A new family of nitrate/nitrite transporters involved in denitrification

Abstract

Denitrifying bacteria carry out nitrate and nitrite respiration inside and outside the cell, respectively. In Thermus thermophilus, nitrate and nitrite transport processes are carried out by major facilitator superfamily (MFS) transporters. The sequence of the nar operon of nitrate-only respiring strains of T. thermophilus includes two tandemly organized MFS transporter genes (narK and narT) of the NarK1 and NarK2 families. Both can function as nitrate/nitrite antiporters, but NarK has been proposed as more specific for nitrate whereas NarT more specific for nitrite. In some nitrate- and nitrite-respiring strains of the same species, a single MFS transporter (NarO) belonging to a different MFS subfamily appears. To analyze the role of this single MFS in the same genetic context, we transferred the two types of nar operon to the aerobic strain HB27, and further included in both of them the ability to respire nitrite. The new denitrifying strains HB27dn, with two MFS, and HB27dp, with a single one, were used to isolate mutants devoid of transporters. Through in trans complementation experiments, we demonstrate that the NarO single MFS works efficiently in the transport of both nitrate and nitrite.

Keywords: Denitrification; MFS; Nitrate; Nitrite; Thermus; Transport.

Fig. 1

One or two MSF transporters are encoded within the nitrate reductase operon. Organization of the nar operon in the indicated strains of Thermus spp.: T. thermophilus NAR1, T. thermophilus Fiji3A1, T. oshimai JL2, T. scotoductus SA01, T. thermophilus PRQ25, T. thermophilus SG0.5JP17-16, and T. thermophilus JL18

Fig. 2

Cladogram of the nitrate/nitrite transporters subfamily. The identification of homologs to the nitrate/nitrite putative transporters was performed by searching with BLAST (Altschul et al., 1990) against a non-redundant protein database. The protein data set was aligned using CLUSTAL OMEGA (Sievers et al., 2011). iTOL (Letunic & Bork, 2016) was used for the visualization of the generated tree. Branch-distance is indicated. The proteins used for the comparison are listed in Supplemental Table S1

Fig. 3

Anaerobic growth with nitrate of the mutants in the nitrate/nitrite transporters. a Anaerobic growth curves of strains HB27, NAR1, HB27dn (27dn), HB27dn ∆narKT (∆narKT), and the HB27dn ∆narKT mutant harboring empty plasmid pMK184 (pMK) or overexpressing NarK (pMKnarK), NarT (pMKnarT), or NarO (pMKnarO). b Anaerobic growth curves of strains HB27, PRQ25, HB27dp (27dp), HB27dp ∆narO (∆narO), and the HB27dp ∆narO mutant harboring empty plasmid pMK184 (pMK) or overexpressing NarK (pMKnarK), NarT (pMKnarT), or NarO (pMKnarO)

Fig. 4

NarO transports both nitrate and nitrite. Nitrite production (mM) in the different strains after 20 min of incubation in the presence of different concentrations of nitrate (0–20 mM). Symbols: ▲ HB27; ♦ 27dn; ▼ 27dp; ● 27dp ∆narO; ○ 27dp ∆narO pMHnarO; ■ 27dn ∆narKT; □ 27dn ∆narKT pMHnarO

Fig. 5

Resistance to nitrite and nitrate provided by MFS transporters on plate assays. Dot plate assays with serial 1:10 dilutions of cultures of T. thermophilus HB27 transformed with the indicated plasmids. TB plates with hygromycin B containing sodium nitrite or potassium nitrate 150 and 200 mM, respectively, were used

Fig. 6

Liquid growth inhibition by nitrite and nitrate. Cultures of the aerobic T. thermophilus HB27 strain transformed with the indicated plasmid were grown for 24 h at 65 °C on TB medium with hygromycin (100 μg/ml) in the presence of the indicated concentrations of nitrite (a) or nitrate (b). The mean percentage of growth (OD₆₀₀) of three different colonies respect to the controls without nitrogen oxide is represented