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Heterologous bacteriocin production in Propionibacterium freudenreichii is described. We developed an efficient system for DNA shuttling between Escherichia coli and P. freudenreichii using vector pAMT1. It is based on the P. freudenreichii rolling-circle replicating plasmid pLME108 and carries the cml(A)/cmx(A) chloramphenicol resistance marker. Introduction of the propionicin T1 structural gene (pctA) into pAMT1 under the control of the constitutive promoter (P 4 ) yielded bacteriocin in amounts equal to those of the wild-type producer Propionibacterium thoenii 419. The P. freudenreichii clone showed propionicin T1 activity in coculture, killing 90% of sensitive bacteria within 48 h. The pamA gene from P. thoenii 419 encoding the protease-activated antimicrobial peptide (PAMP) was cloned and expressed in P. freudenreichii, resulting in secretion of the pro-PAMP protein. Like in the wild type, PAMP activation was dependent on externally added protease. Secretion of the antimicrobial peptide was obtained from a clone in which the pamA signal peptide and PAMP were fused in frame. The promoter region of pamA was identified by fusion of putative promoter fragments to the coding sequence of the pctA gene. The P 4 and P pamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.

-Strains of the genus Propionibacterium, which were recently c1assified within the new c1ass Aetinobaeteria, are grouped into cutaneous (medical) or dairy (c1assical) propionic acid bacteria. 16S rDNA sequences of ail Propionibaeterium type strains and sorne new isolates were completed. A phylogenetic tree was constructed and current trees reexamined. The c1assical species P. thoenii, P. jensenii and P. acidipropionici were c1ustered in a group distinct from both the closely related elusters of P.freudenreiehii and P. eyclohexanieum. Within the cutaneous species, three groups were elustered: P. granulosum forms a distinct c1uster between the c1assical groups and a group containing P. avidum, P. aenes and P. propionicum, whereas P. lymphophilum is placed more distantly from any group in this tree. In order to differentiate Propionibaeterium isolates from other food-isolates a rapid multiplex-PCR (MPCR) method based on 16S rRNA-targeted oligonucleotides and particularly on a 16S rDNA-motif which tumed out to he specifie for the genus Propionibaeterium was developed. The MPCR-amplification could be performed within 1 d and reached detection limits of 10 3 colony forming units or 35 pg of DNA. The MPCR-method was used in various Propionibaeterium screening studies. In the course of screening antifungal and antibacterial activities of propionic acid bacteria, a Propionibaeterium jensenii strain containing a cryptic 7-kb plasmid was identified. This strain produced the bacteriocin Propionicin SM 1. Purification of Propionicin SM 1 and cloning of its genetic locus is currently under way. © Inra/Elsevier, Paris.

-Research in the genetics and molecular biology of propionibacteria is currently making much progress. In order to develop efficient DNA transfer systems for the genus Propionibacterium, dairy and environmental propionibacteria were screened for the presence of suitable plasmids as a first step. Following nucleotide sequence analysis, potential replication functions were identified on several Propionibacterium plasmids such as pLME106/pRGO1, p545 and pLME108. Furthermore, ppnA, the gene encoding the propionicin SM1, was detected on pLME106. Three of these plasmids which had been fused with antibiotic resistance selection markers (ermE, cml, hygB) originating from bacteria with high G+C DNA content were recently successfully used as Escherichia coliPropionibacterium shuttle vectors. DNA restriction/modification systems observed in propionibacteria have to be taken into account since successful DNA transformation at high rates (up to 10 8 Propionibacterium transformants/µg of DNA) succeeds only with plasmid DNA originating from propionibacteria with the same restriction/modification system(s) as the strain to be transformed, and not from E. coli hosts. The basis for an integrating vector has been set up after identification of a potential attP site and an adjacent integrase gene from a Propionibacterium phage/prophage system. Finally, approximately 30 gene sequences with attributed coding functions from propionibacteria are available on databases.

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