Efficient gene transfer in bacterial cell chains

Abstract

Horizontal gene transfer contributes to evolution and the acquisition of new traits. In bacteria, horizontal gene transfer is often mediated by conjugative genetic elements that transfer directly from cell to cell. Integrative and conjugative elements (ICEs; also known as conjugative transposons) are mobile genetic elements that reside within a host genome but can excise to form a circle and transfer by conjugation to recipient cells. ICEs contribute to the spread of genes involved in pathogenesis, symbiosis, metabolism, and antibiotic resistance. Despite its importance, little is known about the mechanisms of conjugation in Gram-positive bacteria or how quickly or frequently transconjugants become donors. We visualized the transfer of the integrative and conjugative element ICEBs1 from a Bacillus subtilis donor to recipient cells in real time using fluorescence microscopy. We found that transfer of DNA from a donor to a recipient appeared to occur at a cell pole or along the lateral cell surface of either cell. Most importantly, we found that when acquired by 1 cell in a chain, ICEBs1 spread rapidly from cell to cell within the chain by additional sequential conjugation events. This intrachain conjugation is inherently more efficient than conjugation that is due to chance encounters between individual cells. Many bacterial species, including pathogenic, commensal, symbiotic, and nitrogen-fixing organisms, harbor ICEs and grow in chains, often as parts of microbial communities. It is likely that efficient intrachain spreading is a general feature of conjugative DNA transfer and serves to amplify the number of cells that acquire conjugative mobile genetic elements. IMPORTANCE Conjugative elements contribute to horizontal gene transfer and the acquisition of new traits. They are largely responsible for spreading antibiotic resistance in bacterial communities. To study the cell biology of conjugation, we visualized conjugative DNA transfer between Bacillus subtilis cells in real time using fluorescence microscopy. In contrast to previous predictions that transfer would occur preferentially from the donor cell pole, we found that transfer of DNA from a donor to a recipient appeared to occur at a cell pole or along the lateral cell surface of either cell. Most importantly, we found that when acquired by 1 cell in a chain, the conjugative DNA spread rapidly from cell to cell within the chain through sequential conjugation events. Since many bacterial species grow naturally in chains, this intrachain transfer is likely a common mechanism for accelerating the spread of conjugative elements within microbial communities.

FIG 1

Map of ICEBs1 and the constructs used. (A) ICEBs1 is ~20 kb and inserted in the 3′ end of trnS-leu2. Large arrows indicate open reading frames and orientation. The shaded boxes with small arrowheads underneath at the ends of ICEBs1 represent the 60-bp direct repeats. Vertical lines with arrows between immR and xis represent the two promoters (PimmR, Pxis) that are controlled by the transcriptional repressor/activator ImmR. (B) Insertion of the lacO array and kan and concomitant removal of part of rapI through yddM. (C) Boundaries of the conG deletion. (D) Insertion of sspB and kan and deletion of rapI-phrI.

FIG 2

Examples of successful mating pairs between donors that contain ICEBs1 with a lacO array (red cells, strain AB86) and recipients that express LacI-GFP (green cells, strain MMB849) visualized by fluorescent microscopy. Transconjugants appear as cells with at least one focus of LacI-GFP. The appearance of LacI-GFP foci in the absence of donors is ≤0.01%, indicating that virtually all of the events we visualized were transconjugants. Arrows point to the donor, recipient, and transconjugant, as indicated (A, B), and to some of the foci of LacI-GFP (D, F to H). (A, B) Mating from the side of the donor cell to the side of the recipient. (C, D) Mating from the pole of the donor cell to a pole of the recipient. (E to H) A single donor transfers ICEBs1 to two recipients. At the beginning of the time course, before visible transfer (A, C, E), 30 min later (B, D, F), and at successive 30-min time points (G, H). In these examples, the transconjugant has multiple green foci, likely due to replication of ICEBs1 in the transconjugant and/or multiple transfer events.

FIG 3

Examples of ICEBs1 transferred to cells in chains. A time course is shown for three different matings. In all cases, the first panel of each set (A, F, K) is the first time point (time 0), followed by images of the same field of cells taken at 30-min intervals. Donors are red, and recipients are green. Arrows point to some of the foci of LacI-GFP in transconjugants. (A to E) Spread of ICEBs1 through a chain of cells. Donors contained ICEBs1 with a lacO array (red cells, strain AB86). Recipients expressed LacI-GFP (green cells, strain MMB849). Transconjugants have at least one focus of LacI-GFP. (F to J) Spreading requires conjugation functions. Donors contained ICEBs1 with a lacO array, a null mutation in conG (an ICEBs1 gene required for conjugation), and a copy of conG⁺ elsewhere in the chromosome (red cells, strain AB101). Recipients and transconjugants were as described above. (K to N) Spreading of ICEBs1 through a chain of cells visualized by conditional protein degradation. Images are merges of phase, green (GFP), and red (mCherry). Red donors (strain CAL1391) contained constitutively expressed sspB in ICEBs1. Green recipients (strain CAL1379) expressed a GFP-ssrA* fusion. Transconjugants turned from green to dark due to instability of GFP-SsrA* in the presence of SspB (29) expressed from the newly transferred ICEBs1.