PCR primers and functional probes for amplification and detection of bacterial genes for extracellular peptidases in single strains and in soil

Abstract

A set of primers and functional probes was developed for the detection of peptidase gene fragments of proteolytic bacteria. Based on DNA sequence data, degenerate PCR primers and internal DIG-labeled probes specific for genes encoding alkaline metallopeptidases (apr) (E.3.4.24), neutral metallopeptidases (npr) (E.3.4.24) and serine peptidases (sub) (E.3.4.21) were derived by multiple sequence alignments. Type strains with known peptidase genes and proteolytic bacteria from a grassland rhizosphere soil, a garden soil and an arable field were investigated for their genotypic proteolytic potential. For 52 out of 53 proteolytic bacterial isolates, at least one of the three peptidase classes could be identified by this approach. The amplified gene fragments were of the expected sizes with each of the three primer sets. The functional probes APR, NPR and SUB have been shown to hybridize specifically to the corresponding gene fragments. sub and npr genes were mainly found in Bacillus species. apr genes were only found in the Pseudomonas fluorescens biotypes and in two morphologically identical Flavobacterium-Cytophaga strains from two different sites. In most of the Bacillus spp., both sub and the npr and in the Flavobacterium-Cytophaga strains even all the three genes could be detected. PCR with DNA isolated from soil led to one main product of the expected size with each primer pair whose identity was additionally confirmed by Southern blot hybridization with the corresponding probes.