Stephan, Roger

The Clostridium estertheticum complex (CEC) consists of closely related bacterial species that are mostly isolated from meat processing environment. Genome mining studies have recently established CEC as a source of class I bacteriocins. However, up until now, class II bacteriocins have not been reported from CEC. In the present study, we determined the presence of class II bacteriocin biosynthetic clusters in 33 CEC genomes through genome mining followed by bacteriocin production through heterologous expression in Escherichia coli . Six biosynthetic gene clusters belonging to class IIa ( n  = 1), IIb ( n  = 2), IId ( n  = 2) and an undefined class containing three precursor peptides were identified in six different CEC strains. Using molecular biology, we developed dedicated expression vectors for the class IIa bacteriocin, clesteriocin A. Its precursor peptide, CleA, and protease, CleB150, were initially expressed in insoluble form in E. coli . Through a systematic analysis of suitable solubility enhancing protein tags, both CleA and CleB150 were expressed in significant amounts of soluble fractions using tandem tags derived from Small Ubiquitin-like Modifier, superfolder Green fluorescent protein, Maltose binding protein and N-utilization substance. Mature clesteriocin A was obtained following in vitro maturation reactions between the tagged CleA and CleB150. The novel bacteriocin displayed antimicrobial activity against Listeria monocytogenes , which was mediated by the mptC gene. By combining genome mining and molecular biology, we have shown CEC is a source of novel class II bacteriocins that have potential for application as food biopreservatives.

Five Gram-negative, facultatively anaerobic, non-spore-forming, coccoid rod-shaped bacterial isolates were obtained from infant formula and an infant formula production environment and were investigated by use of a polyphasic taxonomic study. Biochemical tests and partialrpoBgene sequence analysis of the five isolates revealed that they formed two distinct groups in the familyEnterobacteriaceae, closely related to several species of the generaPantoeaandErwinia, which indicated a phylogenetic position within the genusPantoeaor the genusErwinia. Multilocus sequence analysis of concatenated partialatpD,gyrB,infBandrpoBgene sequences of two of the isolates suggested that they represented two novel species of the genusPantoea, phylogenetically related most closely toPantoea septica. The five isolates had general characteristics consistent with those of the genusPantoea, and DNA–DNA hybridizations between two representatives and the type strains of their phylogenetically closest relatives based on comparative 16S rRNA gene sequence analysis showed that the isolates represented two novel genospecies. These two genospecies could be differentiated from each other based on fermentation of galacturonate, sorbitol and potassium 5-ketogluconate. They could be differentiated from phylogenetically relatedPantoeaspecies based on their ability to ferment lactose and to utilizeβ-gentiobiose and raffinose, their inability to ferment or utilized-arabitol, and their inability to produce indole. On the basis of the results obtained, the five isolates are considered to represent two novel species of the genusPantoea, for which the namesPantoea gaviniaesp. nov. (type strain A18/07T=LMG 25382T=DSM 22758T) andPantoea calidasp. nov. (type strain 1400/07T=LMG 25383T=DSM 22759T) are proposed.