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J. Biol. Chem., Vol. 277, Issue 1, 462-468, January 4, 2002
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From the
Received for publication, July 16, 2001, and in revised form, October 18, 2001
Rhizobium etli CNPAF512 produces an
autoinducer that inhibits growth of Rhizobium
leguminosarum bv. viciae 248 and activates the
Agrobacterium tumefaciens tra reporter system. Production of this compound in R. etli is dependent on two genes,
named cinR and cinI, postulated to code for a
transcriptional regulator and an autoinducer synthase, respectively.
NMR analysis of the purified molecule indicates that the R. etli autoinducer produced by CinI is a saturated long chain
3-hydroxy-acyl-homoserine lactone, abbreviated as 3OH-(slc)-HSL. Using
cin-gusA fusions, expression of
cinI and cinR was shown to be growth
phase-dependent. Deletion analysis of the cinI
promoter region indicates that a regulatory element negatively controls
cinI expression. Mutational analysis revealed that
expression of the cinI gene is positively regulated by the CinR/3OH-(slc)-HSL complex. Besides 3OH-(slc)-HSL, R. etli
produces at least six other autoinducer molecules, for which the
structures have not yet been revealed, and of which the synthesis
requires the previously identified raiI and
raiR genes. At least three different autoinducers,
including a compound co-migrating with 3OH-(slc)-HSL, are produced in
R. etli bacteroids isolated from bean nodules. This is
further substantiated by the observation that cinI and
cinR are both expressed under symbiotic conditions. Acetylene reduction activity of nodules induced by the cin
mutants was reduced with 60-70% compared with wild-type nodules,
indicating that the R. etli 3OH-(slc)-HSL is involved in
the symbiotic process. This was further confirmed by transmission
electron microscopy of nodules induced by the wild type and the
cinI mutant. Symbiosomes carrying cinI mutant
bacteroids did not fully differentiate compared with wild-type
symbiosomes. Finally, it was observed that the cinR
gene and raiR control growth of R. etli.
Although bacteria are unicellular organisms, they often show group
behavior. For this, bacteria have to monitor their own population size.
This can be achieved by means of autoinduction. Cell-cell communication
using N-acyl-homoserine lactone
(AHL)1 signals is one of the
few known mechanisms through which bacteria can communicate with each
other and is a widespread phenomenon in Gram-negative bacteria (1, 2),
including plant-associated bacteria (3, 4). AHLs mainly vary with
respect to the length (4-14 carbons) and the substituent (H, O, or OH)
at the third carbon of the acyl side chain. The AHL signal is released
into the environment, either by passive diffusion, as observed for 3O-C6-HSL in Vibrio fischeri and
Escherichia coli cells (5) or by a combination of diffusion
and active efflux in Pseudomonas aeruginosa (6) and
accumulates with growth of the bacterial population. At least in
V. fischeri, the signal freely diffuses back into the cells
such that its intracellular concentration also rises as a function of
the increase in bacterial population. Transduction of this information
to response regulators of gene expression leads to the elaboration of
an appropriate phenotype at high cell densities.
Using the Agrobacterium tumefaciens tra reporter system to
detect autoinducer molecules, members of the genus Rhizobium
showed the greatest diversity, with some producing as few as one and others producing as many as seven detectable signals (7). In Rhizobium leguminosarum bv. viciae, the
cin locus encodes a master regulatory system. Mutation of
cinIR abolishes the production of
N-(3R)-hydroxy-7-cis-tetradecenoyl-L-homoserine
lactone (3OH-C14:1-HSL), also termed "small", and
reduces the synthesis of AHLs produced by the enzymes encoded by
raiI, traI-like, or rhiI (8). The reduced levels of C6-HSL and C8-HSL and
decreased rhiR expression cause a repression of the
rhizosphere-expressed genes in cinI or cinR
mutants (8-10). Furthermore, 3OH-C14:1-HSL induces the stationary phase (9) whereas mutation of cinI has little
effect on growth or nodulation of the host plant (8, 11).
Rhizobium etli CNPAF512, a nitrogen-fixing symbiont of
Phaseolus vulgaris, produces at least seven different
autoinducer molecules (12). Rosemeyer et al. (12) identified
the raiIR quorum-sensing system in R. etli.
Examination of different rai mutants for nodulation of beans
showed that raiI is involved in the restriction of nodule number, whereas nitrogen-fixing activity per nodule is not affected. The culture supernatant of a raiI mutant revealed only three
different autoinducer molecules. One of them induces a
growth-inhibitory effect on R. leguminosarum bv.
viciae 248, similar to the low molecular weight bacteriocin
"small", which is common in fast-growing rhizobia. The properties,
growth inhibition, and autoinducer activity, are features reported for
3OH-C14:1-HSL, produced by R. leguminosarum bv.
viciae (13).
Here we report on the cin locus, the second quorum-sensing
system in R. etli CNPAF512 that is expressed under both
free-living and symbiotic conditions and is involved in the production
of a 3OH-(slc)-HSL (slc, saturated long chain). Despite high sequence conservation, the cin locus of R. etli and
R. leguminosarum bv. viciae appear to control
different functions. Mutational analysis of R. etli revealed
that the cin system regulates growth and fulfills a key role
in bacteroid differentiation and nitrogen fixation.
Bacterial Strains, Plasmids, and Culture
Conditions--
E. coli was grown in Luria-Bertani (LB)
medium at 37 °C (14). Rhizobium was grown at 30 °C in
TY or AMS medium (15). A. tumefaciens NT1 was grown in AB
medium at 28 °C (16). Antibiotics were added as appropriate. To
study bacterial growth over a long period of time, overnight cultures
of the strains were diluted in 10 mM MgSO4 to
an absorbance at 595 nm (A595) of 0.3 (dilution ~10-fold). Subsequently, these cultures were diluted 10-fold after which 295 µl of growth medium was inoculated with 5 µl of bacterial suspension (total dilution ~6000-fold). Bacteria were grown, and the
absorbance was measured automatically each 30 min during at least 6 days in a BioscreenC (Labsystems Oy). For each time point, the
average optical density was calculated from five independent measurements.
Plant Experiments, Bacteroid Isolation, and Preparation of
Bacteroid-free Supernatant--
Phaseolus vulgaris cv.
Limburgse vroege seedlings were planted in Snoeck medium, which is
optimized for in vitro growth of common
bean.2 Plants were inoculated
and grown essentially as described by Michiels et al. (17).
Acetylene reduction activity was determined 3 weeks after inoculation.
For expression analysis during symbiosis, bacteroids were purified from
plant material by differential centrifugation (17). This protocol was
slightly adapted for the extraction of bacteroid autoinducers. Nodules
from 1-2 plants (± 1.5 g) were collected in a Falcon tube (15 ml) containing 0.2 g of polyvinyl polypyrolidone, and magnesium
phosphate buffer was added to a final volume of 6 ml and subsequently
crushed. Next, the plant material was removed by differential
centrifugation. To lyse the bacteria, the suspension was supplemented
with SDS (final concentration, 1%) and proteinase K (final
concentration, 100 µg/ml) and incubated for 1 h at 37 °C.
After incubation, the cell debris was removed by centrifugation at 6000 rpm after which the cell-free supernatant was immediately extracted to
isolate the autoinducers.
Qualitative and quantitative analysis of Extraction and Detection of
Autoinducers--
Rhizobium strains and A. tumefaciens transformants were tested for activation of the
A. tumefaciens tra reporter system and for bacteriostatic
activity toward the sensitive strain R. leguminosarum bv.
viciae 248 as described by Schripsema et al.
(13). To extract autoinducers, strains were first grown to the
stationary phase. Cell-free supernatant from either a free-living
liquid culture or from symbiotic bacteroids (see above) was extracted,
and the autoinducers were detected on TLC by the tra
reporter system as described by Rosemeyer et al. (12).
Isolation of the R. etli 3OH-(slc)-HSL--
Ethyl acetate
extracts from 10 liters of stationary phase cultures of FAJ4010 were
reconstituted in 50% acetonitrile in water and subjected to
solid-phase extraction using Waters OASIS HLB cartridges. Fractions
were eluted with an increasing concentration of methanol in water
(50-100%, v/v). Positive fractions on the A. tumefaciens
tra reporter and R. leguminosarum bv. viciae
248 growth inhibition assays were collected, dried, and redissolved in
50% acetonitrile in water and applied to a C18 Phenomenex Bondclone HPLC column. Fractions were eluted with a linear gradient of
acetonitrile in water (40-100%, v/v) over a 30-min period at a flow
rate of 1 ml/min and monitored at 200 nm. Positive fractions were
re-chromatographed using an isocratic mobile phase (50% acetonitrile
in water; 1 ml/min), and the active subfraction was analyzed by NMR spectroscopy.
DNA Techniques and Nucleotide Sequencing--
Standard
techniques were used for DNA manipulations (14). Restriction enzymes
were used according to the manufacturer's instructions. DNA probes for
Southern hybridization were labeled with digoxigenin. An ordered series
of sequencing clones was obtained via restriction enzyme mapping of
pFAJ4003 and ExoIII deletion procedures
(Erase-A-Base® Promega). Nucleotide sequencing of
cinR, cinI, and the flanking regions was
accomplished by using the A.L.F. sequencer (Amersham Biosciences).
Cloning of the cinR-cinI Gene Region and Construction of
Mutants--
The 5.3-kb EcoRI fragment from pFAJ4000
containing cinR, cinI, orf123, and
orf140 was first cloned into pBluescriptIIKs+
yielding pFAJ4003. A PstI-EcoRI fragment of
pFAJ4003, lacking the 5'-end of orf140, was
EcoRI-PstI subcloned in the broad host range
vector pLAFR3 (pFAJ4012; Fig. 1). Furthermore, pFAJ4013 was made by
insertion of the 5.3-kb EcoRI fragment from pFAJ4003 in
pPZP200, in which part of the multiple cloning site between XhoI and PacI was deleted.
A fragment containing cinR and cinI was amplified
via PCR using primers Rhi15 (5'-ATGGGAATTCATCCAGTGCCGAGGAGATAC-3') and
Rhi16 (5'-TAGAGGATCCTCGGCATCATCATCACCTCG-3') and pFAJ4003 as template DNA. The resulting 3-kb fragment was digested with EcoRI and
BamHI and cloned in pUCNotI- Construction of Fusions--
Plasmid-borne
PcinR-gusA and
PcinI-gusA fusions were constructed
in the promoter probe vector pFAJ1703 (20). The cinR
promoter region was amplified by PCR with FAJ1337 as a template using
primers Rhi15 and Tn5-B (5'-GGTTCCGTTCAGGACGCTAC-3'). The
resulting 1.2-kb fragment was cloned into the HpaI site of
pFAJ1703 after blunting of the fragments, yielding plasmid pFAJ4011. To
construct pFAJ4014 (cinI-gusA fusion carrying a
mutant cinR gene region), the 1.8-kb EcoRI-SphI fragment of pFAJ4007 was blunt-end
ligated into the HpaI site of pFAJ1703. pFAJ4015 is a
KpnI deletion derivative of the
cinI-gusA fusion pFAJ4014 (Fig. 1).
Identification of the cin Locus of R. etli CNPAF512--
A
Tn5-induced mutant library of R. etli CNPAF512
was screened using the growth inhibition assay (13). The mutant FAJ1337 no longer inhibited growth of R. leguminosarum bv.
viciae 248. By means of inverse PCR on
EcoRI-digested genomic DNA of FAJ1337 with
transposon-specific primers, ~10 kb of the transposon flanking DNA of
FAJ1337 was amplified and cloned. Subsequently, PstI
fragments of the amplified DNA were subcloned into pUC18 and tested for autoinducer production in E. coli using the tra
system of A. tumefaciens as a reporter (7). One positive
clone was obtained, and the corresponding 7.5-kb PstI
fragment, carrying part of the cloning vector, was subsequently used as
a probe in a Southern hybridization of a R. etli CNPAF512
genomic library, constructed in pLAFR1 (21). A 5.3-kb EcoRI
fragment from clone pFAJ4000 was found to give a positive hybridization
signal. FAJ1337 was complemented for growth inhibition of the sensitive
strain R. leguminosarum bv. viciae 248 by
both pFAJ4000 and pFAJ4012 (Fig. 1).
DNA Sequence Analysis of the R. etli CNPAF512 cin Locus--
DNA
sequence analysis of the cloned 5.3-kb EcoRI fragment
revealed five complete open reading frames (ORFs) as illustrated in
Fig. 1. The ORFs were identified and the start codon was assigned on
the basis of the GC content (22), the preferential codon usage
(searchcutg, GCG-package Wisconsin), and similarity with known genes.
One ORF (726 bp) codes for a putative protein of 241 amino acids with a
calculated molecular mass of 27.3 kDa. The putative protein is similar
to several LuxR-type transcriptional activators such as CinR (96%
amino acid identity) of R. leguminosarum bv. viciae (AAF89989), CerR (30% identity) of Rhodobacter
sphaeroides (AAC46021) and RaiR (31% identity) of R. etli (AAC38173). Because of the high amino acid identity of the
R. etli putative protein with CinR, it was given the same
name. Alignment of R. etli CinR with E. coli NarL
(23), designates a helix-turn-helix motif between residues 196 and 220. In silico analysis of the cinR non-coding region
revealed a putative terminator (nucleotides 2615-2650,
A second ORF of 666 bp, which is unidirectional with cinR,
is found 224-bp downstream of cinR. While the deduced amino
acid sequence is most related to CinI (95% identity) of R. leguminosarum bv. viciae (AAF89990), it is also similar
to CerI (33% identity) of R. sphaeroides (AAC46022) and
RaiI of R. etli (39% identity) (AAC38172). The putative
protein with a calculated molecular mass of 25.0 kDa was named CinI.
CinI contains 10 invariant amino acids typical for the LuxI family of
autoinducer synthases (24) of which seven (R24, E43, D45, D48, R70,
E101, R104; numbered with respect to R. etli CinI) may take
part in the S-adenosyl-methionine binding site (25). In the
intergenic region between cinI and ORF123 (see below), two
putative terminators (nucleotides 3520-3576,
Immediately downstream of cinI, a short ORF123 (368 bp),
located on the opposite strand, encodes a putative response regulator of the CheY family with 94% identity to the CheY like protein of
R. leguminosarum bv. viciae (AAF89991), 40%
identity to a probable response regulator of Mesorhizobium
loti (BAB49462) and 35% identity to the FixL receiver domain of
R. etli (AAG00949). The R. etli response
regulator encoded by ORF123 contains the conserved residues D14, D58,
T86 en K106 (numbered with respect to ORF123), which are part of the
essential active site of CheY (26) in which D58 can be phosphorylated.
ORF140 (420 bp) is located upstream of cinR and codes for a
protein similar to a hypothetical protein (AAG2039) of
Halobacterium sp. NRC-1 (51% identity), and an unknown
protein of Bacillus subtilis (CAB11811) (39% identity).
Upstream of ORF123, a Met-tRNA gene (74 bp) (tRNAscan-S.E. v. 1.11) is
found with the anticodon (CAT) located between nucleotides 4364 and
4366 of the 5.3-kb EcoRI fragment. This gene shows perfect
(100%) DNA sequence identity to the Met-tRNA gene of R. leguminosarum bv. viciae (AF210630) and a M. loti sequence (AP002999) and is similar to a Rhizobium sp. NGR234 sequence (AE000079) (91% identity). Analysis of the
intergenic region between ORF123 and the Met-tRNA gene indicates the
presence of a putative terminator sequence downstream of the tRNA gene
(nucleotides 5002-5068, NMR Analysis of the Isolated Compound Produced by R. etli
CinI--
An NMR analysis of the compound produced by R. etli CinI was conducted. As a control, 3OH-C14:1-HSL
was synthesized (data not shown) and analyzed. The NMR data of the
synthetic compound are in agreement with previously recorded data
(13).
The 1H NMR spectrum of the R. etli compound
contains all characteristic signals of a 3-hydroxyacyl-homoserine
lactone. Evidence for the homoserine lactone moiety is constituted by
the signal at 6.33-6.24 ppm (amide NH) and the characteristic
butyrolactone signals at 4.52, 4.47, 4.27, 2.77, and 2.10 ppm. The line
shapes and splitting patterns are in good agreement with those of
synthetic acyl-homoserine lactones. Moreover, the line at 3.98 ppm is
similar to the CH(OH) resonance in 3OH-C14:1-HSL. However,
the characteristic double bond signals between 5 and 6 ppm observed for
3OH-C14:1-HSL, as well as the signals around 2.00 ppm of
the protons on adjacent carbon atoms are absent. On the basis of its
chromatographic properties (TLC, HPLC), the R. etli HSL is
likely to possess a long chain fatty acid group. This allows to
tentatively assign the spectrum of the R. etli autoinducer
produced by CinI to a saturated long chain 3-hydroxy-acyl-homoserine
lactone, which is clearly different from the structure of R. leguminosarum 3OH-C14:1-HSL. In the subsequent part we
will refer to the R. etli autoinducer as 3OH-(slc)-HSL.
Gene Regulation of cinI and cinR--
To study the cell
density-dependent expression of the cin locus,
cinR-gusA (pFAJ4011) and
cinI-gusA (pFAJ4014) fusions were constructed.
The cinR gene in pFAJ4014 was inactivated by site-directed mutagenesis. To determine whether a promoter is present in the cinR-cinI intergenic region, a second
cinI-gusA (pFAJ4015) fusion, containing a 632-bp
upstream region of cinI (Fig. 1), was also constructed. As
shown in Fig. 2A,
cinI expression from pFAJ4014 under free-living conditions
in a wild-type background increased with the cell density and reached a
plateau (1500-2400 units) as the culture entered into the stationary
phase. cinI expression from pFAJ4015 displayed a similar
cell density-dependent pattern of expression (Fig.
2B). However, two differences between the two fusions can be
noticed. Firstly, induction of cinI expression from pFAJ4015
starts at a lower absorbance compared with pFAJ4014. Secondly, the
maximum expression level of cinI from pFAJ4015 is approximately 4-fold higher, compared with that of pFAJ4014. None of
the cinI-gusA fusions are expressed in cinR or
cinI mutant backgrounds (Fig. 2, A and
B), demonstrating that transcription of cinI
requires both CinI and CinR.
A threshold cell density (approximately A595 = 0.6) seems to be required to observe a very low cinR
expression in wild-type and cinR or cinI mutant
backgrounds (Fig. 2C). Although expression of
cinR remains low, it reaches a maximum (~20 units) as soon as cells enter the stationary phase. The observation that both cinR and cinI expression is maximal during the
same stage of growth is in agreement with a role of CinR in the
regulation of cinI expression.
The observation that cinI is expressed in either the
presence or absence of the cinR promoter region,
demonstrates that cinR and cinI are likely
organized into different transcriptional units. Furthermore, the
overall high expression level of cinI (minimal 100-fold
higher than cinR), suggests that transcription of
cinI in both pFAJ4014 and pFAJ4015 is controlled by a
promoter in the cinR-cinI intergenic region. This
is in agreement with the presence of a putative terminator downstream
of cinR. The difference in cinI expression levels
between pFAJ4014 and pFAJ4015, is likely caused by a negative
regulation at the level of the putative cinI promoter.
Expression under Symbiotic Conditions--
Expression of the
cinI-gusA and cinR-gusA
fusions was monitored in isolated bacteroids, obtained from 21-day-old
bean nodules, induced by wild-type CNPAF512, the cinR
(FAJ4007), and cinI mutant (FAJ4006). The presence of the
plasmid-borne gusA-fusions pFAJ4011, pFAJ4014, and pFAJ4015
in the different strains did not affect their symbiotic performance
(data not shown). As illustrated in Fig.
3, expression of the cinR
fusion (pFAJ4011) is low (~40 units) and does not significantly
differ in the three genetic backgrounds. Expression of the short
cinI fusion (pFAJ4015) is significantly higher (~8-fold)
compared with the long cinI fusion (pFAJ4014) in wild-type
background, similar to what was observed under free-living conditions.
However, in contrast to free-living conditions, expression of the short
cinI fusion is less dependent on the presence of CinI
because cinI expression from pFAJ4015 is still observed in cinI mutant (FAJ4006) bacteroids. Similarly to free-living
conditions, cinI gene expression requires the presence of
CinR. Finally, it should be noted that the expression level of
cinI in bacteroids is at least 10-fold lower compared with
free-living conditions.
Shortly after inoculation (24 h), cinI-gusA
expression (pFAJ4015) was observed on the surface of root hairs during
colonization of wild-type and the cinI mutant (FAJ4006)
(data not shown). Light microscopic analysis, 48 h after
inoculation of the roots, localized pFAJ4015 expression in the
infection tread, formed by wild-type and the FAJ4006 mutant (Fig.
4, A and B).
However, no cinI-gusA expression from pFAJ4015
was observed in the cinR mutant (FAJ4007) during
colonization (data not shown) or in the infection tread (Fig.
4C). Based on the intensity of the Gus-staining, the fusion seems less expressed in a cinI mutant background (FAJ4006)
compared with the wild-type background, similarly as observed for
bacteroid expression.
Analysis of Autoinducers--
The cell-free culture supernatant of
the R. etli wild type, the cinR and the
cinI mutant, was extracted with ethyl acetate, and analyzed
by TLC combined with the A. tumefaciens tra reporter system
(3). As was previously published by Rosemeyer et al. (12),
wild-type R. etli CNPAF512 produces at least seven
autoinducers (Fig. 5, lane A).
The raiI mutant, FAJ4010 (Fig. 5, lane C)
produces 3OH-(slc)-HSL (F-AI1) and two other active molecules (F-AI2,
F-AI3), which co-migrate with compounds extracted from sterile,
non-inoculated TY medium (Fig. 5, lane E). Ethyl acetate
extracts from AMS medium or water were negative in this test (data not
shown). Both cinR and cinI mutants (FAJ4006,
FAJ4007, FAJ4009) secrete all of the autoinducers except 3OH-(slc)-HSL
(Fig. 5, data shown for FAJ4006 in lane B) as was also
observed in the growth inhibition assay. As illustrated in Fig. 5
lane D, the extract prepared from a raiIcinI double mutant FAJ4013, contains two active spots (F-AI2, F-AI3), co-migrating with active molecules present in sterile, non-inoculated TY. Taken together, these data suggest that two genetic systems, rai and cin, are required for the synthesis of
all of the autoinducers by R. etli CNPAF512 as detected by
the Agrobacterium tra reporter system.
To confirm that the cin locus is solely responsible for the
production of the 3OH-(slc)-HSL molecule, a plasmid containing the
5.3-kb EcoRI fragment, pFAJ4013, was introduced in the
A. tumefaciens NT1 strain (lacking the Ti plasmid,
containing plasmid pJM749), a derivative lacking synthesis of
autoinducers (Fig. 5, lane G). TLC analysis revealed the
production of a compound co-migrating with the R. etli
3OH-(slc)-HSL (Fig. 5, lane F). Moreover, using this
extract, growth inhibition of R. leguminosarum bv.
viciae 248 was observed in a growth inhibition assay (data not shown).
Finally, the autoinducer production was analyzed during symbiosis. For
this, the lysate, prepared from R. etli bacteroids, was
extracted with ethyl acetate and the extracted autoinducers from one
nodulated plant were analyzed on TLC for compounds activating the
A. tumefaciens tra system. Less different autoinducers seem to be produced under symbiotic conditions compared with free-living growth (Fig. 5, lane H). One of the wild-type bacteroid
autoinducer compounds, S-AI1, co-migrates on TLC with 3OH-(slc)-HSL
(F-AI1). The bacteroid autoinducer S-AI3 co-migrates with a compound,
extracted from root material and able to activate the tra
reporter system (Fig. 5, lane I). Taken together, these
results indicate that at least three different autoinducer molecules
are produced by R. etli during symbiosis (S-AI1, S-AI2,
S-AI4).
Mutation of the rai and cin Autoinducer System Affects Growth of
Rhizobium--
To examine the phenotypic relevance of the
rai and cin system in R. etli, a
rai-cin double mutant was constructed. To monitor growth, absorbance was measured in a BioscreenC over a 6-day period (total dilution 6000-fold). Growth of the raiR mutant
(FAJ1329) and the cinI mutant (FAJ4006) was clearly delayed
compared with wild-type growth in AMS mannitol medium as can be seen
from Fig. 6. The lag phase was prolonged
by 14.5 and 24.5 h, respectively. Noteworthy, the growth pattern
of the cinR mutants, FAJ4007 and FAJ4009, as well as the
growth of a raiI mutant (FAJ4010) was not different from
that of the wild type (Fig. 6). Entry into the exponential phase was
even more delayed (79 h more than the wild type) when growth of the
raiIcinI double mutant, FAJ4013, was examined (Fig. 6).
Furthermore, growth of the cinI (g = 6.8 h) and
raiR (g = 8.3 h) mutants as well as that of the
double raiIcinI mutant (g = 8.8 h) was marked by a
1.5- to 2-fold increase in generation time compared with the wild type
(g = 4.5 h). The observed difference in lag phases was less
pronounced when the culture was only 1000-fold diluted whereas no
difference was obtained with a 100-fold-diluted preculture (data not
shown). Although a similar increase in generation time was also
observed when bacteria were grown in AMS succinate medium (data not
shown), the difference in the lag phases was less pronounced,
indicating that the raicin-dependent regulation
of growth is complex and depends on the growth medium (carbon source)
used.
Symbiotic Phenotype of cinR and cinI Mutants--
The R. etli cinR and cinI mutants were tested for their
ability to nodulate common bean (Phaseolus vulgaris cv.
Limburgse vroege) and to fix nitrogen. No significant differences in
kinetics of appearance of the first nodules were observed (Table
I). Moreover, the plant and nodule dry
weight as well as the nodule number, determined 21 days after
inoculation, were not significantly different between plants nodulated
by the wild-type or the mutant strains (Table I). However, a clear
impact of the cin system on nitrogen fixation could be
observed because inoculation of bean plants with the R. etli
cinR mutants (FAJ4007 and FAJ4009) or the cinI mutant
(FAJ4006) resulted in a statistically lower acetylene reduction activity (ARA) per plant (30-40% of wild-type ARA; Table I). Furthermore, inoculation with the FAJ4013 raiIcinI double
mutant decreased nitrogen fixation per plant even further (27% of
wild-type ARA; Table I). Autoinducer production, growth, and the tested symbiotic features are similar for FAJ4009 and FAJ4007, the polar and
non-polar cinR mutant. This observation supports further the hypothesis that cinR and cinI are likely
organized into different transcriptional units.
To further examine the effect of cin mutations at the
bacteroid level, sections of nodules, formed by wild-type and mutant strains were analyzed by transmission electron microscopy (TEM). This
analysis indicated that cinI mutant bacteroids were always individually packed in the symbiosome membrane (SM) (Fig.
7A), whereas wild-type
symbiosomes usually contained multiple bacteroids (Fig. 7B).
Furthermore, cinI mutant bacteroids were surrounded by a
minimal symbiosome space (SS) compared with wild-type bacteroids. These
results indicate that the cin system fulfills a key role during symbiosome development.
We have characterized the cin locus in R. etli CNPAF512, involved in the synthesis of 3OH-(slc)-HSL,
containing a saturated long acyl chain. In our current model,
cinI codes for the autoinducer synthase and cinR
for the transcriptional regulator that binds the 3OH-(slc)-HSL. The
latter complex activates cinI expression. The
chromatographic properties of 3OH-(slc)-HSL are very similar to
3OH-C14:1-HSL, produced by R. leguminosarum bv.
viciae. Moreover, both compounds induce growth inhibition of
R. leguminosarum bv. viciae 248. In contrast,
major differences between both cin loci with respect to
growth under free-living conditions and symbiotic performance of the
corresponding mutants were observed, indicating that both molecules may
perform different functions in R. etli and R. leguminosarum bv. viciae.
Mutational and expression analysis revealed that cinR and
cinI have distinct promoters. Expression of cinR
is low both under free-living and symbiotic conditions and is cell
density-dependent. Expression of cinI is also
regulated in a cell density-dependent way and reaches a
maximal expression level in the stationary phase. Furthermore, the
cinI gene is expressed in bacteroids. Expression of
cinI requires CinR both under free-living conditions and
during symbiosis. Expression levels of cinI differ depending
on the extent of the upstream region in the plasmid-borne
cinI-gusA fusion construct. Possibly, the DNA
sequence upstream of the putative cinI promoter in the
plasmid construct competes for a trans-acting factor that is required
for cinI transcription, such as CinR. Alternatively, several
reports from the literature suggest complex regulation of genes
involved in quorum-sensing, in particular the occurrence of negative
regulators. Examples of such negative regulators are: EsaR in
Pantoea stewartii (27), TraS in A. tumefaciens
(28) and RsaL in P. aeruginosa (29). Moreover, Lithgow
et al. (8) showed that expression of cinI is
significantly reduced when the symbiotic plasmid pRL1JI is present in
R. leguminosarum bv. viciae, resulting in a
reduction in the level of 3OH-C14:1-HSL. The mechanism of
pRL1JI-mediated repression of cinI expression or the
identity of the factors enhancing or relieving this repression have not yet been identified.
cinI is likely positively autoregulated in the bacteroids
even though a clear expression level could be observed in a
cinI mutant background. CinR may stimulate expression of
cinI even in the absence of 3OH-(slc)-HSL. Whether CinR is
activated through an autoinducer molecule produced by RaiI (see
further) or whether a plant compound is able to activate CinR is yet
unknown. Teplitski et al. (30) noticed that various species
of higher plants can secrete substances, chemically different from
bacterial AHLs but mimicking their activity. Also, extraction of
non-inoculated Phaseolus vulgaris bean roots revealed the
production of a compound activating the Agrobacterium tra
reporter system, as illustrated in this work, making a cross-talk
between both partners in the symbiosis quite reasonable. However, in
contrast to the previously identified plant compounds (30), the
P. vulgaris active molecules were found in the ethyl acetate
fraction. Our suggestion of a possible cross-talk between a prokaryote
and the eukaryotic host, was demonstrated in the case of P. aeruginosa, the opportunistic pathogen of immunocompromised individuals. The P. aeruginosa quorum-sensing signal
molecule 3O-C12-HSL stimulates interleukin-8 production in
pulmonary epithelial cells (31) and may modulate the host immune
response by suppressing cytokine production in macrophages (32). Up to
now, several autoinducer systems have been described in
Rhizobium sp. but the cin system of R. etli is the first proven to be expressed in the infection tread
and in differentiated bacteroids. In contrast, the rhi
system, which is shown to be important for interaction with legumes and
which is specific to R. leguminosarum bv. viciae, is only expressed in the rhizosphere (10). We have illustrated here for
the first time that nitrogen-fixing bacteroids produce autoinducers in
the nodules under conditions that are quite different from free-living
growth. In vivo production and excretion of AHLs was also
observed in lung tissues of mice infected with P. aeruginosa (33). Furthermore, autoinducers other than AHLs, such as quinolones (34) and diketopiperazines (DKP) (35), have been described in
Gram-negative bacteria. The observation that DKP can be generated via
non-enzymatic cyclization of linear dipeptides at extremes of pH and
temperature (36) can offer an explanation for the presence of active
compounds in sterile, non-inoculated TY medium.
Analysis of quorum-sensing in R. etli is further complicated
by the fact that besides the cin system, involved in the
synthesis of 3OH-(slc)-HSL, a second system, rai, regulates
production of several other molecules with autoinducer activity (12).
This is particularly striking when the effect of mutations in the
cin or/and rai genes on growth of R. etli is analyzed: RaiR fulfills a key regulatory role, and it is
shown that CinR negatively affects growth when 3OH-(slc)-HSL is not
produced. Growth was even more delayed in a raiIcinI mutant.
Both systems seem to interact at the level of unknown genes directly or
indirectly controlling growth of the bacteria. The R. etli
autoinduction systems clearly affect the symbiotic properties of the
bacterium. The rai system is involved in restriction of the
nodule number, whereas nitrogen fixation activity is not affected (12).
On the other hand, the decreased acetylene reduction activity of plants
nodulated by cin mutant strains and the bacteroid
morphology, illustrate the important role the cin
quorum-sensing system plays in R. etli during symbiosis. A
disrupted communication, as observed in the cin mutants,
results in an arrest of bacteroid differentiation. Also, in contrast to
wild-type bacteroids, cinI mutants are individually enclosed
in a symbiosome and are devoid of a large symbiosome space. The
cin quorum-sensing system is a prerequisite to complete the
differentiation cycle of the bacterium. In contrast, it was shown that
3OH-C14:1-HSL, produced by CinI in R. leguminosarum bv. viciae, is not required for the
formation of effective nodules (11). This observation was also
confirmed by Lithgow et al.(8) in a plant experiment where
mutation of cinI had little effect on growth or nodulation.
In summary, R. etli 3OH-(slc)-HSL and R. leguminosarum bv. viciae 3OH-C14:1-HSL
share some common characteristics but elicit different phenotypic
changes in their respective producing cells.
We thank Stephen Farrand and Anton van
Brussel for providing the A. tumefaciens autoinducer system
and R. leguminosarum bv. viciae strains,
respectively; Johan Billen for assistance and access to the T.E.M.
facility; Chuanwu Xi for technical assistance during the preparation of
the microscopic samples; and Serge Buellens for helping with the plant experiments.
*
Parts of this study were presented at the American
Society for Microbiology Conference on Cell-Cell Communication in
Bacteria, July 6-9, 2001, Snowbird, UT.The costs of publication of this article were defrayed in part by the
payment of page charges. The article
must therefore be hereby marked
"advertisement" in accordance with 18 U.S.C. Section
1734 solely to indicate this fact.
The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF393621.
§
Recipient of a fellowship from the Instituut voor de Aanmoediging
van Innovatie door Wetenschap en Technologie (IWT).
**
To whom correspondence should be addressed. Tel.: 32 16 321631;
Fax: 32 16 321966; E-mail:
Jozef.Vanderleyden@agr.kuleuven.ac.be.
Published, JBC Papers in Press, October 24, 2001, DOI 10.1074/jbc.M106655200
2
C. Snoeck, unpublished data.
The abbreviations used are:
AHL, N-acyl-homoserine lactone;
TEM, transmission electron
microscopic analysis;
ORF, open-reading frame;
HPLC, high-performance
liquid chromatography;
X-gal, 5-bromo-4-chloro-3-indolyl-
The cin Quorum Sensing Locus of Rhizobium
etli CNPAF512 Affects Growth and Symbiotic Nitrogen Fixation*
§,,,,,,
,**, and
Centre of Microbial and Plant Genetics,
Katholicke Universitat Leuven, Kasteelpark Arenberg 20, B-3001
Heverlee, Belgium, the ¶ Centre for Surface Chemistry and
Catalysis, Katholicke Universitat Leuven, Kasteelpark Arenberg 23, B-3001 Heverlee, Belgium, and the Zoological Institute,
Katholicke Universitat Leuven, Naamsestraat 59, B-3000 Leuven,
Belgium
ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
-glucuronidase (GusA)
activity was performed as described elsewhere (17, 18). For
transmission electron microscopic analysis (TEM), thin sections of
3-week-old nodules were prepared as described by Xi et al. (19), and analyzed in a Zeiss EM 900 electron microscope.
S (pUCNotI derivative lacking
the SphI recognition site via restriction digest and blunt
ligation) yielding plasmid pFAJ4004 (Fig. 1). The 2.2-kb
BamHI fragment from pHP45
-Km containing a Kmr
cassette was ligated into the unique SphI site of
cinI in pFAJ4004 after blunting of the fragments. The
cinRcinI::Km locus was further cloned into the
sacB suicide vector pJQ200uc1 as a 5.2-kb NotI fragment to obtain pFAJ4006. The non-polar cinR point
mutation and an additional frameshift were introduced via the
QuickChangeTM site-directed mutagenesis (Stratagene) using primers
Rhi32 (5'-CTATGCCATGCTGGTACCATGCCCAGAAGCACG-3') and Rhi33
(5'-CGTGCTTCTGGGCATGGTACCAGCATGGCATAG-3') on pFAJ4004. As a result of
the mutation, a new and unique KpnI site
(GGTACC; mutations are shown in bold: insertion
of A; substitution G with C) was created in cinR.
Subsequently, the 3-kb NotI fragment containing the mutated
cinR gene was inserted into pJQ200uc1 creating pFAJ4007. The
cinR::Sp mutation was made by introducing the 2-kb
spectinomycin resistance cartridge from pHP45 into the unique
KpnI site within cinR of pFAJ4007 after blunting
of the fragments, resulting in pFAJ4009. The plasmids pFAJ4006,
pFAJ4007, and pFAJ4009 were introduced into R. etli
CNPAF512, and double recombinants were selected as described previously
(12) creating a cinI mutant (FAJ4006) and two
cinR mutants (FAJ4007, FAJ4009; Fig. 1). A cartridge
containing the promoterless gusA gene and a spectinomycin
resistance gene from pWM5 was removed with SmaI and ligated
into the XhoI site of pFAJ1327 (containing raiI,
raiR, and orf1) after blunting of the vector,
yielding plasmid pFAJ4010. For the construction of a R. etli
raiI mutant strain, pFAJ4010 was introduced into the R. etli CNPAF512 wild-type strain, yielding FAJ4010. Tri-parental conjugation of pFAJ4006 (cinI::Km) into FAJ4010
resulted in the raiI::Sp
cinI::Km double mutant FAJ4013.
RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
View larger version (21K):
[in a new window]
Fig. 1.
Gene organization of the cin
locus in R. etli and construction of gusA
fusions and mutant strains. Panel A, physical and
genetic map of the 5.3-kb fragment containing the cin locus
from R. etli CNPAF512. Arrows indicate directions
of transcription. Restriction sites: A, ApaI;
B, BamHI; C, ClaI;
E, EcoRI; H, HindIII;
P, PstI; S, SalI;
Sc, SacI; Sp, SphI;
V, EcoRV; X, XhoI.
Panel B, cinR and cinI insertional
mutants: cinR::Tn5 (FAJ1337); a
non-polar point mutation in cinR (FAJ4007);
cinR:: Spr (FAJ4009);
cinI:: Kmr (FAJ4006). Panel
C, construction of cinR-gusA (pFAJ4011) and
cinI-gusA fusions (pFAJ4014, pFAJ4015). The
position of the gusA gene is indicated. Panel D,
construction of pFAJ4012 and pFAJ4004. Primers and restriction sites
used for construction are indicated.
G = 
26.3). PCR analysis with a
Tn5-specific primer combined with primers within the coding
sequence of cinR or cinI (see below) indicated
that the transposon in FAJ1337 is inserted between nucleotides 423 and
424 of cinR.
G = 23.8; nucleotide 3694-3735, G = 32.4) were found.
G = 22.9).
View larger version (28K):
[in a new window]
Fig. 2.
Expression of cinR- and
cinI-gusA fusions.
cinI expression from pFAJ4014 (A) or pFAJ4015
(B) and cinR expression from pFAJ4011
(C) was examined quantitatively in a cell
density-dependent manner. Curves representing
the expression in the wild-type background are indicated by a
diamond, in a cinI mutant background (FAJ4006) by
a gray triangle, and in a cinR mutant background
(FAJ4009) by a square. The absorbance at 595 nm of wild-type
R. etli CNPAF512 containing pFAJ4011, pFAJ4014, or pFAJ4015
(cross) is shown. The growth of the other strains is similar
to that of the wild type. The graphs are representative of
experiments carried out independently several times.
View larger version (22K):
[in a new window]
Fig. 3.
Expression of cinR and
cinI during symbiosis. Bacteroid expression of
cinR-gusA (pFAJ4011, light gray) and
cinI-gusA fusions (pFAJ4014, dark
gray; pFAJ4015, white) was monitored in isolated
bacteroids, obtained from 21-day-old bean nodules, induced by wild-type
CNPAF512, the cinR (FAJ4007), and cinI (FAJ4006)
mutants. Values are the means of at least nine plants. Bars
represent mean ± S.D.
View larger version (33K):
[in a new window]
Fig. 4.
Light microscopic analysis of
cinI-gusA (pFAJ4015) expression
during the early steps of symbiosis. Phaseolus vulgaris
roots were inoculated with pFAJ4015/CNPAF512 (A),
pFAJ4015/FAJ4006 (cinI) (B), pFAJ4015/FAJ4009
(cinR) (C). RH, root hair;
IF, infection tread. The scale of the bars is 200 µM.
View larger version (51K):
[in a new window]
Fig. 5.
TLC analysis of R. etli
autoinducers. The ethyl acetate extracts were spotted on
C18 RP-TLC plates and 60% MeOH was used as the liquid
phase. Molecules with autoinducer activity were visualized with a soft
agar overlay containing X-gal and the A. tumefaciens
reporter strain. Autoinducers produced by free-living R. etli CNPAF512 (A), cinI mutant (FAJ4006)
(B), raiI mutant (FAJ4010) (C),
raiIcinI mutant (FAJ4013) (D), A. tumefaciens containing R. etli CNPAF512 cinR
and cinI (pFAJ4013) (F) and the A. tumefaciens negative control (G). Autoinducer F-AI1
corresponds to 3OH-(slc)-HSL and F-AI2 and F-AI3 to spots also detected
in non-inoculated TY medium (lane E). The autoinducers S-AI1
to S-AI4 produced by R. etli bacteroids and by root material
are shown in lanes H and I, respectively.
View larger version (14K):
[in a new window]
Fig. 6.
Growth of R. etli wild-type
and autoinducer mutant strains. Absorbance at 595 nm of R. etli (blue), the raiI mutant FAJ4010
(pink), the raiR mutant FAJ1329
(yellow), the non-polar cinR mutant FAJ4007
(green), the polar cinR mutant FAJ4009
(gray), the cinI mutant FAJ4006 (red),
and the strongly delayed raiIcinI double mutant FAJ4013
(orange) were measured in a BioscreenC over a 6-day
period.
Symbiotic phenotype
View larger version (104K):
[in a new window]
Fig. 7.
TEM of sections of 3-week-old nodules, formed
by R. etli CNPAF512 (B) and the
cinI mutant (FAJ4006) (A) analyzed in
a Zeiss EM 900 electron microscope. SS, symbiosome
space; SM, symbiosome membrane; b, bacteroid. The
scale of the bars is 0.6 µM.
DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
ACKNOWLEDGEMENTS
FOOTNOTES
ABBREVIATIONS
-D-galactopyranoside;
ARA, acetylene reduction activity;
TLC, thin layer chromatography;
DKP, diketopiperasines.
REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES
1.
Fuqua, C.,
Winans, S. C.,
and Greenberg, E. P.
(1996)
Annu. Rev. Microbiol.
50,
727-751[CrossRef][Medline]
[Order article via Infotrieve]
2.
Swift, S.,
Williams, P.,
and Stewart, G. S. A. B.
(1999)
Cell Cell Signaling in Bacteria.
, pp. 291-313, ASM Press, Washington, D.C.
3.
Shaw, P. D.,
Ping, G.,
Daly, S. L.,
Cha, C.,
Cronan, J. E.,
Rinehart, K. L.,
and Farrand, S. K.
(1997)
Proc. Natl. Acad. Sci. U. S. A.
94,
6036-6041 4.
Pierson, L. S.,
Wood, D. W.,
and Pierson, E. A.
(1998)
Annu. Rev. Phytopathol.
36,
207-225[CrossRef][Medline]
[Order article via Infotrieve]
5.
Kaplan, H. B.,
and Greenberg, E. P.
(1985)
J. Bacteriol.
163,
1210-1214 6.
Pearson, J. P.,
Van Delden, C.,
and Iglewski, B. H.
(1999)
J. Bacteriol.
181,
1203-1210 7.
Cha, C.,
Gao, P.,
Chen, Y. C.,
Shaw, P. D.,
and Farrand, S. K.
(1998)
Mol. Plant Microbe Interact.
11,
1119-1129[Medline]
[Order article via Infotrieve]
8.
Lithgow, J. K.,
Wilkinson, A.,
Hardman, A.,
Rodelas, B.,
Wisniewski-Dye, F.,
Williams, P.,
and Downie, J. A.
(2000)
Mol. Microbiol.
37,
81-97[CrossRef][Medline]
[Order article via Infotrieve]
9.
Gray, K. M.,
Pearson, J. P.,
Downie, J. A.,
Boboye, B. E. A.,
and Greenberg, E. P.
(1996)
J. Bacteriol.
178,
372-376 10.
Rodelas, B.,
Lithgow, J. K.,
Wisniewski-Dye, F.,
Hardman, A.,
Wilkinson, A.,
Economou, A.,
Williams, P.,
and Downie, J. A.
(1999)
J. Bacteriol.
181,
3816-3823 11.
van Brussel, A. A.,
Zaat, S. A.,
Wijffelman, C. A.,
Pees, E.,
and Lugtenberg, B. J.
(1985)
J. Bacteriol.
162,
1079-1082 12.
Rosemeyer, V.,
Michiels, J.,
Verreth, C.,
and Vanderleyden, J.
(1998)
J. Bacteriol.
180,
815-821 13.
Schripsema, J.,
de Rudder, K. E.,
van Vliet, T. B.,
Lankhorst, P. P.,
de Vroom, E.,
Kijne, J. W.,
and van Brussel, A. A.
(1996)
J. Bacteriol.
178,
366-371 14.
Sambrook, J.,
Fritsch, E. F.,
and Maniatis, T.
(1989)
Molecular Cloning: a Laboratory Manual
, 2nd Ed.
, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, New York
15.
Michiels, J.,
Van Soom, T.,
D'hooghe, I.,
Dombrecht, B.,
Benhassine, T.,
de Wilde, P.,
and Vanderleyden, J.
(1998)
J. Bacteriol.
180,
1729-1740 16.
Chilton, M. D.,
Currier, T. C.,
Farrand, S. K.,
Bendich, A. J.,
Gordon, M. P.,
and Nester, E. W.
(1974)
Proc. Natl. Acad. Sci. U. S. A.
71,
3672-3676 17.
Michiels, J.,
Moris, M.,
Dombrecht, B.,
Verreth, C.,
and Vanderleyden, J.
(1998)
J. Bacteriol.
180,
3620-3628 18.
Vande Broek, A.,
and Vanderleyden, J.
(1995)
Mol. Plant Microbe Interact.
8,
800-810
19.
Xi, C.,
Schoeters, E.,
Vanderleyden, J.,
and Michiels, J.
(2000)
Proc. Natl. Acad. Sci. U. S. A.
97,
11114-11119 20.
Dombrecht, B.,
Vanderleyden, J.,
and Michiels, J.
(2001)
Mol. Plant Microbe Interact.
14,
426-430[Medline]
[Order article via Infotrieve]
21.
D'hooghe, I.,
Michiels, J.,
Vlassak, K.,
Verreth, C.,
Waelkens, F.,
and Vanderleyden, J.
(1995)
Mol. Gen. Genet.
249,
117-126[CrossRef][Medline]
[Order article via Infotrieve]
22.
Bibb, M. J.,
Findlay, P. R.,
and Johnson, M. W.
(1984)
Gene (Amst.)
30,
157-166[CrossRef][Medline]
[Order article via Infotrieve]
23.
Baikalov, I.,
Schroder, I.,
Kaczor-Grzeskowiak, M.,
Grzeskowiak, K.,
Gunsalus, R. P.,
and Dickerson, R. E.
(1996)
Biochemistry
35,
11053-11061[CrossRef][Medline]
[Order article via Infotrieve]
24.
Parsek, M. R.,
Schaefer, A. L.,
and Greenberg, E. P.
(1997)
Mol. Microbiol.
26,
301-310[CrossRef][Medline]
[Order article via Infotrieve]
25.
Fuqua, C.,
and Greenberg, E. P.
(1998)
Curr. Opin. Microbiol.
1,
183-189[CrossRef][Medline]
[Order article via Infotrieve]
26.
Volz, K.
(1993)
Biochemistry
32,
11741-11753[CrossRef][Medline]
[Order article via Infotrieve]
27.
vonBodman, S. B.,
Majerczak, D. R.,
and Coplin, D. L.
(1998)
Proc. Natl. Acad. Sci. U. S. A.
95,
7687-7692 28.
Zhu, J.,
and Winans, S. C.
(1998)
Mol. Microbiol.
27,
289-297[CrossRef][Medline]
[Order article via Infotrieve]
29.
de Kievit, T.,
Seed, P. C.,
Nezezon, J.,
Passador, L.,
and Iglewski, B. H.
(1999)
J. Bacteriol.
181,
2175-2184 30.
Teplitski, M.,
Robinson, J. B.,
and Bauer, W. D.
(2000)
Mol. Plant Microbe Interact.
13,
637-648[Medline]
[Order article via Infotrieve]
31.
DiMango, E.,
Zar, H. J.,
Bryan, R.,
and Prince, A.
(1995)
J. Clin. Invest.
96,
2204-2210
32.
Telford, G.,
Wheeler, D.,
Williams, P.,
Tomkins, P. T.,
Appleby, P.,
Sewell, H.,
Stewart, G. S.,
Bycroft, B. W.,
and Pritchard, D. I.
(1998)
Infect. Immun.
66,
36-42 33.
Wu, H.,
Song, Z.,
Hentzer, M.,
Andersen, J. B.,
Heydorn, A.,
Mathee, K.,
Moser, C.,
Eberl, L.,
Molin, S.,
Hoiby, N.,
and Givskov, M.
(2000)
Microbiology
146,
2481-2493 34.
Pesci, E. C.,
Milbank, J. B.,
Pearson, J. P.,
McKnight, S.,
Kende, A. S.,
Greenberg, E. P.,
and Iglewski, B. H.
(1999)
Proc. Natl. Acad. Sci. U. S. A.
96,
11229-11234 35.
Holden, M. T. G.,
Chhabra, S. R.,
de Nys, R.,
Stead, P.,
Bainton, N. J.,
Hill, P. J.,
Manefield, M.,
Kumar, N.,
Labatte, M.,
England, D.,
Rice, S.,
Givskov, M.,
Salmond, G. P. C.,
Stewart, G. S. A. B.,
Bycroft, B. W.,
Kjelleberg, S. A.,
and Williams, P.
(1999)
Mol. Microbiol.
33,
1254-1266[CrossRef][Medline]
[Order article via Infotrieve]
36.
Skwierczynski, R. D.,
and Connors, K. A.
(1993)
Pharm. Res.
10,
1174-1180[CrossRef][Medline]
[Order article via Infotrieve]
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 M. J. Soto, J. Sanjuan, and J. Olivares Rhizobia and plant-pathogenic bacteria: common infection weapons. Microbiology, November 1, 2006; 152(Pt 11): 3167 - 3174. [Abstract] [Full Text] [PDF]
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 R. Daniels, S. Reynaert, H. Hoekstra, C. Verreth, J. Janssens, K. Braeken, M. Fauvart, S. Beullens, C. Heusdens, I. Lambrichts, et al. Quorum signal molecules as biosurfactants affecting swarming in Rhizobium etli PNAS, October 3, 2006; 103(40): 14965 - 14970. [Abstract] [Full Text] [PDF]
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H. Zheng, Z. Zhong, X. Lai, W.-X. Chen, S. Li, and J. Zhu A LuxR/LuxI-Type Quorum-Sensing System in a Plant Bacterium, Mesorhizobium tianshanense, Controls Symbiotic Nodulation. J. Bacteriol., March 1, 2006; 188(5): 1943 - 1949. [Abstract] [Full Text] [PDF]
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N. D. Keshavan, P. K. Chowdhary, D. C. Haines, and J. E. Gonzalez L-Canavanine Made by Medicago sativa Interferes with Quorum Sensing in Sinorhizobium meliloti J. Bacteriol., December 15, 2005; 187(24): 8427 - 8436. [Abstract] [Full Text] [PDF]
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M. Moris, K. Braeken, E. Schoeters, C. Verreth, S. Beullens, J. Vanderleyden, and J. Michiels Effective Symbiosis between Rhizobium etli and Phaseolus vulgaris Requires the Alarmone ppGpp J. Bacteriol., August 1, 2005; 187(15): 5460 - 5469. [Abstract] [Full Text] [PDF]
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 R. L. Ulrich Quorum Quenching: Enzymatic Disruption of N-Acylhomoserine Lactone-Mediated Bacterial Communication in Burkholderia thailandensis Appl. Envir. Microbiol., October 1, 2004; 70(10): 6173 - 6180. [Abstract] [Full Text] [PDF]
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 M. Teplitski, H. Chen, S. Rajamani, M. Gao, M. Merighi, R. T. Sayre, J. B. Robinson, B. G. Rolfe, and W. D. Bauer Chlamydomonas reinhardtii Secretes Compounds That Mimic Bacterial Signals and Interfere with Quorum Sensing Regulation in Bacteria Plant Physiology, January 1, 2004; 134(1): 137 - 146. [Abstract] [Full Text] [PDF]
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 J. E. Gonzalez and M. M. Marketon Quorum Sensing in Nitrogen-Fixing Rhizobia Microbiol. Mol. Biol. Rev., December 1, 2003; 67(4): 574 - 592. [Abstract] [Full Text] [PDF]
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J. Zhu, Y. Chai, Z. Zhong, S. Li, and S. C. Winans Agrobacterium Bioassay Strain for Ultrasensitive Detection of N-Acylhomoserine Lactone-Type Quorum-Sensing Molecules: Detection of Autoinducers in Mesorhizobium huakuii Appl. Envir. Microbiol., November 1, 2003; 69(11): 6949 - 6953. [Abstract] [Full Text] [PDF]
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H. Chen, M. Teplitski, J. B. Robinson, B. G. Rolfe, and W. D. Bauer Proteomic Analysis of Wild-Type Sinorhizobium meliloti Responses to N-Acyl Homoserine Lactone Quorum-Sensing Signals and the Transition to Stationary Phase J. Bacteriol., September 1, 2003; 185(17): 5029 - 5036. [Abstract] [Full Text] [PDF]
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C. Tun-Garrido, P. Bustos, V. Gonzalez, and S. Brom Conjugative Transfer of p42a from Rhizobium etli CFN42, Which Is Required for Mobilization of the Symbiotic Plasmid, Is Regulated by Quorum Sensing J. Bacteriol., March 1, 2003; 185(5): 1681 - 1692. [Abstract] [Full Text] [PDF]
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X. He, W. Chang, D. L. Pierce, L. O. Seib, J. Wagner, and C. Fuqua Quorum Sensing in Rhizobium sp. Strain NGR234 Regulates Conjugal Transfer (tra) Gene Expression and Influences Growth Rate J. Bacteriol., February 1, 2003; 185(3): 809 - 822. [Abstract] [Full Text] [PDF]
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J. Loh, R. W. Carlson, W. S. York, and G. Stacey Bradyoxetin, a unique chemical signal involved in symbiotic gene regulation PNAS, October 29, 2002; 99(22): 14446 - 14451. [Abstract] [Full Text] [PDF]
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M. M. Marketon, M. R. Gronquist, A. Eberhard, and J. E. Gonzalez Characterization of the Sinorhizobium meliloti sinR/sinI Locus and the Production of Novel N-Acyl Homoserine Lactones J. Bacteriol., October 15, 2002; 184(20): 5686 - 5695. [Abstract] [Full Text] [PDF]
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J. Loh and G. Stacey Nodulation Gene Regulation in Bradyrhizobium japonicum: a Unique Integration of Global Regulatory Circuits Appl. Envir. Microbiol., January 1, 2002; 69(1): 10 - 17. [Full Text] [PDF]
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