Genomic organization, gene expression and activity profile ofMarinobacter hydrocarbonoclasticusdenitrification enzymes

Abstract

BackgroundDenitrification is one of the main pathways of the N-cycle, during which nitrate is converted to dinitrogen gas, in four consecutive reactions that are each catalyzed by a different metalloenzyme. One of the intermediate metabolites is nitrous oxide, which has a global warming impact greater then carbon dioxide and which atmospheric concentration has been increasing in the last years. The four denitrification enzymes have been isolated and biochemically characterized from Marinobacter hydrocarbonoclasticus in our lab.MethodsBioinformatic analysis of the M. hydrocarbonoclasticus genome to identify the genes involved in the denitrification pathway. The relative gene expression of the gene encoding the catalytic subunits of those enzymes was analyzed during the growth under microoxic conditions. The consumption of nitrate and nitrite, and the reduction of nitric oxide and nitrous oxide by whole-cells was monitored during anoxic and microoxic growth in the presence of 10 mM sodium nitrate at pH 7.5.ResultsThe bioinformatic analysis shows that genes encoding the enzymes and accessory factors required for each step of the denitrification pathway are clustered together. An unusual feature is the co-existence of genes encoding a q- and a c-type nitric oxide reductase, with only the latter being transcribed at similar levels as the ones encoding the catalytic subunits of the other denitrifying enzymes, when cells are grown in the presence of nitrate under microoxic conditions. Using either a batch- or a closed system, nitrate is completely consumed in the beginning of the growth, with transient formation of nitrite, and whole-cells can reduce nitric oxide and nitrous oxide from mid-exponential phase until being collected (time-point 50 h).DiscussionM. hydrocarbonoclasticus cells can reduce nitric and nitrous oxide in vivo, indicating that the four denitrification steps are active. Gene expression profile together with promoter regions analysis indicates the involvement of a cascade regulatory mechanism triggered by FNR-type in response to low oxygen tension, with nitric oxide and nitrate as secondary effectors, through DNR and NarXL, respectively. This global characterization of the denitrification pathway of a strict marine bacterium, contributes to the understanding of the N-cycle and nitrous oxide release in marine environments.

The effect of pH on Marinobacter hydrocarbonoclasticus denitrification pathway and nitrous oxide reductase

“…The decrease in nitrate concentration was concomitant with formation of nitrite, which reached a maximum value close to the initial nitrate concentration. In the growth experiment performed at pH 8.5, nitrite was completely consumed after ~ 10 h (during the initial stages of the second diauxic growth phase) ( Figure 2B), similar to what was previously observed by Carreira and colleagues in cells grown at optimum pH (pH 7.5) [49]. However, in cells grown at pH 6.5, only 27 % of the total nitrite produced was consumed and consequently high levels of nitrite (around 6.2 mM) remained in the medium until the end of the growth period (40 h) (Figure 2A).…”

“…In fact, considering that there is 100 % recovery of N2OR, isolated from cells grown at pH 7.5, 40 µL of cell suspension would have 0.000104 mg of N2OR, thus since this volume of cells have a reduction rate of 3.4 x 10 -2 µmolN2O•min -1 , this gives an estimate of 326 µmolN2O•min -1 •mgN2OR -1 . Another important comment is that the KM for N2O (18 ± 5 µM) of cells grown at pH 7.5 is similar to the one determined for N2OR with "CuZ" center as CuZ(4Cu1S) in the fully reduced state using reduced methyl viologen as an electron donor (14 µM) [49]. Therefore, these results clearly point out that N2OR is activated in vivo through a still unknown mechanism, that seems to include the reduction of "CuZ" center to the hydrocarbonoclasticus cells grown at pH 6.5, NosR might be inhibited, misfolded or incorrectly maturated (e.g., without the flavin group), or its interaction with N2OR impaired (as it is known that in P. aeruginosa these proteins are part of a supramolecular complex [64]).…”

“…In cells grown at pH 7.5 (published work by Carreira and colleagues in [49]) and at pH 8.5, the expression levels of narG were lower than the ones of the other denitrification genes ( Figure 1B). In cells grown at pH 6.5, the gene with the lower expression level was nirS ( Figure 1A).…”

“…Another difference was the expression profile of MARHY1380 (M. hydrocarbonoclasticus nosX homologue, see Figure S2A in Supplementary Material), as its maximum expression occurred at the same time point as the one of genes encoding the catalytic subunits of denitrifying enzymes at pH 6.5 ( Figure 1A). For the growth experiments at pH 7.5 and 8.5, MARHY1380 was still highly expressed during the first 3 h ( Figure S2B and S2C in Supplementary Material), but the maximum gene expression of the genes encoding the catalytic subunits of denitrifying enzymes occurred at 5 h, for the growth experiment performed at pH 7.5 (published data in [49]) and 7 h, for the growth experiment performed at pH 8.5 ( Figure 1B). This might evidence a different regulatory mechanism between MARHY1380 and the denitrifying genes.…”

“…hydrocarbonoclasticus cells grown at pH 7.5, in which the maximum of expression occurred after 5 h of growth [49].…”