Nitrite reductase genes in halobenzoate degrading denitrifying bacteria

Abstract

Diversity of the functional genes encoding dissimilatory nitrite reductase was investigated for the first time in denitrifying halobenzoate degrading bacteria and in two 4-chlorobenzoate degrading denitrifying consortia. Nitrite reductase genes were PCR-amplified with degenerate primers (specific to the two different types of respiratory nitrite reductase, nirS and nirK), cloned and sequenced to determine which type of nitrite reductase was present in each isolate and consortium. Halobenzoate degrading isolates belonging to the genera Ochrobactrum, Ensifer and Mesorhizobium, as well as Pseudomonas mendocina CH91 were found to have nirK genes, which were closely related to the previously published nirK genes of Ochrobactrum anthropi, Achromobacter cycloclastes, Alcaligenes faecalis and Pseudomonas aureofaciens, respectively. The isolates assigned to the genera Acidovorax, Azoarcus and Thauera as well as all other species in the genera Thauera and Azoarcus contained nirS genes, which were closely related to the nirS genes from Pseudomonas stutzeri with some exceptions. In addition, only nirS genes were found in 4-chlorobenzoate degrading denitrifying consortia. Three different major terminal restriction fragments from the nirS genes were detected by terminal restriction fragment length polymorphism analysis of the consortia, and five different nirS genes were cloned from one consortium. Three nirS gene clones were closely related to nirS genes from Thauera chlorobenzoica, Azoarcus tolulyticus and Pseudomonas aeruginosa, respectively. The phylogeny of nir genes was not entirely congruent with the 16S rRNA phylogeny of the genera nor was it correlated with the ecological and geographical origins or isolation substrates used for isolation and enrichment of consortia.