Novel prophage-like sequences in Mycoplasma anserisalpingitidis.

Mycoplasma anserisalpingitidis is a bacterial waterfowl pathogen. In these days of growing antibiotic resistance, it is necessary to search for alternative methods of defense against Mycoplasma impacts in flocks. In order to identify prophage-like sequences, three established bioinformatics tools (PHASTER, PhiSpy, Prophage Hunter) were used in this study for the in silico screening of 82 M. anserisalpingitidis whole genomes. The VIBRANT software was used as a novel approach to further investigate the possibility of prophages in the sequences. The commonly used softwares found prophage-like sequences in the strains, but the results were inconclusive. The VIBRANT search resulted in multiple hits, and many of them were over 10,000 base pairs (bp). These putative prophages are comparable in size to the few described mycoplasma phages. The translated coding DNA sequences of the putative prophages were checked with protein BLAST. The functions of the proteins found by the BLASTP search are common among bacteriophages. The BLASTN search of the sequences found that many of these were more similar to the M. anatis NCTC 10156 strain, rather than the available M. anserisalpingitidis strains. The initial screening pointed at the presence of novel bacteriophages in the M. anserisalpingitidis and M. anatis strains. The VIBRANT search results were very similar to each other and none of these sequences were part of the core genome of M. anserisalpingitidis, with a few exceptions. The VIBRANT analysis explored presumably intact, novel prophages.

Effect of erythromycin on bronchial hyperreactivity and inflammation in ovalbumin-sensitized brown Norway rats.

To evaluate the effect of erythromycin on bronchial hyperreactivity, inflammation, and T-cell related cytokine mRNA expression in rats sensitized to ovalbumin, three experimental groups of 10 brown Norway rats each were sensitized by breathing aerosolized ovalbumin. From day 1 to day 15, one group was given oral erythromycin 80 mg/kg/day, another group oral erythromycin 20mg/kg/day, and the third group oral saline only. A fourth control group of 10 rats breathed aerosolized saline. After sensitization, the three experimental groups were provoked by breathing ovalbumin, with the controls again breathing saline. The rats were then anesthetized and paralyzed, and pulmonary function tests were performed at baseline and after varying doses of acetylcholine. Bronchoalveolar lavage (BAL) fluid and lung tissues were examined for expression of mRNA for T-cell cytokines. Our results showed that erythromycin had no beneficial effects on pulmonary function and lung inflammation in the two erythromycin-treated experimental groups compared with the saline experimental group. Th2-related cytokines and their mRNA expression in the three experimental groups were higher than in controls but did not differ among the experimental groups. In conclusion, erythromycin does not prevent bronchial hyperreactivity or an inflammatory response in ovalbumin-sensitized rats.

Complete nucleotide sequence of the mycoplasma virus P1 genome.

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.

Sequence analysis of the Mycoplasma arthritidis bacteriophage MAV1 genome identifies the putative virulence factor.

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.

Characterization of the lysogenic bacteriophage MAV1 from Mycoplasma arthritidis.

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.

Association of lysogenic bacteriophage MAV1 with virulence of Mycoplasma arthritidis.

Mycoplasma arthritidis causes a severe polyarthritis under natural conditions in rats and under experimental conditions in both rats and mice. Although the disease itself has been extensively studied, M. arthritidis virulence factors remain uncharacterized. Comparison of relative arthritogenicity of 20 strains of M. arthritidis revealed that the strains tended to fall into two groups, a highly arthritogenic group, inducing maximum arthritis scores of > or = 11 in rats, and a low-virulence group, inducing maximum scores of < 6. Chromosomal DNA from the more highly arthritogenic strains possessed sequences that hybridized by Southern analysis with a probe prepared from lysogenic M. arthritidis bacteriophage MAV1, while DNA from low-virulence strains did not. One of the low-virulence strains, 158, was experimentally lysogenized with MAV1. Lysogenized 158 showed a significant increase in arthritogenicity over nonlysogenized 158. These data suggest that MAV1 carries a factor that is important in pathogenesis of M. arthritidis-induced arthritis of rats.

Is LM7 a new human retrovirus or a mycoplasma virion?

A putative retrovirus called LM7 was recently isolated from a patient with MS. This retrovirus was detected in LM7 and LM711 cultured human leptomeningeal cells. In the present work, nucleic acids from LM711 cell culture supernatants were purified and subjected to avian myeloblastosis viral (AMV) reverse transcriptase and to random polymerase chain reaction (rPCR) in order to characterize the genomic material of LM7 virions. Analysis of reverse transcription products allowed the detection of an approximately 14 kb ribonucleic acid in all LM711 cell culture supernatants. However, sequencing of rPCR-amplified molecules as well as RNA blotting data showed essentially that all tested cells producing LM7 particles were infected with mycoplasma. Moreover, purification of LM7 particles onto a linear sucrose density gradient established that the 14 kb nucleic acid was always associated with the 1.19-1.21 g ml-1 sucrose fractions, which are known to correspond to the buoyant density of mycoplasma. In addition, no viral genomic RNA was detected in the 1.17 g ml-1 sucrose fraction containing the low reverse transcription activity. These results strongly suggest that microscopic images and serological data could be related to mycoplasma and/or to a virion associated with the bacteria. The LM7 particle might be a new and additional enveloped virus able to infect Mycoplasma hyorhinis hosts. Thus, for instance, it would be presumptuous to assert, with our current understanding, that the LM7 virion is one of the causal agents of MS in humans.

Mycoplasma virus P1 has a linear, double-stranded DNA genome with inverted terminal repeats.

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.

Links and interactions between mycoplasmas and viruses: past confusions and present realities.

Links between mycoplasmas and viruses are ancient, multiple and complex, from past confusions during the first decades of the virus era to present realities illustrated by the possible implication of mycoplasmas as co-factors in natural infections of AIDS. Mycoplasma viruses (phages) may also be responsible for modifying the pathogenic power of mycoplasmas, at least for plants and insects. In addition, several mycoplasmas are able to act as undesirable cell culture contaminants that induce erroneous results in both applied and general virology. These problems are examined within a historical context.

Regulation of a restriction and modification system via DNA inversion in Mycoplasma pulmonis.

An invertible DNA element of 6.8 kb, designated the hsd1 locus, was identified in the chromosome of Mycoplasma pulmonis. Infection of host cells with mycoplasma virus P1 revealed that the organism's restriction and modification (R-M) properties are controlled by inversion of hsd1. The nucleotide sequence of hsd1 revealed several genes, the predicted amino acids of which bear striking similarity to the subunits of the type I R-M enzymes previously found only in enteric bacteria.