Acid stress response in Escherichia coli: mechanism of regulation of gadA transcription by RcsB and GadE

Abstract

Escherichia coli can survive extreme acid stress for several hours. The most efficient acid resistance system is based on glutamate decarboxylation by the GadA and GadB decarboxylases and the import of glutamate via the GadC membrane protein. The expression of the corresponding genes is controlled by GadE, the central activator of glutamate-dependent acid resistance (GDAR). We have previously shown by genetic approaches that as well as GadE, the response regulator of the Rcs system, RcsB is absolutely required for control of gadA/BC transcription. In the presence of GadE, basal activity of RcsB stimulates the expression of gadA/BC, whereas activation of RcsB leads to general repression of the gad genes. We report here the results of various in vitro assays that show RcsB to regulate by direct binding to the gadA promoter region. Furthermore, activation of gadA transcription requires a GAD box and binding of an RcsB/GadE heterodimer. In addition, we have identified an RcsB box, which lies just upstream of the −10 element of gadA promoter and is involved in repression of this operon.

Interaction of the RcsB Response Regulator with Auxiliary Transcription Regulators in Escherichia coli

“…Constructed and transduced alleles were analyzed by PCR using oligonucleotide primers given in Table 3. type DNA-binding domain (3,8,9). In the E. coli K12 genome, in total, 18 proteins with a FixJ/NarL-type DNA-binding domain are encoded.…”

“…RcsB is a transcriptional regulator that acts as a homodimer or by interaction with auxiliary transcriptional regulators, including RcsA, GadE, and BglJ (3,8,9). RcsB and its auxiliary partners all belong to the FixJ/NarL family of transcriptional regulators characterized by a conserved C-terminal DNA-binding domain family (3,10,11).…”

“…RcsB and its auxiliary partners all belong to the FixJ/NarL family of transcriptional regulators characterized by a conserved C-terminal DNA-binding domain family (3,10,11). The interaction of RcsB with the auxiliary partners alters the DNA binding specificity (8,9,12) and thus extends the regulatory repertoire of the Rcs system to the control of multiple loci related to motility and biofilm formation, various stress responses, cell surface components, and additional functions (3,4). As a homodimer, RcsB activates or represses transcription of several loci, including rprA, encoding the small regulatory RNA RprA (3,13).…”

“…The heterodimer RcsA-RcsB activates loci coding for exopolysaccharide production and represses transcription of flhDC, encoding the flagella master regulator (14 -16). GadE-RcsB activates expression of the gadA gene, encoding a glutamate decarboxylase important in the acidstress response, whereas phosphorylated RcsB represses expression of gadA as a homodimer by binding to a different site (8). BglJ-RcsB activates expression of Ͼ10 loci, including leuO encoding a pleiotropic LysR-type transcription regulator (9,17,18).…”

“…Transcriptional activation by RcsB-RcsB homodimers and RcsA-RcsB heterodimers depends on RcsB phosphorylation and thus on induction of the Rcs signaling cascade (3). In contrast, BglJ-RcsB and GadE-RcsB heterodimers are active independent of RcsB phosphorylation (8,9). Thus, there is a dual control of RcsB activity, by phosphorylation via the Rcs phosphorelay and by interaction with auxiliary partners all belonging to the FixJ/NarL family of transcriptional regulators.…”