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Plant Science 162 (2002) 267–271

The generation of a transposon-mutagenized Burkeholderia glumae
library to isolate novel mutants

Paul A. Nakata *
USDA/ARS Children’s Nutrition Research Center, Department of Pediatrics, Baylor College of Medicine, Houston, TX 77030-2600, USA
Received 10 August 2001; received in revised form 23 October 2001; accepted 24 October 2001
Abstract
The gram-negative bacterium, Burkeholderia glumae, is a pathogen of rice. As a step toward identifying pathogenicity
determinants, a transposon mutagenized B. glumae library was generated. A transposition frequency of 2.1×10
6
cfu/mg of DNA
was achieved using a recently developed Tn5-derived transposon system. Southern-blot analysis of several randomly selected
colonies indicated that the transpositions were single, random events. Two genetic screens were conducted to assess the mutagenic
frequency of the generated library. Screening of 1000 colonies for auxotrophs resulted in the identification of 46 mutants. The 46
mutants were subdivided into five classes based on their ability to grow on supplemented medias. Exposure of 1000 mutagenized
colonies to UV light, as part of a second screen, identified two fluorescent mutants. The transposon library will be a useful tool
in the investigation of metabolic pathways in B. glumae and allow a direct route to the affected gene. Current emphasis is directed
toward identifying the factors required for plant infection. © 2002 Elsevier Science Ireland Ltd. All rights reserved.
Keywords: Transposon; Mutagenesis; Burkeholderia glumae; Auxotroph
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1. Introduction
Burkeholderia glumae, a gram-negative bacterium, is
a causal agent of rice grain and seedling rot [1,2]. B.
glumae was formerly named Pseudomonas glumae [3]. In
recent years, this pathogen has become more widely
observed among rice-growing countries [4,5]. The bac-
terium has also been reported to be the most important
bacterial rice pathogen in Japan [4].
In Japan and other parts of Asia, rice seedlings are
often germinated in nursery boxes before transplanta-
tion to the field [6,7]. Seedling rot is a common problem
during the initial stage of plant growth. In warm and
humid environments (e.g. southwest Japan), grain rot is
also a common problem [7]. Knowledge of the factors
required for B. glumae infection of rice would provide
insights necessary for the rational design of strategies to
combat related crop loss.
As a step toward gaining such insights, we report
here the generation and characterization of a mutagenic
B. glumae library. The production of such a mutagenic
library will be beneficial not only for identifying the
genetic determinants of pathogenicity, but for investi-
gating other metabolic processes. Transposon-tagging
was chosen as the method to mutagenize the B. glumae
genome since it would allow direct identification of the
affected genes. Using a recently developed transposase/
transposon system [8], a B. glumae transposon library
was generated. The system is highly efficient, and elimi-
nates several technical limitations associated with other
transposon systems. The transposition frequency was
determined to be 2.1×10
6
cfu/mg of DNA. Characteri-
zation of the integration event indicated that all trans-
positions were single, independent events. The
mutagenic frequency was assessed by conducting ge-
netic screens for auxotrophs and fluorescent mutants.
The auxotroph mutants were more prevalent and could
be divided into classes based on differences in growth
on supplemented M9 media. Fluorescent mutants were
less prevalent, but easily detectable upon exposure to

The contents of this publication do not necessarily reflect the
views or policies of the US Department of Agriculture, nor does
mention of trade names, commercial products or organizations imply
endorsement by the US Government.
* Tel.: +1-713-798-7013; fax: +1-713-798-7078.
E-mail address: pnakata@bcm.tmc.edu (P.A. Nakata).
0168-9452/02/$ - see front matter © 2002 Elsevier Science Ireland Ltd. All rights reserved.
PII: S0168- 9452( 01) 00566- 0
P.A. Nakata / Plant Science 162 (2002) 267–271 268
UV light. The availability of this transposon-mutage-
nized library will be useful in the study of many aspects
of B. glumae growth and development, including its
infection of rice.
2. Materials and methods
2.1. Strain and culture growth
B. glumae strain ATCC no. 49703 (American Type
Culture Collection, Manassas, VA) was cultured at
30 °C on Lennox Broth (Life Technologies, Rockville,
MD) or PPGA [4] as indicated.
2.2. Pathogenicity test
Assessment of pathogenicity was conducted accord-
ing to a previously published procedure [4]. In brief,
liquid cultures of ATCC 49703 were grown on PPGA
medium at 30 °C. A 1/500 dilution of the saturated
cultures was grown overnight. The culture was then
diluted to OD
600
of 0.1 and added to rice seeds (Ki-
taake). The seeds were incubated in the bacterial sus-
pension for 2 days at 30 °C, and then planted in soil.
After 4 days at 30 °C in the dark, the plants were
moved to the greenhouse.
2.3. Mutagenesis
Electro-competent cells of B. glumae were prepared
by standard protocol. In brief, B. glumae was grown in
5 ml of Lennox Broth (Life Technologies) for 2 days at
30 °C with shaking. An aliquot of this saturated cul-
ture was diluted (1/100) in fresh Lennox Broth (Life
Technologies) and grown to OD
600
of 0.8. The cells
were harvested and washed 3× with ice-cold water and
once with a solution of 10 mM Hepes pH 7.0 and 10%
(v/v) glycerol. Then, 50 ml aliquots of the cells were
frozen and stored at −70 °C until use. Insertional
mutants were generated using the EZ::TN™ ŽR6Kgori/
KAN-2 Tnp Transposome™ kit according to manu-
facturer’s recommendations (Epicentre Technologies,
Madison, WI). Fifty microliters of the electro-com-
petent cells were transformed with 1 ml of Transposome
complex (20 hg of DNA) using a 1-mm gap cuvette at
1720 V. The cells were quickly resuspended in 1 ml of
SOC media (Life Technologies), incubated at 30 °C for
2 h, and plated on Lennox Broth (Life Technologies)
agar plates supplemented with 50 mg/ml kanamycin
sulfate. Colonies containing the insert were visible after
2 days of growth. Transposition frequency was deter-
mined by counting the number of colony-forming units
(cfu).
2.4. Southern-blot analysis
Genomic DNA was isolated from log phase cells
using the MasterPure™ Complete DNA and RNA
(Epicentre Technologies) according to the manufactur-
er’s recommendations with the following additional
steps. After the DNA was resuspended in TE, RNase A
was added to a concentration of 20 hg/ml and incu-
bated at 37 °C for 30–60 min. The sample was then
phenol/chloroform-extracted followed by sodium ace-
tate/ethanol precipitation [9]. The precipitated DNA
was washed once in 80% ethanol and air-dried. The
sample was resuspended in TE (pH 8.0) and quanti-
tated using a spectrophotometer. Five micrograms of
DNA, digested with the appropriate restriction enzyme,
was size-fractionated on a 1% (w/v) agarose gel and
blotted onto nylon. Nylon blots were probed with a
random-primed transposon fragment according to
manufacturer’s recommendations (Amersham Pharma-
cia Biotech, Piscataway, NJ). The transposon fragment
was generated by PCR using primers derived from the
transposon sequence (Tn-1:5%-GGGCTCGCGATAAT-
GTCGGGC-3% and Tn-2:5%-CTTGCATGCCTGCAG-
GTCGAC-3%) and a randomly selected mutagenized
clone as template. All hybridization steps were per-
formed on a MJ Research PTC-200 thermal cycler
(Watertown, MA) using the following parameters:
94 °C for 30 s followed by 30 cycles of 94 °C for 10 s,
58 °C for 30 s and 72 °C for 1.5 min. After completion
of the 30 cycles, a 5 min extension was run at 72 °C.
The identity of the generated transposon fragment was
verified by sequence analysis.
2.5. Mutant screens
Transformed cells were plated onto Lennox Broth
(Life Technologies) agar plates supplemented with 50
mg/ml kanamycin sulfate. One thousand colonies were
replica plated onto Lennox Broth and M9 [10] agar
plates. The colonies that grew well on the Lennox
Broth plates, but not the M9 plates were transferred to
1 ml of Lennox Broth and grown overnight at 30 °C.
Two microliters of each culture was replica spotted
onto M9, Lennox Broth, Media 3, 5, 7, and 8 [10] agar
plates. Media 3, 5, 7, and 8 consisted of M9 media plus
additional supplements [10]. Media 3 was supplemented
with cysteine, isoleucine, tryptophan, uracil, and glu-
tamic acid. Media 5 contained thiamine, valine, proline,
arginine, and glycine. Media 7 was supplemented with
histidine, leucine, isoleucine, lysine, and valine. Media 8
contains phenylalanine, tyrosine, tryptophan,
threonine, and proline. The plates were incubated at
30 °C for 2 days.
To screen for fluorescent mutants, individual colonies
were isolated and grown in liquid PPGA. Aliquots of
saturated culture were spotted onto plastic wrap and
P.A. Nakata / Plant Science 162 (2002) 267–271 269
exposed to UV light. Bacterial mutants that showed
fluorescence were identified and stored at −70 °C.
3. Results and discussion
A common goal among phytopathologists is to de-
sign effective strategies to combat plant disease. Eluci-
dation of the molecular events that lead to plant disease
would be useful in the design of such preventative
strategies. B. glumae, a bacterial phytopathogen, is
responsible for rice yield losses in many parts of Asia as
a result of grain and/or seedling rot (Fig. 1). As an
initial step toward identifying the genetic determinants
of pathogenicity, we generated and characterized a
random transposon B. glumae library.
Transposons, especially Tn5, have been widely uti-
lized to generate random insertional bacterial libraries
[11]. High-frequency transpositions, random insertions,
and lack of high homology to most bacterial systems
have made transposons an attractive resource. Al-
Fig. 2. Southern-blot analysis of B. glumae library. Genomic DNA
was isolated from ATCC 49703 (control) and eight randomly selected
mutagenized colonies (1–8). Five micrograms of DNA was digested
with Sal I, size-fractionated on a 0.7% (w/w) agarose gel and blotted
onto nylon. The blot was probed with the radiolabeled transposon
fragment.
Fig. 1. Pathogenicity of rice seedlings. Control plants (left) and B.
glumae infected plants (right).
though a valuable system, standard transposon mutage-
nesis does have its drawbacks. Such drawbacks include
difficulties expressing the transposase in the host, intro-
ducing the transposon-suicide vector construct into the
host, and genetic instability due to the expression of the
transposase and transposon in subsequent generations
[8]. To alleviate such technical barriers in the generation
of our B. glumae transposon library, we employed a
simple and robust protocol developed by Goryshin and
colleagues [8]. The protocol involves the in vitro assem-
bly of Tn5-derived transposition complexes, followed
by electroporation of these complexes into the target
cells. The transposon event then occurs inside the target
cell in the presence of Mg
2+
. Using this protocol and
subsequent selection for the transposon insertion
(kanamycin selection), we determined an insertion rate
of 2.1×10
6
cfu/mg DNA. In addition to kanamycin
selection, the transposon fragment also encodes a R6K
origin of replication (ori). The presence of the R6K ori
site allows for efficient retrieval of the affected gene
sequence through a single restriction enzyme digestion,
ligation, and transformation procedure. Southern-blot
analysis of several randomly selected colonies indicated
that the transposition events were random and single
(Fig. 2). Stability of the insert was tested by growing
several individual kanamycin-resistant colonies to satu-
ration in the absence of antibiotic selection. After mul-
tiple subcultures, a small aliquot of cells was plated
onto nonselective plates. Several colonies were isolated
P.A. Nakata / Plant Science 162 (2002) 267–271 270
Fig. 3. Auxotroph mutants. Forty-six auxotrophs were identified by their poor growth on M9 minimal media compared with Lennox Broth. The
identified mutants were then divided into classes based on the ability of specific groups of supplements (i.e. amino acids, thiamine and uracil) to
rescue growth. The different media compositions used in this study were: (A) Lennox Broth; (B) M9 agar plate; (C) Media 3. (D) Media 5; (E)
Media 7; (F) Media 8. Two microliters of each mutant (1–46) and control (c) culture was spotted onto the different media plates and grown for
2 days at 30 °C.
and tested for kanamycin resistance. No revertants
were identified, indicating that the transposon insertion
was stably maintained even in the absence of antibiotic
selection.
To demonstrate the ability to isolate mutants from
the generated library, two mutant screens were con-
ducted. The first screen identified auxotroph mutants.
The replica plating of 1000 colonies on M9 media
resulted in the identification of 46 auxotrophs (Fig. 3).
The auxotrophs were then divided into classes. Four
classes were designated based on the ability of different
groups of amino acids to rescue the growth of the
auxotrophs (Fig. 3). A fifth class was assigned to the
mutants that grew on Lennox Broth, but failed to grow
on any of the amino acid supplemented medias (Fig. 3).
The second screen identified fluorescent mutants.
Upon screening 1000 colonies, two mutants were iso-
lated which produced a fluorescent compound that
could be detected using UV light (Fig. 4). The fre-
quency of these mutants was much lower than that of
the auxotrophs, suggesting that a lower number of
genetic determinants contributed to the fluorescent
mutant phenotype. Our findings show that a single
insertion event can dramatically alter these characteris-
tics.
In summary, we show that electroporation of the
preformed transposase–transposon complex is an effi-
cient method of generating transposon insertions in B.
glumae. The use of a transposon will allow direct
identification of the mutated gene. Presumably, this
protocol could be utilized for any microorganism that
can accept DNA via electroporation [8,12,13]. The gen-
erated B. glumae mutant library can now be utilized to
investigate any number of cellular processes. Currently,
we have initiated a study to identify pathogenicity
mutants. The identification of such mutants will allow
us to begin to elucidate the molecular events leading to
B. glumae plant infection and disease.
Fig. 4. Fluorescent mutants. A transposon-tagged ATCC 49703 con-
trol (C) is shown along with two fluorescent mutants (1 and 2) under
UV light illumination.
P.A. Nakata / Plant Science 162 (2002) 267–271 271
Acknowledgements
Thanks go to Thomas Okita for providing the rice
seeds and Leslie Loddeke for manuscript editing. This
research was supported in part by the US Department
of Agriculture, Agricultural Research Service, under
Cooperative Agreement number 58-6250-6-001 and
NIH CHRC 5 P30.
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