The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively, mainly for eukaryotic organisms. This study presents the construction of a Cre-lox-based system for multiple gene deletions in Lactobacillus plantarum that could be adapted for use on gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, doublecrossover gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a lox66-P 32 -cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P 32 -cat is a chloramphenicol resistance cassette. The lox sites serve as recognition sites for the Cre enzyme, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover mutants leads to recombination of the lox66-P 32 -cat-lox71 cassette into a double-mutant loxP site, called lox72, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-lox-based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the melA and bsh1 loci on the chromosome of the gram-positive model organism Lactobacillus plantarum WCFS1. Furthermore, the efficiency of the Cre-lox-based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci melA and lacS2 in L. plantarum WCFS1. The fact that 99.4% of the clones that were analyzed had undergone correct Cre-lox resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background.The development of tools for genetic engineering of grampositive bacteria is highly valuable for research applications. The classic strategy for obtaining gene deletion variants is based on homologous recombination, using double-crossover integration of heterologous nonreplicating vectors such as pUC (34,36,37,42), pACYC184 (6,48,50), or their derivatives in the genome. Several convenient systems that derive from this strategy use conditionally replicating vectors, such as the thermosensitive pG ϩ host system (40) and the RepA-dependent lactococcal pORI system (originating from pWV01) (12, 33, 51) and its broad-host-range derivative (42,51).In addition to systems that derive from the classic strategy, various site-specific recombination systems such as Flp-FRT (52), Gateway (Invitrogen), ParA-res (31), TnpR-res (13), and Cre-lox are used in mutational strategies. To date, however, these systems have not been available for construction of an unlimited number of mutations in the same genetic background in gram-positive bacteria. For this purpo...
Bile salts play an important role in the digestion of lipids in vertebrates and are synthesized and conjugated to either glycine or taurine in the liver. Following secretion of bile salts into the small intestine, intestinal microbes are capable of deconjugating the glycine or taurine from the bile salts, using an enzyme called bile salt hydrolase (Bsh). Intestinal lactobacilli are regarded as major contributors to bile salt hydrolysis in vivo. Since the bile salt-hydrolyzing strain Lactobacillus plantarum WCFS1 was predicted to carry four bsh genes (bsh1, bsh2, bsh3, and bsh4), the functionality of these bsh genes was explored using Lactococcus lactis heterologous overexpression and multiple bsh deletion strains. Thus, Bsh1 was shown to be responsible for the majority of Bsh activity in L. plantarum WCFS1. In addition, bsh1 of L. plantarum WCFS1 was shown to be involved in conferring tolerance to specific bile salts (i.e., glycocholic acid). Northern blot analysis established that bsh1, bsh2, bsh3, and bsh4 are all expressed in L. plantarum WCFS1 during the exponential growth phase. Following biodiversity analysis, bsh1 appeared to be the only bsh homologue that was variable among L. plantarum strains; furthermore, the presence of bsh1 correlated with the presence of Bsh activity, suggesting that Bsh1 is commonly responsible for Bsh activity in L. plantarum strains. The fact that bsh2, bsh3, and bsh4 genes appeared to be conserved among L. plantarum strains suggests an important role of these genes in the physiology and lifestyle of the species L. plantarum. Analysis of these additional bsh-like genes in L. plantarum WCFS1 suggests that they might encode penicillin acylase rather than Bsh activity, indicating their implication in the conversion of substrates other than bile acids in the natural habitat.
Everyone who has ever tried to radically change metabolic fluxes knows that it is often harder to determine which enzymes have to be modified than it is to actually implement these changes. In the more traditional genetic engineering approaches 'bottle-necks' are pinpointed using qualitative, intuitive approaches, but the alleviation of suspected 'rate-limiting' steps has not often been successful. Here the authors demonstrate that a model of pyruvate distribution in Lactococcus lactis based on enzyme kinetics in combination with metabolic control analysis clearly indicates the key control points in the flux to acetoin and diacetyl, important flavour compounds. The model presented here (available at http ://jjj.biochem.sun.ac.za/wcfs.html) showed that the enzymes with the greatest effect on this flux resided outside the acetolactate synthase branch itself. Experiments confirmed the predictions of the model, i.e. knocking out lactate dehydrogenase and overexpressing NADH oxidase increased the flux through the acetolactate synthase branch from 0 to 75 % of measured product formation rates.
Whereas protein engineering of enzymes and structural proteins nowadays is an established research tool for studying structure-function relationships of polypeptides and for improving their properties, the engineering of posttranslationally modified peptides, such as the lantibiotics, is just coming of age. The engineering of lantibiotics is less straightforward than that of unmodified proteins, since expression systems should be developed not only for the structural genes but also for the genes encoding the biosynthetic enzymes, immunity protein and regulatory proteins. Moreover, correct posttranslational modification of specific residues could in many cases he a prerequisite for production and secretion of the active lantibiotic, which limits the number of successful mutations one can apply. This paper describes the development of expression systems for the structural lantibiotic genes for nisin A, nisin Z, gallidermin, epidermin and Pep5, and gives examples of recently produced site-directed mutants of these lantibiotics. Characterization of the mutants yielded valuable information on biosynthetic requirements for production. Moreover, regions in the lantibioties were identified that are of crucial importance for antimicrobial activity. Eventually, this knowledge will lead to the rational design of lantibiotics optimally suited for fighting specific undesirable microorganisms. The mutants are of additional value for studies directed towards the elucidation of the mode of action of lantibiotics.
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