Efficient insertional mutagenesis in lactococci and other gram-positive bacteria

Abstract

In lactococci, the study of chromosomal genes and their regulation is limited by the lack of an efficient transposon mutagenesis system. We associated the insertion sequence ISS1 with the thermosensitive replicon pG ؉ host to generate a mutagenic tool that can be used even in poorly transformable strains. ISS1 transposition is random in different lactococcal strains as well as in Enterococcus faecalis and Streptococcus thermophilus. High-frequency random insertion (of about 1%) obtained with this system in Lactococcus lactis allows efficient mutagenesis, with typically one insertion per cell. After ISS1 replicative transposition, the chromosome contains duplicated ISS1 sequences flanking pG ؉ host. This structure allows cloning of the interrupted gene. In addition, efficient excision of the plasmid leaves a single ISS1 copy at the mutated site, thus generating a stable mutant strain with no foreign markers. Mutants obtained by this transposition system are food grade and can thus be used in fermentation processes.Lactic acid bacteria are important industrial microorganisms because of their role in food fermentations. Lactococcus lactis is widely used in dairy fermentations and also serves as a model organism for biological studies of lactic acid bacteria. Most genes thus far identified in L. lactis have been cloned by (i) complementation (2,10,19,35), (ii) immunoscreening of DNA libraries (11,34,48), (iii) PCR amplification of conserved genes (1,14,15,17,30), and (iv) DNA sequencing of regions adjacent to genes of interest (3,26,36). The chromosome and its genetic regulatory networks, however, remain for the most part unknown.In many bacteria, transposition has been a valuable genetic tool to study chromosomal genes, their functions, and their regulators (4,46,50). In L. lactis, transposition of the conjugative elements Tn916 (41) and Tn919 (22, 23) have been reported. However, their use is limited by a requirement for high-efficiency conjugal transfer and site-specific transposition in certain strains. The transposition of Tn917 has recently been demonstrated in L. lactis MG1614 (28). The vector used in this system is pE194, whose replication is strain specific among lactococci (7), and transposition frequencies appear to be low; also, one-third of the candidates correspond to plasmid integrants. The use of heterologous transposons can be of interest for genetic analyses, but resultant strains containing antibiotic (Ab) resistance markers would be restricted from industrial use, particularly in fermentation.Another class of transposable elements are bacterial insertion sequences (IS). IS elements are small (between 800 and 2,500 bp) and flanked by inverted repeats and generally encode their own transposition functions (16). Three families of IS elements have been defined in lactococci (44), and their host ranges, positions, and frequencies on the chromosome have been shown to vary widely among strains (39, 45). In lactococci, iso-ISS1 elements have been thoroughly characterized (8,18,21,27,39,40). With nonrepli...

Molecular characterization of Bifidobacterium longum biovar longum NAL8 plasmids and construction of a novel replicon screening system

“…To construct the plasmid pGHori, we designed two divergent primers near the extremities of the replication gene of vector pG + host9 (Maguin et al 1996), pGhf (5′-TATCAGTGTGGAGCTCGAGCAAGTT-3′), and pGhr (5′-AGCCATAGATGAGCTCAAACTCTCT-3′), which contained the restriction site SacI (underlined; Fig. 1).…”

Characterization of the haem-uptake system of the equine pathogen Streptococcus equi subsp. equi

“…Utilizing the temperaturesensitive broad-host-range plasmid pG+host9, insertion mutants of shr, shp and htsA were generated by replacing S. equi DNA with the VKm2 interposon (Maguin et al, 1996;Meehan et al, 2001;PerezCasal et al, 1991;Prentki & Krisch, 1984). For S. equi shr : : VKm2, a central 127 bp fragment (nucleotides 619-745) was replaced.…”

Untitled

“…Nevertheless, in some applications it may have the disadvantage that the mutant strains have to be kept at temperatures above 37.5 °C. For more details on the Ts-system the reader is referred to the original literature [2, 18,19]; (ii) a system in which pWV01-derivatives deprived of repA, the gene essential for plasmid replication, are multiplied in strains which provide RepA in trans and are integrated in strains which lack the repA gene of pWV01[Ori + -system; 15,17]. Figure 1 summarises the characteristics of the Ori + -system.…”

“…Lactococcal transposable elements that have been used succesfully in the analysis of the L. lactis chromosome belong to the ISS1 family of insertion sequences [19,21,23,25]. Replicative transposition of an ISS1 element present on a plasmid results in the integration of the entire plasmid in the chromosome, flanked by two copies of the IS element.…”

“…The use of a thermosensitive replicon uncouples transformation and transposition events which results in an important improvement of transposition frequencies. High-frequency random transposition (> 0.5%), allowing for efficient random gene inactivation, has been demonstrated with pGh:ISS1 in L. lactis, Streptococcus thermophilus and E. faecalis [19]. The wide host range of pWV01-based replicons, which replicate both in Gramnegative and Gram-positive bacteria [9], offers the possibility to use this tool in a great variety of bacteria.…”