Nozomu Otsuji

Cloning of colicin E1 tolerant tolC(mtcB) gene of Escherichia coli K12 and identification of its gene product

SummaryA mutation in the tolC(mtcB) gene of Escherichia coli K12 results in increased sensitivity to sodium dodecylsulfate (SDS), sodium deoxycholate, basic dyes, mitomycin C, and bleomycin, and makes the cell tolerant to the killing action of colicin E1. From lysogens with λcI857S7 integrated at a secondary attachment site, a transducing phage (λdtolC+) that transduces a tolC recipient to SDS resistance was isolated. A recombinant DNA molecule was constructed in vitro from plasmid pBR322 as a vector, and an EcoRI−BamHI fragment of λtolC+ DNA. The resulting plasmid, designated pOK1, was 5.6 megadaltons (Md). The tolC bacteria transformed with plasmid pOK1 restored the TolC+ phenotype with regard to mitomycin C, SDS, and colicin E1 sensitivities. A plasmid with an amber mutation in the tolC gene, designated pOK18, was isolated by the same procedure used for the isolation of pOK1. The plasmid had a molecular weight of 5.6 Md and produced the same size of DNA fragments as the tolC+ plasmid, pOK1, after digestion with the indicated restriction enzymes. The plasmid, pOK18, conferred the TolC+ phenotype when introduced into a tolC strain in the presence of, but not the absence of, an amber suppressor. Plasmidspecified polypeptides were determined by using maxicells of strains uvrA recA sup+ and uvr A recA tyrT, containing each plasmid. Three additional proteins of 54,000 (54K), 29K, and 27K were produced in maxicells containing pOK1. These three proteins were synthesized in maxicells of the uvrA recA tyrT strain carrying pOK18, whereas synthesis of the 54K protein by pOK18 did not take place in maxicells of the uvrA recA sup+ strain, although the other two proteins were produced in normal amounts. From these results we concluded that the product of the tolC gene is a protein with a molecular weight of 54K.

Mode of mutagenic action of 4-benzoylamido- and 4-acetamido-4-carboxamido-n(N-nitroso)-butylcyanamide.

The mode of mutagenic action of 4-benzoylamido- and 4-acetamido- 4-carboxamido-n(N-nitroso)-butylcyanamide (BCNBC, ACNBC) was studied using Escherichia coli K12 strains. The strains carrying defects in DNA-repair mechanism, AB2463 (recA) and P3478 (polA) were more sensitive than their parent strains to both compounds, while AB1886 (uvrA) showed the same sensitivity as the parental strain. About 90% of tryptophan revertants from BE1043 (trpambphoamb) by both compounds were due to mutation in suppressor genes. Suppressor analysis by using BE1047 (trpambphooch) revealed that the most frequently occurring reversion was due to a mutation in suppressor gene, supE. This implies that these two alkylnitrosocyanamides predominantly induce GC leads to AT transition.