Characterization of the lysogenic repressor (c) gene of the Pseudomonas aeruginosa transposable bacteriophage D3112

Bacteriophage D3112 is a Mu-like temperate transposable phage of Pseudomona s aeruginosa. Genetic mapping and DNA sequence analysis have identified the left end of the phage genome as encoding the transposase enzyme (A) and th e lysogenic (c) repressor. The c open reading frame (ORF), located at the l eftmost end of the phage genome and transcribed from right to left, has fou r possible GTG initiation codons. Using site-directed mutagenesis, each of the four GTG codons was modified to GTA, which cannot serve as an initiatio n codon. Plasmids were constructed expressing either the wild-type represso r ORF or the ORFs containing the mutated GTA codons. When introduced into P seudomonas aeruginosa, no immunity to superinfection by D3112 was observed when the second GTG had been mutated. Northern blotting analysis demonstrat ed that the D3112 c repressor is transcribed as a 900-nt mRNA. The promoter region was defined by transcriptional lacZ fusions and primer extension an alyses to bp 972-940 from the left end of the phage genome. When the D3112 c repressor was overexpressed and purified as a fusion protein with a C-ter minal six-histidine extension (cts15-His6), it showed high affinity for a 2 61-bp PvuII fragment localized directly upstream of the c repressor ORF. Ou r results indicate that although D3112 c shows higher amino acid similarity to the lambda family of repressors than it does to those of Mu and D108, i t appears that its structure and function more accurately reflect an evolut ionary ancestry with those from transposable coliphages Mu and D108. (C) 20 00 Academic Press.