The expression of propionicin PLG-1 gene (plg-1) by lactic starters

Propionicin PLG-1 is a bacteriocin produced byPropionibacterium thoeniiP127. Such bacteriocin inhibits wide range of food-borne pathogens such as pathogenicEscherichia coli, Pseudomonas aeruginosa, Vibrio parahaemolyticus, Yersinia enterocoliticaand a strain ofCorynebacteriumsp. In the present study,plg-1gene expressing propionicin PLG-1 was isolated, sequenced for the first time and the resulting sequence was analysed using several web-based bioinformatics programs. The PCR product containingplg-1gene was transferred to different lactic acid bacterial (LAB) strains using pLEB590 as a cloning vector to give the modified vector pLEBPLG-1. LAB transformants showed an antimicrobial activity againstEsch. coliDH5α (most affected strain),Listeria monocytogenes18116, andSalmonella enterica25566 as model pathogenic strains. Such LAB transformants can be used in dairy industry to control the food-borne pathogens that are largely distributed worldwide and to feed schoolchildren in the poor countries where dangerous epidemic diseases and diarrhoea prevail.

Development of a Propionibacterium-Escherichia coli Shuttle Vector for Metabolic Engineering of Propionibacterium jensenii, an Efficient Producer of Propionic Acid

ABSTRACTPropionic acid (PA) is an important chemical building block and is widely applied for organic synthesis, food, feedstuff, and pharmaceuticals. To date, the strains that can efficiently produce PA have includedPropionibacterium thoenii,P. freudenreichii, andP. acidipropionici. In this report, we show thatP. jenseniiATCC 4868 is also able to produce PA in much higher yields than the previously reported strains. To further improve the production capacity, aP. jensenii-Escherichia colishuttle vector was developed for the metabolic engineering ofP. jensenii. Specifically, a 6.9-kb endogenous plasmid, pZGX01, was isolated fromP. acidipropioniciATCC 4875 and sequenced. Since the sequencing analysis indicated that pZGX01 could encode 11 proteins, the transcriptional levels of the corresponding genes were also investigated. Then, aP. jensenii-Escherichia colishuttle vector was constructed using the pZGX01 plasmid, theE. colipUC18 plasmid, and a chloramphenicol resistance gene. Interestingly, not only could the developed shuttle vector be transformed intoP. jenseniiATCC 4868 and 4870, but it also could be transformed intofreudenreichiiATCC 6207 subspecies ofP. freudenreichii. Finally, the glycerol dehydrogenase gene (gldA) fromKlebsiella pneumoniaewas expressed inP. jenseniiATCC 4868 with the constructed shuttle vector. In a 3-liter batch culture, the PA production by the engineeredP. jenseniiATCC 4868 strain reached 28.23 ± 1.0 g/liter, which was 26.07% higher than that produced by the wild-type strain (22.06 ± 1.2 g/liter). This result indicated that the constructed vector can be used a useful tool for metabolic engineering ofP. jensenii.

Heterologous Production of Antimicrobial Peptides in Propionibacterium freudenreichii

ABSTRACT 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 (P4) 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 P4 and Ppamp promoters directed constitutive gene expression, and activity of both promoters was enhanced by elements upstream of the promoter core region.

Prevalence of the Genes Encoding Propionicin T1 and Protease-Activated Antimicrobial Peptide and Their Expression in Classical Propionibacteria

ABSTRACT The purpose of this study was to investigate the frequency of production of the bacteriocin propionicin T1 and the protease-activated antimicrobial peptide (PAMP) and their corresponding genes in 64 isolates of classical propionibacteria. This study revealed that these genes are widespread in Propionibacterium jensenii and Propionibacterium thoenii but absent from the remaining species of classical propionibacteria that were studied. The pro-PAMP-encoding gene (pamA) was found in 63% of the P. jensenii strains and 61% of the P. thoenii strains, and all of these strains displayed PAMP activity. The propionicin T1-encoding gene (pctA) was present in 89% of the P. thoenii strains and 54% of the P. jensenii strains. All P. thoenii strains containing the pctA gene exhibited antimicrobial activity corresponding to propionicin T1 activity, whereas only 38% of the pctA-containing P. jensenii strains displayed this activity. Sequencing of the pctA genes revealed the existence of two allelic variants that differed in a single nucleotide in six strains of P. jensenii; in these strains the glycine at position 55 of propionicin T1 was replaced by an aspartate residue (A variant). No strains harboring the A variant showed any antimicrobial activity against propionicin T1-sensitive bacteria. An open reading frame (orf2) located immediately downstream from the pctA gene was absent in three strains containing the G variant of propionicin T1. Two of these strains showed low antimicrobial activity, while the third strain showed no antimicrobial activity at all. The protein encoded by orf2 showed strong homology to ABC transporters, and it has been proposed previously that this protein is involved in the producer immunity against propionicin T1. The limited antimicrobial activity exhibited by the strains lacking orf2 further suggests that this putative ABC transporter plays an important role in propionicin T1 activity.