The presence of intragenically located REP-like elements in Bacillus sporothermodurans is sufficient for REP-PCR typing

Abstract

The technique of repetitive extragenic palindromic polymerase chain reaction (REP-PCR) enables the identification and discrimination of clonally related Bacillus sporothermodurans isolates from ultra-high temperature and sterilized milk. The aim of this study was to investigate the genetic basis for the generation of these highly informative REP-PCR patterns. The major 947-bp REP-PCR fragment of B. sporothermodurans was cloned, together with its 5’ and 3’ flanking sequences. Only partial homology with the REP consensus sequence was established at the borders of the REP-PCR fragment. Moreover, these border sequences were located within two distinct open reading frames, with great homology to the uvrA and uvrB genes of Escherichia coli. The presence of these REP-like elements in B. sporothermodurans was thus sufficient for high resolution REP-PCR typing of this Gram-positive organism. In some cases (and especially with Gram-positives), REP-PCR could thus be considered more as an arbitrary primed PCR, albeit at a somewhat higher annealing temperature and using conserved primers. The random priming effect at the less stringent annealing conditions of REP-PCR was also demonstrated for enterobacterial repetitive intergenic consensus PCR (ERIC-PCR) on another Gram-positive organism, Listeria monocytogenes.