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Previous studies demonstrated that Mycoplasma arthritidis strain 158 acquired a high degree of virulence upon lysogenization with bacteriophage MAV1. In the present study, the association between MAV1 and virulence was reexamined by creating new lysogens of 158 and of a relatively avirulent mutant, strain 158-1. In the absence of lysogenization, 158 was more virulent than expected. The virulence of 158 and 158-1 did not increase upon lysogenization. A major antigenic difference between 158 and 158-1 was identified that is unrelated to MAV1 and could account for the difference in virulence.

The lysogenic bacteriophage MAV1 has been shown to be a virulence factor for the development of arthritis in rats infected with Mycoplasma arthritidis. In the present study, arthritis was evaluated by histopathologic examination to demonstrate that MAV1 is a virulence factor not only in the rat but also in the mouse. Specifically, the MAV1 lysogen 158L3-1 was more virulent than the nonlysogen strain 158 in DBA/2NCr, C3H/HeNCr, C3H/HeJ, and C3Smn.CB17-Prkdc(scid)/J mice, as well as in LEW rats.

Mycoplasma virus P1 is one of only four viruses isolated from the genus Mycoplasma. The host for P1, Mycoplasma pulmonis, possesses complex, phase-variable restriction and modification enzymes and the Vsa family of phase-variable surface proteins. The ability of P1 virus to infect host cells is influenced by these phase-variable systems, rendering P1 a valuable tool for assessing host properties. The double-stranded P1 DNA genome was sequenced (11,660 bp) and 11 ORFs were identified. The predicted P1 DNA polymerase is similar to that of phages that are known to have terminal protein (TP) attached to the 5' end of their genome, consistent with previous studies indicating that P1 DNA has covalently attached TP. Most of the other predicted P1 proteins have little sequence similarity to known proteins, and P1 virus is unrelated to the other mycoplasma virus, MAV1, for which the genome sequence is known. One of the predicted P1 proteins, the ORF 8 gene product, contains a repetitive collagen-like motif characteristic of some bacteriophage tail fiber proteins and is a candidate for interacting with the Vsa proteins., (Copyright 2001 Academic Press.)

The bacteriophage MAV1 is required for the development of arthritis in rats after infection with its host Mycoplasma arthritidis. To identify the phage-encoded virulence factor for this arthritis, the complete nucleotide sequence of MAV1 was determined. The linear double-stranded genome of MAV1 is 15644bp and contains 15 ORFs. Putative protein products from these ORFs were identified by comparison of the deduced amino acid sequences to known proteins and comprise DNA replication, restriction-modification, structural, regulatory, and integration/excision proteins. Eight putative promoters were identified; four of these would produce polycistronic transcripts. Translation of each ORF appears to be initiated independently, with each having its own RBS. A single ORF, vir, was identified on the minus strand of the phage genome. The putative protein product of vir contains a classic prokaryotic lipoprotein signal sequence and is a strong candidate for the MAV1-encoded virulence determinant.

The lysogenic bacteriophage MAV1, which is associated with the arthritogenicity of Mycoplasma arthritidis, was characterized. Several strains of M. arthritidis were examined for their ability to support growth of MAV1. A PFU assay was developed, and the sensitivity of phage to various chemical treatments was assayed. The most notable result was the resistance of MAV1 to proteinase K. The MAV1 genome is a double-stranded, linear DNA molecule of about 16 kb. The site of MAV1 DNA integration in the host chromosome was investigated. The ends of MAV1 DNA were cloned from three independent lysogens shown to have MAV1 DNA inserted at different sites in the host. The nucleotide sequences of the ends of the MAV1 genome and of the MAV1 DNA-chromosomal DNA junctions from each of three lysogens were determined. Sequences flanking the integrated prophage and the ends of native MAV1 DNA were determined, allowing the identification of the phage DNA (attP) and bacterial DNA (attB) recombination sites. Analysis of the left MAV1 DNA-chromosomal DNA junction sites showed a single-base heterogeneity located within MAV1 DNA sequences immediately adjacent to the attB sequence. A model for MAV1 integration-excision is proposed.

Mycoplasma virus P1 is a tailed, polyhedral virus isolated from Mycoplasma pulmonis. To characterize the P1 genome, stocks of virus were prepared free of host cell nucleic acids. A single DNA species of 11.3 kb that was shown by plaque hybridization to be of P1 origin was extracted from the virus. Efficient isolation of P1 DNA required digestion with proteolytic enzymes prior to phenol extraction. Although P1 DNA was double-stranded and linear following such treatment, it was resistant to digestion with the 5'-specific lambda exonuclease. Electron microscopic analysis indicated that globular material is complexed to the ends of P1 DNA. The globular material was not observed on protease-treated P1 DNA molecules, suggesting that it is composed of protein. Removal of the putative terminal protein by chemical treatment allowed the cloning of the P1 DNA ends, and nucleotide sequence analysis revealed that these ends contain a 350-bp inverted terminal repeat.

We isolated two spontaneous variants of mycoplasma virus L2. Both variants, designated L2ins1 and L2ins2, contained a 3.1-kilobase-pair (kbp) insertion in the 11.8-kbp wild-type L2 genome. The insert DNA was shown to be derived from two noncontiguous regions of the L2 genome, and L2ins1 and L2ins2 differed only in the location of the 3.1-kbp insertion. We also isolated L2 miniviruses from serial passages of L2, L2ins1, and L2ins2 viruses. Miniviruses contained circular DNA molecules of 3.1 kbp or multimers of 3.1 kbp. Minivirus 3.1-kbp DNAs had the same sequences as the 3.1-kbp insert DNAs found in L2ins1 and L2ins2 viruses. Miniviruses were not infectious and interfered with the growth of L2, L2ins1, and L2ins2 viruses; hence, L2 miniviruses appeared to be defective interfering particles.

The adsorption of mycoplasma virus P1, a virus which infects some strains of Mycoplasma pulmonis, to host cells was examined. Mutants of M. pulmonis to which P1 virus did not adsorb were isolated. Proteins from the mutants and from wild-type cells were compared by two-dimensional polyacrylamide gel electrophoresis, and the only observed difference was in the surface antigen V-1. The electrophoretic properties of V-1 also correlated with the host range of the virus. These data strongly suggest that the V-1 antigen affects adsorption of P1 virus to host cells.

We have found that mycoplasma virus L172 is an enveloped globular virion containing circular, single-stranded DNA of 14.0 kilobases. L172 has been reported by other workers to have a double-stranded DNA genome of 13 to 17 kilobase pairs and has been classified as a plasmavirus, a group for which mycoplasma virus L2 is the type member. Mycoplasma viruses L172 and L2 differ in genome size and structure, DNA base composition, and protein composition, and they have no detectable DNA homology. As the only reported enveloped virion containing single-stranded DNA, L172 represents a new group of viruses.

The 11.8 kilobase pair (7.8 X 10(6) mol. wt.) genome of mycoplasma virus L2 in lysogenic Acholeplasma laidlawii cells was examined. For this study, DNAs were analysed by agarose gel electrophoresis and viral DNA sequences identified by DNA-DNA hybridization. L2 DNA was found to be integrated into the lysogenic host cell chromosome at a unique site in both viral and cellular DNA. The viral DNA site was roughly mapped and the approximate time of integration during the L2 non-cytocidal infectious cycle was determined.

A virus designated mycoplasma virus P1 has been isolated from Mycoplasma pulmonis. The virus infects M. pulmonis strain UAB 6510, and a plaque-forming unit assay has been developed. P1 has a tailed, polyhedral morphology with a head diameter of about 28 nm. Nucleic acid isolated from crude preparations of P1 virus contains double-stranded RNA, suggesting that P1 may be the first example of an RNA-containing mycoplasma virus.