The viability of the topA mutants lacking DNA topoisomerase I was thought to depend on the presence of compensatory mutations in Escherichia coli but not Salmonella typhimurium or Shigella flexneri. This apparent discrepancy in topA requirements in different bacteria prompted us to reexamine the topA requirements in E. coli. We find that E. coli strains bearing topA mutations, introduced into the strains by DNA-mediated gene replacement, are viable at 37 or 42 degrees C without any compensatory mutations. These topA(-) cells exhibit cold sensitivity in their growth, however, and this cold sensitivity phenotype appears to be caused by excessive negative supercoiling of intracellular DNA. In agreement with previous results (Zhu, Q., Pongpech, P., and DiGate, R. J. (2001) Proc. Natl. Acad. Sci. U. S. A. 98, 9766-9771), E. coli cells lacking both type IA DNA topoisomerases I and III are found to be nonviable, indicating that the two type IA enzymes share a critical cellular function.