The Composition of the Cell Envelope Affects Conjugation in Bacillus subtilis

Abstract

Conjugation in bacteria is the contact-dependent transfer of DNA from one cell to another via donor-encoded conjugation machinery. It is a major type of horizontal gene transfer between bacteria. Conjugation of the integrative and conjugative element ICEBs1 into Bacillus subtilis is affected by the composition of phospholipids in the cell membranes of the donor and recipient. We found that reduction (or elimination) of lysyl-phosphatidylglycerol caused by loss of mpr F caused a decrease in conjugation efficiency. Conversely, alterations that caused an increase in lysyl-phosphatidylglycerol, including loss of ugtP or overproduction of mprF, caused an increase in conjugation efficiency. In addition, we found that mutations that alter production of other phospholipids, e.g., loss of clsA and yfnI, also affected conjugation, apparently without substantively altering levels of lysyl-phosphatidylglycerol, indicating that there are multiple pathways by which changes to the cell envelope affect conjugation. We found that the contribution of mprF to conjugation was affected by the chemical environment. Wild-type cells were generally more responsive to addition of anions that enhanced conjugation, whereas mprF mutant cells were more sensitive to combinations of anions that inhibited conjugation at pH 7. Our results indicate that mprF and lysyl-phosphatidylglycerol allow cells to maintain relatively consistent conjugation efficiencies under a variety of ionic conditions.

Importance: Horizontal gene transfer is a driving force in microbial evolution, enabling cells that receive DNA to acquire new genes and phenotypes. Conjugation, the contact-dependent transfer of DNA from a donor to a recipient by a donor-encoded secretion machine, is a prevalent type of horizontal gene transfer. Although critically important, it is not well understood how the recipient influences the success of conjugation. We found that the composition of phospholipids in the membranes of donors and recipients influences the success of transfer of the integrative and conjugative element ICEBs1 in Bacillus subtilis Specifically, the presence of lysyl-phosphatidylglycerol enables relatively constant conjugation efficiencies in a range of diverse chemical environments.

FIG 1

Pathways of phospholipid biosynthesis that affect conjugation of ICEBs1. Some of the pathways involved in phospholipid biosynthesis are shown. Genes relevant to this work are indicated above the arrows.

FIG 2

Effects of mutations in recipients on acquisition of ICEBs1. The relative conjugation frequency (y axis) is shown for each of the indicated recipients (x axis). The same donor strain (KM250) was used for all experiments, and ICEBs1 was induced in the donor by overproduction of the activator RapI (see Materials and Methods). The relative conjugation frequency (y axis) is the number of transconjugants per donor crossed to the indicated recipient strain, normalized to that of the wild-type (WT) recipient (CMJ161) in each experiment. The wild-type conjugation efficiency was approximately 4% transconjugants per donor in these experiments. Conjugation frequencies measured with recipients that are null for mprF, ugtP, yfnI, and lysA are similar to those previously reported (20) and were included in these experiments to allow direct comparison with the appropriate double mutants. The graph shows means and standard deviation from ≥3 experiments. The conjugation efficiency for each single mutant is statistically different from that for the wild type (P < 0.05). Data for the wild type (CMJ161) and an mprF null mutant recipient (CMJ162) are included in all panels for comparison. (A) The vector (CMJ337) contains Pspank(hy) with no insert; ↑mprF (CMJ222) indicates an mprF null mutant with Pspank(hy) driving expression of mprF. (B) ugtP (CMJ83) and ugtP mprF double mutant (CMJ333) (P < 0.05 versus ugtP). (C) clsA (CMJ86) and clsA mprF double mutant (CMJ332) (P < 0.05 versus clsA and mprF). (D) yfnI (CMJ44) and yfnI mprF double mutant (CMJ132) (P < 0.05 versus yfnI and mprF). (E) lysA (CMJ335) and lysA mprF double mutant (CMJ336) (P < 0.05 versus lysA and mprF). These strains were grown with 40 μg/ml lysine.

FIG 3

Effects of mutations on the level of lysyl-phosphatidylglycerol. The amount of lysyl-phosphatidylglycerol (LPG) recovered from a 1-ml culture of cells at an OD₆₀₀ of 1 was determined for the indicated strains: CMJ161 (wild type [WT]), CMJ162 (mprF), CMJ337 (mprF plus vector), CMJ222 {↑mprF [mprF null with Pspank(hy) driving expression of mprF]}, CMJ44 (yfnI), CMJ83 (ugtP), CMJ86 (clsA), and CMJ335 (lysA) grown with 40 μg/ml lysine (in panel B only). (A) LPG was extracted from cell membranes and examined using thin-layer chromatography (see Materials and Methods). LPG and phosphatidylethanolamine (PE) standards were used to identify the LPG and PE bands. Phosphatidylserine (PS) was added to samples as an internal standard. The locations of the LPG, PE, and PS bands are indicated. The last part of the panel shows the wild-type sample with no added PS. (B) The LPG content of each strain was quantified from ≥3 experiments. Asterisks indicate a significant difference in the amount of LPG recovered compared to that from the wild-type strain (P < 0.05, t test).

FIG 4

The chemical composition of the mating support affects conjugation. Standard filter matings were performed on supports with different chemical compositions. Donor and recipient cells were mixed in equal numbers and then collected on a filter that was placed on a mating support with the indicated composition. KCl was added to 125 mM. Mixed salts contained 106 mM sodium phosphate, 14 mM sodium sulfate, and 3 mM trisodium citrate. The dashed horizontal line in each panel marks the value for mating on TSS. The mean and standard deviation from ≥3 experiments for each condition are shown. Asterisks indicate that the difference in conjugation frequency on the given support compared to conjugation frequency on TSS is statistically significant (P < 0.05, t test). (A and B) The conjugation frequency is shown as transconjugants per donor for a wild-type donor (CMJ348) and recipient (CMJ161) (A) and for an mprF null mutant donor (CMJ476) and recipient (CMJ162) (B). (C) The conjugation frequencies obtained from panels A and B are directly compared. The ratio of the conjugation frequencies of the mprF mutant (B) and the wild-type strain (A) under each of the indicated conditions is shown.

FIG 5

Some salts enhance conjugation of wild-type but not mprF cells. Filter matings were performed as described in Materials and Methods. Equal numbers of donor and recipient cells were mixed, collected on a filter, and placed on a mating support with the indicated composition. Chemical supplements were added at 125 mM. The samples tested with TSS plus NaHPO₄ also contain dibasic sodium phosphate titrated with monobasic sodium phosphate to give a pH of 7.5. The dashed horizontal line in each panel indicates the conjugation frequency on TSS. The mean and standard deviation from ≥3 experiments are shown for each condition. Asterisks indicate that the difference in conjugation frequency on the given support compared to conjugation frequency on TSS is statistically significant (P < 0.05, t test). (A and B) The conjugation frequency (transconjugants per donor) is shown for wild-type donor (CMJ348) and wild-type recipient (CMJ161) (A) and for an mprF null mutant donor (CMJ476) and an mprF null mutant recipient (CMJ162) (B). (C) The conjugation frequencies obtained from panels A and B are directly compared and plotted as the ratio of the conjugation frequencies of the mprF mutant (B) and the wild-type strains (A) under each of the indicated conditions.