E. coli K-12 and EHEC genes regulated by SdiA

Abstract

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 sdiA-dependence. 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 of AHL detection by E. coli.

Figure 1. Antibiotic resistance of S. Typhimurium, E. coli K-12, and EHEC as measured by E-Test strips.

The graphs show the MIC of the wild-type strains and their respective isogenic sdiA mutants (BA612, JNS21, and DL1, respectively). Each bar is the average of two separate experiments performed in triplicate and error bars represent standard deviation.

Figure 2. Antibiotic resistance of E. coli K-12, EHEC and S. Typhimurium grown in motility agar at 37°C.

Strains were grown in LB 0.3% motility agar with either 1 µM oxoC6 or 0.1% EA and a dilution series of each antibiotic tested. The minimum inhibitory concentration was read from the well in which no visible growth was seen at the inoculation point. In panel A, S. Typhimurium was not tested because the sdiA plasmid carries a gene for chloramphenicol resistance. Each strain was assayed in triplicate and error bars represent standard deviation.

Figure 3. Antibiotic resistance of E. coli K-12, EHEC and S. Typhimurium grown in motility agar at 30°C.

Strains were grown in LB 0.3% motility agar with either 1 µM oxoC6 or 0.1% EA and a dilution series of each antibiotic tested. The minimum inhibitory concentration was read from the well in which no visible growth was seen at the inoculation point. In panel A, S. Typhimurium was not tested because the sdiA plasmid carries a gene for chloramphenicol resistance. Each strain was assayed in triplicate and error bars represent standard deviation.

Figure 4. Regulation of acrA by sdiA.

A chromosomal merodiploid acrA ⁺/acrA-lacZY fusion was constructed in a Δlac mutant E. coli strain (JLD370), and in an isogenic sdiA mutant (JLD373). Additionally, derivatives of the sdiA mutant were constructed that contained either a low copy number vector expressing sdiA (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.

Figure 5. Regulation of ftsQAZ by sdiA.

A chromosomal ftsQAZ-lacZ fusion was constructed in a Δlac mutant E. coli strain (JLD3011), and an isogenic sdiA mutant (JLD3013). Additionally, derivatives of the sdiA mutant were constructed that contained either a low copy number vector expressing sdiA (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.

Figure 6. Regulation of AHL-regulated genes of E. coli K-12 and EHEC in motility agar containing either 100 nM oxo-C6 or the solvent control, EA at 37°C.

Luminescence in relative light units (RLU) was 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/JLD800 (gadW), B) JLD604/JLD803 (fliE), C) JLD605/JLD804 (gadE), D) JLD607/JLD806 (yhiD), E) JLD610/JLD809 (hdeA).

Figure 7

A) Acid fitness island of E. coli. The transposon insertion in E. coli K-12, AL4001, is within gadW 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 gadE at nucleotide 4401036; JLD607 is within yhiD at nucleotide 4397949; JLD610 is within hdeA at nucleotide 4398821. B) JLD604 is just upstream of ECs2675 in the anti-sense orientation at nucleotide 3662317.

Figure 8. Regulation of AHL-regulated genes of E. coli K-12 and EHEC in liquid cultures at 37°C containing either 1 µM oxo-C6 or the solvent control, EA.

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/JLD800 (gadW), B) JLD604/JLD803(fliE), C) JLD605/JLD804 (gadE), D) JLD607/JLD806 (yhiD), E) JLD610 /JLD809 (hdeA).

Figure 9. Regulation of AHL-regulated genes of E. coli K-12 and EHEC in liquid cultures at 30°C containing either 1 µM oxo-C6 or the solvent control, EA.

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/JLD800 (gadW), B) JLD604/JLD803 (fliE), C) JLD605/JLD804 (gadE), D) JLD607/JLD806 (yhiD), E) JLD610 /JLD809 (hdeA).

Figure 10. Cross streak assays of the E. coli K-12 and EHEC lux fusions.

The chromosomal lux fusions and their respective sdiA mutants were grown in broth overnight. The strains were dripped down the plate perpendicular to 20 µl of EA then 20 µl of 10 µM oxoC6 (diagrammed in Panel A for all panels). Plates were incubated at 37°C for 7 hours then light emission was imaged using a C2400-32 intensified charge-coupled device camera with an Argus 20 image processor. A) AL4001/JLD800 (gadW), B) JLD604/JLD803 (fliE), C) JLD605/JLD804 (gadE), D) JLD607/JLD806 (yhiD), E) JLD610 /JLD809 (hdeA).

Figure 11. Acid resistance of E. coli K-12 and EHEC.

Cells were grown in LB glucose with 1 µM oxo-C6 or 0.1% EA at either 37°C or 30°C and then subcultured into pre-warmed MEM with glucose and glutamate at pH 2.0 with continued incubation at the same temperature. Resistance to the acid challenge was determined by plating for cfu/ml every hour for two hours. E. coli K-12 wild-type MG1655 and sdiA mutant JNS21 at 37°C (A) and 30°C (B). EHEC wild-type 700927 and sdiA mutant DL1 at 37°C (C) and 30°C (D). Each strain was assayed in triplicate and error bars represent standard deviation.