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Pseudomonas aeruginosa MifS-MifR Two-Component System Is Specific for α-Ketoglutarate Utilization.

Tatke G, Kumari H, Silva-Herzog E, Ramirez L, Mathee K - PLoS ONE (2015)

Bottom Line: The loss of mifSR had no effect on the antibiotic resistance profile.We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport.These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, College of Arts & Sciences, Florida International University, Miami, Florida, United States of America; Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States of America.

ABSTRACT
Pseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (σ54). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a ß-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO∆mifS, PAO∆mifR and PAO∆mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO∆mifS and PAO∆mifR revealed that these mutants were unable to utilize C5-dicarboxylate α-ketoglutarate (α-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except α-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport. The inability of the mutants to utilize α-KG was rescued by expressing PA5530, encoding C5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, α-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

No MeSH data available.


Related in: MedlinePlus

Expression of PA5530 in response to α-KG.PA5530 gene expression was determined in the wild type PAO1 with varying concentrations of α-KG (1 h) (A). In addition, the expression of PA5530 was tested in mifSR mutants relative to PAO1, with cells exposed to 30 mM α-KG for 1 h (B). Data was normalized to expression in PAO1 under the respective conditions. Bars above or below the line represents up- and down-regulation, respectively and the bars indicate standard errors. The clpX gene (PA1802) was used as the housekeeping control. Statistically significant difference between the wild type and mutants as determined by one-way ANOVA with Bonferroni’s post-hoc test, ** p-value < 0.001.
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pone.0129629.g011: Expression of PA5530 in response to α-KG.PA5530 gene expression was determined in the wild type PAO1 with varying concentrations of α-KG (1 h) (A). In addition, the expression of PA5530 was tested in mifSR mutants relative to PAO1, with cells exposed to 30 mM α-KG for 1 h (B). Data was normalized to expression in PAO1 under the respective conditions. Bars above or below the line represents up- and down-regulation, respectively and the bars indicate standard errors. The clpX gene (PA1802) was used as the housekeeping control. Statistically significant difference between the wild type and mutants as determined by one-way ANOVA with Bonferroni’s post-hoc test, ** p-value < 0.001.

Mentions: In a recent study using transposon mutagenesis; PA5530 was identified as the functional α-KG transporter [48]. To confirm the role of P. aeruginosa PA5530 in α-KG uptake and identify the role of mifSR genes, the gene was amplified and subcloned downstream of the inducible PlacUV5 promoter. The plasmid pPA5530 was introduced into PAO1 and the mifSR mutants. Expression of PA5530 in trans in PAO∆mifS, PAO∆mifR, PAO∆mifSR mutants restored their growth to a level similar to the wild-type PAO1 in M9 minimal media with α-KG (30 mM) as the sole carbon source (Fig 7B). Expression of an extra copy of PA5530 gene in the wild-type PAO1 did not affect its growth (Fig 5E). This finding suggests that expression of PA5530 is likely regulated by MifSR and/or α-KG. In fact, expression of PA5530 is regulated by α-KG, as seen in qRT-PCR analysis when PAO1 was grown in M9 media with varying amounts α-KG (Fig 11A). The loss of mifS, mifR and mifSR results in a significant decrease in PA5530 expression as compared to the wild type PAO1 in the presence of α-KG (Fig 11B). Thus, α-KG-dependent PA5530 expression requires MifS and MifR.


Pseudomonas aeruginosa MifS-MifR Two-Component System Is Specific for α-Ketoglutarate Utilization.

Tatke G, Kumari H, Silva-Herzog E, Ramirez L, Mathee K - PLoS ONE (2015)

Expression of PA5530 in response to α-KG.PA5530 gene expression was determined in the wild type PAO1 with varying concentrations of α-KG (1 h) (A). In addition, the expression of PA5530 was tested in mifSR mutants relative to PAO1, with cells exposed to 30 mM α-KG for 1 h (B). Data was normalized to expression in PAO1 under the respective conditions. Bars above or below the line represents up- and down-regulation, respectively and the bars indicate standard errors. The clpX gene (PA1802) was used as the housekeeping control. Statistically significant difference between the wild type and mutants as determined by one-way ANOVA with Bonferroni’s post-hoc test, ** p-value < 0.001.
© Copyright Policy
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4482717&req=5

pone.0129629.g011: Expression of PA5530 in response to α-KG.PA5530 gene expression was determined in the wild type PAO1 with varying concentrations of α-KG (1 h) (A). In addition, the expression of PA5530 was tested in mifSR mutants relative to PAO1, with cells exposed to 30 mM α-KG for 1 h (B). Data was normalized to expression in PAO1 under the respective conditions. Bars above or below the line represents up- and down-regulation, respectively and the bars indicate standard errors. The clpX gene (PA1802) was used as the housekeeping control. Statistically significant difference between the wild type and mutants as determined by one-way ANOVA with Bonferroni’s post-hoc test, ** p-value < 0.001.
Mentions: In a recent study using transposon mutagenesis; PA5530 was identified as the functional α-KG transporter [48]. To confirm the role of P. aeruginosa PA5530 in α-KG uptake and identify the role of mifSR genes, the gene was amplified and subcloned downstream of the inducible PlacUV5 promoter. The plasmid pPA5530 was introduced into PAO1 and the mifSR mutants. Expression of PA5530 in trans in PAO∆mifS, PAO∆mifR, PAO∆mifSR mutants restored their growth to a level similar to the wild-type PAO1 in M9 minimal media with α-KG (30 mM) as the sole carbon source (Fig 7B). Expression of an extra copy of PA5530 gene in the wild-type PAO1 did not affect its growth (Fig 5E). This finding suggests that expression of PA5530 is likely regulated by MifSR and/or α-KG. In fact, expression of PA5530 is regulated by α-KG, as seen in qRT-PCR analysis when PAO1 was grown in M9 media with varying amounts α-KG (Fig 11A). The loss of mifS, mifR and mifSR results in a significant decrease in PA5530 expression as compared to the wild type PAO1 in the presence of α-KG (Fig 11B). Thus, α-KG-dependent PA5530 expression requires MifS and MifR.

Bottom Line: The loss of mifSR had no effect on the antibiotic resistance profile.We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport.These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

View Article: PubMed Central - PubMed

Affiliation: Department of Biological Sciences, College of Arts & Sciences, Florida International University, Miami, Florida, United States of America; Department of Molecular Microbiology and Infectious Diseases, Herbert Wertheim College of Medicine, Florida International University, Miami, Florida, United States of America.

ABSTRACT
Pseudomonas aeruginosa is a Gram-negative, metabolically versatile opportunistic pathogen that elaborates a multitude of virulence factors, and is extraordinarily resistant to a gamut of clinically significant antibiotics. This ability, in part, is mediated by two-component regulatory systems (TCS) that play a crucial role in modulating virulence mechanisms and metabolism. MifS (PA5512) and MifR (PA5511) form one such TCS implicated in biofilm formation. MifS is a sensor kinase whereas MifR belongs to the NtrC superfamily of transcriptional regulators that interact with RpoN (σ54). In this study we demonstrate that the mifS and mifR genes form a two-gene operon. The close proximity of mifSR operon to poxB (PA5514) encoding a ß-lactamase hinted at the role of MifSR TCS in regulating antibiotic resistance. To better understand this TCS, clean in-frame deletions were made in P. aeruginosa PAO1 creating PAO∆mifS, PAO∆mifR and PAO∆mifSR. The loss of mifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAO∆mifS and PAO∆mifR revealed that these mutants were unable to utilize C5-dicarboxylate α-ketoglutarate (α-KG), a key tricarboxylic acid cycle intermediate. This finding was confirmed using growth analyses, and the defect can be rescued by mifR or mifSR expressed in trans. These mifSR mutants were able to utilize all the other TCA cycle intermediates (citrate, succinate, fumarate, oxaloacetate or malate) and sugars (glucose or sucrose) except α-KG as the sole carbon source. We confirmed that the mifSR mutants have functional dehydrogenase complex suggesting a possible defect in α-KG transport. The inability of the mutants to utilize α-KG was rescued by expressing PA5530, encoding C5-dicarboxylate transporter, under a regulatable promoter. In addition, we demonstrate that besides MifSR and PA5530, α-KG utilization requires functional RpoN. These data clearly suggests that P. aeruginosa MifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism.

No MeSH data available.


Related in: MedlinePlus