The glucose-specific phosphocarrier protein (HIGIC) of the bacterial phosphoenolpyruvate: glycose phosphotransferase system (PTS) is a major signal transducer that mediates the intricate interplay among extracellular signals (PTS and non-PTS sugars), cytoplasmic and membrane proteins (PTS and non-PTS transporters), and adenylate cyclase. To further define the central role of 11GIc in these multiplex signaling mechanisms, we have used site-directed mutagenesis to construct three mutant iGkc proteins containing single amino acid changes; Phe-3 was replaced with tryptophan ([Trp3JIIIG1%, wild-type crr; SR1602, His-90 -* Gln; SR1603, His-75 -* Gln; SR1604, Phe-3 -* Trp. Cells were grown aerobically at 37°C in LB medium (11), supplemented with appropriate antibiotics.Mutagenesis. Kits for mutagenesis and dideoxy-nucleotide sequencing were from Bio-Rad and Bethesda Research Laboratories, respectively. The Muta-Gene in vitro mutagenesis kit (Bio-Rad) was used with the modifications of the manufacturer's procedure indicated below. Mutagenic oligonucleotides were assembled on a Biosearch 8750 automated DNA synthesis apparatus using ,-cyanoethylphosphoramidite methodology (12). The template for mutagenesis was pBluescript SK-containing the EcoRI-Pst I restriction fragment of pDS45 (10); this 1.5-kilobase fragment contains the structural gene for IIIGIc, but lacks the genes for HPr and enzyme I.Recombinant phagemids were introduced into the dut ung strain CJ236 by transformation (13). Packaged singlestranded DNA was generated by superinfecting transforAbbreviations: 1j1GIc, the phosphocarrier protein of the phosphotransferase system specific for glucose and methyl a-D-glucopyranoside; PTS, phosphoenolpyruvate:glycose phosphotransferase system; [Trp3]IIIGlc, [Gln75]IIIGlc, and fGln90IIIGic, the Phe-3 --Trp, His-75--+ Gln, and His-90--+ Gln 111G c mutant proteins, respectively; IIBGiC, glucose-specific integral membrane protein of the PTS; HPr, histidine-containing phosphocarrier protein of the PTS; aMeGlc, methyl a-D-glucopyranoside. 4052 The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact.

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Several potential target sites for multiple regulatory mechanisms were previously identified in the 5' flanking region of the pts operon. We have investigated the in vitro interactions of the cAMP receptor protein (CRP)'cAMP regulatory complex with two DNA binding sites, by gel mobility-shift assays, and report the analysis of the functional role of each of the binding sites in vivo. Promoter-reporter gene fusion studies identified two CRPcAMP-dependent promoters (the previously identified P1 and another promoter, PO) upstream of ptsH. The crr promoters (P2) within ptsl may be negatively regulated by CRP'cAMP.The phosphoenolpyruvate:glycose phosphotransferase system (PTS) catalyzes the phosphorylation and transport of its sugar substrates and acts as a major signal-transduction system in bacterial cells. Thus, the PTS must be responsive to multiple and diverse external signals (for a review, see ref. 1).The PTS has been extensively studied in Escherichia coli and Salmonella typhimurium and consists of a complex array of reversibly phosphorylated cytoplasmic and membrane proteins. Two general, cytoplasmic proteins are required for the phosphorylation ofall PFTS substrates: enzyme I, encoded by ptsI, and the histidine-containing phosphoprotein HPr, encoded byptsH. Specific membrane complexes are required for translocation of the individual PTS sugars, and one of these, the glucose-specific complex, comprises a soluble protein (IIIGic) and a membrane-bound enzyme (IIBGlc) encoded by the crr and ptsG genes, respectively.The ptsH, ptsl, and crr genes constitute an operon ( Fig. 1) that is located at 52 min on the E. coli chromosome; ptsG is unlinked at 25 min. The PTS, itself, is stringently regulated and the mechanisms underlying the genetic regulation ofpts expression are unclear and apparently complex. The complete DNA sequence of ptsH, ptsI, crr and their flanking regions suggested two canonical binding regions for CRP cAMP upstream of the first gene, ptsH, and a promoter for crr, the last gene, within and toward the 3' terminus of ptsl (5). Transcriptional analysis of the region (4) suggested one promoter upstream of ptsH, designated Pi, and two withinptsIthat regulated transcription of crr, designated P2-I and P2-II. The designations P1 and P2 will be employed in this paper for the previously described promoters.In the present studies, we have measured CRP binding to the two putative binding sites upstream of ptsH by gel mobility-shift assays. We also report CRP-cAMP-independent and CRP-cAMP-dependent promoter activities in the DNA regions preceding and within the pts operon, including another promoter, designated PO, upstream of P,.