Acholeplasma laidlawii PG8 culture adapted to unfavorable growth conditions shows an expressed phytopathogenicity.

Abstract

Mycoplasmas are the smallest, self-replicating, prokaryotic organisms with avid biochemical potential and spreading in higher eukaryotes in nature. In this study, Acholeplasma laidlawii PG8 cells were cultivated on a deficient medium for 480 days resulting in a mycoplasma culture that was adapted in vitro to unfavorable growth conditions. Cells that survive this condition had decreased sizes (about 0.2 microm) and increased phytopathogenicity. This resulted in more frequent appearance of various morphological alterations when plants of vinca (Vinca minor L.) were infected by adapted mycoplasma cells. The increasing pathogenicity was accompanied by changes in genome expression in these adapted cells. Further studies are needed to explore the exact mechanisms that permit adaptation to unfavorable growth conditions and changes in phytopathogenic potential.

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Research Article
TheScientificWorldJOURNAL (2007) 7, 1–6
ISSN 1537-744X; DOI 10.1100/tsw.2007.25


*Corresponding author.
©2007 with author.
Published by TheScientificWorld, Ltd.; www.thescientificworld.com


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Acholeplasma laidlawii PG8 Culture
Adapted to Unfavorable Growth Conditions
Shows an Expressed Phytopathogenicity
Vladislav M. Chernov, Natalia E. Moukhametshina, Yurii V. Gogolev,
Tatiana N. Nesterova, Maxim V. Trushin*, and Olga A. Chernova
Kazan Institute of Biochemistry and Biophysics, Russian Academy of Sciences,
Kazan, Russia
E-mail: mtrushin@mail.ru
Received August 23, 2006; Revised December 19, 2006; Accepted December 20, 2006; Published January 10, 2007
Mycoplasmas are the smallest, self-replicating, prokaryotic organisms with avid
biochemical potential and spreading in higher eukaryotes in nature. In this study,
Acholeplasma laidlawii PG8 cells were cultivated on a deficient medium for 480 days
resulting in a mycoplasma culture that was adapted in vitro to unfavorable growth
conditions. Cells that survive this condition had decreased sizes (about 0.2 μm) and
increased phytopathogenicity. This resulted in more frequent appearance of various
morphological alterations when plants of vinca (Vinca minor L.) were infected by adapted
mycoplasma cells. The increasing pathogenicity was accompanied by changes in
genome expression in these adapted cells. Further studies are needed to explore the
exact mechanisms that permit adaptation to unfavorable growth conditions and changes
in phytopathogenic potential.
KEYWORDS: Acholeplasma laidlawii PG8, phytopathogenicity, ultramicroforms

INTRODUCTION
Mycoplasmas are the smallest, self-replicating, prokaryotic organisms. Despite restricted biochemical
properties, these bacteria spread in various biotic communities and are able to persist in higher eukaryotes
through their circulation in nature. It seems that the strong adaptability of these organisms resides in
molecular genetic principles that permit them to adapt to unfavorable growth conditions (UGC). In our
previous reports, we took some initial steps in exploring this adaptability[1,2], however, our knowledge
of this mycoplasma potential is still in preliminary stages.
Acholeplasma laidlawii, a “ubiquitous” mycoplasma, is found in soil, compost, and wastewaters, as
well as in tissues of humans, animals, and plants[3]. Previously, we showed that the adaptive reactions of
A. laidlawii PG8 to UGC were connected with transformation of the vegetative forms of the mycoplasma
cells into ultramicroforms (UMF) resistant to biotic and abiotic stress factors[1,2], a process that is called
nanotransformation. Nanotransformation is not limited to mycoplasmas and has been shown in a number
of bacteria. In some bacteria, adaptation to UGC was followed by distortions of pathogenicity. In our
prior model of plant infection by mycoplasmas, we showed that A. laidlawii PG8 could cause

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phytomycoplasmosis[4,5]. In this study, we tried to show whether adaptation to UGC could affect the
phytomycoplasmosis capability of A. laidlawii PG8 culture cells.
MATERIALS AND METHODS
Acholeplasma laidlawii PG8, a reference strain, was obtained from the N.F. Gamalei Research Institute of
Epidemiology and Microbiology (Moscow, Russia). Mycoplasma cells were cultivated for 2 days at 37°C
on Edward’s medium (1 l of this medium consisted of tryptic extract of bovine heart – 850 ml, serum of
horse blood – 100 ml, fresh yeast extract (5%) – 40 ml, glucose [40% solution] – 5 ml, benzylpenicillin
[1,000,000 IE/ml] – 5 ml) to obtain a culture unadapted to UGC. To obtain a mycoplasma culture adapted
to UGC, glucose and yeast extracts were eliminated from Edward’s medium; microorganisms were kept
in this condition for 480 days as was previously described[1]. To detect the titer of the colony-forming
units (CFU), semifluid and solid-state variants of the Edward’s medium with addition of 0.5% and 1.2%
agarose were used. Two-dimensional protein electrophoresis was performed as was described by us[2].
PCR analysis of DNA was done using specific AL16LF and A23LR primers for amplifying nucleotide
sequences of A. laidlawii PG8 rRNA operons as was previously reported[2].
The phytopathogenicity of the mycoplasma culture cells adapted and unadapted to UGC was
investigated on substantive stalks of vinca (Vinca minor L.), a specific indicator of
phytomycoplasmosis[6]. Both control and experimental groups consisted of 20 plants with similar size
and age of the root. Plant seedlings were infected with mycoplasmas on the 15th day after rooting. It was
hypothesized that adapted culture of A. laidlawii PG8 has an increased phytopathogenic potential. To
compare a level of its phytopathogenicity, we knowingly used a lesser amount of A. laidlawii PG8 cells
adapted to UGCs. Our presupposition that a decreased number of cells may cause increased
phytopathogenic alterations (in comparison with unadapted culture) was totally confirmed in our study
(see results). We injected 10 μL of A. laidlawii PG8 culture (105 CFU of adapted culture and 107 CFU of
unadapted mycoplasma culture) by microsyringe into the root waist. In the control group, plants were
injected with 10 μL of sterile nutrient medium. Control and experimental plants were monitored for 6–8
weeks, a time necessary for manifestation of phytomycoplasmosis. The alterations in plant characteristics
were recorded. These alterations were previously described[6] and included plant delay in bine growth,
chlorosis, necrosis, leaf marcescence, and abnormalities in bine development.
A Hitachi-110 transmission electron microscope (Hitachi, Japan) was used for analysis of the in vitro
cultivating mycoplasma cells. Ultrathin sections were obtained using LKB-III ultramicrotome (Sweden).
The material under study was fixed with glutaraldehyde (2.5%) prepared on a 0.1 M phosphate buffer (pH
7.2) for 12 h. Then, the material was dehydrated using an acetone series and postfixed in 0.1% OsO4 with
addition of 34 mg/ml of saccharose.
Numerical data are presented as mean ± standard deviation. A probability level of p < 0.05 was
considered significant[7].
RESULTS
The adapted A. laidlawii PG8 cells showed significant reduction in size compared to unadapted cells (Fig.
1). On the solid medium, the UMF formed specific microcolonies 50–300 μm in size. This is in contrast
to typical mycoplasma cells that form “fried eggs” colonies. The UMF were able to revert into original
vegetative forms of A. laidlawii PG8 cells when transferring to the full Edward’s medium, showing an
evident viability and resistance to stress factors.

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FIGURE 1. Electron micrography of A. laidlawii PG8 cells. Cultures were
grown on Edward’s medium for 2 days (A) and on the impoverished medium
for 480 days (B).

FIGURE 2. Morphosis in plants of V. minor L. infected by cells of A. laidlawii
PG8 adapted culture. (a) Chlorosis and abnormalities of bine development
(bulging of plant ribs); (b) necrosis (of the leaf edge); (c) abnormalities of bine
development; (d) necrosis (of the apical leaves); (e) abnormalities of bine
development (microplasia of the infected plant, right; normal plant, left).
Infecting plants of V. minor L. by A. laidlawii PG8 culture adapted in vitro to UGC resulted in the
appearance of plant infection (Fig. 2). Plants infected by A. laidlawii PG8 culture unadapted to UGC
showed significantly less signs of infection. For example, chlorosis was seen in 75% of plants infected
with adapted mycoplasma in comparison with 40% in those with infection caused by unadapted

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mycoplasma. The other signs of infection were also more prevalent in adapted mycoplasma culture, such
as necrosis by (50 vs. 25%), leaf marcescence (50 vs. 25%), and abnormalities of bine development (30
vs. 0%) (Table 1).
TABLE 1
A Number of V. minor L. Plants with Various Morphological Deviations in Control and
Experimental Groups*
Type of Phytomycoplasmosis
Variant of
Experiment
Chlorosis NecrosisLeaf
Marcescence
Abnormalities
of Bine
Development
Mean ± SD
Control 3 (15) 0 (0) 0 (0) 0 (0) 0.75 ± 1.5
(3.75 ± 7.5%)
4.50 ± 3.32
(22.5±16.58%)
10.25 ± 3.68
(51.25 ± 18.43%)
Unadapted culture 8 (40) 5 (25) 5 (25) 0 (0)
Adapted culture 15 (75) 10 (50) 10 (50) 6 (30)
* Absolute number of plants is indicated (percentage of the respective morphological deviations is shown
in brackets).
The obtained results favor the hypothesis that adaptation in vitro of A. laidlawii PG8 culture to UGC
was followed by a significant increase of its pathogenic potential.
DISCUSSION
Previously, we showed that adaptation of A. laidlawii PG8 culture to UGC was followed by
nanotransformation, transformation of the vegetative form of cells of the mycoplasma into UMF resistant
to biotic and abiotic stress factors[1,2]. There were noticeable differences in the morphological,
biochemical, and genetic molecular properties between the vegetative form of cells of the mycoplasma
and its UMF.
As a result of the analysis of two-dimensional electrophoresis data, significant differences in the cell
polypeptide spectra of A. laidlawii PG8 culture adapted to UGC and the unadapted one were registered
(Fig. 3). This might be evidence for an essential reorganization of the mycoplasma genome expression
during nanotransformation, entering the vegetative form of cells of the mycoplasma into UMF due to
UGC.
Other researchers have shown that there is a reverse attenuation of PCR signals for DNA of bacterial
cells during cultivation in UGC[8]. Appearance of differential amplification of A. laidlawii PG8 rrnA and
rrnB nucleotide sequences due to dissociation of the cell culture population was reported in our recent
study[9]. The data were considered to be useful to detect dissociation in cell population of the
mycoplasma culture and to confirm transformation of the vegetative form of A. laidlawii PG8 cells into
UMF in plant tissues.
In our previous experiments, we demonstrated that infecting by A. laidlawii PG8 culture unadapted to
UGC was followed by the appearance of ultrastructural, morphophysiological, and biochemical
deviations correlated with transformation of the vegetative form of the mycoplasma cells into UMF[1,2].
The data from our present study show that A. laidlawii PG8 culture adapted to UGC has higher expressive
phytopathogenicity than the unadapted one. Even a 100-fold difference in a number of cells could not

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FIGURE 3. Electrophoregram of polypeptides of vegetative cells
(A) and ultramicroforms (B) of A. laidlawii PG8. The arrows mark
the polypeptides different in the mycoplasma cells.
influence the situation. In the plants infected by A. laidlawii PG8 unadapted to UGC culture, the
symptoms of the mycoplasma infection were detected somehow later, and were less evident in
comparison with the adapted mycoplasma culture (Fig. 4). A delayed manifestation of the mycoplasma
infection by plants infected by unadapted mycoplasma could be related to the time that is needed for
transformation of the vegetative form of cells into UMF. A comparison of the injected-adapted
mycoplasma strain and the isolated one from the plants on biological differences remains to perform.











FIGURE 4. Detection of A. laidlawii PG8 by PCR with primers LA16LF
and LA23LR in plants (V. minor L.). The lanes 1–4 contain PCR products
of DNA extracted from plants infected with cells of adapted (1, 2) and
unadapted (3, 4) culture of the mycoplasma through 4 days (1, 3) and 21
days (2, 4) after injecting. The lanes 5–7 correspond to negative (5) and
positive (6, 7) controls – PCR products of DNA (230 ng) extracted from
uninfected plants (5), mixed DNAs (230 ng/10 ng) of uninfected plants
and cells of unadapted culture of the mycoplasma (6) and DNA (10 ng)
extracted from cells of unadapted culture of the mycoplasma (7). The lane
M corresponds to marker for length of DNA fragments (b.p.).
Changes in genome expression reorganization in bacteria adapting to UGC may explain the shift of
bacterial metabolism and distortion or disappearance of virulence of some microorganisms[9,10,11,12].
However, this is the first report that shows that, at least in mycoplasma, adaptive processes were followed
by an increase in pathogenicity. Further studies are needed to explore the exact mechanisms that permit
adaptation to unfavorable growth conditions and changes in phytopathogenic potential.