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Appl Environ Microbiol, May 1998, p. 1657-1662, Vol. 64, No. 5
0099-2240/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
A Transposable Partitioning Locus Used To Stabilize
Plasmid-Borne Hydrogen Oxidation and Trifolitoxin Production Genes
in a Sinorhizobium Strain
Angela D.
Kent,1,2
Michelle L.
Wojtasiak,2,3
Eduardo A.
Robleto,2,3 and
Eric W.
Triplett1,2,3,*
Department of
Bacteriology,1
Center for the Study of
Nitrogen Fixation,2 and
Department of
Agronomy,3 University of Wisconsin-Madison,
Madison, Wisconsin 53706
Received 6 November 1997/Accepted 19 February 1998
 |
ABSTRACT |
Improved nitrogen-fixing inoculum strains for leguminous crops must
be able to effectively compete with indigenous strains for nodulation,
enhance legume productivity compared to the productivity obtained with
indigenous strains, and maintain stable expression of any added genes
in the absence of selection pressure. We constructed a transposable
element containing the tfx region for expression of
increased nodulation competitiveness and the par locus for plasmid stability. The transposon was inserted into tetA of
pHU52, a broad-host-range plasmid conferring the H2 uptake
phenotype. The resulting plasmid, pHUTFXPAR, conferred the plasmid
stability, trifolitoxin production, and H2 uptake
phenotypes in the broad-host-range organism Sinorhizobium
sp. strain ANU280. The broad applications of a transposon conferring
plasmid stability are discussed.
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INTRODUCTION |
An important goal in nitrogen
fixation research is genetic improvement of inoculum strains of root
nodule bacteria sold commercially for the formation of nitrogen-fixing
root nodules on leguminous crops. Three obstacles have prevented the
achievement of this goal (25). First, genes that enhance
nitrogen fixation must be identified. Second, mechanisms which enhance
the ability of an inoculum strain to compete for infection sites with
indigenous root nodule bacteria must be discovered. And third,
sufficient knowledge of these genes must be available in order to make
constructs that allow stable expression of the genes in the absence of
selection pressure. The progress made in these areas was recently
reviewed by Maier and Triplett (25).
Hydrogen is an obligate product of the nitrogenase reaction
(37). One phenotype expressed by microsymbionts that has
been shown to increase legume yield is the H2 uptake
phenotype (commonly referred to as the Hup phenotype). With the Hup
phenotype, the root nodule bacteria recover the energy lost in the
production of the H2 evolved during the nitrogenase
reaction (12, 46). The increases in soybean yields are as
great as 17% (11). However, most indigenous strains of root
nodule bacteria that infect leguminous crops do not possess uptake
hydrogenase activity. In Bradyrhizobium japonicum, less than
25% of the isolates collected from the soybean-growing region of the
northeastern United States were Hup+ (17, 44).
Very few strains of the alfalfa microsymbiont, Sinorhizobium
meliloti, are Hup+, and even the strains that are
Hup+ are not efficient at recovering the energy obtained
from hydrogen oxidation (22, 23). In addition, very few
strains of Rhizobium leguminosarum bv. viceae, the pea
microsymbiont, are Hup+, and most of these strains are
unable to efficiently couple H2 oxidation to ATP formation
(27, 28, 34).
Transfer of the yield-enhancing Hup phenotype to Rhizobium,
Sinorhizobium, and Bradyrhizobium inoculum
strains requires the isolation of the genes involved in this process.
Significant progress has been made in elucidating the genetics of the
uptake hydrogenases of B. japonicum and R. leguminosarum (for a review, see reference 25).
Lambert et al. (19) isolated a cosmid clone, pHU52, from a
B. japonicum 122DES gene bank, and this cosmid clone
conferred uptake hydrogenase activity and chemolithotrophic growth to
Hup
strains of B. japonicum, S. meliloti, R. leguminosarum bv. viceae, and R. leguminosarum bv. trifolii. However, as pHU52 is not stable in the
absence of selection pressure, legume plants inoculated with
pHU52-containing root nodule bacteria expressed low levels of hydrogen
uptake activity (20, 21).
As a result, pHU52 has no commercial value for enhancing legume
productivity despite the fact that it contains all of the genes
necessary for full Hup phenotype expression in
Bradyrhizobium, Rhizobium, and
Sinorhizobium strains. Weinstein et al. have described a set
of genes from RK2 that is capable of conferring complete plasmid
stability in the absence of selection pressure in both free-living and
nodule bacteroids of S. meliloti (47). We
describe here the construction and use of a transposon that includes
the plasmid stability locus from RK2. This transposon was used to stabilize pHU52 both in free-living cells and in nodule bacteroids in
the absence of selection pressure. Similarly, we constructed a
broad-host-range plasmid that includes genes that enhance nodulation competitiveness, as well as hydrogen uptake and plasmid stability.
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MATERIALS AND METHODS |
Strains and plasmids.
The strains and plasmids used in this
study are shown in Table 1.
Escherichia coli strains were maintained on Luria-Bertani medium (35) at 37°C, while Rhizobium and
Sinorhizobium strains were cultured on the synthetic medium
of Bergersen et al. (1) at 28°C. Antibiotics were used at
the following concentrations: 100 µg/ml for ampicillin, 50 µg/ml
for kanamycin and spectinomycin, 20 µg/ml for nalidixic acid and
chloramphenicol, and 2.5 µg/ml for tetracycline. Streptomycin was
used at a concentration of 25 µg/ml for E. coli strains
and at a concentration of 50 µg/ml for Rhizobium and
Sinorhizobium strains.
Construction of transposons.
The transposable par
locus of pTn3PAR was constructed by excising par
from pTR102 on a 3.2-kb KpnI-BamHI fragment and
cloning this fragment into the ClaI site of pHoKmGus by
using blunt-end ligation. pTn3TFXPAR carries a transposable
element which includes both tfx and par. This
transposon was constructed by cloning the 3.2-kb par locus
into the XhoI site of pTFX24 by using blunt-end ligation. A
10.4-kb ApaI-SacI fragment containing both the
par locus and the tfx operon was then inserted
into the ClaI site of pHoKmGus.
Transposition into tetA of pHU52 and pLAFR1.
To
accomplish transposition into the tetA gene of the cosmids,
pHoKmGus, pTn3PAR, and pTn3TFXPAR were each
transformed into HB101(pSshe, pHU52) and HB101(pSshe, pLAFR1). To
enhance the transposition frequency of Tn3Gus,
Tn3PAR, and Tn3TFXPAR, the transformation reaction mixtures were incubated at 28°C (18, 43).
Transformants were selected on plates containing only kanamycin.
Kanamycin-resistant transformants from each transformation were pooled
and used as the donors in triparental matings in which E. coli C2110 was used as the recipient and HB101(pRK2073) was used
as the helper strain. Transconjugants were selected for
Nalr and Kmr and then screened for
Tcs. Since pHoKmGus and its derivatives are not able to
replicate in a polA strain, such as C2110, this procedure
allows isolation of pLAFR1 and pHU52 with an insertion in the
tet region.
The transpositions of pTn
3Par and pTn
3TFXPAR into
tetA of pHU52 and pLAFR1 were verified by performing PCR
that included primers
for
tetA found on the cosmids and
uidA found on the transposon.
Each reaction mixture
contained primer
uidA-R (5'-TTGGGGTTTCTACAGGACG-3')
and either primer
tetA-F
(5'-GTGAAACCCAACATACCC-3') or primer
tetA-R
(5'-CGGCTCGTTGCCCTGCG-3'). Amplification products that
were
0.3 to 1.2-kb long indicated that successful transposition
into
tetA had occurred. This procedure also allowed us to
determine
the orientation of each transposon within
tetA.
The identities
of the
hup-containing plasmids were verified
by performing PCR
for the
hupS region of each cosmid with
primers
hupS-F (5'-ATGGGCGCGGCGACGGAAAC-3')
and
hupS-R (5'-TCAGCTGTTGTGGTCGGCGT-3').
Plant culture, hydrogen evolution, and nitrogenase assays.
Seeds were germinated in Leonard jars and were inoculated 3 days after
planting. Plants were cultured in a growth chamber as described by
Datta et al. (8). Hydrogen evolution by whole root systems
of Vigna unguiculata (L.) Walp. cv. Blackeye was determined
by gas chromatography 24 days after inoculation with Sinorhizobium sp. strain ANU280 or a derivative of this
strain carrying pHU52, pHUTn3, pHUPAR, pHUTFXPAR, pLAFR1,
pLAFR1::Tn3, pLAFR1::PAR, or
pLAFR1::TFXPAR. Gas chromatography was performed as described
by Hanus et al. (15) with a Shimadzu gas chromatograph, and
N2 was used as the carrier gas. Nitrogenase assays were
performed by the acetylene reduction method as described by Rasche and
Arp (30) by using the same root systems used for the
H2 evolution determination. The relative efficiency of root
nodule nitrogenase activity was determined as described by Van Kessel
and Burris (45) by measuring C2H2
reduction and H2 evolution during acetylene reduction, as
well as in the absence of C2H2.
Plasmid stability and trifolitoxin production assays.
Sinorhizobium sp. strain ANU280 was the background strain
used for the plasmids used in the plasmid stability and trifolitoxin production assays. Plasmid stability was determined in free-living cells as described by Weinstein et al. (47), except that
cells were grown in hydrogen uptake medium broth (24).
Trifolitoxin production was determined by the plate assay described by
Breil et al. (5) by using R. leguminosarum bv.
viceae 128C1 as the sensitive strain, except that the assay was done on
hydrogen uptake medium. Plasmid maintenance in planta was assessed by
collecting nodules from the root systems used for hydrogen evolution
assays and determining the percentages of cells that contained the
plasmid found in the inoculum strain.
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RESULTS |
Cloning and transposition strategies.
The cloning and
transposition strategies used in this study are shown in Fig.
1. The strategy which we used to
stabilize hydrogenase expression involved inserting the 3.2-kb
par locus into pHU52. This was accomplished by inserting the
par locus into the inverted repeats of a Tn3
transposon and then transposing the par locus into the
tetracycline resistance gene of pHU52, tetA. The possibility that the par locus would transpose out of pHU52 after
insertion was greatly reduced by providing a transposase in
trans. In addition, as the Tn3 transposon which
we used for this study contains kanamycin and ampicillin resistance
genes, selection for pHU52 remained after tetA was
interrupted. The resulting plasmid is referred to below as pHUPAR.
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FIG. 1.
Strategy used to construct pHUTFXPAR. The par
locus from pTR102 was cloned into a unique XhoI site of
pTFX24. An ApaI-SacI fragment containing
tfx and par was then cloned into a unique
ClaI site of pHoKmGus to create pTn3TFXPAR (A).
The Tn3 derivative containing tfx and
par was transposed into tetA of pHU52 to generate
pHUTFXPAR (B). pHUPAR was constructed in a similar fashion by using a
transposon containing par but not tfx.
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|
The transposable partitioning system also allowed us to insert other
genes of interest into the inverted repeats of the Tn
3 transposon and then transpose them into pHU52. Of particular interest
was the cassette of the
tfxABCDEFG genes from
R. leguminosarum bv. trifolii T24 that confers trifolitoxin
production and resistance
(
5). Trifolitoxin is a peptide
antibiotic that inhibits the
growth of a specific group of the
subclass of the
Proteobacteria (
42).
Rhizobium strains with the ability to produce
trifolitoxin
have been shown to exhibit increased nodulation
competitiveness
(
33,
39-41). Thus, by adding both
tfxABCDEFG and the partitioning
locus in the inverted
repeats of Tn
3GUS, we were able to add the
trifolitoxin
production phenotype to pHU52 and to stabilize pHU52
by creating
pHUTFXPAR. As in the construction of pHUPAR, we screened
for
transposition events within
tetA.
Insertion of the transposons into
tetA had a number of
purposes. First, it allowed us to easily screen for insertion into
a
specific gene within pHU52. Second, it ensured that the genes
of
interest had not transposed into a gene essential for plasmid
replication or hydrogen uptake. And third, it removed tetracycline
resistance from this plasmid, eliminating a potential regulatory
hurdle
to the commercialization of this technology.
Plasmid stability in vitro and in planta.
The stability of
each plasmid was assessed in vitro by serial passage of each strain for
more than 60 generations without selection for the plasmid marker. The
addition of the par locus from RK2 by transposition into
pHU52 completely stabilized this plasmid in ANU280. The resulting
plasmid, pHUPAR, was stable in the absence of selection pressure for
more than 60 generations (Fig. 2). The
addition of the tfx region had no effect on this stabilization since both pHUPAR and pHUTFXPAR were very stable in the
absence of selection pressure (Fig. 2). In contrast, pHU52 was absent
from 90 and 99% of the ANU280 cells after 30 and 66 generations,
respectively (Fig. 2). The stability of the plasmids in nodule
bacteroids was characterized by determining the percentage of the
bacteria in a nodule that contained the plasmid present in the
inoculum. The presence of the transposeable par locus
conferred plasmid stability throughout the infection process (Fig.
3).
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FIG. 2.
Proportions of free-living Sinorhizobium sp.
strain ANU280 cultured in the absence of selection pressure that
expressed the appropriate antibiotic resistance characteristics for the
plasmid present in the inoculum strain.
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FIG. 3.
Proportions of Sinorhizobium sp. strain
ANU280 recovered from nodules that expressed the appropriate antibiotic
resistance characteristics for the plasmid present in the inoculum
strain.
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|
Trifolitoxin production.
Trifolitoxin was produced by
any Rhizobium or Sinorhizobium strain
carrying either pHUTFXPAR or pLAFR1::TFXPAR. Trifolitoxin production by ANU280 (pHUTFXPAR) is illustrated in Fig.
4. Strains lacking the tfx
region failed to inhibit the trifolitoxin-sensitive strain R. leguminosarum bv. viceae 128C1.
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FIG. 4.
Lack of trifolitoxin production by ANU280(pHUPAR) (a)
and trifolitoxin production by ANU280(pHUTFXPAR) (b). In both cases the
inhibited strain was R. leguminosarum bv. viceae 128C1.
Identical results were obtained with these plasmids with several other
Sinorhizobium and Rhizobium strains.
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Hydrogen evolution and acetylene reduction by root nodules.
Hydrogen is an obligate product of the nitrogenase reaction. Bacteroid
hydrogen oxidation has been shown to improve the efficiency of nitrogen
fixation (46). This phenotype is often referred to as the
Hup phenotype. Cowpea nodules infected with any strain lacking the
uptake hydrogenase region from pHU52 evolved significant amounts of
H2 (Fig. 5). As expected,
pHU52-infected nodules also evolved significant amounts of
H2 as a result of plasmid loss during the infection and
nodule development processes. However, nodules infected with either
ANU280(pHUPAR) or ANU280(pHUTFXPAR) evolved very low amounts of
H2 (Fig. 5) since each of these strains harbors a plasmid
that contains both the uptake hydrogenase genes from B. japonicum 122DES and the plasmid stability locus from RK2. As the
stabilization of pHU52 reduced H2 evolution in root nodules
to nearly undetectable levels, the relative efficiency of electron
allocation in bacteroids of ANU280 containing either pHUPAR or
pHUTFXPAR as defined by Van Kessel and Burris (45) was
significantly enhanced compared with the relative efficiency of
electron allocation in ANU280 or its derivatives lacking either the
uptake hydrogenase genes or the stability locus (Fig.
6).
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FIG. 5.
Hydrogen evolution from cowpea root systems. The values
for bars with the same letter were statistically similar at the 5%
confidence level. Each value is the mean of the values from three
replicates. gfw, gram (fresh weight).
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FIG. 6.
Relative efficiency of nitrogenase electron allocation.
The values for bars with the same letter were statistically similar at
the 5% confidence level. Each value is the mean of the values from
three replicates. Relative efficiency (RE) was determined by the
formula of Van Kessel and Burris (45): RE = 1  [(H2 evolution in air)/(C2H2
reduction + H2 evolution in 10%
C2H2)].
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DISCUSSION |
Rationale for the cloning and transposition strategies used.
We used a transposable partitioning locus to stabilize a large cosmid
clone in gram-negative bacteria. Our strategy was based on the
discovery of a plasmid partitioning locus in the broad-host-range plasmid RK2 (47). A 3.2-kb par locus from RK2,
which contains five genes in two divergently transcribed operons,
forces complete plasmid partitioning to occur during cell division.
Each operon codes for an independent plasmid stability mechanism. The
parCBA operon includes a resolvase mechanism; the
parDE operon encodes a toxin (ParE)-antitoxin (ParD)
mechanism (16). The par locus confers plasmid
stability regardless of the replicon containing the 3.2-kb region
(9, 31, 32). This region has also been shown to confer
complete plasmid stability in S. meliloti during root nodule
development (47). The ability of this par region to confer plasmid stability in other species of root nodule bacteria has been confirmed by workers in our laboratory (33, 42).
The
hup-containing cosmid pHU52 is unstable in the absence
of selection and does not increase legume productivity in agricultural
situations. Addition of the
par locus to this cosmid allows
stable
expression of the Hup phenotype to occur.
Benefits of the approach presented here.
Stabilization of a
cosmid clone harboring commercially useful genes is important in many
situations. For our purposes, we were faced with the dilemma of finding
a method to transfer and stably express a large set of genes that
confer a yield-enhancing phenotype present in certain root nodule
bacteria for leguminous crops. As the complete nucleotide sequence of
the uptake hydrogenase region is not known, it is very difficult to
identify unique restriction sites for the cloning of this large (30-kb)
set of genes into a vector useful for marker exchange in the
chromosome. In addition, cloning these genes into a chromosome or
megaplasmid would require identification of symbiotically silent sites
in the genome of every species in which the uptake hydrogenase
phenotype is desired.
Identification of symbiotically silent sites is difficult. During the
1980s, geneticists at Biotechnica International identified
such sites
in
S. meliloti by cloning antibiotic resistance genes
into
regions thought to be symbiotically silent. Alfalfa greenhouse
yield
trials were performed to determine the effects of the insertions
on the
symbiosis (
3). In these greenhouse experiments, no effect
was observed when cloning into either the inositol catabolism
locus or
a site between the
nif and
fix regions called P3
was
performed. However, multiple-year and multiple-site alfalfa yield
trials performed in the field revealed significantly altered yields
after insertion into these sites (
36). Thus, at least for
alfalfa,
the effects of gene deletions or insertions in a microsymbiont
on the crop yield of a rhizobium-legume symbiotic system can often
be
observed only if the plants are grown to maturity or under
proper
agricultural conditions (
11,
36).
In addition to the practical problems of identifying symbiotically
silent insertion sites, such sites have to be identified
for every
microsymbiont species of interest. A symbiotically silent
site in
S. meliloti may not be present or may not be symbiotically
silent in
R. leguminosarum. Thus, it may be necessary to
develop
new insertion strategies for each species.
To avoid these problems, we decided to construct a stably maintained,
broad-host-range plasmid which could be used to improve
the symbiotic
properties of root nodule bacteria. pHUTFXPAR can
be transferred easily
to any Km
s or Ap
s strain of root nodule
bacteria by conjugation. In addition, it
is stably maintained in the
absence of selection pressure and
provides two phenotypes that provide
improved efficacy to an inoculum
strain. First, as a result of the
presence of the trifolitoxin
production and resistance genes, this
multicopy plasmid confers
high levels of trifolitoxin production.
Trifolitoxin production
has been shown to increase nodulation
competitiveness in soil
(
33). The presence of the
tfx genes on a multicopy plasmid,
such as pHUTFXPAR, results
in much higher levels of trifolitoxin
production than the level of
production that would occur if single
copies of the genes were present
on the chromosome (
39,
40).
The addition of nodulation
competitiveness on a multicopy plasmid
is better than chromosomal
integration of multiple copies of competitiveness
genes in tandem, as
described recently (
26). Concatamers containing
repeated
regions of nodulation competitiveness genes can easily
recombine with
one another and be lost over time. This is especially
true of many
Rhizobium genomes that are known to be unstable as
a result
of recombination events (
6,
29).
Second, the presence of the uptake hydrogenase genes on pHUTFXPAR
is expected to enhance legume yield compared to the legume
yield
obtained after infection with rhizobia that lack this phenotype.
The presence of these genes on pHUTFXPAR and pHUPAR was confirmed
by
the significant reductions in H
2 evolution from ANU280
(pHUTFXPAR)-
and ANU280(pHUPAR)-infected nodules;
H
2 evolution was reduced
by 91.5 and 95.3%, respectively.
Thus, the addition of pHUTFXPAR
to a wide variety of root nodule
bacteria can be used to enhance
the productivity of many legumes
worldwide. All of this was done
without the need for complex cloning
strategies that include symbiotically
silent sites as described
previously (
3).
Limitations to the current approach and their solutions.
The
limitations of the approach described here are few, and all of them can
be overcome. For example, pTn3PAR is limited in that it
confers kanamycin resistance. Thus, our approach is limited to target
plasmids that lack kanamycin resistance. This limitation is easily
overcome by excising the kanamycin resistance gene in pHoKmGus and
replacing it with either a gene that confers resistance to a different
antibiotic or a reporter gene, such as the gene for the green
fluorescent protein. Also, pTn3PAR does include a
promoterless reporter gene, uidA, that is expressed if it is transposed in the proper orientation into an expressed gene.
Another limitation of the technology described here is also one of its
advantages. This limitation is the fact that the genes
provided to root
nodule bacteria by pHUTFXPAR are present on a
broad-host-range plasmid
rather than inserted into the chromosome.
The advantages of this are
described above. The primary disadvantage
is that plasmid pHUTFXPAR can
be mobilized from one strain to
another in the environment. In our
specific example, this may
not necessarily be a disadvantage as the
improved symbiotic properties
of the plasmid may be transferred to
indigenous rhizobia, albeit
at a low frequency, and improve their
characteristics. However,
a way to restrict the movement of a plasmid
such as pHUTFXPAR
can be envisioned with one modification. One of the
two mechanisms
of plasmid stability conferred by the
par
locus of RK2 is a toxin-antitoxin
system expressed by
parE
and
parD. Spatial separation of these
two genes such that
parD is on the chromosome and
parE is on the
broad-host-range plasmid should substantially reduce the transfer
of
the plasmid to other bacteria, as the plasmid would express
only
production of the toxin.
Thus, any shortcomings of the strategy described here can be overcome
with additional refinements of the system. These refinements
should
also enhance the number of useful applications of this
system.
Other applications for the transposable stability locus.
The
approach described here for stabilization of pHU52 has broad
applications for any plasmid used in biotechnology for which providing
antibiotic selection pressure is very expensive or, as is the case in
our example, impossible. The transposable stability locus constructed
here, pTn3PAR, can provide stability to any plasmid
maintained in any bacterium which can serve as a host for RK2. As RK2
has the broadest host range of any known plasmid, the applicability of
this approach is extensive. Once stability is provided to a plasmid,
the transposon remains stable since the transposase is provided in
trans on a nonreplicating plasmid (2, 38).
As we did here with pHUTFXPAR, additional valuable genes can be added
to a plasmid along with the plasmid stability region
by simply cloning
into the transposon another set of genes along
with the
par
locus from RK2. The entire cassette can then be transposed
into the
plasmid of interest.
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ACKNOWLEDGMENTS |
We thank the USDA NRICGO, LiphaTech, Inc. (Milwaukee, Wis.), and
the University of Wisconsin-Madison College of Agricultural and Life
Sciences for providing support for this work.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: Center for the
Study of Nitrogen Fixation, Department of Agronomy, University of
Wisconsin-Madison, 1575 Linden Drive, Madison, WI 53706. Phone: (608)
262-9824. Fax: (608) 262-5217. E-mail:
ewtriple{at}facstaff.wisc.edu.
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Appl Environ Microbiol, May 1998, p. 1657-1662, Vol. 64, No. 5
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Copyright © 1998, American Society for Microbiology. All rights reserved.
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Abstract
Introduction
