Quorum sensing in Sinorhizobium meliloti :
AHL-induced expression of sinI
Rupika Madhavan
July 18, 2012
Abstract
Sinorhizobium meliloti is a species of quorum-sensing bacteria that has a symbiotic
relationship with its plant host, Medicago sativa. Quorum sensing allows bacteria to communicate by excreting and uptaking chemical signals, called autoinducers, and change the
behavior of the entire population once a critical concentration of autoinducer is reached.
S. meliloti is predicted to have at least 2 quorum sensing networks (QSNs): the hypothesized Mel system and the known Sin system. The Sin QSN has a synthase (SinI) which
produces several long-chain AHLs with a wide range of carbon-chain length (C12 to C18 ).
Though it has been studied for a while, not much is known about the specifics of the Sin
QSN. In this paper, we test the effects of several AHLs (C4-C18) on various strains of S.
meliloti to see if more can be understood about the complex behavioral network in these
small bacteria.
1
Introduction
ficial bacteria around us.
Quorum sensing (QS) is a process through
which bacteria communicate with each other
[1]. It is facilitated by three main parts: (1)
autoinducers, which are the chemical signals
used by the system for effective communcation; (2) synthases (which produce the chemical signals); and (3) receptors (which bind to
the autoinducers). In ram negative bacteria,
like Sinorhizobium meliloti, N -acyl homoserine lactons (or AHLs) are the preferred autoinducer. The bacteria ”learn” about and
respond to environmental changes by preducing, emitting, binding to, and responding
to these autoinducers.This system allows for
population-wide behavioral changes, which
are essential for survival. Uncovering the
mechanisms used by different quorum sensing networks (QSNs) will allow us to better
manipulate the behavior of harmful or bene-
One of the best understood (QSNs) is that
of the bacteria Vibrio fischeri, the bioluminescent bacteria that live in a Hawaiian squid
[1]. It has a fairly simple system; there are
only two proteins involved (See Figure 1).
LuxI (the synthase) produces the AHL 3-oxoC6 , which leaves the cell. In a population
of bacteria, these AHLs increase in concentration and absorbed by the cells. Once in
the cell, an autoinducer attaches to the receptor, LuxR. The LuxR-AHL complex then
causes the transcription of the luxICDABE
operon, which encodes for luciferase, the bioluminescent enzyme. In addition, luxI is
also encoded within this operon, further producing AHLs, which, in turn, bind to LuxR.
This positive feedback loop accelerates the
process, and causes the whole population to
produce light. This bacterial circuit is sim1
at the center of QS research on S. meliloti.
The Sin system is essential for symbiosis with
the host plant; strains without a functional
synthase cannot successfully invade the host
plant’s roots [4]. In addition, Medicago sativa
produces AHL-like signals that can interfere
with the activities of the Sin QSN [5].
Three proteins work within the framework
of the Sin system: SinI, SinR, and ExpR
[6, 3]. Sin I is a LuxI homolog; i.e., SinI
is the synthase which produces the autoinducers used by the system. The position of
sinR with respect to sinI on the genome (directly upstream; see Figure 2) would imply
that SinR is a LuxR homolog [7]; however, it
has been shown that ExpR is the protein that
actually binds to the AHLs produced by SinI
[4]. Though ExpR is an LuxR solo (expR is
not near sinI on the bacterial genome) [8],
it does have a binding site is located in the
intergenic space between sinI and sinR [7].
Several AHLs have been shown to be used
by the rhizobia. Of these, those with carbon
chains of lengths between 12 and 18 have been
shown to be synthesized by the Sin system;
though there is evidence of other AHLs being produced by the bacteria [6]. This wide
range of carbon-chain length is rather peculiar; these lengths correspond directly to the
ability of the AHL to diffuse. Though much
is known about S. meliloti, a lot is still left to
discover, such as what the purpose of sinR is,
whether SinR interacts with Sin AHLs, and
whether all the AHLs predicted to be produced by SinI are actually used by the Sin
QSN. Once this is known, the bigger purpose
of this experiment may be carried out: to discover the diffusion patterns of AHLs in the
bacteria.
In this paper, we try to seek the answers
to these questions. Our experiment testing
the purpose of SinR is running and will provide answers within the next week, but we
have shown that of the many AHLs produced
by SinI, only 3-oxo-C14 , C16:1 , and 3-oxo-C16:1
Figure 1: The well-known QSN of V. fischeri.
LuxI is a synthase that produces the autoinducer, 3-oxo-C6 . It is released from the cell,
where more AHLs have been excreted. It
is then taken in by a cell and attaches to
LuxR, the receptor protein. These two together bind to the ”lux-box” (an operon with
several genes including luxI and the gene for
luciferase) and promote the production of luciferase, the bioluminescent protein. In addition, luxI is also promoted, and thus creates
a positive feedback loop that accelerates the
quorum sensing process.
ple, but provides a nice insight into the way
in which bacteria work. The goal of a scientist studying a quorum sensing network is to
discover the circuit through which his or her
bacteria communicates.
The bacteria discussed in this paper are
Sinorhizobium meliloti, gram-negative bacteria who fix nitrogen for their host, the alfalfa
plant Medicago sativa [2]. The system is more
complicated than the V. fischeri. It is suspected that there are two QSNs at work in
S. meliloti: the Sin system and the hypothesized Mel system [3]. As the Mel system has
not been located, the Sin system has been
2
and then retired when colonies from a single
clone were no longer able to be picked. Clonal
colonies were picked with a pipette tip and
dropped into 5 mL of Ty and grown overnight
(around 9 hours) until the optical density was
between 0.2 and 0.4. These colonies were diluted 100-fold, returning them to an OD between 0.002 and 0.004, so the fluorescence due
to expression of the sinI promoter could be
seen at all stages of growth.
The autoinducers were treated in four
ways:
Figure 2: The arrangement of the quroumsensing-related genes in the S. meliloti chromosome. It has been predicted that the ExpR
binding site is in the intergenic region between sinR and sinI. The lux-box -like gene
is predicted to be the binding site for SinR.
change the response behavior of the sinI mutant strain when grown in Ty medium.
2
1. Originally, 400 µL of diluted bacteria
were added to each well. The AHLs
(stored in ethyl acetate) were added directly to the bacteria in the well plates
after the bacteria were placed in the
wells; however, as ethyl acetate kills bacteria and the effects from this were noticed, it was decided that a new method
needed to be used.
Methods and Materials
The fluorescence and optical density curves
were produced by using a BioTek machine.
Gen5 software was used to program the machine to take measurements of the optical
density and fluorescence every 10 minutes for
48 hours. Polystyrene 48-well plates and 96well plates were primarily used in these experiments.
Five strains of bacteria were studied in
these series of experiments: 8530 (wild
type), 1021 (expR mutant), MG32 (sinI mutant), MG75 (sinI, expR double mutant), and
MG170 (sinR mutant). Each of these strains
contained a psinI-gfp plasmid−i.e., when the
sinI promoter was activated (psinI ), GFP
would fluoresce green when blue light was
shone on it. Glycerol stock of these strains
were kept in a -80 ◦ C freezer.
Ice from the glycerol stock cultures was
scraped onto a pipette tip and dropped into
5 mL of Ty medium and grown overnight
(around 14 hours) in a 30 ◦ C incubator.
These new cultures were used to streak plates
of 1.5% agar (made with antibiotics streptomycin at 400 µg/mL and spectinomycin at
50 µg/mL) and then incubated at 30 ◦ C until enough colonies were formed, but before
to many colonies started to merge. These
agar plates were used for several experiments,
2. Phosphate buffer dilutions were used
next: The AHLs in ethyl acetate were
evaporated in polycarbonate tubes, leaving just the AHLs. Phosphate buffer was
then added to the appropriate concentration and mixed throroughly. Since
AHLs do not mix well with phosphate
buffer, the concentration of the AHLs
was doubted.
3. Further experiments were conducted by
placing the AHLs directly into the well
plate, and then evaporating. Since the
ethyl acetate ate up the polystyrene,
150-200 µL of deionized water were
added to the bottom of each well,
followed by pipetting 4 µL of the
autoinducer-ethyl acetate mixture on
top of the water. The wells were placed
in a 60 ◦ C oven and left to evaporate until all the liquid was gone from the wells.
As the ethyl acetate is lighter and evaporates more quickly, the water was a pro3
Strain
Rm8530
Rm1021
MG32
MG75
MG170
Relevant Characteristics
1021 expR+ SmR
expR102 ::ISRm2011-1 expR, SmR
8530 δsinI, SmR
1021 δsinI, SmR
8530 δsinR, SmR
Source
Pellock et al., 2002
Galibert et al., 2001
Gao et al., 2005
Gao et al., 2005
Gao, et. al, 2012
Table 1: The various names and characteristics of the strains studied in these experiments.
tective barrier between the ethyl acetate
and the water. The plate was allowed to
cool to room temperature, and the 100fold dilution of colonies mixed with antibiotics was added to the wells. However, some wells were still affected negatively by the ethyl acetate.
ing sinR were transformed into E. coli cells
via electroporation. The results of these experiments have not yet been delivered, so further detail will be provided when the results
come in.
3
4. Final experiments were conducted by diluting the AHLs with ethanol. Ethanol
does not eat away polystyrene, and so
did not have a negative affect on the well
plates. The AHLs were serially diluted in
ethanol, and 4 µL of the diluted solution
was added to the appropriate wells. The
well plate was left under the hood until
all the ethanol was evaporated. Ty and
the bacteria were then added to the evaporated wells, and mixed with a pipette.
Results
In our experiments, we see that only MG32
(sinI mutant) has any response to a variation in the concentration of AHL (Figure 3);
it is the only one of the strains not producing
AHL (it lacks sinI ) but also has expR (the receptor). The psinI activity in MG32 is significantly lower than in MG75, as shown in Figure 7. However, with the right AHLs, psinI
activity increases in MG32, with 3-oxo-C16
raising psinI activity to a level greater than
the MG75. The C16:1 and 3-oxo-C14 raise the
psinI activity, but not enough to overcome
the psinI activity of MG75. As Figure 3 and
Figure 5 show, of all the AHLs believed to be
produced by the Sin system (C12 -C18 ), only
3-oxo-C16 , C16:1 , and 3-oxo-C14 showed any
increase in psinI activity in the MG32 strain.
Looking at Figure 3 and Figure 4, we see
that none of the AHLs have any effect on
psinI expression in MG75, the only other
strain that does not produce its own AHLs.
Furthermore, MG75 (sinI, expR double mutant) and 1021 (expR mutant) have fluorescence curves that increase continuously despite the leveling-off of the optical density.
The most significant impact on psinI expression aside from removing sinI itself was
After adding both autoinducers and bacteria to each of the wells, the well plates were
placed in the BioTek machine and data was
collected for 48 hours at 30 ◦ C.
Two experiments were conducted testing
the effects of phosphate on psinI activity.
The first experiment used a 100 mM phosphate buffer and Ty medium mixture. The
second experiment used a 2 mM phosphate
buffer and Ty medium mixture. To add phosphate to the Ty medium, a 1 M solution of
phosphate buffer at a pH of 7 was diluted
using Ty medium to the appropriate concentrations. The various strains were then grown
in this mixture.
Furthermore, to test the purpose of SinR,
the psinI-gfp plasmid and a plasmid contain4
Fluorescence of Strains at OD=0.5, 1.5 µM AHL
5
4
x 10
C4
C6
3oC6
C8
C12
C14
3oC14
C16
C16:1
3oC16:1
C18
No AHL
3.5
Fluorescence (counts)
3
2.5
2
1.5
1
0.5
0
8530
1021
MG32
MG75
MG170
Figure 3: sinI promoter activity of various strains of S.meliloti with all AHLs. 8530 is the
wild-type strain with psinI-gfp; 1021 is the expR mutant with psinI-gfp; MG32 is the sinI
mutant with psinI-gfp; MG75 is the sinI, expR double mutant with psinI-gfp; MG170 is the
sinR mutant with psinI-gfp. It is important to note that data from the 8530 is not reliable;
as it forms EPS, a colony forms at the bottom of the wells, making the fluorescence hard
to read. Fluorescence curves of the 8530 (not shown) show the eccentric, and hard to map
behavior of the 8530.
5
5
Optical Density of 1021 and MG75 with short−chain AHLs
0.8
4
x 10
Fluorescence of 1021 and MG75 with short−chain AHLs
−expR
0.7
−expR/−sinI
3.5
0.6
0.5
Fluorescence (counts)
Optical Density (AU)
3
0.4
0.3
2.5
2
1.5
0.2
1
0.1
0
−10
0
10
20
Time (h)
30
40
0.5
−10
50
0
10
20
Time (h)
30
40
50
Figure 6: sinI promoter activity of 1021 (expR mutant) and MG75 (sinI, expR double
mutant) with various AHLs (C12 , 3-oxo-C14 , 3-oxo-C16 , C16:1 , and C18 ). The two have
identical fluorescence curves, in spite of the fact that MG75 does not have a functional sinI.
Optical Density of MG32 and MG75 strains
5
1
5
0.9
4.5
Fluorescence of MG32 and MG75 strains
MG32+3oC16:1
0.8
4
MG75
0.7
3.5
Fluorescence
Optical Density (AU)
x 10
0.6
0.5
0.4
3
2
0.3
0.2
1.5
0.1
1
0
0
MG32+3oC14, C16:1
2.5
10
20
30
Time (h)
40
50
0.5
0
MG32
10
20
30
Time (h)
40
50
Figure 7: sinI promoter activity of MG32 (sinI mutant) and MG75 (sinI, expR double
mutant) with various AHLs (C12 , 3-oxo-C14 , 3-oxo-C16 , C16:1 , and C18 ). the psinI activity
of MG75 is significantly greater than that of MG32, unless an autoinducer binds to ExpR
6
5
4
Fluorescence of MG32 at OD=0.5
x 10
1.5 µM
150 nM
15 nM
1.5 nM
3.5
Fluorescence of MG75 at OD=0.5
5
x 10
3
100 µM
10 µM
1 µM
100 nM
No AHL
3.5
Fluorescence (counts)
3
Fluorescence (counts)
4
2.5
2.5
2
1.5
2
1
1.5
0.5
0
1
C14
3oC14
C16
C16:1
3oC16
No AHL
0.5
Figure 9: sinI promoter activity of MG32
(sinI mutant) with long-chain AHLs, with
AHLs prepared by way of method 4. The
Figure 4: sinI promoter activity of MG75 concentration of AHL needed for a response
(sinI, expR double mutant) with several long- is lower (1.5 µM).
chain AHLs, with AHLs prepared by way of
method 4. MG75 does not respond to any of
removing sinR, with around a 3-fold decrease
the AHLs.
in fluorescence. Disrupting expR reduced the
fluorescence by a factor of two. Once expR
was removed, disrupting sinI from the expR
mutant had little to no effect; the expR mutant and expR, sinI double mutant had the
same fluorescence curves (Figure 6). As this
Fluorescence of MG32 at OD=0.5
x 10
4.5
result was surprising, PCR was done to con100 µM
4
10 µM
firm that these two strains were in fact differ1 µM
3.5
100 nM
ent; the results showed that they were.
No AHL
3
As the experimental technique improved,
2.5
the concentration of AHL required to induce
2
a response decreased significantly (compare
1.5
Figure 5 and Figure 9). The final experiments
1
show a response to concentrations around 150
0.5
nM.
0
No AHL C12
3oC14 3oC16 C16:1
C18
When the phosphate concentration was increased to 2 mM, no effect was detected; howFigure 5: sinI promoter activity of MG32 ever, when the phosphate concentration was
(sinI mutant) with several long-chain AHLs, increased to 100 mM, a significant change in
with AHLs prepared by way of method 3. the behavior of the bacteria was detected.
The concentration of AHL needed for a re- The fluorescence curves had an interesting
pattern (Figure 8).
sponse is extremely high (100 µM!)
0
No AHL
C12
3oC14
3oC16
C16:1
C18
Fluorescence (counts)
5
7
Optical Density of MG32 in 100 mM phosphate
5
0.7
9
0.4
0.3
0.2
0.1
7
Fluorescence (counts)
C6
3oC6
C8
C12
C14
3oC14
C16
C16:1
3oC16:1
C18
NoAHL
No AHL
0.5
Optical Density (AU)
Fluorescence of MG32 in 100 mM phosphate
8
0.6
0
0
x 10
6
5
4
3
2
20
40
60
Time (h)
80
1
0
100
20
40
60
Time (h)
80
100
Figure 8: sinI promoter activity of MG32 (sinI mutant) with various AHLs (C6 , 3-oxo-C6 ,
C8 , C12 , C14 , 3-oxo-C14 , C16 , 3-oxo-C16 , C16:1 , and C18 )
4
Discussion
[6]. They also discovered AHLs with shorter
chains: 3-oxo-C6 and C6 ; however, these
AHLs were unaffected by a disruption in the
sinI gene, implying that the Sin QSN does
not produce these AHLs. Teplitski, et al.
(2003) found a response from C14 , 3-oxo-C14 ,
C16 , 3-oxo-C16 and C16:1 , but did not find a
response from C8 , C12 , or C18 [9]. We only
found a response in 3-oxo-C14 , 3-oxo-C16 and
C16:1 . It should be noted that the environment of the rhizobia has a profound effect on
the way that these bacteria interact [10]; the
Teplitski group found a response from C14 -HL
and C16 -HL in different growth media than
the one used in this experiment (Ty).
We are unsure of why S. meliloti would
produce so many AHLs without using all of
them. What is clear, however, is that the
primary AHLs seem to be 3-oxo-C14 , 3-oxoC16:1 , and C16:1 in that order. This is further supported by an experiment conducted
by Gao et. al (2007), in which a gene encoding for AiiA (which inactivates AHLs)
was inserted into Rm1021 [11]. The exper-
The main interest of our physics group in
S. meliloti has to do with the wide range of
AHLs it produces. As this group previously
did an experiment looking at the QS diffusion patterns of Vibrio fischeri, we thought
it would be interesting to see how an organism interacted with a wide range of autoinducers. Our hypothesis was that the shortchain AHLs would diffuse more quickly and
over a larger area than the long-chain AHLs,
and thereby control bacterial population behavior on a grand scale, while the long-chain
AHLs would control behavior at a more local range. However, in the process of learning more about this species, we have discovered that there is much left to understand
about the interesting quorum sensing network of these bacteria before diffusion experiments can be conducted.
Marketon et al. (2002) identified six AHLs
being produced by the Sin QSN: C8 -HL, C12 HL, 3-oxo-C14 , 3-oxoC16 : 1, C16:1 , and C18
8
Figure 10: SinI produces several AHLs; of these, only 3-oxo-C14 , C16:1 , and 3-oxo-C16:1 bind
to ExpR, which represses sinR activity, but promotes sinI activity when bound to AHL. SinR
constantly upregulates sinI, but is repressed by ExpR and promoted by PhoB. A positive
feedback loop with expR allows for the autoregulation of expR.
9
iment showed that without AiiA, 3-oxo-C16:1
was shown to be the most abundant QS signal, followed by C16:1 and C16 (in minimal
medium), and finally 3-oxo-C14 . With the
AiiA insertion, however, C16:1 was the only
AHL detected, with its signal only reduced by
90%. At these levels, this paper also showed
that even at these low concentrations of AHL,
the bacteria still swarmed, indicating that
this AHL allows for quorum sensing in even
the direst circumstances.
Not much is known about SinR; it is not
very soluble. McIntosh et al. (2009) showed
that eliminating sinR reduces sinI expression to background levels, while the overexpression of sinR increases sinI expression 8fold, implying that SinR is the primary promoter of sinI [10]. Looking at the fluorescence of the sinR mutant in our experiments,
we see the same thing; the fluorescence drops
down to the same levels as the sinI mutant.
However, looking at Figure 3, and considering the fact that sinI and sinR transcribe
in the same direction, there is the possibility that disrupting sinR also disrupts sinI.
As Figure 3 and Figure 5show, eliminating
expR increases psinI activity two-fold (compare the sinI mutant with the expR mutant
and sinI /expR double mutant). Since SinR
seems to be the biggest promoter of sinI [10],
this implies that ExpR represses sinR expression somehow, an idea suggested by McIntosh
et al. (2009).
Comparing MG32 with the other strains in
Figure 5, we see that while disrupting sinI
reduces psinI activity to background levels,
activity is regained with the addition of AHL.
This implies that ExpR regulates itself. Our
experiments show that without expR, we see
no response in psinI activity due to increased
AHL concentrations. Furthermore, looking
at Figure 6, we can see that once expR has
been disrupted, removing sinI has no effect
on the activity of psinI ; i.e., ExpR is necessary for AHL-dependent quorum sensing.
We see that phosphate has some clear effect
on the system. McIntosh et al. (2009) showed
that in phosphate-limiting conditions, sinR is
significantly induced [10]. This dependence of
sinR on phosphate levels is mediated through
the Pho regulon. One study found that there
are around 96 Pho boxes in the S. meliloti
genome [12]. Another study located one Pho
box upstream of sinR [13].
Putting all of this together, we can organize
a new circuit diagram for S. meliloti (Figure 4). The next step for this experiment
(once we know which autoinducers create a
response in psinI activity and what sinR is
doing) is to calculate the diffusion curves for
the quorum sensing response. As this paper
is being written, we are conducting an experiment that will test if SinR interacts with any
of the Sin AHLs; we have transformed a plasmid encoding sinR with the psinI-gfp gene
and one with expR and psinI-gfp into E. coli.
Using the BioTek machine, we will add AHL
and see if the fluorescence increases with any
of these AHLs. This will decouple sinR and
expR from the QS circuit, and we will be able
to see which AHLs, if any, are the ligands for
SinR and see if ExpR can bind to AHL without SinR. If our E. coli experiments show that
SinR does not interact with AHL, we will be
able to confirm our suspicions that ExpR, and
only ExpR, binds to the Sin AHLs.
5
Conclusion
Out of the five strains used in this experiment, only the psinI activity of MG32 responds to an increase in autoinducer concentration. The only autoinducers that induce this increased response are the 3-oxoC14 , C16:1 , and 3-oxo-C16:1 . ExpR appears to
repress sinR expression. SinR has the biggest
effect on psinI activity. These three autoinducers will be used to perform diffusion experiments once more data is collected.
10
6
Acknowledgements
The research conducted in this paper was
funded by an NSF grant: NSF DMR1156737.
7
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