SECTION B
STRAIN IDENTIFICATION INTRODUCTION
Rhizobia that have dramatic differences in such important traits
as host specificity, infectiveness (invasiveness) and
effectiveness are indistinguishable from each other under the
microscope.
However, there are many circumstances in which
recognition of a particular rhizobial strain and monitoring its
occurrence following introduction to a soil environment is
important in ecological studies.
Indirect procedures are
available for this purpose.
Serological markers
Any substance which provokes an immune response when introduced
into the tissue of an animal or human is referred to as an
antigen.
In work with rhizobia, rabbits are commonly used for
immunization and the antigens are rhizobial cell preparations.
As a result of antigen injections, complex immunological
reactions result in the rabbit producing special proteins called
globular antibodies (immunoglobulins).
These antibodies are
found in the serum portion of the blood, and the study of the
reactions of the immune serum with the antigens outside the
animal is known as serology.
Antigen-antibody reactions are
highly specific in that the antibody reacts only with the antigen
that elicited its formation.
As in other bacteria, antigens of rhizobia can be categorized
into somatic, flagellar, and capsular, depending on their
derivation.
Somatic antigens are closely related to the
rhizobial cell-wall and usually designated by the letter "O".
Some somatic antigens may be tightly bound to the cell wall, in
which case they are not removed by washing of the cells;
therefore, these antigens are only detected when whole cells of
rhizobia react with the antibody as in agglutination or
immunofluorescence.
The somatic antigens that are soluble and
easily removed by washing are detected by precipitation in gel,
as in the Ouchterlony double-diffusion process.
are also heat stable.
Somatic antigens
They are the most specific of the three
groups of antigens.
The tiny whip-like appendages (flagella) of the rhizobia are also
antigenic and appropriately called flagellar or H-antigens.
They
are heat labile and are commonly detected by agglutination or
immunofluorescence.
The capsular (extracellular) antigens are surface antigens and
are found outside the cell itself.
They are usually designated
by the letter "K."
In rhizobial serology, both cultured cells and nodule antigens
(bacteroids) are used for strain identification.
Basic concepts
on some serological methods for identification of rhizobia are
described below.
Agglutination.
The process in which the antigens are linked
together by their corresponding antibodies is called
agglutination.
The linked antigens may be microscopically or
macroscopically visible as clumps, agglutinates or aggregates.
The agglutination reaction depends on a firm structural
relationship between an exposed bacterial antigen and the
antibody.
Linus Pauling's lattice hypothesis (Figure B.1) is the
widely accepted concept for explaining the agglutination
reaction.
Pauling postulated that the antibody is bivalent and
the antigen is multivalent, and that the antigen-antibody
complexes are molded into a lattice or framework of alternating
antigen-antibody particles.
Figure B.1.
Lattice formation in an antigen-antibody reaction
Precipitin reaction.
In recent years the precipitation reactions
of somatic antigens have been used extensively for work with
rhizobia.
The precipitation reaction occurs when certain soluble
antigens are brought into contact with the corresponding
antibody. Precipitation differs from agglutination in that the
precipitating antigens are not whole bacterial cells (cellular)
but are proteins or polysaccharide molecules in solution.
In the
double-diffusion technique, gels, usually clarified agar, are
used as matrices for combining diffusion with precipitation.
The
reactants simply diffuse through the gel towards each other and
precipitation results when the equivalence points have been
reached.
Antigen preparation of a rhizobial strain will give
rise to one or more lines of precipitation in the presence of the
homologous antibody.
When two antigens are present in a system,
they behave independently of one another.
The different types of
precipitation reactions are illustrated in Figure B.2.
Rhizobial strains that share some of or all their antigens will
cross-react with respective antisera.
These cross-reactions may
be encountered in both agglutinations and precipitations.
Immunofluorescence.
Certain chemical dyes (fluorescein
isothiocynate and lissamine rhodamine) have the property of
fluorescing when excited by near ultraviolet light.
Rhizobial
antibodies developed in rabbits can be conjugated to these
fluorescing chemical dyes or fluorochromes.
In work with
rhizobia, the chemical dye commonly used for labeling the
specific antibody is fluorescein isothiocyanate (FITC) which has
an apple-green fluorescence upon irradiation with blue light.
In
practice, a smear of rhizobial cells (cultured, or from a nodule)
is made on a microscope slide, and this smear is allowed to react
or "stain" with the specific antibody labeled with FITC.
After
appropriate washing to remove uncombined and excess labeled
antibody, the smear may be viewed through a UV-microscope fitted
with appropriately complementary filters, and an apple-green
fluorescence of the bacterial (rhizobial) cells would mean that
the antigen smear has reacted with the FITC-labeled antibody.
There are two types of fluorescent antibody techniques, namely
the direct- and indirect-immunofluorescence.
In the direct
method the specific antiserum is conjugated and is used as a
"stain" in the procedure.
This is different from the indirect
method where the unconjugated (unlabeled) specific or primary
antibody is first reacted with the antigen smear, and after
sufficient time is allowed for antigen-antibody reaction, the
smear is then washed free of excess antiserum.
This step is
followed by "staining" with the FITC-labeled secondary antibody.
In serological work with rhizobia, the specific or primary
antibody against the rhizobial strain is most often developed in
rabbits.
The secondary antibody is developed by immunization of
goats or sheep with purified rabbit immunoglobulins from a
previously unimmunized rabbit.
Thus, the rabbit immunoglobulin
serves as an antigen for immunization of the goat or sheep.
Therefore, the antibody produced in the goat or sheep will not
only react with the rabbit antiserum, but also with rhizobial
Figure B.2.
Precipitation reactions
antigen with specific unlabeled rabbit antibody attached when the
indirect procedure is employed.
Though the results are the same,
the indirect method is considered more sensitive.
The indirect
method requires the labeling of only the immune serum from the
goat or sheep, and involves two reaction steps; but the indirect
method is also known to give more nonspecific staining
reactions.
In the direct method, each rabbit antiserum developed
against each rhizobial strain must be conjugated.
The two
methods are illustrated diagrammatically in Figures B.3 and B.4.
Antibiotic resistance markers
When high density inocula of a rhizobial strain are inoculated
into media containing an antibiotic, a few cells may exhibit
resistance as a result of spontaneous genetic changes or
mutations.
The resistance of a rhizobial strain to a particular
antibiotic is a useful marker.
If the mutant strain is used to
inoculate a legume then nodules occupied by that strain may be
identified by plating nodule isolates on media containing the
respective antibiotic.
The mutant rhizobial strain will grow on
the antibiotic media and other bacteria will be suppressed.
It
is important that antibiotic-resistant mutants that are selected
for inoculation experiments have not lost their infectiveness
(ability to form nodules) nor their effectiveness (ability to fix
nitrogen) in the symbiosis with the host plant.
The symbiotic
capacity of the mutant should be compared with its parent culture
from time to time.
The mutant should be stable throughout the
steps of infection, nodulation, nitrogen-fixation and subsequent
re-isolation.
Streptomycin resistance is frequently used as a marker for
rhizobia.
Mutants resistant to this aminoglycoside are stable,
have a low incidence of cross-resistance, and infrequently lose
their symbiotic capacity.
Besides streptomycin, spectinomycin
and rifampicin have also been used.
Highly resistant mutants
with single or double markers (streptomycin-spectinomycin or
streptomycin-rifampicin) can be obtained with one exposure of the
rhizobia to low concentrations of these antibiotics or by
successive selection for resistance.
Figure B.3.
Direct immunofluorescence
Cross-resistance is a phenomenon whereby a bacterium develops
resistance to a second antibiotic as a result of resistance to
the first.
This may happen if the antibiotics are closely
related.
The parallel use of antibiotic and serological markers, both
relatively stable in themselves, provides a means of confirming
the stability of each marker independently in ecological research
with rhizobia.
Compared to the serological marker techniques
(fluorescent antibody, enzyme immunoassays, gel-diffusion and
agglutination) the development and use of antibiotic resistant
Figure B.4.
Indirect immunofluorescence
markers is relatively inexpensive and does not require
sophisticated equipment.
Bacteriophage markers: phage typing
Viruses that infect bacteria (bacteriophage) were independently
discovered by Twort and by d'Herelle in 1917 and 1919,
respectively.
Since then, the processes of infection and
multiplication have been well defined.
The first step involves
the adsorption of the virus to specific receptors on the
bacterial cell (somatic), flagella or pilli.
This is followed by
the injection of the viral nucleic acid into the bacteria.
The
nucleic acids utilize the machinery of the host cell to
replicate, leading to the accumulation of several copies of the
viral nucleic acid.
These nucleic acids are packaged
newly-synthesized viral coat protein and are then released by
lysis of the host cell, liberating many infective viruses.
Susceptibility of a certain bacterial strain to a particular
bacteriophage forms the basis for phage-typing.
One approach in
a phage-typing scheme is the use of a group of phages with
different host-specificities.
The bacteria can then be placed
into groups (lysotypes) if they are susceptible to some of the
phages and not others.
Through this means, bacteriophage-marked
rhizobia can be indirectly traced in soil, in isolates from
nodules and in laboratory experimentation.