Identification, characterization and benefits of an exclusion system in an integrative and conjugative element of Bacillus subtilis

Abstract

Integrative and conjugative elements (ICEs) are mobile genetic elements that transfer from cell to cell by conjugation (like plasmids) and integrate into the chromosomes of bacterial hosts (like lysogenic phages or transposons). ICEs are prevalent in bacterial chromosomes and play a major role in bacterial evolution by promoting horizontal gene transfer. Exclusion prevents the redundant transfer of conjugative elements into host cells that already contain a copy of the element. Exclusion has been characterized mostly for conjugative elements of Gram-negative bacteria. Here, we report the identification and characterization of an exclusion mechanism in ICEBs1 from the Gram-positive bacterium Bacillus subtilis. We found that cells containing ICEBs1 inhibit the activity of the ICEBs1-encoded conjugation machinery in other cells. This inhibition (exclusion) was specific to the cognate conjugation machinery and the ICEBs1 gene yddJ was both necessary and sufficient to mediate exclusion by recipient cells. Through a mutagenesis and enrichment screen, we identified exclusion-resistant mutations in the ICEBs1 gene conG. Using genes from a heterologous but related ICE, we found that the exclusion specificity was determined by ConG and YddJ. Finally, we found that under conditions that support conjugation, exclusion provides a selective advantage to the element and its host cells.

Fig.1.. Genetic map of ICEBs1.

Organization of ICEBs1 open reading frames, indicated by horizontal arrows pointing in the direction of transcription, with the name of the gene indicated below. The color and patterns of each arrow indicates the gene’s function as DNA processing (diagonal stripes), regulation (black), conjugation (gray), and unknown (white). Conjugation genes encoding proteins homologous/analogous to the VirB/D type IV secretion system are indicated by the corresponding protein names in bold above the arrows. The positions of the promoters for immR, xis, yddJ, yddK, rapI, phrI, and an uncharacterized small antisense RNA are indicated by vertical arrows with the arrow head pointing in the direction of transcription. Black boxes indicate the 60 bp repeats marking the ends of the element (Auchtung et al., 2016).

Fig. 2.. ICEBs1 in recipient cells inhibits acquisition of pC194 mobilized by ICEBs1, but not by Tn916.

The percent mobilization of pC194 (Cm^R) by indicated donors and recipients. Left two bars: donors with ICEBs1 (MA116; ICEBs1 Δ(rapI-phrI)342::kan, Pxyl-rapI; pC194(cat), Str^S). Right two bars: donors with Tn916 (MA1100; ICEBs1⁰ Tn916; pC194(cat), Spc^S). White bars: mobilization into recipients without ICEBs1 (CAL89; ICEBs1⁰ str-84 comK::spc). Black bars: mobilization into recipients with ICEBs1 (CAL88; ICEBs1 str-84 comK::spc). Mobilization was calculated as the percent number of transconjugants (Cm^R Str^R cells for ICEBs1 donors, and Cm^R Spc^R cells for Tn916 donors) per number of initial donors. Data presented are averages from three independent experiments, with error bars depicting standard deviations.

Fig. 3.. In recipient cells, ICEBs1 gene yddJ is necessary and sufficient for exclusion.

A. yddJ is necessary and sufficient in the recipient cell for exclusion of pC194 mobilized by ICEBs1. The percent mobilization of pC194 by ICEBs1 (MA116) donors into various recipients: without ICEBs1 (CAL89), white bars; with ICEBs1 (CAL88), black bars; with ICEBs1 with yddJ deleted (MA665; ICEBs1 ΔyddJ str-84), gray bars; without ICEBs1 and yddJ expressed from its own promoter (MA996; ICEBs1⁰ lacA::PyddJ-yddJ str-84), light dashed bars; without ICEBs1 and yddJ over-expressed from the Pspank(hy) promoter (MA982; ICEBs1⁰ lacA::Pspank(hy)-yddJ str-84), dark dashed bars. Mobilization was calculated as the percent number of transconjugants (Cm^R Str^R cells) per number of initial donors. Data presented are averages from three independent experiments, with error bars depicting standard deviations. B. yddJ is sufficient in the recipient cell and not required in the donor cell for exclusion of ICEBs1. Left two bars: percent transfer of ICEBs1 with yddJ (MMB970; ICEBs1 Δ(rapI-phrI)342::kan, Pxyl-rapI). Right two bars: percent transfer of ICEBs1 without yddJ (MA11; ICEBs1 ΔyddJ Δ(rapI-phrI)342::kan, Pxyl-rapI). White bars: transfer into recipients without ICEBs1 (CAL89). Black bars: transfer into recipients without ICEBs1 and overexpressing yddJ (MA982). Transfer was calculated as the percent number of transconjugants (Kan^R Str^R cells) per number of initial donors. Data presented are averages from three independent experiments, with error bars depicting standard deviations.

Fig. 4.. Isolation of exclusion-resistant conG mutations in ICEBs1.

A. Schematic of the mutagenesis and enrichment screen for exclusion-resistant mutations in ICEBs1 (described in the text and Methods). B. Schematic of YddJ and ConG predicted topologies. YddJ is a putative lipoprotein. Results from proteomic fractionation studies indicated that YddJ is associated with the cell membrane but that it is not a transmembrane protein (Otto et al., 2010). ConG is predicted to have seven transmembrane regions. Residues 285–305 of the extracellular loop between the third and fourth transmembrane regions are shown with the residues (288 and 292) identified in the screen for exclusion-resistance circled. C. ICEBs1 conG-E288K donors are resistant to yddJ-mediated exclusion. Left two bars: percent transfer of ICEBs1 (MA1049; ICEBs1 Δ(rapI-phrI)342::kan, Pxyl-rapI). Right two bars: percent transfer of exclusion-resistant ICEBs1 (MA1089; ICEBs1 conG-E288K Δ(rapI-phrI)342::kan, Pxyl-rapI). White bars: recipients without ICEBs1 (CAL89). Dashed bars: recipients without ICEBs1 and overexpressing yddJ (MA982). Transfer was calculated as the percent number of transconjugants (Kan^R Str^R cells) per number of initial donors. Data presented are averages from three independent experiments, with error bars depicting standard deviations.

Fig. 5.. ICEBs1 and ICEBat1 homology and exclusion specificity.

Alignments of ConG (A) and YddJ (B) homologs from ICEBs1-like elements from five Bacillus species, including B. subtilis (ICEBs1) and B. atrophaeus (ICEBat1), were generated using Jalview (www.jalview.org). A. Schematic alignment of ConG homologs. Gray indicates regions that are identical, while white indicates regions that are dissimilar. Alignment of the dissimilar internal region that includes the residues mutated in exclusion-resistant ConG from ICEBs1 (circled) is shown in detail. B. Alignment of YddJ homologs. C. The percent transfer of ICEBs1 strains with conG from either ICEBs1 (left panel; donor strain KPD225) or ICEBat1 (right panel; donor strain KPD224). White bars: recipients without ICEBs1 (CAL89). Dashed bars: recipients with yddJ from ICEBs1 over-expressed (MA982). Hatched bars: recipients with yddJ from ICEBat1 over-expressed (KPD219; ICEBs1⁰ lacA::Pspank(hy)-yddJ_ICEBat1 str-84). Transfer was calculated as the percent number of transconjugants (Kan^R Str^R cells) per number of initial donors. Data presented are averages from three independent experiments, with error bars depicting standard deviations.

Fig. 6.. Exclusion is beneficial to ICEBs1 and its host cells by preventing loss of viability due to redundant transfer.

Data depicted in A-C. are from experiments where cells (monocultures) were grown in minimal medium, induced (as indicated) with 1% xylose for 2 hours, and placed at high cell density on filters for 3 hours. Cells were sampled before and after plating on filters to determine CFU/ml pre- and post-mating conditions. The y-axis shows the number of viable cells recovered post-mating per number of input cells pre-mating on a log₂ scale. Each dot represents a value from an independent experiment (n=6). The middle bars represent averages and the shorter bars depict standard deviations. P-values were calculated by an ordinary one-way ANOVA with Dunnett’s correction for multiple comparisons (**** indicates p-value <0.0001) using GraphPad Prism version 6. A and B. Cells containing exclusion-defective ICEBs1 exhibit a loss of viability under conditions that favor conjugation. Cell recovery is shown for cells containing wildtype (MA1049; ICEBs1, Pxyl-rapI) or exclusion-deficient ICEBs1 (MA1050; ICEBs1 ΔyddJ, Pxyl-rapI, and MA1089; ICEBs1 conG-E288K, Pxyl-rapI). A. Cell viability following activation (induction) of ICEBs1. B. Cell viability with NO activation (uninduced) of ICEBs1. C. Loss of viability from defective exclusion depends on a functional conjugation machinery. Cell recovery is shown for induced cells containing wildtype (MA1049) and transfer-deficient ICEBs1 with exclusion (MA1070; ICEBs1 ΔconQ, Pxyl-rapI) and without exclusion (MA1069; ICEBs1 ΔconQ ΔyddJ, Pxyl-rapI). D. Loss of viability depends on high cell density. Cells containing wildtype (MA1049) and exclusion-deficient ICEBs1 (MA1050 and MA1089) were grown in minimal medium, induced with 1% xylose for 2 hours, and placed at high and low cell density on filters for 3 hours. CFU/ml were determined before and after incubation on the filter. The y-axis depicts the number of viable cells recovered from the filter per number of input cells pre-filtration on a log₂ scale. Each dot represents a value from an independent experiment (n=8). The middle bars represent averages and the shorter bars depict standard deviations. P-values were calculated by an unpaired two-tailed t-test with Welch’s correction (*** indicates p-value<0.0005; **** indicates p-value <0.0001) using GraphPad Prism version 6.