DNA microarrays revealed that expression ofycfR, which encodes a putative outer membrane protein, is significantly induced inEscherichia colibiofilms and is also induced by several stress conditions. We show that deletion ofycfRincreased biofilm formation fivefold in the presence of glucose; the glucose effect was corroborated by showing binding of the cyclic AMP receptor protein to theycfRpromoter. It appears that YcfR is a multiple stress resistance protein, since deletingycfRalso rendered the cell more sensitive to acid, heat treatment, hydrogen peroxide, and cadmium. Increased biofilm formation through YcfR due to stress appears to be the result of decreasing indole synthesis, since a mutation in thetnaAgene encoding tryptophanase prevented enhanced biofilm formation upon stress and adding indole prevented enhanced biofilm formation upon stress. DeletingycfRalso affected outer membrane proteins and converted the cell from hydrophilic to hydrophobic, as well as increased cell aggregation fourfold. YcfR seems to be involved in the regulation ofE. coliK-12 biofilm formation by decreasing cell aggregation and cell surface adhesion, by influencing the concentration of signal molecules, and by interfering with stress responses. Based on our findings, we propose that this locus be namedbhsA, for influencingbiofilm throughhydrophobicity andstress response.

The roles of toxin-antitoxin (TA) systems in bacteria have been debated. Here, the role of five TA systems in regard to biofilm development was investigated (listed as toxin/antitoxin: MazF/MazE, RelE/RelB, ChpB, YoeB/YefM, and YafQ/DinJ). Although these multiple TA systems were reported previously to not impact bacterial fitness, we found that deletion of the five TA systems decreased biofilm formation initially (8 h) on three different surfaces and then increased biofilm formation (24 h) by decreasing biofilm dispersal. Whole-transcriptome profiling revealed that the deletion of the five TA systems induced expression of a single gene, yjgK , which encodes an uncharacterized protein; quantitative real-time PCR (qRT-PCR) confirmed consistent induction of this gene (at 8, 15, and 24 h). Corroborating the complex phenotype seen upon deleting the TA systems, overexpression of YjgK decreased biofilm formation at 8 h and increased biofilm formation at 24 h; deletion of yjgK also affected biofilm formation in the expected manner by increasing biofilm formation after 8 h and decreasing biofilm formation after 24 h. In addition, YjgK significantly reduced biofilm dispersal. Whole-transcriptome profiling revealed YjgK represses fimbria genes at 8 h (corroborated by qRT-PCR and a yeast agglutination assay), which agrees with the decrease in biofilm formation upon deleting the five TA systems at 8 h, as well as that seen upon overexpressing YjgK. Sand column assays confirmed that deleting the five TA systems reduced cell attachment. Furthermore, deletion of each of the five toxins increased biofilm formation at 8 h, and overexpression of the five toxins repressed biofilm formation at 8 h, a result that is opposite that of deleting all five TA systems; this suggests that complex regulation occurs involving the antitoxins. Also, the ability of the global regulator Hha to reduce biofilm formation was dependent on the presence of these TA systems. Hence, we suggest that one role of TA systems is to influence biofilm formation.

Summary: 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.