The region of the Pf3 virus genome encoding its major coat protein and its single-stranded DNA-binding protein is organized somewhat like the corresponding region of the fd (M13, fl) genome. Nevertheless, the major coat protein is unique among the major coat proteins of fd and the other filamentous phages studied in that it lacks a signal sequence and appears to be a direct translation product and in that it has fewer basic amino acid residues than its equivalent of DNA phosphates in the virion. These features are relevant to considerations of both protein insertion into membranes and DNA structure in filamentous viruses. The single-stranded DNAbinding protein also has a sequence that is different from the sequences of single-stranded DNA-binding proteins from other filamentous viruses.Bacteriophage Pf3 infects Pseudomonas aeruginosa PAO1 bearing the RP1 plasmid (1). It is one of a variety of filamentous viruses of Gram-negative bacteria, the best known of which are the closely related fd, fl, and M13 phages, all of which infect male strains of Escherichia coli. Other filamentous phages are Pfl (P. aeruginosa, strain K), Xf (Xanthomonas oryzae), and IKe and Ifl, both of which grow on E. coli strains bearing N and I plasmids, respectively. These viruses each contain a single-stranded circular DNA molecule encased in a slender protein sheath composed of thousands of identical protein subunits of molecular weight about 5,000 (the major coat protein) and a few copies each of a few coat proteins located at the ends of the virions (the minor coat proteins). Various comparative physical studies have shown that the packing of the DNA inside the different viruses is not the same, and also that intracellular complexes between the DNAs and their single-stranded DNA (ss DNA)-binding proteins are different (2-13).The best-characterized of these systems is that of fd (fl, M13), and it serves as the basis for comparisons (see ref. 12 for reviews). Of particular relevance to the present study is that the major coat protein subunits offd span the membrane of the infected. cell before they form a coat around the viral DNA. Their insertion into the membrane involves the processing of a signal sequence from a precursor protein. Dur-ing virus assembly, which occurs at the membrane, the viral DNA leaves its complex with its single-stranded DNA-binding protein as it becomes covered by major coat protein subunits and is extruded into the medium. The assembly and extrusion process does not cause cell lysis.In this paper, we present the results of an investigation of a region on the genome of Pf3 that encodes the DNA-binding protein and the coat protein of this virus. Comparisons of this region of DNA in Pf3 with its fd counterpart indicate close similarities in overall organization yet quite different amino acid sequences for the structural proteins themselves and the absence of a signal sequence for the major coat protein. MATERIALS AND METHODSBacteria and Bacteriophage. Pf3 bacteriophage and its host P. aeruginosa (PAO1) beari...

The circular, single-stranded DNA genome of the Pf3 bacteriophage was sequenced in its entirety by each of two methods, the M13-dideoxy chain termination method and the chemical degradation method. It consists of 5,833 nucleotides. With respect to both the DNA and the protein sequences, there is virtually no homology between Pf3 and the phages Ff (M13, f1, and fd) and IKe. However, similarities between these phages were noted with respect to their overall genome organizations. The gene for the single-stranded DNA-binding protein is followed by the gene for the major coat protein and then by a transcription termination signal. Open reading frames for seven other proteins were predicted, and their sizes and order show a fair correspondence to the sizes and order of the genes of the Ff phages and IKe. In addition, several regions have the potential to form stem and loop structures similar to those in the intergenic region of the Ff phage genome, but in Pf3 some are within open reading frames. Evolutionary relationships between Pf3 and the Ff phages and IKe are thus apparent through the correspondence of overall gene order rather than through primary sequence homologies.

The genome of Pf3, a filamentous single-stranded DNA bacteriophage of Pseudomonas aeruginosa (a gram-negative organism) was cloned into pBD214, a plasmid cloning vector of Bacillus subtilis (a gram-positive organism). Cloning in the gram-positive organism was done to avoid anticipated lethal effects. The entire Pf3 genome was inserted in each orientation at a unique BclI site within a thymidylate synthetase gene (from B. subtilis phage 322) on the plasmid. Additional clones were made by inserting EcoRI fragments of Pf3 DNA into a unique EcoRI site within this gene.