Control regions within the argECBH gene cluster of Escherichia coli K12

Summary In Escherichia coli K12, four of the nine structural genes involved in the biosynthesis of arginine (argE, C, B and H) form a tight cluster within which a clockwise-polarized unit of expression (argCBH) had previously been identified. From a mutant carrying an argCB deletion that greatly lowers the rate of expression of argE but falls short of known argE markers, we have isolated several derivatives in which the expression of argE is partly restored. In about a third of these strains repression of both E and H enzymes by arginine is almost abolished. The mutations responsible appear to be cis-dominant and to map to the right of argE, probably between argE and C. One mutant in which control of argE alone is affected has also been found; it is shown to carry a duplication of argE in addition to the argCB deletion of the parental strain. We discuss the hypothesis that argE and argCBH form two operons transcribed in opposite directions from an internal promoter-operator complex.It is also suggested that a secondary promoter exists at or near the argB-H boundary.     

An Escherichia coli enterobactin cluster gene with sequence homology to trpE and pabB

Abstract Oxaloacetate decarboxylase from Klebsiella pneumoniae is a membrane bound sodium-pumping biotin enzyme. In electron microscopic samples, the enzyme particle appeared rod-like, with a length of about 12.9 nm and a width of about 7.4 nm, and with two submasses. Based on electron microscopic comparison of full-size enzyme molecules and free α-subunits, it is concluded that oxaloacetate decarboxylase contains only one α-subunit per enzyme particle. The α-subunit of the enzyme revealed a subdivision into two domains of different sizes forming a 'cleft'. Electron microscopic affinity labeling with avidin demonstrated that the biotin prosthetic group present on the α-subunit is located in this cleft, close to the complex formed by the β- and γ-subunits. The fact that 'pairs' but no higher specific aggregates could be observed after incubation with avidin, also indicates that only one copy of the α-subunit is present in an oxaloacetate decarboxylase particle.     

Stereospecificity of the ferric enterobactin receptor of Escherichia coli K-12

Synthetic enterobactin and enantioenterobactin (D-seryl enterobactin) have been examined for the ability to transport iron in Escherichia coli. Failure of the unnatural, D-serine-derived material to support growth of E. coli mutants indicates outer membrane receptor specificity for the naturally occurring complex having an L-seryl backbone and the delta-cis configuration of the Fe(III).catecholate center. Enantioenterobactin was markedly less effective in protecting cells against colicin B compared to synthetic or natural enterobactin.     

Surface topology of the Escherichia coli K-12 ferric enterobactin receptor.

Monoclonal antibodies (MAb) were raised to the Escherichia coli K-12 ferric enterobactin receptor, FepA, and used to identify regions of the polypeptide that are involved in interaction with its ligands ferric enterobactin and colicins B and D. A total of 11 distinct FepA epitopes were identified. The locations of these epitopes within the primary sequence of FepA were mapped by screening MAb against a library of FepA::PhoA fusion proteins, a FepA deletion mutant, and proteolytically modified FepA. These experiments localized the 11 epitopes to seven different regions within the FepA polypeptide, including residues 2 to 24, 27 to 37, 100 to 178, 204 to 227, 258 to 290, 290 to 339, and 382 to 400 of the mature protein. Cell surface-exposed epitopes of FepA were identified and discriminated by cytofluorimetry and by the ability of MAb that recognize them to block the interaction of FepA with its ligands. Seven surface epitopes were defined, including one each in regions 27 to 37, 204 to 227, and 258 to 290 and two each in regions 290 to 339 and 382 to 400. One of these, within region 290 to 339, was recognized by MAb in bacteria containing intact (rfa+) lipopolysaccharide (LPS); all other surface epitopes were susceptible to MAb binding only in a strain containing a truncated (rfaD) LPS core, suggesting that they are physically shielded by E. coli K-12 LPS core sugars. Antibody binding to FepA surface epitopes within region 290 to 339 or 382 to 400 inhibited killing by colicin B or D and the uptake of ferric enterobactin. In addition to the FepA-specific MAb, antibodies that recognized other outer membrane components, including Cir, OmpA, TonA, and LPS, were identified. Immunochemical and biochemical characterization of the surface structures of FepA and analysis of its hydrophobicity and amphilicity were used to generate a model of the ferric enterobactin receptor's transmembrane strands, surface peptides, and ligand-binding domains.     

Purification of the NusB gene product of Escherichia coli K12

Summary The nucleotide sequence of the entire nusB gene of Escherichia coli has recently been determined and the amino acid sequence of its product deduced (Ishii et al. 1984; Swindle et al. 1984). The NusB protein was purified by chromatography on Sephadex G-100, phosphocellulose and hydroxylapatite. Purification of the protein was monitored using ¹⁴C-labelled NusB protein, which was synthesized in a maxicell containing an nusB plasmid as a marker. The final product, which was at least 95% pure as judged by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulphate, had a molecular weight of about 16,000 and an isoelectric point of about 7.3. Analytical data on the amino acid composition of the purified protein agreed with that deduced from the DNA sequence and indicated that this protein was indeed the product of the nusB gene.Abbreviations SDS sodium dodecyl sulphate - kDa kilodaltons - bp base pair(s) - kbp kilobase pair(s)     

Characterization of the Escherichia coli K12 gltS glutamate permease gene

Summary The gltS gene is known to encode a sodium-dependent, glutamate-specific permease. We have localized the Escherichia coli K12 gltS gene with respect to the spoT gene, sequenced it, and recombined a null insertion-deletion allele into the chromosome without loss of viability. The gltS null allele gives a Glt^– phenotype, i.e. it abolishes the ability of a gltC ^c host to grow on glutamate as sole carbon and nitrogen source and also confers -methylglutamate resistance. A multicopy plasmid expressing the gltS gene can reverse the Glt^– phenotype of gltS ^– or wild-type strains while other plasmids show host-dependent complementation patterns. Induction of gltS gene overexpression under control of isopropyl-d-thiogalactoside (IPTG)-inducible promoters severely inhibits growth. The G1tS protein is deduced to be a 42425 dalton hydrophobic protein with 2 sets of 5 possible integral protein domains, each flanking a central hydrophilic, flexible region.