The TetR-Type MfsR Protein of the Integrative and Conjugative Element (ICE) ICE clc Controls both a Putative Efflux System and Initiation of ICE Transfer

Abstract

Integrative and conjugating elements (ICE) are self-transferable DNAs widely present in bacterial genomes, which often carry a variety of auxiliary genes of potential adaptive benefit. One of the model ICE is ICEclc, an element originally found in Pseudomonas knackmussii B13 and known for its propensity to provide its host with the capacity to metabolize chlorocatechols and 2-aminophenol. In this work, we studied the mechanism and target of regulation of MfsR, a TetR-type repressor previously found to exert global control on ICEclc horizontal transfer. By using a combination of ICEclc mutant and transcriptome analysis, gene reporter fusions, and DNA binding assays, we found that MfsR is a repressor of both its own expression and that of a gene cluster putatively coding for a major facilitator superfamily efflux system on ICEclc (named mfsABC). Phylogenetic analysis suggests that mfsR was originally located immediately adjacent to the efflux pump genes but became displaced from its original cis target DNA by a gene insertion. This resulted in divergence of the original bidirectional promoters into two separated individual regulatory units. Deletion of mfsABC did not result in a strong phenotype, and despite screening a large number of compounds and conditions, we were unable to define the precise current function or target of the putative efflux pump. Our data reconstruct how the separation of an ancestor mfsR-mfsABC system led to global control of ICEclc transfer by MfsR.

Highly variable individual donor cell fates characterize robust horizontal gene transfer of an integrative and conjugative element

“…S3A). This is consistent with previously measured proportions of initiated tc cells in stationary-phase cultures of P. putida ICEclc and P. knackmussii B13, estimated from EGFP expression of singlecopy P int -egfp insertions located outside ICEclc (17,37). In P. putida carrying the same ICEclc-intB13-egfp bicistronic fusion but with an additional deletion of the mfsR global negative regulator [strain 4612 (27)], 45 ± 5% of individual cells expressed EGFP after 72 h in stationary phase (Fig.…”

“…Time-lapse microscopy was performed as described previously with minor changes (37). Briefly, all derivatives of UWC1 carrying ICEclc were pregrown overnight in LB medium supplemented with antibiotics to select for maintenance of their respective traits.…”

The ICEXTD of Azoarcus sp. CIB, an integrative and conjugative element with aerobic and anaerobic catabolic properties

“…The integrase gene ( int XTD , AzCIB_4396), whose product should catalyze the site‐specific integration and excision of the ICE XTD element from the chromosome (Gaillard et al ., ), is located next to the attL sequence and oriented to the inward direction. In the vicinity of the int XTD gene is located a regulatory module (AzCIB_4397‐4399) that may be involved in the control of the expression of the integrase gene (Pradervand et al ., ) (Fig. A, Supporting Information Table S1).…”

A four-step regulatory cascade controls bistable transfer competence development of the integrative and conjugative element ICEclcinPseudomonas

“…Exponentially growing cells of both P. putida and P. knackmussii B13 containing ICE clc show no signs of transfer competent cells, which we have attributed to MfsR autorepression of tciR transcription [24]. Since we could so far not find any ligand specifically derepressing MfsR [24,51], we assume that basal production of TciR in exponentially growing cells is simply too low or its degradation too fast to activate the next node (the bisR promoter, as simulated in the model of Fig. 6B).…”