Isolation of conditionally lethal amber mutations affecting synthesis of the nusA protein of Escherichia coli.

Abstract

Amber mutants (am3 and am4) of Escherichia coli K12 defective in the synthesis of nusA protein (Friedman 1971) were isolated from a strain harboring an amber suppressor (sup-126) that is active only at low temperatures. These mutants grew at low temperature (30 degrees C) but did not grow at temperatures above 38 degrees C. Complementation experiments with plasmids carrying the nusA+ gene and its derivatives or with plasmids carrying the nusA1 or am4 mutation indicated that the mutations am3, am4 and nusA1 affected the same gene function. Analysis of proteins produced by minicells containing a plasmid demonstrated that the plasmid pYN87, which can complement the nusA1 and amber mutations, codes for three bacterial proteins, a truncated nusA gene product (61 K), argG gene product (48 K) and a 21 K dalton protein, and that the am4 mutation affects the synthesis of only NusA protein. lambda Nam7 (and lambda Nam7Nam53) phages could grow on these amber mutants at 32 degrees C but not on the parental strain. Spontaneous temperature-resistant revertants of the amber mutants simultaneously lost the ability to permit lambda Nam7 phage development, indicating that the two phenotypes are due to a single mutation. These results suggest that the nusA gene function is essential for the growth of E. coli, and that the lambda N function is dispensable for phage development if the nusA gene is defective.