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

^a Laboratory for Microbiology, State University, Catharijnesingel 59, Utrecht, The Netherlands

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.

¹ Present address: University of Connecticut Health Center, Department of Microbiology, Farmington, Conn. 06032.