Conjugative transfer of the Lactococcus lactis chromosomal sex factor promotes dissemination of the Ll.LtrB group II intron

Abstract

The Ll.LtrB group II intron from the low-G+C gram-positive bacterium Lactococcus lactis was the first bacterial group II intron shown to splice and mobilize in vivo. This retroelement interrupts the relaxase gene (ltrB) of three L. lactis conjugative elements: plasmids pRS01 and pAH90 and the chromosomal sex factor. Conjugative transfer of a plasmid harboring a segment of the pRS01 conjugative plasmid including the Ll.LtrB intron allows dissemination of Ll.LtrB among L. lactis strains and lateral transfer of this retroelement from L. lactis to Enterococcus faecalis. Here we report the dissemination of the Ll.LtrB group II intron among L. lactis strains following conjugative transfer of the native chromosomally embedded L. lactis sex factor. We demonstrated that Ll.LtrB dissemination is highly variable and often more efficient from this integrative and conjugative element than from an engineered conjugative plasmid. Cotransfer among L. lactis strains of both Ll.LtrB-containing elements, the conjugative plasmid and the sex factor, was detected and shown to be synergistic. Moreover, following their concurrent transfer, both mobilizable elements supported the spread of their respective copies of the Ll.LtrB intron. Our findings explain the unusually high efficiency of Ll.LtrB mobility observed following conjugation of intron-containing plasmids.

FIG. 1.

Mobility assay and probes. (A) Scoring of intron mobility efficiency after conjugation (colony patch hybridization). Plasmid mixes (donor, recipient, and mobility products) from 10 independent transconjugants were recovered (1) and retransformed in E. coli DH5α cells (LB/Spc) (2). One-hundred colonies were patched on LB/Spc plates (3) and transferred onto a nylon membrane that was then hybridized with an intron-specific probe (4) to calculate the ratio of recipient plasmids that received the intron. (B) Different probes used to identify the two categories of Ll.LtrB mobility products. The mobility products observed were generated either from the plasmid (twintron Ll.LtrB) or the sex factor (wild-type Ll.LtrB). The wild-type intron generates only one kind of mobility product, while the twintron potentially generates two types of mobility products, harboring or lacking the td intron. The group II probe (gpII) recognizes the three types of mobility products. The other three probes (gpI SJ, gpI, and WT gpII) are specific and recognize only one type of mobility product. The td group I intron splice junction probe (gpI SJ) recognizes the two td ligated exons (12 nucleotides for each exon) and gives a positive signal only upon group I intron loss. The group I probe (gpI) hybridizes within the td group I intron and reveals twintron mobility products still harboring it. The wild-type group II probe (WT gpII) recognizes wild-type Ll.LtrB mobility products and does not hybridize with either of the two twintron mobility products. Ll.LtrB group II intron, black; Ll.LtrB exons, E1 and E2; td intron, I; td exons, grey.

FIG. 2.

Conjugation/retrohoming assays. (A) Schematic of the assays. The first step (1) represents the transfer of pLE12I by conjugation from donor (NZ9800 or NZ9800ΔltrB) to recipient cell (MMS372). The second step (2) shows the invasion (retrohoming) of some homing sites (E1-E2) present on the recipient plasmid (pMNHS) by the Ll.LtrB intron expressed from donor plasmid (pLE12I). Intron-harboring recipient plasmids (pMNHS+) are represented in the transconjugant cell. Recipient plasmid used in these assays contained polymorphic sites in both exons (E1 and E2) (pMNHS-CR). Mobility products harboring the td intron are not depicted. Ll.LtrB group II intron, black; Ll.LtrB exons, E1 and E2; td intron, I; td exons, grey. (B and C) Agarose (0.5%) gels containing undigested plasmid mixes recovered from the progeny of 10 transconjugant cells (Spc^r/Cam^r). Bands corresponding to donor plasmid (D), recipient plasmid (R), and mobility product (M) are highlighted.

FIG. 3.

Conjugation/retrohoming assays. (A and B) Schematic of the assays. The first step (1) represents the transfer of sex factor (A) or both sex factor and pLE12I plasmid (B) by conjugation from donor (NZ9800) to recipient cell (MMS372). The second step (2) shows invasion (retrohoming) of some homing sites (E1-E2) present on the recipient plasmid (pMNHS) by Ll.LtrB introns expressed either from pLE12I or sex factor. Intron-harboring recipient plasmids (pMNHS+) are also represented in the transconjugant cell. Chromosomes are depicted as circular double-stranded helices. Ll.LtrB group II intron, black; Ll.LtrB exons, E1 and E2. (C and D) Agarose (0.5%) gels containing undigested plasmid mixes recovered from transconjugants containing sex factor (C) or both sex factor and pLE12I plasmid (D). Bands corresponding to donor plasmid (D), recipient plasmid (R), and mobility product (M) are highlighted. Ll.LtrB mobility efficiency, % Mob; +, presence of sex factor (Sex F); −, absence of sex factor.

FIG. 4.

Plasmid- and sex factor-based Ll.LtrB dissemination between L. lactis strains. A conjugation assay between the donor NZ9800/pLE12I and recipient MMS372/pMNHS strains can generate three types of transconjugant cells. In the first step (1), the recipient cell acquires pLE12I plasmid (1a), sex factor (1b), or both mobilizable elements (1c) by conjugation. The second step (2) represents mobility of the Ll.LtrB intron from its carrying element(s) (pLE12I, sex factor) to the homing site on the recipient plasmid (pMNHS). The percentage and nature of Ll.LtrB mobility products (pMNHS+) depends on which Ll.LtrB-harboring element(s) is present within the recipient cell. Ll.LtrB group II intron, black; Ll.LtrB exons, E1 and E2; td intron, I; td exons, grey.