Effect of metabolic inhibitors on entry of exogenous deoxyribonucleic acid into Ca2+-treated Escherichia coli cells

The addition of various metabolic inhibitors (uncouplers, cyanide, arsenate, ionophores) separately or together (for example, arsenate and an uncoupler) or even harsher methods of energy depletion did not prevent bacteriophage T5 from injecting its first-step-transfer DNA (a DNA segment 3 ,um long) into the cytoplasm of host cells. The same indifference to metabolic energy was observed if first-step-transfer DNA was decapsidated and uncoiled before injection, thus precluding any energetic help from the phage capsid or from some tension stored in DNA tightly packed in the head. Penetration of the second-step-transfer DNA across the cytoplasmic membrane was studied by determining injection of superinfecting T5 A2-amber phages into Sup-bacteria containing proteins Al and A2 previously encoded by the first-step-transfer DNA of a primary wild-type phage. The addition of various metabolic inhibitors after synthesis of proteins Al and A2 but before superinfection did not prevent this penetration of second-steptransfer DNA. Thus, we conclude that traversal of the cytoplasmic membrane by the entire T5 DNA (a molecule 34 ,m long) must occur by diffusion through protein channels.

Section: Discussionmentioning

confidence: 93%

mentioning

confidence: 99%

Host cell metabolic energy is not required for injection of bacteriophage T5 DNA

Maltouf¹,

Labedan²

1983

“…Since gram-negative cells are surrounded by an outer and an inner membrane, much effort has been expended to develop conditions for making these membranes permeable to such large molecules (21,22,34). The best-known example is transformation, during which gram-negative bacteria take up DNA by passive diffiision after Ca2+ treatment combined with a temperature shift (6,22,30). Until recently, Ca2+ treatment was only used for DNA import.…”

mentioning

confidence: 99%

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

Brass¹,

Ehmann²,

Bukau³

1983

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°C and decreased sharply with increasing temperature. The time necessary for optimal reconstitution at 0°C and 250 mM Ca was about 1 min. The competence for reconstitution was highest in exponentially growing cultures with cell densities up to 1 x 109/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 puM). Vmax at saturating maltose-binding protein concentration was 125 pmol per min per 7.5 x 107 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 histidinebinding 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.

“…The same individual cells preferentially took up a second plasmid (1,13). The amount of DNA introduced into the periplasm was much higher than that found in the cytoplasm (29).…”

mentioning

confidence: 90%

Ca2+-induced permeabilization of the Escherichia coli outer membrane: comparison of transformation and reconstitution of binding-protein-dependent transport

Bukau¹,

Brass²,

Boos³

1985

Ca2+ treatment renders the outer membrane of Escherichia coli reversibly permeable for macromolecules. We investigated whether Ca2+-induced uptake of exogenous protein into the periplasm occurs by mechanisms similar to Ca2+-induced uptake of DNA into the cytoplasm during transformation. Protein import through the outer membrane was monitored by measuring reconstitution of maltose transport after the addition of shock fluid containing maltose-binding protein. DNA import through the outer and inner membrane was measured by determining the efficiency of transformation with plasmid DNA. Both processes were stimulated by Preparation of shock fluid. Shock fluid of maltose-induced cells of strain poplO80 [HfrG6 lamB102(Am) metA trpE(Am) galE galY] (18), containing between 30 and 50% of total protein as MBP, was prepared by osmotic shock by the 61 on August 5, 2020 by guest