Cloning of gene lon (capR) of Escherichia coli K-12 and identification of polypeptides specified by the cloned deoxyribonucleic acid fragment

Abstract

A mutation in the lon (capR) gene of Escherichia coli K-12 results in overproduction of capsular polysaccharide and increased sensitivity to ultraviolet and ionizing radiations. The lon (capR) gene deoxyribonucleic acid was cloned from a new F' factor. The new plasmids, designated pBZ201 and pBZ203, (i) contained an additional 8.2-megadalton (Md) EcoRI fragment that had the same mobility as one of the EcoRI fragments of the F', and (ii) conferred repression of capsular polysaccharide synthesis and repression of sensitivity to ultraviolet radiation in a bacterial transformation experiment with capR mutant recipient strains. A capR9 mutant plasmid, pBZ201M9, was also isolated and conferred expression of mucoidy and ultraviolet sensitivity to a capR(+) (lon(+)) strain, indicating that the capR9 allele was dominant. Plasmids pBZ201M80, pBZ201M9-INSA, and pBZ201M9-INSB were characterized by transformation as containing recessive capR mutant alleles. Heteroduplex analyses and agarose gel electrophoresis of restriction endonuclease digests of plasmid DNA preparations revealed that (i) pBZ201M9-INSA and pBZ201M9-INSB each contains a 0.5-Md insertion (probably IS1) in the cloned DNA fragment at the same site, and (ii) pBZ201 and pBZ203, both capR(+) plasmids, contain the same 8.2-Md fragment cloned in opposite orientations with respect to the cloning vehicle, pSC101. Plasmid-specified polypeptides were determined by using strain CSR603 maxicells containing each plasmid. Two new polypeptides were coded by the lon(+) (capR(+)) 8.2-Md DNA fragment: Z1, 94 kilodaltons (94K), and Z2, 67K. The maxicells containing recessive capR mutant plasmids were deficient only in synthesis of the 94K polypeptide, and the dominant (capR9) mutant plasmid specified 5 to 10 times more of the 94K polypeptide than the maxicells containing the capR(+) plasmid. Other data indicated that the capR9-specified "94K polypeptide" was not identical to the capR(+)-specified "94K polypeptide." Thus the altered mutant polypeptide was synthesized in increased quantities, suggesting a defective mode of autogenous regulation for the capR9 polypeptide and effective autogenous regulation of the capR(+) polypeptide.