Sensitivity of Escherichia coli to Viral Nucleic Acid, XII. Ca²⁺-or Ba²⁺-Facilitated Transfection of Cell Envelope Mutants

Abstract

Viral DNA, Transfection, Envelope Mutants, Escherichia coliUsing various envelope mutants of Escherichia coli, the relationship between cell surface struc ture and the Ca2*-or Ba2+-dependent competence for transfection was investigated. In contrast with rough strains, smooth bacteria treated with Ca2+ or Ba2+ were incompetent for the trans fection by 0 A RF. In E. coli K12 D21 derivatives, Ca2+-dependent competence remarkably in creased by Ip sA l mutation and the highest level of competence was attained by further deficiency in glucose units of the LPS. Upon treatment with BaCl2 , strain D21 and its Ip s A l mutant became highly competent for 0 A RF. The effect of Ba2* was, however, feeble for Ip s A l mutants further deficient in heptose units and/or glucose units. Among different LPS mutants of E. coli B, variation of the Ca2*-or Ba2+-dependent competence was relatively small and even the competence of strain BB12, whose LPS core contained only two KDO units, was nearly equal to that of wild type bacteria. However, the level of cellular competence induced by Ba2+ was not allways parallel to that induced by Ca2+. In mutants deficient in outer membrane protein I, either Ca2*-or Ba2+-dependent competence increased several-fold, whereas in mutants devoid of outer membrane II*, the com petence decreased considerably. Unlike nucleoside transport, the uptake of DNA was not affected by tsx mutation.

Transformation of Salmonella typhimurium with plasmid DNA: differences between rough and smooth strains

“…Transformation occurs in two stages: uptake, which includes both binding of the DNA to the cell surface and its transport across the cell envelope, and establishment. The particular surface components involved in DNA binding and the means by which transport is effected are currently unknown, although outer membrane proteins involved in cobalamin transport (9) and the ompA (35) protein have been implicated. The structure of lipopolysaccharide (LPS), a major surface component of the gram-negative cell, affects the transfection of both Escherichia coli (34) and Salmonella typhimurium (5).…”

“…The very high transformability of galE or rfaF mutants may be due to increased exposure of sites responsible for DNA binding. The most probable DNA binding sites are the outer membrane proteins (9,35,36,38). However, the lack of a correlation between LPS core chain length and transformability, especially among the Rc, Rd1, and Rd2 chemotypes, argues strongly against simple steric hindrance as the limiting determinant.…”