SdiA, an N-Acylhomoserine Lactone Receptor, Becomes
Active during the Transit of Salmonella enterica through
the Gastrointestinal Tract of Turtles
Jenee N. Smith1., Jessica L. Dyszel1., Jitesh A. Soares1, Craig D. Ellermeier2¤a, Craig Altier3¤b, Sara D.
Lawhon4, L. Garry Adams4, Vjollca Konjufca5¤d, Roy Curtiss III5¤c, James M. Slauch2, Brian M. M. Ahmer1*
1Department of Microbiology, The Ohio State University, Columbus, Ohio, United States of America, 2Department of Microbiology and College of Medicine, University of
Illinois, Urbana, Illinois, United States of America, 3Department of Population Health and Pathobiology, North Carolina State University, Raleigh, North Carolina, United
States of America, 4Department of Veterinary Pathobiology, College of Veterinary Medicine, Texas A&M University, College Station, Texas, United States of America,
5Department of Biology, Washington University, St. Louis, Missouri, United States of America
Background: LuxR-type transcription factors are typically used by bacteria to determine the population density of their own
species by detecting N-acylhomoserine lactones (AHLs). However, while Escherichia and Salmonella encode a LuxR-type AHL
receptor, SdiA, they cannot synthesize AHLs. In vitro, it is known that SdiA can detect AHLs produced by other bacterial species.
Methodology/Principal Findings: In this report, we tested the hypothesis that SdiA detects the AHL-production of other
bacterial species within the animal host. SdiA did not detect AHLs during the transit of Salmonella through the gastrointestinal
tract of a guinea pig,a rabbit,a cow,5 mice, 6 pigs, or 12chickens. However,SdiA was activated during the transit of Salmonella
through turtles. All turtles examined were colonized by the AHL-producing species Aeromonas hydrophila.
Conclusions/Significance: We conclude that the normal gastrointestinal microbiota of most animal species do not produce
AHLs of the correct type, in an appropriate location, or in sufficient quantities to activate SdiA. However, the results
obtained with turtles represent the first demonstration of SdiA activity in animals.
Citation: Smith JN, Dyszel JL, Soares JA, Ellermeier CD, Altier C, et al. (2008) SdiA, an N-Acylhomoserine Lactone Receptor, Becomes Active during the Transit of
Salmonella enterica through the Gastrointestinal Tract of Turtles. PLoS ONE 3(7): e2826. doi:10.1371/journal.pone.0002826
Editor: Frederick M. Ausubel, Massachusetts General Hospital, United States of America
Received June 23, 2008; Accepted July 7, 2008; Published July 30, 2008
Copyright: ? 2008 Smith et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits
unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
Funding: B.M.M.A. was supported by Public Health Service grants AI0500002 and AI073971. J.M.S. and C.D.E. were supported by grant 00-25 from the Roy J.
Carver Charitable Trust and by Public Health Service grant AI63230. S.D.L was supported by Public Health Service grant 5K08AI060933-02. L.G.A. was supported by
USDA NRICGP grant 2002-35204-11624. C.A. was supported by the National Research Initiative of the USDA Cooperative State Research, Education and Extension
Service, grant number 2005-35201-16270. R.C. was supported by Public Health Service grant AI24533. The funders had no role in study design, data collection and
analysis, decision to publish, or preparation of the manuscript.
Competing Interests: The authors have declared that no competing interests exist.
* E-mail: firstname.lastname@example.org
¤a Current address: Department of Microbiology, The University of Iowa, Iowa City, Iowa, United States of America
¤b Current address: Department of Population Medicine, Cornell University, Ithaca, New York, United States of America
¤c Current address: The Biodesign Institute, Arizona State University, Tempe, Arizona, United States of America
¤d Current address: Department of Pathology and Immunology, Washington University School of Medicine, St. Louis, Missouri, United States of America
. These authors contributed equally to this work.
The functions of more than half of the genes present in
Escherichia coli and Salmonella enterica serovar Typhimurium
(hereafter referred to as S. Typhimurium) have never been
determined, despite decades of genetic screens and selections. This
failure is hypothesized to result from a lack of gene expression or
gene function in the laboratory environment. A major component
of the natural environment missing from most laboratory
experiments is the presence of other microbial species. E. coli
and S. Typhimurium colonize and propagate in the intestines of
animals in the presence of large numbers of other microbial
species. We have previously identified genes of S. Typhimurium
that are expressed specifically in the presence of signaling
molecules produced by other microbes [1–4]. These seven genes
(two loci) are activated by SdiA, a member of the LuxR family of
transcription factors, in response to N-acylhomoserine lactones
(AHLs) produced by other species.
The use of LuxR homologs to detect the AHL production of
other species is unusual. The typical function of LuxR homologs is
‘‘quorum sensing’’, a phenomenon by which bacteria sense their
own population density [5–7]. Many Gram-negative bacteria use
this information to regulate the production of host colonization
factors. For instance, Vibrio fischeri uses this information to regulate
genes that play a role in colonization of the squid, Euprymna scolopes
[8–10]. Numerous plant and animal pathogens use quorum
sensing to regulate host interaction genes [11,12]. Presumably, this
prevents the bacteria from alerting host immune responses before
a population sufficient to overcome host defenses has been
PLoS ONE | www.plosone.org1 July 2008 | Volume 3 | Issue 7 | e2826
AHLs are synthesized using enzymes of the LuxI or LuxM
family [13–18]. AHLs vary widely in the length of their acyl chain
(from four to 18 carbons) and can be modified at the 3-carbon
position to have a carbonyl or hydroxyl group. Each LuxI
homolog produces predominantly a single AHL variant, along
with smaller quantities of closely related variants . The LuxR
family member of a species can detect nanomolar concentrations
of the AHL produced by that species, providing some species
specificity to the system. However, LuxR homologs can often
detect other related AHLs with lower sensitivity .
Salmonella encodes a LuxR homolog, SdiA, but does not encode
an AHL synthase [3,4]. Instead, SdiA responds to AHLs produced
by other microbial species [1,2]. SdiA detects a much wider range
of AHLs than other LuxR homologs, although with varying
sensitivities. The most potent AHL is 3-oxo-octanoyl-homoserine
lactone (oxoC8) and SdiA responds to this molecule at
concentrations of 1 nM or higher [3,21]. The closely related
AHL, 3-oxo-hexanoyl-homoserine lactone (oxoC6) can be detect-
ed at concentrations above 5 nM, and at 50 nM SdiA responds to
oxoC4, oxoC10, oxoC12, and the unmodified AHLs C6 and C8.
At concentrations above 1 mM SdiA responds to C4, C10, and
C12 [3,21]. The three-dimensional structure of the N-terminus of
E. coli SdiA bound to C8 has been determined using NMR,
confirming that detection of AHLs by SdiA is direct .
Upon detection of AHL, SdiA activates the expression of two srg
(SdiA regulated gene) loci: the rck operon located on the S.
Typhimurium virulence plasmid, pSLT, and the srgE gene located
in the chromosome at 33.6 centisomes [1,2]. The rck operon is
directly downstream of the pef operon that encodes plasmid-
encoded fimbriae . These fimbriae play a role in adhesion to
the small intestine of mice . The rck operon contains six genes:
pefI, srgD, srgA, srgB, rck, and srgC [3,23]. PefI is a regulator of the
upstream pef operon . SrgD is a putative transcription factor
with a helix-turn-helix motif of the LuxR family, but its target
genes have not been identified [3,23]. SrgA is a DsbA homolog
that catalyzes disulfide bond formation of the Pef fimbrial subunit
and other periplasmic proteins [26,27]. SrgB is a lipoprotein of
unknown function . Rck is an 8-stranded b-barrel protein
localized to the outer membrane . This protein has dual
functions. The first function provided its name: resistance to
complement killing [29,30]. More specifically, Rck prevents the
polymerization of complement component C9 on the bacterial
surface . Second, Rck serves as an adhesin to fibronectin and
laminin . The last gene in the rck operon, srgC, is a regulator of
the AraC-family of transcription factors whose target genes are
unknown . The second srg locus consists of a single gene, srgE,
which is predicted to encode a protein containing a coiled-coil
domain [2,4]. As with the rck operon, srgE is not present in E. coli
and was probably acquired horizontally by Salmonella. Despite the
regulation of putative virulence genes by SdiA, we have previously
reported that an sdiA mutant of S. Typhimurium is not attenuated
in mouse, chicken, or cow models of infection .
The mammalian intestinal community is comprised of approx-
imately 800 microbial species . We hypothesized that
members of this community utilize AHL-type quorum sensing
and that the presence of these AHLs would provide an effective
way for S. Typhimurium to detect the intestinal environment
[1,4]. In this report we have tested this hypothesis. S. Typhimur-
ium did not detect AHLs during transit through the intestinal tract
of several species of animals, indicating that the normal microbiota
of these hosts did not produce AHLs of the correct type or at a
sufficient concentration to activate SdiA. However, SdiA was
active during transit through turtles colonized by the AHL-
producing organism, Aeromonas hydrophila.
Construction and testing of RIVET strains
To test the hypothesis that Salmonella uses SdiA to detect the
AHL production of other microorganisms within the gastrointes-
tinal tract, we used the RIVET method (Recombination-based In
Vivo Expression Technology) to record SdiA activity in vivo [34–
37]. We constructed a srgE-tnpR-lacZY transcriptional fusion in a
strain that carries a res1-tetRA-res1 cassette elsewhere in the
chromosome (strains are listed in Table 1). The tnpR-lacZY fusion
was placed immediately after the srgE stop codon so that srgE
remains functional. The tnpR gene encodes a resolvase that
catalyzes a site-specific recombination event at res1 target
sequences. Thus any condition or event that induces expression
of srgE-tnpR-lacZY is permanently recorded in the genome by the
deletion of the tetRA cassette. The deletion event is detected by
screening the bacteria for a loss of tetracycline resistance.
To obtain the optimal fusion sensitivity for the srgE promoter
three versions of this strain were constructed, each with a different
tnpR ribosome binding site . Once these strains were
constructed, an sdiA1::mTn3 mutation was transduced into each
of them using phage P22HTint to create isogenic sdiA mutant
controls. We have previously reported that even in the presence of
AHLs, SdiA is not active when Salmonella is grown on LB agar
plates . However, SdiA is slightly active when grown in liquid
broth, and is highly active when grown in motility agar .
Therefore, the functionality of the RIVET strains was tested under
these same conditions. Either synthetic AHL (1 mM oxoC6) or the
solvent control, acidified ethyl acetate (EA), was added to each
medium. After overnight growth in liquid medium at 37uC, serial
dilutions were plated on agar plates to obtain individual colonies.
Bacteria were recovered from motility agar by stabbing the agar
with a sterile toothpick and streaking to isolation. The resulting
colonies were then screened for loss of tetracycline resistance. The
strains containing mutated tnpR ribosome binding sites showed no
resolution under any condition and were not used further. The
strains containing the wild-type tnpR ribosome binding site showed
resolution and are the strains used for the remainder of the
experiments in this report (JNS3206 is sdiA+and JNS3226 is
sdiA::mTn3, hereafter referred to as the RIVET strains). In media
containing AHL, resolution was observed in 23% of wild-type
bacteria recovered from motility agar, 2.5% of wild-type bacteria
recovered from broth, and from 0.6% of bacteria grown on solid
agar. No resolution was observed under any growth condition in
the absence of AHL or from sdiA mutant colonies. The reason that
only 23% of colonies resolve after overnight growth in vitro is not
known, but indicates that the srgE gene is not highly expressed, or
that truly optimal conditions have not yet been discovered.
Regardless, resolution in the sdiA+bacteria but not the sdiA mutant
bacteria indicates SdiA activity. The observation that the highest
resolution was observed after growth in motility agar is similar to
what was observed previously using a lacZY fusion to srgE .
Therefore, expression of the srgE-tnpR-lacZY fusion can be
recorded as a percentage of tetracycline susceptible colonies and
this expression is dependent upon sdiA and AHL.
SdiA is not active during the transit of Salmonella
through mice, chickens, pigs, a rabbit, a guinea pig, or a
To determine if SdiA is active during the transit of Salmonella
through the gastrointestinal tract, the wild-type and sdiA mutant
RIVET strains were mixed together in a 1:1 ratio and orally
administered to six CBA/J mice. These mice are resistant to
Salmonella infection and provide a long term model of intestinal
SdiA Activation in Turtles
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colonization and persistence . Inoculating a mixture of the
wild-type and sdiA mutant allows a comparison of the resolvase
activity of these two strains within the gut of the same animal.
Fecal samples were collected, homogenized and plated on XLD
indicator plates containing kanamycin to recover the Salmonella
RIVET strains. These colonies were confirmed to be Salmonella by
their black color on the XLD indicator plates and their sensitivity
to phage P22. No Salmonella were recovered from the feces of
uninfected mice. Each recovered colony was then screened for
ampicillin and tetracycline resistance. Wild-type and sdiA mutant
are distinguished by the ampicillin sensitivity of the colony (the
sdiA1::mTn3 mutation encodes ampicillin resistance) and resolvase
activity is detected by the loss of tetracycline resistance. Sufficient
numbers of bacteria for screening were recovered from five of the
six mice. Only one of 717 colonies recovered from the five mice
was tetracycline susceptible, indicating that SdiA was not active
during transit through the mice (Table 2). Consistent with the lack
of SdiA activity, both the wild-type and sdiA mutant were
recovered in ratios that were not significantly different from the
ratio inoculated indicating that the sdiA mutation did not confer a
fitness phenotype in these mice (Table 2 and Figure 1).
The lack of SdiA activity in the mouse gut was surprising, but
since compounds that activate AHL biosensor strains have been
chemically extracted from the bovine rumen , we hypothe-
sized that the presence of AHLs might differ among animal
species. Therefore, the RIVET strains were orally administered to
cows, pigs, rabbits, guinea pigs, and chickens, as described in the
Materials and Methods. For some individual animals, we failed to
recover the RIVET strains from their feces. However, the RIVET
strains were recovered from the feces of one rabbit (of two), one
guinea pig (of four), one cow (of three), six pigs (of six), and 12
chickens (of 12). No SdiA activity was observed during the transit
of Salmonella through any of these animals as indicated by the fact
all recovered RIVET strains were tetracycline resistant (Table 2).
Consistent with the lack of SdiA activity, the sdiA mutant did not
appear to have a significant fitness defect during the transit
through any of the animals (Table 2 and Figure 1). It should be
noted that the sdiA mutant actually faired slightly better than the
wild-type during transit through chicks. The sdiA+:sdiA2ratio was
57:43 at inoculation while it was 48:52 at recovery. This is a
competitive index of 1.4. While this result was statistically
significant, competitive indices of even 3 to 5 are considered
SdiA is active during the transit of Salmonella through
Having determined that Salmonella did not detect AHLs in the
gastrointestinal tracts of the birds and mammals tested above, we
decided to test a reptile that is commonly associated with Salmonella,
the turtle. Preliminary experiments indicated that SdiA was indeed
activated during transit through turtles (not shown). To determine if
AHL-producing bacteria could be recovered from turtles, cloacal
swab samples were obtained from a group of eight turtles and the
recovered material was plated on several media in order to culture a
wide variety of bacterial species (LB, blood agar, XLD, lactose
MacConkey). The recovered colonies were then tested in a cross-
streak assay against the bioluminescent SdiA activity reporter strains
14028/pJNS25 (sdiA+srgE-luxCDABE) and BA612/pJNS25 (sdiA2
srgE-luxCDABE) . Luminescence of the wild-type reporter but not
the sdiA mutant at the junction indicates SdiA activity . Bacteria
that can activate SdiA in the cross-streak assay were found in each of
the eight turtles (Figure 2). Eighteen isolates were submitted to the
a Dade Microscan WalkAway 96si. All 18 isolates were found to be
After determining that all eight of the turtles contained
Aeromonas hydrophila, the Salmonella RIVET strains were orally
administered to these turtles and cloacal swab samples were
collected over time. When the recovered colonies from all turtles at
all time points were screened, it was observed that the ratio of
wild-type to sdiA mutant recovered was changed during transit
Table 1. Strains and plasmids used.
Strain or plasmid Genotype Source or reference
14028Wild type Salmonella enterica serovar Typhimurium American Type Culture Collection
BA61214028 sdiA1::mTn3 (ampr) 
JNS3206 JS246 srgE10-tnpR-lacZY (kanr) This study
JNS3226 JS246 srgE10-tnpR-lacZY sdiA1::mTn3 (kanrampr)This study
JS198 LT2 metE551 metA22 ilv452 trpB2 hisC527(am) galE496 xyl-404 rpsL120 flaA66 hsdL6 hsdSA29
JS24614028 zjg8103::res1-tetRA-res1 
S17-1lpirE. coli recA pro hsdR ,RP4-2-tet::Mu-1kan::Tn7. lpir 
W3110E. coli K-12 F2IN(rrnD-rrnE)1 
pCE71FRT-tnpR-lacZY thisoriR6K (kanr). Contains wild type tnpR Shine Dalgarno. FRT orientation B.
pCE73 FRT-tnpR-lacZY thisoriR6K (kanr). Contains mutated tnpR Shine Dalgarno mut168. FRT orientation B. 
pCE75 FRT-tnpR-lacZY thisoriR6K (kanr). Contains mutated tnpR Shine Dalgarno mut135. FRT orientation B.
pCP20cI857 lPRflp pSC101 oriTS (amprcamr) 
pJNS25PsrgE-luxCDABE p15A ori (tetr) 
pKD4FRT-kan-FRT oriR6K (ampr) 
pKD46PBADgam bet exo pSC101 oriTS (ampr)
SdiA Activation in Turtles
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through the turtles (a 49:51 ratio inoculated, a 56:44 ratio
recovered, Table 2 and Figure 1). However, while statistically
significant, this is only a 1.3-fold difference in competitive index
that is not likely to be biologically relevant, especially when looking
at the time course plotted in Figure 1B. Therefore, we do not feel
that the sdiA mutant of Salmonella serovar Typhimurium has a
significant fitness phenotype in turtles. However, the striking result
is that the resolution observed in wild-type Salmonella was greater
than in the sdiA mutant, indicating that SdiA was active during the
transit of Salmonella through these turtles (Table 2 and Figure 3).
This is the first observation of SdiA activity in vivo.
To test the hypothesis that Aeromonas hydrophila is indeed the
organism responsible for activation of SdiA during the transit of
Salmonella through turtles, we hoped to obtain a group of turtles
that lacked AHL-producing bacteria. Half of these turtles would
then be inoculated with Aeromonas hydrophila and half would be
inoculated with LB prior to performing the RIVET experiment.
Unfortunately, the next two groups of turtles that we obtained (one
group of 12 and another group of 17) all had Aeromonas in their
feces. Next, we reasoned that newly hatched turtles would be less
likely to have picked up Aeromonas from the environment and we
would have a better chance of obtaining Aeromonas-free animals if
Table 2. SdiA activity and fitness during transit through different animal species.
Mean of the
5 mice 52%717 72%0.41
1 guinea pig 45% 621 54%0.70
1 rabbit45% 70950%0.82
45%1349 53% 0.73
20.105+/20.1760 0% 0%
89%1000 96% 0.34
57% 245448% 1.44 0.176+/20.150*
0 0% 0%
49%6793 56%0.7571525.9% 3.0%
1Animals were inoculated with a 1:1 mixture of the wild-type and sdiA mutant Salmonella RIVET strains (a total of 9.76108cfu for the mice, 6.06109cfu for the guinea
pigs and rabbits, 1.86109cfu for the pigs, 1.361011cfu for the calves, 7.06109cfu for the chicks, and 3.16109cfu for turtles).
2The inoculum was plated for single colonies and screened for ampicillin resistance to determine the actual percent wild-type inoculated.
3The ‘‘Total colonies screened’’ represents a compilation of all colonies recovered (sum of all time points) from all animals of each species. The colonies were recovered
by plating fecal samples, unless noted otherwise, on XLD-kan and then screened for ampicillin and tetracycline resistance. Wild-type and sdiA mutant Salmonella are
differentiated by ampicillin resistance and resolution of the res1-tetRA-res1 cassette is indicated by tetracycline sensitivity.
4The competitive index equals the output ratio (cfu of mutant/cfu of wild type) divided by the input ratio (cfu of mutant/cfu of wild type). A competitive index of 1.0
indicates that the wild-type and sdiA mutant had equal fitness during transit through the animal. The log of the competitive index represents a normal distribution so
the mean of this value was calculated. A value of zero indicates that the wild-type and sdiA mutant had equal fitness during transit through the animal. For those
animal species in which colonies were recovered from more than one animal, the standard deviation is shown. The wild-type was statistically more fit than the sdiA
mutant in turtles, while the sdiA mutant was more fit than wild-type in chicks (student’s t test, p,0.05). However, the differences are so small (competitive index of less
than 3) that they are not likely to be biologically significant. For those animal species where only one individual shed colonies, the standard deviation could not be
calculated, but a competitive index that is less than 3-fold different than 1.0 is unlikely to be biologically significant (0.33 to 3.0).
5Of the total screened, 700 colonies were from fecal samples, 582 were from the ileum, and 67 were from the cecum.
6Of the total screened, 782 colonies were from fecal samples, 150 were from the ileum, 2 were from the cecum, and 66 were from the Peyer’s patches.
7All colonies from the chicks were obtained from the cecum and distal large intestine. No colonies were from fecal samples.
8All colonies from the turtles were obtained from cloacal swabs. These data were also used to generate Figures 1 and 3.
*Statistically significant fitness difference between wild-type and sdiA mutant (p,0.05).
Figure 1. Fitness of the sdiA mutant in different animal species. (A) The log of the competitive index for each individual animal is plotted. The
mean is indicated by the large horizontal line while the 95% confidence intervals are indicated by the smaller horizontal lines. A value of zero
indicates that the wild-type and the sdiA mutant were of equal fitness during transit through the animal. (*) The wild-type was statistically more fit
than the sdiA mutant during transit through turtles, while the sdiA mutant was statistically more fit than the wild-type during transit through chicks
(p,0.05). (B) Fitness of the sdiA mutant in turtles over time. The data from (A) is separated into time points. (*) The wild-type was statistically more fit
than the sdiA mutant on day 7 (p,0.05).
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we ordered hatchlings. A group of 20 hatchlings was obtained and,
unfortunately, all of these animals were colonized with Aeromonas.
In a final attempt at obtaining Aeromonas-free turtles, we ordered 30
turtle eggs and hatched them under sterile conditions. More
specifically, we placed the eggs on sterile vermiculite until
hatching, and then placed them in sterile cages containing sterile
water. The hatchlings were fed standard non-sterile turtle food to
promote the development of a normal microbiota (the food was
tested and shown to be free of Aeromonas). Remarkably, all of the
hatchlings were found to have Aeromonas in their feces. We are not
sure if the Aeromonas is transmitted from the mother to the embryo
during the development of the egg, or if there was Aeromonas on the
egg shell, or some other source of environmental contamination,
but we were not able to obtain turtles that lacked Aeromonas.
Next, we attempted to clear the Aeromonas from adult turtles
using Amikacin as described in the Materials and Methods. This
was not successful. Aeromonas numbers would drop temporarily but
the organism was never completely eradicated. Therefore, we can
only conclude that SdiA was indeed activated during the transit of
S. Typhimurium through turtles, and that the probable source of
the AHLs was Aeromonas hydrophila.
We have determined that SdiA of S. Typhimurium is activated
in turtles, but not in mice, pigs, chickens, a cow, a guinea pig, and
a rabbit. It appears that the normal microbiota of these particular
animals did not produce AHLs, at least not in a location or at a
concentration sufficient to activate SdiA. The lack of SdiA activity
during the transit of Salmonella through the cow was particularly
surprising because there is a report of chemical extraction of AHLs
from the rumens of six of eight bovines . It is certainly possible
that AHLs are a normal component of the bovine rumen and
certain other gastrointestinal regions of particular animals. We
may not have observed SdiA activity in our bovine experiment (or
other experiments) due to the age or the diet of the animals. Our
calves were young, only three to four months old. While the
animals have a functioning rumen at this point, AHL-producing
species may not be part of the ecosystem at this age. The ages of
the animals in the Erickson study were not reported. Diet may also
play a role. Our calves were fed free choice grass and hay and a
pelleted feed that was 12% protein and 3% fat that is primarily
comprised of corn and cottonseed meal. The Erickson study used
four different combinations of alfalfa hay and barley, but did not
observe a statistically significant correlation between diet and
AHLs. Further study will be required to determine the
circumstances in which AHL production occurs in the bovine
Figure 2. Cross-streak assay demonstrates that the S. Typhi-
murium srgE gene is activated in an sdiA-dependent fashion in
the vicinity of Aeromonas hydrophila. (A) A turtle isolate of
Aeromonas hydrophila was struck across the bottom of an LB agar
plate. On the left side, a wild-type S. Typhimurium strain carrying a srgE-
luxCDABE fusion (14028/pJNS25) was struck in duplicate and perpen-
dicular to the Aeromonas. On the right side, an isogenic sdiA mutant
was struck in the same fashion (BA612/pJNS25). Light intensity is
pseudocolored with blue being the weakest and red being the most
intense. (B) Same as panel A except that an AHL-negative organism was
struck across the bottom of the plate (E. coli K-12 strain W3110).
Figure 3. Resolution over time in eight turtles that were culture positive for Aeromonas hydrophila. A 1:1 mixture of the sdiA+ and
sdiA::mTn3 RIVET strains (JNS3206 and JNS3226, respectively) were orally administered to the eight turtles (3.16109 total cfu). Plating of cloacal swabs
was used to recover the strains at time points. Each colony recovered was then screened for ampicillin resistance to determine if it is sdiA+ or
sdiA::mTn3, and tetracycline resistance to determine if it had resolved. The percentage of sdiA+ ($) and sdiA::mTn3 (&) resolution is plotted over
time. Turtles 2, 3, and 8 were euthanized before the final time point.
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It is possible that AHLs were indeed synthesized by the normal
microbiota of some of the animals in this study, but that the AHLs
were rapidly degraded by either host enzymes or enzymes
produced by other microbes. While AHL-degrading enzymes
have not been reported in the intestine, they have been found in
soil and in the termite hindgut, and it would not be surprising to
find them in the intestine [41–43]. While enzymatic degradation
may occur, the Salmonella SdiA activity observed in turtles infected
with Aeromonas argues that AHLs can survive the turtle intestinal
environment in a concentration sufficient to activate SdiA. It will
be interesting to determine the half-life of AHLs in the intestines of
various animal species and to determine the distances over which
signaling can occur.
The SdiA activity observed during the transit of Salmonella
through turtles was likely due to the AHL production of Aeromonas
hydrophila. This organism is a major fish pathogen that causes
septicemia and Ulcerative Disease Syndrome and is among the
most troublesome of bacterial pathogens in aquaculture [44–46].
It also causes gastroenteritis, peritonitis, and meningitis in humans
. Many of the Aeromonas virulence factors are regulated by the
AhyR/AhyI quorum sensing system in which AhyI is an AHL
synthase. When a high population density is achieved at a focus of
infection, a positive feedback loop is initiated in which AhyR
activates the ahyI gene, thus making more AHL signaling
molecules to activate AhyR. In this way, the population of
organisms can simultaneously express virulence factors via AhyR
and attack the host [48–50].
The activation of the srgE-tnpR fusion during the transit of
Salmonella through turtles was dependent on the sdiA gene. This is
consistent with in vitro cross-streak assays in which Salmonella can
detect Aeromonas in an sdiA-dependent fashion (Figure 2). However,
the fitness phenotype in turtles was extremely small. When a 49:51
mixture of sdiA+and sdiA2RIVET strains was given to turtles, a
56:44 mixture was recovered in the feces indicating that the sdiA
mutant faired slightly worse than the wild-type in this environ-
ment. This is a competitive index of 0.75 which is very close to 1.0.
While this result was statistically significant, it is not considered
significant in the field of bacterial pathogenesis where even 3 to 5-
fold differences in competitive indices are considered minimal.
Given that S. Typhimurium is not a serovar that is typically
associated with turtles, S. Typhimurium may not have sdiA-
regulated genes that play a role in this animal. It would be
interesting to determine if sdiA mutants of turtle-associated
serovars such as Muenchen, Arizonae, or Newport [51–53] have
a phenotype in Aeromonas-infected turtles. While not typically
associated with turtles, S. Typhimurium is an exceptionally broad
host-range pathogen, causing gastroenteritis in humans, cattle,
pigs, poultry, horses, and sheep. It is quite common in humans,
accounting for 26% of all U.S. isolates . It will be interesting to
determine if serovar Typhimurium detects AHL-producing
pathogens in any of these other hosts and if sdiA confers a fitness
advantage in any of these coinfection situations.
Materials and Methods
Bacterial strains and media
All bacterial strains and plasmids used are listed in Table 1. E.
coli, Salmonella, and Aeromonas were grown in Luria-Bertani (LB)
broth at 37uC unless otherwise indicated (EMD chemicals,
Gibbstown NJ). LB agar plates and LB motility agar were made
by adding agar to 1.2% or 0.25%, respectively (EMD chemicals).
For recovery of Salmonella and Aeromonas from animal tissues or
feces, xylose-lysine-deoxycholate (XLD) agar plates were used
(EMD chemicals). The antibiotics ampicillin (amp), kanamycin
(kan), or tetracycline (tet) were added to concentrations of 150,
100, and 20 ug/ml respectively, when needed (Sigma-Aldrich, St.
Louis MO). N-(3-oxo-hexanoyl)-L-homoserine lactone (oxoC6)
was obtained from Sigma-Aldrich and dissolved in ethyl acetate
that had been acidified by the addition of 0.1 ml glacial acetic acid
per liter (EA) . The stock concentration of oxoC6 was 1 mM
and it was used at a final concentration of 1 mM. Solvent controls
were performed by using EA alone at 0.1%. All plasmids were
prepared from JS198, a r2m+Salmonella, before electroporation
into restriction-proficient Salmonella strains .
Construction of srgE-tnpR-lacZY fusions
A FRT site was inserted between the stop codons and the
transcription terminator of the srgE gene using l Red recombination
with PCR primers BA723 (TTGTATGGGGCATATAAAAA-
GAAATAGTAACATATGAATATCCTCCTTAG) and BA908
CTGGAGCTGCTTC). The first 30 nucleotides of BA723 bind the
end of the srgE gene (nt 15344-15315 of Genbank accession number
AE008767), pointing downstream, and ending with the two stop
codons which are underlined. (The srgE gene has two adjacent stop
codons.) The next 20 nucleotides match the Priming Site 2 sequence
of Datsenko and Wanner’s mutagenesis system . The first 30
nucleotides of BA908 bind downstream of the srgE gene (nt 15275-
15304 of Genbank accession number AE008767), pointing up-
stream. The next 20 nucleotides match the Priming Site 1 sequence
of Datsenko and Wanner’s mutagenesis system .The insertion of
the antibiotic resistance marker was verified using PCR with two
different primer sets, each containing a primer that binds within the
antibiotic resistance gene and a second that binds either upstream or
downstream of the intended insertion site. The antibiotic resistance
cassette was then removed using the temperature sensitive plasmid
pCP20 carrying the FLP recombinase . Kanamycin susceptible
strains containing pCP20 were electroporated with the pCE71,
pCE73,andpCE75 suicide plasmidsfollowedbyselection onLBkan
platesat37uC.Thesesuicideplasmidsencodea FRT sitefollowed by
a promoterless tnpR-lacZY . A Flp-mediated site-specific
recombination recombines the FRT site on the suicide vector with
vector into the chromosome . Growth of the resulting colonies at
37uC was sufficient to cure pCP20 and this was confirmed by
screening for ampicillin sensitivity. Integration of the tnpR-lacZY
fusion plasmid into the srgE10-FRT site was confirmed using PCR.
Once the srgE10-tnpR-lacZY fusion strains were confirmed, an
sdiA1::mTn3 mutation was introduced from BA612 by transduction
using P22HTint .
Female CBA/J mice (8–10 weeks old) were obtained from
Jackson Laboratories (Bar Harbor, ME). Female Hartley albino
guinea pigs (retired breeders) and female New Zealand albino
Rabbits (250 g) were obtained from Charles River Laboratories
(Wilmington, MA). All animals were caged separately. Red-eared
slider turtles (Trachemys scripta elegans) of mixed sex were obtained
from Concordia Turtle Farms (Wildsville, LA). Turtles were kept
in individual cages containing water and a basking support. The
cages, water, and basking supports were sterilized and changed
daily. All experiments performed with mice, guinea pigs, rabbits,
and turtles were performed at Ohio State University with IACUC
approved protocol #2006A0037.
Six domestic female pigs (30–35 pounds) were obtained from
North Carolina State University Farms. Pigs were housed three to
a stall and food and water were provided ad libitum. This
SdiA Activation in Turtles
PLoS ONE | www.plosone.org6 July 2008 | Volume 3 | Issue 7 | e2826
experiment was performed at North Carolina State University
with IACUC approved protocol #02-018-B.
Three Holstein calves, 2 male and 1 female, were obtained from
Texas A&M University Farms. Calves were three to four months
old and weighed from 190–300 lbs. Calves were housed together
and food (a pelleted feed that was 12% protein and 3% fat that is
primarily comprised of corn and cottonseed meal) and water were
provided ad libitum. Calves were also allowed to graze. The calf
experiment was performed at Texas A&M University with AUP
approved protocol #2003-178.
Chicks were obtained as Fertilized SPAFAS Specific pathogen-
free layer eggs (Spafas, Inc., (Roanoke, IL)). The hatchlings were
housed in a single Horsefall isolator with 45–50% humidity under
constant light at 35uC. Food and water were provided ad libitum.
Salmonella infections were performed one week after hatching at
Washington University under Animal Welfare Assurance number
All animals (except the certified specific pathogen-free chicks)
were pre-tested for the presence of kanamycin resistant Salmonella
by serially diluting and plating fecal samples onto XLD-kan. No
animals were positive for kanamycin resistant Salmonella after
overnight incubation, and no other microbes grew on the plates
for several days.
Measurement of resolvase activity in vitro
Liquid broth assay.
strains were inoculated in triplicate from glycerol stocks into
5 ml of LB kan broth containing either 1 mM oxoC6 or the solvent
control 0.1% EA and incubated overnight with shaking at 37uC.
The resulting cultures were then serially diluted onto LB kan
plates. Isolated colonies were screened for tetracycline resistance.
Motility agar assay.
Wild-type or sdiA mutant RIVET strains
were stabbed into LB motility agar containing 1 mM oxoC6 or the
solvent control 0.1% EA and incubated overnight at 37uC. Colonies
were recovered by stabbing the motility agar with a sterile wooden
inoculating stick and streaking to isolation on LB kan plates. Isolated
colonies were screened for tetracycline resistance.
Solid agar assay.
Wild-type or sdiA mutant RIVET strains
were struck to isolation on LB kan plates containing 1 mM oxoC6
or 0.1% EA and incubated overnight at 37uC. Isolated colonies
were screened for tetracycline resistance.
Wild-type or sdiA mutant RIVET
Measurement of resolvase activity in animals
Overnight cultures of the wild-type and sdiA mutant RIVET
strains (JNS3206 and JNS3226) were grown in LB kan tet at 37uC.
The next morning the overnight cultures were centrifuged at
5,0006g and resuspended in fresh LB lacking antibiotics. A 1:1
mixture of the cultures was used to inoculate the animals
intragastrically (200 ml for mice, 100 ml of concentrate for chicks
(1 ml resuspended in 100 ml), 4 ml for pigs (1 ml diluted to 4 ml),
10 ml for calves, 1 ml for turtles, guinea pigs, and rabbits. Dilution
plating of the inoculum was used to determine the actual dose
administered. Screening the isolated colonies for tetracycline
resistance indicated that in each experiment no colonies resolved
before the beginning of the experiment. The percentage of wild-
type Salmonella in each inoculum was determined by screening the
colonies for ampicillin resistance with the wild-type being amp
sensitive and the sdiA1::mTn3 mutant being amp resistant.
For most animals, fresh fecal samples were collected at time
points and resuspended in phosphate-buffered saline (PBS)
followed by serial dilution and plating on XLD-kan agar. Isolated
colonies were then screened for ampicillin and tetracycline
resistance. For the turtle experiments, cloacal swabs were
collected, vortexed in 1 ml PBS, and plated on XLD-kan. For
pigs, a Jorvet fecal sampling loop (Livestock Concepts) was inserted
into the rectum, withdrawn, and the feces removed by agitating
the loop in a 15 ml conical tube containing 1 ml PBS. Chicks were
sampled in groups of three each day post-infection for a total of
four days. They were euthanized and fecal samples were collected
directly from the cecum and distal large intestine.
Clearance of Aeromonas from turtles
In an attempt to eradicate Aeromonas from turtles, the following
four steps were performed every 48 hours for two weeks. First, a
cloacal swab from each turtle was collected and tested for the
presence of Aeromonas and Salmonella by plating on XLD, LB, and
blood agar. Second, the turtles received an IM injection in the
front leg of the antibiotic Amikacin at a dose of 5 mg/kg. The
injections were performed in alternating legs. Third, immediately
after the injection the turtle was bathed in 0.2% chlorhexidine
gluconate (except around the eyes) and rinsed with sterile water to
remove any external Aeromonas or Salmonella from the animal.
Fourth, the animal was placed into a new sterile container
containing sterile water. On the days when no injections were
performed the turtles were moved to a new sterile container
containing sterile water.
We gratefully acknowledge the technical assistance of Shinichiro Shoji,
Amber Lindsay, and Darren Lucas at OSU, Mitsu Suyemoto at North
Carolina State, and Alan Patranella at Texas A&M. We thank Roberta
Pugh at Texas A&M for incredible hospitality. We thank Michael
McClelland, Andreas Baumler, and Renee Tsolis for numerous helpful
Conceived and designed the experiments: CA LGA RCI BMMA.
Performed the experiments: JNS JLD JAS CA SL VK. Analyzed the
data: JNS JLD BMMA. Contributed reagents/materials/analysis tools:
CDE JS. Wrote the paper: JNS JLD BMMA.
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