No abstract

The previously described hybrid plasmid pC7 which carries lacI+ O+Δ(Z)Y+ A+ on a 12.3 × 106‐Mr DNA fragment [Teather et al. (1978) Mol. Gen. Genet. 159, 239–248] was partially digested with the restriction endonuclease EcoRI under conditions reducing the recognition sequence to ↓ d(A‐A‐T‐T) and ligated to the vector pBR322. lacY‐carrying inserts of various sizes (Mr, 1.5–4.7 × 106) were obtained. Hybrid plasmid pTE18 (2300‐base‐pair insert) carries part of the I (repressor) gene, the promotor‐operator region, part of the Z (β‐galactosidase) gene, the Y (lactose carrier) gene and part of the A (transacetylase) gene. Upon induction of pTE18‐harbouring strains the Y‐gene product is expressed at a nearly constant rate for several generations and accumulates to a level of 12–16% of the total cytoplasmic membrane protein. Integration into the membrane leads to active carrier as judged by binding and transport measurements.

The binding of galactosides to the lactose carrier of cytoplasmic membrane vesicles derived from Escherichia coli strains T185 or T206 which harbor plasmids bearing the lacy gene is measured and compared to the active transport of galactosides in vesicles or cells of the haploid strain ML308-225 in terms of the apparent affinity. Lactose and other galactosides bind only to a single site on the carrier protein, The affinity of lactose for this site is low (KO = 14 f 5 mM), while the half-saturation constant for active transport, KT, is 0.085 f 0.007 mM, approximately 160-fold smaller. Other galactosides exhibit smaller differences between KD and KT, the ratio of KD to KT ranging from 48 to 1. The turnover number of the carrier for lactose transport is 2.9 s-' in membrane vesicles and 48 s-I in EDTA-treated cells. Galactoside binding and transport parameters are independent of the bulk pH in the range 5.5-8.0. The binding of 6-Dgalactosyl 1 -thio-P-D-galactoside to the lactose carrier elicits the binding of less than 0.1 proton per galactoside. The fatty acid composition of the plasmid-harboring unsaturated fatty acid auxotroph strain T200Ela is manipulated by supplementation with elaidate, palmitelaidate, or oleate. The midpoint of the ordered -fluid transition of the membrane phospholipids lies at 32, 27, or 14 "C, respectively. Arrhenius plots of the rate of o-nitrophenyl /3-D-galactoside transport (in vivo) exhibit corresponding downward changes in slope at 34, 26, or 15 "C, respectively. In contrast to the effect upon transport, the lipid phase transition does not affect substrate binding to the carrier. Over the same temperature interval (40-1 4 "C), the dissociation constant, K,, for p-nitrophenyl

The OmpA protein is one of the major outer membrane proteins of Escherichia coli. Among other functions the protein serves as a receptor for several phages and increases the efficiency of F-mediated conjugation when present in recipient cells. TraT is an F-factor-coded outer membrane lipoprotein involved in surface exclusion, the mechanism by which E. coli strains carrying F-factors become poor recipients in conjugation.To determine a possible interaction of TraT with OmpA, the influence of TraT on phage binding to cells was measured. Because TraT inhibits inactivation of OmpA-specific phages it is suggested that TraT interacts directly with OmpA. Sequence homology of TraT with proteins 38, the phage proteins recognizing outer membrane proteins, supports this finding. A model of protein interactions is discussed. TraTprotein OmpA protein Protein interaction

Short tail fibres of T-even like phages are involved in host recognition. To determine the specificity of the fibres, the region containing gene 12 of phages T2, K3, and K3hx was cloned. The genes 11, 12, wac, and 13, coding for the baseplate outer wedge, short tail fibres, collar wishes, and a head completion component, respectively, were localized on the cloned fragments. Plasmid-encoded gene 12 could be expressed without helper phage. Efficient expression of gene 12 from T2 and K3hx made an extraction of protein 12 possible. Hybrid phages obtained by in vitro complementation, recombination analysis and protein 12 binding to host range mutant bacteria excluded a role of the short tail fibres from T2, K3 or K3hx in the recognition of outer membrane proteins. Binding patterns of protein 12 to different Escherichia coli lipopolysaccharide mutants and inhibition of binding of protein 12 by a monoclonal antibody against the core region of E. coli K12 lipopolysaccharide suggested that heptose residues are necessary for efficient binding. The binding site of the same monoclonal antibody is different from the short tail fibre binding site in an E. coli B strain suggesting different binding specificities of protein 12. Thus, the ability of different bacterial strains to inactivate phage could be related to differences in the binding specificity of the short tail fibres for the lipopolysaccharides of these bacteria.