The Tn3-family of Replicative Transposons.

Transposons of the Tn3 family form a widespread and remarkably homogeneous group of bacterial transposable elements in terms of transposition functions and an extremely versatile system for mediating gene reassortment and genomic plasticity owing to their modular organization. They have made major contributions to antimicrobial drug resistance dissemination or to endowing environmental bacteria with novel catabolic capacities. Here, we discuss the dynamic aspects inherent to the diversity and mosaic structure of Tn3-family transposons and their derivatives. We also provide an overview of current knowledge of the replicative transposition mechanism of the family, emphasizing most recent work aimed at understanding this mechanism at the biochemical level. Previous and recent data are put in perspective with those obtained for other transposable elements to build up a tentative model linking the activities of the Tn3-family transposase protein with the cellular process of DNA replication, suggesting new lines for further investigation. Finally, we summarize our current view of the DNA site-specific recombination mechanisms responsible for converting replicative transposition intermediates into final products, comparing paradigm systems using a serine recombinase with more recently characterized systems that use a tyrosine recombinase.

Enantioselective regulation of lactate racemization by LarR in Lactobacillus plantarum.

Lactobacillus plantarum is a lactic acid bacterium that produces a racemic mixture of l- and d-lactate from sugar fermentation. The interconversion of lactate isomers is performed by a lactate racemase (Lar) that is transcriptionally controlled by the l-/d-lactate ratio and maximally induced in the presence of l-lactate. We previously reported that the Lar activity depends on the expression of two divergently oriented operons: (i) the larABCDE operon encodes the nickel-dependent lactate racemase (LarA), its maturases (LarBCE), and a lactic acid channel (LarD), and (ii) the larR(MN)QO operon encodes a transcriptional regulator (LarR) and a four-component ABC-type nickel transporter [Lar(MN), in which the M and N components are fused, LarQ, and LarO]. LarR is a novel regulator of the Crp-Fnr family (PrfA group). Here, the role of LarR was further characterized in vivo and in vitro. We show that LarR is a positive regulator that is absolutely required for the expression of Lar activity. Using gel retardation experiments, we demonstrate that LarR binds to a 16-bp palindromic sequence (Lar box motif) that is present in the larR-larA intergenic region. Mutations in the Lar box strongly affect LarR binding and completely abolish transcription from the larA promoter (PlarA). Two half-Lar boxes located between the Lar box and the -35 box of PlarA promote LarR multimerization on DNA, and point mutations within one or both half-Lar boxes inhibit PlarA induction by l-lactate. Gel retardation and footprinting experiments indicate that l-lactate has a positive effect on the binding and multimerization of LarR, while d-lactate antagonizes the positive effect of l-lactate. A possible mechanism of LarR regulation by lactate enantiomers is proposed.

The fast milk acidifying phenotype of Streptococcus thermophilus can be acquired by natural transformation of the genomic island encoding the cell-envelope proteinase PrtS.

BACKGROUND: In industrial fermentation processes, the rate of milk acidification by Streptococcus thermophilus is of major technological importance. The cell-envelope proteinase PrtS was previously shown to be a key determinant of the milk acidification activity in this species. The PrtS enzyme is tightly anchored to the cell wall via a mechanism involving the typical sortase A (SrtA) and initiates the breakdown of milk casein into small oligopeptides. The presence or absence of PrtS divides the S. thermophilus strains into two phenotypic groups i.e. the slow and the fast acidifying strains. The aim of this study was to improve the milk acidification rate of slow S. thermophilus strains, and hence optimise the fermentation process of dairy products. RESULTS: In the present work, we developed for the first time a strategy based on natural transformation to confer the rapid acidification phenotype to slow acidifying starter strains of S. thermophilus. First, we established by gene disruption that (i) prtS, encoding the cell-envelope proteinase, is a key factor responsible for rapid milk acidification in fast acidifying strains, and that (ii) srtA, encoding sortase A, is not absolutely required to express the PrtS activity. Second, a 15-kb PCR product encompassing the prtS genomic island was transferred by natural transformation using the competence-inducing peptide in three distinct prtS-defective genetic backgrounds having or not a truncated sortase A gene. We showed that in all cases the milk acidification rate of transformants was significantly increased, reaching a level similar to that of wild-type fast acidifying strains. Furthermore, it appeared that the prtS-encoded activity does not depend on the prtS copy number or on its chromosomal integration locus. CONCLUSION: We have successfully used natural competence to transfer the prtS locus encoding the cell-envelope proteinase in three slow acidifying strains of S. thermophilus, allowing their conversion into fast acidifying derivatives. The efficient protocol developed in this article will provide the dairy industry with novel and optimised S. thermophilus starter strains.

Development of a versatile procedure based on natural transformation for marker-free targeted genetic modification in Streptococcus thermophilus.

A versatile natural transformation protocol was established for and successfully applied to 18 of the 19 Streptococcus thermophilus strains tested. The efficiency of the protocol enables the use of in vitro-amplified mutagenesis fragments to perform deletion or insertion of large genetic fragments. Depending on the phenotype linked to the mutation, markerless mutants can be selected either in two steps, i.e., resistance marker insertion and excision using an adapted Cre-loxP system, or in one step using a powerful positive screening procedure as illustrated here for histidine prototrophy.