ABSTRACT
Bacteriophage D3112 is a Mu-like temperate transposable phage of Pseudomonas aeruginosa. Genetic mapping and DNA sequence analysis have identified the left end of the phage genome as encoding the transposase enzyme (A) and the lysogenic (c) repressor. The c open reading frame (ORF), located at the leftmost end of the phage genome and transcribed from right to left, has four possible GTG initiation codons. Using site-directed mutagenesis, each of the four GTG codons was modified to GTA, which cannot serve as an initiation codon. Plasmids were constructed expressing either the wild-type repressor ORF or the ORFs containing the mutated GTA codons. When introduced into Pseudomonas aeruginosa, no immunity to superinfection by D3112 was observed when the second GTG had been mutated. Northern blotting analysis demonstrated that the D3112 c repressor is transcribed as a 900-nt mRNA. The promoter region was defined by transcriptional lacZ fusions and primer extension analyses 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-terminal six-histidine extension (cts15-His6), it showed high affinity for a 261-bp PvuII fragment localized directly upstream of the c repressor ORF. Our 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, it appears that its structure and function more accurately reflect an evolutionary ancestry with those from transposable coliphages Mu and D108.
Subject(s)
DNA Transposable Elements/genetics , Genes, Viral/genetics , Lysogeny/genetics , Pseudomonas Phages/genetics , Repressor Proteins/genetics , Viral Proteins/genetics , Amino Acid Sequence , Base Sequence , Cloning, Molecular , Codon, Initiator/genetics , DNA, Viral/genetics , DNA, Viral/metabolism , DNA-Binding Proteins/chemistry , DNA-Binding Proteins/genetics , DNA-Binding Proteins/isolation & purification , DNA-Binding Proteins/metabolism , Genes, Reporter/genetics , Lysogeny/immunology , Molecular Sequence Data , Mutation/genetics , Open Reading Frames/genetics , Operator Regions, Genetic/genetics , Promoter Regions, Genetic/genetics , Pseudomonas Phages/physiology , Pseudomonas aeruginosa/virology , RNA, Messenger/analysis , RNA, Messenger/genetics , RNA, Viral/analysis , RNA, Viral/genetics , Recombinant Fusion Proteins/chemistry , Recombinant Fusion Proteins/genetics , Recombinant Fusion Proteins/isolation & purification , Recombinant Fusion Proteins/metabolism , Repressor Proteins/chemistry , Repressor Proteins/isolation & purification , Repressor Proteins/metabolism , Sequence Alignment , Viral Proteins/chemistry , Viral Proteins/isolation & purification , Viral Proteins/metabolismABSTRACT
The left end DNA of Mu-like transposable bacteriophage D3112 was sequenced from bp 2521 to bp 5483. Two large open reading frames were identified: ORF A (bp 2539-4611) and ORF B (bp 4626-5378). ORF A can encode a 690 amino acid, 78 kDa protein which is 44.4% similar to Mu transposase and ORF B can encode a 250 amino acid, 27 kDa protein, which is 46.4% similar to, though 62 amino acids shorter than, the Mu B protein. The cloned D3112 A gene exhibited activity on a mini-D3112-containing plasmid in Pseudomonas aeruginosa.
