D-ribose metabolism in Escherichia coli K-12: genetics, regulation, and transport.

We have isolated mutants defective in high-affinity D-ribose transport. The mutations map in rbsT or rbsB , the structural gene for ribose binding protein. rbsT consists of at least one gene coding for a protein required for high-affinity transport. The high-affinity transport-defective mutants were able to utilize D-ribose, indicating that at least a second, low-affinity transport system for D-ribose is present in Escherichia coli K-12. rbsT and rbsB are located at min 84 on the E. coli genetic map and, together with rbsK , the gene coding for ribokinase , constitute an rbs operon. The order of genes is rbsP /O rbsT rbsB rbsK . The rbs operon is subject to negative control by the product of the rbsR gene. rbsR is located distal to the rbs operon and appears to form a separate transcriptional unit.

Relation of therapeutic response to nifedipine to coronary anatomy and motion of S-T segment during unstable angina pectoris.

Of 77 patients hospitalized for unstable angina pectoris and failure of oral, dermal, or intravenous nitrates and/or beta blockade, 81 percent with negligible or single-vessel disease and 55 percent with two- or three-vessel disease showed response (p less than 0.05) to nifedipine therapy. Patients with either S-T elevation or no change during pain responded better (31 of 45) than those with any S-T depression (16 of 32; p less than 0.05). Patients with negligible or single-vessel disease had a higher prevalence of S-T elevation (13 of 16) than patients with two- or three-vessel disease (15 of 31; p = 0.004). S-T motion did not predict response in patients with two- or three-vessel disease, but did predict response in patients with negligible or single-vessel disease. On follow-up study at 9 +/- 8 (range one to 33) months, 39 of 42 who had shown response were free from pain. Three died from infarction without unstable angina. (range one to 33) months, 39 of 42 who had shown response were free from pain. Three died from infarction without unstable angina. Five who showed response had elective bypass surgery. The addition of nifedipine abolished or reduced pain episodes by more than 50 percent in 61 percent of patients with refractory unstable angina pectoris. Patients with negligible or single-vessel disease with S-T elevation benefit most. In patients with two- or three-vessel disease, the type of S-T motion did not predict response. Follow-up of all those with response indicated sustained amelioration by nifedipine therapy. Failure of nifedipine therapy should not be accepted until a dose of 120 mg per day has been achieved, or until intolerable side effects appear.

Positions of early nonsense and deletion mutations in lacZ.

The positions of three Escherichia coli lacZ operator-proximal nonsense mutations and one deletion mutation have been determined. The nonsense mutations were suppressed with supF, resulting in the production of active beta-galactosidase by each strain. Amino acid sequencing identified the positions of the tyrosine residues inserted by supF, and thereby established that nonsense mutations lacZ2, lacZ2246, and lacZU131 are at sites corresponding to amino acids 23, 36, and 41 of beta-galactosidase, respectively. The deletion mutant, lacZM112, produced a dimeric beta-galactosidase protein missing amino acid residues 23 through 31 of the native enzyme.

Sites within gene lacZ of Escherichia coli for formation of active hybrid beta-galactosidase molecules.

We describe the genetic analysis of 21 Escherichia coli strains in which the amino-terminal sequence of beta-galactosidase has been removed and replaced by an amino-terminal sequence from one or another of the proteins involved in maltose transport. Genetic mapping of the lacZ end of these fused genes indicates that only those fusions in which fewer than 41 amino acids are removed from the amino-terminal sequence of beta-galactosidase result in enzymatically active molecules. Within the region between amino acid 17 and amino acid 41 there are at least four or five sites where enzymatically active hybrid proteins can be formed.

Use of gene fusion to study secretion of maltose-binding protein into Escherichia coli periplasm.

We have employed the technique of gene fusion to fuse the LacZ gene encoding the cytoplasmic enzyme beta-galactosidase with the malE gene encoding the periplasmic maltose binding protein (MBP). Strains were obtained which synthesize malE-lacZ hybrid proteins of various sizes. These proteins have, at their amino terminus, a portion of the MBP and at their carboxyl terminus, enzymatically active beta-galactosidase. When the hybrid protein includes only a small, amino-terminal portion of the MBP, the hybrid protein residues in the cytoplasm. When the hybrid protein contains enough of the MBP to include an intact MBP signal sequence, a significant portion of the hybrid protein is found in the cytoplasmic membrane, suggesting that secretion of the hybrid protein has been initiated. However, in no case is the hybrid protein secreted into the periplasm, even when the hybrid protein includes almost the entire MBP. In the latter case, the synthesis and attempted export of the hybrid protein interferes with the export of at least certain normal envelope proteins, which accumulate in the cell in their precursor forms, and the cell dies. These results suggest that a number of envelope proteins may be exported at a common site, and that there are only a limited number of such sites. Also, these results indicate that it is not sufficient to simply attach an amino-terminal signal sequence to a polypeptide to assure its export.

Structure of the malB region in Escherichia coli K12. II. Genetic map of the malE,F,G operon.

Starting with a strain containing a malK-lacZ fusion, a series of lambda plaque-forming phages which carry varying amounts of the malE,F operon have been isolated. We have used these phages to construct a deletion map of the malE,F operon. The construction of this deletion map has led to the identification of a new gene, malG. The malG gene is located distal to malF. The malG gene product is a protein required for the active transport of maltose and maltodextrins.

Position of the mutation in beta-galactosidase ochre mutant U118.

The Escherichia coli lacZ ochre mutant strain U118 was converted to an amber mutant and suppressed with supF, which inserts tyrosine. Enzymatically active beta-galactosidase was isolated. It contained tyrosine at residue number 17 instead of glutamic acid as in wild type.

Conversion of beta-galactosidase to a membrane-bound state by gene fusion.

We have isolated a series of strains in which the lacZ gene has been fused to one of the maltose operons, such that the synthesis of beta-galactosidase (beta-D-galactoside galactohydrolase; EC 3.2.1.23) is inducible by maltose. The most frequent event that generates such fusions results in strains in which an intact lacZ gene has become a part of the malE,F operon. By using a special selection procedure, we have detected much rarer fusion events resulting in an altered beta-galactosidase molecule. In these strains, we presume that there is a hybrid protein molecule produced, comprised of an NH2-terminal amino acid sequence from a maltose transport protein (malF) and a COOH-terminal amino acid sequence from beta-galactosidase. The hybrid protein, which still retains some beta-galactosidase activity, is found in the cytoplasmic membrane. These results provide information on the component of the malF gene essential for incorporation of its product into the membrane.

Transposition of the lac region to the gal region of the Escherichia coli chromosome: isolation of lambda-lac transducing bacteriophages.

Defective and plaque-forming lambdalac transducing phages have been isolated from bacterial strains in which the lactose operon has been genetically transposed to the galactose locus.

An adenosine 3':5'-cyclic monophosphate-binding protein that acts on the transcription process.

An assay system for the in vitro transcription of the lac operon is described. A protein factor (CAP) and cyclic AMP, which are essential for the lac expression in vivo, also stimulate lac transcription in vitro.

Fusions of the lac and trp Regions of the Escherichia coli Chromosome.

Two classes of strains were studied in which the lac operon is transposed to a chromosomal site close to the tonB and trp loci. The two classes differ in the orientation of the lac region on the chromosome. In both types of strains, tonB mutants were selected in which deletions removing the tonB locus also caused a fusion of the lac and trp regions. The study of the properties of such fusion strains provides information on the control of both the lac and trp operons.