JOURNAL OF BACTERIOLOGY, Feb. 2003, p. 1357–1366 Vol. 185, No.

4
0021-9193/03/$08.00⫹0 DOI: 10.1128/JB.185.4.1357–1366.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detection of Other Microbial Species by Salmonella: Expression

of the SdiA Regulon
Jenée N. Smith and Brian M. M. Ahmer*
Department of Microbiology, The Ohio State University, Columbus Ohio 43210
Received 4 September 2002/Accepted 22 November 2002

Salmonella, Escherichia, and Klebsiella do not encode any recognized type of N-acylhomoserine lactone (AHL)
synthase, and consistent with this, they do not synthesize AHLs under any conditions tested. However, they do
encode an AHL receptor of the LuxR family, named SdiA. MudJ fusions in four loci are known to respond to
plasmid-encoded sdiA in Salmonella, but only the rck locus has been described. Here we report the location and
sequence analysis of the remaining three loci. The srg-6::MudJ is within gtgA of the gifsy-2 prophage, and the
srg-7::MudJ is within PSLT61 of the virulence plasmid. Both fusions are in the antisense orientation. The third
fusion, srgE5::MudJ, is within a horizontally acquired gene of unknown function at 33.6 centisomes that we
have named srgE. Previously, sdiA expressed from its natural position in the chromosome was demonstrated
to activate a plasmid-based transcriptional fusion to the rck promoter in response to AHL production by other
bacterial species. However, the MudJ fusions did not respond to chromosomal sdiA. Here we report that MudJ
fusions to three of the four loci (not srg-6) are activated by AHL in an sdiA-dependent manner during growth
in motility agar (0.25% agar) but not during growth in top agar (0.7% agar) or on agar plates (1.2% agar). In
motility agar, the srgE promoter responds to sdiA at 30°C and higher while the rck and srg-7 promoters respond
only at 37 or 42°C. Substantial AHL-independent SdiA activity was observed at 30°C but not at 37°C.

Many species of bacteria use cell-to-cell signaling as an input
for gene regulation (10, 24). Small signal molecules are re-
leased into the environment and can be detected by bacterial
sensory systems. In the gram-negative bacteria, the most com-
monly studied signals are N-acylhomoserine lactones (AHLs),
with the corresponding sensor being a LuxR homolog (36).
The LuxR homologs have an N-terminal domain for binding
AHL and a C-terminal helix-tum-helix domain for binding
DNA (33, 44). In most cases it is hypothesized that the bacteria
use the concentration of signal molecules as an indicator of the
population density of their own species, a phenomenon known
as quorum sensing (24). Many bacteria regulate secondary
an unusual case. The existing DNA sequence databases for
these organisms show that each species carries a single luxR
homolog named sdiA but does not carry a luxI family member.
Other types of AHL synthase enzymes have been identified in
Vibrio and Pseudomonas (luxLM and hdtS [5, 14, 20]), but
Escherichia, Salmonella, and Klebsiella do not encode ho-
mologs of these enzymes either. Consistent with the genome
sequence data, representatives of these genera fail to produce
detectable levels of AHLs in standard bioassays under labora-
tory growth conditions (22, 36). Since these organisms contain
an AHL receptor but do not synthesize AHLs, they cannot be
using SdiA to determine the population density of their own

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metabolism and/or host interaction genes in response to this
information (24).
The final step in AHL synthesis is catalyzed by enzymes of
the LuxI family (13, 39). Each LuxI family member typically
generates a single AHL, along with smaller amounts of related
molecules. The primary difference between the AHLs synthe-
sized by different LuxI enzymes is the length of the acyl chain,
which usually varies between 4 and 14 carbons. AHLs can also
vary at the 3-carbon position by the presence of a carbonyl or
hydroxyl group. The AHLs discussed in this report are abbre-
viated C6, C8, etc. to denote chain length, and oxoC6, oxoC8,
etc. to denote a carbonyl modification. Many bacteria encode
more than one LuxI-type enzyme and synthesize several AHLs.
The AHL production and response profile of different species
can overlap, providing the potential for cross talk between
species (27, 30, 31, 40).
The genera Escherichia, Salmonella, and Klebsiella provide
* Corresponding author. Mailing address: Department of Microbi-
ology, The Ohio State University, 484 West 12th Ave., 376 Biological
Sciences Building, Columbus, OH 43210. Phone: (614) 292-1919. Fax:
(614) 292-8120. E-mail: ahmer.1@osu.edu.
species. Instead, they may use SdiA to detect AHLs produced
by other microbial species (22).
Before the signals for SdiA had been identified plasmid-
based expression of sdiA was used to identify responsive tran-
scriptional fusions in Salmonella enterica serovar Typhimurium
(1). The fusions were created with the MudJ transposable
element, which creates lacZY fusions on insertion into a coding
sequence (8, 17). Seven fusions were isolated in the rck operon
(for “resistance to complement killing”) on the serovar Typhi-
murium virulence plasmid. Three additional fusions were ob-
tained, but the DNA sequence flanking the MudJ insertions
did not match the existing database entries. Here we report the
locations of these three fusions now that the serovar Typhi-
murium genome sequence has been completed. These 10 fu-
sions were isolated based on their response to plasmid-based
expression of sdiA, but they failed to show any sdiA-dependent
expression when placed into isogenic wild-type and sdiA mu-
tant backgrounds (1). It was hypothesized that there were no
AHLs in the culture and that overexpression of sdiA had by-
passed the AHL requirement (1). Evidence supporting this
hypothesis was obtained when a plasmid-based luciferase fu-
sion to the rck operon promoter was found to be activated in

1357
1358 SMITH AND AHMER J. BACTERIOL.

TABLE 1. Strains and plasmids used in this study

Strain or
Genotype Source or reference
plasmid

Strains
DH5␣ E. coli ␾80 lacZ⌬M15 deoR endA1 gyrA96 hsdR17 recA1 Life Technologies
relA1 supE44 thi-1 ⌬(lacZYA-argF)U169
JM109 E. coli F⬘[traD36 proAB lacIqlacZ⌬M15] endA1 gyrA96 43
hsdR17 recA1 relA1 supE44 thi-1 ⌬(lac-proAB)
14028 Wild-type S. enterica serovar Typhimurium American Type Culture Collection
BA612 14028 sdiA1::mTn3 1
BA1101 14028 srgE5::MudJ 1
BA1103 14028 srgA1::MudJ 1
BA1110 14028 srg-6::MudJ 1
BA1112 14028 srg-7::MudJ 1
BA1301 BA612 srgE5::MudJ 1
BA1303 BA612 srgA1::MudJ 1
BA1310 BA612 srg-6::MudJ 1
BA1312 BA612 srg-7::MudJ 1
10460 Wild-type Y. enterocolitica 37
10460 yenI 10460 yenI::Kan 37
OSU239 Wild-type H. alvei Ohio State University Department of
Microbiology culture collection
PAO1 Wild-type P. aeruginosa 16
PAO-JP2 PAO1 ⌬lasI::tet ⌬rhlI::Tn501-2 29
97021 Wild-type S. marcescens Jean Guard-Petter
CVOblu C. violaceum cviI::Tn5xylE 34

Plasmids
pSB401 luxR⫹ PluxI-luxCDABE Tetr p15A ori 40
pSB536 ahyR⫹ PahyI-luxCDABE Ampr ColE1 ori 34
pSB1075 lasR⫹ PlasI-luxCDABE Ampr ColE1 ori 40
pBA428 Prck-luxCDABE Tetr 22
pJNS25 PsrgE-luxCDABE Tetr pSB401⌬EcoRI carrying the srgE-
STM1555 intergenic region
pJNS35 PSTM1555-luxCDABE Tetr Same as pJNS25 but the intergenic
region is in the opposite
orientation

an sdiA-dependent manner in response to synthetic oxoC6 and and oxoC8 (22) were dissolved in acidified ethyl acetate (EA) (28) and used at
oxoC8 at concentrations of 1 to 10 nM. The fusion also re- final concentrations of 10 nM and 1 ␮M, respectively. EA was used as a solvent
control at a final concentration of 0.1%. The strains and plasmids used are
sponded to C6 and C8 when the concentrations were approxi- described in Table 1.

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mately 10-fold higher (22). However, responses to AHL were
not observed when the chromosomal MudJ fusions were used
(we refer to all of the MudJ fusions, including those on the
virulence plasmid, as chromosomal to distinguish them from
the multicopy plasmid-based luciferase fusions [22]). The
MudJ fusions failed to respond in cross-streak assays on 1.5%
agar plates, in filter disk assays performed with 0.7% top agar,
and in early-exponential-phase liquid cultures (22). In this re-
port, we show that growth in motility agar or to the late expo-
nential phase in liquid culture allows the sdiA-dependent ac-
tivation of chromosomal fusions in response to AHL or to
bacterial species producing AHL. This is a definitive demon-
stration that SdiA detects and responds to AHL production by
other bacterial species. We also reveal the locations of three
previously uncharacterized MudJ fusions that are regulated by
SdiA.
MATERIALS AND METHODS
Bacterial strains and media. Bacteria were grown in Luria-Bertani (LB) broth
(EM Science). Agar was added to 0.25% (motility agar), 0.7% (top agar), or
1.2% (agar plates) as indicated. Tetracycline and kanamycin were used at 20 and
60 ␮g/ml, respectively, when appropriate. 5-Bromo-4-chloro-3-indolyl-␤-D-galac-
topyranoside (X-Gal) was used at a final concentration of 40 ␮g/ml. C6 (Sigma)
Plasmid constructions. To isolate the promoter of srgE, a plasmid-based
transcriptional fusion was constructed (see Fig. 1). The putative promoter region
was amplified from S. enterica serovar Typhimurium strain 14028 using Pfu
Turbo DNA polymerase (Stratagene). Each primer contained a BglII restriction
site at the 5⬘ end. The PCR product spans nucleotides 16638 to 17114 of
Genbank accession number AE008767. The resulting PCR product was cloned
using pCR-Blunt II Topo (Invitrogen). The insert was removed from pCR-Blunt
II Topo by using EcoRI and cloned into pSB401 from which the luxR-containing
EcoRI fragment had been removed. Clones were screened for insert and orien-
tation using PCR. One isolate forming a srgE-luxCDABE fusion was named
pJNS25, while a clone in the opposite orientation was named pJNS35. These
plasmids were electroporated into strains 14028 and BA612 using a Bio-Rad
Gene Pulser II.
Measurement of luciferase and ␤-galactosidase activity. Liquid cultures were
grown in 5-ml volumes in 18- by 150-mm tubes placed near horizontal in a drum
roller to maintain agitation. The optical density (OD) of the cultures was mea-
sured with a Spectronic 20D⫹ instrument at 550 nm. The light production of
10-␮l samples was integrated for 10 s with a Turner Designs TD-20/20 luminom-
eter and expressed as Light/OD. Expression of luciferase by bacteria on plates or
in motility agar was imaged and quantitated using a C2400-32 intensified charge-
coupled device camera with an Argus 20 image processor (Hamamatsu Photon-
ics) (9).
Expression of lacZY reporters was measured using ␤-galactosidase assays as
described by Miller with the following modifications (23). The liquid cultures
were grown as described above for luciferase assays, except that at each time
point a 300-␮l sample was taken and placed on ice until all samples had been
collected. A 20-␮l portion of each sample was placed into a polystyrene flat-
VOL. 185, 2003
FIG. 1. (A) ORF maps of the rck and srgE regions of the S. enterica
serovar Typhimurium genome derived from GenBank accession num-
bers L08613 and AE008767, respectively. Numbers along the bottom
of each map represent nucleotide positions. The locations of the
srgA1::MudJ and srgE5::MudJ insertions are indicated by flags. The
regions of DNA cloned upstream of a promoterless luxCDABE operon
to form plasmids pBA428 and pJNS25 are indicated by hatched boxes.
(B) Wild-type Salmonella strain 14028 carrying pJNS25 responds to
AHL in a cross-streak assay, while the isogenic sdiA mutant strain,
BA612/pJNS25, does not. An sdiA-dependent response to AHL from
strains carrying pBA428 has been previously demonstrated (22).
bottom 96-well plate containing 180 ␮l of LB for reading of the OD at 570 nm
with a Dynatech Instruments MR700 plate reader. The readings using this
instrument were multiplied by 3.47 to account for path length differences be-
tween the MR700 and the Spectronic 20D⫹ used for the luciferase assays. A
100-␮l volume of sample was placed into a polypropylene round-bottom 96-well
plate containing 100 ␮l of Z-buffer and solubilized with 4 ␮l of chloroform and
4 ␮l of 0.1% sodium dodecyl sulfate. A 40-␮l volume of 4-mg/ml o-nitrophenyl-
␤-D-galactopyranoside (ONPG) was added to start the reaction, and 100 ␮l of
1M NaCO3 was added to stop it. The plate was then centrifuged at 5,000 ⫻ g for
10 min to pellet the chloroform and bacteria. Supernatant (100 ␮l) was trans-
ferred to a polystyrene flat-bottom 96-well plate for determination of A410 with
the Dynatech MR700 plate reader. Units of activity were calculated as (1,000 ⫻
A410)/[reaction time (minutes) ⫻ sample volume (milliliters) ⫻ OD570].
Comparisons of lacZY fusions in motility agar were qualitative, with each
EXPRESSION OF THE SdiA REGULON 1359
strain being stabbed, via a pipette tip, into agar containing 40 ␮g of X-Gal per ml.
This was followed by an overnight incubation, during which the bacteria spread
across the plate. However, in some experiments the agar concentration was
increased to a point at which the bacteria could no longer swim through it. For
those experiments, the plates were seeded with bacteria when the plates were
poured. Each reporter strain (100 ␮l of an overnight culture) was added to 25 ml
of molten LB agar (cooled to 50°C) containing X-Gal and AHL, poured into a
petri plate, allowed to solidify at room temperature, and then incubated over-
night at 37°C.
RESULTS
Locations of sdiA-regulated MudJ insertions. Previously we
identified 10 MudJ fusions that respond to plasmid-encoded
sdiA (1). Inverse PCR was performed to obtain small frag-
ments of DNA flanking the MudJ insertion sites, and DNA
sequencing revealed that seven of the fusions are located
within the rck operon while three are in uncharacterized genes
at unknown map locations (1, 22). For this study, we deter-
mined their locations by comparing the flanking DNA se-
quence of the fusions to that of the recently completed S.
enterica serovar Typhimurium genome (21).
Of the three MudJ fusions located outside the rck operon,
the first is srg-5::MudJ (Fig. 1A). According to the nomencla-
ture of the recently completed serovar Typhimurium genome
(21), this insertion is within an open reading frame (ORF)
named STM1554 at 33.6 centisomes (between nucleotides
15335 and 15336 of accession number AE008767). The inser-
tion is at the extreme 3⬘ end of the gene so that only the last 5
codons are disrupted and the lacZ fusion is in the sense ori-
entation. Because this gene is strongly regulated by sdiA and
because the promoter region has been isolated and confirmed
to be sdiA dependent (see below), we propose the name srgE
for STM1554 (for “sdiA-regulated gene E”) and the allele
number srgE5::MudJ for the insertion. srgE appears to be
within a 39-kb “island” because eight ORFs upstream of srgE
(STM1555 to STM1562) and 26 ORFs downstream of srgE
(STM1553 to STM1528) are predominantly Salmonella spe-
cific. The boundaries of the island are the osmC gene in the
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clockwise direction and the yne genes in the counterclockwise
direction. However, the G ⫹ C content varies widely among
the ORFs, suggesting that the island is a mosaic of acquisition
and/or deletion events. The organization of the region suggests
that srgE is expressed from a monocistronic transcript. Three
ORFs are divergently transcribed from the srgE promoter re-
gion (STM1555 to STM1557), but these are not regulated by
sdiA (see below). The G ⫹ C content of Salmonella is typically
53%, while the G ⫹ C content of srgE is much lower at 36%
(21). The G ⫹ C content abruptly increases on both sides of
srgE, suggesting that srgE may have been a single-gene acqui-
sition.
The molecular mass of SrgE is predicted to be 55.7 kDa,
with a pI of 6.8. The annotation of the serovar Typhimurium
genome predicts that SrgE is an inner membrane protein based
on identification of a transmembrane domain between residues
418 and 435, using the program PSORT (25). However, an-
other transmembrane helix prediction program, TMHMM 2.0,
suggests that there are no transmembrane helices (19), leaving
the predicted subcellular localization of SrgE uncertain. The
genome annotation suggests that SrgE has a coiled-coil domain
(21). The PairCoil program also suggests that SrgE has a
1360 SMITH AND AHMER
coiled-coil domain, located between residues 344 and 374 (6).
The function and significance of this domain are unknown. The
only similar protein in the current databases, using BLAST
programs, is an apparent ortholog of srgE in the S. enterica
serovar Typhi genome, ORF STY1509 (3, 4, 26). STY1509 is
98% identical to SrgE throughout the majority of the protein.
However, STY1509 lacks 15 N-terminal residues (possibly due
to a start codon prediction discrepancy) and 82 C-terminal
residues compared to SrgE. The PairCoil program predicts a
coiled-coil domain in the serovar Typhi protein between resi-
dues 329 and 359.
The second fusion, srg-6::MudJ, is located within STM1026
(gtgA) in the antisense orientation (nucleotide 29215 of acces-
sion number AE008743). This gene lies within the gifsy-2 pro-
phage at 24 centisomes (12). GtgA has 97% amino acid iden-
tity to GogA of the gifsy-1 phage and 71% identity to PipA of
SPI5 (12, 41). The function of this gene and the significance of
the antisense regulation by sdiA are unknown.
The third fusion, srg-7::MudJ, resides on the virulence plas-
mid of serovar Typhimurium. The fusion is in the antisense
orientation within ORF PSLT61 (nucleotide 50951 of acces-
sion number AE006471) (21). PSLT61 has a high G ⫹ C
content (61%) and is predicted to encode an inner membrane
protein of 28.5 kDa with a pI of 5.2. This protein is at least 38%
identical to the products of numerous plasmid-located genes of
unknown function including ycfA from plasmid ColIb-P9, pro-
tein L7076 from enterohemorrhagic Escherichia coli plasmid
pO157, and ORF248 of the E. coli F plasmid. As with srg-6, the
function of this gene and the significance of the antisense
regulation by sdiA are unknown.
Cloning of the srgE promoter region. The intergenic region
between srgE and the divergently transcribed gene was ampli-
fied by PCR and cloned into pSB401 to place the putative
promoter region upstream of the promoterless luxCDABE
operon. A clone with the srgE promoter in the appropriate
orientation was named pJNS25, while a clone with the frag-
ment in the opposite orientation was named pJNS35. These
two reporter constructs were electroporated into the wild-type
and sdiA mutant serovar Typhimurium strains 14028 and
BA612, respectively. Cross-streak assays were performed with
these strains to detect sdiA-dependent responses to AHL. A
20-␮l volume of 10 ␮M C6 was streaked across one end of an
LB plate, and the reporter strains were streaked perpendicular
to the AHL. Increases in light production of at least 12-fold
were observed from the wild-type strain carrying pJNS25 near
the C6, while the sdiA mutant strain did not respond (Fig. 1B).
This confirms that the region cloned within pJNS25 does con-
tain an sdiA-dependent promoter. The strains carrying pJNS35
did not respond to AHL (data not shown), demonstrating that
the sdiA-dependent activation of the srgE promoter is unidi-
rectional. Similar attempts to clone the srg-6 and srg-7 promot-
ers were unsuccessful.
Conditions that allow chromosomal fusions to respond to
SdiA and AHL. Previously we determined that SdiA expressed
from the chromosome could activate a plasmid-based Prck-
luxCDABE reporter in the presence of particular AHLs (22).
This activation occurred under a variety of conditions includ-
ing cross-streak assays on solid LB agar plates (1.2 or 1.5%
agar), filter disk assays in 0.7% top agar, and liquid culture.
However, the MudJ insertions in the rck operon and the other
J. BACTERIOL.
three srg loci did not respond to chromosomally encoded SdiA
and AHL (they responded only to plasmid-encoded SdiA).
Since this observation cast doubt on the ability of SdiA to truly
detect and respond to AHL production by other species, our
recent focus has been on identifying growth conditions that
allow the activation of chromosomal fusions by chromosomal
sdiA. It was recently discovered that the regulator adjacent to
sdiA, named sirA, is much more active during growth in mo-
tility agar (LB broth containing 0.25% agar) than in liquid
culture (15). Therefore, we tested the ability of SdiA to acti-
vate chromosomal fusions in motility agar. All plates contained
either 1 ␮M C6 or 0.1% acidified EA as a solvent control.
MudJ fusions to all four sdiA-regulated loci were tested in each
type of medium at four different incubation temperatures.
While this is a qualitative assay, the results clearly demon-
strated that three of the four loci can be activated under par-
ticular growth conditions. srgA1::MudJ (representing the rck
operon), srgE5::MudJ, and srg-7::MudJ responded to AHL in
an sdiA-dependent manner, while srg-6::MudJ was not regu-
lated by sdiA under any condition (Fig. 2). Activation of the
three fusions was primarily sdiA and AHL dependent, al-
though both sdiA- and AHL-independent expression was ob-
served.
Expression of the srgE5::MudJ fusion was observed at 30, 37,
and 42°C but not at 22°C. At each temperature the expression
was completely sdiA dependent. Consistent with the sdiA de-
pendence, the expression at 37°C was almost entirely AHL
dependent. However, at 30°C, sdiA-dependent activation was
observed in the absence of AHL.
The srgA1::MudJ fusion differs from the srgE5::MudJ in two
respects. First, the rck operon is more tightly temperature
dependent in that expression was observed only at 37 and 42°C.
Second, the rck operon can be expressed in the absence of sdiA
under certain conditions. For example, in Fig. 2 the sdiA mu-
tant shows an increase in srgA1::MudJ expression at 37 and
42°C compared to 22 and 30°C.
The regulation of srg-7::MudJ by sdiA was very weak, but it
had an expression pattern similar to that of srgA1::MudJ (Fig.
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2). This is interesting given that both fusions are present on the
serovar Typhimurium virulence plasmid. Like srgA1, the srg-7
fusion was not expressed at 22°C or 30°C. Also like srgA1, there
was expression in the absence of sdiA and AHL at higher
temperatures.
The assays throughout this report used C6 at a concentration
of 1 ␮M. SdiA detects oxoC8 at much lower concentrations
than C6, but oxoC8 is not commercially available and is not
easy to synthesize. To determine if the use of oxoC8 would give
different results from the use of C6, the motility agar assays in
this section were performed once with oxoC8 at a final con-
centration of 10 nM. The results were essentially identical to
those obtained using 1 ␮M C6 (data not shown).
Agar percentages that allow sdiA-dependent activation of
chromosomal fusions. To examine the effect of agar concen-
tration more closely, we seeded plates of various agar concen-
trations with each reporter strain and 1 ␮M C6 and incubated
them overnight at 37°C. SdiA-dependent activation of srgE was
observed with 0.3, 0.4, and 0.5% agar but not with 0.7% agar
(data not shown). The response in 0.6% agar was very weak.
The srgA1::MudJ and srg-7::MudJ fusions were both activated
in an sdiA-dependent manner in 0.3 and 0.4% agar. Interest-
VOL. 185, 2003
FIG. 2. MudJ fusions to three of the four srg loci respond to AHL
in motility agar. Bacteria were stabbed into LB motility agar containing
X-Gal and incubated overnight at one of four different temperatures as
indicated above each column. All of the bacteria in each photograph
carry a particular MudJ fusion that is listed in the top left of the
photograph. The strain stabbed into the left side of each plate is wild
type (except for the MudJ fusion), while the strain stabbed into the
right side is an isogenic sdiA mutant. Within each photograph, the top
row of plates contained 1 ␮M C6 while the bottom row contained the
solvent control acidifie EA. (A) Serovar Typhimurium strain BA1101
is on the left side of each plate and strain BA1301 is on the right side.
(B) Strain BA1103 is on the left and strain BA1303 is on the right.
(C) Strain BA1110 is on the left and strain BA1310 is on the right.
(D) Strain BA1112 is on the left and strain BA1312 is on the right.
ingly, both fusions were more active using 0.5, 0.6, and 0.7%
agar, but this was not an sdiA-dependent activation, and in fact
the presence of sdiA seemed to inhibit the increased response
(the sdiA mutant was slightly darker blue than was the wild
type [data not shown]). The srg-6::MudJ fusion did not respond
to AHL under any conditions. To summarize, all of the chro-
mosomal MudJ fusions except srg-6 respond to AHL in an
sdiA-dependent manner when the bacteria are grown in 0.25 to
0.4% motility agar but not in 0.7% top agar or on 1.2% agar
plates.
Quantitation of sdiA-dependent responses in liquid media.
Attempts to measure ␤-galactosidase activity in motility agar
were unsuccessful. Therefore, we evaluated the response of the
chromosomal and plasmid-based fusions in liquid culture. Mild
responses were observed for chromosomal fusions at late-ex-
EXPRESSION OF THE SdiA REGULON 1361
ponential and stationary phases (maximum of 4.4-fold for
srgE5::MudJ at 37°C [Fig. 3; Table 2]). These responses al-
lowed us to quantitate and compare the levels of AHL-depen-
dent and AHL-independent SdiA activity. The pattern of re-
sponse in liquid culture was similar to that obtained in motility
agar in that srgE5::MudJ was the strongest responder (with
regard to fold differences), followed by srgA1::MudJ and
srg-7::MudJ (Fig. 3 and Table 2 for srgE5 and srgA1; data not
shown for srg-7). srg-6::MudJ failed to respond (data not
shown). One difference between liquid and motility agar is that
the rck operon was activated weakly in liquid at 30°C (2.4-fold)
while no expression was detected in motility agar at the same
temperature. Another difference is that the AHL-independent
SdiA activity observed at 30°C was much stronger in motility
agar than in liquid (Fig. 3; Table 2). Overall, fold induction
values in the presence of AHL increased as the temperature
was increased from 30 to 37°C (Fig. 3; Table 2). However, as
the temperature was reduced from 37 to 30°C, SdiA became
active in the absence of AHL (Fig. 3; Table 2).
SdiA can activate chromosomal fusions in response to other
species of bacteria. A plasmid-based Prck-luxCDABE fusion
was previously demonstrated to respond in an sdiA-dependent
fashion to the presence of other bacterial species in a cross-
streak assay on a standard 1.5% agar LB plate (22). Two
species that were potent activators of SdiA in this assay are
Yersinia enterocolitica and Hafnia alvei (22). However, the
chromosomal MudJ fusions to sdiA-regulated loci did not re-
spond in this assay (22), which cast doubt on the ability of SdiA
to detect and respond to other species. To determine if the
MudJ fusions would respond to Y. enterocolitica or H. alvei in
motility agar, we stabbed one side of a motility agar plate with
an S. enterica serovar Typhimurium reporter strain and the
other side with Y. enterocolitica or H. alvei. The best results
were obtained when the Yersinia and Hafnia strains were given
a 1-day head start at either 22 or 30°C, respectively, before the
Salmonella was added to the plate. Once the Salmonella was
stabbed into the plates, the plates were incubated for a further
24 h at 37°C. During this time, the serovar Typhimurium cells
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swam outward and surrounded the Y. enterocolitica or H. alvei
cells. As shown in Fig. 4, the chromosomal MudJ fusions were
activated in an sdiA-dependent manner in the region immedi-
ately surrounding the AHL-producing species. The fusions did
not respond to a yenI mutant of Y. enterocolitica that cannot
synthesize AHLs (Fig. 4). The sdiA-dependent response of the
serovar Typhimurium chromosomal MudJ fusions in this assay
conclusively demonstrates that Salmonella can detect and re-
spond to other bacterial species that are synthesizing AHLs.
SdiA-dependent expression in the absence of exogenously
added AHL is not due to AHL production by Salmonella. We
have previously reported that the three genera known to en-
code SdiA (Escherichia, Klebsiella, and Salmonella) do not syn-
thesize AHLs at concentrations that can be detected by bio-
sensors (22). However, those studies were performed at 37°C.
The studies performed at 30°C in this report show SdiA activity
in the absence of exogenously supplied AHL. It was possible
that sufficient concentrations of AHL are generated by Salmo-
nella at 30°C to partially activate SdiA.
To test this hypothesis, we grew two Salmonella strains at
30°C in LB motility agar containing X-Gal, adjacent to four
different AHL biosensor strains (Fig. 5). The Salmonella strain
 1362
SMITH AND AHMER
J. BACTERIOL.

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VOL. 185, 2003 EXPRESSION OF THE SdiA REGULON 1363

TABLE 2. Maximal sdiA-dependent activation in liquid culture in

the presence of either EA or C6a

Temp Fold activation in:

Fusion
(°C) EA C6

srgE5::MudJ 30 2.0 4.0

37 1.3 4.4
PsrgE-luxCDABE 30 4.9 12.3
37 1.6 14.6
srgA1::MudJ 30 1.2 1.9
37 1.3 2.4
Prck-luxCDABE 30 2.5 3.6
37 1.5 7.2
a
Data from Fig. 3.

BA1101 (14028 srgE5::MudJ) and the isogenic sdiA mutant,

BA1301, were used to demonstrate that there was indeed
AHL-independent SdiA activity during the assay (BA1101 was
light blue, while BA1301 was white). The four reporters were
CviR, LasR, LuxR, and AhyR. The CviR reporter strain was
Chromobacterium violaceum strain CVOblu, while the other
three reporters are E. coli based: DH5␣/pSB536 (AhyR),
JM109/pSB401 (LuxR), and JM109/pSB1075 (LasR). CVOblu
synthesizes the purple pigment violacein when short-chain
AHLs are detected (C4 and C6 [34]). The pSB536 reporter
encodes AhyR from Aeromonas hydrophila, which responds to
C4 (34, 35). The pSB401 plasmid encodes LuxR from Vibrio
fischeri, which detects oxoC6 with highest sensitivity but also FIG. 4. Chromosomal MudJ fusions respond to AHL-producing
bacterial species in an sdiA-dependent manner. All plates contain LB
detects oxoC8, C6, and C8, the same AHLs detected by SdiA motility agar and X-Gal. Either H. alvei, Y. enterocolitica, or an isogenic
(40). Plasmid pSB1075 encodes the Pseudomonas aeruginosa yenI mutant of Y. enterocolitica was stabbed into the right side of each
LasR protein, which detects oxoC12 with highest sensitivity but plate as indicated. Wild-type Salmonella containing the srgE5::MudJ
also detects C10, C12, oxoC10, and oxoC14 (40). We also at- insertion (BA1101) was stabbed into the left side of each plate in the
left column. The isogenic sdiA mutant (BA1301) was stabbed into the
tempted to use the TraR reporter Agrobacterium tumefaciens
left side of each plate in the right column. The Yersinia strains were
strain NT1/pDCI41E33 (31), but it failed to detect positive stabbed and incubated at room temperature for 24 h before the Sal-
control strains under the conditions used, and so it was not monella strains were stabbed. The Hafnia was stabbed and incubated at
used. 30°C for 24 h before the Salmonella strains were stabbed. After the
The CviR reporter was able to detect the positive control Salmonella strains had been stabbed, the plates were incubated for
another 24 h at 37°C. The Salmonella cells are more motile than the H.

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species Serratia marcescens and Hafnia alvei but showed abso-
lutely no response to the Salmonella strains (Fig. 5A). The
lux-based reporters all showed a basal level of light in the
presence of Salmonella but responded strongly to control
strains (Fig. 5B). To be sure that the low levels of light emitted
in the presence of Salmonella are indeed basal levels of re-
porter expression, indicative of no signal, negative control
strains were used. For the LuxR reporter, the positive control
species was Y. enterocolitica and the negative control was an
isogenic yenI mutant which cannot synthesize AHLs. For the
AhyR and LasR reporters, the positive control species was P.
aeruginosa and the negative control was an isogenic lasI rhlI
double mutant of P. aeruginosa that cannot synthesize AHLs.
The response of these three reporter systems to Salmonella was
alvei cells and much more motile than the Y. enterocolitica cells under
these conditions, resulting in the Salmonella cells completely surround-
ing the Y. enterocolitica cells. A blue halo indicating activation of
srgE5::MudJ is seen near the AHL-producing strains H. alvei and Y.
enterocolitica but not near the yenI mutant. The activation does not
occur in the sdiA mutant. Salmonella carrying the srgA1::MudJ also
responds in this assay (data not shown).
less than or equal to the response generated by the negative
control strains. Given that all of these reporters can detect
AHLs at nanomolar concentrations, similar to SdiA (1 to 10
nM for SdiA [22], 1 nM for CviR [7], 1 to 10 nM for LuxR [40],
and 0.1 to 1 nM for LasR [40]), we conclude that Salmonella is

FIG. 3. Expression of the rck and srgE promoters measured with chromosomal MudJ fusions and plasmid-based luxCDABE fusions in liquid
LB medium at 30 and 37°C. An sdiA-dependent response to AHL can be seen under all conditions with both promoters. However, AHL-
independent SdiA activity is present at 30°C and to a lesser extent at 37°C. A key for each pair of graphs is located between the graphs. Expression
is indicated by bars, with units indicated on the left y axis (the units are Miller units of ␤-galactosidase for the MudJ fusions and Light/OD for the
luxCDABE fusions). The growth curves of the cultures are superimposed with units indicated on the right y axis (the units are OD570 for the MudJ
fusions and OD550 for the luxCDABE fusions).
1364 SMITH AND AHMER
FIG. 5. Activity of SdiA in the absence of exogenously supplied
AHL is not the result of AHL production by Salmonella. In LB motility
agar containing X-Gal at 30°C, the Salmonella strain BA1101 (14028
srgE5::MudJ) is a darker blue than the isogenic sdiA mutant BA1301.
To determine if Salmonella is synthesizing AHLs under these condi-
tions, four different AHL reporter strains were inoculated adjacent to
BA1101 and BA1301. All strains (reporters and positive and negative
controls) were stabbed and incubated at room temperature for 24 h
before the Salmonella strains were stabbed into the plates, at which
point the plates were incubated at 37°C. (A) The CviR reporter
(CVOblu) fails to detect AHL production by Salmonella, although it
responds strongly to the positive control species H. alvei and S. marc-
escens under these conditions (indicated by purple pigment produc-
tion). (B) The LuxR reporter (JM109/pSB401) shows only basal levels
of light production in response to Salmonella strains BA1101 and
BA1301 and the Y. enterocolitica yenI mutant, although it responds
strongly to the positive control species Y. enterocolitica (luminescence
from four plates is shown). The LasR and AhyR reporters also fail to
detect AHL production from Salmonella although they respond
strongly to positive control species (data not shown).
not producing AHL at the nanomolar concentrations required
to activate SdiA. Because low levels of SdiA activity were
observed during the assay (BA1101 was light blue, while
BA1301 was white), we conclude that this is a basal level of
SdiA activity that does not represent AHL detection.
DISCUSSION
Quorum sensing in gram-negative bacteria typically utilizes a
LuxI homolog to generate a signal molecule and a LuxR ho-
molog to detect it. In Salmonella, Escherichia, and Klebsiella,
J. BACTERIOL.
there is no LuxI homolog; consistent with this, biosensors fail
to detect AHL production by these organisms (22, 36; this
report). However, these genera do encode a LuxR homolog
named SdiA. In this study we determined the locations of three
previously uncharacterized MudJ fusions that respond to plas-
mid-encoded sdiA and demonstrated conclusively that Salmo-
nella uses SdiA to detect AHL production by other species of
bacteria.
We have demonstrated that chromosomal MudJ fusions to
srgE, srg-7, and the rck operon respond to exogenous AHL in
an sdiA-dependent fashion when sdiA is expressed from its
natural position in the chromosome. This is the first study in
which chromosomal fusions have been activated by chromo-
somal sdiA. The chromosomal fusions respond in both liquid
medium and in motility agar (0.25%) but do not respond in top
agar (0.7% agar) or on solid agar plates (1.2 or 1.5% agar). The
plasmid-based reporter systems for srgE and rck are not limited
in this way and respond to SdiA and AHL under all these
conditions. Therefore, the chromosomal fusions are the more
relevant reporters, although as a simple AHL detection system
the plasmid-based fusions are more useful since they can be
used in a filter disk assay or in a cross-streak assay. There is a
regulator downstream of sdiA that has requirements that seem
inverted compared to sdiA. The regulator is named sirA in
Salmonella, and it regulates motility and invasion functions (2,
15, 18). SirA is active on solid agar and in motility agar but is
only weakly active in liquid (15). Therefore, motility agar con-
taining AHL is currently the in vitro medium of choice to
ensure that both regulators are active.
The significance of two of the srg loci with regard to the SdiA
regulon is questionable at this point. The MudJ insertions for
both are in the antisense orientation. One of these, srg-7, can
be activated slightly by chromosomal sdiA in response to ex-
ogenous AHL. The srg-7::MudJ fusion is located within
PSLT61 on the S. enterica serovar Typhimurium virulence plas-
mid which does not have a known function. There are ho-
mologs of PSLT61 on many plasmids including the F plasmid
of E. coli and pO157 of enterohemorrhagic E. coli. The second
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antisense fusion is srg-6::MudJ. This fusion resides within the
gtgA gene of the gifsy-2 prophage. The function of this gene is
not known. This fusion is the weakest in response to plasmid-
located sdiA (1) and is the only fusion for which we have not
yet detected a response to chromosomal sdiA and exogenous
AHL. Our preliminary attempts to isolate the sdiA-dependent
promoters for both srg-6 and srg-7 were unsuccessful.
In contrast, the srg-5::MudJ is in the sense orientation within
ORF STM1554 and is strongly activated by sdiA. The promoter
region for this gene was successfully isolated and confirmed to
be sdiA dependent (Fig. 1B). Therefore, we propose the name
srgE (for “sdiA-regulated gene”) for ORF STM1554 and the
allele name srgE5::MudJ for the insertion. In addition, we
propose a name change within the rck operon. We recently
reported that the sdiA-dependent promoter of the rck operon
was further upstream than expected (22). This added two
genes to the operon, pef1 and ORF7 (Fig. 1a). Because other
genes within this operon are named srgA, srgB, and srgC, we
propose that ORF7 be named srgD.
The rck operon and srg-7 are carried on the serovar Typhi-
murium virulence plasmid and share several traits that differ
from srgE. Fusions to both the rck operon and srg-7 are tightly
VOL. 185, 2003
temperature dependent in motility agar in that they fail to
respond to AHL at 30°C and instead respond only at 37°C and
higher. In contrast, the srgE promoter responds strongly to
AHL at 30°C and higher. However, this difference between the
rck operon and srgE largely disappears in liquid culture, where
both loci can be activated at 30°C. None of the fusions are
active at room temperature, and the mechanism of tempera-
ture-dependence is unknown. However, the 37°C optimal tem-
perature for the SdiA regulon suggests that the mammalian
gastrointestinal tract may be the location where SdiA detects
and responds to AHL. Consistent with this hypothesis is the
recent detection in the bovine rumen of compounds that can
activate AHL biosensors (11). A second trait shared by the rck
operon and srg-7 is that both can be expressed in the absence
of sdiA, a characteristic that complicates studies of sdiA. Under
the conditions examined to date, the srgE promoter is not
expressed in the absence of sdiA and therefore provides the
simplest and strongest reporter of SdiA activity.
The srgE promoter is the third sdiA-dependent promoter to
be isolated. The other two promoters are the rck operon pro-
moter in Salmonella and the ftsQ promoter in E. coli. The
promoter for the rck operon resides between ORF6 and pef1
(22) and is shown in Fig. 1A. In E. coli, the ftsQP2 promoter
cloned on a multicopy plasmid as a fusion to lacZY is activated
5- to 13-fold in response to plasmid-encoded sdiA but only
1.3-fold in response to chromosomal sdiA (38). Addition of
AHL to the culture increases expression by twofold but only
when using plasmid-encoded sdiA (32). Therefore, unlike the
rck and srgE promoters, the E. coli ftsQP2 promoter has never
been shown to respond to AHL using sdiA expressed from its
natural position in the chromosome, and our preliminary re-
sults indicate that it does not (unpublished data). However,
biochemical experiments using purified SdiA and the ftsQP2
promoter (in the absence of AHL) revealed a putative SdiA
binding site (42). This sequence is not present within the pro-
moter regions of either srgE or the rck operon, suggesting that
this is either not a specific SdiA binding site or SdiA of E. coli
has a different binding site than SdiA of Salmonella or that rck
and srgE are only indirectly affected by SdiA. We feel that
prediction of SdiA binding sites is premature at this point and
should await the results of biochemical experiments with more
promoters.
To date, SdiA is the only LuxR homolog that is hypothesized
to detect only the AHL production of other species. For this to
be true, it must be clear that Salmonella fails to synthesize
AHLs and that SdiA is active only when AHLs are supplied
exogenously. This has been demonstrated conclusively in this
study at 37°C. In liquid culture we observed a 1.3-fold differ-
ence between the wild type and the sdiA mutant when mea-
suring the regulation of the srgA1::MudJ and srgE5::MudJ fu-
sions (Table 2). However, there is no detectable AHL
production by S. enterica serovar Typhimurium at this temper-
ature (22), so we ascribe this 1.3-fold difference to basal levels
of SdiA activity. Furthermore, as shown in Fig. 4, Salmonella
can detect and respond to the presence of H. alvei or Y. en-
terocolitica. This detection event requires that Salmonella carry
sdiA and that the other species produce AHL (the yenI mutant
of Yersinia is not detected). We feel that this combination of
evidence conclusively demonstrates that at 37°C Salmonella
EXPRESSION OF THE SdiA REGULON 1365
fails to synthesize AHLs but detects and responds to AHLs
produced by other species.
The conclusions to be drawn from results obtained at 30°C
are not so clear. At this temperature there is substantial SdiA
activity in the absence of AHL (Fig. 2 and 3; Table 2). This
activity is strongest in motility agar, where the expression of
srgE in the presence and absence of AHL is nearly identical but
still entirely sdiA dependent (this is seen only with srgE, since
the other fusions are not expressed at 30°C [Fig. 2]). In liquid
culture, the AHL-independent SdiA activity is less intense but
is seen with both srgE and the rck operon (Fig. 3; Table 2).
Since Salmonella is not producing AHL at this temperature
(Fig. 5), there are two potential explanations for this activity.
First, SdiA may be detecting some type of molecule synthe-
sized by Salmonella at 30°C that is not an AHL. While we
cannot eliminate this possibility, we feel that it is unlikely
because the molecule would have to be detected by SdiA but
not by the other four LuxR-type biosensors, it would have to be
produced only at 30°C, and it would have to be produced by a
currently unrecognized type of synthase. We favor the second
possibility, in which the activity does not represent signal de-
tection at all but simply represents high basal levels of SdiA
activity in the absence of signal. This immediately raises the
question of why the basal level of activity is so much higher at
30°C than at 37°C. We hypothesize that this could be due to
either increased sdiA expression or increased oligomerization
of SdiA at 30°C. Both hypotheses need to be tested, but we
already know that expression of sdiA from a plasmid increases
the expression of SdiA-regulated genes in the absence of signal
(1, 22). Regardless, this is the first demonstration of chromo-
somally encoded sdiA activating chromosomal fusions. At
37°C, this activation requires the addition of AHL from an-
other source. We conclude that SdiA is used to detect AHLs
that are synthesized by other species.
ACKNOWLEDGMENTS
We thank Joe Krzycki for critical reading of the manuscript, Jean
Downloaded from https://journals.asm.org/journal/jb on 20 June 2023 by 132.248.207.67.
Guard-Petter for the Serratia marcescens isolate, and Eb Pesci and
Barbara Iglewski for strain PAO-JP2.
This publication was made possible by grant 1 R01 AI50002-01 from
the National Institute of Allergy and Infectious Diseases.
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