1. Production of Latent Viruses from Nitrogen-Fixing Bacterial Symbionts of Soybean
Tessa R. Jarvis1, Suhair Najhm1, Jeffry J. Fuhrmann2, K. Eric Wommack1
1Delaware Biotechnology Institute, University of Delaware, Newark, Delaware
2Department of Plant and Soil Sciences, University of Delaware, Newark, Delaware
Bradyrhizobium is a genus of gram-negative soil bacteria. Bradyrhizobium elkanii and B.
diazoefficiens form a symbiotic relationship with soybean by attaching to the root hairs and
eventually forming nodules on the roots. In the nodules, Bradyrhizobium cells fix atmospheric,
plant-unavailable dinitrogen gas (N2) into a plant-useful form (ammonia [NH3]). In exchange,
the bacteria receive sugars and other growth factors from the plant. Some subspecies (strains)
of these Bradyrhizobium have lysogenic prophages (i.e., latent or temperate viruses) that
incorporate their genome into that of the bacterium. In this way the viral genome is
maintained as part of the host bacterial genome. It is of interest to determine if these
incorporated Bradyrhizobium viruses (prophages) can again become free viruses through either
the addition of inducing agents (e.g., mitomycin C or norfloxacin) or spontaneously without an
external chemical cue. To determine the nature of their induction, two species of
Bradyrhizobium (B. diazoefficens USDA 122 and B. elkanii USDA 76) were grown in laboratory
culture for 48 hours either with or without the addition of chemical inducing agents. Two
replicates of the treatment group and control group for each strain were sampled after 0, 6, 12,
24, 48 hours of growth. Significant changes in the abundance of free viruses within cultures of
B. elkanii USDA 76 demonstrated that the associated prophages induce spontaneously . Viral
populations were considerably higher than the bacterial populations (at least 10-fold larger) at
each sample time. This finding may have important implications for our understanding of the
life cycles of Bradyrhizobium and how its species interact with soybean in agricultural
environments.
Introduction
USDA 76 Bradyrhizobium elkanii and USDA 122 B. diazoefficiens are found within the root
nodules of legumes. These bacteria transform atmospheric nitrogen into ammonia, thus
providing legumes with usable sources of nitrogen.
Bacteriophages (phages) are viruses that infect bacteria. Phages replicate either through a lytic
(virulent phages) or lysogenic (temperate phages) life cycle. USDA 76 and USDA 122 are
believed to harbor lysogenic phages. Lysogenic phages integrate the viral genome within the
host DNA genome. In this state, the lysogenic phage is called a prophage or provirus. The
integrated prophage genome replicates along with replication of host genome so that a single
prophage genome copy is transferred to the daughter cell. During this process the cellular
machinery of the host is only slightly disturbed. So, the host cell in lysogeny does not get lysed
and the virus particles are liberated only rarely.
Integrated prophage can be induced to become free viruses. The viral induction process excises
the prophage from the bacterial chromosome under certain conditions such as exposure to UV
light or antibiotics like Mitomycin C and Norfloxacin. Induction can also occur spontaneously
with a loss of the regulatory machinery that represses the expression of prophage genes. After
induction takes place, lytic viral replication begins ultimately lysing the bacterial cell to release
viral particles. The rate of lytic viral production from induction of prophage can be calculated
through timecourse experiments monitoring changes in viral and bacterial cell abundance.
In previous experiments spontaneously inducing prophage have been detected in USDA 761.
Our work sought to determine the frequency and spontaneous induction rate of prophages
from broth cultures of USDA 122 B. diazoefficiens and USDA 76 Bradyrhizobium elkanii.
Methods
Conclusion
Abstract
Results
Acknowledgements:
This publication (or program) was made possible by the National Science Foundation EPSCoR Grant No. IIA-
1301765 and the State of Delaware.
References:
1 Fuhrman, C. A. (2010). Enumeration of virus particles in aquatic or sediment samples by epifluorescence microscopy. Manual of
Aquatic Viral Ecology , 9.
Two soybean nodulating USDA strains of Bradyrhizobium species were obtained from Dr.
Fuhrmann of the department of Plant and Soil Science: USDA 76 B. elkanii and USDA 122 B.
diazoefficiens. These bacteria were grown in a prepared modified arabinose gluconate media
(MAG). Each strain was then innoculated into four centrifuge tubes (two control and two
treatment°). The treatment groups were exposed to Mitomycin C and Norfloxacin. All samples
were then centrifuged and then transferred to autoclaved beakers with fresh MAG on a rotary
shaker for the remaining of the experiment. Ten milliliters of each of the eight cultures were
sampled at 0, 6, 12, 24, and 48 hours post induction. The bacterial samples were taken directly
from the culture. The viral samples were centrifuged and filtered through a 0.22 µm syringe
filter at each time point to remove bacteria from the sample. These samples were then stored
at -80°C until they were counted. The viral samples were thawed then filtered a second time to
remove any bacteria that may have passed through the initial filtering. Viral particles within
each sample were captured on a 13mm anodisc filter (0.02µm) using a vacuum manifold and
stained with a 2.5X dilution of SYBR Gold in a Tris-HCl buffer (pH 8.14).1 These filters were
placed on slides and observed using epifluorscence microscopy under FTIC excitation (Fig. 5).
The average number of virus like particles for each filter were calculated using pictures taken
on the microscope.
Table 1: Experimental design
Image 1. Growth comparison of non-
nodulated soybeans (left) and
nodulated soybeans (right), courtesy of
Dr. Fuhrmann.
USDA
strains
(Be 76 &
Bd 122)
in MAG
Inoculated
strains
into 8
labeled
beakers
Exposed
treatment groups
to inducers, then
put all reps in
fresh MAG
Sampled
at
0,6,12,24
and 48
hours
Viral
samples
stored at
-80°C
Thawed, filtered,
and stained
samples
Counted using
epifluorescence
microscopy
Future Work
• Continue to count virus like particles for the 6 hour timepoint samples
• Reevaluate USDA 122 B. diazoeffficiens data to determine the cause of bacteria in the
viral samples and low inconsistent viral counts
• Possibly reproduce the procedure using Mitomycin C alone as an inducer to eliminate
any error from using both inducers at once
• Obtain more data to prove B. elkanii spontaneously induces
• There were significantly more viruses than bacteria (USDA 76 Bradyrhizobium elkanii) at each
time point in the control treatment which indicates spontaneous induction.
• For both USDA strains, there were less viruses in the treatment groups indicating the
Mitomycin C and Norfoxacin had a negative effect on viral induction and replication.
Fig. 2: Change in abundance of viruses and bacteria within control and treatment groups for
USDA 122. Red lines (bacterial abundance), black lines (viral abundance), solid lines (control),
dashed lines (treatment), bars (virus to bacteria ratio).
Fig. 1: Flow chart of
basic steps of
procedure
Fig. 5: Sample image from
epifluorescence microscope
for USDA 76 B. elkanii control
replication 1 at hour 12
(arrows indicate viruses)
Isolate B. elkanii 76 B. diazoefficiens 122
Treatment Control Treatment Control Treatment
Rep 1 2 1 2 1 2 1 2
Fig. 3: Change in abundance of viruses and bacteria within control
treatment for USDA 76. Red lines (bacterial abundance), black lines
(viral abundance), solid lines (control), dashed lines (treatment), bars
(virus to bacteria ratio).
• Over time there were more bacteria than
viruses in the USDA 122 control and
treatment samples, making the virus to
bacteria ratio relatively low at all time
points (Fig 2).
• Unlike USDA 122 control, at almost all
sampling time points for the USDA 76
control and treatment samples there were
more viruses than bacteria and relatively
high virus to bacteria ratios (Fig 3).