Novel fimbrial subunit genes of Dichelobacter nodosus: recombination in vivo or in vitro?

Polymerase chain reaction (PCR) was used to amplify the variable region of the fimbrial subunit encoding gene (fimA) of Dichelobacter nodosus from she ep and goats infected with footrot. Two amplimers (designated X and Y) gene rated single-strand conformation polymorphism (SSCP) patterns different to those of previously identified serogroups and serotypes. DNA sequencing rev ealed that these two fragments were novel. The upstream of X (nt 1-183) was identical to serotype M1 while its downstream (nt 223-414) was identical t o serotype F1; the upstream of Y (nt 1-116) was identical to serotype El, w hereas its downstream (nt 148-423) was identical to serotype Fl. A 14-mer s equence consisting of two partially overlapping Chi-like sequences, 5'-GCTG GTGCTGGTGA-3', was also found in these fragments. Two primer sets with the downstream primer specific for serotype Fl and the upstream primer specific for serotype M or El, generated PCR products of the expected sizes from th e footrot samples from which fragments X and Y were isolated, respectively. These primer sets did not appear to amplify artificially mixed genomic DNA from serotypes M and Fl or El and Fl. However, when the reactions were rea mplified, PCR recombination artifacts were observed, suggesting that PCR re combination does occur, but at a low frequency. It, therefore, seems more l ikely that fragments X and Y reflect genuine fimA genes of D. nodosus which have resulted from in vivo DNA recombination rather than from a PCR recomb ination artifact. (C) 2000 Elsevier Science B.V. All rights reserved.