Rob J.M. van Spanning

Expression of nitrite reductase in Nitrosomonas europaea involves NsrR, a novel nitrite-sensitive transcription repressor

Production of nitric oxide (NO) and nitrous oxide (N2O) by ammonia (NH3)‐oxidizing bacteria in natural and man‐made habitats is thought to contribute to the undesirable emission of NO and N2O into the earths atmosphere. The NH3‐oxidizing bacterium Nitrosomonas europaea expresses nitrite reductase (NirK), an enzyme that has so far been studied predominantly in heterotrophic denitrifying bacteria where it is involved in the production of these nitrogenous gases. The finding of nirK homologues in other NH3‐oxidizing bacteria suggests that NirK is widespread among this group; however, its role in these nitrifying bacteria remains unresolved. We identified a gene, nsrR, which encodes a novel nitrite (NO2–)‐sensitive transcription repressor that plays a pivotal role in the regulation of NirK expression in N. europaea. NsrR is a member of the Rrf2 family of putative transcription regulators. NirK was expressed aerobically in response to increasing concentrations of NO2– and decreasing pH. Disruption of nsrR resulted in the constitutive expression of NirK. NsrR repressed transcription from the nirK gene cluster promoter (Pnir), the activity of which correlated with NirK expression. Reconstruction of the NsrR‐Pnir system in Escherichia coli revealed that repression by NsrR was reversed by NO2– in a pH‐dependent manner. The findings are consistent with the hypothesis that N. europaea expresses NirK as a defence against the toxic NO2– that is produced during nitrification.

Nitrite Reductase of Nitrosomonas europaea Is Not Essential for Production of Gaseous Nitrogen Oxides and Confers Tolerance to Nitrite

A gene that encodes a periplasmic copper-type nitrite reductase (NirK) was identified in Nitrosomonas europaea. Disruption of this gene resulted in the disappearance of Nir activity in cell extracts. The nitrite tolerance of NirK-deficient cells was lower than that of wild-type cells. Unexpectedly, NirK-deficient cells still produced nitric oxide (NO) and nitrous oxide (N(2)O), the latter in greater amounts than that of wild-type cells. This demonstrates that NirK is not essential for the production of NO and N(2)O by N. europaea. Inactivation of the putative fnr gene showed that Fnr is not essential for the expression of nirK.

The terminal oxidases of Paracoccus denitrificans

Three distinct types of terminal oxidases participate in the aerobic respiratory pathways of Paracoccus denitrificans. Two alternative genes encoding sub unit I of the aa3‐type cytochrome c oxidase have been isolated before, namely ctaDI and ctaDII. Each of these genes can be expressed separately to complement a double mutant (ActaDI, ActaDII), indicating that they are isoforms of subunit I of the aa3‐type oxidase. The genomic locus of a quinol oxidase has been isolated: cyoABC. Thisprotohaem‐containing oxidase, called cytochrome bb3, is the oniy quinoi oxidase expressed under the conditions used, in a triple oxidase mutant (ActaDI, ActaDII, cyoB::KmR) an alternative cyto‐chrome c oxidase has been characterized; this cbb3‐type oxidase has been partially purified. Both cytochrome aa3 and cytochrome bb3 are redox‐driven proton pumps. The proton‐pumping capacity of cytochrome cbb3 has been analysed; arguments for and against the active transport of protons by this novel oxidase complex are discussed.

Introduction to the biochemistry and molecular biology of denitrification.

Publisher Summary This chapter provides an overview of the biochemistry and genetics of denitrification in such organisms. It considers the aspects of denitrification that occur in archaea and certain fungi. Denitrification has been mostly studied in Paracoccus denitrificans and Pseudomonas stutzeri and so it describes denitrification for each of these organisms in turn before considering to what extent general principles can be discerned. In recent years, high-resolution crystal structures have become available for these enzymes with the exception of the structure for NO-reductase. In general, the proteins required for denitrification are only produced under (close to) anaerobic conditions, and if anaerobically grown, cells are exposed to O2 and then the activities of the proteins are inhibited. Specialized denitrifiers, such as P. denitrificans and the denitrifying Pseudomonads, contain more than 40 genes, which encode the proteins that make up a full denitrification pathway. They include the structural genes for the enzymes and e− donors, their regulators as well as many accessory genes required for assembly, cofactor synthesis, and insertion into the enzymes. In contrast, some denitrifiers can only carry out the two central reactions of the pathway and use these activities to support growth, but the cost of maintaining this capability is a very small amount of genome space. It provides insights into the regulation of gene expression and the way in which some denitrification enzymes play different roles in bacteria.

Nitrite and nitric oxide reduction in Paracoccus denitrificans is under the control of NNR, a regulatory protein that belongs to the FNR family of transcriptional activators.

The nir and nor genes, which encode nitrite and nitric oxide reductase, lie close together on the DNA of Paracoccus denitrificans. We here identify an adjacent gene, nnr, which is involved in the expression of nir and nor under anaerobic conditions. The corresponding protein of 224 amino acids is homologous with the family of FNR proteins, although it lacks the N‐terminal cysteines. A mutation in the nnr gene had a negative effect on the expression of nitrite and nitric oxide reductase. Synthesis of membrane bound nitrate reductase, of nitrous oxide reductase, and of the cbb 3‐type cytochrome c oxidase were not affected by mutation of this gene. These results suggest that denitrification in P. denitrificans may be governed by a signal transduction network that is similar to that involved in oxygen regulation of nitrogen metabolism in other organisms.

Improved nitrogen removal by application of new nitrogen-cycle bacteria

In order to meet increasingly stringentEuropean discharge standards, new applicationsand control strategies for the sustainableremoval of ammonia from wastewater have to beimplemented. In this paper we discuss anitrogen removal system based on the processesof partial nitrification and anoxic ammoniaoxidation (anammox). The anammox process offersgreat opportunities to remove ammonia in fullyautotrophic systems with biomass retention. Noorganic carbon is needed in such nitrogenremoval system, since ammonia is used aselectron donor for nitrite reduction. Thenitrite can be produced from ammonia inoxygen-limited biofilm systems or in continuousprocesses without biomass retention. Forsuccessful implementation of the combinedprocesses, accurate biosensors for measuringammonia and nitrite concentrations, insight inthe complex microbial communities involved, andnew control strategies have to be developed andevaluated.

Mutagenesis of the gene encoding amicyanin of Paracoccus denitrificans and the resultant effect on methylamine oxidation

The gene encoding the blue‐copper protein amicyanin was isolated from a genomic bank of Pracoccus denitrificans by using a synthetic oligonucleotide. It is located directly downstream of the gene encoding the small subunit of methylmine dehydrogenase. Amicyanin is transcribed as a presursor protein with a signal sequence, typical for periplasmic proteins. Specific inactivation of amicyanin by means of gene replacement techniques resulted in the complete loss of the ability to grow on methylamine.

Nitrosomonas europaea Expresses a Nitric Oxide Reductase during Nitrification

In this paper, we report the identification of a norCBQD gene cluster that encodes a functional nitric oxide reductase (Nor) in Nitrosomonas europaea. Disruption of the norB gene resulted in a strongly diminished nitric oxide (NO) consumption by cells and membrane protein fractions, which was restored by the introduction of an intact norCBQD gene cluster in trans. NorB-deficient cells produced amounts of nitrous oxide (N2O) equal to that of wild-type cells. NorCB-dependent activity was present during aerobic growth and was not affected by the inactivation of the putative fnr gene. The findings demonstrate the presence of an alternative site of N2O production in N. europaea.

Structural and functional analysis of aa3-type and cbb3-type cytochrome c oxidases of Paracoccus denitrificans reveals significant differences in proton-pump design

In Paracoccusdenitrificans the aa3‐type cytochrome c oxidase and the bb3‐type quinol oxidase have previously been characterized in detail, both biochemically and genetically. Here we report on the isolation of a genomic locus that harbours the gene cluster ccoNOQP, and demonstrate that it encodes an alternative cbb3‐type cytochrome c oxidase. This oxidase has previously been shown to be specifically induced at low oxygen tensions, suggesting that its expression is controlled by an oxygen‐sensing mechanism. This view is corroborated by the observation that the ccoNOQP gene cluster is preceded by a gene that encodes an FNR homologue and that its promoter region contains an FNR‐binding motif. Biochemical and physiological analyses of a set of oxidase mutants revealed that, at least under the conditions tested, cytochromes aa3, bb3. and cbb3 make up the complete set of terminal oxidases in P. denitrificans. Proton‐translocation measurements of these oxidase mutants indicate that all three oxidase types have the capacity to pump protons. Previously, however, we have reported decreased H+/e coupling efficiencies of the cbb3‐type

Effects of probiotic Lactobacillus salivarius W24 on the compositional stability of oral microbial communities

Probiotics are microorganisms beneficial to gastrointestinal health. Although some strains are also known to possess positive effects on oral health, the effects of most intestinal probiotics on the oral microflora remain unknown. We assessed the ability of the intestinal probiotic Lactobacillus salivarius W24 to incorporate into and to affect the compositional stability and cariogenicity of oral microbial communities. Microtiter plates with hydroxyapatite discs were incubated with W24 (+W24) or without W24 (-W24) and saliva from four individuals in plain (-sucrose) or sucrose-supplemented (+sucrose) medium. Biofilms were subjected to community profiling by 16S rRNA gene-based Denaturing Gradient Gel Electrophoresis (DGGE) after 72h growth. Diversity (Shannon-Weaver index) and similarities (Pearson correlation) between biofilm communities were calculated. Microcosms +sucrose were less diverse and more acidic than -sucrose microcosms (p<0.001). The effects of W24 on the community profiles were pH dependent: at pH 4 (+sucrose), the respective +W24 and -W24 microcosms differed significantly more from each other than if the pH was approximately 7 (-sucrose). The pH of +W24/+sucrose microcosms was lower (p<0.05) than the pH of the microcosms supplemented with sucrose alone (-W24/+sucrose). Although not able to form a monospecies biofilm, L. salivarius W24 established itself into the oral community if inoculated simultaneously with the microcosm. In the presence of sucrose and low pH, W24 further lowered the pH and changed the community profiles of these microcosms. Screening of probiotics for their effects on oral microbial communities allows selecting strains without a potential for oral health hazards.