The missing enzymatic link in syntrophic methane formation from fatty acids.

The microbial production of methane from organic matter is an essential process in the global carbon cycle and an important source of renewable energy. It involves the syntrophic interaction between methanogenic archaea and bacteria that convert primary fermentation products such as fatty acids to the methanogenic substrates acetate, H2, CO2, or formate. While the concept of syntrophic methane formation was developed half a century ago, the highly endergonic reduction of CO2 to methane by electrons derived from ß-oxidation of saturated fatty acids has remained hypothetical. Here, we studied a previously noncharacterized membrane-bound oxidoreductase (EMO) from Syntrophus aciditrophicus containing two heme b cofactors and 8-methylmenaquinone as key redox components of the redox loop-driven reduction of CO2 by acyl-coenzyme A (CoA). Using solubilized EMO and proteoliposomes, we reconstituted the entire electron transfer chain from acyl-CoA to CO2 and identified the transfer from a high- to a low-potential heme b with perfectly adjusted midpoint potentials as key steps in syntrophic fatty acid oxidation. The results close our gap of knowledge in the conversion of biomass into methane and identify EMOs as key players of ß-oxidation in (methyl)menaquinone-containing organisms.

Functionality of the Na+-translocating NADH:quinone oxidoreductase and quinol:fumarate reductase from Prevotella bryantii inferred from homology modeling.

Members of the family Prevotellaceae are Gram-negative, obligate anaerobic bacteria found in animal and human microbiota. In Prevotella bryantii, the Na+-translocating NADH:quinone oxidoreductase (NQR) and quinol:fumarate reductase (QFR) interact using menaquinone as electron carrier, catalyzing NADH:fumarate oxidoreduction. P. bryantii NQR establishes a sodium-motive force, whereas P. bryantii QFR does not contribute to membrane energization. To elucidate the possible mode of function, we present 3D structural models of NQR and QFR from P. bryantii to predict cofactor-binding sites, electron transfer routes and interaction with substrates. Molecular docking reveals the proposed mode of menaquinone binding to the quinone site of subunit NqrB of P. bryantii NQR. A comparison of the 3D model of P. bryantii QFR with experimentally determined structures suggests alternative pathways for transmembrane proton transport in this type of QFR. Our findings are relevant for NADH-dependent succinate formation in anaerobic bacteria which operate both NQR and QFR.

Efficacy and Safety of Tirzepatide in Adults With Type 2 Diabetes: A Perspective for Primary Care Providers.

This article reviews the efficacy and safety data of tirzepatide, a once-weekly, novel glucose-dependent insulinotropic polypeptide and glucagon-like peptide 1 (GLP-1) receptor agonist approved in the United States, the European Union, and other regions for the treatment of type 2 diabetes. All doses of tirzepatide demonstrated superiority in reducing A1C and body weight from baseline versus placebo or active comparators. The safety profile of tirzepatide was consistent with that of the GLP-1 receptor agonist class, with mild to moderate and transient gastrointestinal side effects being the most common adverse events. With clinically and statistically significant reductions in A1C and body weight without increased risk of hypoglycemia in various populations, tirzepatide has demonstrated potential as a first-in-class treatment option for many people with type 2 diabetes.

Variation across operating sites in urinary and sexual outcomes after radical prostatectomy in localized and locally advanced prostate cancer.

PURPOSE: The extent of variation in urinary and sexual functional outcomes after radical prostatectomy (RPE) between prostate cancer (PC) operating sites remains unknown. Therefore, this analysis aims to compare casemix-adjusted functional outcomes (EPIC-26 scores incontinence, irritative/obstructive function and sexual function) between operating sites 12 months after RPE. MATERIALS AND METHODS: Analysis of a cohort of 7065 men treated with RPE at 88 operating sites (prostate cancer centers, "PCCs") between 2016 and 2019. Patients completed EPIC-26 and sociodemographic information surveys at baseline and 12 months after RPE. Survey data were linked to clinical data. EPIC-26 domain scores at 12 months after RPE were adjusted for relevant confounders (including baseline domain score, clinical and sociodemographic information) using regression analysis. Differences between sites were described using minimal important differences (MIDs) and interquartile ranges (IQR). The effects of casemix adjustment on the score results were described using Cohen's d and MIDs. RESULTS: Adjusted domain scores at 12 months varied between sites, with IQRs of 66-78 (incontinence), 89-92 (irritative/obstructive function), and 20-29 (sexual function). Changes in domain scores after casemix adjustment for sites ≥ 1 MID were noted for the incontinence domain (six sites). Cohen's d ranged between - 0.07 (incontinence) and - 0.2 (sexual function), indicating a small to medium effect of casemix adjustment. CONCLUSIONS: Variation between sites was greatest in the incontinence and sexual function domains for RPE patients. Future research will need to identify the factors contributing to this variation. TRIAL REGISTRY: The study is registered at the German Clinical Trial Registry ( https://www.drks.de/drks_web/ ) with the following ID: DRKS00010774.

Sequence analysis and specificity of distinct types of menaquinone methyltransferases indicate the widespread potential of methylmenaquinone production in bacteria and archaea.

Menaquinone (MK) serves as an essential membranous redox mediator in various electron transport chains of aerobic and anaerobic respiration. In addition, the composition of the quinone/quinol pool has been widely used as a biomarker in microbial taxonomy. The HemN-like class C radical SAM methyltransferases (RSMTs) MqnK, MenK and MenK2 have recently been shown to facilitate specific menaquinone methylation reactions at position C-8 (MqnK/MenK) or C-7 (MenK2) to synthesize 8-methylmenaquinone, 7-methylmenaquinone and 7,8-dimethylmenaquinone. However, the vast majority of protein sequences from the MqnK/MenK/MenK2 family belong to organisms, whose capacity to produce methylated menaquinones has not been investigated biochemically. Here, representative putative menK and menK2 genes from Collinsella tanakaei and Ferrimonas marina were individually expressed in Escherichia coli (wild-type or ubiE deletion mutant) and the corresponding cells were found to produce methylated derivatives of the endogenous MK and 2-demethylmenaquinone. Cluster and phylogenetic analyses of 828 (methyl)menaquinone methyltransferase sequences revealed signature motifs that allowed to discriminate enzymes of the MqnK/MenK/MenK2 family from other radical SAM enzymes and to identify C-7-specific menaquinone methyltransferases of the MenK2 subfamily. This study will help to predict the methylation status of the quinone/quinol pool of a microbial species (or even a microbial community) from its (meta)genome and contribute to the future design of microbial quinone/quinol pools in a Synthetic Biology approach.

A Sodium-Translocating Module Linking Succinate Production to Formation of Membrane Potential in Prevotella bryantii.

Ruminants such as cattle and sheep depend on the breakdown of carbohydrates from plant-based feedstuff, which is accomplished by the microbial community in the rumen. Roughly 40% of the members of the rumen microbiota belong to the family Prevotellaceae, which ferments sugars to organic acids such as acetate, propionate, and succinate. These substrates are important nutrients for the ruminant. In a metaproteome analysis of the rumen of cattle, proteins that are homologous to the Na+-translocating NADH:quinone oxidoreductase (NQR) and the quinone:fumarate reductase (QFR) were identified in different Prevotella species. Here, we show that fumarate reduction to succinate in anaerobically growing Prevotella bryantii is coupled to chemiosmotic energy conservation by a supercomplex composed of NQR and QFR. This sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex was enriched by blue native PAGE (BN-PAGE) and characterized by in-gel enzyme activity staining and mass spectrometry. High NADH oxidation (850 nmol min-1 mg-1), quinone reduction (490 nmol min-1 mg-1), and fumarate reduction (1,200 nmol min-1 mg-1) activities, together with high expression levels, demonstrate that SNFR represents a charge-separating unit in P. bryantii. Absorption spectroscopy of SNFR exposed to different substrates revealed intramolecular electron transfer from the flavin adenine dinucleotide (FAD) cofactor in NQR to heme b cofactors in QFR. SNFR catalyzed the stoichiometric conversion of NADH and fumarate to NAD+ and succinate. We propose that the regeneration of NAD+ in P. bryantii is intimately linked to the buildup of an electrochemical gradient which powers ATP synthesis by electron transport phosphorylation. IMPORTANCE Feeding strategies for ruminants are designed to optimize nutrient efficiency for animals and to prevent energy losses like enhanced methane production. Key to this are the fermentative reactions of the rumen microbiota, dominated by Prevotella spp. We show that succinate formation by P. bryantii is coupled to NADH oxidation and sodium gradient formation by a newly described supercomplex consisting of Na+-translocating NADH:quinone oxidoreductase (NQR) and fumarate reductase (QFR), representing the sodium-translocating NADH:fumarate oxidoreductase (SNFR) supercomplex. SNFR is the major charge-separating module, generating an electrochemical sodium gradient in P. bryantii. Our findings offer clues to the observation that use of fumarate as feed additive does not significantly increase succinate production, or decrease methanogenesis, by the microbial community in the rumen.

Functional Outcome and Complications following Ileal Neobladder Reconstruction in Male Patients without Tumor Recurrence. More than 35 Years of Experience from a Single Center.

PURPOSE: There is a lack of data on true long-term functional outcome of orthotopic bladder substitution. The primary study objective was to report our 35-year clinical experience. MATERIALS AND METHODS: Since October 1985, 259 male patients from a large single center radical cystectomy series with complete followup of more than 60 months (median 121, range 60-267) without recurrence, irradiation or undiversion that might have affected the functional outcome, were included. RESULTS: Median age at radical cystectomy and at survey was 63 (range 23-81) and 75 (range 43-92) years, respectively. Overall 87% of patients voided spontaneously and residual-free. This rate decreased with increasing age at the time of surgery (less than 50 years old 94%, 70 years old or older 82%). Overall day/nighttime continence rates were 90%/82%. These rates decreased with increasing age at the time of surgery from 100%/88% to 87%/80%. The overall pad-free rate was 71%/47%. Bicarbonate use decreased from 51% (5 years) to 19% (25 years). Patients with a followup of more than 20 years had the lowest rate of residual urine and clean intermittent catheterization (0.0%) as well as use of more than 1 pad at daytime/nighttime (6.3%/12.5%) and mucus obstruction (0.0%). Serum creatinine showed only the age related increase. The surgical complication rate was 27% and correlated inversely with functional results (chi-squared 11.227, p <0.005), even when the younger age at the time of surgery (younger than 60 years) was related to higher rates of surgical complications (chi-squared 6.80, p <0.05). CONCLUSIONS: The ileal neobladder represents an excellent long-term option for urinary diversion with an acceptable complication rate.

The octahaem MccA is a haem c-copper sulfite reductase.

The six-electron reduction of sulfite to sulfide is the pivot point of the biogeochemical cycle of the element sulfur. The octahaem cytochrome c MccA (also known as SirA) catalyses this reaction for dissimilatory sulfite utilization by various bacteria. It is distinct from known sulfite reductases because it has a substantially higher catalytic activity and a relatively low reactivity towards nitrite. The mechanistic reasons for the increased efficiency of MccA remain to be elucidated. Here we show that anoxically purified MccA exhibited a 2- to 5.5-fold higher specific sulfite reductase activity than the enzyme isolated under oxic conditions. We determined the three-dimensional structure of MccA to 2.2 Å resolution by single-wavelength anomalous dispersion. We find a homotrimer with an unprecedented fold and haem arrangement, as well as a haem bound to a CX15CH motif. The heterobimetallic active-site haem 2 has a Cu(I) ion juxtaposed to a haem c at a Fe-Cu distance of 4.4 Å. While the combination of metals is reminiscent of respiratory haem-copper oxidases, the oxidation-labile Cu(I) centre of MccA did not seem to undergo a redox transition during catalysis. Intact MccA tightly bound SO2 at haem 2, a dehydration product of the substrate sulfite that was partially turned over due to photoreduction by X-ray irradiation, yielding the reaction intermediate SO. Our data show the biometal copper in a new context and function and provide a chemical rationale for the comparatively high catalytic activity of MccA.

Leydig-cell tumour of the testis: retrospective analysis of clinical and therapeutic features in 204 cases.

PURPOSE: Leydig-cell tumours (LCT) of the testis are poorly understood clinically. The aim of this report is to analyse the clinical characteristics of LCT in a large patient sample and to compare these findings with corresponding data of germ-cell tumours (GCT). METHODS: In a sample of 208 patients treated during 1995-2017 in 33 institutions, the following characteristics were registered: age, presenting symptoms, primary tumour size, testis-sparing surgery (TSS) or orchiectomy, malignancy, laterality, medical history, and outcome. Data analysis included descriptive statistical methods and logistic regression analysis. RESULTS: The ratio LCT:GCT is 1:23 (4.4%). The findings are as follows: median age 41 years, undescended testis 8%, bilateral LCTs 3%, malignant LCT 2.5%, contralateral GCT 2.5%, incidental detection 28%, scrotal symptoms 43%, infertility 18%, elevated estradiol levels 29%. TSS was performed in 56% with no local relapse. Of the patients with malignant LCT, one was cured through surgery. CONCLUSION: LCT is rare, with a relative frequency (relative to GCT) of 1:23. Malignancy is found in 2.5%. LCT and GCT share a number of clinical features, e.g. bilaterality, history of undescended testis, and presenting age. TSS is safe in benign LCT. Surgery is the treatment of choice in malignant LCT.

Major complications of post-chemotherapy retroperitoneal lymph node dissection in a contemporary cohort of patients with testicular cancer and a review of the literature.

BACKGROUND: Post-chemotherapy retroperitoneal lymph node dissection (pc-RPLND) is one cornerstone in the clinical management of patients with nonseminomatous testicular germ cell tumours (GCT). A wide range of complication rates in this type of surgery is reported so far. We retrospectively evaluated the frequency of major complications by using the Clavien-Dindo classification and analysed the influence of various clinical factors on complication rates in pc-RPLND. METHODS: We retrospectively analysed 146 GCT patients undergoing pc-RPLND. Complications of grade III-V according to the Clavien-Dindo classification occurring within 30 days after surgery were registered along with the following clinical factors: age, body mass index (BMI), duration of surgery, number of anatomic fields resected, side of primary tumour, histology of surgical specimen, histology of primary tumour, and total dose of cisplatin applied prior to surgery. For comparison, we also evaluated 35 chemotherapy-naïve patients with primary RPLND and 19 with laparoscopic RPLND. We analysed types and frequencies of the various complications as well as associations with clinical factors using descriptive statistical methods. RESULTS: A total of 14.4% grade III-IV complications were observed in pc-RPLND, and 8.6% and 5.3% in primary and in laparoscopic RPLND, respectively. There was no perioperative mortality. Lymphocele was the most frequent adverse event (16% of grade III-IV complications). Operation time > 270 min (p = 0.001) and vital cancer in the resected specimen (p = 0.02) were significantly associated with higher complication rates. Left-sided resection fields involved two-fold higher complication rates, barely missing statistical significance (p = 0.06). CONCLUSIONS: Pc-RPLND involves a grade III-V complication rate of 14.4%. Prolonged operation time and vital cancer in the residual mass are significantly associated with higher complication rates. The Clavien-Dindo classification system may allow inter-observer variation in rating complication grades, which may represent one reason for the wide range of reported RPLND complication rates. RPLND represents major surgery and surgeons active in this field must be competent to manage adverse events.

Two dedicated class C radical S-adenosylmethionine methyltransferases concertedly catalyse the synthesis of 7,8-dimethylmenaquinone.

Dimethylmenaquinone (DMMK), a prevalent menaquinone (MK) derivative of uncertain function, is characteristic for members of the class Coriobacteriia. Such bacteria are frequently present in intestinal microbiomes and comprise several pathogenic species. The coriobacterial model organism Adlercreutzia equolifaciens was used to investigate the enzymology of DMMK biosynthesis. A HemN-like class C radical S-adenosylmethionine methyltransferase (MenK2) from A. equolifaciens was produced in Wolinella succinogenes or Escherichia coli cells and found to methylate MK specifically at position C-7. In combination with a previously described MK methyltransferase (MqnK/MenK) dedicated to MK methylation at C-8, 7,8-DMMK6 was produced in W. succinogenes. The position of the two methyl groups was confirmed by two-dimensional NMR and midpoint redox potentials of 7-MMK6, 8-MMK6 and 7,8-DMMK6 were determined by cyclic voltammetry. A phylogenetic tree of MenK, MenK2 and HemN proteins revealed a Coriobacteriia-specific MenK2 clade. Using chimeric A. equolifaciens MenK/MenK2 proteins produced in E. coli it was shown that the combined linker and HemN domains determined the site-specificity of methylation. The results suggest that the use of MenK2 as a biomarker allows predicting the ability of DMMK synthesis in microbial species.

A class C radical S-adenosylmethionine methyltransferase synthesizes 8-methylmenaquinone.

The membranous quinone/quinol pool is essential to the majority of life forms and has been widely used as an important biomarker in microbial taxonomy. In the anaerobic world, the most important quinones are menaquinone (MK) and a methylated form of MK, designated methylmenaquinone (MMK), which is anticipated to serve specifically in low-potential electron transport chains involved in anaerobic respiration. However, it has remained unclear how MMK is generated. Here, we show that a novel enzyme homologous to class C radical SAM methyltransferases (RSMTs) synthesizes MMK using MK as substrate. Such enzymes, termed either MenK or MqnK, are present in MMK-producing bacteria (and some archaea) that possess either the classical MK biosynthesis pathway (Men) or the futalosine pathway (Mqn). An mqnK deletion mutant of the model Epsilonproteobacterium Wolinella succinogenes was unable to produce MMK6 but its formation was restored upon genomic complementation using either the native mqnK gene or menK from the human gut bacterium Adlercreutzia equolifaciens or Shewanella oneidensis. Moreover, any of the menK genes enabled Escherichia coli cells to produce MMK8 and a methylated form of 2-demethylmenaquinone8 (DMK8 ). The results expand the knowledge on quinone synthesis and demonstrate an unprecedented function for a class C RSMT-type enzyme in primary cell metabolism.

Epsilonproteobacterial hydroxylamine oxidoreductase (εHao): characterization of a 'missing link' in the multihaem cytochrome c family.

Members of the multihaem cytochrome c family such as pentahaem cytochrome c nitrite reductase (NrfA) or octahaem hydroxylamine oxidoreductase (Hao) are involved in various microbial respiratory electron transport chains. Some members of the Hao subfamily, here called εHao proteins, have been predicted from the genomes of nitrate/nitrite-ammonifying bacteria that usually lack NrfA. Here, εHao proteins from the host-associated Epsilonproteobacteria Campylobacter fetus and Campylobacter curvus and the deep-sea hydrothermal vent bacteria Caminibacter mediatlanticus and Nautilia profundicola were purified as εHao-maltose binding protein fusions produced in Wolinella succinogenes. All four proteins were able to catalyze reduction of nitrite (yielding ammonium) and hydroxylamine whereas hydroxylamine oxidation was negligible. The introduction of a tyrosine residue at a position known to cause covalent trimerization of Hao proteins did neither stimulate hydroxylamine oxidation nor generate the Hao-typical absorbance maximum at 460 nm. In most cases, the εHao-encoding gene haoA was situated downstream of haoC, which predicts a tetrahaem cytochrome c of the NapC/NrfH family. This suggested the formation of a membrane-bound HaoCA assembly reminiscent of the menaquinol-oxidizing NrfHA complex. The results indicate that εHao proteins form a subfamily of ammonifying cytochrome c nitrite reductases that represents a 'missing link' in the evolution of NrfA and Hao enzymes.

Gender Specific Differences in Disease-Free, Cancer Specific and Overall Survival after Radical Cystectomy for Bladder Cancer: A Systematic Review and Meta-Analysis.

PURPOSE: We summarize the evidence on gender specific differences in disease-free, cancer specific and overall survival after radical cystectomy for bladder cancer. MATERIALS AND METHODS: We performed a systematic literature search of MEDLINE®, Embase® and the Cochrane Library in July 2017. Studies evaluating gender specific differences in disease-free, cancer specific or overall survival after radical cystectomy for bladder cancer were included in study. Analyses included random effect meta-analysis, subgroup analyses, meta-influence and cumulative meta-analyses. Funnel plots and the Egger test were used to assess publication bias. RESULTS: Of the 3,868 studies identified during the literature search 59 published between 1998 and 2017 were included in analysis. Of the studies 30 in a total of 38,321 patients evaluated disease-free survival, 44 in a total of 69,666 evaluated cancer specific survival and 26 in a total of 30,039 evaluated overall survival. Random effect meta-analyses revealed decreased disease-free, cancer specific survival and overall survival in female patients than in their male counterparts. Pooled estimates showed a HR of 1.16 (95% CI 1.06-1.27, p = 0.0018) for disease-free survival, 1.23 (95% CI 1.15-1.31, p <0.001) for cancer specific survival and 1.08 (95% CI 1.03-1.12, p = 0.0004) for overall survival. Subgroup analyses confirmed impaired disease-free, cancer specific and overall survival in female patients in all strata. Publication bias was evident only for studies of cancer specific survival (Egger test p = 0.0029). After adjusting for publication bias by the trim and fill method the corrected pooled estimated HR of cancer specific survival was 1.13 (95% CI 1.05-1.21, p = 0.0012). CONCLUSIONS: Female patients who underwent radical cystectomy for bladder cancer demonstrated worse disease-free, cancer specific and overall survival than their male counterparts. The multifactorial etiology might include epidemiological differences, gender specific health care discrepancies and hormonal influences.

Impact of herpes zoster and postherpetic neuralgia on the quality of life of Germans aged 50 or above.

BACKGROUND: Herpes zoster (HZ) is a painful dermatomal rash caused by reactivation of latent varicella zoster virus surviving in the patient's sensory ganglia after a previous episode of varicella. The incidence of HZ increases markedly with age as does the proportion of HZ patients who develop postherpetic neuralgia (PHN) with often severe and debilitating pain persisting for months and even years. This prospective study aimed to assess the impact of HZ and PHN on the quality of life (QoL) of individuals aged ≥ 50 years in Germany. METHODS: Patients were recruited when consulting primary care physicians for a first HZ episode. PHN was defined as a 'worst' pain score ≥ 3 on the Zoster Brief Pain Inventory (ZBPI) scale persisting or appearing 90 days or more after rash onset. PHN-cases were followed for up to nine months after rash onset. The interference of pain with patients' ability to carry out normal activities was assessed by the ZBPI activities of daily living (ADL) scale and QoL by the EuroQoL five-dimension scale (EQ-5D) utility score. RESULTS: Of 513 patients enrolled, 61 (11.9%) developed PHN. At HZ onset, the mean ZBPI worst pain score of all patients was 5.1, the least square (LS)means estimates of the ZBPI ADL and EQ-5D utility scores were 2.970 and 0.740, respectively. Over three months follow-up, the pain scores decreased and the QoL increased monotonically across all age groups. At Day 90, the mean ZBPI worst pain score of the PHN patients was 4.4, while the LSmeans estimates of the ZBPI ADL and EQ-5D utility scores were 2.899 and 0.826, respectively. For patients with PHN persisting at nine months, the pain scores and QoL remained unchanged over the six months following the development of PHN. CONCLUSION: HZ and PHN had a substantial impact on the patients' QoL and ability to function in their normal activities. There was a clear association in time between the evolution of pain and estimated QoL. The impact on ADL and QoL did not vary with age.

Clade II nitrous oxide respiration of Wolinella succinogenes depends on the NosG, -C1, -C2, -H electron transport module, NosB and a Rieske/cytochrome bc complex.

Microbial reduction of nitrous oxide (N2 O) is an environmentally significant process in the biogeochemical nitrogen cycle. However, it has been recognized only recently that the gene encoding N2 O reductase (nosZ) is organized in varying genetic contexts, thereby defining clade I (or 'typical') and clade II (or 'atypical') N2 O reductases and nos gene clusters. This study addresses the enzymology of the clade II Nos system from Wolinella succinogenes, a nitrate-ammonifying and N2 O-respiring Epsilonproteobacterium that contains a cytochrome c N2 O reductase (cNosZ). The characterization of single non-polar nos gene deletion mutants demonstrated that the NosG, -C1, -C2, -H and -B proteins were essential for N2 O respiration. Moreover, cells of a W. succinogenes mutant lacking a putative menaquinol-oxidizing Rieske/cytochrome bc complex (QcrABC) were found to be incapable of N2 O (and also nitrate) respiration. Proton motive menaquinol oxidation by N2 O is suggested, supported by the finding that the molar yield for W. succinogenes cells grown by N2 O respiration using formate as electron donor exceeded that of fumarate respiration by about 30%. The results demand revision of the electron transport chain model of clade II N2 O respiration and challenge the assumption that NosGH(NapGH)-type iron-sulfur proteins are menaquinol-reactive.

Three transcription regulators of the Nss family mediate the adaptive response induced by nitrate, nitric oxide or nitrous oxide in Wolinella succinogenes.

Sensing potential nitrogen-containing respiratory substrates such as nitrate, nitrite, hydroxylamine, nitric oxide (NO) or nitrous oxide (N2 O) in the environment and subsequent upregulation of corresponding catabolic enzymes is essential for many microbial cells. The molecular mechanisms of such adaptive responses are, however, highly diverse in different species. Here, induction of periplasmic nitrate reductase (Nap), cytochrome c nitrite reductase (Nrf) and cytochrome c N2 O reductase (cNos) was investigated in cells of the Epsilonproteobacterium Wolinella succinogenes grown either by fumarate, nitrate or N2 O respiration. Furthermore, fumarate respiration in the presence of various nitrogen compounds or NO-releasing chemicals was examined. Upregulation of each of the Nap, Nrf and cNos enzyme systems was found in response to the presence of nitrate, NO-releasers or N2 O, and the cells were shown to employ three transcription regulators of the Crp-Fnr superfamily (homologues of Campylobacter jejuni NssR), designated NssA, NssB and NssC, to mediate the upregulation of Nap, Nrf and cNos. Analysis of single nss mutants revealed that NssA controls production of the Nap and Nrf systems in fumarate-grown cells, while NssB was required to induce the Nap, Nrf and cNos systems specifically in response to NO-generators. NssC was indispensable for cNos production under any tested condition. The data indicate dedicated signal transduction routes responsive to nitrate, NO and N2 O and imply the presence of an N2 O-sensing mechanism.

Resolution of key roles for the distal pocket histidine in cytochrome C nitrite reductases.

Cytochrome c nitrite reductases perform a key step in the biogeochemical N-cycle by catalyzing the six-electron reduction of nitrite to ammonium. These multiheme cytochromes contain a number of His/His ligated c-hemes for electron transfer and a structurally differentiated heme that provides the catalytic center. The catalytic heme has proximal ligation from lysine, or histidine, and an exchangeable distal ligand bound within a pocket that includes a conserved histidine. Here we describe properties of a penta-heme cytochrome c nitrite reductase in which the distal His has been substituted by Asn. The variant is unable to catalyze nitrite reduction despite retaining the ability to reduce a proposed intermediate in that process, namely, hydroxylamine. A combination of electrochemical, structural and spectroscopic studies reveals that the variant enzyme simultaneously binds nitrite and electrons at the catalytic heme. As a consequence the distal His is proposed to play a key role in orienting the nitrite for N-O bond cleavage. The electrochemical experiments also reveal that the distal His facilitates rapid nitrite binding to the catalytic heme of the native enzyme. Finally it is noted that the thermodynamic descriptions of nitrite- and electron-binding to the active site of the variant enzyme are modulated by the prevailing oxidation states of the His/His ligated hemes. This behavior is likely to be displayed by other multicentered redox enzymes such that there are wide implications for considering the determinants of catalytic activity in this important and varied group of oxidoreductases.

Diversity and evolution of bioenergetic systems involved in microbial nitrogen compound transformations.

Nitrogen is an essential element of life that needs to be assimilated in its most reduced form, ammonium. On the other hand, nitrogen exists in a multitude of oxidation states and, consequently, nitrogen compounds (NCs) serve as electron donor and/or acceptors in many catabolic pathways including various forms of microbial respiration that contribute to the global biogeochemical nitrogen cycle. Some of these NCs are also known as reactive nitrogen species able to cause nitrosative stress because of their high redox reactivity. The best understood processes of the nitrogen cycle are denitrification and ammonification (both beginning with nitrate reduction to nitrite), nitrification (aerobic oxidation of ammonium and nitrite) and anaerobic ammonium oxidation (anammox). This review presents examples of the diverse architecture, either elucidated or anticipated, and the high degree of modularity of the corresponding respiratory electron transport processes found in Bacteria and Archaea, and relates these to their respective bioenergetic mechanisms of proton motive force generation. In contrast to the multiplicity of enzymes that catalyze NC transformations, the number of proteins or protein modules involved in connecting electron transport to and from these enzymes with the quinone/quinol pool is comparatively small. These quinone/quinol-reactive protein modules consist of cytochromes b and c and iron-sulfur proteins. Conclusions are drawn towards the evolutionary relationships of bioenergetic systems involved in NC transformation and deduced aspects of the evolution of the biogeochemical nitrogen cycle are presented. This article is part of a Special Issue entitled: The evolutionary aspects of bioenergetic systems.

Production and consumption of nitrous oxide in nitrate-ammonifying Wolinella succinogenes cells.

Global warming is moving more and more into the public consciousness. Besides the commonly mentioned carbon dioxide and methane, nitrous oxide (N2O) is a powerful greenhouse gas in addition to its contribution to depletion of stratospheric ozone. The increasing concern about N2O emission has focused interest on underlying microbial energy-converting processes and organisms harbouring N2O reductase (NosZ), such as denitrifiers and ammonifiers of nitrate and nitrite. Here, the epsilonproteobacterial model organism Wolinella succinogenes is investigated with regard to its capacity to produce and consume N2O during growth by anaerobic nitrate ammonification. This organism synthesizes an unconventional cytochrome c nitrous oxide reductase (cNosZ), which is encoded by the first gene of an atypical nos gene cluster. However, W. succinogenes lacks a nitric oxide (NO)-producing nitrite reductase of the NirS- or NirK-type as well as an NO reductase of the Nor-type. Using a robotized incubation system, the wild-type strain and suitable mutants of W. succinogenes that either produced or lacked cNosZ were analysed as to their production of NO, N2O and N2 in both nitrate-sufficient and nitrate-limited growth medium using formate as electron donor. It was found that cells growing in nitrate-sufficient medium produced small amounts of N2O, which derived from nitrite and, most likely, from the presence of NO. Furthermore, cells employing cNosZ were able to reduce N2O to N2. This reaction, which was fully inhibited by acetylene, was also observed after adding N2O to the culture headspace. The results indicate that W. succinogenes cells are competent in N2O and N2 production despite being correctly grouped as respiratory nitrate ammonifiers. N2O production is assumed to result from NO detoxification and nitrosative stress defence, while N2O serves as a terminal electron acceptor in anaerobic respiration. The ecological implications of these findings are discussed.