Post‐transcriptional enhancement of Escherichia coli bgl operon silencing by limitation of BglG‐mediated antitermination at low transcription rates

Abstract

Summary The silent bgl operon of Escherichia coli is activated by spontaneous mutations that derepress its promoter. In addition, expression depends on specific transcriptional antitermination within the operon by the antiterminator protein BglG. Here, we show that BglG‐mediated antitermination limits expression of the bgl operon when the cellular transcription rate is low. The expression levels of chromosomally encoded activated bgl operon alleles are low but increase significantly when BglG protein is provided in trans or when the expression is rendered inde‐pendent of BglG‐mediated antitermination by mutation of the terminator. Plasmid‐encoded activated bgl operon alleles are expressed at high levels. Moreover, a moderate (threefold) further increase in the transcription rate of chromosomally encoded activated bgl operon alleles in an rpoS mutant can result in high (up to 50‐fold increased) expression levels. These data show that the expression of the bgl operon does not correlate linearly with its cellular transcription rate. Moderate differences in the transcription initiation rate are amplified post‐transcriptionally into large changes in the expression level of the operon by the requirement of a threshold for BglG‐mediated antitermination. Implications for bgl operon regulation by global regulators H‐NS, RpoS and others are discussed.

Regulation of the Escherichia coli Antiterminator Protein BglG by Phosphorylation at Multiple Sites and Evidence for Transfer of Phosphoryl Groups between Monomers

“…The resulting truncated protein showed considerable activity and was not subject to negative control any more (Table III). It may be relevant in this context that small changes in initial concentrations of active BglG species encoded downstream of bglt1 can result in high bglG expression levels due to auto-amplification of bglG expression when a certain threshold limit is exceeded (40). In conclusion, it appears possible that inactivity of the H208R mutant was masked by antitermination activity of the truncated BglG protein in the above study.…”

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

“…Strain construction included transductions with phage T4GT7 and phage P1vir (27,28), integration of reporter constructs at the attachment site attB (29,30), and generation of chromosomal replacements and deletions by Red-Gam-mediated recombination (31). Mutants were analyzed by allele-specific PCRs, whereas replacements of the matA and rcsA promoters, respectively, were in addition characterized by sequencing.…”

Silencing of the Escherichia coli bgl operon by RpoS requires Crl

“…RpoS, the key regulator in the stress response of E. coli and the H-NS homologue StpA are necessary for silencing of the wild-type bgl operon by a truncated H-NS protein lacking its C-terminal DNA-binding domain (Free et al, 1998(Free et al, , 2001Ohta et al, 1999). Furthermore, RpoS downregulates the expression of activated bgl operon alleles (Dole et al, 2002). Interestingly, the up to 50-fold repression of the bgl operon by RpoS is based on the amplification of a moderate (two-to threefold) repression of the transcription rate by RpoS via a second, post-transcriptional level of regulation involving the specific antiterminator protein BglG.…”

“…At low transcription rates BglG is limiting and transcription halts at rho-independent transcriptional terminators t1 in the leader and t2 within the operon. If the transcription rate increases above a threshold the basal synthesis of BglG is sufficient for antitermination and, as a result, bglG and the operon are expressed at high levels (Dole et al, 2002). RpoS activity is controlled by multiple signals and at various levels.…”

“…Mutations causing activation of the silent bgl operon include bgl-CRP (a C to T exchange in the CRP binding site at position k66 relative to the transcription start), bgl ::IS1-R1243 (integration of IS1 in orientation II generating a target site duplication from k88 to k80), bgl ::IS5-H3 (an integration of IS5 in orientation II generating a target site duplication from position k92 to k89) and bgl-∆2 (a deletion of the upstream silencer, extending from position k77). Terminator mutation t1-L carries a three base exchange in the left stem of the inverted repeat structure (Dole et al, 2002). bgl!…”