SummaryAmino sugars are essential precursor molecules for the biosynthesis of bacterial cell walls. Their synthesis pathway is initiated by glucosamine-6-phosphate (GlcN-6-P) synthase (GlmS) which catalyses the rate limiting reaction. We report here that expression of the Escherichia coli glmS gene is negatively feedback regulated by its product GlcN-6-P at the posttranscriptional level. Initially, we observed that mutants defective for yhbJ, a gene of the rpoN operon, overproduce GlmS. Concomitantly, a glmS mRNA accumulates that is derived from processing of the primary glmUS transcript at the glmU stop codon by RNase E. A transposon mutagenesis screen in the yhbJ mutant identified the small RNA GlmZ (formerly RyiA or SraJ) to be required for glmS mRNA accumulation. GlmZ, which is normally processed, accumulates in its full-length form in the yhbJ mutant. In the wild type, a decrease of the intracellular GlcN-6-P concentration induces accumulation of the glmS transcript in a GlmZ-dependent manner. Concomitantly, GlmZ accumulates in its unprocessed form. Hence, we conclude that the biological function of GlmZ is to positively control the glmS mRNA in response to GlcN-6-P concentrations and that YhbJ negatively regulates GlmZ. As in yhbJ mutants GlcN-6-P has no effect, YhbJ is essential for sensing this metabolite.

(34,35). Indeed, out of about 1,000 spontaneous Bgl+ mutations isolated in E. coli K-12 carrying the wild-type operon on a plasmid, only 17 were not due to transposition of one of these elements (H. Ronecker, K. Schnetz, and B. Rak, unpublished data). Activation could, at least in the case of IS5, be caused by specific sequences internal to the element, exerting their effect in an orientationindependent manner from positions upstream as well as downstream of the promoter, analogous to the eucaryotic enhancing sequences (Schnetz and Rak, in preparation).In this communication we present the nucleotide sequence of a 5,270-base-pair (bp) segment of the E. coli chromosome which includes all functions necessary for regulated uptake and degradation of aryl-,-glucosides but possibly not the 3' end of the bgl operon. These data extend the known sequence information in the region of oriC to a total of >25 kbp, the longest contiguous sequence of the E. coli genome reported to date.Our

The bgl operon of Escherichia coli encodes all functions necessary for the regulated uptake and utilization of aryl beta‐glucosides. The operon is unusual, however, in that it is cryptic in wild‐type strains, requiring activation by mutational events. The vast majority of these mutations are due to transposition of insertion elements into the promoter region of the operon. In this report we show that integration of IS5 into the vicinity of the bgl promoter (P0) enhances its activity by greater than 60‐fold thereby activating the operon. In the activated state the operon is subject to induction by substrate. Recent studies have shown that induction of the bgl operon by substrate involves antitermination within the leader of the operon. We now show that substrate‐dependent regulation involves specific termination/antitermination of transcription at two signal structures flanking the first gene of the operon, bglG. Antitermination is mediated by the product of gene bglG. In the absence of substrate this antitermination is prevented by the action of the product of gene bglF (the second gene of the operon), which encodes the beta‐glucoside‐specific transport protein (enzymeIIBgl of the phosphoenolpyruvate‐dependent phosphotransferase system, PTS) resulting in repression of the operon. The bgl promoter (P0) is not subject to substrate‐dependent regulation. The bgl operon has two additional promoters (P1 and P2) located within the terminators, which could also participate in regulation.

The cryptic bgl operon of Escherichia coli is activated by the spontaneous insertion of mobile DNA elements. Screening of a collection of such mutations revealed insertion of the 1195-base-pair element ISS into various positions both upstream and downstream of the bgl promoter Po.Activation of the operon was in all cases attributable'to enhancement of PO activIty. Introduction of internal deletions into ISS almost completely abolished PO enhancement, demonstrating that enhancement is not simply the result ofmutational inactivation of some inhibitory sequences. Intact copies of IS5 in trans restored the enhancing activity of the deletion derivatives. The trans-activator is encoded by ISS gene ins5A, an essential transposition function. Activation of gene expression by means ofinteraction of a defective mobile element in cis with functions encoded by a nondefective element in trans has so far been described only for a maize controlling element.Mobile DNA elements were first discovered in maize by Barbara McClintock, who named them "controlling elements" (see refs. 1-3 for reviews). Much later such elements were found and analyzed in bacteria (see ref. 4 for a recent review), which triggered their discovery in a wide range of species (5) and also led to "rediscovery" of the maize controlling elements.The inactivation of genes by insertion of mobile DNA elements into coding and control regions and the activation of gene expression by promoter activity provided by mobile elements are well-known phenomena in both eukaryotic and prokaryotic organisms (5). Modulation of expression of adjacent genes due to the presence of enhancers or silencers within mobile elements, however, has thus far been detected only in eukaryotes (1-3, 6-11). In all cases in which mobile elements interfere with the expression of adjacent genes, this interference leads to disturbance of their normal regulatory regime.The bgl operon of Escherichia coli (responsible for t3-glucoside utilization) is unique in that it depends on integration of mobile elements to be active: the operon is silent in the wild-type state and is activated by spontaneous transposition of the mobile insertion elements IS] or IS5 into a proximal region termed bglR (12). In independent isolates of activated mutants both elements were found to be integrated in both orientations at different sites within a region of 49 base pairs (bp) (13,14). Once activated, the operon is coordinately regulated by its substrate and the catabolite gene activator protein (CAP)-cAMP complex (12,15). The basis for activation is a drastic enhancement of activity (up to 60-fold; ref. 14) of the major bgl promoter (PO) (14-16). The scattered and orientation-independent occurrence of activating mutations made it conceivable that in the wild-type state, activity of promoter Po may be somehow inhibited by its upstream sequences and that activation by IS elements is based on the uncoupling of these sequences from the promoter (13) or on their insertional inactivation. However, upon systematic exam...

In bacteria various sugars are taken up and concomitantly phosphorylated by sugar-specific enzymes II (EII) of the phosphoenolpyruvate:sugar phosphotransferase system (PTS). The phosphoryl groups are donated by the phosphocarrier protein HPr. BglG, the positively acting regulatory protein of the Escherichia coli bgl (beta-glucoside utilization) operon, is known to be negatively regulated by reversible phosphorylation catalyzed by the membrane spanning beta-glucoside-specific EIIBgl. Here we present evidence that in addition BglG must be phosphorylated by HPr at a distinct site to gain activity. Our data suggest that this second, shortcut route of phosphorylation is used to monitor the state of the various PTS sugar availabilities in order to hierarchically tune expression of the bgl operon in a physiologically meaningful way. Thus, the PTS may represent a highly integrated signal transduction network in carbon catabolite control.