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.

Four major clusters, designated A, B, C and D, were distinguished in Western Blots by a monoclonal antibody specific for the "antigen family at 90 kDa" after two-dimensional electrophoretic analysis on immobilized pH gradient of chlamydial elementary bodies of abortifacient C. psittaci. Clusters B, C, and D were closely related with molecular mass (kDa) pI values of 91.5/5.2-5.4, 90/5.0-5.2 and 90.5/5.6-5.8, respectively. Cluster A was larger, with molecular mass/pI of 104.7/5.1-5.3. Evidence for the antigenic relationship between cluster A and clusters B, C and D was further supported by immunological cross-reaction with affinity-purified antibodies from serum of ewes with chlamydial-induced abortion. The experimental values obtained for size and pI of the four clusters correlated well with the calculated values from known sequences coded by multiple chlamydial genes. Direct evidence for the correspondence between the immunoreactive clusters B, C and D and the retrieved genes was provided by antibody binding experiments to recombinant polypeptides representing fragments of the deduced proteins. The 4-member antigen family at 90/104 kDa is the first example of proteins coded by multiple genes within the genus Chlamydia.

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