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Genes important for growth of Pseudomonas stutzeri PDA on chlorate were identified using a randomly DNA bar-coded transposon mutant library. During chlorate reduction, mutations in genes encoding the chlorate reductase clrABC, predicted molybdopterin cofactor chaperon clrD, molybdopterin biosynthesis and two genes of unknown function (clrE, clrF) had fitness defects in pooled mutant assays (Bar-seq). Markerless in-frame deletions confirmed that clrA, clrB and clrC were essential for chlorate reduction, while clrD, clrE and clrF had less severe growth defects. Interestingly, the key detoxification gene cld was essential for chlorate reduction in isogenic pure culture experiments, but showed only minor fitness defects in Bar-seq experiments. We hypothesized this was enabled through chlorite dismutation by the community, as most strains in the Bar-seq library contained an intact cld. In support of this, Δcld grew with wild-type PDA or ΔclrA, and purified Cld also restored growth to the Δcld mutant. Expanding on this, wild-type PDA and a Δcld mutant of the perchlorate reducer Azospira suillum PS grew on perchlorate in co-culture, but not individually. These results demonstrate that co-occurrence of cld and a chloroxyanion reductase within a single organism is not necessary and raises the possibility of syntrophic (per)chlorate respiration in the environment., (© 2015 Society for Applied Microbiology and John Wiley & Sons Ltd.)

Cytochrome c nitrite reductase (ccNiR) from Shewanella oneidensis, which catalyzes the six-electron reduction of nitrite to ammonia in vivo, was shown to oxidize hydroxylamine in the presence of large quantities of this substrate, yielding nitrite as the sole free nitrogenous product. UV-visible stopped-flow and rapid-freeze-quench electron paramagnetic resonance data, along with product analysis, showed that the equilibrium between hydroxylamine and nitrite is fairly rapidly established in the presence of high initial concentrations of hydroxylamine, despite said equilibrium lying far to the left. By contrast, reduction of hydroxylamine to ammonia did not occur, even though disproportionation of hydroxylamine to yield both nitrite and ammonia is strongly thermodynamically favored. This suggests a kinetic barrier to the ccNiR-catalyzed reduction of hydroxylamine to ammonia. A mechanism for hydroxylamine reduction is proposed in which the hydroxide group is first protonated and released as water, leaving what is formally an NH2(+) moiety bound at the heme active site. This species could be a metastable intermediate or a transition state but in either case would exist only if it were stabilized by the donation of electrons from the ccNiR heme pool into the empty nitrogen p orbital. In this scenario, ccNiR does not catalyze disproportionation because the electron-donating hydroxylamine does not poise the enzyme at a sufficiently low potential to stabilize the putative dehydrated hydroxylamine; presumably, a stronger reductant is required for this.

Shewanella oneidensis cytochrome c nitrite reductase (soNrfA), a dimeric enzyme that houses five c-type hemes per protomer, conducts the six-electron reduction of nitrite and the two-electron reduction of hydroxylamine. Protein film voltammetry (PFV) has been used to study the cytochrome c nitrite reductase from Escherichia coli (ecNrfA) previously, revealing catalytic reduction of both nitrite and hydroxylamine substrates by ecNrfA adsorbed to a graphite electrode that is characterized by "boosts" and attenuations in activity depending on the applied potential. Here, we use PFV to investigate the catalytic properties of soNrfA during both nitrite and hydroxylamine turnover and compare those properties to the properties of ecNrfA. Distinct differences in both the electrochemical and kinetic characteristics of soNrfA are observed; e.g., all detected electron transfer steps are one-electron in nature, contrary to what has been observed in ecNrfA [Angove, H. C., Cole, J. A., Richardson, D. J., and Butt, J. N. (2002) J. Biol. Chem. 277, 23374-23381]. Additionally, we find evidence of substrate inhibition during nitrite turnover and negative cooperativity during hydroxylamine turnover, neither of which has previously been observed in any cytochrome c nitrite reductase. Collectively, these data provide evidence that during catalysis, potential pathways of communication exist between the individual soNrfA monomers comprising the native homodimer.

The high-yield expression and purification of Shewanella oneidensis cytochrome c nitrite reductase (ccNiR) and its characterization by a variety of methods, notably Laue crystallography, are reported. A key component of the expression system is an artificial ccNiR gene in which the N-terminal signal peptide from the highly expressed S. oneidensis protein "small tetraheme c" replaces the wild-type signal peptide. This gene, inserted into the plasmid pHSG298 and expressed in S. oneidensis TSP-1 strain, generated approximately 20 mg crude ccNiR per liter of culture, compared with 0.5-1 mg/L for untransformed cells. Purified ccNiR has nitrite and hydroxylamine reductase activities comparable to those previously reported for Escherichia coli ccNiR, and is stable for over 2 weeks in pH 7 solution at 4 °C. UV/vis spectropotentiometric titrations and protein film voltammetry identified five independent one-electron reduction processes. Global analysis of the spectropotentiometric data also allowed determination of the extinction coefficient spectra for the five reduced ccNiR species. The characteristics of the individual extinction coefficient spectra suggest that, within each reduced species, the electrons are distributed among the various hemes, rather than being localized on specific heme centers. The purified ccNiR yielded good-quality crystals, with which the 2.59-Å-resolution structure was solved at room temperature using the Laue diffraction method. The structure is similar to that of E. coli ccNiR, except in the region where the enzyme interacts with its physiological electron donor (CymA in the case of S. oneidensis ccNiR, NrfB in the case of the E. coli protein)., (© SBIC 2012)

Unfractionated heparin is a widely utilized anticoagulant during peripheral angioplasty procedures (PTA). In contrast to heparin, bivalirudin is a direct thrombin inhibitor with predictable anticoagulation, does not activate platelets, and inhibits both soluble and bound thrombin. The experience with bivalirudin during PTA remains limited. In this single-center prospective study, 48 consecutive patients (60.4% males, mean age 70.0 12.1) received bivalirudin as the primary anticoagulant during PTA (0.75 mg/kg bolus, 1.75 mg/kg/h during the procedure). Thirty-four (70.8%) had claudication and 6 (12.5%) had ulceration. Thrombus was angiographically seen in 3 (6.3%) patients. In-hospital serious procedural complications were limited to 2 (4.2%) (exact 95% confidence interval: (0.5%,14.3%]) patients with major bleeding; none (0.0%) of the other following endpoints occurred: death, limb loss, emergent need for revascularization of the same vessel, embolic strokes, and vascular complications (exact 95% confidence intervals: [0.0%,6.1%]). This compared favorably to previously reported data using unfractionated heparin and the same serious procedural complications endpoints from our group at the same institution (9.2%). We conclude that the use of bivalirudin during PTA appears feasible and safe. Large prospective registries are needed to confirm these findings.

Unfractionated heparin is the current antithrombotic of choice in peripheral vascular interventions. The rate of in-hospital major complications during peripheral angioplasty procedures (PTA) using heparin as the primary anticoagulant has not been well defined. In this single-center study, the charts of 213 consecutive PTA procedures in a 1-year period were reviewed. Of unstaged procedures, a total of 131 patients (57.3% males; mean age, 66.4 12.1 years) met inclusion criteria. Forty-five patients (34.4%) had recent onset of claudication and 15 (11.5%) had ulceration. Thrombus was angiographically visualized in 16.7% of patients. Unfractionated heparin was administered at a mean of 4,672 1,238 U (59.1 20.0 U/kg) during the procedure. The highest activated clotting time (ACT) during the procedure was recorded in 114 patients. ACTs were < 300, 300 400 and > 400 seconds in 29.0%, 29.0% and 42.1%, respectively. In-hospital clinical events occurred in 12 patients (9.2%) who met any one of the following endpoints: death (0.8%), limb loss (1.5%), major bleeding (4.6%), emergent need for repeat revascularization of the same vessel (7.6%), embolic stroke (0.0%) and vascular complications (1.5%). The best model associated with salvage revascularization included cigarette smoking within the past year, recent onset of claudication and PTA treatment below the knee. Increased dosages of heparin (U/kg) were associated with a trend toward higher rates of complications. A significant number of patients have in-hospital major complications following PTA procedures using unfractionated heparin as the primary anticoagulant. Current ongoing registries are evaluating the feasibility of direct thrombin inhibitors bivalirudin instead of heparin as a primary anticoagulant during PTA.