction of hexavalent chromium, Cr(VI), can generate reactive Cr intermediates and various types of oxidative stress. The potential role of human microsomal enzymes in free radical generation was examined using reconstituted proteoliposomes (PLs) containing purified cytochrome b(5) and NADPH:P450 reductase. Under aerobic conditions, the PLs reduced Cr(VI) to Cr(V) which was confirmed by ESR using isotopically pure (53)Cr(VI). When 5-diethoxyphosphoryl-5-methyl-1-pyrroline-N-oxide (DEPMPO) was included as a spin trap, a very prominent signal for the hydroxyl radical (HO()) adduct was observed as well as a smaller signal for the superoxide (O(2)(-)) adduct. These adducts were observed even at very low Cr(VI) concentrations (10 muM). NADPH, Cr(VI), O(2), and the PLs were all required for significant HO() generation. Superoxide dismutase eliminated the O(2)(-) adduct and resulted in a 30% increase in the HO() adduct. Catalase largely diminished the HO() adduct signal, indicating its dependence on H(2)O(2). Some sources of catalase were found to have Cr(VI)-reducing contaminants which could confound results, but a source of catalase free of these contaminants was used for these studies. Exogenous H(2)O(2) was not needed, indicating that it was generated by the PLs. Adding exogenous H(2)O(2), however, did increase the amount of DEPMPO/HO() adduct. The inclusion of formate yielded the carbon dioxide radical adduct of DEPMPO, and experiments with dimethyl sulfoxide (DMSO) plus the spin trap alpha-phenyl-N-tert-butylnitrone (PBN) yielded the methoxy and methyl radical adducts of PBN, confirming the generation of HO(). Quantification of the various species over time was consistent with a stoichiometric excess of HO() relative to the net amount of Cr(VI) reduced. This also represents the first demonstration of a role for cytochrome b(5) in the generation of HO(). Overall, the simultaneous generation of Cr(V) and H(2)O(2) by the PLs and the resulting generation of HO() at low Cr(VI) concentrations could have important implications for Cr(VI) toxicity.

What Can a Primary Care Physician Discuss With Older Patients to Improve Advance Directive Completion Rates? A Clin-IQ.

Advance directives (ADs) provide patients with the opportunity to indicate their preferences for medical care while they still maintain the capacity to express their wishes, thus retaining autonomy. ADs increase the likelihood that patients will receive the care they desire, as their family members and physicians will better understand the level of care desired. Despite this, the AD completion rate by elderly patients continues to be low, especially for patients not facing serious illnesses. Primary care physicians (PCPs) are uniquely positioned to engage patients in discussions about ADs before a health crisis arises yet often do not due to time constraints. Using assets associated with the PCP relationship to and longitudinal care for patients, findings reveal that PCPs who emphasize the importance of ADs and who normalize the discussion during office visits by asking questions to understand patients' health goals and holding short conversations over several visits can improve AD completion rates.

Redox activation of Fe(III)-thiosemicarbazones and Fe(III)-bleomycin by thioredoxin reductase: specificity of enzymatic redox centers and analysis of reactive species formation by ESR spin trapping.

Thiosemicarbazones such as Triapine (Tp) and Dp44mT are tridentate iron (Fe) chelators that have well-documented antineoplastic activity. Although Fe-thiosemicarbazones can undergo redox cycling to generate reactive species that may have important roles in their cytotoxicity, there is only limited insight into specific cellular agents that can rapidly reduce Fe(III)-thiosemicarbazones and thereby promote their redox activity. Here we report that thioredoxin reductase-1 (TrxR1) and glutathione reductase (GR) have this activity and that there is considerable specificity to the interactions between specific redox centers in these enzymes and various Fe(III) complexes. Site-directed variants of TrxR1 demonstrate that the selenocysteine (Sec) of the enzyme is not required, whereas the C59 residue and the flavin have important roles. Although TrxR1 and GR have analogous C59/flavin motifs, TrxR is considerably faster than GR. For both enzymes, Fe(III)(Tp)2 is reduced faster than Fe(III)(Dp44mT)2. This reduction promotes redox cycling and the generation of hydroxyl radical (HO) in a peroxide-dependent manner, even with low-micromolar levels of Fe(Tp)2. TrxR also reduces Fe(III)-bleomycin and this activity is Sec-dependent. TrxR cannot reduce Fe(III)-EDTA at significant rates. Our findings are the first to demonstrate pro-oxidant reductive activation of Fe(III)-based antitumor thiosemicarbazones by interactions with specific enzyme species. The marked elevation of TrxR1 in many tumors could contribute to the selective tumor toxicity of these drugs by enhancing the redox activation of Fe(III)-thiosemicarbazones and the generation of reactive oxygen species such as HO.

The intracellular redox stress caused by hexavalent chromium is selective for proteins that have key roles in cell survival and thiol redox control.

Hexavalent chromium [Cr(VI)] compounds (e.g. chromates) are strong oxidants that readily enter cells where they are reduced to reactive Cr intermediates that can directly oxidize some cell components and can promote the generation of reactive oxygen and nitrogen species. Inhalation is a major route of exposure which directly exposes the bronchial epithelium. Previous studies with non-cancerous human bronchial epithelial cells (BEAS-2B) demonstrated that Cr(VI) treatment results in the irreversible inhibition of thioredoxin reductase (TrxR) and the oxidation of thioredoxins (Trx) and peroxiredoxins (Prx). The mitochondrial Trx/Prx system is somewhat more sensitive to Cr(VI) than the cytosolic Trx/Prx system, and other redox-sensitive mitochondrial functions are subsequently affected including electron transport complexes I and II. Studies reported here show that Cr(VI) does not cause indiscriminant thiol oxidation, and that the Trx/Prx system is among the most sensitive of cellular protein thiols. Trx/Prx oxidation is not unique to BEAS-2B cells, as it was also observed in primary human bronchial epithelial cells. Increasing the intracellular levels of ascorbate, an endogenous Cr(VI) reductant, did not alter the effects on TrxR, Trx, or Prx. The peroxynitrite scavenger MnTBAP did not protect TrxR, Trx, Prx, or the electron transport chain from the effects of Cr(VI), implying that peroxynitrite is not required for these effects. Nitration of tyrosine residues of TrxR was not observed following Cr(VI) treatment, further ruling out peroxynitrite as a significant contributor to the irreversible inhibition of TrxR. Cr(VI) treatments that disrupt the TrxR/Trx/Prx system did not cause detectable mitochondrial DNA damage. Overall, the redox stress that results from Cr(VI) exposure shows selectivity for key proteins which are known to be important for redox signaling, antioxidant defense, and cell survival.

The iron-chelating drug triapine causes pronounced mitochondrial thiol redox stress.

Triapine (Tp) is an iron chelator with activity against several types of cancer. Iron-Tp [Fe(III)(Tp)(2)] can be redox-cycled to generate reactive oxygen species that may contribute to its cytotoxicity. However, evidence for this mechanism in cells is limited. The cytosolic and mitochondrial thioredoxins (Trx1 and Trx2, respectively) are essential for cell survival. They are normally maintained in the reduced state, and support the function of many intracellular proteins including the peroxiredoxins (Prxs). Their redox status can indicate oxidant stress in their respective subcellular compartments. Tp treatment of human lung A549 cells caused almost complete oxidation of Trx2 and its dependent peroxiredoxin (Prx3), but there was no effect on Trx1 redox status. Significant inhibition of total TrxR activity did not occur until Tp levels were 4-fold above those needed to cause Trx2 oxidation. While Tp caused a 36-45% decline in reduced glutathione (GSH) levels, GSH accounted for >99% of the total glutathione in the absence and presence of Tp. In vitro studies demonstrated that cysteine reduces Fe(III)(Tp)(2) to Fe(II)(Tp)(2), and cysteine was faster and more efficient than reduced glutathione (GSH) in this regard. Fe(III)(Tp)(2) also mediated the oxidation of purified Trx2 in vitro. Thus, Fe(III)(Tp)(2) itself, and/or various reactive species that may result from its redox cycling, could account for Trx2 and Prx3 oxidation in Tp-treated cells. The striking difference between the effects on Trx2 and Trx1 implies a pronounced thiol redox stress that is largely directed at the mitochondria. These previously unrecognized effects of Tp could contribute to its overall cytotoxicity.

The effects of acrolein on the thioredoxin system: implications for redox-sensitive signaling.

The reactive aldehyde acrolein is a ubiquitous environmental pollutant and is also generated endogenously. It is a strong electrophile and reacts rapidly with nucleophiles including thiolates. This review focuses on the effects of acrolein on thioredoxin reductase (TrxR) and thioredoxin (Trx), which are major regulators of intracellular protein thiol redox balance. Acrolein causes irreversible effects on TrxR and Trx, which are consistent with the formation of covalent adducts to selenocysteine and cysteine residues that are key to their activity. TrxR and Trx are more sensitive than some other redox-sensitive proteins, and their prolonged inhibition could disrupt a number of redox-sensitive functions in cells. Among these effects are the oxidation of peroxiredoxins and the activation of apoptosis signal regulating kinase (ASK1). ASK1 promotes MAP kinase activation, and p38 activation contributes to apoptosis and a number of other acrolein-induced stress responses. Overall, the disruption of the TrxR/Trx system by acrolein could be significant early and prolonged events that affect many aspects of redox-sensitive signaling and oxidant stress.

The pro-oxidant chromium(VI) inhibits mitochondrial complex I, complex II, and aconitase in the bronchial epithelium: EPR markers for Fe-S proteins.

Hexavalent chromium (Cr(VI)) compounds (e.g., chromates) are strong oxidants that readily enter cells, where they are reduced to reactive Cr species that also facilitate reactive oxygen species generation. Recent studies demonstrated inhibition and oxidation of the thioredoxin system, with greater effects on mitochondrial thioredoxin (Trx2). This implies that Cr(VI)-induced oxidant stress may be especially directed at the mitochondria. Examination of other redox-sensitive mitochondrial functions showed that Cr(VI) treatments that cause Trx2 oxidation in human bronchial epithelial cells also result in pronounced and irreversible inhibition of aconitase, a TCA cycle enzyme that has an iron-sulfur (Fe-S) center that is labile with respect to certain oxidants. The activities of electron transport complexes I and II were also inhibited, whereas complex III was not. Electron paramagnetic resonance (EPR) studies of samples at liquid helium temperature (10K) showed a strong signal at g=1.94 that is consistent with the inhibition of electron flow through complex I and/or II. A signal at g=2.02 was also observed, which is consistent with oxidation of the Fe-S center of aconitase. The g=1.94 signal was particularly intense and remained after extracellular Cr(VI) was removed, whereas the g=2.02 signal declined in intensity after Cr(VI) was removed. A similar inhibition of these activities and analogous EPR findings were noted in bovine airways treated ex vivo with Cr(VI). Overall, the data support the hypothesis that Cr(VI) exposure has deleterious effects on a number of redox-sensitive core mitochondrial proteins. The g=1.94 signal could prove to be an important biomarker for oxidative damage resulting from Cr(VI) exposure. The EPR spectra simultaneously showed signals for Cr(V) and Cr(III), which verify Cr(VI) exposure and its intracellular reductive activation.

The effects of hexavalent chromium on thioredoxin reductase and peroxiredoxins in human bronchial epithelial cells.

Inhalational exposure to hexavalent chromium (Cr(VI)) compounds (e.g., chromates) is of concern in many Cr-related industries and their surrounding environments. The bronchial epithelium is directly exposed to inhaled Cr(VI). Cr(VI) species gain easy access inside cells, where they are reduced to reactive Cr species, which may also contribute to the generation of reactive oxygen species. The thioredoxin (Trx) system promotes cell survival and has a major role in maintaining intracellular thiol redox balance. Previous studies with normal human bronchial epithelial cells (BEAS-2B) demonstrated that chromates cause dose- and time-dependent oxidation of Trx1 and Trx2. The Trx's keep many intracellular proteins reduced, including the peroxiredoxins (Prx's). Prx1 (cytosolic) and Prx3 (mitochondrial) were oxidized by Cr(VI) treatments that oxidized all, or nearly all, of the respective Trx's. Prx oxidation is therefore probably the result of a lack of reducing equivalents from Trx. Trx reductases (TrxR's) keep the Trx's largely in the reduced state. Cr(VI) caused pronounced inhibition of TrxR, but the levels of TrxR protein remained unchanged. The inhibition of TrxR was not reversed by removal of residual Cr(VI) or by NADPH, the endogenous electron donor for TrxR. In contrast, the oxidation of Trx1, Trx2, and Prx3 was reversible by disulfide reductants. Prolonged inhibition of TrxR in Cr(VI)-treated cells might contribute to the sustained oxidation of Trx's and Prx's. Reduced Trx binds to an N-terminal domain of apoptosis signaling kinase (ASK1), keeping ASK1 inactive. Cr(VI) treatments that significantly oxidized Trx1 resulted in pronounced dissociation of Trx1 from ASK1. Overall, the effects of Cr(VI) on the redox state and function of the Trx's, Prx's, and TrxR in the bronchial epithelium could have important implications for redox-sensitive cell signaling and tolerance of oxidant insults.

Reductive activation of hexavalent chromium by human lung epithelial cells: generation of Cr(V) and Cr(V)-thiol species.

Chromium(VI) compounds (e.g. chromates) are cytotoxic, mutagenic, and potentially carcinogenic. The reduction of Cr(VI) can yield reactive intermediates such as Cr(V) and reactive oxygen species. Bronchial epithelial cells are the primary site of pulmonary exposure to inhaled Cr(VI) and are the primary cells from which Cr(VI)-associated human cancers arise. BEAS-2B cells were used here as a model of normal human bronchial epithelium for studies on the reductive activation of Cr(VI). Cells incubated with Na(2)CrO(4) exhibited two Cr(V) ESR signals, g=1.979 and 1.985, which persisted for at least 1h. The g=1.979 signal is similar to that generated in vitro by human microsomes and by proteoliposomes containing P450 reductase and cytochrome b(5). Unlike many cells in culture, these cells continued to express P450 reductase and cytochrome b(5). Studies with the non-selective thiol oxidant diamide indicated that the g=1.985 signal was thiol-dependent whereas the g=1.979 signal was not. Pretreatment with phenazine methosulfate eliminated both Cr(V) signals suggesting that Cr(V) generation is largely NAD(P)H-dependent. ESR spectra indicated that a portion of the Cr(VI) was rapidly reduced to Cr(III). Cells incubated with an insoluble chromate, ZnCrO(4), also generated both Cr(V) signals, whereas Cr(V) was not detected with insoluble PbCrO(4). In clonogenic assays, the cells were very sensitive to Na(2)CrO(4) and ZnCrO(4), but considerably less sensitive to PbCrO(4).

Genetic complementation of an outer membrane cytochrome omcB mutant of Shewanella putrefaciens MR-1 requires omcB plus downstream DNA.

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.

Shewanella oneidensis MR-1 restores menaquinone synthesis to a menaquinone-negative mutant.

The mechanisms underlying the use of insoluble electron acceptors by metal-reducing bacteria, such as Shewanella oneidensis MR-1, are currently under intensive study. Current models for shuttling electrons across the outer membrane (OM) of MR-1 include roles for OM cytochromes and the possible excretion of a redox shuttle. While MR-1 is able to release a substance that restores the ability of a menaquinone (MK)-negative mutant, CMA-1, to reduce the humic acid analog anthraquinone-2,6-disulfonate (AQDS), cross-feeding experiments conducted here showed that the substance released by MR-1 restores the growth of CMA-1 on several soluble electron acceptors. Various strains derived from MR-1 also release this substance; these include mutants lacking the OM cytochromes OmcA and OmcB and the OM protein MtrB. Even though strains lacking OmcB and MtrB cannot reduce Fe(III) or AQDS, they still release a substance that restores the ability of CMA-1 to use MK-dependent electron acceptors, including AQDS and Fe(III). Quinone analysis showed that this released substance restores MK synthesis in CMA-1. This ability to restore MK synthesis in CMA-1 explains the cross-feeding results and challenges the previous hypothesis that this substance represents a redox shuttle that facilitates metal respiration.

MtrB is required for proper incorporation of the cytochromes OmcA and OmcB into the outer membrane of Shewanella putrefaciens MR-1.

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.

Vanadium(V) reduction by Shewanella oneidensis MR-1 requires menaquinone and cytochromes from the cytoplasmic and outer membranes.

The metal-reducing bacterium Shewanella oneidensis MR-1 displays remarkable anaerobic respiratory plasticity, which is reflected in the extensive number of electron transport components encoded in its genome. In these studies, several cell components required for the reduction of vanadium(V) were determined. V(V) reduction is mediated by an electron transport chain which includes cytoplasmic membrane components (menaquinone and the tetraheme cytochrome CymA) and the outer membrane (OM) cytochrome OmcB. A partial role for the OM cytochrome OmcA was evident. Electron spin resonance spectroscopy demonstrated that V(V) was reduced to V(IV). V(V) reduction did not support anaerobic growth. This is the first report delineating specific electron transport components that are required for V(V) reduction and of a role for OM cytochromes in the reduction of a soluble metal species.

Identification of the gene encoding the sole physiological fumarate reductase in Shewanella oneidensis MR-1.

Shewanella oneidensis MR-1 is a Gram-negative, nonfermentative rod with a complex electron transport system which facilitates its ability to use a variety of terminal electron acceptors, including fumarate, for anaerobic respiration. CMTn-3, a mutant isolated by transposon (TnphoA) mutagenesis, can no longer use fumarate as an electron acceptor; it lacks fumarate reductase activity as well as a 65-kDa soluble tetraheme flavocytochrome c. The sequence of the TnphoA-flanking genomic DNA of CMTn-3 did not align to those for fumarate reductase or related electron transport genes from other bacteria. Sequence analysis of the MR-1 genomic database demonstrated that an open reading frame encoding a 65-kDa tetraheme cytochrome c with sequence similarity to the fumarate reductase from S. frigidimarina NCIMB400 was found 8 kb away from the TnphoA-flanking genomic DNA of CMTn-3. PCR analysis demonstrated that a large deletion (>or=9.2 kb and