Denitrifying metabolism of the methylotrophic marine bacterium <i>Methylophaga nitratireducenticrescens</i> strain JAM1

Background Methylophaga nitratireducenticrescens strain 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 (${\mathrm{NO}}_{3}^{-}$) to nitrite (${\mathrm{NO}}_{2}^{-}$). These physiological traits are attributed to gene clusters that encode two dissimilatory nitrate reductases (Nar). Strain JAM1 also contains gene clusters encoding two nitric oxide (NO) reductases and one nitrous oxide (N 2 O) reductase, suggesting that NO and N 2 O can be reduced by strain JAM1. Here we characterized further the denitrifying activities of M. nitratireducenticrescens JAM1. Methods Series of oxic and anoxic cultures of strain JAM1 were performed with N 2 O, ${\mathrm{NO}}_{3}^{-}$ or sodium nitroprusside, and growth and N 2 O, ${\mathrm{NO}}_{3}^{-}$, ${\mathrm{NO}}_{2}^{-}$ and N 2 concentrations were measured. Ammonium (${\mathrm{NH}}_{4}^{+}$)-free cultures were also tested to assess the dynamics of N 2 O, ${\mathrm{NO}}_{3}^{-}$ and ${\mathrm{NO}}_{2}^{-}$. Isotopic labeling of N 2 O was performed in 15 NH 4 + -amended cultures. Cultures with the JAM1Δ narG1narG2 double mutant were performed to assess the involvement of the Nar systems on N 2 O production. Finally, RT-qPCR was used to measure the gene expression levels of the denitrification genes cytochrome bc -type nitric oxide reductase ( cnorB1 and cnorB2 ) and nitrous oxide reductase ( nosZ ), and also nnrS and norR that encode NO-sensitive regulators. Results Strain JAM1 can reduce NO to N 2 O and N 2 O to N 2 and can sustain growth under anoxic conditions by reducing N 2 O as the sole electron acceptor. Although strain JAM1 lacks a gene encoding a dissimilatory ${\mathrm{NO}}_{2}^{-}$ reductase, ${\mathrm{NO}}_{3}^{-}$-amended cultures produce N 2 O, representing up to 6% of the N-input. ${\mathrm{NO}}_{2}^{-}$ was shown to be the key intermediate of this production process. Upregulation in the expression of c norB1 , cnorB2, nnrS and norR during the growth and the N 2 O accumulation phases suggests NO production in strain JAM1 cultures. Discussion By showing that all the three denitrification reductases are active, this demonstrates that M. nitratireducenticrescens JAM1 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 an incomplete pathway could be related to the specific role of strain JAM1 in the denitrifying biofilm of the denitrification reactor from which it originates. The production of N 2 O in strain JAM1 did not involve Nar, contrary to what was demonstrated in Escherichia coli . M. nitratireducenticrescens JAM1 is the only reported Methylophaga species that has the capacity to grow under anoxic conditions by using ${\mathrm{NO}}_{3}^{-}$ and N 2 O 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.

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