Transcript of Quorum sensing in Sinorhizobium meliloti AHL-induced ...
Rupika Madhavan
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 com- municate 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 hypoth- esized 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
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 communca- tion; (2) synthases (which produce the chem-
ical signals); and (3) receptors (which bind to the autoinducers).
In ram negative bacteria, like Sinorhizobium meliloti, N -acyl
homoser- ine lactons (or AHLs) are the preferred au- toinducer. The
bacteria ”learn” about and respond to environmental changes by pre-
ducing, 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 sens- ing networks (QSNs) will
allow us to better manipulate the behavior of harmful or
bene-
ficial bacteria around us.
One of the best understood (QSNs) is that of the bacteria Vibrio
fischeri, the biolumi- nescent 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-oxo- C6, which leaves the cell. In a population of bacteria,
these AHLs increase in concen- tration and absorbed by the cells.
Once in the cell, an autoinducer attaches to the re- ceptor, LuxR.
The LuxR-AHL complex then causes the transcription of the luxICDABE
operon, which encodes for luciferase, the bi- oluminescent enzyme.
In addition, luxI is also encoded within this operon, further pro-
ducing 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 sim-
1
Figure 1: The well-known QSN of V. fischeri. LuxI is a synthase
that produces the autoin- ducer, 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 to-
gether bind to the ”lux-box” (an operon with several genes
including luxI and the gene for luciferase) and promote the
production of lu- ciferase, the bioluminescent protein. In addi-
tion, 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 scien- tist 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 bacte- ria who fix nitrogen for their host, the
alfalfa plant Medicago sativa [2]. The system is more complicated
than the V. fischeri. It is sus- pected that there are two QSNs at
work in S. meliloti : the Sin system and the hypothe- sized Mel
system [3]. As the Mel system has not been located, the Sin system
has been
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 autoin- ducers used by the system. The
position of sinR with respect to sinI on the genome (di- rectly
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 be- ing produced by the bacteria [6]. This wide range
of carbon-chain length is rather pecu- liar; 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 pro- duced by
SinI are actually used by the Sin QSN. Once this is known, the
bigger purpose of this experiment may be carried out: to dis- cover
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 pro-
vide 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
2
Figure 2: The arrangement of the quroum- sensing-related genes in
the S. meliloti chro- mosome. It has been predicted that the ExpR
binding site is in the intergenic region be- tween sinR and sinI.
The lux-box -like gene is predicted to be the binding site for
SinR.
change the response behavior of the sinI mu- tant strain when grown
in Ty medium.
2 Methods and Materials
The fluorescence and optical density curves were produced by using
a BioTek machine. Gen5 software was used to program the ma- chine
to take measurements of the optical density and fluorescence every
10 minutes for 48 hours. Polystyrene 48-well plates and 96- well
plates were primarily used in these ex- periments.
Five strains of bacteria were studied in these series of
experiments: 8530 (wild type), 1021 (expR mutant), MG32 (sinI mu-
tant), 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 strepto- mycin at 400
µg/mL and spectinomycin at 50 µg/mL) and then incubated at 30 C un-
til enough colonies were formed, but before to many colonies
started to merge. These agar plates were used for several
experiments,
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 di- luted 100-fold, returning them to an OD be- tween 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:
1. Originally, 400 µL of diluted bacteria were added to each well.
The AHLs (stored in ethyl acetate) were added di- rectly to the
bacteria in the well plates after the bacteria were placed in the
wells; however, as ethyl acetate kills bac- teria and the effects
from this were no- ticed, it was decided that a new method needed
to be used.
2. Phosphate buffer dilutions were used next: The AHLs in ethyl
acetate were evaporated in polycarbonate tubes, leav- ing just the
AHLs. Phosphate buffer was then added to the appropriate concen-
tration 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 un- til all the
liquid was gone from the wells. As the ethyl acetate is lighter and
evapo- rates more quickly, the water was a pro-
3
Strain Relevant Characteristics Source Rm8530 1021 expR+ SmR
Pellock et al., 2002 Rm1021 expR102 ::ISRm2011-1 expR, SmR Galibert
et al., 2001 MG32 8530 δsinI, SmR Gao et al., 2005 MG75 1021 δsinI,
SmR Gao et al., 2005 MG170 8530 δsinR, SmR 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 100- fold dilution
of colonies mixed with an- tibiotics was added to the wells. How-
ever, some wells were still affected nega- tively by the ethyl
acetate.
4. Final experiments were conducted by di- luting 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 evap- orated wells, and mixed with a pipette.
After adding both autoinducers and bacte- ria 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 phos- phate
buffer and Ty medium mixture. The second experiment used a 2 mM
phosphate buffer and Ty medium mixture. To add phos- phate to the
Ty medium, a 1 M solution of phosphate buffer at a pH of 7 was
diluted using Ty medium to the appropriate concen- trations. The
various strains were then grown in this mixture.
Furthermore, to test the purpose of SinR, the psinI-gfp plasmid and
a plasmid contain-
ing sinR were transformed into E. coli cells via electroporation.
The results of these ex- periments have not yet been delivered, so
fur- ther detail will be provided when the results come in.
3 Results
In our experiments, we see that only MG32 (sinI mutant) has any
response to a varia- tion 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 re- ceptor). The psinI activity in MG32 is
signif- icantly lower than in MG75, as shown in Fig- ure 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
mu- tant) and 1021 (expR mutant) have fluores- cence curves that
increase continuously de- spite the leveling-off of the optical
density.
The most significant impact on psinI ex- pression aside from
removing sinI itself was
4
0.5
1
1.5
2
2.5
3
3.5
C4
C6
3oC6
C8
C12
C14
3oC14
C16
C16:1
3oC16:1
C18
No AHL
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
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8 Optical Density of 1021 and MG75 with short−chain AHLs
Time (h)
e n s it y (
A U
1
1.5
2
2.5
3
3.5
5 Fluorescence of 1021 and MG75 with short−chain AHLs
Time (h)
F lu
o re
c o u n ts
)
−expR −expR/−sinI
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.
0 10 20 30 40 50 0
0.1
0.2
0.3
0.4
0.5
0.6
0.7
0.8
0.9
Time (h)
O p
1
1.5
2
2.5
3
3.5
4
4.5
Time (h)
F lu
o re
MG32
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
0.5
1
1.5
2
2.5
3
3.5
100 µM
10 µM
1 µM
100 nM
No AHL
Figure 4: sinI promoter activity of MG75 (sinI, expR double mutant)
with several long- chain AHLs, with AHLs prepared by way of method
4. MG75 does not respond to any of the AHLs.
No AHL C12 3oC14 3oC16 C16:1 C18 0
0.5
1
1.5
2
2.5
3
3.5
4
c o u n ts
)
100 µM
10 µM
1 µM
100 nM
No AHL
Figure 5: sinI promoter activity of MG32 (sinI mutant) with several
long-chain AHLs, with AHLs prepared by way of method 3. The
concentration of AHL needed for a re- sponse is extremely high (100
µM!)
C14 3oC14 C16 C16:1 3oC16 No AHL 0
0.5
1
1.5
2
2.5
3
3.5
1.5 µM
150 nM
15 nM
1.5 nM
Figure 9: sinI promoter activity of MG32 (sinI mutant) with
long-chain AHLs, with AHLs prepared by way of method 4. The
concentration of AHL needed for a response is lower (1.5 µM).
removing sinR, with around a 3-fold decrease 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 mu- tant and expR, sinI double mutant had
the same fluorescence curves (Figure 6). As this result was
surprising, PCR was done to con- firm that these two strains were
in fact differ- ent; the results showed that they were.
As the experimental technique improved, the concentration of AHL
required to induce a response decreased significantly (compare
Figure 5 and Figure 9). The final experiments show a response to
concentrations around 150 nM.
When the phosphate concentration was in- creased to 2 mM, no effect
was detected; how- ever, when the phosphate concentration was
increased to 100 mM, a significant change in the behavior of the
bacteria was detected. The fluorescence curves had an interesting
pattern (Figure 8).
7
0.1
0.2
0.3
0.4
0.5
0.6
Time (h)
O p
2
3
4
5
6
7
8
Time (h)
F lu
o re
No AHL
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
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 diffu- sion patterns of Vibrio
fischeri, we thought it would be interesting to see how an organ-
ism interacted with a wide range of autoin- ducers. Our hypothesis
was that the short- chain AHLs would diffuse more quickly and over
a larger area than the long-chain AHLs, and thereby control
bacterial population be- havior on a grand scale, while the
long-chain AHLs would control behavior at a more lo- cal range.
However, in the process of learn- ing more about this species, we
have discov- ered that there is much left to understand about the
interesting quorum sensing net- work of these bacteria before
diffusion exper- iments 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
[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 environ- ment 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-oxo- C16:1, and C16:1 in that order. This
is fur- ther supported by an experiment conducted by Gao et. al
(2007), in which a gene en- coding for AiiA (which inactivates
AHLs) was inserted into Rm1021 [11]. The exper-
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 sig- nal, 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 expres- sion
to background levels, while the overex- pression of sinR increases
sinI expression 8- fold, implying that SinR is the primary pro-
moter of sinI [10]. Looking at the fluores- cence 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 consider- ing the fact that sinI and sinR
transcribe in the same direction, there is the possibil- ity that
disrupting sinR also disrupts sinI. As Figure 3 and Figure 5show,
eliminating expR increases psinI activity two-fold (com- pare 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 expres- sion 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 neces- sary 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 (Fig- ure 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 exper- iment that will test if SinR
interacts with any of the Sin AHLs; we have transformed a plas- mid
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 with- out 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 experi- ment, only the psinI
activity of MG32 re- sponds to an increase in autoinducer con-
centration. The only autoinducers that in- duce this increased
response are the 3-oxo- C14, C16:1, and 3-oxo-C16:1. ExpR appears
to repress sinR expression. SinR has the biggest effect on psinI
activity. These three autoin- ducers will be used to perform
diffusion ex- periments once more data is collected.
10
6 Acknowledgements
The research conducted in this paper was funded by an NSF grant:
NSF DMR- 1156737.
7 References
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