Genomic organization, gene expression and activity profile of Marinobacter hydrocarbonoclasticus denitrification enzymes

Background Denitrification 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. Methods Bioinformatic 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. Results The 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). Discussion M. 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.


S1 -List of denitrification genes identified in different bacteria
In Table S1 are listed the genes that have been identified to encode catalytic enzymes/subunits that catalyze the different steps of the denitrification pathway.
Table S1 -Genes that encode the catalytic subunits of the enzymes involved in the denitrification pathway.
Step Enzyme Gene encoding the catalytic subunit Ref.
NO3 -→ NO2 -Membrane nitrate reductase (NaR) narG (Ghiglione et al. 1999 cnosZ (Simon et al. 2004) Notes: * This is a hexameric nitrite reductase isolated from Hyphomicrobium denitrificans that contains an additional N-terminal cupredoxin domain. ** The gene coding for this enzyme has been identified at the genome level and is proposed to present an additional C-terminal class I c-type cytochrome domain.

S2 -Bioinformatic analysis of denitrification gene cluster of M. hydrocarbonoclasticus
The identification of putative gene functions was performed by BLAST search using the web platforms of blastp and blastn suites (NCBI) (Altschul et al. 1997) (statistic data are provided in Table S1), gene and protein alignments using ClustalOmega (McWilliam et al. 2013), and comparison of gene organization with other denitrifying microorganisms.

S6 -NO and N2O reduction by the whole-cells -Data Analysis
The rate of NO and N2O reduction by the whole-cells was indirectly measured through oxidation of methyl viologen. In the curve obtained, linear regressions were used to fit regions immediately before and after substrate addition. The slope of the fitting before substrate addition was subtracted to the slope obtained after substrate addition. The rates were determined taking into consideration the calculated slope and the extinction coefficient of methyl viologen at 600 nm (ε600nm = 11.4 mM -1 cm -1 ) (Kristjansson & Hollocher 1980). We also consider that methyl viologen re-oxidation requires one electron while N2O reduction involves two electrons and the reduction of two molecules of NO involves two electrons. Note that in this assay, the subtraction of the slope prior to the addition of substrate is particularly important as other enzymes might also use the reduced methyl viologen, causing partial oxidation even before the addition of substrate. Rates of NO and N2O reduction were reported as micromoles of NO or N2O reduced per minute per optical density (µmolNO or N2O min -1 OD -1 ).

S7 -Genome organization of M. hydrocarbonoclasticus denitrification genes
A search in the genome of M. hydrocarbonoclasticus SP17 for genes involved in denitrification showed that several genes encoding proteins involved in the different steps of this pathway (see Figure S2). The nosRZDFYL gene cluster, containing the catalytic unit of N2OR, encoded by nosZ (Philippot 2002;Zumft & Kroneck 2007). This gene cluster presents nosR, which encodes a transmembrane protein containing Fe-S centers and a periplasmic domain that binds a flavin (Cuypers et al. 1992;Wunsch & Zumft 2005). Although its function remains unclear, NosR seems to be important for nosZ transcription, as well as for enzyme full activity in vivo, as evidenced by mutational studies in P. stutzeri (Cuypers et al. 1992;Wunsch & Zumft 2005). The nosD gene encodes a protein that together with NosFY is proposed to form an ABC-transporter (NosDFY) and be involved in sulfur transport for CuZ center assembly (Wunsch et al. 2003;Zumft 2005a), while nosL encodes a putative copper chaperone (McGuirl et al. 2001). Upstream nos cluster is the NaR gene cluster, narLXKGHJV (see Figure S2) containing the genes narG, S6 narH and narV (also designated as narI) that encode the α,  and  subunits of the enzyme, respectively (Philippot 2002). The narJ gene encodes a chaperone-like component involved in the maturation and assembly of the enzyme complex (Lanciano et al. 2007), and narXL encode NarXL a two-component system. Several nark type genes have been identified in this genome, with one being associated with the nar cluster (MARHY3029). Two other narK genes are located upstream the nir operon, which correspond to MARHY3076, annotated as narK2, and MARHY3078, annotated as narK. Additionally, a narU (MARHY3080), encoding in E. coli a nitrate/nitrite transporter (Jia et al. 2009;Yan et al. 2013), is also found in this genome (see Figure S2). Further upstream is norBC, encoding the two subunits of the short-chain membrane-bound c-NOR (see Figure S2). NorB (MARHY3054) is the catalytic subunit, while NorC (MARHY3053) is a small membrane bound c-type cytochrome functioning as an electron transfer subunit (Philippot 2002;Zumft 2005b). The accessory genes norQ (MARHY3060, annotated in other organisms as napH) and norD (MARHY3056), together with two other ORFs (MARHY3057 and MARHY3058) are located upstream this operon. NorE and NorF, are predicted to be membrane associated, though their exact function remains unknown (Bergaust et al. 2014). It has been shown that NorEF are involved in denitrification as its inactivation slows nitrate reduction during denitrification, with accumulation of micromolar concentrations of nitric oxide (Bergaust et al. 2014;de Boer et al. 1996). In contrast, norQ and norD are always found linked to or in the vicinity of norBC (Zumft 2005b). The encoded proteins are suggested to be involved in the maturation of NorBC in either heme insertion, multisubunit assembly and/or insertion into the membrane (Zumft 1997;Zumft 2005b). After the norBC genes, there is nirFCSDLGHJEN. The gene nirS encodes the cytochrome cd1NiR, a homodimeric enzyme with c-and d1-type hemes (Lopes et al. 2001 (Zajicek et al. 2009) and nirN (Adamczack et al. 2014) encode enzymes involved in the biosynthesis of heme-d1, all located in the cytoplasm, with the exception of NirN. NirF has been proposed to be a chaperone involved in the uptake and transport of dihydro-heme d1 precursor from the cytoplasm to NirN located in the periplasm. The exact role of NirC remains unknown, but has been shown to be essential for the synthesis of heme-d1 in P. denitrificans (Bali et al. 2014;de Boer et al. 1994). It is predicted to be a periplasmic c-type cytochrome, playing a role in the biosynthesis of heme-d1 as an electron acceptor from NirN (Adamczack et al. 2014;Zajicek et al. 2009). Finally, a gene annotated as nnrS, was identified upstream the nir cluster, which might be a membranebound NO sensor with a role in nitrosative stress, yet poorly explored (Stern et al. 2013).