Physical and genetic characterization of the cloned sbcB (exonuclease I) region of the Escherichia coli genome

Abstract

A 17-kilobase (kb) HindIII fragment containing the structural gene for exonuclease I (sbcB) from Escherichia coli K-12 was physically and genetically characterized. The monomeric molecular weight of exonuclease I was 53,700, based on sodium dodecyl sulfate-polyacrylamide gel electrophoresis of 35S-labeled E. coli mini- and maxicells. The gene was in close proximity to two unidentified proteins with molecular weights of 15,200 and 13,100. No other polypeptides appeared to be constitutively synthesized from the 17-kb fragment. Genetically, no portion of the histidine operon or the shikimic acid transport gene (shiA) was detected on the fragment. Although the entire 17-kb fragment in the vector pMB9 was too unstable to be useful, a 7.6-kb BamHI-EcoRI fragment inserted into a variety of vectors was stable. A detailed restriction map of the fragment is presented. Several derivatives in the runaway-replication vectors pMB06 and pMOB45 yielded 20- to 52-fold increases in exonuclease I activity after a switch in growth temperature to 40 degrees C. Of six exonuclease I mutants examined by DNA-DNA hybridization, one (xonA6) appeared to have arisen from a 1.2-kb insertion into the structural gene for exonuclease I.