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Applied and Environmental Microbiology, August 2001, p. 3728-3731, Vol. 67, No. 8
MSU-DOE Plant Research
Laboratory,1 NSF Center for Microbial
Ecology,2 and Department of
Microbiology,3 Michigan State University, East
Lansing, Michigan 48824
Received 29 January 2001/Accepted 30 May 2001
Eleven Sinorhizobium meliloti 1021 loci whose
expression was induced under low oxygen concentrations were identified
in a collection of 5,000 strains carrying
Tn5-1063 (luxAB)
transcriptional reporter gene fusions. The 11 Tn5-1063-tagged loci were cloned and
characterized. The dependence of the expression of the tagged loci on
the FixL/FixJ oxygen-sensing two-component regulatory system was
examined. Three of the loci were found to be dependent upon
fixL and fixJ for their expression, while
one locus showed a partial dependence. The remaining seven loci showed
fixL- and fixJ-independent induction of
expression in response to oxygen limitation. This suggests that in
S. meliloti, additional regulatory system(s) exist that
respond either directly or indirectly to oxygen limitation conditions.
Bacteria in soil environments are
continuously exposed to changing environmental conditions, including
oxygen limitation (23). Oxygen levels in the soil
fluctuate with time, often resulting in microaerobic and anaerobic
microsites (15). In the rhizosphere, the soil adjacent to
and influenced by the plant root system, root and microbial respiration
can result in oxygen levels lower than those found in bulk soil
(4). Microorganisms have developed mechanisms to sense and
adapt to environmental changes, such as oxygen limitation. These
responses may be important for the persistence of and competition
between microorganisms in the soil.
The aerobic soil bacterium Sinorhizobium meliloti encounters
oxygen limitation conditions in two different ecological niches: while
in a free-living state in the soil or when in symbiotic association
with the legume alfalfa (Medicago sativa) inside
microaerobic nitrogen-fixing root nodules. Microaerobic conditions
inside legume nodules are necessary to maintain nitrogen fixation
activity due to the oxygen sensitivity of the enzyme nitrogenase.
S. meliloti coordinates the expression of genes required for
nitrogen fixation and for respiration inside nodules via a
two-component regulatory system, FixL/FixJ, that senses microaerobic
conditions and controls target genes accordingly (3, 6).
Towards the goal of examining the importance of the oxygen limitation
response for S. meliloti survival in the soil environment, we have initiated a characterization of the molecular response of
free-living S. meliloti to oxygen limitation using a gene
reporter system. The involvement of FixL/FixJ in the regulation of the oxygen limitation response was also examined.
Isolation of S. meliloti loci expressed in response
to oxygen limitation.
A previously described (16)
collection of 5,000 S. meliloti 1021 strains containing
transposon Tn5-1063 insertions was screened for strains
that luminesced when oxygen levels became limiting. Tn5-1063 functions as a transcriptional reporter system as
it contains promoterless luxA luxB genes which encode the
enzyme luciferase (24). The collection was screened by
first spotting the strains onto duplicate plates containing solid
GTS medium (12) lined with a filter membrane as
previously described (16). The duplicate plates' contents
were incubated at 28°C for 36 h followed by the incubation
(28°C) of one plate's contents with atmospheric oxygen
concentrations (~21%) and the incubation of the second plate's
contents in an airtight jar under continuous flushing with an ~1%
oxygen gas mixture. The gas mixture was generated by mixing nitrogen
and compressed air using a Multigas System (Hotpack, Philadelphia, Pa.)
and was monitored periodically using an oxygen electrode
(Microelectrodes Inc., Londonderry, N.H.). After 6 to 8 h of
incubation, the strains were examined for luciferase activity using a
photonic camera system as described previously (16). A
total of 11 strains containing Tn5-1063 fusions
that showed significantly increased luminescence after incubation under 1% oxygen, compared to incubation under 21% oxygen, were identified (Fig. 1). The loci tagged in these
strains have been designated loe (low-oxygen expressed).
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3728-3731.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
Isolation and Regulation of Sinorhizobium
meliloti 1021 Loci Induced by Oxygen Limitation
ABSTRACT
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FIG. 1.
Induction of expression of the loe
fusions after 6 h of incubation under decreased oxygen
concentrations. Shown are the average levels of luminescence generated
by the loe fusion strains on solid medium when incubated
for 6 h with 1% oxygen (white bars) or 21% oxygen (black bars).
The
cv-2::Tn5-1063
fusion is expressed at both oxygen concentrations; therefore, the
strain harboring the cv-2 fusion was included to serve
as a positive control in these studies. Shown atop each bar is the
standard deviation observed in four or five independent trials. The
pixel values correlate to the level of luminescence observed for each
strain and were obtained using the University of Texas Health Science
Center, San Antonio, Image Tool program. A standard t
test of the data indicated that the difference observed between the two
conditions for each loe fusion strain was significant.
Molecular characterization of the loe strains. A Southern analysis using pRL1063a (24) as probe revealed that the 11 loe strains each contained a single Tn5-1063 fusion and that the location of each fusion was distinct (data not shown).
The 11 Tn5-1063 fusions were excised from genomic DNA and cloned, and at least 200 bp of the DNA sequence flanking each end of the Tn5-1063 insertions was determined as described previously (5). The sequence obtained for each locus was assembled using the program Sequencher (Gene Code Corporation, Ann Arbor, Mich.) and compared to GenBank databases using the gapped BLASTX and BLASTN programs (2). The results are summarized in Table 1.
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Regulation of the loe fusions by fixL and fixJ. An important objective of this research was to determine if regulatory systems in addition to FixL/FixJ exist that help to mediate the physiological response of S. meliloti to changes in oxygen availability. Towards this goal, the luciferase expression patterns of the strains carrying the loe fusions were examined in genetic backgrounds where the oxygen regulatory genes fixL and fixJ were inactivated.
To generate loe::Tn5-1063 fixL or fixJ double mutants, it was necessary to first generate insertions within fixL and fixJ that contained a selectable marker different from the markers carried by Tn5-1063. Therefore, we obtained S. meliloti strains GMI5705 (fixL2.66::Tn5) and GMI5704 (fixJ2.3::Tn5) (6) and replaced the Tn5 insertions in these strains with Tn5-233 via homologous recombination using the method described by De Vos et al. (9). The exact replacement of Tn5 with Tn5-233 within fixL and fixJ in the resulting strains (FdB3463 and FdB3464) was verified by Southern analysis (data not shown.) By using protocols described in reference 10, phage
M12 lysates of FdB3463 and FdB3464 were generated and used to transfer the fixL::Tn5-233 and the
fixJ::Tn5-233 insertion mutations into the 11 loe fusion strains. The resulting double mutants were
examined by Southern analysis of genomic DNA to ensure that the
positions of the Tn5-1063 and Tn5-233 markers
within the genome had been maintained (data not shown). The double
mutants were then examined for changes in the expression of the
loe fusions relative to the parent strains after a switch to
oxygen limitation conditions. The expression of the loe-1,
loe-10, and loe-11 fusions was found to be
dependent upon both fixL and fixJ, while
loe-7 fusion expression was found to be partially dependent
upon fixL and fixJ (Fig.
3). fixL- and
fixJ-dependent expression of loe-1
(fixN) was the expected result, as it had been demonstrated
previously in S. meliloti that fixN expression is
controlled via the FixL/FixJ regulatory system (6). The
remaining loe fusions demonstrated increased expression in a
background of fixL and fixJ. This increase may have been an artifact of the luciferase reporter system and assay, since the positive control fusion, cv-2, also showed this
increase. It was clear, however, that the induction of the expression
of the remaining loe fusions (loe-2,
loe-3, loe-4, loe-5, loe-6, loe-8, and loe-9) was independent of
fixL and fixJ. These results support the idea
that at least one additional regulatory system exists in S. meliloti that responds to low-oxygen conditions. Whether the
regulator(s) responds to oxygen levels directly or indirectly (e.g., a
change in redox or growth rate caused by oxygen depletion) remains to
be determined.
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The symbiotic phenotype of the loe fusion strains. Because the physiological environment inside alfalfa nodules is microaerobic (0.03 µM oxygen) (3), we hypothesized that the loci identified in this study may have roles important in the formation of functional nitrogen-fixing nodules on alfalfa. To test this hypothesis, the 11 loe fusion strains were inoculated onto alfalfa seedlings and examined for nodule formation and for nitrogenase activity (via acetylene reduction assays) as described previously (16). All 11 loe fusion strains generated nodules with nitrogenase activity (data not shown) comparable to those for the reference strain S. meliloti 1021 (data not shown), suggesting that the loci disrupted by Tn5-1063 in these strains were not essential for symbiotic nitrogen fixation. Although not essential, the loe loci may still be involved in symbiotic nitrogen fixation, since our assay may not have been sensitive enough to detect small decreases in nitrogenase activity. Lack of a nitrogen fixation phenotype may also be due to the presence of additional copies of the loci within the genome. For example, the finding that the strain carrying loe-1(Tn5-1063 insertion within fixN) was symbiotically proficient was expected, as it had been noted in the literature that a second functional copy of fixN exists within the genome of S. meliloti (7, 19).
In conclusion, the approach presented here has clearly permitted the identification of loci affected by oxygen availability. In addition we have provided evidence for the existence of additional regulatory systems in S. meliloti that are responsive either directly or indirectly to oxygen limitation conditions. Future studies in our laboratory will focus on isolating the genes involved in the regulation of the FixL/FixJ-independent loe loci and on examining a possible role for the regulatory genes in microbial persistence in the soil environment.Nucleotide sequence accession numbers. The GenBank nucleotide sequence accession numbers for the loe loci are AF336863 (loe-2), AF336864 (loe-3), AF336865 (loe-4), AF336866 (loe-5), AF336867 (loe-6), AF336868 (loe-7), AF336869 (loe-8, loe-9), AF336870 (loe-10), and AF336871 (loe-11).
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ACKNOWLEDGMENTS |
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We thank G. Walker and P. Boistard for strains and U. Rossbach for computer support. We also thank C. P. Wolk and de Bruijn lab members for helpful suggestions and discussions.
This work was supported by grant DE-FG02-91ER20021 from the U.S. Department of Energy. J. Trzebiatowski was supported by National Research Service Award 5-F32-GM19412-03 from the National Institute of Health.
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FOOTNOTES |
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* Corresponding author. Mailing address: Center for Molecular Medicine and Genetics, Wayne State University, 540 E. Canfield St., Detroit, MI 48201. Phone: (313) 577-1627. Fax: (313) 577-5218. E-mail: jparrish{at}genetics.wayne.edu.
Present address: Laboratoire de Biologie Moléculaire des
Relations Plantes-Microorganismes, BP27, 31326 Castanet Tolosan Cedex, France.
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Applied and Environmental Microbiology, August 2001, p. 3728-3731, Vol. 67, No. 8
0099-2240/01/$04.00+0 DOI: 10.1128/AEM.67.8.3728-3731.2001
Copyright © 2001, American Society for Microbiology. All rights reserved.
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