Nature of transforming deoxyribonucleic acid in calcium-treated Escherichia coli.

A study of the reactivation of ultraviolet-irradiated plasmid and phage deoxyribonucleic acid molecules after transformation into Escherichia coli strains indicated that, when double-stranded deoxyribonucleic acid was used as the donor species, it was taken up without conversion to the single-standed form.     

Sensitivity of Escherichia coli to viral nucleic acid. VI. Capacity of dna mutants and DNA polymerase-less mutants for multiplication of phiA and phiX 174

Summary Seven groups of dna mutants were tested for the capacity to support the growth of A and X174, using a calcium-dependent transfection system. At the restrictive temperature, two groups of mutants, dnaA and dnaF, allowed the viral multiplication. Group B, C, D, E and G mutants were nonpermissive at 43°C to SS¹ DNA as well as to double-stranded RF molecule. Evidence showing the dispensability for the viral growth of DNA polymerase I and recombination function was also presented. Double mutant deficient in DNA polymerase I and II supported the growth of A sufficiently.     

Competence in Escherichia coli cells. V. Interaction of the competence factor with the cells]

Some trends of the interaction of the competence factor with the cells are described on the model of phage lambda transfection. It is shown that this process depends on the composition of the recipients growth media, the temperature regime in the interaction processand on the concentration of the competence factor and the recipient cells. The preliminary treatment of the recipient by EDTA (0.0001 M), CaCl2 (less than 0.02 M), MgCl2 (less than 0.01 M), and NaCl (less than 0.01 M) resulted in the increaze of their sensitivity to subsequent treatment by the factor. Data are obtained demonstrating that after the treatment of cells by the competence factor their sensitivity to decreased CaCl2 concentration is increasing.     

Transport of nucleic acid bases into Escherichia coli

The uptake and retention of purine bases and of uracil requires both a protonmotive force and intracellular conversion to nucleotides. Inhibition of uptake by arsenate does not imply that ATP is required for the transport process because arsenate caused a rapid fall in the level of 5-phosphoribosyl 1-pyrophosphate. A mutation defective in high-affinity adenine transport has been identified and is designated purP. This mutation has been found to lie in the neighbourhood of mtl. Competition experiments indicate that at least two other systems are used to transport guanine, xanthine and hypoxanthine.     

Reconstitution of maltose chemotaxis in Escherichia coli by addition of maltose-binding protein to calcium-treated cells of maltose regulon mutants.

Maltose chemotaxis was reconstituted in delta malE cells lacking maltose-binding protein (MBP). Purified MBP was introduced into intact cells during incubation with 250 mM CaCl2 in Tris-hydrochloride buffer at 0 degrees C. After removal of extracellular CaCl2 and MBP, chemotaxis was measured with tethered bacteria in a flow chamber or with free-swimming cells in a capillary assay. About 20% of tethered cells responded to 10(-4) M maltose; the mean response times were about half those of CaCl2-treated wild-type cells (100 s as opposed to 190 s). In capillary tests, the maltose response of reconstituted cells was between 15 and 40% of the aspartate response, about the same percentage as in wild-type cells. The best reconstitution was seen with 0.5 to 1 mM MBP in the reconstitution mixture, which is similar to the periplasmic MBP concentration estimated for maltose-induced wild-type cells. Strains containing large deletions of the malB region and malT mutants lacking the positive regulator gene of the mal regulon also could be reconstituted for maltose chemotaxis, showing that no product of the mal regulon other than MBP is essential for maltose chemotaxis.     

Sensitivity of Escherichia coli cells to seawater closely depends on their growth stage.

Sensitivity of Escherichia coli cells in seawater, considered in terms of culturability loss, was examined after different growth periods in a mineral medium supplemented with glucose (M9) at 37 degrees C under aerobic or anaerobic conditions. Their sensitivity varied considerably during the different growth phases and differed when cells were grown under aerobic or anaerobic conditions. Sensitivity of aerobic cells rapidly increased during the lag phase, then decreased during the exponential phase and became minimal during the stationary phase. Coliforms isolated from human faeces showed a similar sensitivity after incubation in wastewater at 37 degrees C for 3 h. The sensitivity phase was completely eliminated when cells were incubated with chloramphenicol. Variation of sensitivity in anaerobic cells according to their growth phase was comparable with that found for aerobic cells which had been left in seawater for a long period (6 d). However, for shorter periods in this medium (1-2 d), cells grown until the mid-exponential phase remained resistant to seawater. During the second half of the growth phase, they were as sensitive as aerobic cells at lag phase. Escherichia coli cells grown under anaerobic conditions, such as found in the intestine, progressively adapt to aerobic conditions after their transfer into aerated seawater and their sensitivity to seawater increases. On a practical level, these observations show that it is necessary to control accurately the age of cells before inoculation in seawater microcosms to conserve a comparative value in results. The importance of this factor is vital as all variations in sensitivity of cells to seawater according to their prior growth phase proved to be logarithmic functions of time.     

Endonuclease V of Escherichia coli.

A small endodeoxyribonuclease )2.3 S) that is active on single-stranded DNA has been extensively purified from Escherichia coli so as to be free of other known DNases. It has an alkaline pH optimum (9.5), requires Mg2+, and makes 3'-hydroxy and 5'-phosphate termini. The nuclease nicks duplex DNA, particularly if treated with OsO4, irradiated with ultraviolet light, or exposed to pH 5. The uracil-containing duplex DNA from the Bacillus subtilis phage PBS-2 is an especially good substrate; it is made acid-soluble by levels of the enzyme which fail to produce any acid-soluble material in other single-stranded or duplex DNAs. Neither RNA nor RNA-DNA hybrid are degraded by the enzyme. The enzyme specificity suggests that it might act at abnormal regions in DNA, so that its in vivo function could be to initiate an excision repair sequence. Its high activity on uracil-containing DNA could imply that the enzyme provides an alternative mechanism for excising uracil residues from DNA to the pathway utilizing uracil-DNA N-glycosidase. We suggest that this enzyme be designated as endonuclease V of E. coli.     

Reconstitution of maltose transport in Escherichia coli: conditions affecting import of maltose-binding protein into the periplasm of calcium-treated cells.

The reconstitution of active transport by the Ca2+ -induced import of exogenous binding protein was studied in detail in whole cells of a malE deletion mutant lacking the periplasmic maltose-binding protein. A linear increase in reconstitution efficiency was observed by increasing the Ca2+ - concentration in the reconstitution mixture up to 400 mM. A sharp pH optimum around pH 7.5 was measured for reconstitution. Reconstitution efficiency was highest at 0 degree C and decreased sharply with increasing temperature. The time necessary for optimal reconstitution at 0 degree C and 250 mM Ca2+ was about 1 min. The competence for reconstitution was highest in exponentially growing cultures with cell densities up to 1 X 10(9)/ml and declined when the cells entered the stationary-growth phase. The apparent Km for maltose uptake was the same as that of wild-type cells (1 to 2 microM). Vmax at saturating maltose-binding protein concentration was 125 pmol per min per 7.5 X 10(7) cells (30% of the wild-type activity). The concentration of maltose-binding protein required for half-maximal reconstitution was about 1 mM. The reconstitution procedure appears to be generally applicable. Thus, galactose transport in Escherichia coli could also be reconstituted by its respective binding protein. Maltose transport in E. coli was restored by maltose-binding protein isolated from Salmonella typhimurium. Finally, in S. typhimurium, histidine transport was reconstituted by the addition of shock fluid containing histidine-binding protein to a hisJ deletion mutant lacking histidine-binding protein. The method is fast and general enough to be used as a screening procedure to distinguish between transport mutants in which only the binding protein is affected and those in which additional transport components are affected.