Molecular Characterization of a Bacteriophage (Chp2) from Chlamydia psittaci

Abstract

Comparisons of the proteome of abortifacient Chlamydia psittaci isolates from sheep by two-dimensional gel electrophoresis identified a novel abundant protein with a molecular mass of 61.4 kDa and an isoelectric point of 6.41. C-terminal sequence analysis of this protein yielded a short peptide sequence that had an identical match to the viral coat protein (VP1) of the avian chlamydiaphage Chp1. Electron microscope studies revealed the presence of a 25-nm-diameter bacteriophage (Chp2) with no apparent spike structures. Thin sections of chlamydia-infected cells showed that Chp2 particles were located to membranous structures surrounding reticulate bodies (RBs), suggesting that Chp2 is cytopathic for ovine C. psittaci RBs. Chp2 double-stranded circular replicative-form DNA was purified and used as a template for DNA sequence analysis. The Chp2 genome is 4,567 bp and encodes up to eight open reading frames (ORFs); it is similar in overall organization to the Chp1 genome. Seven of the ORFs (1 to 5, 7, and 8) have sequence homologies with Chp1. However, ORF 6 has a different spatial location and no cognate partner within the Chp1 genome. Chlamydiaphages have three viral structural proteins, VP1, VP2, and VP3, encoded by ORFs 1 to 3, respectively. Amino acid residues in the X174 procapsid known to mediate interactions between the viral coat protein and internal scaffolding proteins are conserved in the Chp2 VP1 and VP3 proteins. We suggest that VP3 performs a scaffolding-like function but has evolved into a structural protein.

Isolation, Molecular Characterisation and Genome Sequence of a Bacteriophage (Chp3) from Chlamydophila pecorum

“…48-72 h post infection monolayers were washed in PBS, fixed in 3% glutaraldehyde in 0.1% cacodylate buffer and processed for negatively stained thin-section grids [3]. A partially purified preparation of bacteriophage Chp3 was adsorbed onto a formvar coated carbon grid for 2 min, then negatively stained with 0.75% phospho-tungstic acid pH 6.0 for 10 s and air dried.…”

“…The first chlamydial bacteriophage to be characterised (Chp1) was found by thin section transmission EM of avian C. psittaci RBs [1] where it formed characteristic paracrystalline arrays. Although Chp1 was later lost a second bacteriophage (ØCPG1) [2] was found infecting C. caviae and a third bacteriophage (Chp2) was isolated from C. abortus [3]. The genome sequencing project of C. pneumoniae strain AR39 revealed the presence of a dsDNA extrachromosomal element [4] which was subsequently shown to be the replicative form of bacteriophage ØCPAR39 [5].…”

“…The genome sequencing project of C. pneumoniae strain AR39 revealed the presence of a dsDNA extrachromosomal element [4] which was subsequently shown to be the replicative form of bacteriophage ØCPAR39 [5]. These four bacteriophages share similar features, they are small icosahedral T ¼ 1 particles containing circular, single-stranded DNA genomes and molecular characterisation showed that they belong to the virus family Microviridae [3,4,6,7].…”

“…the prototypical coliphage ØX174 [8] and related bacteriophages; secondly those that infect obligate intracellular bacteria or mollicutes, including chlamydiaphages [1][2][3][4], a bacteriophage of spiroplasma (SpV4) [9] and a novel bacteriophage, ØMH2K [10] recently isolated from Bdellovibrio bacteriovorus. This second sub-family also has a similar overall genome organisation although the genomes of bacteriophages in this sub-family are smaller and do not encode homologues to the major spike and the external scaffold proteins [3] of the ØX174 subfamily.…”

“…The four chlamydiaphages that have been characterised were all discovered by chance [1][2][3][4]. The purpose of this work was to define the nature and extent of chlamydiaphage diversity by searching for new chlamydial microviruses using a systematic approach by screening a large collection of viable chlamydiae representing the six species of Chlamydophila and three species of Chlamydia.…”