A mini-Tn5Cm insertion has been identified that significantly reduced the amount of an extracellular activating signal for a lacZ fusion (cma37::lacZ) in Providencia stuartii. The transposon insertion was located immediately upstream of an open reading frame encoding a putative CysE ortholog. The CysE enzyme, serine acetyltransferase, catalyzes the conversion of serine to O-acetyl-L-serine (OAS). This activating signal was also produced by Escherichia coli, and production was abolished in a strain containing a null allele of cysE. Products of the CysE enzyme (OAS, N-acetyl-L-serine [NAS], O-acetyl-L-threonine, and N-acetyl-L-threonine) were individually tested for the ability to activate cma37::lacZ. Only OAS was capable of activating the cma37::lacZ fusion. The ability of OAS to activate the cma37::lacZ fusion was abolished by pretreatment at pH 8.5, which converts OAS to NAS. However, the activity of the native signal in conditioned medium was not decreased by treatment at pH 8.5. In contrast, conditioned medium prepared from cells grown at pH 8.5 exhibited a 4-to 10-fold-higher activity, relative to pH 6.0. Additional genes regulated by the CysE-dependent signal and OAS were identified in P. stuartii and E. coli. The response to the extracellular signal in E. coli was dependent on CysB, a positive activator that requires NAS as a coactivator. In E. coli, a cysE mutant formed biofilms at an accelerated rate compared to the wild type, suggesting a physiological role for this extracellular signal.Cell-to-cell communication in bacteria is mediated by a wide variety of distinct chemical signals, and a number of reviews on this subject have been compiled (11,12,17,22). In gramnegative bacteria, these signals include the N-acylhomoserine lactones, autoinducer 2 (AI-2), quinolones, cyclic dipeptides, and the tryptophan derivative indole (2,12,26,32,38). In gram-positive bacteria, peptides are the primary mode of communication (11,17,22). Although peptide-mediated signals have not been isolated from gram-negative bacteria, recent data suggest that such systems are present (13,29,30).Several extracellular signals have been identified in Escherichia coli. Indole, a product of the tryptophanase-mediated catabolism of tryptophan, is capable of activating the astD, tnaB, and gabT genes (38). Studies using a tnaA mutant unable to produce indole have indicated that a second signal is required for full activation of the astD and gabT genes (38). A second signal in E. coli, the AI-2 furanone signal, has been shown by microarray analysis to control the expression of a large number of genes in E. coli (8,34). Additional genes in E. coli that are regulated by undefined extracellular signals include rpoS, sdiA, cysK, and ftsQAZ (1,14,23,33,37).The cysteine regulon in E. coli comprises a set of genes involved in the biosynthesis of cysteine from L-serine (18,19). These genes are positively controlled by the CysB activator in concert with the molecule N-acetyl-L-serine (NAS) (19). The production of NAS occurs in two steps. First, se...