Previous Article | Next Article 
J Bacteriol, February 1998, p. 815-821, Vol. 180, No. 4
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
luxI- and luxR-Homologous
Genes of Rhizobium etli CNPAF512 Contribute to Synthesis of
Autoinducer Molecules and Nodulation of Phaseolus
vulgaris
Viola
Rosemeyer,
Jan
Michiels,
Christel
Verreth, and
Jos
Vanderleyden*
F. A. Janssens Laboratory of Genetics,
B-3001 Heverlee, Belgium
Received 1 August 1997/Accepted 16 December 1997
 |
ABSTRACT |
Autoinduction plays an important role in intercellular
communication among symbiotic and pathogenic gram-negative bacteria. We
report here that a nitrogen-fixing symbiont of Phaseolus
vulgaris, Rhizobium etli CNPAF512, produces at least
seven different autoinducer molecules. One of them exhibits a
growth-inhibitory effect like that of the bacteriocin small
[N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone]. At least two of the other autoinducers are synthesized by a
LuxI-homologous autoinducer synthase. The corresponding
luxI homologous gene (raiI) and a
luxR homolog (raiR) have been identified and
characterized. Enhanced expression of raiI is dependent on cell density and on the presence of one or more autoinducer molecules synthesized by R. etli CNPAF512. A raiI mutant
was shown to release only three different autoinducer molecules; a
raiR mutant releases four different autoinducer molecules.
Examination of different mutants for nodulation of beans showed that
raiI is involved in the restriction of nodule number,
whereas nitrogen-fixing activity in terms of acetylene reduction per
nodule was not affected.
| |
INTRODUCTION |
Autoinduction is a highly conserved
mechanism of differential gene expression in many gram-negative
bacteria (20, 48). The key trigger of this system is the
concentration of small diffusible molecules, termed autoinducers for
their biological activity. All autoinducers so far identified are
N-acyl homoserine lactones (AHLs) (4, 8, 13, 43, 50,
56, 63). They are synthesized by an autoinducer synthase, the
product of a luxI-homologous gene, and are thought to bind
to a protein belonging to the LuxR family of transcriptional
activators. This autoinducer-protein complex activates the expression
of defined genes or sets of genes. As this gene activation occurs only
when a required threshold concentration of AHLs is attained, the onset
of specific genes is dependent on the cell density of bacteria.
Consequently, autoinduction allows bacteria to monitor their own
population density and to discriminate between high and low cell
density. It can also be understood as a cell-cell communication system
(48).
The physiological processes regulated by autoinduction are diverse, as
exemplified by the following systems: bioluminescence in Vibrio
fischeri (10, 13, 14), plasmid conjugal transfer in
Agrobacterium tumefaciens (18, 30, 44, 63),
antibiotic production in Erwinia carotovora (1, 2, 45,
55), and synthesis of exoenzymes in plant and animal
pathogens such as E. carotovora (32) and
Pseudomonas aeruginosa (21, 41, 42). In
Escherichia coli, a LuxR homolog is involved in the
regulation of cell division (22). Rhizobium
leguminosarum bv. viciae contains a
transcriptional-activator protein, RhiR, belonging to the LuxR family
(11). RhiR is encoded by the symbiotic plasmid pRL1JI and
regulates transcription of an operon of three rhizosphere-expressed genes of unknown function (the rhiABC operon). The
bacteriocin small, produced by fast-growing R. leguminosarum strains (60), has been shown to be an AHL
[N-(3R-hydroxy-7-cis-tetradecanoyl)-L-homoserine lactone] (50). Gray (24) has reported that
R. leguminosarum produces two additional autoinducer
compounds, activating rhiABC together with RhiR.
small increases production of these signals independently
from RhiR. A gene encoding an autoinducer synthase has so far not been
identified.
Rhizobium etli CNPAF512 (formerly classified as R. leguminosarum bv. phaseoli CNPAF512) forms
nitrogen-fixing nodules on the roots of the common bean. Within this
structure bacteria are densely packed and differentiate into their
symbiotic state, that of the bacteroids, able to reduce atmospheric
dinitrogen into ammonia. In view of the high cell density during
Rhizobium-plant interaction, autoinduction may be involved
at some stage of symbiosis.
We demonstrate in this study that R. etli CNPAF512 produces
at least seven different autoinducer molecules. We have identified luxI- and luxR-homologous genes and demonstrate
their contribution to the synthesis of autoinducers and to the
nodulation of Phaseolus vulgaris.
(Parts of the results described here have been presented on posters at
the 8th International Congress on Molecular Plant-Microbe Interactions, Knoxville, Tenn., 14 to 19 July 1996, at the
Euroconference on Microbial Response to Stress, Sesimbra,
Portugal, 15 to 18 March 1997, at the International Congress on
Marker/Reporter Genes in Microbial Ecology, Stockholm, Sweden, 14 to 17 June 1997, and at the 11th International Congress on Nitrogen Fixation,
Paris, France, 20 to 25 July 1997.)
| |
MATERIALS AND METHODS |
Bacterial strains, plasmids, media, and culture conditions.
Bacterial strains and plasmids used in this study are listed in Table
1. Antibiotic concentrations were as
follows (in micrograms per milliliter): nalidixic acid, 30; ampicillin,
100; kanamycin, 25; neomycin, 60; tetracycline, 10; gentamicin, 25;
carbenicillin, 100; and rifampin, 100. E. coli DH5
was
grown in Luria-Bertani medium (49) at 37°C, R. etli CNPAF512 was grown in TY medium (6), and A. tumefaciens NT1(pJM749, pSVB33) was grown in AB medium
(9) at 28°C.
Extraction and detection of autoinducers.
Rhizobium
was grown for 48 h and E. coli was grown for 24 h
in 500 ml of medium to stationary phase. After centrifugation, the
supernatant was extracted twice with an equal volume of ethyl acetate
containing 1.5 ml of acetic acid per liter. The extract was evaporated
to dryness by vacuum rotation at 42°C and redissolved in a small
volume of ethyl acetate. One microliter of this suspension was spotted
on a C18 reversed-phase thin-layer chromatography (TLC)
plate (RP-18F254S; Merck), which was then developed with 60% methanol. The air-dried plate was overlaid with AB soft agar (9) containing
5-bromo-4-chloro-3-indolyl-
-galactopyranoside (X-Gal) and A. tumefaciens NT1(pJM749, pSVB33) indicator cells (44,
52). This strain contains a Tn3HoHo1-generated
lacZ fusion to a tra gene, the expression of
which is dependent on TraR and autoinducer. As the clone lacks the Ti
plasmid, it does not produce an autoinducer detectable with this
system. Consequently, the lacZ reporter fusion is expressed
only upon exogenous supply of autoinducer molecules (44).
Incubation of the TLC plates at 28°C allowed the visualization of
compounds activating the A. tumefaciens tra system
expression. For comparison, we used purified autoinducer molecules from
V. fischeri (VAI)
[N-(3-oxohexanoyl)-L-homoserine lactone] and
from P. aeruginosa (PAI)
[N-(3-oxododecanoyl)-L-homoserine lactone],
kindly provided by E. P. Greenberg.
For fractionation, the ethyl acetate extract has been diluted 1:10 with
30% methanol in water, loaded on a C
18 reversed-phase
high-performance liquid chromatography column (Bondclone 10 C
18;
Phenomenex), and eluted isocratically with 30%
methanol. The fractions
were tested for activation of the
A. tumefaciens tra system and
for bacteriostatic activity towards the
sensitive strain
R. leguminosarum bv.
viciae 248 as described by Schripsema et al. (
50).
Screening of an
R. etli CNPAF512 gene library in
E. coli HB101 comprising 5,000 clones was performed in microtiter
plates.
Each 100 µl of AB medium supplemented with 0.003% leucine,
0.023%
proline, 0.004% thymine, and 0.0017% thiamine, containing
A. tumefaciens indicator cells and X-Gal, was pipetted into
the wells of a microtiter
plate. The clones of the gene library were
added, and the plates
were incubated at 28°C with shaking.
DNA techniques and nucleotide sequencing.
Standard
techniques were used for isolation and manipulation of DNA
(49). Enzymes were purchased from Boehringer Mannheim. Hybridization experiments were carried out with digoxigenin-labeled probes according to the instructions of the manufacturer (Boehringer Mannheim). High-stringency hybridization was performed at 68°C, and
low-stringency hybridization was performed at 40°C. Nucleotide sequencing was accomplished by using the A.L.F. sequencer (Pharmacia Biotech). Sequences were compiled and compared with the aid of the
PCGENE software package (IntelliGenetics Inc.).
Construction of raiI and raiR
mutants.
A 4-kb region from pFAJ1322 comprising raiI,
raiR, and open reading frame 1 (ORF1) has been amplified by
PCR. NotI sites were introduced by designing appropriate
primers (5'-CGCGCGGCCGCCATAGCCATCGCTGGTGATGTTGC-3' and
5'-CGCGCGGCCGCATGATAGGCATCGCCGAGAAAGAGG-3'). The product was cloned into the pCR2.1 TA cloning vector (pFAJ1326). For verification, the terminal regions were sequenced. Subsequently, the fragment was
excised with NotI and ligated into pJQ200uc1 (pFAJ1327)
(47). For mutagenesis of raiI, this construct was
linearized with XhoI, allowing the insertion of a
promoterless glucuronidase (gusA) gene coupled to a
kanamycin resistance gene from pWM6 (37) 28 bp downstream of
the predicted raiI translation start, resulting in a
nonpolar mutation (pFAJ1328). The same cassette was inserted in reverse
orientation into raiR via a unique NdeI site, 126 bp downstream of the predicted translation start (pFAJ1329) (see Fig.
2B). These plasmids were conjugated into R. etli CNPAF512 with the helper E. coli HB101/pRK2013, yielding
cis merodiploid recombinants with a
raiI::gusA fusion
(CNPAF512::pFAJ1328) or a gusA-Km insertion in
raiR. Selection for double homologous recombination using
the sacRB-based positive selection system on sucrose
(38) led to a raiI mutant with a
raiI::gusA fusion (FAJ1328) or a
raiR mutant (FAJ1329), respectively. The genotypes of the
mutants were verified by hybridization of a raiI-raiR probe
and a probe of the pWM6 cassette to the same R. etli EcoRI
fragment.
Expression analyses.
Cell density-dependent expression of
raiI was monitored by using a
raiI::gusA fusion. Each 5 ml of TY
medium was inoculated with a different volume of a stationary-phase
culture and grown for 12 h. Prior to inoculation, the cells were
washed twice with 10 mM MgSO4 to remove autoinducers.
Quantitative analysis of GusA activity was carried out with
p-nitrophenyl--D-glucuronide as the substrate
by the method of Miller (39) in microtiter plates.
Plant experiments.
Surface-sterilized and germinated bean
seedlings (58) were planted under sterile conditions in
0.5× Jenssen medium (59) and were subsequently inoculated
with 200 µl of stationary-phase bacterial cultures. The plants were
grown at 26°C for 18 days with a 12-h day length. Nitrogen fixation
was measured in terms of acetylene reduction by gas chromatography
(5890A; Hewlett-Packard).
Nucleotide sequence accession numbers.
The nucleotide
sequences of raiI and raiR have been deposited
under accession no. U92712 and U92713, respectively, in the GenBank
Sequence Data Library.
| |
RESULTS AND DISCUSSION |
Detection of autoinducers.
Among several reporter systems
available for detection of autoinducers, such as activation of
lasB (26), the lux operon (56), or the tra system (44), the last
recognized the broadest spectrum of different molecules in our
hands. With the exception of N-butanoyl-,
N-(3-hydroxyoctanoyl)-, and
N-(3-hydroxydecanoyl)-L-homoserine lactone,
activation of tra gene expression has been shown with 3-oxo-, 3-hydroxy-, and 3-unsubstituted side chains with even chain
lengths of C4 up to C12 (52).
R. etli CNPAF512 was grown in 500 ml of TY medium for
48 h to stationary phase. The ethyl acetate extract of the
cell-free spent medium was analyzed on a TLC plate for compounds
activating the A. tumefaciens tra system expression (Fig.
1, lane D). Seven distinct spots could
be detected, indicating that there are at least seven
different autoinducer types (AI-1 through AI-7) present in
R. etli CNPAF512. P. aeruginosa has been
shown to produce six different autoinducers (52, 61). The existence of seven autoinducers in one species is
described here for Rhizobium for the first time. Purified
PAI and VAI were used for comparison (Fig. 1, lane A). VAI migrated
between AI-1 and AI-2, and PAI exhibited chromatographic features
similar to those of AI-7. The amount of autoinducer molecules necessary
to activate the A. tumefaciens test system depends on
the recognition of autoinducers by TraR and thus on the type of
molecule. In Fig. 1, lane A, 10 ng of PAI and 20 pg of VAI were
applied. Although about 3 orders of magnitude more PAI than VAI has
been spotted on the plate, VAI evokes a bigger spot. This demonstrates
that the amounts of different autoinducers are incomparable, as the assay used is based on biological activity. Therefore, absolute quantification of unknown molecules by measuring spot intensity is not
possible.
View larger version (81K):
[in this window]
[in a new window]
|
FIG. 1.
Purified VAI and PAI (A) and autoinducers produced by
E. coli DH5 (B), E. coli DH5 containing the
R. etli CNPAF512 raiI and raiR in
pUC18 (FAJ1323) (C), wild-type R. etli CNPAF512 (D), the
R. etli CNPAF512 mutant with disrupted raiI
(FAJ1328) (E), and the R. etli CNPAF512 mutant with
disrupted raiR (FAJ1329) (F). Ethyl acetate extracts (lanes
B to E) were spotted on a C18 reversed-phase TLC plate, and
60% methanol was used as the liquid phase. Molecules with autoinducer
activity were visualized with a soft agar overlayer containing X-Gal
and A. tumefaciens indicator cells.
|
|
The
R. etli CNPAF512 ethyl acetate extract has been
fractionated on a C
18 reversed-phase high-performance
liquid chromatography
column. Assaying the fractions for
tra
activation confirmed the
presence of at least seven different
autoinducer molecules, as
expected from the results of the TLC plates
(data not shown).
In addition to the list of bacteriocin-producing
rhizobia published
by Hirsch (
29), we observed that
R. etli CNPAF512 inhibits the
growth of
R. leguminosarum
bv.
viciae 248, too. Therefore, the
fractions from
high-performance liquid chromatography were tested
for bacteriostatic
activity toward this sensitive strain. Only
the fractions containing
AI-7 exhibited a growth-inhibitory effect
like that of the bacteriocin
small (
27,
50). Although we cannot
exclude a
coelution of two different molecules, one showing the
features of a
bacteriocin and the other activating
tra expression
in
A. tumefaciens, we have evidence that the
A. tumefaciens system
also recognizes
small: TLC analysis
of an extract from the
small-producing
strain
R. leguminosarum bv.
viciae RBL1309 (
57)
revealed the
presence of three autoinducer molecules. The molecule with
the
highest hydrophobicity, which was missing in the
small
mutant
R. leguminosarum bv.
viciae RBL1376,
comigrated with AI-7 (data
not shown).
Identification and characterization of luxI- and
luxR-homologous genes.
An R. etli
CNPAF512 gene library in E. coli HB101, previously
constructed in the cosmid pLAFR1 by partial EcoRI digestion of genomic R. etli CNPAF512 DNA, was screened for clones
activating the A. tumefaciens tra system in microtiter
plates. After 12 h, eight wells were blue. Restriction analysis of
the corresponding clones and hybridization with a V. fischeri
luxI probe from pHV200 (25) revealed that they all
contained an identical 3.8-kb SalI-fragment. E. coli DH5 containing this SalI fragment in the pUC18
vector (FAJ1323) could still induce the A. tumefaciens
test system to the same extent as the positive E. coli HB101
clones from the library. Sequencing of pFAJ1323 revealed two open
reading frames, the deduced amino acid sequences of which showed
similarity to the autoinducer synthases of the LuxI family and to
transcriptional activators of the LuxR family. Downstream of these
genes are two complete open reading frames and the beginning of a
third. The deduced ORF1 protein shows homology to members of the Lrp
family of transcriptional regulators (46). The deduced ORF2
protein shows homology to a catabolic alanine racemase (DadX), and the deduced ORF3 protein shows homology to the small subunit of
D-amino acid dehydrogenase (DadA) from E. coli
(34). The racemase converts L-alanine to the
D isomer, which is then oxidatively deaminated by the
D-amino acid dehydrogenase to pyruvate and ammonia
(16). In E. coli, dadA and
dadX form an operon of which Lrp is a direct repressor
(36). In Pseudomonas putida, the lrp
homolog bkdR is required for the expression of the
bkd operon, which is necessary for the metabolism of the
branched-chain amino acids (35). Comparable to the gene
order in R. etli CNPAF512, bkdR is located
immediately upstream of the bkd operon in the opposite
orientation. In Bacillus subtilis, the lrp
homolog azlB is the first gene of an operon involved in
branched-chain amino acid transport (5). Figure 2A shows the physical map of this region,
including the two BamHI fragments that overlap the 3.8-kb
SalI fragment.
View larger version (16K):
[in this window]
[in a new window]
|
FIG. 2.
(A) Physical and genetic map of the region containing
raiI and raiR from R. etli CNPAF512.
Coding regions are shaded. Arrows indicate directions of transcription.
The open arrow represents the beginning of an open reading frame. Light
shading illustrates a part of ORF3 that has not been sequenced. The
boxed SalI sites have been used for construction of
pFAJ1323. The small arrows indicate the primers with NotI
sites used for amplification of a 4-kb fragment. Restriction sites: B,
BamHI; H, HindIII; P, PstI; S,
SphI; Sl, SalI; Sm, SmaI. (B)
Construction of the raiI (FAJ1328) and raiR
(FAJ1329) mutant. The PCR product was cloned into pCR2.1 TA, excised
with NotI, and inserted into pJQ200uc1. A cassette with a
promoterless gusA and kanamycin resistance from pWM6 was
introduced in the XhoI (raiI mutant) or
NdeI (raiR mutant) site by blunt-end ligation.
|
|
The
luxI-homologous gene, termed
raiI
(
Rhizobium autoinducer synthase), consists of a 639-bp open
reading frame that possibly
codes for an autoinducer synthase with 212 amino acids. Similarities
of the deduced amino acid sequence with those
of TraI, RhlI, and
LuxI are relatively low (Table
2). The highest similarities are
clustered in the N-terminal region, especially from residue 22
to 36 and from residue 67 to 84 (referring to RaiI), which is
a
characteristic feature of LuxI homologs described so far (Fig.
3). Amino acid residues found to be
conserved in 12 LuxI homologs
(
28) are also present in RaiI
(Arg-24, Phe-28, Trp-34, Asp-48,
Arg-70, and Arg-104). However, instead
of a cysteine at position
68 of LuxI, which is thought to be the active
site of the autoinducer
synthase, RaiI contains a serine. Autoinducer
synthesis is proposed
to involve transfer of the fatty acyl substrate
from the acyl
carrier protein to a cysteine residue on the LuxI
homolog, forming
a covalent bond with the sulfhydryl group
(
40). Given the structural
and chemical similarity of serine
and cysteine, it can be speculated
that the hydroxyl group of the
serine at the active site could
also form a covalent bond with the
fatty acyl group, as proposed
by Hanzelka et al. (
28).
raiI is followed by the
luxR homolog
raiR, separated by 144 bp.
raiR consists of a
729-bp open reading
frame and codes for a protein of 242 amino acids.
Identities and
similarities of RaiR with RhlR, TraR, and RhiR are given
in Table
2. The LuxR-related transcriptional activators consist of an
N-terminal regulator domain and a C-terminal activator domain.
In spite
of the overall low similarity, the regions for autoinducer
binding and
the helix-turn-helix motif for DNA binding are conserved
(Fig.
4). The organization of
raiI
and
raiR differs from that
of other species, as both are
transcribed unidirectionally and
the
luxI homolog precedes
the
luxR homolog. In
P. aeruginosa the
gene order
of the two autoinduction systems (
las and
rhl) is
reversed,
and in
A. tumefaciens traI and
traR are not clustered. In all
other identified
autoinduction systems, both genes are either
convergently or
divergently transcribed (
48).
View larger version (58K):
[in this window]
[in a new window]
|
FIG. 3.
Alignments of members of the LuxI family. Shaded amino
acid residues are identical in at least three of the aligned sequences.
Amino acid residues that are conserved in all the homologs aligned are
shown below the sequences. The sequences for RhlI (accession no.
U40458) and RaiI (U92712) are in the GenBank Sequence Data Library;
those for LuxI (accession no. P12747) and TraI (P33907) are in the
Swiss-Prot Protein Sequence Database.
|
|
View larger version (78K):
[in this window]
[in a new window]
|
FIG. 4.
Alignments of members of the LuxR family. Shaded amino
acid residues are identical in at least three of the aligned sequences.
Amino acid residues that are conserved in all the homologs aligned are
shown below the sequences. Diamonds indicate LuxR positions at which
mutations affect interaction with VAI (51, 54). The
helix-turn-helix motif (19) is given in bold letters.
Accession numbers are S25491 (PIR Protein Database) for TraR, P12746
(Swiss-Prot Protein Sequence Database) for LuxR, and, in the GenBank
Sequence Data Library, U40458 for RhlR, U92713 for RaiR, and M98835 for
RhiR.
|
|
E. coli DH5
containing the 3.8-kb
SalI
fragment in pUC18 (FAJ1323) was grown in 500 ml for 24 h to
stationary phase. Cell-free
culture supernatant was extracted with
ethyl acetate. Analysis
on TLC plates revealed the presence of four
different molecules
(Fig.
1, lane C). The extract from
E. coli DH5
evoked two spots
on the TLC plate (Fig.
1, lane B),
indicating that this strain
produced molecules able to activate
tra expression in
A. tumefaciens.
The other
spots in Fig.
1, lanes B and C, are due to brownish
contaminating
material in the extract. There have been indications
that
E. coli produces extracellular factors, which are involved
in
cell-to-cell signalling and transcriptional regulation (
22,
53). We demonstrate here for the first time that these molecules
trigger the autoinducer-dependent activation of
tra genes in
A. tumefaciens.
The pattern of autoinducers produced by FAJ1323 (Fig.
1, lane C)
indicates that RaiI directly catalyzes the synthesis of AI-2,
AI-3,
AI-4, and AI-5. It has been described for
V. fischeri that
one autoinducer synthase (LuxI) can direct the synthesis of two
different autoinducer molecules [
N-hexanoyl- and
N-(3-oxohexanoyl)-
L-homoserine
lactone]
(
33). Comparison with the autoinducers synthesized
by
an
raiI mutant (Fig.
1, lane E) (see below) suggests
that the
spots of AI-3 and AI-5 consist of comigrating molecules.
Thus,
AI-3 and AI-5 produced by FAJ1323 could be synthesized by RaiI,
and the corresponding autoinducers produced by the
raiI
mutant
could be synthesized by an autoinducer synthase(s) other than
RaiI. However, at this stage we cannot exclude the possibility
that the
increased intensities of AI-3 and AI-5 from FAJ1323 in
comparison to
E. coli DH5
could be due to enhanced synthesis
of
E. coli DH5
molecules with autoinducer activity.
The 3.8-kb
SalI fragment was inserted downstream of the
lac promoter into the pUC18 vector, which is induced in the
presence
of isopropyl-
-
D-thiogalactopyranoside. However,
the synthesis
of AI-3 and AI-5 also occurs in the absence of the
inducer. It
can therefore be concluded that the cloned fragment
contained
all necessary promoter elements.
Hybridization of total
R. etli CNPAF512 DNA in a Southern
blot with a
raiI probe at low stringency gave rise to only
one band.
This indicates that the homology between
raiI and
the gene(s)
encoding the other autoinducer synthase(s) is less than the
homology
shared by
raiI and
luxI (Table
2), as
these are cross-hybridizing
(see above). It is also possible that the
other autoinducer synthase
gene(s) does not belong to the
luxI family, as is the case for
ainS in
V. fischeri (
23) or
luxL and
luxM in
V. harveyi (
3).
Analysis of raiI and raiR mutants.
In
order to examine the phenotypic relevance of raiI and
raiR in R. etli, mutants have been constructed as
described in Materials and Methods.
Spent culture supernatants of the mutants FAJ1328 and FAJ1329 were
extracted and analyzed on TLC plates. The chromatogram
revealed that
the
raiI mutant does not produce AI-1, AI-2, AI-4,
and AI-6
in amounts detectable with the assay used (Fig.
1, lane
E). The absence
of AI-2 and AI-4 confirmed that
raiI is directly
responsible
for the synthesis of these two molecules. The failure
to detect AI-1
and AI-6 suggests that their synthesis by an autoinducer
synthase(s)
other than RaiI is dependent on autoinducers made
by RaiI. However, we
cannot exclude the possibility that the synthesis
of AI-1 and AI-6 is
directly dependent on
raiI and that in
E. coli
these compounds are not produced. The intensities of the
spots
representing AI-3 and AI-5 are clearly lower than in the
wild type.
This supports the above-mentioned hypothesis that the
AI-3 and AI-5
spots consist of comigrating molecules. In the
raiR mutant,
four molecules could be detected (Fig.
1, lane F): AI-7
and AI-5 in
about the same concentration as in the
raiI mutant,
AI-3 in
a concentration higher than that in the
raiI mutant, and
AI-4, which could not be detected in the
raiI mutant. AI-1,
AI-2,
and AI-6 could not be detected, indicating that RaiR is necessary
for their synthesis.
Expression studies of raiI.
raiI expression
in a wild-type (CNPAF512::pFAJ1328) and a mutant
(FAJ1328) background was examined quantitatively in a cell density-dependent way. As shown in Fig.
5, expression of raiI in a
wild-type background increased with cell density. In the mutant,
raiI expression was nearly negligible, indicating that at
least one of the autoinducers AI-1, AI-2, AI-4, and AI-6 is necessary
for direct or indirect activation of raiI. raiI expression in FAJ1328 was restored in filter-sterilized spent medium from R. etli CNPAF512. At low cell densities, expression levels
under these conditions exceeded those of the wild type
in fresh medium (data not shown).
View larger version (19K):
[in this window]
[in a new window]
|
FIG. 5.
Comparison of cell density-dependent expression of
raiI in wild-type and raiI mutant backgrounds.
Expression of the raiI::gusA fusions
was monitored by using
p-nitrophenyl--D-glucuronide as a substrate.
Values for optical density and Miller units were based on measurement
in microtiter plates. Curves show averages from three
separate experiments. Squares, CNPAF512::pFAJ1328
(raiI::gusA in a wild-type background);
triangles, FAJ1328 (raiI knockout mutant); open symbols,
optical density at 595 nm; solid symbols, GusA activity in Miller
units.
|
|
Mutation of raiI affects number of nodules of
inoculated bean plants.
The phenotypic relevance of the
rai genes for symbiosis with beans and for nitrogen
fixation was examined. Germinated bean seedlings were planted under
sterile conditions and subsequently inoculated with 200 µl of dense
cultures (optical density, >1) of R. etli CNPAF512, FAJ1328
(raiI mutant), FAJ1329 (raiR mutant), or
CNPAF512::pFAJ1334 (merodiploid with respect to
raiI and raiR). The plants were grown at 26°C
for 18 days. While no significant differences in delay of the
appearance of the first nodules and dry weight of the shoot were
observed (Table 3), the number of nodules
per plant inoculated with the raiI mutant, FAJ1328, was about twice as high as that for the plants inoculated with the wild
type. The raiR mutant, FAJ1329, did not differ significantly from the wild type. Inoculation with the merodiploid
CNPAF512::pFAJ1334 resulted in significantly lower acetylene
reduction per plant and a reduced number of nodules, although a
relatively high variation was observed. Values for dry weight of
nodules and for per-plant nitrogen fixation illustrate this
observation. However, nitrogen-fixing activity in terms of acetylene
reduction per nodule remained unchanged. We therefore conclude that
raiI, but not raiR, is involved in the
restriction of the number of nodules, whereas nitrogen-fixing activity
is not affected. We further conclude by comparison of the patterns of
autoinducers produced by the raiI and raiR
mutants (Fig. 1, lanes E and F) that the molecules representing AI-3, which is synthesized at wild-type levels in the raiR mutant
but is present only in a lower amount in the raiI mutant,
and/or AI-4, which could not be detected in the raiI mutant,
are involved in the regulation of the number of nodules. This
observation adds a new element in the signalling pathways between
Rhizobium and the host plant and is currently being further
explored.
| |
ACKNOWLEDGMENTS |
We thank Stephen Farrand for the gift of the A. tumefaciens autoinducer detection system; Anton van Brussel for
the small-sensitive strain R. leguminosarum bv.
viciae 248, the small-producing strain R. leguminosarum bv. viciae RBL1309, and the
small mutant R. leguminosarum bv.
viciae RBL1376; and E. Peter Greenberg for purified PAI and VAI and for the V. fischeri ES184 lux regulon.
V.R. is a fellow of the Training and Mobility of Researchers Program
(no. ERBFMBICT 950142), which is financed by the European Commission.
J.M. is a postdoctoral fellow of the Fund for Scientific Research
Flanders.
| |
FOOTNOTES |
*
Corresponding author. Mailing address: F. A. Janssens Laboratory of Genetics, Kardinaal Mercierlaan 92, B-3001
Heverlee, Belgium. Phone: 32-16-32 96 79. Fax: 32-16-32 19 66. E-mail:
Jozef.vanderleyden{at}agr.kuleuven.ac.be.
| |
REFERENCES |
| 1.
|
Bainton, N. J.,
B. W. Bycroft,
S. R. Chhabra,
P. Stead,
L. Gledhill,
P. J. Hill,
C. E. D. Rees,
M. K. Winson,
G. P. C. Salmond,
G. S. A. B. Stewart, and P. A. Williams.
1992.
A general role for the lux autoinducer in bacterial cell signalling: control of antibiotic biosynthesis in Erwinia.
Gene
116:87-91[Medline].
|
| 2.
|
Bainton, N. J.,
P. Stead,
S. R. Chhabra,
B. W. Bycroft,
G. P. C. Salmond,
G. S. A. B. Stewart, and P. Williams.
1992.
N-(3-oxohexanoyl)-L-homoserine lactone regulates carbapenem antibiotic production in Erwinia carotovora.
Biochem. J.
288:997-1004.
|
| 3.
|
Bassler, B. L.,
M. Wright,
R. E. Showalter, and M. R. Silverman.
1993.
Intercellular signalling in Vibrio harveyi: sequence and function of genes regulating expression of luminescence.
Mol. Microbiol.
9:773-786[Medline].
|
| 4.
|
Beck von Bodman, S., and S. K. Farrand.
1995.
Capsular polysaccharide biosynthesis and pathogenicity in Erwinia stewartii require induction by an N-acylhomoserine lactone autoinducer.
J. Bacteriol.
177:5000-5008[Abstract/Free Full Text].
|
| 5.
|
Belitsky, B. R.,
M. C. U. Gustafsson,
A. L. Sonenshein, and C. von Wachenfeldt.
1997.
An lrp-like gene of Bacillus subtilis involved in branched-chain amino acid transport.
J. Bacteriol.
179:5448-5457[Abstract/Free Full Text].
|
| 6.
|
Beringer, J. E.
1974.
R-factor transfer in Rhizobium leguminosarum.
J. Gen. Microbiol.
120:421-429.
|
| 7.
|
Brint, J. M., and D. E. Ohman.
1995.
Synthesis of multiple exoproducts in Pseudomonas aeruginosa is under control of RhlR-RhlI, another set of regulators in strain PAO1 with homology to the autoinducer-responsive LuxR-LuxI family.
J. Bacteriol.
177:7155-7163[Abstract/Free Full Text].
|
| 8.
|
Cao, J.-G., and E. A. Meighen.
1989.
Purification and structural identification of an autoinducer for the luminescence system of Vibrio harveyi.
J. Biol. Chem.
264:21670-21676[Abstract/Free Full Text].
|
| 9.
|
Chilton, M.-D.,
T. C. Currier,
S. K. Farrand,
A. J. Bendich,
M. P. Gordon, and E. W. Nester.
1974.
Agrobacterium tumefaciens DNA and PS8 bacteriophage DNA not detected in crown gall tumors.
Proc. Natl. Acad. Sci. USA
71:3672-3676[Abstract/Free Full Text].
|
| 10.
|
Choi, S. H., and E. P. Greenberg.
1992.
Genetic dissection of DNA binding and luminescence gene activation by the Vibrio fischeri LuxR protein.
J. Bacteriol.
174:4064-4069[Abstract/Free Full Text].
|
| 11.
|
Cubo, M. T.,
A. Economou,
G. Murphy,
A. W. B. Johnston, and J. A. Downie.
1992.
Molecular characterization and regulation of the rhizosphere-expressed genes rhiABCR that can influence nodulation of Rhizobium leguminosarum biovar viciae.
J. Bacteriol.
174:4026-4035[Abstract/Free Full Text].
|
| 12.
|
Devine, J. H.,
C. Countryman, and T. O. Baldwin.
1988.
Nucleotide sequence of the luxR and luxI genes and the structure of the primary regulatory region of the lux regulon of Vibrio fischeri ATCC 7744.
Biochemistry
27:837-842.
|
| 13.
|
Eberhard, A.,
A. L. Burlingame,
C. Eberhard,
G. L. Kenyon,
K. H. Nealson, and N. J. Oppenheimer.
1981.
Structural identification of autoinducer of Photobacterium fischeri luciferase.
Biochemistry
20:2444-2449[Medline].
|
| 14.
|
Engebrecht, J.,
K. Nealson, and M. Silverman.
1983.
Bacterial bioluminescence: isolation and genetic analysis of functions from Vibrio fischeri.
Cell
32:773-781[Medline].
|
| 15.
|
Figurski, D. H., and D. R. Helinski.
1979.
Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans.
Proc. Natl. Acad. Sci. USA
76:1648-1652[Abstract/Free Full Text].
|
| 16.
|
Franklin, F. C. H., and W. A. Venables.
1976.
Biochemical, genetic and regulatory studies of alanine catabolism in Escherichia coli K12.
Mol. Gen. Genet.
149:229-237[Medline].
|
| 17.
|
Friedman, A. M.,
S. R. Long,
S. E. Brown,
W. J. Buikema, and F. M. Ausubel.
1982.
Construction of a broad host range cosmid cloning vector and its use in the genetic analysis of Rhizobium mutants.
Gene
18:289-296[Medline].
|
| 18.
|
Fuqua, W. C., and S. C. Winans.
1994.
A LuxR-LuxI type regulatory system activates Agrobacterium Ti plasmid conjugal transfer in the presence of a plant tumor metabolite.
J. Bacteriol.
176:2796-2806[Abstract/Free Full Text].
|
| 19.
|
Fuqua, W. C.,
S. C. Winans, and E. P. Greenberg.
1994.
Quorum sensing in bacteria: the LuxR-LuxI family of cell density-responsive transcriptional regulators.
J. Bacteriol.
176:269-275[Free Full Text].
|
| 20.
|
Fuqua, W. C.,
S. C. Winans, and E. P. Greenberg.
1996.
Census and consensus in bacterial ecosystems: the LuxR-LuxI family of quorum-sensing transcriptional regulators.
Annu. Rev. Microbiol.
50:727-751[Medline].
|
| 21.
|
Gambello, M. J., and B. H. Iglewski.
1991.
Cloning and characterization of the Pseudomonas aeruginosa lasR gene, a transcriptional activator of elastase expression.
J. Bacteriol.
173:3000-3009[Abstract/Free Full Text].
|
| 22.
|
García-Lara, J.,
L. H. Shang, and L. I. Rothfield.
1996.
An extracellular factor regulates expression of sdiA, a transcriptional activator of cell division genes in Escherichia coli.
J. Bacteriol.
178:2742-2748[Abstract/Free Full Text].
|
| 23.
|
Gilson, L.,
A. Kuo, and P. V. Dunlap.
1995.
AinS and a new family of autoinducer synthesis proteins.
J. Bacteriol.
177:6946-6951[Abstract/Free Full Text].
|
| 24.
| Gray, K. M. 1996. Identification of multiple
autoinducer signals in Rhizobium leguminosarum. Presented as
poster X15 at the 8th International Congress on Molecular Plant-Microbe
Interactions, Knoxville, Tenn., 14 to 19 July 1996.
|
| 25.
|
Gray, K. M., and E. P. Greenberg.
1992.
Physical and functional maps of the luminescence gene cluster in an autoinducer-deficient Vibrio fischeri strain isolated from a squid light organ.
J. Bacteriol.
174:4384-4390[Abstract/Free Full Text].
|
| 26.
|
Gray, K. M.,
L. Passador,
B. H. Iglewski, and E. P. Greenberg.
1994.
Interchangeability and specificity of components from the quorum-sensing regulatory systems of Vibrio fischeri and Pseudomonas aeruginosa.
J. Bacteriol.
176:3076-3080[Abstract/Free Full Text].
|
| 27.
|
Gray, K. M.,
J. P. Pearson,
J. A. Downie,
B. E. A. Boboye, and E. P. Greenberg.
1996.
Cell-to-cell signaling in the symbiotic nitrogen-fixing bacterium Rhizobium leguminosarum: autoinduction of a stationary phase and rhizosphere-expressed genes.
J. Bacteriol.
178:372-376[Abstract/Free Full Text].
|
| 28.
|
Hanzelka, B. L.,
A. M. Stevens,
M. R. Parsek,
T. J. Crone, and E. P. Greenberg.
1997.
Mutational analysis of the Vibrio fischeri LuxI polypeptide: critical regions of an autoinducer synthase.
J. Bacteriol.
179:4882-4887[Abstract/Free Full Text].
|
| 29.
|
Hirsch, P. R.
1979.
Plasmid-determined bacteriocin production by Rhizobium leguminosarum.
J. Gen. Microbiol.
113:219-228.
|
| 30.
|
Hwang, I.,
D. M. Cook, and S. K. Farrand.
1995.
A new regulatory element modulates homoserine lactone-mediated autoinduction of Ti plasmid conjugal transfer.
J. Bacteriol.
177:449-458[Abstract/Free Full Text].
|
| 31.
|
Hwang, J.,
P.-L. Li,
L. Zhang,
K. R. Piper,
D. M. Cook,
M. E. Tate, and S. K. Farrand.
1994.
TraI, a LuxI homologue, is responsible for production of conjugation factor, the Ti plasmid N-acyl homoserine lactone autoinducer.
Proc. Natl. Acad. Sci. USA
91:4639-4643[Abstract/Free Full Text].
|
| 32.
|
Jones, S.,
B. Yu,
N. J. Bainton,
M. Birdsall,
B. W. Bycroft,
S. R. Chhabra,
A. J. R. Cox,
P. Golby,
P. J. Reeves,
S. Stephens,
M. K. Winson,
G. P. C. Salmond,
G. S. A. B. Stewart, and P. Williams.
1993.
The lux autoinducer regulates the production of exoenzyme virulence determinants in Erwinia carotovora and Pseudomonas aeruginosa.
EMBO J.
12:2477-2482[Medline].
|
| 33.
|
Kuo, A.,
N. V. Blough, and P. V. Dunlap.
1994.
Multiple N-acyl-L-homoserine lactone autoinducers of luminescence in the marine symbiotic bacterium Vibrio fischeri.
J. Bacteriol.
176:7558-7565[Abstract/Free Full Text].
|
| 34.
|
Lobocka, M.,
J. Hennig,
J. Wild, and T. Klopotowski.
1994.
Organization and expression of the Escherichia coli K-12 dad operon encoding the smaller subunit of D-amino acid dehydrogenase and the catabolic alanine racemase.
J. Bacteriol.
176:1500-1510[Abstract/Free Full Text].
|
| 35.
|
Madhusudhan, K. T.,
D. Lorenz, and J. R. Sokatch.
1993.
The bkdR gene of Pseudomonas putida is required for expression of the bkd operon and encodes a protein related to Lrp of Escherichia coli.
J. Bacteriol.
175:3934-3940[Abstract/Free Full Text].
|
| 36.
|
Mathew, E.,
J. Zhi, and M. Freundlich.
1996.
Lrp is a direct repressor of the dad operon in Escherichia coli.
J. Bacteriol.
178:7234-7240[Abstract/Free Full Text].
|
| 37.
|
Metcalf, W. W., and B. L. Wanner.
1993.
Construction of new -glucuronidase cassettes for making transcriptional fusions and their use with new methods for allele replacements.
Gene
129:17-25[Medline].
|
| 38.
|
Michiels, J.
1993.
.
Regulation of nitrogen fixation in Rhizobium leguminosarum biovar phaseoli CNPAF512. Dissertationes de agricultura, vol. 244.
K. U. Leuven, Leuven, Belgium.
|
| 39.
|
Miller, J. H.
1972.
, p. 354-358.
Experiments in molecular genetics
Cold Spring Harbor Laboratory, Cold Spring Harbor, N.Y.
|
| 40.
|
Moré, M. I.,
D. Finger,
J. L. Stryker,
C. Fuqua,
A. Eberhard, and S. C. Winans.
1996.
Enzymatic synthesis of a quorum-sensing autoinducer through the use of defined substrates.
Science
272:1655-1658[Abstract].
|
| 41.
|
Ochsner, U. A., and J. Reiser.
1995.
Autoinducer-mediated regulation of rhamnolipid biosurfactant synthesis in Pseudomonas aeruginosa.
Proc. Natl. Acad. Sci. USA
92:6424-6428[Abstract/Free Full Text].
|
| 42.
|
Passador, L.,
J. M. Cook,
M. J. Gambello,
L. Rust, and B. H. Iglewski.
1993.
Expression of Pseudomonas aeruginosa virulence genes requires cell-to-cell communication.
Science
260:1127-1130[Abstract/Free Full Text].
|
| 43.
|
Pearson, J. P.,
K. M. Gray,
L. Passador,
K. D. Tucker,
A. Eberhard,
B. H. Iglewski, and E. P. Greenberg.
1994.
Structure of the autoinducer required for expression of Pseudomonas aeruginosa virulence genes.
Proc. Natl. Acad. Sci. USA
91:197-201[Abstract/Free Full Text].
|
| 44.
|
Piper, K. R.,
S. Beck von Bodman, and S. K. Farrand.
1993.
Conjugation factor of Agrobacterium tumefaciens regulates Ti plasmid transfer by autoinduction.
Nature
362:448-450[Medline].
|
| 45.
|
Pirhonen, M.,
D. Flego,
R. Heikinheimo, and E. T. Palva.
1993.
A small diffusible signal molecule is responsible for the global control of virulence and exoenzyme production in the plant pathogen Erwinia carotovora.
EMBO J.
12:2467-2476[Medline].
|
| 46.
|
Platko, J. V.,
D. A. Willins, and J. M. Calvo.
1990.
The ilvIH operon of Escherichia coli is positively regulated.
J. Bacteriol.
172:4563-4570[Abstract/Free Full Text].
|
| 47.
|
Quandt, J., and M. F. Hynes.
1993.
Versatile suicide vectors which allow direct selection for gene replacement in Gram-negative bacteria.
Gene
127:15-21[Medline].
|
| 48.
|
Salmond, G. P. C.,
B. W. Bycroft,
G. S. A. B. Stewart, and P. Williams.
1995.
The bacterial `enigma': cracking the code of cell-cell communication.
Mol. Microbiol.
16:615-624[Medline].
|
| 49.
|
Sambrook, J.,
E. F. Fritsch, and T. Maniatis.
1989.
.
Molecular cloning: a laboratory manual, 2nd ed.
Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.
|
| 50.
|
Schripsema, J.,
K. E. E. de Rudder,
T. B. van Vliet,
P. P. Lankhorst,
E. de Vroom,
J. W. Kijne, and A. A. N. van Brussel.
1996.
Bacteriocin small of Rhizobium leguminosarum belongs to the class of N-acyl-L-homoserine lactone molecules, known as autoinducers and as quorum sensing co-transcription factors.
J. Bacteriol.
178:366-371[Abstract/Free Full Text].
|
| 51.
|
Shadel, G. S.,
R. Young, and T. O. Baldwin.
1990.
Use of regulated cell lysis in a lethal genetic selection in Escherichia coli: identification of the autoinducer-binding region of the LuxR protein from Vibrio fischeri ATCC 7744.
J. Bacteriol.
172:3980-3987[Abstract/Free Full Text].
|
| 52.
|
Shaw, P. D.,
G. Ping,
S. L. Daly,
C. Cha,
J. E. Cronan, Jr.,
K. L. Rinehart, and S. K. Farrand.
1997.
Detecting and characterizing N-acyl-homoserine lactone signal molecules by thin-layer chromatography.
Proc. Natl. Acad. Sci. USA
94:6036-6041[Abstract/Free Full Text].
|
| 53.
|
Sitnikov, D. M.,
J. B. Schineller, and T. O. Baldwin.
1996.
Control of cell division in Escherichia coli: regulation of transcription of ftsQA involves both rpoS and SdiA-mediated autoinduction.
Proc. Natl. Acad. Sci. USA
93:336-341[Abstract/Free Full Text].
|
| 54.
|
Slock, J.,
D. VanRiet,
D. Kolibachuk, and E. P. Greenberg.
1990.
Critical regions of the Vibrio fischeri LuxR protein defined by mutational analysis.
J. Bacteriol.
172:3974-3979[Abstract/Free Full Text].
|
| 55.
|
Swift, S.,
M. K. Winson,
P. F. Chan,
N. J. Bainton,
M. Birdsall,
P. J. Reeves,
C. E. D. Rees,
S. R. Chhabra,
P. J. Hill,
J. P. Throup,
B. W. Bycroft,
G. P. C. Salmond,
P. Williams, and G. S. A. B. Stewart.
1993.
A novel strategy for the isolation of luxI homologues: evidence for widespread distribution of a LuxR:LuxI superfamily in enteric bacteria.
Mol. Microbiol.
10:511-520[Medline].
|
| 56.
|
Throup, J. P.,
M. Camara,
G. S. Briggs,
M. K. Winson,
S. R. Chhabra,
B. W. Bycroft,
P. Williams, and G. S. A. B. Stewart.
1995.
Characterisation of the yenI/yenR locus from Yersinia enterocolitica mediating the synthesis of two N-acylhomoserine lactone signal molecules.
Mol. Microbiol.
17:345-356[Medline].
|
| 57.
|
van Brussel, A. A. N.,
S. A. J. Zaat,
C. A. Wijffelman,
E. Pees, and B. J. J. Lugtenberg.
1985.
Bacteriocin small of fast-growing rhizobia is chloroform soluble and is not required for effective nodulation.
J. Bacteriol.
162:1079-1082[Abstract/Free Full Text].
|
| 58.
|
van Rhijn, P. J. S.,
B. Feys,
C. Verreth, and J. Vanderleyden.
1993.
Multiple copies of nodO in Rhizobium tropici CIAT899 and BR816.
J. Bacteriol.
175:438-447[Abstract/Free Full Text].
|
| 59.
|
Vincent, J. M.
1970.
.
A manual for the practical study of the root-nodule bacteria.
Blackwell Scientific Publications, Oxford, United Kingdom.
|
| 60.
|
Wijffelman, C. A.,
E. Pees,
A. A. N. van Brussel, and P. J. J. Hooykaas.
1983.
Repression of small bacteriocin excretion in Rhizobium leguminosarum and Rhizobium trifolii by transmissible plasmids.
Mol. Gen. Genet.
192:171-176.
|
| 61.
|
Winson, M. K.,
M. Camara,
A. Latifi,
M. Foglino,
S. R. Chhabra,
M. Daykin,
M. Bally,
V. Chapon,
G. P. C. Salmond,
B. W. Bycroft,
A. Lazdunski,
G. S. A. B. Stewart, and P. Williams.
1995.
Multiple N-acyl-L-homoserine lactone signal molecules regulate production of virulence determinants and secondary metabolites in Pseudomonas aeruginosa.
Proc. Natl. Acad. Sci. USA
92:9427-9431[Abstract/Free Full Text].
|
| 62.
|
Yanisch-Perron, C.,
J. Vieira, and J. Messing.
1985.
Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors.
Gene
33:103-119[Medline].
|
| 63.
|
Zhang, L.,
P. J. Murphy,
A. Kerr, and M. E. Tate.
1993.
Agrobacterium conjugation and gene regulation by N-acyl-L-homoserine lactones.
Nature
362:446-448[Medline].
|
J Bacteriol, February 1998, p. 815-821, Vol. 180, No. 4
0021-9193/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
This article has been cited by other articles:
-
Edwards, A., Frederix, M., Wisniewski-Dye, F., Jones, J., Zorreguieta, A., Downie, J. A.
(2009). The cin and rai Quorum-Sensing Regulatory Systems in Rhizobium leguminosarum Are Coordinated by ExpR and CinS, a Small Regulatory Protein Coexpressed with CinI. J. Bacteriol.
191: 3059-3067
[Abstract]
[Full Text]
-
Sanchez-Contreras, M., Bauer, W. D, Gao, M., Robinson, J. B, Allan Downie, J
(2007). Quorum-sensing regulation in rhizobia and its role in symbiotic interactions with legumes. Phil Trans R Soc B
362: 1149-1163
[Abstract]
[Full Text]
-
Daniels, R., Reynaert, S., Hoekstra, H., Verreth, C., Janssens, J., Braeken, K., Fauvart, M., Beullens, S., Heusdens, C., Lambrichts, I., De Vos, D. E., Vanderleyden, J., Vermant, J., Michiels, J.
(2006). Quorum signal molecules as biosurfactants affecting swarming in Rhizobium etli. Proc. Natl. Acad. Sci. USA
103: 14965-14970
[Abstract]
[Full Text]
-
Zheng, H., Zhong, Z., Lai, X., Chen, W.-X., Li, S., Zhu, J.
(2006). A LuxR/LuxI-Type Quorum-Sensing System in a Plant Bacterium, Mesorhizobium tianshanense, Controls Symbiotic Nodulation.. J. Bacteriol.
188: 1943-1949
[Abstract]
[Full Text]
-
Moris, M., Braeken, K., Schoeters, E., Verreth, C., Beullens, S., Vanderleyden, J., Michiels, J.
(2005). Effective Symbiosis between Rhizobium etli and Phaseolus vulgaris Requires the Alarmone ppGpp. J. Bacteriol.
187: 5460-5469
[Abstract]
[Full Text]
-
Gonzalez, J. E., Marketon, M. M.
(2003). Quorum Sensing in Nitrogen-Fixing Rhizobia. Microbiol. Mol. Biol. Rev.
67: 574-592
[Abstract]
[Full Text]
-
Tun-Garrido, C., Bustos, P., Gonzalez, V., Brom, S.
(2003). Conjugative Transfer of p42a from Rhizobium etli CFN42, Which Is Required for Mobilization of the Symbiotic Plasmid, Is Regulated by Quorum Sensing. J. Bacteriol.
185: 1681-1692
[Abstract]
[Full Text]
-
He, X., Chang, W., Pierce, D. L., Seib, L. O., Wagner, J., Fuqua, C.
(2003). Quorum Sensing in Rhizobium sp. Strain NGR234 Regulates Conjugal Transfer (tra) Gene Expression and Influences Growth Rate. J. Bacteriol.
185: 809-822
[Abstract]
[Full Text]
-
Loh, J., Carlson, R. W., York, W. S., Stacey, G.
(2002). Bradyoxetin, a unique chemical signal involved in symbiotic gene regulation. Proc. Natl. Acad. Sci. USA
99: 14446-14451
[Abstract]
[Full Text]
-
Marketon, M. M., Gronquist, M. R., Eberhard, A., Gonzalez, J. E.
(2002). Characterization of the Sinorhizobium meliloti sinR/sinI Locus and the Production of Novel N-Acyl Homoserine Lactones. J. Bacteriol.
184: 5686-5695
[Abstract]
[Full Text]
-
Pellock, B. J., Teplitski, M., Boinay, R. P., Bauer, W. D., Walker, G. C.
(2002). A LuxR Homolog Controls Production of Symbiotically Active Extracellular Polysaccharide II by Sinorhizobium meliloti. J. Bacteriol.
184: 5067-5076
[Abstract]
[Full Text]
-
Gram, L., Grossart, H.-P., Schlingloff, A., Kiorboe, T.
(2002). Possible Quorum Sensing in Marine Snow Bacteria: Production of Acylated Homoserine Lactones by Roseobacter Strains Isolated from Marine Snow. Appl. Environ. Microbiol.
68: 4111-4116
[Abstract]
[Full Text]
-
Marketon, M. M., Gonzalez, J. E.
(2002). Identification of Two Quorum-Sensing Systems in Sinorhizobium meliloti. J. Bacteriol.
184: 3466-3475
[Abstract]
[Full Text]
-
Wisniewski-Dye, F., Jones, J., Chhabra, S. R., Downie, J. A.
(2002). raiIR Genes Are Part of a Quorum-Sensing Network Controlled by cinI and cinR in Rhizobium leguminosarum. J. Bacteriol.
184: 1597-1606
[Abstract]
[Full Text]
-
Loh, J., Lohar, D. P., Andersen, B., Stacey, G.
(2002). A Two-Component Regulator Mediates Population-Density-Dependent Expression of the Bradyrhizobium japonicum Nodulation Genes. J. Bacteriol.
184: 1759-1766
[Abstract]
[Full Text]
-
FRAY, R. G.
(2002). Altering Plant-Microbe Interaction Through Artificially Manipulating Bacterial Quorum Sensing. ANN BOT (LOND)
89: 245-253
[Abstract]
[Full Text]
-
Daniels, R., De Vos, D. E., Desair, J., Raedschelders, G., Luyten, E., Rosemeyer, V., Verreth, C., Schoeters, E., Vanderleyden, J., Michiels, J.
(2002). The cin Quorum Sensing Locus of Rhizobium etli CNPAF512 Affects Growth and Symbiotic Nitrogen Fixation. J. Biol. Chem.
277: 462-468
[Abstract]
[Full Text]
-
Zhang, Z., Pierson, L. S. III
(2001). A Second Quorum-Sensing System Regulates Cell Surface Properties but Not Phenazine Antibiotic Production in Pseudomonas aureofaciens. Appl. Environ. Microbiol.
67: 4305-4315
[Abstract]
[Full Text]
-
de Kievit, T. R., Iglewski, B. H.
(2000). Bacterial Quorum Sensing in Pathogenic Relationships. Infect. Immun.
68: 4839-4849
[Full Text]
-
Sourjik, V., Muschler, P., Scharf, B., Schmitt, R.
(2000). VisN and VisR Are Global Regulators of Chemotaxis, Flagellar, and Motility Genes in Sinorhizobium (Rhizobium) meliloti. J. Bacteriol.
182: 782-788
[Abstract]
[Full Text]
-
Rodelas, B., Lithgow, J. K., Wisniewski-Dye, F., Hardman, A., Wilkinson, A., Economou, A., Williams, P., Downie, J. A.
(1999). Analysis of Quorum-Sensing-Dependent Control of Rhizosphere-Expressed (rhi) Genes in Rhizobium leguminosarum bv. viciae. J. Bacteriol.
181: 3816-3823
[Abstract]
[Full Text]
-
Thorne, S. H., Williams, H. D.
(1999). Cell Density-Dependent Starvation Survival of Rhizobium leguminosarum bv. phaseoli: Identification of the Role of an N-Acyl Homoserine Lactone in Adaptation to Stationary-Phase Survival. J. Bacteriol.
181: 981-990
[Abstract]
[Full Text]
Top
Abstract
Introduction
