Escherichia coli Hha is an environmental-response regulator of the pathogenic hemolysin operon, and Hha and the contiguous YbaJ are both induced 30-fold in E. coli biofilms (Appl. Microbiol. Biotechnol. 64:515, 2004). Here it is shown that Hha and YbaJ regulate biofilm formation since the hha/ybaJ deletion reduces biofilm mass in microtitre plates (81% in minimal medium, 50% in complex medium) and in flow cells (1,000-fold less surface coverage in minimal medium). The addition of the derepressed conjugative plasmid R1drd19, which increases significantly biofilm formation, eliminated motility completely in wild-type E. coli K12, promoted cell aggregation 27.18 +/- 0.05-fold, and produced a flatter biofilm. Deletion of hha/ybaJ or ybaJ restored motility (this motility phenotype may be complemented by providing hha(+)/ybaJ(+) or ybaJ(+) in trans) and reduced cell aggregation to that of the wild-type strain that lacks the conjugation plasmid. This increase in motility due to deleting hha/ybaJ was found to be due to 8-fold induction of fliA transcription. In addition, deletion of ompA reduced biofilm mass by 80% in both LB medium and LB medium with glucose. Also, Hha/YbaJ promotes conjugation since there was five-fold less conjugation in the hha/ybaJ mutant. It appears that conjugation plasmids promote biofilm formation by promoting cell aggregation, and that Hha and YbaJ increase biofilm formation by increasing conjugation and by decreasing motility when a conjugative plasmid (R1drd19) is present (YbaJ plays the most important role in this regulation of motility). When hha/ybaJ are deleted, there is less conjugation, less aggregation, more motility, and less biofilm.