Class IIa bacteriocin resistance in Enterococcus faecalis V583: The mannose PTS operon mediates global transcriptional responses

<p>Abstract</p> <p>Background</p> <p>The class IIa bacteriocin, pediocin PA-1, has clear potential as food preservative and in the medical field to be used against Gram negative pathogen species as <it>Enterococcus faecalis </it>and <it>Listeria monocytogenes</it>. Resistance towards class IIa bacteriocins appear in laboratory and characterization of these phenotypes is important for their application. To gain insight into bacteriocin resistance we studied mutants of <it>E. faecalis </it>V583 resistant to pediocin PA-1 by use of transcriptomic analyses.</p> <p>Results</p> <p>Mutants of <it>E. faecalis </it>V583 resistant to pediocin PA-1 were isolated, and their gene expression profiles were analyzed and compared to the wild type using whole-genome microarray. Significantly altered transcription was detected from about 200 genes; most of them encoding proteins involved in energy metabolism and transport. Glycolytic genes were down-regulated in the mutants, but most of the genes showing differential expression were up-regulated. The data indicate that the mutants were relieved from glucose repression and putative catabolic responsive elements (<it>cre</it>) could be identified in the upstream regions of 70% of the differentially expressed genes. Bacteriocin resistance was caused by reduced expression of the <it>mpt </it>operon encoding the mannose-specific phosphoenolpyruvate:carbohydrate phosphotransferase system (PTS), and the same transcriptional changes were seen in a <it>mptD</it>-inactivated mutant. This mutant also had decreased transcription of the whole <it>mpt </it>operon, showing that the PTS is involved in its own transcriptional regulation.</p> <p>Conclusion</p> <p>Our data confirm the important role of mannose PTS in class IIa bacteriocin sensitivity and we demonstrate its importance involving global carbon catabolite control.</p

Genomic Characterization of Dairy Associated Leuconostoc Species and Diversity of Leuconostocs in Undefined Mixed Mesophilic Starter Cultures

Undefined mesophilic mixed (DL-type) starter cultures are composed of predominantly Lactococcus lactis subspecies and 1–10% Leuconostoc spp. The composition of the Leuconostoc population in the starter culture ultimately affects the characteristics and the quality of the final product. The scientific basis for the taxonomy of dairy relevant leuconostocs can be traced back 50 years, and no documentation on the genomic diversity of leuconostocs in starter cultures exists. We present data on the Leuconostoc population in five DL-type starter cultures commonly used by the dairy industry. The analyses were performed using traditional cultivation methods, and further augmented by next-generation DNA sequencing methods. Bacterial counts for starter cultures cultivated on two different media, MRS and MPCA, revealed large differences in the relative abundance of leuconostocs. Most of the leuconostocs in two of the starter cultures were unable to grow on MRS, emphasizing the limitations of culture-based methods and the importance of careful media selection or use of culture independent methods. Pan-genomic analysis of 59 Leuconostoc genomes enabled differentiation into twelve robust lineages. The genomic analyses show that the dairy-associated leuconostocs are highly adapted to their environment, characterized by the acquisition of genotype traits, such as the ability to metabolize citrate. In particular, Leuconostoc mesenteroides subsp. cremoris display telltale signs of a degenerative evolution, likely resulting from a long period of growth in milk in association with lactococci. Great differences in the metabolic potential between Leuconostoc species and subspecies were revealed. Using targeted amplicon sequencing, the composition of the Leuconostoc population in the five commercial starter cultures was shown to be significantly different. Three of the cultures were dominated by Ln. mesenteroides subspecies cremoris. Leuconostoc pseudomesenteroides dominated in two of the cultures while Leuconostoc lactis, reported to be a major constituent in fermented dairy products, was only present in low amounts in one of the cultures. This is the first in-depth study of Leuconostoc genomics and diversity in dairy starter cultures. The results and the techniques presented may be of great value for the dairy industry

The CWPS Rubik's cube: Linking diversity of cell wall polysaccharide structures with the encoded biosynthetic machinery of selected Lactococcus lactis strains

The biosynthetic machinery for cell wall polysaccharide (CWPS) production in lactococci is encoded by a large gene cluster, designatedcwps. This locus displays considerable variation among lactococcal genomes, previously prompting a classification into three distinct genotypes (A-C). In the present study, thecwpsloci of 107 lactococcal strains were compared, revealing the presence of a fourthcwpsgenotype (type D). Lactococcal CWPSs are comprised of two saccharidic structures: a peptidoglycan-embedded rhamnan backbone polymer to which a surface-exposed, poly/oligosaccharidic side-chain is covalently linked. Chemical structures of the side-chain of seven lactococcal strains were elucidated, highlighting their diverse and strain-specific nature. Furthermore, a link betweencwpsgenotype and chemical structure was derived based on the number of glycosyltransferase-encoding genes in thecwpscluster and the presence of conserved genes encoding the presumed priming glycosyltransferase. This facilitates predictions of several structural features of lactococcal CWPSs including (a) whether the CWPS possesses short oligo/polysaccharide side-chains, (b) the number of component monosaccharides in a given CWPS structure, (c) the order of monosaccharide incorporation into the repeating units of the side-chain (for C-type strains), (d) the presence of Galfand phosphodiester bonds in the side-chain, and (e) the presence of glycerol phosphate substituents in the side-chain

Unprecedented diversity of lactococcal group 936 bacteriophages revealed by amplicon sequencing of the portal protein gene

Lactococcus lactis is one of the most important bacteria in dairy fermentations, being used in the production of cheese and buttermilk. The processes are vulnerable to phage attacks, and undefined mixtures of lactococcal strains are often used to reduce the risk of bacteriophage caused fermentation failure. Other preventive measures include culture rotation to prevent phage build-up and phage monitoring. Phage diversity, rather than quantity, is the largest threat to fermentations using undefined mixed starter cultures. We have developed a method for culture independent diversity analysis of lytic bacteriophages of the 936 group, the phages most commonly found in dairies. Using, as a target, a highly variable region of the portal protein gene, we demonstrate an unprecedented diversity and the presence of new 936 phages in samples taken from cheese production. The method should be useful to the dairy industry and starter culture manufacturers in their efforts to reduce phage problems.publishedVersio

Unprecedented diversity of lactococcal group 936 bacteriophages revealed by amplicon sequencing of the portal protein gene

Lactococcus lactis is one of the most important bacteria in dairy fermentations, being used in the production of cheese and buttermilk. The processes are vulnerable to phage attacks, and undefined mixtures of lactococcal strains are often used to reduce the risk of bacteriophage caused fermentation failure. Other preventive measures include culture rotation to prevent phage build-up and phage monitoring. Phage diversity, rather than quantity, is the largest threat to fermentations using undefined mixed starter cultures. We have developed a method for culture independent diversity analysis of lytic bacteriophages of the 936 group, the phages most commonly found in dairies. Using, as a target, a highly variable region of the portal protein gene, we demonstrate an unprecedented diversity and the presence of new 936 phages in samples taken from cheese production. The method should be useful to the dairy industry and starter culture manufacturers in their efforts to reduce phage problems.publishedVersio

Increased serum osteocalcin levels and vitamin K status by daily cheese intake

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Determination of maintenance Jarlsberg® cheese dose to keep the obtained serum osteocalcin level; a response surface pathway designed de-escalation dose study with individual starting values

Background: Daily maximum effective dose (MED) of Jarlsberg® increased the serum osteocalcin (tOC) level, vitamin K2 and affected the lipid pattern positively. The aim of the study was to estimate and verify a daily maintenance dose. Methods: 12 healthy female volunteers (HV) were included in a de-escalation study after a six week run-in period on the daily MED of 57 g Jarlsberg® cheese. A 3-level within-patient response surface pathway (RSP) design with individual starting values was developed. Another 12 HVs were included in a new study with a six week run-in period on MED followed with six weeks on the estimated maintenance dose. All HVs were premenopausal female between 20 and 52 years of age. The main variable in the studies was the tOC level. Results: tOC, cOC and the vitamin K2 variants increases significantly (p<0.01) during the run-in period on daily MED of Jarlsberg® in both studies. The maintenance daily dose was estimated to 45 g (95% CI: 38-52 g/day) and used in the new study. The tOC level was reduced from 19.8 ng/ml (95% CI: 12.0-27.6) obtained in the run-in period to 18.5 ng/ml (95% CI: 11.7-25.3) during the maintenance part. This represents a reduction of 6.6%. The sum of vitamin K2 variants changed from 0.58 ng/ml on MED of Jarlsberg® to 0.59 ng/ml (95% CI: 0.37-0.82) during the maintenance period. Conclusions: Daily MED of Jarlsberg® cheese increases tOC, cOC and the vitamin K2 level. The maintenance Jarlsberg® dose was estimated to 45 g/day and verified as sufficient.publishedVersio

Engineering Lactococcus lactis for Increased Vitamin K2 Production

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High-Frequency Transformation, by Electroporation, of Lactococcus lactis subsp. cremoris Grown with Glycine in Osmotically Stabilized Media

An efficient method for genetic transformation of lactococci by electroporation is presented. Highly competent lactococci for electrotransformation were obtained by growing cells in media containing high concentrations of glycine and 0.5 M sucrose as the osmotic stabilizers. These cells could be stored at −85°C without loss of competence. With Lactococcus lactis subsp. cremoris BC101, a transformation frequency of 5.7 × 10(7) transformants per μg of pIL253 DNA was obtained, which represents 5% of the surviving cells. All the lactococcal strains tested could be transformed by the present method