RcsB‐BglJ activates the Escherichia coli leuO gene, encoding an H‐NS antagonist and pleiotropic regulator of virulence determinants

Abstract

SummaryThe LysR-type transcription factor LeuO is involved in regulation of pathogenicity determinants and stress responses in Enterobacteriaceae, and acts as antagonist of the global repressor H-NS. Expression of the leuO gene is repressed by H-NS, and it is upregulated in stationary phase and under amino acid starvation conditions. Here, we show that the heterodimer of the FixJ/NarL-type transcription regulators RcsB and BglJ strongly activates expression of leuO and that RcsB-BglJ regulates additional loci. Activation of leuO by RcsB-BglJ is independent of the Rcs phosphorelay system. RcsB-BglJ binds to the leuO promoter region and activates one of two leuO promoters mapped in vivo. Moreover, LeuO antagonizes activation of leuO by RcsB-BglJ and acts as negative autoregulator in vivo and in vitro. Further, the H-NS paralogue StpA causes repression of leuO in addition to H-NS. Together, our data suggest a complex arrangement of regulatory elements and they indicate a feedback control mechanism of leuO expression.

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

“…However, the leuO gene is also repressed by H-NS at standard laboratory growth conditions. Although the leuO gene can be activated by BglJ-RcsB and the mutual regulation of leuO and bglJ resembles a double-positive feedback regulation, inducing conditions are not known (17). The dctR gene is encoded in the acid stress island and presumably activated by YdeO, a regulator of genes involved in the cellular response to acid resistance.…”

“…Values of bars marked with 1 are from Ref. 17. Cultures for ␤-galactosidase assays were grown to A 600 of 0.5 in LB medium supplemented with 1 mM IPTG and 25 mg/ml kanamycin.…”

“…Total RNA was isolated using the RNeasy minikit system, including an on-column DNase I treatment according to the manufacturer's instructions (Qiagen, Germany). For each strain background, three biological replicates of the RNA preparations were used for microarray analysis, which was performed by the Cologne Center for Genomics using Affymetrix GeneChip E. coli Genome 2.0 microarrays, as described (17). Differential expression levels were calculated as -fold change relative to the empty vector control.…”

“…For measuring MatA-RcsB activity, the PmatA CFT073 P L -matA ⌬rcsB ⌬lacZ strain T1987 was used, as described above. For BglJ-RcsB, we used a PleuO bglJ C ⌬rcsB ⌬lacZ reporter strain (T572), in which bglJ is expressed constitutively (17). For analyzing the RcsB homodimer activity, we constructed a PrprA lacZ fusion, as described (13), and for analyzing RcsA-RcsB activity, we constructed a fusion of the RcsA-RcsB-activated Pwza to lacZ (45).…”

“…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).…”