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An approach to characterizing uncultivated prokaryotes: the Grey Lung agent and proposal of a Candidatus taxon for the organism, ‘Candidatus Mycoplasma ravipulmonis’

Harold Neimark¹, Deborah Mitchelmore² and Ronald H. Leach^2,

¹Department of Microbiology and Immunology, Morse Institute for Molecular Biology, State University of New York, Health Sciences Center at Brooklyn, Brooklyn, NY 11203, USA
²Central Public Health Laboratory, Colindale, London NW9 5HT, UK

Author for correspondence: Harold Neimark. Tel: +1 718 270 1242. Fax: +1 718 270 2656.

ABSTRACT

An approach to characterizing uncultivated bacteria which combines a PFGE procedure for obtaining purified full-length chromosomes with PCR amplification is described. Isolated chromosomes from uncultivated organisms provide a specifically identifiable source material for hybridization, amplification and cloning. The availability of purified chromosomes for DNA amplification should aid in examining the diversity of microbial populations and should also reduce the possibility of forming hybrid DNA artifact molecules. The approach is illustrated by isolating the chromosome of the uncultivated agent of rodent Grey Lung disease and using the purified chromosomes to amplify and directly sequence the evolutionarily conserved 16S rRNA gene. The Grey Lung agent (GLA) contains a 650 kb chromosome and is shown to be a Mycoplasma sp. located phylogenetically in the hominis group of mycoplasmas. If a simple genomic lesion(s) is responsible for the unculturability of GLA, it is conceivable that complementation with DNA from a close relative could permit growth on artificial media.

Key Words: ‘Candidatus Mycoplasma ravipulmonis’ • Grey Lung disease

The GenBank accession number for the nucleotide sequence reported in this paper is AF001173.

Present address: 50 Links Drive, Radlett, Herts WD7 8BG, UK.

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