1022-7954/04/4004- © 2004
Russian Journal of Genetics, Vol. 40, No. 4, 2004, pp. 356–362. Translated from Genetika, Vol. 40, No. 4, 2004, pp. 454–461.
Original Russian Text Copyright © 2004 by Roumiantseva, Andronov, Sagulenko, Onishchuk, Provorov, Simarov.
Native populations of nodule bacteria have a high
level of genetic diversity, but the largest portion of these
bacteria were rhizobial strains of high competitiveness
but low effectiveness . It is known that some biolog-
ical and nonbiological factors may signiﬁcantly affect
the level of natural polymorphism of populations.
Legume host plants play an important role in this,
because most stable and numerous populations of nod-
ule bacteria are formed in the presence of these exact
plants . The duration of legume cultivation also
directly affect the population structure of rhizobia .
Strains isolated from the soil under alfalfa grown for
several years exhibits elevated plasmid diversity with
respect to both the number and molecular size, as com-
pared with those from soils, where the host plant was
not cultivated. Note that in the course of studies of the
interaction between plants and agrobacteria that are
taxonomically close to rhizobia, mutations may arise in
these microorganisms . Such studies were virtually
not conducted with alphalpha nodulated rhizobia. It is
very likely that the host plant may affect the genetic
structure of the microsymbiont during legume–rhizobia
symbiosis as well.
The process of inoculation of legume plant with
rhizobia consists of several steps, the ﬁrst of which is
the penetration of bacteria into the host plant root and
formation of infection thread. It has been recently ascer-
tained that upon growth of infection thread accompanied
by vigorous bacteria division, drastic alkalization of
plant cell cytoplasm is provoked by Nod factors .
These conditions are stressful for rhizobia, so they must
have developed tolerance for them. After the release of
the infection thread into the cytoplasm of plant cell,
rhizobia are converted to nitrogen-ﬁxing bacteroids fol-
lowing a number of cytomorphological alterations .
The formation of bacteroids includes at least four
stages, which results in the loss of division ability and
in cessation of chromosomal and plasmid DNA replica-
tion. Rhizobial cells retain their usual size and cyto-
plasm composition characteristic of free-living forms
only during the ﬁrst stage of bacteroid formation .
Meanwhile, despite the fact that the majority of rhizo-
bial cells are transformed irreversibly into bacteroids,
the release of viable rhizobial cells from old degraded
nodules into soil occurs at completion of the vegetative
period [6, 7].
It is known that bacteria in the nodule usually result
from propagation of one cell initially penetrated into
the root of host plant [2, 5] and, therefore, they have the
same genotype. Nevertheless, it was shown that some
clones isolated from nodules may have phenotypic dif-
ferences  and altered symbiotic properties [9–11].
Analysis of rhizobial strains after passing them through
host plant nodules also revealed that strains stably
retained the plasmids, which they contained earlier .
However, rearrangements of plasmids, deletions, or
even plasmid loss were detected in some cases [10, 11].
It should be noted that alterations of plasmid proﬁle
were observed, as a rule, in genetically modiﬁed strains
containing foreign plasmid DNA [12–14]. Remember
that no systematic studies on genetic stability of native
Instability of Cryptic Plasmids
P108 in the Course of Symbiosis
M. L. Roumiantseva, E. E. Andronov, V. V. Sagulenko, O. P. Onishchuk,
N. A. Provorov, and B. V. Simarov
All-Russia Research Institute of Agricultural Microbiology,
St. Petersburg, Pushkin, 196608 Russia; fax: (812) 470-43-62; e-mail: firstname.lastname@example.org, email@example.com
Received July 9, 2003
—Instability of cryptic plasmids in
laboratory strains SXM1, DM7-R, and
P108 as well as in their clones isolated from nodules of alfalfa grown during a long-term microvegetation exper-
iment (120 days) was studied. The isolated clones of strains SXM1 and DM7-R manifested stable inheritance
of plasmids, whereas 12.7–14.0% of clones with changed plasmid proﬁle were detected in a population of
clones from strain P108. These segregants were designated as P108c. Segregants P108c exhibited signiﬁcantly
decreased symbiotic effectiveness, nitrogenase activity, and the competitiveness with respect to alfalfa, com-
pared to the original strain P108. It was established that a 80-kb deletion occurred in a larger of two cryptic
plasmids (240 and 230 kb) of segregants P108c. It was concluded that genetic rearrangements are possible in
rhizobial clones that did not undergo structural transformation and retained viability in the nodule during the
natural vegetation period of alfalfa.