High-efficiency gene inactivation and replacement system for gram-positive bacteria

Abstract

A system for high-efficiency single-and double-crossover homologous integration in gram-positive bacteria has been developed, with Lactococcus lactis as a model system. The system is based on a thermosensitive broad-host-range rolling-circle plasmid, pG'host5, which contains a pBR322 replicon for propagation in Escherichia coli at 37°C. A nested set of L. lactis chromosomal fragments cloned onto pG'host5 were used to show that the single-crossover integration frequency was logarithmically proportional to the length of homology for DNA fragments between 0.35 and 2.5 kb. Using random chromosomal 1-kb fragments, we showed that homologous integration can occur along the entire chromosome. We made use of the reported stimulatory effect of rolling-circle replication on intramolecular recombination to develop a protocol for gene replacement. Cultures were first maintained at 37°C to select for a bacterial population enriched for plasmid integrants; activation of the integrated rolling-circle plasmid by a temperature shift to 28°C resulted in efficient plasmid excision by homologous recombination and replacement of a chromosomal gene by the plasmid-carried modified copy. More than 50% of cells underwent replacement recombination when selection was applied for the replacing gene. Between 1 and 40%o of cells underwent replacement recombination when no selection was applied. Chromosomal insertions and deletions were obtained in this way. These results show that gene replacement can be obtained at an extremely high efficiency by making use of the thermosensitive rolling-circle nature of the delivery vector. This procedure is applicable to numerous gram-positive bacteria.Numerous gram-positive bacteria are targets of study as biological models (e.g., Bacillus subtilis), industrially important fermenter strains (the lactic acid bacteria), or pathogens (e.g., clostridia, listeria, staphylococci, and streptococci). Many strains of industrial or medical importance have been characterized physiologically, but relatively few have been studied or modified genetically. The study or modification of strains could be facilitated by the use of delivery vectors for the introduction of directed or nonspecific insertions in the bacterial chromosome. Delivery systems that rely on nonreplicative vectors are limited to bacteria that can be transformed at a high frequency, and those with conditionally active replicons are often restricted in their host range. Thus, the construction of recombinant strains requires substantial effort and can be applied efficiently only to particular organisms.We previously described a broad-host-range thermosensitive (Ts) plasmid, pVE6002 (22), which was isolated from pGK12 (15). Such plasmids replicate by a rolling-circle (rc) mechanism (rc plasmids) (21). pVE6002 was shown to have potential use as a delivery vector in numerous gram-positive bacteria (22). Ts plasmids are nonreplicative at 37°C, making them particularly useful in bacteria with a low-temperature growth range or when a drastic thermal shock is un...

BglR protein, which belongs to the BglG family of transcriptional antiterminators, is involved in beta-glucoside utilization in Lactococcus lactis

“…Since bglR, like E. coli bglG, is controlled by P-glucoside sugars, we suspected that BglR might be involved in regulating ,-glucoside utilization. To test this hypothesis, bglR was disrupted by insertion of a 10-nucleotide HindIII linker at the unique Swal site and replacement of the wild-type gene by the disrupted gene, using a recently described strategy (9,23). Clones containing the disrupted bglR were distinguished from those containing the wild-type bglR by comparing the profiles of their HindIIIcleaved DNAs hybridized with the bglR DNA.…”

Role of the nhaC-encoded Na+/H+ antiporter of alkaliphilic Bacillus firmus OF4

“…fragment was ligated with HindIII-restricted pG ϩ host4 to generate the plasmid pGC800. Plasmid pG ϩ host4 is sensitive to temperatures above 37°C and has previously been used to obtain single-crossover integrants with as little as 500 bp of homology (4). The recombinant plasmid pGC800 was established in E. coli XL-1 Blue MR (Stratagene, La Jolla, Calif.) and then transformed into B. firmus OF4811M.…”

“…Transformed cells were resistant to both erythromycin (1 g/ml) and chloramphenicol (3 g/ml). Gene replacement was achieved by the approach of Biswas et al (4). For isolation of single-crossover integrants, transformants growing at 28°C were diluted and plated onto complex medium containing erythromycin and incubated at 39°C.…”

Genetic characterization of the conjugative DNA processing system of enterococcal plasmid pCF10

“…The improved efficiency of conjugation over electroporation will be particularly helpful in the delivery of suicide vectors for mutagenesis of gram positive bacteria. Allelic exchange in gram positive bacteria often requires a two-step process (Biswas et al, 1993;Leenhouts et al, 1996). The first step involves selection of plasmid transformants under conditions that permit replication of the plasmid.…”