Correlation of Antagonistic Regulation of leuO Transcription with the Cellular Levels of BglJ-RcsB and LeuO in Escherichia coli

Abstract

LeuO is a conserved and pleiotropic transcription regulator, antagonist of the nucleoid-associated silencer protein H-NS, and important for pathogenicity and multidrug resistance in Enterobacteriaceae. Regulation of transcription of the leuO gene is complex. It is silenced by H-NS and its paralog StpA, and it is autoregulated. In addition, in Escherichia coli leuO is antagonistically regulated by the heterodimeric transcription regulator BglJ-RcsB and by LeuO. BglJ-RcsB activates leuO, while LeuO inhibits activation by BglJ-RcsB. Furthermore, LeuO activates expression of bglJ, which is likewise H-NS repressed. Mutual activation of leuO and bglJ resembles a double-positive feedback network, which theoretically can result in bi-stability and heterogeneity, or be maintained in a stable OFF or ON states by an additional signal. Here we performed quantitative and single-cell expression analyses to address the antagonistic regulation and feedback control of leuO transcription by BglJ-RcsB and LeuO using a leuO promoter mVenus reporter fusion and finely tunable bglJ and leuO expression plasmids. The data revealed uniform regulation of leuO expression in the population that correlates with the relative cellular concentration of BglJ and LeuO. The data are in agreement with a straightforward model of antagonistic regulation of leuO expression by the two regulators, LeuO and BglJ-RcsB, by independent mechanisms. Further, the data suggest that at standard laboratory growth conditions feedback regulation of leuO is of minor relevance and that silencing of leuO and bglJ by H-NS (and StpA) keeps these loci in the OFF state.

Interference of transcription across H‐NS binding sites and repression by H‐NS

“…To ensure a gradual induction of P BAD with arabinose and to circumvent feedback regulation of the arabinose system (Khlebnikov et al ., ), the araC‐P BAD ‐proU allele was inserted into strain U65 (Supporting Information Table S1). This strain carries araE encoding a arabinose proton symporter under control of a constitutive promoter ( P cp8 ) for constitutive uptake of arabinose, while araBAD encoding the catalytic enzymes and araFGH encoding an arabinose ABC transporter are deleted (Breddermann and Schnetz, ). Cultures of the strain carrying the araC‐P BAD ‐proU allele were grown in low osmolarity medium (LB 10 mM NaCl) without and with arabinose concentrations ranging from 10 µM to 1.3 mM for gradual induction of the P BAD promoter.…”

Effects of Global and Specific DNA-Binding Proteins on Transcriptional Regulation of the E. coli bgl Operon

“…RcsB and BglJ, harboring similar DNA binding domains at their C-termini, can form heterodimers, binding to an upstream promoter region and relieving H-NS-mediated repression of the bgl operon [ 29 , 33 ]. LeuO is a global transcription factor that not only regulates the leucine biosynthesis operon of E. coli but is also involved in the regulation of stress responses [ 34 , 35 ]. Its overexpression interferes with the silencing of bgl by H-NS and thereby activates bgl operon transcription, although it is not required for bgl activation upon IS insertional mutation or the absence of H-NS [ 16 ].…”

Activation of leuO by LrhA in Escherichia coli

“…BglJ-RcsB is a heterodimer of the FixJ/NarL-type transcription factors BglJ, a response regulator-like protein, and RcsB, the response regulator of the Rcs (regulation of capsule synthesis) twocomponent phosphorelay (Giel et al, 1996;Majdalani and Gottesman, 2005). In addition, positive autoregulation of leuO was reported (Fang et al, 2000), but LeuO seems to work predominantly as an autorepressor, since it represses leuO transcription in hns stpA mutants and also counteracts activation by BglJ-RcsB Breddermann and Schnetz, 2016). Transcription of leuO is presumably directed by several promoters.…”