Multiplicity of oligopeptide transport systems in Escherichia coli.

The ability of Escherichia coli K-12 4212 to utilize a variety of oligopeptides as sources of required amino acids was examined. Triornithine-resistant mutants of this strain were oligopeptide permease deficient (Opp-) as judged by their inability to utilize (Lys)3 and (Lys)4 as sources of lysine and their resistance to the toxic tripeptide (Val)3. These same mutants were able to grow when Met-Met-Met, Met-Gly-Met, Met-Gly-Gly, Gly-Met-Gly, Gly-Gly-Met, Gly-Met-Met, Met-Met-Gly, or Leu-Leu-Leu were supplied in place of the requisite amino acid. The system mediating the uptake of these peptides, herein designated Opr I, was not able to transport N-alpha-acetylated peptides, nor the tetrapeptides Met-Gly-Met-Met, Met-Met-Gly-Met, or Met-Met-Met-Gly. Competition experiments indicated that trimethionine and trileucine enter E. coli K-12 via either Opp or Opr I. Analogous results were found using the methionine, leucine-requiring auxotroph E. coli B163. It appears that more than one oligopeptide transport system exists in E. coli and that the system mediating peptide uptake is complex.     

Crystal structure of the dipeptide binding protein from Escherichia coli involved in active transport and chemotaxis.

The Escherichia coli periplasmic dipeptide binding protein functions in both peptide transport and taxis toward peptides. The structure of the dipeptide binding protein in complex with Gly-Leu (glycyl-L-leucine) has been determined at 3.2 A resolution. The binding site for dipeptides is designed to recognize the ligand's backbone while providing space to accommodate a variety of side chains. Some repositioning of protein side chains lining the binding site must occur when the dipeptide's second residue is larger than leucine. The protein's fold is very similar to that of the Salmonella typhimurium oligopeptide binding protein, and a comparison of the structures reveals the structural basis for the dipeptide binding protein's preference for shorter peptides.     

The gene for a small stable RNA (10Sa RNA) of Escherichia coli

A gene that codes for a small stable RNA (362 nucleotides) has been sequenced. It is a monocistronic gene, with its own promoter and terminator. It produces a precursor that is about 100 nucleotides longer than the mature RNA with all the extra nucleotides at the 3' end. The gene contains an open reading frame that corresponds to a small protein 25 amino acids long.     

Genetic analysis of Escherichia coli oligopeptide transport mutants.

The composition of the outer membrane channels formed by the OmpF and OmpC porins is important in peptide permeation, and elimination of these proteins from the Escherichia coli outer membrane results in a cell in which the primary means for peptide permeation through this cell structure has been lost. E. coli peptide transport mutants which harbor defects in genes other than the ompF/ompC genes have been isolated on the basis of their resistance to toxic tripeptides. The genetic defects carried by these oligopeptide permease-negative (Opp-) strains were found to map in two distinct chromosomal locations. One opp locus was trp linked and mapped to the interval between att phi 80 and galU. Complementation studies with F'123 opp derivatives indicated that this peptide transport locus resembles that characterized in Salmonella typhimurium as a tetracistronic operon (B. G. Hogarth and C. F. Higgins, J. Bacteriol. 153:1548-1551, 1983). The second opp locus, which we have designated oppE, was mapped to the interval between dnaC and hsd at 98.5 min on the E. coli chromosome. The differences in peptide utilization, sensitivity and resistance to toxic peptides, and the L-[U-14C]alanyl-L-alanyl-L-alanine transport properties observed with these Opp-E. coli strains demonstrated that the transport systems encoded by the trp-linked opp genes and by the oppE gene(s) have different substrate preferences. Mutants harboring defects in both peptide transport loci defined in this study would not grow on nutritional peptides except for tri-L-methionine, were totally resistant to toxic peptides, and would not actively transport L-[U-14C]alanyl-L-alanyl-L-alanine.     

Photoaffinity inhibition of dipeptide transport in Escherichia coli.

A dipeptide containing a nitrene precursor, glycyl-4-azido-2-nitro-L-phenylalanine, has been synthesized. This compound is a photoaffinity inhibitor of dipeptide transport in E. coli. In the dark, the dipeptide is a reversible inhibitor of glycylglycine uptake by live E. coli W cells. The 14C-labeled compound is a substrate for the transport system, with a Km of 7 micrometer and V max of 5 x 10(3) molecules cell-1 s-1 (compare 9 micrometer and 1 x 10(4) molecules cell-1 s-1, respectively, for the transport of glycylglycine under the same conditions). When intact E. coli cells are photolyzed at approximately 350 nm in the presence of the photolabile dipeptide, their ability to transport either glycylglycine or unphotolyzed glycyl-4-azido-2-nitro-L-phenylalanine is irreversibly inhibited, but their ability to transport arginine is unaffected. The presence of glycylglycine in the medium during photolysis protects the cells against the light-dependent inactivation of dipeptide transport.     

The Escherichia coli dsbC (xprA) gene encodes a periplasmic protein involved in disulfide bond formation.

We have identified and functionally characterized a new Escherichia coli gene, dsbC, whose product is involved in disulfide bond formation in the periplasmic space. It corresponds to a previously sequenced open reading frame mapping upstream of recJ with no previously assigned function. Null mutations in dsbC were obtained using a screen for dithiothreitol (DTT)-sensitive mutants and were shown to result in the accumulation of reduced forms of a variety of disulfide bond-containing periplasmic proteins. This defect could be rescued by the addition of either oxidized DTT or cystine or by multicopy expression of dsbA, a known periplasmic disulfide oxidase. The DsbC protein is synthesized as a precursor form of 25.5 kDa which is processed to a 23.3 kDa mature species located in the periplasmic space. The DsbC protein was overexpressed, purified to homogeneity and shown to catalyse the reduction of insulin in a DTT-dependent manner at levels comparable with those of purified DsbA. The replacement of either cysteine residue of the predicted active site, F-(X4)-C-G-Y-C, completely inactivates DsbC protein function. We have further shown that in vivo overexpression of DsbC can functionally substitute for a loss of DsbA function. Taken together, all of our results demonstrate that DsbC acts in vivo as a disulfide oxidase.     

The birA gene of Escherichia coli encodes a biotin holoenzyme synthetase

Mutations in the birA gene of Escherichia coli cause defects in biotin operon repression, biotin uptake and retention of intracellular biotin (Campbell et al., 1972: Barker &, Campbell, 1980). We report here that the birA gene encodes the major biotin-fixing enzyme of this organism, the acetyl-CoA carboxylase biotin holoenzyme synthetase (EC 6.3.4.15). Unlike the situation in wild-type E. coli extracts, measurements of labeled biotin incorporation into protein in sonicated extracts reveal no in vitro activity. Three different mutants exhibit altered holoenzyme synthetase activity, including one clear instance of a thermolabile activity specified by birA361.Amplification of birA gene expression by infection of cells with a λ phage bearing an EcoRI fragment of the E. coli chromosome which includes the gene results in a 20- to 40-fold increase in specific activity. When the λbirA phage carries the birA85 mutation, no activity increase is observed. Infection of cells with a λbirA361 transducing phage results in a 20- to 40-fold increase in temperature-sensitive activity. We have purified the activity specified by birA361 approximately 1000-fold and have shown that the purified enzyme is more thermolabile than similarly purified wild-type enzyme.Measurements of holoenzyme synthetase in extracts and biotin uptake by whole cells indicate that certain mutations located at the same chromosomal position as birA mutations but initially characterized as defective only in bio repression are also deficient in biotin holoenzyme synthetase and biotin uptake. This result indicates that all mutations at this location affect the same enzyme, and we have redesignated these “bioR” mutations as birA. Results of complementation analysis of birA mutations and biochemical characterization of the gene and its product, presented in the accompanying paper, support the view that the birA product functions both as the bio repressor and biotin holoenzyme synthetase.     

Phaseolotoxin transport in Escherichia coli and Salmonella typhimurium via the oligopeptide permease.

Phaseolotoxin [(N delta-phosphosulfamyl)ornithylalanylhomoarginine], a phytotoxic tripeptide produced by Pseudomonas syringae pv. phaseolicola that inhibits ornithine carbamoyltransferase, is transported into Escherichia coli and Salmonella typhimurium via the oligopeptide transport system (Opp). Mutants defective in oligopeptide permease (Opp-) were resistant to phaseolotoxin. Spontaneous phaseolotoxin-resistant mutants (Toxr) lacked the Opp function as evidenced by their cross-resistance to triornithine and failure to utilize glycylhistidylglycine as a source of histidine. Growth inhibition by phaseolotoxin was prevented by peptides known to be transported via the Opp system and by treatment of the toxin with L-aminopeptidase. In both E. coli and S. typhimurium, Toxr mutations were cotransducible with trp, suggesting that the opp locus occupies similar positions in genetic maps of these bacteria.