ISLpl1 is a functional IS30-related insertion element in Lactobacillus plantarum that is also found in other lactic acid bacteria

Abstract

We describe the first functional insertion sequence (IS) element in Lactobacillus plantarum. ISLpl1, an IS30-related element, was found on the pLp3 plasmid in strain FB335. By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene upp on the FB335 chromosome. The plasmid-carried IS element was also sequenced, and a second potential IS element was found: ISLpl2, an IS150-related element adjacent to ISLpl1. When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of ISLpl1-related sequences in different L. plantarum strains as well as in Pediococcus strains. The ISLpl1 pattern changed over many generations of the strain L. plantarum NCIMB 1406. This finding strongly supports our hypothesis that ISLpl1 is a mobile element in L. plantarum. Database analysis revealed five quasi-identical ISLpl1 elements in Lactobacillus, Pediococcus, and Oenococcus strains. Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes. In some cases, ISLpl1 was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance. ISLpl1 is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment.

FIG. 1.

Genetic organization of two ISLpl1 loci. ISLpl1 is represented by a gray rectangle, and adjacent white rectangles represent ISLpl2 (A) or the upp gene (B). The ORF orientations are designated with thick arrows. Proposed initiation codons were TTG for ISLpl1 transposase and GTG for ISLpl2 OrfB. Only the C-terminal part of OrfA was determined. Primer names and orientations are indicated with small arrows for each PCR product; continuous lines and broken lines represent PCR products amplified directly from native DNA templates or by inverse PCR, respectively. Dashed lines represent inverse PCR-amplified fragments. (A) In the pLp3 plasmid in strains FB335 and HN38, ISLpl1 is contiguous to ISLpl2 (GenBank accession no. AF459445). Relevant restriction enzyme sites are indicated. (B) In the chromosome of HN38, ISLpl1 is also inserted in the upp gene. The location of the 1-kb specific ISLpl1 probe used in the Southern hybridization is shown.

FIG. 2.

Simplified diagram of the metabolic pathways involved in the selection of the spontaneous mutant HN38. The intracellular UMP pool depends on pyrimidine synthesis and utilization of preformed pyrimidine supplied from RNA degradation or pumped from the culture medium. The upp gene catalyzed the key reaction in preformed uracil utilization (19). The UMP pool controls expression of the pyr genes involved in the de novo pyrimidine biosynthesis (shown with thick arrows). PyrR, an RNA binding protein, senses the concentration of UMP in the cell and regulates pyr gene expression through an attenuator mechanism. This regulation mechanism was demonstrated for Bacillus subtilis (24) and has been proposed for L. plantarum (11). Carbamoyl phosphate (CP) is a common intermediate for both arginine and pyrimidine biosynthesis. In L. plantarum, two CP synthetases (CPS) are present: a pyrimidine-regulated CPS encoded by the pyrAaAb genes (12) and an arginine-repressed CPS encoded by the carAB genes (20). In strain FB335, the carAB genes were deleted (20); growth relied on pyrAaAb expression to provide CP for both pathways. When uracil is provided, the pyr genes are not transcribed and FB335 cannot grow since it lacks CP for arginine biosynthesis. However, spontaneous uracil-resistant derivatives of FB335 were selected and one of these, mutant HN38, harbored the upp::ISLpl1 allele. HN38 grew in the presence of uracil, since the inactivation of upp may have generated a low UMP pool, which favored pyrAaAb expression.

FIG. 3.

ISLpl1-related element distribution in L. plantarum and Pediococcus strains. Southern hybridization blots with probe ISLpl1 (arrow b) or with probe Lambda DNA (arrow a). Lane 1, Lambda DNA digested with HindIII; lane 2, Lambda digested with HindIII and EcoRI. LAB genomes were digested with restriction enzymes HaeIII (lanes 3 to 8) and HindIII (lanes 9 to 15). Lanes 3 and 9, L. plantarum strain HN38; lanes 4 and 11, L. plantarum NCIMB 8826; lanes 5 and 13, L. plantarum ATCC 14917^T; lanes 6 and 10, L. plantarum FB335; lanes 7 and 14, L. plantarum CNRZ 1891; lanes 8 and 15, L. plantarum NCFB 1406; lane 12, Pediococcus strain H.

FIG. 4.

Plasmid and chromosomal localization of ISLpl1. (A) Ethidium bromide-stained DNA after agarose gel electrophoresis; (B) Southern blot DNA bands hybridized with the digoxigenin-labeled specific ISLpl1 probe. Lanes 1, 3, and 5, native plasmid DNA extracted from L. plantarum strains FB335, NCIMB 1406, and NCIMB 8826, respectively; lanes 2 and 4, HindIII-digested CsCl gradient linear DNA preparations of strain FB335 and NCIMB 1406, respectively. Linear DNA molecular mass markers (Raoul marker; Appligene) are indicated in kilobases. Native plasmid band sizes were deduced from the three plasmids harbored by strain NCIMB 8826 (36, 2.3, and 1.9 kb). Native plasmid bands detected by the IS probe are shown by circles representing strains FB335 (circle in panel B, lane 1) and NCIMB 1406 (circles in panel B, lane 3).

FIG. 5.

Stability over time of the ISLpl1-related element patterns in L. plantarum strain NCIMB 1406. Insertion sites were evaluated with ISLpl1 DNA probed against HaeIII digests of NCIMB 1406-derived strains, using Southern hybridization under low-stringency conditions. The IS profile was analyzed in four clones isolated after 110 generations of NCIMB 1406 grown in liquid MRS medium. Arrows indicate the 18 bands detected in strain NCIMB 1406 at the beginning of the experiment. Clones 1 to 4 were isolated from a liquid MRS culture obtained by serial cultivation of strain NCIMB 1406 as described in Materials and Methods. Circles show detected bands, which differ from those of the parent strain. (A) DNA after agarose gel electrophoresis. (B) Southern blot. MW marker, molecular mass marker (Raoul marker; Appligene) bands (indicated in kilobases).

FIG. 6.

Proposed role of ISLp1l in LAB genome plasticity. In a P. pentosaceus isolate, ISLpl1 homologous sequences were present 9 kb upstream of an iso-IS30 (GenBank accession no. Z32771) region. Short sequences shaded in gray (shown at the bottom of the figure) are identical to those of an ISLpl1 left arm. The arrows delimit the IRL defined for ISLpl1. The proposed genetic rearrangement at the origin of this structure is a recombination between two short directly repeated sequences (indicated by small boxes at the top and in the middle of the figure) of two divergently oriented IS. The sequence of the repeats is GCTGt/aTC (shown at the top of the figure), with only one mismatch (lowercase characters) between the two IS elements.