Physical and genetic characterization of the glucitol operon in Escherichia coli.

The glucitol (gut) operon has been identified in the colony bank of Clark and Carbon (A. Sancar and W. D. Rupp, Proc. Natl. Acad. Sci. USA 76:3144-3148, 1979). We subcloned the gut operon by using paCYC184, pACYC177, and pBR322. The operon, which is encoded in a 3.3-kilobase nucleotide fragment, consists of the gutC, gutA, gutB, and gutD genes. The repressor of the gut operon seemed to be encoded in the region downstream from the operon. The gene products of the gut operon were identified by using maxicells. The apparent molecular weights of the glucitol-specific enzyme II (product of the gutA gene), enzyme III (product of the gutB gene), and glucitol-6-phosphate dehydrogenase (product of the gutD gene) were about 46,000, 13,500, and 27,000, respectively, as estimated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis.     

Nucleotide sequence of the promoter region of the melibiose operon of Escherichia coli

The nucleotide sequence of the promoter region of the melibiose operon of E. coli was determined. Consensus sequences for the -35 region, the Pribnow box and the binding site for cyclic AMP receptor protein were found in this region. The possible secondary structure of this DNA region was very similar to that of the promoter region of the lactose operon. A possible initiation ATG preceded by a Shine-Dalgarno sequence with proper spacing was present just downstream of the promoter region. The possible sequence of 52 amino acid residues in the NH2 terminus of the alpha-galactosidase were determined.     

Genetic and molecular characterization of the Escherichia coli secD operon and its products.

The secD operon of Escherichia coli is required for the efficient export of proteins. We have characterized this operon, and found that, in addition to secD and secF, it contains the upstream gene yajC, but not the genes queA or tgt, in contrast to previous reports. An analysis of yajC mutations constructed in vitro and recombined onto the chromosome indicates that yajC is neither essential nor a sec gene. The secD operon is not induced in response to either secretion defects or temperature changes. TnphoA fusions have been used to analyze the topology of SecD in the inner membrane; the protein contains six transmembrane stretches and a large periplasmic domain. TnphoA fusions to SecD and SecF have also been recombined onto the chromosome and used to determine the level of these proteins within the cell. Our results indicate that there are fewer than 30 SecD and SecF molecules per cell.     

Sequence of the Escherichia coli pheST operon and identification of the himA gene.

The complete nucleotide sequence of the Escherichia coli pheST operon coding for the two subunits of phenylalanyl-tRNA synthetase (an alpha 2 beta 2-type enzyme) has been determined. Another open reading frame (prp) was revealed downstream from pheT which was identified as himA, the gene for the alpha subunit of the integration host factor.     

Kinetic modeling of ace operon genetic regulation in Escherichia coli

A family of kinetic models has been developed that takes into account available experimental information on the regulation of ace operon expression in Escherichia coli. This has allowed us to study and analyze possible versions of regulation of the ace operon and to test their possibilities. Based on literature analysis, we found that there is an ambiguity of properties of IclR (main repressor of ace operon). The main aspect of this ambiguity are two different forms of IclR purified from E. coli K strain and different coeffector sets for IclR purified from E. coli K and B strains. It has been shown that the full-length form of IclR is physiologically relevant and that IclR truncation is a result of purification of the protein from E. coli K strains. We also found that the IclR protein purified from E. coli B strain carries two coeffector binding sites. Using model-developed levels of steady state aceBAK expression against physiological ranges of coeffectors, concentration has been predicted.     

Reconstitution of the melibiose carrier of Escherichia coli

Different conditions were studied for optimal solubilization and reconstitution of the melibiose carrier of Escherichia coli. Several alpha- and beta-galactosides, known to be substrates for the melibiose carrier, were found to inhibit [3H]-melibiose uptake by proteoliposomes. In the presence of 10 mM Na+ the Km for melibiose counterflow was 0.42 mM. Melibiose and raffinose were good substrates for counterflow, while thiomethyl-beta-galactoside and p-nitrophenyl-alpha-galactoside were accumulated very poorly. Although the latter two sugars are known to be substrates for the carrier, they showed a very rapid rate of passive diffusion across the liposome membrane. The proton ionophore carbonylcyanidechlorophenylhydrazone had no effect on uptake, suggesting that a proton motive force is not essential for the counterflow phenomenon.     

Molecular cloning and characterization of the purE operon of Escherichia coli

The purE operon of Escherichia coli has been cloned and localized to a 1.7-kb HpaI fragment. The operon has been further characterized by subcloning into the lac fusion vector, pMC1403, and by the construction of BAL 31-generated deletions. The purE regulation region has been identified by assay of beta-galactosidase produced by pur-lac fusion plasmids and by RNA polymerase binding to end-labelled restriction fragments. Two purE promoters have been identified; one strong that is regulated by purines, the other weaker which is not regulated. The latter may be internal to the purE1 structural gene.     

Regulatory mechanism of the tryptophan operon in Escherichia coli: possible interaction between trpR and trpS gene products

Summary The trpS5 mutation (a mutation in the structural gene for tryptophanyl-tRNA synthetase (TRSase) in E. coli), when present in the genetic background of strain KY913 (HfrH), results in the failure to grow at high temperature (42° C) in a complete medium. The rel (RC relaxed) marker present in this strain was found to be partly responsible for this temperature sensitivity. TRSase in such a strain was rapidly inactivated during growth at 42° C in rich media, but not in minimal media or in the presence of chloramphenicol. A partial derepression of anthranilate synthetase formation took place in the presence of excess tryptophan at growth-restricting temperatures. When some of the trpR mutations (including amber mutations) were combined with trpS5, the resulting double mutants (trpR trpS5) were temperature-insensitive, and TRSase was not inactivated at high temperature, in contrast to the trpR ⁺trpS5 strain. This effect of trpR mutations on temperature sensivity was shown not to be a secondary consequence of the constitutive expression of the trp operon. These findings suggest that the trpR ⁺ product interacts with the TRSase of the trpS5 mutant so as to bring about the growth-dependent inactivation of the enzyme. Furthermore, a special class of trpR mutants was obtained whose constitutivity with respect to the trp operon is manifested only in strains carrying trpS5 (but not trpS ⁺) grown at high temperatures. It is proposed that TRSase participates in repression trrough direct interaction with the product of the trpR gene.