Functional genomics provides insights into the role of Propionibacterium freudenreichii ssp shermanii JS in cheese ripening

Propionibacterium freudenreichii is a commercially important bacterium that is essential for the development of the characteristic eyes and flavor of Swiss-type cheeses. These bacteria grow actively and produce large quantities of flavor compounds during cheese ripening at warm temperatures but also appear to contribute to the aroma development during the subsequent cold storage of cheese. Here, we advance our understanding of the role of P. freudenreichii in cheese ripening by presenting the 2.68-Mbp annotated genome sequence of P. freudenreichii ssp. shermanii JS and determining its global transcriptional profiles during industrial cheese-making using transcriptome sequencing. The annotation of the genome identified a total of 2377 protein-coding genes and revealed the presence of enzymes and pathways for formation of several flavor compounds. Based on transcriptome profiling, the expression of 348 protein-coding genes was altered between the warm and cold room ripening of cheese. Several propionate, acetate, and diacetyl/acetoin production related genes had higher expression levels in the warm room, whereas a general slowing down of the metabolism and an activation of mobile genetic elements was seen in the cold room. A few ripening-related and aminoacid catabolism involved genes were induced or remained active in cold room, indicating that strain JS contributes to the aroma development also during cold room ripening. In addition, we performed a comparative genomic analysis of strain JS and 29 other Propionibacterium strains of 10 different species, including an isolate of both P. freudenreichii subspecies freudenreichii and shermanii. Ortholog grouping of the predicted protein sequences revealed that close to 86% of the ortholog groups of strain JS, including a variety of ripening-related ortholog groups, were conserved across the P. freudenreichii isolates. Taken together, this study contributes to the understanding of the genomic basis of P. freudenreichii and sheds light on its activities during cheese ripening. (C) 2016 Elsevier B.V. All rights reserved.Peer reviewe

Metagenomic and metatranscriptomic analysis of the microbial community in Swiss-type Maasdam cheese during ripening

In Swiss-type cheeses, characteristic nut-like and sweet flavor develops during the cheese ripening due to the metabolic activities of cheese microbiota. Temperature changes during warm and cold room ripening, and duration of ripening can significantly change the gene expression of the cheese microbiota, which can affect the flavor formation. In this study, a metagenomic and metatranscriptomic analysis of Swiss-type Maasdam cheese was performed on samples obtained during ripening in the warm and cold rooms. We reconstructed four different bacterial genomes (Lactococcus lactis, Lactobacillus rhamnosus, Lactobacillus helveticus, and Propionibacterium freudenreichii subsp. shermanii strain JS) from the Maasdam cheese to near completeness. Based on the DNA and RNA mean coverage, Lc. lactis strongly dominated (similar to 80-90%) within the cheese microbial community. Genome annotation showed the potential for the presence of several flavor forming pathways in these species, such as production of methanethiol, free fatty acids, acetoin, diacetyl, acetate, ethanol, and propionate. Using the metatranscriptomic data, we showed that, with the exception of Lc. lactis, the central metabolism of the microbiota was downregulated during cold room ripening suggesting that fewer flavor compounds such as acetoin and propionate were produced. In contrast, Lc. lactis genes related to the central metabolism, including the vitamin biosynthesis and homolactic fermentation, were upregulated during cold room ripening.Peer reviewe

Cloning and characterization of a prolinase gene (pepR) from Lactobacillus rhamnosus

vokEK

Cloning and Characterization of a Prolinase Gene (pepR) from Lactobacillus rhamnosus

A peptidase gene expressing l-proline-β-naphthylamide-hydrolyzing activity was cloned from a gene library of Lactobacillus rhamnosus 1/6 isolated from cheese. Peptidase-expressing activity was localized in a 1.5-kb SacI fragment. A sequence analysis of the SacI fragment revealed the presence of one complete open reading frame (ORF1) that was 903 nucleotides long. The ORF1-encoded 34.2-kDa protein exhibited 68% identity with the PepR protein from Lactobacillus helveticus. Additional sequencing revealed the presence of another open reading frame (ORF2) following pepR; this open reading frame was 459 bp long. Northern (RNA) and primer extension analyses indicated that pepR is expressed both as a monocistronic transcriptional unit and as a dicistronic transcriptional unit with ORF2. Gene replacement was used to construct a PepR-negative strain of L. rhamnosus. PepR was shown to be the primary enzyme capable of hydrolyzing Pro-Leu in L. rhamnosus. However, the PepR-negative mutant did not differ from the wild type in its ability to grow and produce acid in milk. The cloned pepR expressed activity against dipeptides with N-terminal proline residues. Also, Met-Ala, Leu-Leu, and Leu-Gly-Gly and the chromogenic substrates l-leucine-β-naphthylamide and l-phenylalanine-β-naphthylamide were hydrolyzed by the PepR of L. rhamnosus

Peptide Utilization Encoded by Lactococcus lactis subsp. lactis SSL135 Chromosomal DNA

A cloned chromosomal fragment of Lactococcus lactis subsp. lactis SSL135 on plasmid p VS8 in an L. lactis subsp. lactis MG1614 background enabled proteinase-negative strain MG1614 to grow in autoclaved milk. The strain (VS230) did not, however, degrade milk proteins and did not grow in pasteurized milk. In contrast, a strain (VS150) carrying p VS9, the proteinase plasmid of SSL135, in an MG1614 background degraded β-casein but did not grow in milk. VS230 was shown to utilize peptides produced by VS150 in growth experiments in pasteurized milk preincubated with the latter strain. The peptide utilization phenotype linked with p VS8 was further confirmed by growth of VS230 on tryptic peptide fractions, on which the plasmid-free but otherwise isogenic strain MG1614 failed to grow. Plasmid p VS8 produced 69-, 42-, 38-, and 36-kilodalton proteins, as determined by in vitro transcription-translation. At least three of these proteins affected the peptide utilization phenotype. We suggest that there could be a coupled peptidase-peptide transport system encoded by the chromosomal fragment