E. coli K-12 and EHEC Genes Regulated by SdiA

Background: Escherichia and Salmonella encode SdiA, a transcription factor of the LuxR family that regulates genes in response to N-acyl homoserine lactones (AHLs) produced by other species of bacteria. E. coli genes that change expression in the presence of plasmid-encoded sdiA have been identified by several labs. However, many of these genes were identified by overexpressing sdiA on a plasmid and have not been tested for a response to sdiA produced from its natural position in the chromosome or for a response to AHL. Methodology/Principal Findings: We determined that two important loci reported to respond to plasmid-based sdiA, ftsQAZ and acrAB, do not respond to sdiA expressed from its natural position in the chromosome or to AHLs. To identify genes that are regulated by chromosomal sdiA and/or AHLs, we screened 10,000 random transposon-based luciferase fusions in E. coli K-12 and a further 10,000 in E. coli O157:H7 for a response to AHL and then tested these genes for sdiAdependence. We found that genes encoding the glutamate-dependent acid resistance system are up-regulated, and fliE is down-regulated, by sdiA. Gene regulation by sdiA of E. coli is only partially dependent upon AHL. Conclusions/Significance: The genes of E. coli that respond to plasmid-based expression of sdiA are largely different than those that respond to chromosomal sdiA and/or AHL. This has significant implications for determining the true function o

SdiA, an N-Acylhomoserine Lactone Receptor, Becomes Active during the Transit of Salmonella enterica through the Gastrointestinal Tract of Turtles

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..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

Salmonella enterica Serovar Typhimurium Can Detect Acyl Homoserine Lactone Production by Yersinia enterocolitica in Mice▿

LuxR-type transcription factors detect acyl homoserine lactones (AHLs) and are typically used by bacteria to determine the population density of their own species. Escherichia coli and Salmonella enterica serovar Typhimurium cannot synthesize AHLs but can detect the AHLs produced by other bacterial species using the LuxR homolog, SdiA. Previously we determined that S. Typhimurium did not detect AHLs during transit through the gastrointestinal tract of a guinea pig, a rabbit, a cow, 5 mice, 6 pigs, or 12 chickens. However, SdiA was activated during transit through turtles colonized by Aeromonas hydrophila, leading to the hypothesis that SdiA is used for detecting the AHL production of other pathogens. In this report, we determined that SdiA is activated during the transit of S. Typhimurium through mice infected with the AHL-producing pathogen Yersinia enterocolitica. SdiA is not activated during transit through mice infected with a yenI mutant of Y. enterocolitica that cannot synthesize AHLs. However, activation of SdiA did not confer a fitness advantage in Yersinia-infected mice. We hypothesized that this is due to infrequent or short interactions between S. Typhimurium and Y. enterocolitica or that the SdiA regulon members do not function in mice. To test these hypotheses, we constructed an S. Typhimurium strain that synthesizes AHLs to mimic a constant interaction with Y. enterocolitica. In this background, sdiA+ S. Typhimurium rapidly outcompetes the sdiA mutant in mice. All known members of the sdiA regulon are required for this phenotype. Thus, all members of the sdiA regulon are functional in mice

Figure 7

<p>A) Acid fitness island of <i>E. coli</i>. The transposon insertion in <i>E. coli</i> K-12, AL4001, is within <i>gadW</i> at nucleotide 3662317 of Genbank accession number U00096. The transposon insertions in the EHEC strains are shown on the same map but the nucleotide positions are from Genbank accession number BA000007. JLD605 is within <i>gadE</i> at nucleotide 4401036; JLD607 is within <i>yhiD</i> at nucleotide 4397949; JLD610 is within <i>hdeA</i> at nucleotide 4398821. B) JLD604 is just upstream of ECs2675 in the anti-sense orientation at nucleotide 3662317.</p

Oligonucleotides used.

<p>Oligonucleotides used.</p

Regulation of <i>ftsQAZ</i> by <i>sdiA</i>.

<p>A chromosomal <i>ftsQAZ</i>-<i>lacZ</i> fusion was constructed in a Δ<i>lac</i> mutant <i>E. coli</i> strain (JLD3011), and an isogenic <i>sdiA</i> mutant (JLD3013). Additionally, derivatives of the <i>sdiA</i> mutant were constructed that contained either a low copy number vector expressing <i>sdiA</i> (pCX16) or the vector control (pGB2). The strains were subcultured 1∶100 into LB broth containing either 1 µM oxoC6 or EA. The cultures were incubated with shaking at 30°C (A) and at 37°C (B). Samples were removed from the cultures at time points for β-galactosidase assays. Each strain was assayed in triplicate and error bars represent standard deviation. * denotes p<0.05 compared to the adjacent solvent control.</p

Regulation of AHL-regulated genes of <i>E. coli</i> K-12 and EHEC in liquid cultures at 30°C containing either 1 µM oxo-C6 or the solvent control, EA.

<p>Luminescence in relative light units (RLU) and OD590 were measured using a Wallac Victor² 1420 multimode plate reader at the time intervals noted. Each strain was assayed in triplicate and error bars represent standard deviation. A) AL4001<i>/</i>JLD800 (<i>gadW</i>), B) JLD604/JLD803 (<i>fliE</i>), C) JLD605/JLD804 (<i>gadE</i>), D) JLD607/JLD806 (<i>yhiD</i>), E) JLD610 /JLD809 (<i>hdeA</i>).</p

Regulation of AHL-regulated genes of <i>E. coli</i> K-12 and EHEC in liquid cultures at 37°C containing either 1 µM oxo-C6 or the solvent control, EA.

<p>Luminescence in relative light units (RLU) and OD590 were measured using a Wallac Victor² 1420 multimode plate reader at the time intervals noted. Each strain was assayed in triplicate and error bars represent standard deviation. A) AL4001<i>/</i>JLD800 (<i>gadW</i>), B) JLD604/JLD803(<i>fliE</i>), C) JLD605/JLD804 (<i>gadE</i>), D) JLD607/JLD806 (<i>yhiD</i>), E) JLD610 /JLD809 (<i>hdeA</i>).</p

Regulation of <i>acrA</i> by <i>sdiA</i>.

<p>A chromosomal merodiploid <i>acrA</i>⁺/<i>acrA</i>-<i>lacZY</i> fusion was constructed in a Δ<i>lac</i> mutant <i>E. coli</i> strain (JLD370), and in an isogenic <i>sdiA</i> mutant (JLD373). Additionally, derivatives of the <i>sdiA</i> mutant were constructed that contained either a low copy number vector expressing <i>sdiA</i> (pCX16) or the vector control (pGB2). The strains were subcultured 1:100 into LB broth containing either 1 µM oxoC6 or EA. The cultures were incubated with shaking at 30°C (A) and 37°C (B). Samples were removed from the cultures at time points for β-galactosidase assays. Each strain was assayed in triplicate and error bars represent standard deviation. * denotes p<0.05 compared to the adjacent solvent control.</p