Primers for overlooked nirK, qnorB, and nosZ genes of thermophilic Gram-positive denitrifiers

Although efforts have been made the past few years, knowledge on genomic and phenotypic diversity and occurrence of the denitrification ability in Gram-positive bacteria are still fragmentary. Many environmental monitoring approaches have used nir, nor, and nos genes as marker genes for detection of denitrification or denitrifying bacteria. However, primers used in these methods often fail to detect the genes in specific bacterial taxa, such as Gram-positive denitrifiers. In this study, novel primer sets specifically targeting nirK, qnorB, and nosZ genes of the Firmicute genus Geobacillus were developed by genomic mining and tested in parallel with commonly used primers on a set of phylogenetically closely related denitrifying geobacilli. Novel nirK and qnorB sequences were recovered from all strains tested, whereas nosZ was detected in part of the strain set, which was in agreement with observed phenotypes. Interspecies and modest intraspecies variations in amplified fragment length polymorphism (AFLP) patterns were observed, verifying presence of genomic variation within the strain set. Our study shows that closely related Gram-positive denitrifiers may differ in denitrification phenotype and genotype. But foremost, novel primers targeting very divergent nirK, qnorB, and nosZ gene sequences of Gram-positive denitrifiers, are now available for cultivation-independent environmental surveys.