Decreased conjugation efficiency by Fla-mutants of Escherichia coli K-12 bearing F-like plasmids.

Fla-, Pil-mutants of Escherichia coli K-12 were found to have decreased transfer efficiency of F-like resistance plasmids as compared to the parent strains. This was accompanied by decreased production of conjugation pili and decreased resistance level to some, but not all, of the antibiotics to which resistance was conferred. There was no reduction in pilus production or transfer efficiency in any of the mutants when the plasmid was F'gal. This host-mediated influence on conjugation pilus production is discussed with reference to a possible loss of cell envelope integrity which causes the simultaneous loss of all cellular appendage structures.     

A stochastic model of recombination during conjugation in Escherichia coli K-12.

A mathematical model is constructed in which recombination in E. coli K-12 is considered as a stochastic Markov process. The model takes into account the possibility of inclusion in the recombinant structure of the origin of the donor chromosome and makes it possible to correctly describe results of irradiation of the donor Hfr before crossing. Formulae are deduced for frequencies of unselected markers for cases when the counter-selected maternal marker lies in the distal or proximal region of the chromosome.     

Cross-resistance between bacteriophages and colicins in Escherichia coli K-12.

Cross-resistance between bacteriophages and colicins was studied using collections of bacteriophage- and colicin-resistant mutants of Escherichia coli K-12. No new examples were found of highly specific one-to-one cross-resistance of the type suggestive of common receptors. However, several groups of mutants showed tolerance to colicins and resistance to bacteriophages. Mutants known to be very defective in lipopolysaccharides composition were found to commonly show tolerance to certain colicins in addition to their bacteriophage resistance. Another group of mutants showed varying patterns of resistance to colicins E2, E3, K, L, A, S4, N, and X and bacteriophages E4, K2, K20, K21, K29, and H+. However, many bacteriophage-resistant mutants were fully colicin sensitive, and most colicin-resistant mutants were fully sensitive to bacteriophages.     

Iron uptake and iron limited growth of Escherichia coli K-12

Cells of Escherichia coli K-12 could grow aerobically at an iron concentration as low as 0.05 M without any of the known iron ionophores present. The growth rate increased between 0.05 and 2 M iron. Supplementation with the iron ligands ferrichrome and citrate resulted in optimal growth already at 0.05 M iron. Under certain conditions iron uptake preceded growth of cells by more than an hour. During logarithmic growth the rate of iron uptake matched the growth rate. The radioactive tracer method revealed a cellular iron content of 4 nmol/mg dry weight.After consumption of the iron in the medium cells continued to grow with high rate for 1–2 generations. The iron uptake activity was increased during iron starvation.     

Novel UGA-suppressors in Escherichia coli K-12

UGA-specific nonsense suppressors from Escherichia coli K-12 were isolated and characterized. One of them (Su+UGA-11) was identified as a mutant of the prfB gene for the peptide releasing factor RF2. It appears that in this strain, while peptide release at sites of UGA mutations is retarded, the UGA stop codon is read through even in the absence of a tRNA suppressor, exhibiting a novel type of passive nonsense suppression. Three suppressors (Su+UGA-12, -16 and -34) were capable of restoring the streptomycin sensitive phenotype in resistant bacteria (strAr). Because of their drug-related phenotype, these are possibly mutations in the components of the ribosomal machinery, particularly those concerned with peptide release at UGA nonsense codons. A tRNA suppressor was also obtained which was derived from the tRNA(Trp) gene. In this strain, a long region between rrnC (84.5 min) and rrnB (89.5 min) was duplicated and one of the duplicated genes of tRNA(Trp) was mutated to the suppressor. The mechanism of UGA-suppression is discussed in terms of translation termination at the nonsense codon in both active and passive fashions.     

Iron transport in Escherichia coli K-12

The study of iron uptake promoted by 2,3-dihydroxybenzoate (DHB) into Escherichia coli K-12 aroB mutants allowed some dissection of outer and cytoplasmic membrane functions. These strains are unable to produce the iron-transporting chelate enterochelin, unless fed with a precursor such as DHB. When added to the medium, enterochelin and its natural breakdown products, the linear dimer and trimer of 2,3-dihydroxybenzoylserine (DBS), efficiently transported iron via the feuB, tonB and fep gene products. Thus mutants in these genes were defective in transport of the above chelates. However, feuB and tonB mutants were able to take up iron when DHB was added to the medium. Thus DHB-promoted iron uptake bypassed two functions required for the transport of ferric-enterochelin from the medium. One of these functions, feuB, has been shown to be an outer membrane protein. In contrast to three other iron transport systems including ferric-enterochelin uptake, DHB-promoted iron uptake was little affected by the uncoupler 2,4-dinitrophenol. Dissipation of the energized state of the cytoplasmic membrane apparently only affects those iron transport systems which require an outer membrane protein. Since DHB-promoted iron uptake bypasses the feuB outer membrane protein and the tonB function, it is concluded that, in ferricenterochelin transport, the tonB gene may function in coupling the energized state of the cytoplasmic membrane to the protein-dependent outer membrane permeability. DHB-promoted iron uptake required the synthesis and enzymatic breakdown of enterochelin as judged by the effects of the entF and fesB mutations. A fep mutant was not only deficient in the transport of the ferric chelates of enterochelin and its breakdown products, but was also deficient in DHB-promoted iron uptake. A scheme is presented in which iron diffuses as DHB-complex through the outer membrane, and is subsequently captured by enterochelin or DBS dimer or trimer and translocated across the cytoplasmic membrane.List of Abbreviations DHB 2,3-dihydroxybenzoate - DBS 2,3-dihydroxybenzoylserine - NTA nitrilotriacetate - DNP 2,4-dinitrophenol     

Escherichia coli K-12 mutants with an increased efficiency of plasmid transformation

Escherichia coli K-12 mutants with an enhanced efficiency of plasmid transformation were obtained. In all the mutants, the efficiency of transfection with lambda vir phage DNA was changed, in comparison to the parent strain. However, these changes did not always correlate strictly with plasmid transformation alterations. For instance, two mutants with an increased plasmid transformation efficiency demonstrated 50-fold decrease in the level of transfection with lambda phage DNA. Polyacrylamide gel electrophoresis points to both quantitative and qualitative differences in protein composition of the mutant cell envelopes, as compared with the parent strain.     

Two proline porters in Escherichia coli K-12

Escherichia coli mutants defective at putP and putA lack proline transport via proline porter I and proline dehydrogenase activity, respectively. They retain a proline uptake system (proline porter II) that is induced during tryptophan-limited growth and are sensitive to the toxic L-proline analog, 3,4-dehydroproline. 3,4-Dehydroproline-resistant mutants derived from a putP putA mutant lack proline porter II. Auxotrophic derivatives derived from putP+ or putP bacteria can grow if provided with proline at low concentration (25 microM); those derived from the 3,4-dehydroproline-resistant mutants require high proline for growth (2.5 mM). We conclude that E. coli, like Salmonella typhimurium, possesses a second proline porter that is inactivated by mutations at the proP locus.     

L-Serine-sensitive mutants of Escherichia coli K-12

While attempting to isolate d-serine-sensitive mutants of Escherichia coli K-12, we found a class of mutants sensitive to low concentrations of l-serine (10 to 25 mug/ml).     

Ozone-induced damage of Escherichia coli K-12

Escherichia coli K-12 transformed with pACYC184 plasmid DNA was exposed to ozone (O₃) in aqueous solution. The damage to the membrane, protein, plasmid DNA, and cell survival were investigated. Cell viability was unaffected by short-term O₃ exposure (1–5 min) but membrane permeability was compromised as indicated by protein and nucleic acid leakage and lipid oxidation. The intracellular components, protein and DNA, remained intact. With longer durations of O₃ exposure (up to 30 min) cell viability decreased with a more significant increase in lipid oxidation and protein and nucleic acid leakage. The proteins leaking out were further oxidized by O₃. The total intracellular proteins run on sodium dodecyl sulfate/polyacrylamide gel electrophoresis, and plasmid DNA run on agarose gel, showed progressive degradation corresponding to the decrease in cell viability. The data indicate that membrane components are the primary targets of O₃ damage with subsequent reactions involving the intracellular components, protein and DNA. Received: 18 Apirl 1996 / Received revision: 26 July 1996 / Accepted: 5 August 1996