Construction of signature-tagged mutant library in Mesorhizobium loti as a powerful tool for functional genomics.

Construction of Signature-tagged Mutant Library in Mesorhizobium loti
as a Powerful Tool for Functional Genomics
Yoshikazu SHIMODA1, Hisayuki MITSUI2, Hiroko KAMIMATSUSE2, Kiwamu MINAMISAWA2, Eri NISHIYAMA2,
Yoshiyuki OHTSUBO2, Yuji NAGATA2, Masataka TSUDA2, Sayaka SHINPO1, Akiko WATANABE1, Mitsuyo KOHARA1,
Manabu YAMADA1, Yasukazu NAKAMURA1, Satoshi TABATA1, and Shusei SATO1,*
Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan1 and Graduate School of
Life Sciences, Tohoku University, Katahira, Aoba-ku, Sendai 980-8577, Japan2
(Received 30 April 2008; accepted 25 June 2008; published online 25 July 2008)
Abstract
Rhizobia are nitrogen-fixing soil bacteria that establish endosymbiosis with some leguminous plants.
The completion of several rhizobial genome sequences provides opportunities for genome-wide func-
tional studies of the physiological roles of many rhizobial genes. In order to carry out genome-wide pheno-
typic screenings, we have constructed a large mutant library of the nitrogen-fixing symbiotic bacterium,
Mesorhizobium loti, by transposon mutagenesis. Transposon insertion mutants were generated using the
signature-tagged mutagenesis (STM) technique and a total of 29 330 independent mutants were
obtained. Along with the collection of transposon mutants, we have determined the transposon insertion
sites for 7892 clones, and confirmed insertions in 3680 non-redundant M. loti genes (50.5% of the total
number of M. loti genes). Transposon insertions were randomly distributed throughout the M. loti genome
without any bias toward G1C contents of insertion target sites and transposon plasmids used for the
mutagenesis. We also show the utility of STM mutants by examining the specificity of signature tags and
test screenings for growth- and nodulation-deficient mutants. This defined mutant library allows for
genome-wide forward- and reverse-genetic functional studies of M. loti and will serve as an invaluable
resource for researchers to further our understanding of rhizobial biology.
Key words: Mesorhizobium loti; signature-tagged mutagenesis; mutant library; reverse genetics
1. Introduction
Nitrogen-fixing rhizobia are of great agronomic
benefit, allowing many leguminous crops to be grown
without nitrogen fertilizer by forming endosymbiotic
relationships with the plants. In the course of the sym-
biosis, rhizobia elicit formation of specialized organs,
‘root nodules’, on the roots of compatible host
legumes. Inside the nodules, the bacteria convert
inert atmospheric dinitrogen into biologically
usable ammonia. Since this symbiotic relationship is
established by the results of highly regulatedmolecular
dialogues between host plant and rhizobia, the system
of symbiotic nitrogen fixation is therefore a very suit-
able model for studies of plant–microbe interactions.
The agronomic and biological importance of the
rhizobia has accelerated the determination of the full
genome sequences of several rhizobial species.1–6 The
availability of complete genome sequences has drasti-
cally changed the strategy for studying rhizobial genetics.
Gene identification and functional assignment have
been accelerated by utilizing the genome sequences.
Simultaneously with the completions of rhizobial
genome sequences, post-genomic researches on several
rhizobia have rapidly progressed. Comprehenzsive tran-
scriptome or proteome analyses have been conducted
to examine physiological states of rhizobia under a
Edited by Naotake Ogasawara
* To whom correspondence should be addressed. Tel. þ81 438-
52-3935. Fax. þ81 438-52-3934. E-mail: ssato@kazusa.or.jp
# The Author 2008. Kazusa DNA Research Institute.
The online version of this article has been published under an open access model. Users are entitled to use, reproduce, disseminate, or display the
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DNA RESEARCH 15, 297–308, (2008) doi:10.1093/dnares/dsn017

variety of conditions, including symbiosis with the
host legume or nutrient-depleted conditions.7–12 In
addition, we previously conducted a large-scale interac-
tome analysis in order to propose functional relation-
ships between known and unknown rhizobial genes.13
These advances in rhizobial genetics have generated a
vast amount of information about gene function and
regulation; however, the advances have simultaneously
raised the need for development of novel molecular
tools which make use of the results of these functional
analyses.
One of the most utilized genetic approaches to reveal
gene function is the disruption of particular genes
and subsequent phenotypic characterization of the
mutants. Comprehensive collections of single gene dis-
ruptants have been systematically constructed in
several model organisms by transposon random muta-
genesis or targeted gene disruption approaches.14–18
These analyses have contributed to the discovery of
essential genes of the organisms and have provided fun-
damental tools for functional studies of many known
and unknown genes. In rhizobia, recent genome-wide
analyses have provided various intriguing and useful
starting points for more detailed functional studies;7–13
however, there are still large gaps between such sys-
tematic analyses and the availability of the corre-
sponding gene mutants. Such gaps have resulted
primarily because targeted gene disruption achieved
by homologous recombination is not suitable for sys-
tematic generation of mutants in rhizobia because of
its low efficiency. In addition, screening of mutants in
some test conditions is problematic because pheno-
types of individual mutants have to be evaluated
one-by-one. Therefore, more effective approaches to
generate large mutant collections and allow simul-
taneous screening of many mutants are needed.
In this study, we conducted the first large-scale
random mutagenesis of Mesorhizobium loti, an endo-
symbiont of the model legume Lotus japonicus, using a
signature-tagged mutagenesis (STM) technique. STM
is based on transposon insertional mutagenesis that
allows large numbers of mutants to be analyzed simul-
taneously. This is accomplished by tagging eachmutant
with a unique short DNA sequence so that it can sub-
sequently be distinguished from other mutants by
detecting unique signature tags. This system has been
used with a variety of bacteria, and, in particular, has
been applied to discovery of virulence genes in several
microbial pathogens.19 Recently, a large STM library
was constructed in Sinorhizobium meliloti strain
1021,20a symbiontofMedicago truncatulaandcontrib-
uted to thediscoveryofmany genes relevant to symbio-
sis and competitiveness.21 However, genome-wide
generation of disruption mutants in M. loti, in addition
to the set of S. meliloti, is also very valuable for several
reasons. First, M. loti and S. meliloti exhibit different
aspects as symbiont for legumes. They naturally
exhibit distinct host-specificity and form different
types of nodules (determinate and indeterminate
nodules) on their respective model legume hosts, L.
japonicus andM. truncatula.With respect to this symbio-
sis, previous studies have discovered several rhizobial
factors and metabolic cycles required for nodulation
and nitrogen-fixation.22–24 However, comparative ver-
ifications of the significance of these factors on respect-
ive symbioses have not been fully explored. Second,
comparative genomics analyses have revealed the exist-
ence of genes that are conserved, as well as many that
are specific to each species,2,25 but the molecular
tools available for the functional comparisons of these
genes are extremely limited. Therefore, large collections
of disruption mutants on the individual species are
necessary to reveal common and unique biological
aspects of the two species.
In this report, we developed a large signature-
tagged mutant library of M. loti in order to provide
fundamental tools for functional studies of M. loti
genes and characterized it with the gene coverage
and transposon insertion distribution of the library.
Sequencing of transposon insertion sites enabled us
to collect defined sets of transposon mutants.
Furthermore, pilot experiments showed the utility of
the mutants and provide basic experimental con-
ditions that can be used for future mutant screenings.
This large signature-tagged mutant library will be an
important and powerful resource for future functional
genomics in M. loti.
2. Materials and methods
2.1. Bacterial strains and growth conditions
Escherichia coli strains DH5a and MT616 were used
as hosts for the transposon plasmids and pRK600
helper plasmid,26 respectively. Transposon insertion
mutants were generated in the wild-type M. loti
strain MAFF303099. Mesorhizobium loti was grown
at 308C in tryptone yeast (TY) extract medium27
supplemented with phosphomycin (100 mg/ml).
Escherichia coli was grown at 378C in Luria-Bertani
(LB) medium. LB medium was supplemented
with streptomycin (100 mg/ml) and spectinomycin
(100 mg/ml) for DH5a donor cells harboring transpo-
son plasmids and with chloramphenicol (20 mg/ml)
for MT616 helper cells. Transposon insertion
mutants were selected on TY medium supplemented
with streptomycin, spectinomycin and phosphomycin
at the same concentration as above.
2.2. Construction of signature-tagged transposon
plasmids
The backbone of the transposon plasmid was
pTnMod-OGm.28 The V interposon cassette was
298 Construction of Signature-tagged Mutant Library in Mesorhizobium loti [Vol. 15,

excised from pHP45V29 and inserted by replacing a
SacI fragment containing the gentamicin resistance
gene cassetteof pTnMod-OGm. The resultant construct
was designated pTnMod-OV. Twenty-seven different
21-base oligonucleotides (Supplementary Table S1)
were annealed to complementary molecules to make
double-strand tags. Each oligonucleotide tag was
inserted individually into the blunted KpnI site of
pTnMod-OV (Fig. 1). These 27 signature-tagged
constructs were designated pTnMod-OV-tag1 to
pTnMod-OV-tag41.
2.3. Transposon mutagenesis
Introduction of tagged transposon plasmids into
M. loti was carried out by tri-parental mating.30
Escherichia coli strains DH5a and MT616 (containing
the pRK600 helper plasmid) were used as the con-
jugation donor and helper, respectively. An overnight
culture of donor and helper (0.5 ml each) was mixed
with 1 ml of a 2-day culture of M. loti. After washing
several times with sterilized water, the cell mixture
was suspended in 50 ml of sterilized water and spotted
onto a sterilized membrane filter (MILLIPORE, Billerica,
MA, USA, pore size 0.45 mm, Cat. No. HAWG047S3)
that was placed on TY agar medium without antibiotics.
After an 8 h incubation on the membrane filter at 308C,
all the cells were collected and suspended in 1 ml of
sterilized water. Suspended cells were plated on TY
selection medium supplemented with phosphomycin,
spectinomycin and streptomycin, and grown further
until positive colonies appeared. Finally, individual
positive colonies were picked up randomly in 96-well
plates and stored at 2808C.
2.4. Determination of transposon insertion sites
Transposon insertion sites were determined by
direct sequencing of transposon-genome flanking
region. Cell culture and genomic DNA isolation were
carried out in 96-well plate format. Five microlitter
of stocked mutant clones were inoculated into 1 ml
of TY medium containing phosphomycin (100 mg/ml),
streptomycin (100 mg/ml) and spectinomycin
(100 mg/ml), and the cultures were grown in 96-
well deep-well plate (Axygen Bioscience, Union City,
CA, USA) at 308C for 2 days. After centrifugation of
cultured cells at 4000 rpm for 15 min, genomic
DNA of mutants was prepared using AquaPure
Genomic DNA isolation kit (Bio-Rad Laboratories,
Tokyo, Japan). Collected cells in each well were sus-
pended in 150 ml of Genomic DNA Lysis Solution
and were incubated at 808C for 15 min. RNaseA sol-
ution (0.75 ml) was added to cell lysate, and the
mixture was incubated at 378C for 60 min. After the
incubation on ice for 15 min, 50 ml of protein precipi-
tation solution was added to each well and the
samples were mixed well by vortex. Samples were
then transferred individually to 1.5 ml microcentri-
fuge tube and were centrifuged at 15 000 rpm for
15 min. Supernatants (100–125 ml) were trans-
ferred to new 96-well PCR plate containing 100 ml
of isopropanol and were mixed well by inverting
plate. Genomic DNA precipitated by centrifugation
(4000 rpm, 30 min) was rinsed three times with
200 ml of 70% ethanol. Genomic DNA was then
dried and dissolved in 20 ml of DNA hydration sol-
ution. After heating the samples at 658C for 60 min,
concentration of genomic DNA was measured and
�2 mg of genomic DNA was used for direct sequence.
Direct sequencing was performed with the STM-seq
primer (50-TTTGCTGGCCTTTTGCTCACATGTTCTTTC-
30) using the following PCR conditions: 988C for
3 min, followed by 70 cycles of 978C for 15 s and
608C for 4 min. The composition of reaction mixture
for direct sequence was as follows. Two micrograms
of genomic DNA, 1 ml of STM-seq primer (3.2 mM),
1 ml of BigDye Terminator premix (Applied
Biosystem, Foster City, CA, USA), 3.5 ml of 5� sequen-
cing buffer (Applied Biosystems) and sterilized water
were added up to 20 ml.
For some clones which were not successfully identi-
fied by direct sequencing, we determined the transpo-
son integration sites by two-round inverse PCR. For
inverse PCR, genomic DNA (�1 mg) isolated from
each mutant was digested with SalI and XhoI, and
then self-ligated using a DNA ligation kit (TAKARA
Figure 1. Vector construct used for STM of M. loti. The tagged
transposon plasmids were constructed from the backbone of
pTnMod-OGm.28 The omega-interposon cassette (Spr/Smr)
was excised from pHP45V29 and inserted into the SacI site of
pTnMod-OGm. Oligonucleotide tags (21 bp) were incorporated
into the KpnI site. Arrows indicate the locations of the STM
common primer and the tag-specific primer (Supplementary
Table S1). OriT is an RP4 origin of transfer. IR and tnp
represent the inverted repeat and Tn5 transposase, respectively.
No. 5] Y. Shimoda et al. 299

Bio, Shiga, Japan). Using a self-ligated DNA template
and a primer pair (50-TTCGCCACCTCTGACTTG
AGCGTCG-30 and 50-GAATTGATCCGGTGGATGAC-30),
first round PCR was performed using the following
cycling conditions: 988C for 2 min, followed by 35
cycles of 978C for 15 s, 558C for 45 s, 728C for
3 min and a final extension at 728C for 10 min.
A 1 ml aliquot from the first round PCR product was
added to a secondary PCR reaction mixture contain-
ing a nested primer pair (50-TTTGCTGGCCTTTTGCT
CACATGTTCTTTC-30 and 50-ACGGTTTACAAGCATAA
AGC-30). Second round PCR was performed using
the following PCR conditions: 988C for 2 min, fol-
lowed by 35 cycles of 978C for 15 s, 578C for 45 s,
728C for 3 min and a final extension at 728C for
10 min. After the secondary PCR, products were
purified by polyethylene glycol precipitation and
suspended in 10 ml distilled water. The composition
of sequence reaction mixture with amplified PCR
product was as follows. Two microliters of PCR
product, 1 ml of sequencing primer (50-ACGGTTTA
CAAGCATAAAGC-30) (3.2 mM), 1 ml of BigDye
Terminator premix (Applied Biosystems), 3.5 ml of
5� sequencing buffer (Applied Biosystems) and
12.5 ml of sterilized water. Cycle sequencing was per-
formed at 968C for 1 min and then at 968C for 10 s,
508C for 5 s and 608C for 4 min for 25 cycles. All
sequencings were analyzed by ABI 3730 autosequen-
cer. The resulting sequences were subsequently
subjected to BlastN search against the M. loti
genome sequence (http://www.kazusa.or.jp/rhizobase/
Mesorhizobium/index.html) to determine the transpo-
son insertion sites.
2.5. PCR and real-time PCR
The specificity of oligonucleotide tags was examined
by PCR. Tag-containing DNA fragments (160 bp) were
amplified from genomic DNA using the Herculase II
Fusion DNA Polymerase (STRATAGENE, La Jolla, CA,
USA), and forward (Supplementary Table S1) and
reverse (STM-common, 50-TTCGCCACCTCTGACTTG
AGCGTCG-30) primers were used for amplification.
PCR reactions were conducted in GeneAmp PCR
system 9700 (Applied Biosystems) using the following
conditions: initial denaturation at 988C for 2 min, fol-
lowed by 25 cycles of 978C for 15 s, 688C for 1 min
and a final extension at 728C for 10 min. Amplified
products were analyzed on 2% agarose gels.
The population of growth-deficient mutant within a
mutant pool was examined by real-time PCR.
Genomic DNA isolated from input and output pools
was used as the DNA template. Real-time PCR was
performed using a DyNAmo HS SYBR Green qPCR kit
(Finnzyme, Espoo, Finland) and reactions were
detected by a DNA Engine Opticon system (Bio-Rad).
The conditions used for real-time PCR are as follows:
15 min at 958C, followed by 35 cycles of 948C for
30 s, 588C for 30 s and 728C for 30 s and a final
extension at 728C for 10 min. Quantitative and speci-
ficity (melting curve) analyses of amplified PCR pro-
ducts were carried out using associated software in
the Opticon system (Opticon monitor3) by following
the manufacture’s protocol.
2.6. Detection of signature tags from plant samples
Seeds of L. japonicus (MG-20 Miyakojima) were ster-
ilized and germinated on half-strength B&D agar
medium. Six-day-old seedlings were transferred to
B&D agar medium or inoculation pots filled with ver-
miculite andwere then inoculatedwith1 � 107 cell/ml
of mutant mixture.
For tag detection from nodules, detached nodules
were washed twice with a solution of 0.5%-SDS-
100 mM NaCl and then soaked in 70% ethanol for
5 min. After five times washes with sterilized water,
total DNA was isolated using a DNeasy plant mini kit
(Qiagen, Hilden, Germany). For amplification of a
tag-containing fragment from a single nodule,
nodule was washed as above and crushed in 700 ml
sterilized water. After boiling the samples at 958C
for 5 min, 5 ml of supernatant was used for PCR. PCR
reactions with nodule samples used the following
conditions: initial denaturation at 988Cfor 2 min, fol-
lowed by 25 cycles of 978C for 15 s, 688C for 1 min
and a final extension at 728C for 10 min. Amplified
products were analyzed on 2% agarose gels.
3. Results and discussion
3.1. Construction of a signature-tagged mutant library
In this study, we adapted transposon-based STM
techniques to genome-wide mutagenesis of the sym-
biotic bacterium, M. loti. As a transposon construct,
the plasposon plasmid pTnMod-OGm which carries a
Tn5-based mini-transposon and conditional origin of
replication28 was used for mutagenesis (Fig. 1). In
order to screen for genes which were inactivated by
transposon insertion and those affected polarity effect
of the inserted transposon, we introduced the V inter-
poson cassette by replacing the gentamycin resistance
gene (aacC1) of pTnMod-OGm (Fig. 1). Since pTnMod
is a broad host range suicide vector and the insertion
of the V fragment confers spectinomycin and strepto-
mycin resistance, only mutants in which the transpo-
son fragment is integrated into the M. loti genome
can be selected effectively by their antibiotic resistance.
In order to distinguish individual transposon
mutants, we introduced 21 bp short oligonucleotide
tags that have been used in a previous study.31 Since
these oligonucleotide tags were designed to have
similar GþC content and melting temperature, they
300 Construction of Signature-tagged Mutant Library in Mesorhizobium loti [Vol. 15,

are feasible for PCR-based mutant screening.
Considering the isolation of symbiotic mutants from
root nodules in which reduction of variation was
expected, number of tag variation need to be restricted
to obtain reliable results of screening froma reasonable
number of plant materials (root nodules). Therefore,
we further selected tags from the original 41 tags31
by analyzing their specificity of PCR amplification. We
selectedatotal of29unique tagsbasedon the reprodu-
cibility of PCR amplification and the plasposon plas-
mids tagged with each of 29 tags (pTnMod-OV-tagn)
were then individually mobilized into wild-type M. loti
by tri-parental mating. Among the 29 tags, two tag
plasmids which yielded significantly small numbers of
transposon mutant colonies were eliminated, and the
remaining 27 tags (Supplementary Table S1) were
used for further analysis (name of tags are designated
according to Hunt et al.31). In order to construct
independent mutant libraries, tri-parental mating
and collection of transposon mutants were carried
out separately for each oligonucleotide tag. As the
result of 63 independent tri-parental mating exper-
iments, 29 330 independent transposon mutants
were obtained (Table 1). Individual mutants were
stored in 96-well plates and mutant names (ID) were
assigned according to their position in the plate with
prefix indicating tag number followed by ‘T’ (i.e.
07T02d09 is a mutant integrated with tag7 plasmid).
The coverage of the collected mutants is approxi-
mately fourfold with respect to the 7281 ORFs of
M. loti.1 If the transposon insertions are evenly distrib-
uted throughout the M. loti genome, the mean dis-
tance between independent insertions is estimated
to be 259 bp, and thus 91% of M. loti genes are
expected to carry at least one insertion since M. loti
genes shorter than 259 bp account for 9% of all genes.
3.2. Utility of STM mutants
The STM technique allows for the simultaneous
screening of multiple mutants in mixed populations
by detecting unique DNA tags. However, previous
studies have reported that non-specific amplification
or failure in tag amplification from mutant chromo-
somal DNA is a major inherent problem that affects
the efficiency and reproducibility of the screening pro-
cedure.31,32 Therefore, the specificity and efficiency of
tag amplification after integration into the genome
needs to be tested in the respective targeted organ-
isms. To investigate the possibility of cross-detection
among the 27 signature tags used in this study, we
conducted test amplifications by PCR. Fig. 2A shows
representative results in which PCR was performed
with genomic DNA from three individual tagged
mutants (Tag2, Tag9 and Tag11). When a tag-specific
primer (Supplementary Table S1) and the STM
common primer (50-TTCGCCACCTCTGACTTGAGCGT
CG-30) that anneals 120 bp downstream of the inte-
grated oligonucleotide tag were used for PCR, specific
amplification was observed only in reactions contain-
ing the appropriate tag-specific primer. A series of PCR
reactions performed on the other oligonucleotide tags
produced the same results as Fig. 2A, indicating that
there are no cross-amplifications among the designed
oligonucleotide tags. In addition, all 27 oligonucleo-
tide tags were amplified specifically when genomic
DNA from a mixture of mutants was used as the tem-
plate (Fig. 2B). These test amplifications demon-
strated that the applied oligonucleotide tags can be
recovered specifically from the M. loti genome and
that the 27 individually tagged mutants can be distin-
guished from one another by detecting these signa-
ture tags.
In order to test the utility of our mutant sets, we
carried out additional pilot experiments using both
in vitro and in vivo conditions. In vitro experiments
were conducted with a pool of mutants that contains
a mutant with an attenuated growth on rich medium.
To make a mutant set for the test experiment, one
mutant was selected randomly from each 27-tag
library and cultured individually. One growth-
deficient clone (07T02d09) was included in the
pool (Fig. 3A). Independently cultured clones were
then mixed at same concentration to make an input
pool. As for an output pool, the input pool was
diluted 500-fold and cultured further for 2 days.
After isolation of genomic DNA from both pools, the
tag population was examined by real-time PCR. Real-
time PCR was performed using the SYBR Green detec-
tion system with the same primer set used to test the
cross-reactivity (Fig. 2). Fig. 3 illustrates the results of
real-time PCR of the input (Fig. 3B) and output
(Fig. 3C) pool samples. Consistent with the growth
on agar medium (Fig. 3A), the quantity of PCR
product derived from the growth-deficient mutant
(07T02d09) was significantly lower than the other
tags which were all amplified to similar levels
Table 1. Summary of experimental results
Mutants collected 29 330
Mutants sequenced for mapping of transposon
insertion site 9344
Mapped insertion locations 7892
Unique insertion locations 7586
Insertion between ORFs (intergenic region) 1156
Chromosome 1018
pMLa 80
pMLb 58
Insertion inside ORFs 6430
Chromosome 6000
pMLa 318
pMLb 112
Insertion inside ORFs (non-redundant) 3680
Genes supported by more than two mutant alleles 1592
No. 5] Y. Shimoda et al. 301