Temperature-Sensitive Mutant of Escherichia coli K-12 with an Impaired d -Alanine: d -Alanine Ligase

Abstract

The temperature-sensitive Escherichia coli mutant strain ST-640 lyses at the restrictive temperature except when an osmotic stabilizer or a high concentration of d -alanine is present. The presence of dl -alanyl- dl -alanine does not prevent lysis. The rate of murein synthesis, followed in a wall medium, is decreased at both 30 and 42 C. d -Alanyl- d -alanine and uridine diphosphate- N -acetyl-muramyl (UDP-MurNAc)-pentapeptide are synthesized in decreased amounts, accompanied by accumulation of UDP-MurNAc-tripeptide at 42 C but not at 30 C. Uridine nucleotide precursors leak into the medium, especially out of the mutant cells. This leakage is prevented when NaCl is present. The d -alanine: d -alanine ligase (ADP) (EC 6.3.2.4) of the mutant strain, assayed in crude extracts, is temperature sensitive. The impaired ligase is relatively resistant to d -cycloserine and other inhibitors of the enzyme. Combined genetic and enzymatic results show that the low ligase activity is due to a mutation in the ddl gene, the structural gene for d -alanine: d -alanine ligase.