Sugar transport by the bacterial phosphotransferase system. Molecular cloning and structural analysis of the Escherichia coli ptsH, ptsI, and crr genes

Abstract

Specialized lambda-transducing phages that carry the Escherichia coli genes ptsH, ptsI, crr, cysM, and cysA have been isolated, and the genes were subcloned in plasmid pBR322. Subcloning and restriction mapping data gave the following clockwise order of genes located at about 52 min on the E. coli genetic map: lig, cysK, ptsH, ptsI, crr, cysM, cysA. The nucleotide sequences of ptsH, ptsI, and crr and the corresponding flanking regions have been determined. These genes encode three cytoplasmic proteins of the phosphoenol-pyruvate:glycose phosphotransferase system: HPr, Enzyme I, and IIIGlc, respectively. The deduced amino acid sequences are consistent with amino acid composition and Edman degradation analyses obtained with the purified proteins. The calculated subunit molecular weight values (9,109 for HPr, 63,489 for Enzyme I, and 18,099 for IIIGlc) also agree well with values obtained with the proteins. Results of gamma delta-transposon insertional studies provided definitive evidence that IIIGlc is the gene product of crr, and therefore that IIIGlc plays a critical role in regulating the metabolism and uptake of certain non-PTS sugars (see accompanying papers: Mitchell, W.J., Saffen, D.W., and Roseman, S. (1987) J. Biol. Chem. 16254-16260; Misko, T.P., Mitchell, W.J., Meadow, N.D., and Roseman, S. (1987) J. Biol. Chem. 16261-16266). The gamma delta transposon studies also suggest that crr is transcribed from an independent promoter located within the ptsI gene. Putative regulatory sequence features include a catabolite gene activator protein-cAMP-binding site and two regions of 2-fold rotational symmetry adjacent to the potential promoter upstream from the HPr structural gene, several ribosome-binding sites, and a rho-independent RNA polymerase termination site downstream from crr. In addition, the ptsI gene contains two highly conserved direct repeats. The significance of these sequence features is discussed with respect to possible multiple forms of pts regulation.