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

Dyszel JL, Soares JA, Swearingen MC, Lindsay A, Smith JN, Ahmer BM - PLoS ONE (2010)

Bottom Line: We found that genes encoding the glutamate-dependent acid resistance system are up-regulated, and fliE is down-regulated, by sdiA.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, The Ohio State University, Columbus, Ohio, United States of America.

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.

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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).
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pone-0008946-g006: 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).

Mentions: One mutant in E. coli K-12 and four in EHEC showed a consistent change in luciferase expression in the presence of oxoC6 compared to the solvent control, EA. One of the fusions was down-regulated 7-fold by oxoC6 while four were up-regulated by up to 7-fold (Figure 6). The DNA sequence of each transposon insertion site was determined using the mutant chromosomal DNA as the template and two different sequencing primers that bind within the transposon sequence (primers are listed in Table 2). The two sequencing reactions yielded the same result in each case with regard to the transposon insertion point (Figure 7). The single AHL-regulated fusion in E. coli K-12 (AL4001) was inserted in the gadW gene. In EHEC there were three transposon-based fusions that were up-regulated in response to AHL. JLD605 contained an insertion in ECs4392 (ortholog of E. coli K-12 gadE/yhiE); JLD607 contained an insertion in ECs4388 (ortholog of E. coli K-12 yhiD); and JLD610 contained an insertion in ECs4390 (ortholog of E. coli K-12 hdeA). The one fusion in EHEC that was down-regulated, JLD604, was inserted just upstream of ECs2675, encoding a hypothetical protein, but the transposon orientation was anti-sense suggesting that expression was being driven from the ECs2676 gene promoter (ortholog of E. coli K-12 fliE). Because of the unusual location of the fusion in JLD604, we cloned the fliE promoter region into pSB401 to form a fliE-luxCDABE transcriptional fusion. When this construct, pJLD1203, was placed into wild-type and sdiA mutant E. coli, we observed that the fliE promoter is indeed repressed by sdiA (data not shown).


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

Dyszel JL, Soares JA, Swearingen MC, Lindsay A, Smith JN, Ahmer BM - PLoS ONE (2010)

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).
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2812512&req=5

pone-0008946-g006: 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).
Mentions: One mutant in E. coli K-12 and four in EHEC showed a consistent change in luciferase expression in the presence of oxoC6 compared to the solvent control, EA. One of the fusions was down-regulated 7-fold by oxoC6 while four were up-regulated by up to 7-fold (Figure 6). The DNA sequence of each transposon insertion site was determined using the mutant chromosomal DNA as the template and two different sequencing primers that bind within the transposon sequence (primers are listed in Table 2). The two sequencing reactions yielded the same result in each case with regard to the transposon insertion point (Figure 7). The single AHL-regulated fusion in E. coli K-12 (AL4001) was inserted in the gadW gene. In EHEC there were three transposon-based fusions that were up-regulated in response to AHL. JLD605 contained an insertion in ECs4392 (ortholog of E. coli K-12 gadE/yhiE); JLD607 contained an insertion in ECs4388 (ortholog of E. coli K-12 yhiD); and JLD610 contained an insertion in ECs4390 (ortholog of E. coli K-12 hdeA). The one fusion in EHEC that was down-regulated, JLD604, was inserted just upstream of ECs2675, encoding a hypothetical protein, but the transposon orientation was anti-sense suggesting that expression was being driven from the ECs2676 gene promoter (ortholog of E. coli K-12 fliE). Because of the unusual location of the fusion in JLD604, we cloned the fliE promoter region into pSB401 to form a fliE-luxCDABE transcriptional fusion. When this construct, pJLD1203, was placed into wild-type and sdiA mutant E. coli, we observed that the fliE promoter is indeed repressed by sdiA (data not shown).

Bottom Line: We found that genes encoding the glutamate-dependent acid resistance system are up-regulated, and fliE is down-regulated, by sdiA.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.

View Article: PubMed Central - PubMed

Affiliation: Department of Microbiology, The Ohio State University, Columbus, Ohio, United States of America.

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.

Show MeSH
Related in: MedlinePlus