International Journal of Systematic Bacteriology (1998), 48, 389-394
Printed in Great Britain
An approach to characterizing uncultivated
prokaryotes: the Grey Lung agent and proposal
of a Candidatus taxon for the organism,
’Candidatus Mycoplasma ravipulmonis’
Harold Neimark,’ Deborah Mitchelmore’ and Ronald H. Leach2t
Author for correspondence: Harold Neimark. Tel:
1
Department of
Microbiology and
Immunology, Morse
Institute for Molecular
Biology, State University of
New York, Health Sciences
Center at Brooklyn,
Brooklyn, NY 11203, USA
2
Central Public Health
Laboratory, Colindale,
London NW9 5HT, UK
+ 1 718 270 1242. Fax: + 1 718 270 2656.
An approach t o 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 t o amplify and directly sequence the evolutionarily conserved
165 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.
Keywords: ‘ Candidatus Mycoplasma ravipulmonis ’, Grey Lung disease
INTRODUCTION
Uncultivated bacteria have long been recognized in
natural environments, and similarly, pathogenic or
commensal bacteria that have defied all efforts at
cultivation inhabit specialized niches in animal or
plant hosts. Uncultivated bacteria have been observed
in a wide variety of hosts including protozoa (34),
insects (38), other invertebrates including marine
bryozoa (44), vertebrates ( 5 , 14) and plants (36, 16).
Molecular methods have permitted identification of
uncultivated bacteria from some of these habitats but
obstacles to characterizing uncultivated bacteria remain [for a review see (l)].
Here we describe an approach to characterizing uncultivated bacteria, which combines a PFGE procedure for obtaining purified full-length chromosomes
(27) with PCR amplification. Isolated chromosomes
from uncultivated organisms provide a specifically
..................................................................................................................................................
tPresent address: 50 Links Drive, Radlett, Herts WD7 8BG, UK.
Abbreviation: GLA, Grey Lung agent.
The GenBank accession number f o r t h e nucleotide sequence reported in
this paper is AFOOI 173.
identifiable source material for hybridization, amplification and cloning. The utility of this approach is
illustrated by isolating the chromosome of the uncultivated agent of Grey Lung disease of mice and
using the purified chromosomes, for example here, to
amplify and directly sequence the evolutionarily conserved 16s rRNA gene (20). The use of purified
chromosomes for PCR templates should aid in
examining the diversity of microbial populations and
also should reduce the possibility of forming artifact
hybrid DNA molecules.
The Grey Lung agent (GLA), initially described as the
‘Grey Lung virus’, causes a pneumonia that appeared
spontaneously in mice during passage of respiratory
tract materials from different mammalian sources (2).
Its pathogenicity seems to be confined to mice and, to
a lesser degree, other rodents (2). GLA appears to be
strictly pneumotropic with no evident effects on other
organs ; its name derives from the characteristic
greyish-pink oedematous consolidation produced in
the lungs ( 2 , 12,31). The histopathologic picture shows
mononuclear ‘cuffing ’ of bronchioles and pulmonary
blood vessels. Numerous pleomorphic particles, 300600 nm in diameter, are evident in Giemsa-stained
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H. Neimark, D. Mitchelmore and R. H. Leach
impression smears of infected lung. Grey Lung disease
is not known to occur naturally in mice, but a similar
form of pneumonia occurs in laboratory and wild rats,
and is caused by an aetiological agent(s) closely
resembling the GLA (3, 10, 29, 30, 41). Originally
regarded as a virus because of its filterability, its
resistance to antibacterial agents (including penicillin,
sulfonamide and chloramphenicol), and its failure to
grow in bacteriological culture media (2,4), GLA was
later recognized by Marmion and colleagues to possess
some of the characteristics of a mycoplasma (13, 23).
Extensive efforts during the past 30 years t o cultivate
the GLA from infectious lung materials have failed to
demonstrate growth or even survival of the agent in
vitro (21; Leach, unpublished data). No active immunity nor any serological response has been detected
in infected mice or in Grey Lung-immunized mice or
rabbits by use of standard serological methods (2,
11); more recent attempts to detect antibody to the
G L A (or to Mycoplasma pulmonis or Mycoplasma
arthritidis) in infected mice by ELISA, colonyimmunofluorescence, or by an immuno-binding assay
(W18) also have failed (21). The enzootic chronic
pneumoniae agent from laboratory rats also resembles
GLA in its failure to elicit an immune response in
infected animals (3, 11, 41).
The GLA characterized here has been maintained by
mouse serial passage for more than 50 years, and aside
from the agents of leprosy and syphilis, is one of the
oldest examples of a n uncultivated bacterial pathogen.
METHODS
Mouse inoculation and infectivity-titration procedures.
These were carried out with the technical collaboration of
David Conway. White Schofield or BALB/c mice housed
under specificpathogen-free conditions in the animal facility
of the Central Public Health Laboratory, Colindale, UK,
were used in all experiments; all-male or all-female mice
were used for individual experiments. Mice under light
anaesthesia (Halothane) were inoculated with one drop in
each nostril of an infected-lung suspension or lung-wash
fluid (see below). Development of pulmonary disease was
observable at post-mortem examination within about 12
weeks with high-titre inocula, or longer for lower-titre
inocula. Some animals showed transient or permanent
external signs of illness, in the form of increased respiration
or physical appearance, but others showed no overt signs
and remained relatively normal despite the fact that this
infection does not resolve naturally and the lung lesions
persist throughout the animal's lifetime. The agent can thus
be maintained by occasional mouse-to-mouse passages at
intervals of many months or even years.
High-infectivity suspensions of the GLA were prepared by
lightly homogenizing small portions (10-100 p1) of infectedlung tissue in Hartley's broth (Oxoid; 5-lo%, w/v) and
using either the whole suspension or the supernate obtained
after low-speed centrifugation. Clearer suspensions, relatively free of mouse tissue cells, could be obtained by
employing a lung-washing method (39) ;this entails inserting
a tracheal cannula into a mouse killed by cervical dislocation
and gently inflating the lungs with 0.5-1 ml Hartley's broth,
followed by withdrawal of the resulting lung-wash fluid for
390
use as an infective suspension. By either of these means,
Grey Lung suspensions with high mouse-infectivity titres
(up to lo6 ml-l) could be obtained. Infectivity levels of
suspensions were titred by inoculating dilutions into mice
and subsequent post-mortem examination for characteristic
' Grey Lung ' consolidation.
Electron microscopy. Electron microscopy was carried out
by Kitty Plaskitt at the John Innes Institute, Norwich, UK.
Lung samples for electron microscopy were fixed in glutaraldehyde, post-fixed in 1 % osmic acid, dehydrated in ethanol,
and embedded in araldite; sections were stained with uranyl
acetate and lead acetate. Microscopy was done with a
Siemens 101 or 1A microscope.
PFGE. All steps were carried out with ice-cold reagents;
centrifugations were at 4 "C. Lung-wash fluids from 12 to 22
mice, 4-8 weeks post-infection (titre 104-106 ml-l), were
pooled and centrifuged at 1500g for 3 min and the resulting
supernatant was centrifuged at 17000 g for 30 min. The
pellet containing the GLA was resuspended in 20 ml PBS
and centrifuged again at 17000 g for 30 min. The final pellet
was resuspended in 100-200 p1 TES buffer (0.1 M Tris,
10 mM disodium EDTA, 0.1 M NaCl, pH 8.0). From
electron microscopy studies or other experience, these
buffers are known to be suitable for suspending GLA cells.
The suspension was quickly warmed to 37 "C and mixed
with an equal volume of previously melted agarose [lo mg
InCert grade agarose (FMC) ml-l TES buffer kept at 40 "C].
Aliquots of this mixture were rapidly dispensed into a plastic
mould, the mould was chilled, and the resulting 25 p1 blocks
were extruded into lysis buffer (0.5 M disodium EDTA, 1 YO
SDS and 1 mg proteinase K ml-l) and incubated at 52 "C for
48-72 h to release GLA chromosomes. (Many walled
bacteria must be treated with wall lytic enzymes such as
lysozyme and mutanolysin to obtain satisfactory lysis with
detergents.) The agarose blocks were washed in TE (10 mM
Tris, 1 mM disodium EDTA, pH 8.0) three times for at least
15 min each, resuspended in TE and gamma-irradiated
(12500 rad) to produce one (on average) random doublestrand break per chromosome (28). The use of gamma
irradiation to produce linearized chromosomes for PFGE
was also devised independently by Van der Bliek et al. (40).
Blocks were then subjected to PFGE, the DNA bands were
stained with ethidium bromide and photographed, and the
chromosome size was estimated by comparing DNA band
mobilities to yeast chromosome size markers as described
previously (28).
Chromosome isolation, PCR amplification and DNA
sequencing. Rigorous precautions were taken to avoid PCR
contamination. Deionized water for solutions from a flushed
apparatus was filtered through a 0.22 nm filter and solutions
made with this water were filter sterilized immediately after
preparation. Chromosome bands were cut from gels with a
flame sterilized scalpel and the DNA extracted from the gel
with glass powder (BiolOl) and eluted with 0 . 5 ~TE.
Controls were gel slices cut from unused lanes. 16s rRNA
genes were amplified (31 cycles: 94 "C, 1 min; 55 "C, 1 min;
72 "C 3 min, with a 10 min extension for the last cycle) using
two conserved 16s rRNA sequence primers: 8F (5'-AGAGTTTGATCMTGGCTCAG-3') and 1492R (5'-CGGTTACCTTGTTACGACTT-3'). The 50 or 100 pl reaction mix
contained 10 mM Tris/HCl, pH 8.3; 50 mM KC1; 2.5 mM
MgC1,; 200 pM (each) dATP, dCTP, dGTP and dTTP;
50pM each primer and 2.5 U Taq polymerase (PerkinElmer). The PCR products were subjected to electrophoresis, the band was extracted from the gel with glass
powder or a spin-column (Qiagen), and the DNA was
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Characterizing uncultivated prokaryotes
Fig. I # Transmission electron micrographs of lung thin sections from Grey-Lung-infected mice. (a) Numerous extracellular
bodies evident in the mucoid layer lining a pulmonary air-space, 3-4 weeks post-infection. Bar, 2 pm. (b) High-power
magnification of the extracellular bodies, showing their characteristic structure, absence of a cell wall and presence of a
bilaminar cell membrane. Bar, 0.2 pm.
sequenced directly with a dye terminator cycle sequencing
core kit and an automated sequencer (Applied Biosystems).
Amplification and sequencing primers were 8F, 339R, 5 19R,
536F, 786F, 907R, 1175F and 1492R (8,20,43).
Phylogenetic analysis. 16s rRNA gene sequences were
obtained from the GenBank database. Alignments of 16s
rRNA sequences were performed with the program
MULTALIN (http://www. toulouse.inra.fr/mutalin.html) and
further adjusted manually. Alignments were aided by comparison to the secondary structure models of the 16s rRNA
from the Gram-positive bacterium Bacillus subtilis (42) and
from Mycoplasma hyopneumoniae ( 1 5). Phylogenetic analysis of the sequence alignment was by the neighbour-joining
method of the program MEGA, version 1.01 (19).
Nucleotide sequence accession numbers. The following
Mycoplasma species 16s rRNA gene sequences were used in
this study: Mycoplasma hominis PG21T, accession no.
M24473 ;Mycoplasma mobile 163KT,M24480; Mycoplasma
mycoides subsp. mycoides UM30847, M23943 ; Mycoplasma
orale CH19299T, M24659; M . pulmonis PG34T, M23941;
and Mycoplasma sualvi Mayfield BT, M23936.
RESULTS AND DISCUSSION
Electron microscopic studies of lung sections (Fig. 1)
show numerous extracellular organisms in the alveoli
only of infected lungs and demonstrate that the agent
present has the morphology of a wall-less prokaryote
which closely resembles earlier descriptions of the
' Grey Lung virus ' (1 0 , l l ) : the pleomorphic organisms
lack q cell wall, are bound by a single unit membrane
(1 10 A), a n d the size is about 650 nm in diameter.
Furthermore, the infectious cells pass 0.45 pm filters.
A genomic probe kit reported t o be specific for
mycoplasmal r RNA (Mycoplasma TC ; Gen-Probe)
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391
H. Neimark, D. Mitchelmore and R. H. Leach
Fig. 2. Isolation of full-length chromosomes from the GLA by
PFGE. Lung washings from uninfected and GLA-infected mice
were prepared and subjected to PFGE as described in Methods.
Lanes: 1 and 5, Saccharomyces cerevisiae YPH149 chromosome
size markers; 2, lung-wash preparation from GLA-infected
Schofield strain mice; 3, control lung-wash preparation from
healthy Schofield strain mice; 4, preparation from GLA-infected
BALBk strain mice. Electrophoresis was in 1% agarose a t 11 "C
with a current of 150 mA and a switching interval of 60 s for
18 h in a transverse-field gel electrophoresis apparatus.
was used to examine mouse lung-wash fluids (21); on
high-titre infective preparations from consolidated
lungs at 4-23 weeks post-infection, hybridization
values of 7-39% were obtained as compared with
negative values (d 0.2 %) for uninfected lung or early
post-infection samples with no evident lung lesions
(< 3 weeks). These results provided the first molecular
evidence for the mycoplasmal nature of the GLA and
indicated that GLA is not a stable bacterial L-form.
Thus, the GLA appeared to be an animal mycoplasmalike organism (2 1, 27) analogous to the uncultured
100
80
M. hominis PG21T
67
94
-
Grey Lung agent
M. pulmonis PG34T
~
0.05
392
plant pathogenic mycoplasma-like organisms (MLOs,
now called phytoplasmas).
GLA suspensions embedded in agarose and then
gamma-irradiated produced linear chromosomes
which entered pulsed-field gels and migrated as a
single, discrete DNA band (Fig. 2). Identical size
bands were obtained with lung washings from infected
BALB/c and Schofield strain mice; no DNA bands
were detected in lung washings from healthy control
mice. The GLA chromosome size is 650 kb. The DNA
in unirradiated sample blocks did not travel
appreciably into the gel which indicates that only GLA
chromosomes which have sustained one DNA doublestrand break migrate in the gel. Thus, the electrophoretic behaviour of the GLA chromosome is identical to that of circular prokaryotic chromosomes (28),
and indicates that the GLA chromosome is circular.
The pulsed-field switching protocol used here separates
DNA molecules up to about 1600 kb in size while
larger DNA molecules form a band with a mobility
near that of the largest yeast chromosome size marker;
larger DNA molecules are resolved by employing
longer interval switching protocols. In natural or manmade environments where several different microorganisms are present, chromosomes may be isolated
by employing one or more different pulsed-field
switching protocols. Although the possibility exists
that more than one organism in a population could
have exactly the same chromosome size, this possibility
does not nullify the approach.
The GLA 16s rRNA gene was amplified from gelpurified chromosomes, sequenced directly in both
directions and the sequence compared to the GenBank
database. All the high score sequences found were
from Mycoplasma species which affirms that the cell
wall-less GLA is a mollicute and a member of the
genus Mycoplasma. Several of the high-score Mycoplasma species were members of the hominis group of
mycoplasmas (42), and the GLA 16s rRNA gene was
found to contain the characteristic hominis group
motif of a T residue at position 912 in the segment
[Escherichia coli
907-9 15 (-AAACTTAAA-)
numbering (7)] instead of the C residue found in
virtually all eubacteria (42). Additionally, characteristic dominating residues which occur in the hominis
.................................................................................. ...................
Fig. 3. Phylogenetic tree showing the
relationship of GLA to selected members of
the hominis group of mycoplasmas. The tree
was estimated by the neighbour-joining
method using 1321 positions in the 165
rRNA sequences from GLA and mycoplamas
obtained from GenBank. All alignment gaps
and missing information sites were deleted
before analysis. The sequence from M.
mycoides subsp. mycoides UM30847 was
used as the outgroup. Bootstrap confidence
level percentages obtained from 500
resamplings of the data set are shown a t
several nodes.
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Characterizing uncultivated prokaryotes
group with no more than one exception (33, 42) were
found at positions 157.164 (U-A), 158.163 ( G X ) ,
746 (U) and 824.876 (COG).However, position 610
was the dominating G common to mycoplasmas rather
than a dominating U ; also it is worth noting that the
GLA 16s rRNA gene does not contain an additional
loop-proximal nucleotide pair in the helix located
between positions 416 and 427, a character which
occurs in several members of the hominis group (42).
A phylogenetic tree (Fig. 3) produced by a neighbourjoining method (19) shows GLA is related more closely
to the hominis cluster than to any other recognized
cluster in the hominis group, however the length of the
GLA branch indicates that the GLA 16s rRNA gene
has diverged considerably from the sequences in the
hominis cluster. The similarity between the sequences
from GLA and M . orale CH19299T, the closest
mycoplasma sequence found, is only 87% while the
similaritybetween GLA and M . pulmonis is just a little
less at 84%; in contrast, the similarity between M .
orale CH19299Tand M. hominis PG21T,two members
of the hominis cluster, is 94%. A phylogenetic tree
with essentially identical structure (not shown) was
produced with the maximum-likelihood program
DNAML of the PHYLIP 3% package of programs (9).
Although the GLA 650 kb chromosome is small, it is
still within the size range of 580-2200 kb known for
cultured mollicutes (28, 35; P. Carle & H. Neimark,
unpublished), so small genome size alone does not
explain the inability to cultivate this wall-less
prokaryote; all the other known members of the
hominis group are culturable.
If a simple genomic lesion(@ is responsible for the
unculturability of GLA, it is conceivable that
complementation with DNA from a close relative
could permit GLA to grow on artificial media. This
notion should be applicable to many other uncultivated bacteria.
The procedure described here permits isolation of
individual chromosomes and amplification from these
templates should obviate formation of hybrid molecule
PCR artifacts (‘ shuffled ’ molecules). PCR products
can usually be assumed to be homogeneous, but
artifact DNA products can result when related target
sequences are co-amplified ; the artifact product is a
chimeric molecule composed of parts of two different
template sequences (6,22,24,26,32,37). In the case of
rRNA, secondary structure analysis does not necessarily detect chimeric structures (17). Of course, the use
of any purified chromosome does not alter the fact that
sequencing problems may occur where more than one
rRNA operon exists and the operons contain alternate
nucleotides at one or a few positions in the rRNA
genes (33).
The single 650 kb chromosomal DNA band isolated
and amplified here almost certainly came from the
wall-less GLA, but conclusive evidence would require
in situ hybridization to lung thin sections with a specific
DNA probe.
The Grey Lung disease agent appears to be a newly
recognized mycoplasma, still it and other uncultivated
bacteria should not be given specific names only on the
basis of 16s rRNA sequence data, since previously
recognized species whose 16s rRNA has not been
sequenced could contain the same sequence. In concurrence with guidelines suggested for recording the
properties of uncultivated prokaryotes (25), we propose giving the GLA a Candidatus designation with
the following description : ‘ Candidatus Mycoplasma
ravipulmonis’ (M.L. adj. ravus greyish; M.L. n. pulmo
the lung; M.L. compound gen. n. ravipulmonis of a
grey lung) [(Mollicutes) NC; NA; 0 , wall-less; NAS
(GenBank no. AFOOll73), oligonucleotide sequence
complementary to unique region of 16s rRNA 5’CGCTTGGCAACCCATTGTAACCGC-3‘; S (MUS
musculus, lung) ; MI.
ACKNOWLEDGEMENTS
This paper is dedicated to the late Dr D. G. ff. Edward, who
instigated our first attempts (R.H.L., 1961) to confirm the
mycoplasmal nature of the Grey Lung agent and whose
enthusiasm for this question engendered our own persisting
commitment to resolving it. We are grateful to David
Conway for skilled collaboration in the nasal inoculation
and lung washing of mice at the Central Public Health
Laboratory and also to Kitty Plaskitt for her expertise in
carrying out the electron microscopy and for providing
electron micrographs. We also thank Christopher Lange for
use of the gamma radiation source and PFGE equipment.
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