Escherichia coli toxin/antitoxin pair MqsR/MqsA regulate toxin CspD

Abstract

Previously we identified that the Escherichia coli protein MqsR (YgiU) functions as a toxin and that it is involved in the regulation of motility by quorum sensing signal autoinducer-2 (AI-2). Furthermore, MqsR is directly associated with biofilm development and is linked to the development of persister cells. Here we show that MqsR and MqsA (YgiT) are a toxin/antitoxin (TA) pair, which, in significant difference to other TA pairs, regulates additional loci besides its own. We have recently identified that MqsR functions as an RNase. However, using three sets of whole-transcriptome studies and two nickel-enrichment DNA binding microarrays coupled with cell survival studies in which MqsR was overproduced in isogenic mutants, we identified eight genes (cspD, clpX, clpP, lon, yfjZ, relB, relE and hokA) that are involved in a mode of MqsR toxicity in addition to its RNase activity. Quantitative real-time reverse transcription polymerase chain reaction (qRT-PCR) showed that (i) the MqsR/MqsA complex (and MqsA alone) represses the toxin gene cspD, (ii) MqsR overproduction induces cspD, (iii) stress induces cspD, and (iv) stress fails to induce cspD when MqsR/MqsA are overproduced or when mqsRA is deleted. Electrophoretic mobility shift assays show that the MqsA/MqsR complex binds the promoter of cspD. In addition, proteases Lon and ClpXP are necessary for MqsR toxicity. Together, these results indicate the MqsR/MqsA complex represses cspD which may be derepressed by titrating MqsA with MqsR or by degrading MqsA via stress conditions through proteases Lon and ClpXP. Hence, we demonstrate that the MqsR/MqsA TA system controls cell physiology via its own toxicity as well as through its regulation of another toxin, CspD.

Fig. 1

Growth curves for BW25113 mqsR and BW25113 mqsR mqsA containing pBS(Kan) (empty plasmid), pBS(Kan)-mqsR and pBS(Kan)-mqsR-mqsA at 37°C in LB with 1 mM IPTG induction (A). Normalized biofilm formation (total biofilm/growth) with 96-well polystyrene plates at 37°C in LB with 1 mM IPTG induction for 24 h for E. coli BW25113 mqsR, BW25113, MG1655 mqsR and MG1655 containing pCA24N, pCA24N-mqsA, pCA24N-mqsR (B). Growth data are the average of two independent cultures, biofilm data are the average of 10 replicate wells from two independent cultures, and one standard deviation is shown.

Fig. 2

Electrophoretic mobility shift assay (EMSA) to confirm the specific DNA binding of the MqsR/MqsA complex. MqsR/MqsA (full length of MqsA) (A) and MqsR/MqsA-N (N-terminal of MqsA) (B) were tested for binding to the promoter regions of cspD (PcspD) and bssR (PbssR). Lane 1: labelled promoter regions (6 ng), lane 2: protein complex (200 ng) and labelled promoter regions (6 ng), and lane 3: protein complex (200 ng) with labelled (6 ng) and unlabelled (specific competitor) promoter regions (600 ng). The unspecific competitor poly(dI-dC) was used for all samples.

Fig. 3

Growth curves and cell viability (cfu ml⁻¹) for BW25113 cspD/pCA24N and BW25113 cspD/pCA24N-cspD (A) and for BW25113 hokA/pCA24N and BW25113 hokA/pCA24N-hokA (B). Cells were grown to a turbidity of 0.5 at 600 nm at 37°C in LB medium, then 1 mM IPTG was added to induce the pCA24N-based genes. Data are the average of two independent cultures, and one standard deviation is shown.

Fig. 4

Growth curves (A) and normalized biofilm formation (total biofilm/growth; B) of isogenic deletion strains (clpX, clpP, lon, yfjZ, cspD, relB, relE and hokA) with production of MqsR via pCA24N-mqsR and IPTG at 37°C in LB medium. Growth data are the average of two independent cultures, biofilm data are the average of 10 replicate wells from two independent cultures, and one standard deviation is shown.

Fig. 5

Schematic of MqsR toxicity via CspD. cspD is repressed by the MqsR/MqsA complex. Under stress, MqsA is degraded by proteases (ClpXP and Lon), and cspD transcription is derepressed by the breakdown of the MqsR/MqsA complex. Stress also elevates the level of free-MqsR toxin. → indicates induction, ⊥ indicates repression, and dotted lines indicate regulatory pathways that are not active whereas solid lines indicate active regulatory pathways.