An epigenetic switch activates bacterial quorum sensing and horizontal transfer of an integrative and conjugative element

Abstract

Horizontal transfer of the integrative and conjugative element ICEMlSymR7A converts non-symbiotic Mesorhizobium spp. into nitrogen-fixing legume symbionts. Here, we discover subpopulations of Mesorhizobium japonicum R7A become epigenetically primed for quorum-sensing (QS) and QS-activated horizontal transfer. Isolated populations in this state termed R7A* maintained these phenotypes in laboratory culture but did not transfer the R7A* state to recipients of ICEMlSymR7A following conjugation. We previously demonstrated ICEMlSymR7A transfer and QS are repressed by the antiactivator QseM in R7A populations and that the adjacently-coded DNA-binding protein QseC represses qseM transcription. Here RNA-sequencing revealed qseM expression was repressed in R7A* cells and that RNA antisense to qseC was abundant in R7A but not R7A*. Deletion of the antisense-qseC promoter converted cells into an R7A*-like state. An adjacently coded QseC2 protein bound two operator sites and repressed antisense-qseC transcription. Plasmid overexpression of QseC2 stimulated the R7A* state, which persisted following curing of this plasmid. The epigenetic maintenance of the R7A* state required ICEMlSymR7A-encoded copies of both qseC and qseC2. Therefore, QseC and QseC2, together with their DNA-binding sites and overlapping promoters, form a stable epigenetic switch that establishes binary control over qseM transcription and primes a subpopulation of R7A cells for QS and horizontal transfer.

Figure 1.

Model of excision and transfer regulation by genes encoded on ICEMlSym^R7A. (A) Gene maps of the traR-qseC2 and rdfS-rlxS regions present on ICEMlSym^R7A are shown with a schematic detailing the molecular activation and repression of QS and ICEMlSym^R7A excision and transfer. Proteins with a positive effect on QS, excision and conjugative transfer are shown in blue, while the QseM antiactivator is shown in red. In cells active for QS, AHLs produced by TraI1 activate TraR which activates transcription from two tra boxes, located upstream of traI1 and the likely pseudogene traI2 (16). traI2 forms an operon with two open-reading frames msi172 and msi171. A single polypeptide product, FseA, is translated from msi172-msi171 following a programmed + 1 ribosomal frameshift encoded within the 3′ end of msi172 (18). FseA activates transcription from the rdfS promoter and RdfS stimulates ICEMlSym^R7A excision and transfer (17,20). rdfS is coded upstream of genes encoding the conjugative prepilin peptidase TraF, the lytic transglycosylase Msi107 and the conjugative relaxase RlxS. In cells repressed for QS, the antiactivator QseM binds TraR and FseA, inhibiting their ability to act as transcriptional activators. The QseC protein controls expression of QseM. QseC both activates its own gene promoter from a leaderless mRNA and represses qseM expression. A schematic illustrating the overlapping -35 regions of the qseC and qseM promoters (PqseC and PqseM) and the QseC binding regions O_L and O_R is shown in the shaded box. Note the role of qseC2 (shown in yellow) is not incorporated in this model yet as it had not been studied prior to this work. (B) Illustration and summary of the features of cells that are either in a transfer-repressed state expressing QseM (shaded red) or those repressed for qseM expression (in blue), which participate in QS and maintain an excised ICE in stationary-phase populations and exhibit elevated rates of conjugation.

Figure 2.

Quorum sensing and ICEMlSym^R7A excision phenotypes of R7A* and derivatives. (A) Chromobacterium violaceum CV026 bioassays were used to detect AHLs in broth-culture supernatants. Three 25-cm plates are shown. Supernatants from R7A* exconjugants in which ICEMlSym^R7A was reintroduced into the R7ANS* background are labeled as ‘Donor X R7ANS*’. Vector-only (pPR3 and pFAJ1700), positive (10 μM 3-oxo-C6-HSL) and negative controls (DMSO, R7ANS, TY) are included for comparison. (B) Quantitative PCR assays of ICEMlSym^R7A excision. Strains were grown in liquid TY medium and sampled in log phase (24 h) and late stationary phase (64 h). DNA was extracted and assayed by quantitative PCR. PCR primers amplify products that span the circular ICEMlSym^R7A DNA (attP) and vacant chromosomal insertion sites (attB) and these are presented as the percentage of total chromosomes in the sample, measured by amplification of the chromosomal melR gene (17). Bars indicate the average percentage and standard deviation (error bars) of three replicate experiments. The y-axis is in log-scale. A one-way ANOVA followed by Tukey’s post-hoc test was carried out for comparisons between R7A and every other strain and for comparisons between R7A* and every other strain. Green asterisks indicate the mean is statistically different from R7A* at the same time point (* 0.05, **P < 0.01) and orange asterisks indicate the mean is statistically different from R7A at the same time point (*P < 0.05, **P < 0.01). Vector-only controls are presented in Supplementary Figure S3.

Figure 3.

Transcriptome sequencing of R7A and R7A* in the traR-qseC2 region. Gene maps of the traR-qseC2 region are shown between coverage maps (y-axis represents moving 20-bp average of reads per nucleotide) for strand-specific RNA-seq reads from (A) R7A and (B) R7A* cells, together with a schematic highlighting proteins involved in activation (blue) and repression (red) of quorum sensing and ICEMlSym^R7A excision and transfer. Genes are indicated as gray blocks and RNA-seq read-depth averages from two experiments are shaded on the y-axis in blue for the forward strand and orange for the reverse strand. Positions of QseC and QseC2 operator sites are indicated as vertical rectangles. Numbers underneath genes in (A) represent average mRNA abundance fold-change relative to R7A* and numbers above genes in (B) represent abundance fold-change relative to R7A.

Figure 4.

QseC2 binds O2_L and O2_R and represses transcription from PqseC2 and PasqseC. (A) A gene map of the qseM-qseC2 region (to scale). DNA operator sequences are shown below the map. The promoters PqseM and PqseC were mapped previously (15). Approximate transcription start positions for asqseC and qseC2 were manually estimated from transcriptome alignments (gray highlight). The positions of the constructed ΔqseC2 and ΔPasqseCΔqseC2 deletions are indicated with dashed lines. (B) Electrophoretic Mobility Shift Assays (EMSAs) using purified 6H-QseC2 with fluorescently labeled dsDNA oligonucleotides containing the O2_L-O2_R sequence. The top EMSA labeled O2_L-O2_R was carried out using dsDNA containing the wild-type O2_L-O2_R sequence; the O2_L EMSA used DNA carrying a scrambled O2_R sequence; the O2_R EMSA used DNA carrying a scrambled O2_L sequence and both operators were scrambled in DNA for the RDM2 EMSA. All assays were carried out using a final concentration of 5 nM labeled dsDNA. (C) SPR responses for purified 6H-QseC2 (i) and 6H-QseC (ii) with their cognate operator sequences and mutated derivatives as described in (B). R_max is the theoretical maximum binding response assuming 6H-QseC and 6H-QseC2 each bind as dimers to an individual operator (i.e. dimers bound to both operators would have a theoretical R_max of 200%). Proteins were added at either 0.1 or 1 μM concentration as indicated. (D) Partial gene map (not to scale) of pSDz containing cloned PasqseC or PqseC2 upstream of lacZ, and qseC2 (when present) under control of Plac, which exhibits leaky expression in the absence of IPTG (dashed arrow) or elevated expression in the presence of IPTG. (E) β-Galactosidase activity of stationary-phase R7ANS cultures containing pSDz plasmid constructs with PqseC2 (i) or PasqseC (ii) oriented to drive transcription of the lacZ gene. Modified versions of these constructs harboring qseC2 under control of the lac promoter were also assayed, with or without 1 mM IPTG as indicated. β-Galactosidase activity was measured using the fluorescent substrate 4-methylumbelliferyl-β-D-galactopyranoside (40). Bars represent the mean of four biological replicates and error bars represent standard error of the mean. Statistical significance values from Student's t-test are represented by asterisks (< 0.05 * or < 0.01 **). The mean expression and standard error values for pSDz lacking any cloned promoter (indicated approximately by a dashed line in both (i) and (ii)) were 3,374 ± 127 and 3,542 ± 1,128 with IPTG.

Figure 5.

Model of the molecular steps leading to the R7A* state. Abridged gene maps (not to scale) illustrate regulatory steps involved in transition into the R7A* state. In the absence of any of the protein regulators (for example in a recipient upon receiving ICEMlSym^R7A immediately post-transfer), PqseM, PasqseC and PqseC2 promoters are active. Once QseC2 concentrations increase above a certain threshold, QseC2 binds O2_L and represses PasqseC. Repression of PasqseC derepresses the translation of QseC through an unknown antisense mechanism. QseC activates expression from its own promoter through binding O_L. QseC also represses PqseM and, based on the position of the PqseM -35 region, this repression may involve QseC-dimer occupancy of both O_L and O_R (indicated by a question mark) (15). Once QseM concentrations decrease, TraR is free to activate QS through activation of the PtraI1. TraR activation of PtraI2 leads to expression of FseA, which in turn leads to expression of PrdfS.