256 research outputs found

    Lactococcal 936-type phages and dairy fermentation problems: from detection to evolution and prevention

    Get PDF
    The so-called 936-type phages are the most frequently encountered lactococcal phage species in dairy fermentations, where they cause slow or even failed fermentations with concomitant economic losses. Several dairy phage population studies, performed in different geographical locations, have detailed their dominance in dairy phage populations, while various phage-resistance mechanisms have been assessed in a bid to protect against this virulent phage group. The impact of thermal and chemical treatments on 936 phages is an important aspect for dairy technologists and has been assessed in several studies, and has indicated that these phages have adapted to better resist such treatments. The abundance of 936 phage genome sequences has permitted a focused view on genomic content and regions of variation, and the role of such variable regions in the evolution of these phages. Here, we present an overview on detection and global prevalence of the 936 phages, together with their tolerance to industrial treatments and anti-phage strategies. Furthermore, we present a comprehensive review on the comparative genomic analyses of members of this fascinating phage species

    Expanding the molecular toolbox for Lactococcus lactis: construction of an inducible thioredoxin gene fusion expression system

    Get PDF
    <p>Abstract</p> <p>Background</p> <p>The development of the Nisin Inducible Controlled Expression (NICE) system in the food-grade bacterium <it>Lactococcus lactis </it>subsp. <it>cremoris </it>represents a cornerstone in the use of Gram-positive bacterial expression systems for biotechnological purposes. However, proteins that are subjected to such over-expression in <it>L. lactis </it>may suffer from improper folding, inclusion body formation and/or protein degradation, thereby significantly reducing the yield of soluble target protein. Although such drawbacks are not specific to <it>L. lactis</it>, no molecular tools have been developed to prevent or circumvent these recurrent problems of protein expression in <it>L. lactis</it>.</p> <p>Results</p> <p>Mimicking thioredoxin gene fusion systems available for <it>E. coli</it>, two nisin-inducible expression vectors were constructed to over-produce various proteins in <it>L. lactis </it>as thioredoxin fusion proteins. In this study, we demonstrate that our novel <it>L. lactis </it>fusion partner expression vectors allow high-level expression of soluble heterologous proteins Tuc2009 ORF40, Bbr_0140 and Tuc2009 BppU/BppL that were previously insoluble or not expressed using existing <it>L. lactis </it>expression vectors. Over-expressed proteins were subsequently purified by Ni-TED affinity chromatography. Intact heterologous proteins were detected by immunoblotting analyses. We also show that the thioredoxin moiety of the purified fusion protein was specifically and efficiently cleaved off by enterokinase treatment.</p> <p>Conclusions</p> <p>This study is the first description of a thioredoxin gene fusion expression system, purposely developed to circumvent problems associated with protein over-expression in <it>L. lactis</it>. It was shown to prevent protein insolubility and degradation, allowing sufficient production of soluble proteins for further structural and functional characterization.</p

    A general method for selection of riboflavin-overproducing food grade micro-organisms

    Get PDF
    BACKGROUND: This study describes a strategy to select and isolate spontaneous riboflavin-overproducing strains of Lactobacillus (Lb.) plantarum, Leuconostoc (Lc.) mesenteroides and Propionibacterium (P.) freudenreichii. RESULTS: The toxic riboflavin analogue roseoflavin was used to isolate natural riboflavin-overproducing variants of the food grade micro-organisms Lb. plantarum, Lc. mesenteroides and P. freudenreichii strains. The method was successfully employed for strains of all three species. The mutation(s) responsible for the observed overproduction of riboflavin were identified for isolates of two species. CONCLUSION: Selection for spontaneous roseoflavin-resistant mutants was found to be a reliable method to obtain natural riboflavin-overproducing strains of a number of species commonly used in the food industry. This study presents a convenient method for deriving riboflavin-overproducing strains of bacterial starter cultures, which are currently used in the food industry, by a non-recombinant methodology. Use of such starter strains can be exploited to increase the vitamin content in certain food products

    Stimulation of reverse transcriptase generated cDNAs with specific indels by template RNA structure: retrotransposon, dNTP balance, RT-reagent usage

    Get PDF
    RNA dependent DNA-polymerases, reverse transcriptases, are key enzymes for retroviruses and retroelements. Their fidelity, including indel generation, is significant for their use as reagents including for deep sequencing. Here, we report that certain RNA template structures and G-rich sequences, ahead of diverse reverse transcriptases can be strong stimulators for slippage at slippage-prone template motif sequence 3′ of such ‘slippage-stimulatory’ structures. Where slippage is stimulated, the resulting products have one or more additional base(s) compared to the corresponding template motif. Such structures also inhibit slippage-mediated base omission which can be more frequent in the absence of a relevant stem–loop. Slippage directionality, base insertion and omission, is sensitive to the relative concentration ratio of dNTPs specified by the RNA template slippage-prone sequence and its 5′ adjacent base. The retrotransposon-derived enzyme TGIRT exhibits more slippage in vitro than the retroviral enzymes tested including that from HIV. Structure-mediated slippage may be exhibited by other polymerases and enrich gene expression. A cassette from Drosophila retrotransposon Dme1_chrX_2630566, a candidate for utilizing slippage for its GagPol synthesis, exhibits strong slippage in vitro. Given the widespread occurrence and importance of retrotransposons, systematic studies to reveal the extent of their functional utilization of RT slippage are merited