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

A set of primers and functional probes was developed for the detection of p eptidase 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 metallopepti dases (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 gra ssland rhizosphere soil, a garden soil and an arable field were investigate d for their genotypic proteolytic potential. For 52 out of 53 proteolytic b acterial isolates, at least one of the three peptidase classes could be ide ntified by this approach. The amplified gene fragments were of the expected sizes with each of the three primer sets. The functional probes APR, NPR a nd SUB have been shown to hybridize specifically to the corresponding gene fragments, sub and npr genes were mainly found in Bacillus species. apr gen es were only found in the Pseudomonas fluorescens biotypes and in two morph ologically identical Flavobacterium-Cytophaga strains from two different si tes. In most of the Bacillus spp.. both sub and the npr and in the Flavobac terium-Cytophaga strains even all the three genes could be detected. PCR wi th DNA isolated from soil led to one main product of the expected size with each primer pair whose identity was additionally confirmed by Southern blo t hybridization with the corresponding probes. (C) 2001 Elsevier Science B. V. All rights reserved.