Denitrifying metabolism of the methylotrophic marine bacteriumMethylophaga nitratireducenticrescensstrain JAM1

Methylophaga nitratireducenticrescensstrain JAM1 is a methylotrophic, marine bacterium that was isolated from a denitrification reactor treating a closed-circuit seawater aquarium. It can sustain growth under anoxic conditions by reducing nitrate (NO3−) to nitrite (NO2−), which accumulates in the medium. These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (NarGHJI). M.nitratireducenticrescensstrain JAM1 also contains gene clusters encoding two putative nitric oxide reductase (NO) reductases and one putative nitrous oxide (N2O) reductase, suggesting that NO and N2O can be reduced by strain JAM1. In this study, we show that strain JAM1 can reduce NO to N2O and N2O to N2 and can sustain growth under anoxic conditions by reducing N2O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory copper-(NirK) or cytochrome cd1-type (NirS) NO2−reductase, NO3−-amended strain JAM1 cultures produce N2O, representing up to 6% of the N-input. NO2−was shown to be the key intermediate of this production process. In NO3−amended cultures, we analyzed denitrification genes in succession of net N2O-production and -consumption phases at the gene expression level. These phases were found to correlate with changes in the expression levels of the NO reductase genecnorB1andnnrS, which indicated NO production in the cultures.ImportanceBy showing that all the three denitrification reductases are active, this demonstrates thatMethylophaga nitratireducenticrescensJAM1 is one of many bacteria species that maintain genes associated primarily with denitrification, but not necessarily related to the maintenance of the entire pathway. The reason to maintain such incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of a denitrification reactor from which it originates. The small production of N2O via NO in strain JAM1 did not involve Nar contrary to what was demonstrated inEscherichia coli. M. nitratireducenticrescensJAM1 is the only reportedMethylophagaspecies that has the capacity to grow under anoxic conditions by using NO3−and N2O as sole electron acceptors for its growth. It is also one of a few marine methylotrophs that is studied at the physiological and genetic levels in relation to its capacity to perform denitrifying activities.

The functional role of the structure of the dioxo-isobacteriochlorin in the catalytic site of cytochrome cd1 for the reduction of nitrite

We studied the functional role of the unique heme d1 in the catalytic nitrite reduction using synthetic model complexes.

Maturation of the cytochrome cd1 nitrite reductase NirS from Pseudomonas aeruginosa requires transient interactions between the three proteins NirS, NirN and NirF

The periplasmic cytochrome cd1 nitrite reductase NirS occurring in denitrifying bacteria such as the human pathogen Pseudomonas aeruginosa contains the essential tetrapyrrole cofactors haem c and haem d1. Whereas the haem c is incorporated into NirS by the cytochrome c maturation system I, nothing is known about the insertion of the haem d1 into NirS. Here, we show by co-immunoprecipitation that NirS interacts with the potential haem d1 insertion protein NirN in vivo. This NirS–NirN interaction is dependent on the presence of the putative haem d1 biosynthesis enzyme NirF. Further, we show by affinity co-purification that NirS also directly interacts with NirF. Additionally, NirF is shown to be a membrane anchored lipoprotein in P. aeruginosa. Finally, the analysis by UV–visible absorption spectroscopy of the periplasmic protein fractions prepared from the P. aeruginosa WT (wild-type) and a P. aeruginosa ΔnirN mutant shows that the cofactor content of NirS is altered in the absence of NirN. Based on our results, we propose a potential model for the maturation of NirS in which the three proteins NirS, NirN and NirF form a transient, membrane-associated complex in order to achieve the last step of haem d1 biosynthesis and insertion of the cofactor into NirS.

Observation of fast release of NO from ferrous d1 haem allows formulation of a unified reaction mechanism for cytochrome cd1 nitrite reductases

Cytochrome cd1 nitrite reductase is a haem-containing enzyme responsible for the reduction of nitrite into NO, a key step in the anaerobic respiratory process of denitrification. The active site of cytochrome cd1 contains the unique d1 haem cofactor, from which NO must be released. In general, reduced haems bind NO tightly relative to oxidized haems. In the present paper, we present experimental evidence that the reduced d1 haem of cytochrome cd1 from Paracoccus pantotrophus releases NO rapidly (k=65–200 s−1); this result suggests that NO release is the rate-limiting step of the catalytic cycle (turnover number=72 s−1). We also demonstrate, using a complex of the d1 haem and apomyoglobin, that the rapid dissociation of NO is largely controlled by the d1 haem cofactor itself. We present a reaction mechanism proposed to be applicable to all cytochromes cd1 and conclude that the d1 haem has evolved to have low affinity for NO, as compared with other ferrous haems.

Partial and complete denitrification in Thermus thermophilus: lessons from genome drafts

We have obtained draft genomic sequences of PD (partial denitrificant) and CD (complete denitrificant) strains of Thermus thermophilus. Their genomes are similar in size to that of the aerobic strains sequenced to date and probably contain a similar megaplasmid. In the CD strain, the genes encoding a putative cytochrome cd1 Nir (nitrite reductase) and ancillary proteins were clustered with a cytochrome c-dependent Nor (nitric oxide reductase), and with genes that are probably implicated in their regulation. The Nar (nitrate reductase) and associated genes were also clustered and located 7 kb downstream of the genes coding for the Nir. The whole nar–nir–nor denitrification supercluster was identified as part of a variable region of a megaplasmid. No homologues of NosZ were found despite nitrogen balance supports the idea that such activity actually exists.