The complete coenzyme B12 biosynthesis gene cluster of Lactobacillus reuteri CRL1098

1 Kluyver Centre for Genomics of Industrial Fermentation, TI Food and Nutrition, and NIZO Food Research, Kernhemseweg 2, PO Box 20, 6710 BA Ede, The Netherlands 2 Centro de Referencia para Lactobacilos (CERELA-CONICET), Chacabuco 145 (4000), San Miguel de Tucumán, Tucumán, Argentina 3 Center for Molecular and Biomolecular Informatics, Radboud University Nijmegen, Toernooiveld 1, 6525 ED Nijmegen, The Netherlands Correspondence Jeroen Hugenholtz jeroen.hugenholtz{at}nizo.nl The coenzyme B 12 production pathway in Lactobacillus reuteri has been deduced using a combination of genetic, biochemical and bioinformatics approaches. The coenzyme B 12 gene cluster of Lb. reuteri CRL1098 has the unique feature of clustering together the cbi , cob and hem genes. It consists of 29 ORFs encoding the complete enzymic machinery necessary for de novo biosynthesis. Transcriptional analysis showed it to be expressed as two tandem transcripts of approximately 22 and 4 kb, carrying cobD, cbiABCDETFGHJ , cobA/hemD , cbiKLMNQOP , sirA , hemACBL , and cobUSC , hemD, cobT, respectively. Both transcripts appear to be similarly regulated, and under the conditions assayed are induced in the late-exponential growth phase. Evidence for a regulatory mechanism of negative feedback inhibition by vitamin B 12 itself was observed. Comparative genomics analysis of the coding sequences showed them to be most similar to those coding for the anaerobic coenzyme B 12 pathways previously characterized in a few representatives of the genera Listeria and Salmonella . This contrasts with the trusted species phylogeny and suggests horizontal gene transfer of the B 12 biosynthesis genes. G+C content and codon adaptation index analysis is suggestive that the postulated transfer of these genes was not a recent event. Additional comparative genomics and transcriptional analysis of the sequences acquired during this study suggests a functional link between coenzyme B 12 biosynthesis and reuterin production, which might be implicated in Lb. reuteri 's success in colonizing the gastrointestinal tract. This information on gene organization, gene transcription and gene acquisition is relevant for the development of (fermented) foods and probiotics enriched in B 12 . Abbreviations: 3-HPA, 3-hydroxypropionaldehyde; RT-PCR, reverse transcriptase PCR; Q-RT-PCR, quantitative reverse transcriptase PCR; GI tract, gastrointestinal tract; GRAS, generally regarded as safe The GenBank accession number for the sequence re