DNA sequence analysis of spontaneous and beta-methoxy-acrolein-induced mutations in Salmonella typhimurium hisD3052

The histidine auxotroph hisD3052 results from a single base-pair deletion (C:G) from the 298th codon (alanine) in the D gene of the histidine operon of S. typhimurium LT-2. Bacteria carrying this mutation revert to histidine prototrophy by frameshift mutations (additions or deletions) that restore the correct reading frame. Among the compounds that revert hisD3052 are the naturally occurring dicarbonyl compound malondialdehyde and a structural analog, beta-methoxy-acrolein. To determine the sequence changes responsible for reversion, spontaneous and beta-methoxy-acrolein induced revertants were isolated, male derivatives constructed, and infected with M13Ho167.18, a phage carrying partial O, D, and C genes of the histidine operon. M13hisD+ recombinants were selected by complementation in TA2890, single-stranded DNA was isolated, and the reverted D genes were sequenced using Sanger dideoxy chain-termination sequencing with a synthetic oligonucleotide primer. Analysis of 37 spontaneous revertants revealed 41% additions and 59% deletions with 22% of the mutations occurring as two base-pair (-CG-) deletions in a -CG- rich 'hot spot'. The profile of additions was +1 (30%), +4 (8%), +7 (3%); the profile of deletions was -2 (32%), -5 (11%), -8 (8%), and -11 (8%). Analysis of 27 beta-methoxy-acrolein-induced revertants revealed 96% additions and 4% deletions. The profile of induced additions was +1 (70%), +4 (22%), +7 (4%). No additions occurred in the -CG- rich hot-spot. Frameshift reversions of the hisD3052 gene demonstrate a surprising degree of sequence diversity and reveal the ability of the hisD3052 tester strain to detect a wide variety of frameshift reversion events. In addition, the results demonstrate that beta-methoxy-acrolein induces a high percentage of additions in this reversion system.