Phenotypic and genotypic heterogeneity among closely related soil-borne N2- and N2O-producing Bacillus isolates harboring the nosZ gene

Little is known about the genetic and phenotypic diversity of Gram-positive denitrifying bacteria. We compared the production of gaseous denitrification products for 14 closely related Bacillus soil isolates at pH 6 and 7 during 48-h batch incubations using a robotic gas-sampling apparatus. Primers targeting the nosZ gene encoding the nitrous oxide reductase were designed to confirm the presence of this gene in the isolates. The variation in the production of gaseous nitrogen products was compared with the genetic variation based on 16S rRNA gene sequences, genomic fingerprinting and nosZ sequences. The nosZ gene was detected in all isolates and all produced N-2 as the dominant end product at pH 7. Production of gaseous nitrogen products was more variable at pH 6, with different levels of NO and N2O production among the isolates, although minimal variation was observed among the 16S rRNA and nosZ gene sequences. One isolate was more divergent from the others based on genomic fingerprinting, and had two different nosZ gene copies, which coincided with the highest production of N-2 at pH 7 and the lack of intermediates at pH 6. Overall, our analysis suggests that genetic variation plays a role in the variation in N2O and N-2 production, but the variation in activity caused by acidification can be substantially greater than genotypic variation among closely related Bacillus.