Dissimilatory reduction of nitrate in seawater by a Methylophaga strain containing two highly divergent narG sequences

Methylophaga spp. are methylotrophs commonly associated with marine environments and have been defined as strict aerobic methylotrophs. They have been shown previously to represent 50–70% of the bacterial population in the biofilm of the methanol-fed denitrification reactor treating a large seawater aquarium at the Montreal Biodome. It was therefore surprising to find such a high concentration of Methylophaga spp. in anoxic conditions. In this study, we showed by cultivation-independent and -dependent approaches that one Methylophaga strain present in the anoxic biofilm is involved in the denitrification process. DNA stable-isotope probing (SIP) experiments in which the biofilm was cultured under denitrifying conditions with 13 C-methanol have revealed the enrichment of one particular taxon. By screening a 16S ribosomal RNA gene library derived from a 13 C-DNA fraction of the SIP gradients, 62% of the library was composed of one sequence affiliated with the genus Methylophaga . One strain, named JAM1, representing this Methylophaga species was isolated. It grows aerobically but also under denitrifying conditions by reducing nitrate into nitrite. The nitrate-reducing activity was correlated with the presence and the expression of two highly divergent narG genes ( narG 1 and narG 2). narG 1 showed a high percentage of identity with the corresponding part of narG found in Thiobacillus denitrificans, which suggests a recent acquisition of narG in strain JAM1 by horizontal gene transfer. This study provides the first direct evidence of the adaptation of a Methylophaga species to an oxygen-limited environment.