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Applied and Environmental Microbiology, June 2003, p. 3299-3307, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3299-3307.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Consensus-Degenerate Hybrid Oligonucleotide Primers for Amplification of Priming Glycosyltransferase Genes of the Exopolysaccharide Locus in Strains of the Lactobacillus casei Group

Cathy Provencher,1,2 Gisèle LaPointe,2 Stéphane Sirois,1 Marie-Rose Van Calsteren,1 and Denis Roy1*

Food Research and Development Centre, Agriculture and Agri-Food Canada, Saint-Hyacinthe, Quebec, Canada J2S 8E3,1 STELA Dairy Research Centre, Laval University, Quebec, Canada G1K 7P42

Received 4 September 2002/ Accepted 18 March 2003

A primer design strategy named CODEHOP (consensus-degenerate hybrid oligonucleotide primer) for amplification of distantly related sequences was used to detect the priming glycosyltransferase (GT) gene in strains of the Lactobacillus casei group. Each hybrid primer consisted of a short 3' degenerate core based on four highly conserved amino acids and a longer 5' consensus clamp region based on six sequences of the priming GT gene products from exopolysaccharide (EPS)-producing bacteria. The hybrid primers were used to detect the priming GT gene of 44 commercial isolates and reference strains of Lactobacillus rhamnosus, L. casei, Lactobacillus zeae, and Streptococcus thermophilus. The priming GT gene was detected in the genome of both non-EPS-producing (EPS-) and EPS-producing (EPS+) strains of L. rhamnosus. The sequences of the cloned PCR products were similar to those of the priming GT gene of various gram-negative and gram-positive EPS+ bacteria. Specific primers designed from the L. rhamnosus RW-9595M GT gene were used to sequence the end of the priming GT gene in selected EPS+ strains of L. rhamnosus. Phylogenetic analysis revealed that Lactobacillus spp. form a distinctive group apart from other lactic acid bacteria for which GT genes have been characterized to date. Moreover, the sequences show a divergence existing among strains of L. rhamnosus with respect to the terminal region of the priming GT gene. Thus, the PCR approach with consensus-degenerate hybrid primers designed with CODEHOP is a practical approach for the detection of similar genes containing conserved motifs in different bacterial genomes.

* Corresponding author. Mailing address: Food Research and Development Centre, Agriculture and Agri-Food Canada, 3600 Casavant Blvd. West, Saint-Hyacinthe, Quebec, Canada J2S 8E3. Phone: (450) 773-1105. Fax: (450) 773-8461. E-mail: royd{at}agr.gc.ca.

Applied and Environmental Microbiology, June 2003, p. 3299-3307, Vol. 69, No. 6
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.6.3299-3307.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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