Modification of the interaction between<Emphasis Type="Italic">Escherichia coli</Emphasis> and bacteriophage in saline sediment

The effects of various combinations of light intensity, oxygen concentration, and CO₂ concentration on photosynthesis and growth in several algal types were studied. The results suggest the following. (1) Different algae show different responses to high oxygen concentrations and high light intensities. (2) Inhibition of photosynthesis (CO₂ fixation and growth), if seen, increases with increasing oxygen concentration and with increasing light intensity (at light intensities greater than saturation). (3) The inhibition of net photosynthesis observed cannot be attributed to high light intensity alone. (4) The inhibition cannot be attributed to increased rates of excretion of organic materials under conditions of high oxygen concentration and high light intensity. (5) Increased concentrations of CO₂ can decrease the effect of high oxygen and light in some algae. (6) The decrease in net photosynthesis observed is probably the result of photorespiration. (7) The effect of light intensity, oxygen concentration, or CO₂ concentration on algal photosynthesis should not be studied without considering the effect of the other factors. Some implications of these results, as related to primary productivity measurements, are also discussed.     

Interaction ofEscherichia coli O55 hybrids with bacteriophage lambda. A new type of host specificity betweenEscherichia coli O55 and urinaryEscherichia coli

Escherichia coli O55 hybrids able to adsorb the lambda phage were obtained by mating anEscherichia coli O55 recipient with anEscherichia coli K12HfrC donor. λ mutants, capable of forming plaques on these hybrids, were not isolated. A new type of host specificity betweenEscherichia coli O55 and urinaryEscherichia coli J was established. For efficient reduction of the phage plating ability more growth steps on the new strain are required in this type. Host specificities O55 and J proved to be different from K specificity.     

Construction of a shuttle vector betweenEscherichia coli andZymomonas anaerobia

Summary A 1.7-kb cryptic plasmid was isolated fromZymomonas anaerobia and used to construct a shuttle vector inserting useful parts of pUC9, pBR322, and pRK2501.Escherichia coli was employed to clone the new plasmid designated pSR12. The 7.7-kb plasmid pSR12 reisolated from the host cells could transform competent cells ofZ.anaerobia at 2×10^–7 frequency. This shuttle vector contains two antibiotic resistance markers, Kan^r and Tet^r, as well as restriction sites such as EcoRI, PstI, and XhoI, suitable for DNA recombinations.     

Metabolic differences betweenEscherichia coli cultures growing aerobically and anaerobically in the presence of fluoroacetate

The amount of citrate and pyruvate increased during the stationary phase of anEscherichia coli B culture growing in a synthetic medium, aerobically in the presence of glucose. In an anaerobic culture the amount of citrate was minute and did not rise during the stationary phase; the level of pyruvate in the stationary phase rose only slightly. Fluoroacetate blocked the growth of cells both aerobically and anaerobically. Cultures growing aerobically in the presence of fluoroaeetate displayed an increased accumulation of citrate as compared with the control. In anaerobic cultures citrate did not accumulate in the presence of inhibitory concentrations of fluoroacetate. In the presence of 2mm fluoroacetate cells grew aerobically somewhat more slowly at first, then inhibition ceased and finally the growth yield was greater than in the control. The obtained data indicate that citrate accumulated at first was partly utilized for growth under these conditions. The results are discussed from the point of view of differences in the metabolism of aerobically and anaerobically grown cells.     

Bacterial conjugation betweenEscherichia coli andPseudomonas spp. donor and recipient cells in soil

Summary Experiments conducted in microcosms containing loam soil samples inoculated with eitherE. coli orPseudomonas spp. donor and recipient cells showed that bacterial cells survived and conjugated over a 24-h incubation period.E. coli transconjugants were detected 6 h after donor and recipient strains were introduced into sterile soil samples. In non-sterile soil samples, transconjugants were detected between 8 and 24 h incubation.Pseudomonas transconjugants were recovered from sterile soil samples between 6 and 12 h after their introduction and as early as 2 h in non-sterile soil. The results show that genetic interactions occur in non-sterile soil in relatively short periods of time at relatively high transfer frequencies (10^–3 to 10^–4). Studies on genetic interactions in soil are becoming necessary in risk assessment/environmental impact studies prior to the release of genetically engineered or modified organisms into uncontained environments.     

The interaction of bacteriophage P2 B protein with Escherichia coli DnaB helicase

Bacteriophage P2 requires several host proteins for lytic replication, including helicase DnaB but not the helicase loader, DnaC. Some genetic studies have suggested that the loading is done by a phage-encoded protein, P2 B. However, a P2 minichromosome containing only the P2 initiator gene A and a marker gene can be established as a plasmid without requiring the P2 B gene. Here we demonstrate that P2 B associates with DnaB. This was done by using the yeast two-hybrid system in vivo and was confirmed in vitro, where (35)S-labeled P2 B bound specifically to DnaB adsorbed to Q Sepharose beads and monoclonal antibodies directed against the His-tagged P2 B protein were shown to coprecipitate the DnaB protein. Finally, P2 B was shown to stabilize the opening of a reporter origin, a reaction that is facilitated by the inactivation of DnaB. In this respect, P2 B was comparable to lambda P protein, which is known to be capable of binding and inactivating the helicase while acting as a helicase loader. Even though P2 B has little similarity to other known or predicted helicase loaders, we suggest that P2 B is required for efficient loading of DnaB and that this role, although dispensable for P2 plasmid replication, becomes essential for P2 lytic replication.     

Modification of RNA polymerase from Escherichia coli by pre-early gene products of bacteriophage T5.

Replication of early region deletion mutations in polyoma in the kidneys of mice was in most cases reduced by 10- to 10,000-fold, as compared with wild-type polyoma, and the mutants failed to produce the persistent infection observed with wild-type polyoma.     

Delineating the site of interaction on the pIII protein of filamentous bacteriophage fd with the F-pilus of Escherichia coli

The minor coat protein pIII at one end of the filamentous bacteriophage fd, mediates the infection of Escherichia coli cells displaying an F-pilus. pIII has three domains (D1, D2 and D3), terminating with a short hydrophobic segment at the C-terminal end. Domain D2 binds to the tip of F-pilus, which is followed by retraction of the pilus and penetration of the E. coli cell membrane, the latter involving an interaction between domain D1 and the TolA protein in the membrane. Surface residues on the D2 domain of pIII were replaced systematically with alanine. Mutant virions were screened for D2-pilus interaction in vivo by measuring the release of infectious virions from E. coli F(+) cells infected with the mutants. A competitive ELISA was developed to measure in vitro the ability of mutant phages to bind to purified pili. This allowed the identification of amino acid residues involved in binding to F and to EDP208 pili. These residues were found to cluster on the outer rim of the 3D structure of the D2 domain, unexpectedly identifying this as the F-pilus binding region on the pIII protein.     

Molecular interaction between bacteriophage and the gram-negative cell envelope

Conclusions Accumulation of molecular data on rceptor recognition by phages over the last years has provided some insight into the underlying molecular mechanisms. Some common motifs emerge. The receptor-recognizing areas of phage proteins seem to be confined to single regions on the polypeptide chains. The repeat structure of these regions, as found in T-even-type phages, seems to be specific for this group since it is not found in other phages such as lambda, T5, and BF23. The maximal number of surface exposed loops of receptor proteins recognized by the phages appears to be limited to four loops. The knowledge of X-ray structures and multiple alignment calculations of related receptor proteins will soon provide exact topological models of these proteins. These models will make possible the positioning of the receptor-binding proteins with respect to the surface-exposed loops involved in phage reception. The knowledge of the exact nature of mutations with new host-ranges will be of great value for such positioning.The TonB-dependent receptor proteins are a group of transport proteins which appear able to undergo considerable conformational changes not only during transport but also during phage adsorption. The isolation of receptor missense mutations impaired solely in phage reception should be very rewarding for TonB-dependent phages, since this could possibly lead to the identification of transiently surface-exposed loops of the receptor proteins.As for DNA uptake, the biochemical and biophysical characterization of pore-forming phage proteins should help elucidate the molecular mechanism of membrane penetration. Studies of this mechanism will be very much facilitated by the accessibilities of the corresponding genes to modern molecular biology techniques.Abbreviation aa amino acid(s)     

A mutant form of maltose-binding protein of Escherichia coli deficient in its interaction with the bacteriophage lambda receptor protein.

In one malE mutant known to be deficient in the transport of maltose and maltodextrins across the outer membrane, the altered MalE protein was shown to be defective in its interaction with the phage lambda receptor, or LamB protein, of the outer membrane.     

Determinants of DNA sequence divergence betweenEscherichia coli andSalmonella typhimurium: Codon usage,map position,and concerted evolution

Summary The nature and extent of DNA sequence divergence between homologous proteincoding genes fromEscherichia coli andSalmonella typhimurium have been examined. The degree of divergence varies greatly among genes at both synonymous (silent) and nonsynonymous sites. Much of the variation in silent substitution rates can be explained by natural selection on synonymous codon usage, varying in intensity with gene expression level. Silent substitution rates also vary significantly with chromosomal location, with genes nearoriC having lower divergence. Certain genes have been examined in more detail. In particular, the duplicate genes encoding elongation factor Tu,tufA andtufB, fromS. typhimurium have been compared to theirE. coli homologues. As expected these very highly expressed genes have high codon usage bias and have diverged very little between the two species. Interestingly, these genes, which are widely spaced on the bacterial chromosome, also appear to be undergoing concerted evolution, i.e., there has been exchange between the loci subsequent to the divergence of the two species.Presented at the NATO Advanced Research Workshop on Genome Organization and Evolution, held in Spetses, Greece, September 1990