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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 poxB (PA5514) in mifSR mutants.Expression of poxB (PA5514) was tested in mifSR mutants relative to PAO1. Data was normalized to expression in PAO1. 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. There was no statistically significant difference (p-value > 0.05) between the wild type PAO1 and mifSR mutant strains as determined by one-way ANOVA and student’s unpaired t test.
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pone.0129629.g002: Expression of poxB (PA5514) in mifSR mutants.Expression of poxB (PA5514) was tested in mifSR mutants relative to PAO1. Data was normalized to expression in PAO1. 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. There was no statistically significant difference (p-value > 0.05) between the wild type PAO1 and mifSR mutant strains as determined by one-way ANOVA and student’s unpaired t test.

Mentions: Previous studies in our lab postulated that the MifSR TCS system, found 81-bp upstream of the pox operon, may contribute to P. aeruginosa ß-lactam resistance [43] as the genes regulated by TCS tend to be co-located on the chromosome [30]. However, MIC analyses using E-test and micro-dilution methods showed that the loss of these genes did not affect the antibiotic resistance profile when compared to the parent strain, P. aeruginosa PAO1(Data not shown). Further, qRT-PCR studies showed that deletion of mifS, mifR and mifSR had no effect on the expression of poxB compared to the parent PAO1 (Fig 2).


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 poxB (PA5514) in mifSR mutants.Expression of poxB (PA5514) was tested in mifSR mutants relative to PAO1. Data was normalized to expression in PAO1. 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. There was no statistically significant difference (p-value > 0.05) between the wild type PAO1 and mifSR mutant strains as determined by one-way ANOVA and student’s unpaired t test.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129629.g002: Expression of poxB (PA5514) in mifSR mutants.Expression of poxB (PA5514) was tested in mifSR mutants relative to PAO1. Data was normalized to expression in PAO1. 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. There was no statistically significant difference (p-value > 0.05) between the wild type PAO1 and mifSR mutant strains as determined by one-way ANOVA and student’s unpaired t test.
Mentions: Previous studies in our lab postulated that the MifSR TCS system, found 81-bp upstream of the pox operon, may contribute to P. aeruginosa ß-lactam resistance [43] as the genes regulated by TCS tend to be co-located on the chromosome [30]. However, MIC analyses using E-test and micro-dilution methods showed that the loss of these genes did not affect the antibiotic resistance profile when compared to the parent strain, P. aeruginosa PAO1(Data not shown). Further, qRT-PCR studies showed that deletion of mifS, mifR and mifSR had no effect on the expression of poxB compared to the parent PAO1 (Fig 2).

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