Major coat protein and single-stranded DNA-binding protein of filamentous virus Pf3.

Abstract

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...

A CD determination of the α‐helix contents of the coat proteins of four filamentous bacteriophages: fd, IKe, Pf1, and Pf3

“…This result disagreed with the relative a-helix contents of these phage determined using Raman ~pectroscopy,~ which indicated that fd had about 20% less a-helix content than Pfl. 3. Since sample concentration can affect the a-helix content of some filamentous phages," and Raman studies are typically performed a t much higher concentrations ( = 100 mg/mL) than those we used ( = 0.1-0.3 mg/mL), we measured the CD spectra of samples of each phage differing by at least 100-fold in concentration (0.2-2.2 mg/mL for fd, 0.02-1.9 mg/mL for IKe, 0.03-3.7 mg/mL for Pfl, and 0.02-1.2 mg/mL for Pf3).…”

Nucleotide sequence determination, characterization and purification of the single-stranded DNA-binding protein and major coat protein of filamentous phage Lf of Xanthomonas campestris pv. campestris

“…4). In addition, all major coat proteins, except that of Pf3 (Luiten et al, 1983;Putterman et al, 1984), are synthesized as a precoat protein containing a hydrophobic N-terminal signal peptide. This signal peptide is cleaved off by the signal peptidase with concomitant deposition of the mature coat protein into the inner membrane (Webster & Cashman, 1978;Chang et al, 1979).…”

“…It is known from the studies on Ff and Pf that each filamentous phage particle consists of more than 2500 copies of the major coat protein (Pratt et al, 1969;Newman et al, 1977;Luiten et al, 1983;Putterman et al, 1984). In this study, a concentrated eLf suspension prepared by ultracentrifugation was subjected to SDS-PAGE.…”

“…However, it was interesting to note that the major coat protein of ~bLf appeared to be synthesized as a mature protein instead of a pre-coat protein which is processed post-translation. Among all known major coat proteins, only that of phage Pf3 is synthesized as a mature protein (Luiten et al, 1983;Putterman et al, 1984).…”

Insertion and glycosylation of Pf3‐derived membrane proteins in microsomes

“…It is a small protein of only 44 amino acids, which is stored in the inner membrane of the bacterium during the infection cycle. In contrast to coat proteins of closely related phages such as M13, fd or Pf1 (for a review about filamentous phages, see [1]), it is synthesized without a leader sequence [2]. Its membrane insertion is independent of Sec proteins [3], but depends on the proton motive force, which translocates the negatively charged N‐terminus across the membrane [4].…”