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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

Quantification of rpoN, acnA, idh, icd, sucA, and Ipd3 mRNA by qRT-PCR.RNA was isolated from cells grown in M9 minimal media supplemented with citrate (30 mM), reverse transcribed to cDNA and the presence of specific transcripts was analyzed by qPCR using gene-specific primers (Table 5). The expression of genes encoding aconitate hydratase 1 (acnA (PA1562)) isocitrate dehydrogenase (idh (PA2623)) isocitrate dehydrogenase, α-KG dehydrogenase complex (icd (PA2623)), sucA (PA1585) and lpd3 (PA4829), and σ54 (rpoN (PA4462)) were analyzed in mifSR mutants relative to PAO1 (log10 RQ = 1). Bars above or below the line represents up- and down-regulation, respectively and the bars are standard errors. The clpX (PA1802) gene 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. Difference in the expression levels of genes is not statistically significant at p-value < 0.05.
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pone.0129629.g010: Quantification of rpoN, acnA, idh, icd, sucA, and Ipd3 mRNA by qRT-PCR.RNA was isolated from cells grown in M9 minimal media supplemented with citrate (30 mM), reverse transcribed to cDNA and the presence of specific transcripts was analyzed by qPCR using gene-specific primers (Table 5). The expression of genes encoding aconitate hydratase 1 (acnA (PA1562)) isocitrate dehydrogenase (idh (PA2623)) isocitrate dehydrogenase, α-KG dehydrogenase complex (icd (PA2623)), sucA (PA1585) and lpd3 (PA4829), and σ54 (rpoN (PA4462)) were analyzed in mifSR mutants relative to PAO1 (log10 RQ = 1). Bars above or below the line represents up- and down-regulation, respectively and the bars are standard errors. The clpX (PA1802) gene 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. Difference in the expression levels of genes is not statistically significant at p-value < 0.05.

Mentions: The absence of growth in the presence of exogenous α-KG could be due to either failure to enter the cells or loss of the mutants’ ability to convert α-KG to succinate. The latter is likely if the mutants failed to express a functional α-KG dehydrogenase complex. The ability of mifSR mutants to grow effectively in the presence of citrate and succinate suggests that these mutants are likely to harbor a functional α-KG dehydrogenase complex, unless the mutants bypass it using the glyoxylate shunt (Fig 8). The former is likely as qPCR analysis of genes encoding isocitrate dehydrogenase (idh, icd) and α-KG dehydrogenase complex (sucA, sucB, lpd3) revealed no difference in the expression levels in the wild-type PAO1 and mifSR mutants (Fig 10).


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)

Quantification of rpoN, acnA, idh, icd, sucA, and Ipd3 mRNA by qRT-PCR.RNA was isolated from cells grown in M9 minimal media supplemented with citrate (30 mM), reverse transcribed to cDNA and the presence of specific transcripts was analyzed by qPCR using gene-specific primers (Table 5). The expression of genes encoding aconitate hydratase 1 (acnA (PA1562)) isocitrate dehydrogenase (idh (PA2623)) isocitrate dehydrogenase, α-KG dehydrogenase complex (icd (PA2623)), sucA (PA1585) and lpd3 (PA4829), and σ54 (rpoN (PA4462)) were analyzed in mifSR mutants relative to PAO1 (log10 RQ = 1). Bars above or below the line represents up- and down-regulation, respectively and the bars are standard errors. The clpX (PA1802) gene 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. Difference in the expression levels of genes is not statistically significant at p-value < 0.05.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129629.g010: Quantification of rpoN, acnA, idh, icd, sucA, and Ipd3 mRNA by qRT-PCR.RNA was isolated from cells grown in M9 minimal media supplemented with citrate (30 mM), reverse transcribed to cDNA and the presence of specific transcripts was analyzed by qPCR using gene-specific primers (Table 5). The expression of genes encoding aconitate hydratase 1 (acnA (PA1562)) isocitrate dehydrogenase (idh (PA2623)) isocitrate dehydrogenase, α-KG dehydrogenase complex (icd (PA2623)), sucA (PA1585) and lpd3 (PA4829), and σ54 (rpoN (PA4462)) were analyzed in mifSR mutants relative to PAO1 (log10 RQ = 1). Bars above or below the line represents up- and down-regulation, respectively and the bars are standard errors. The clpX (PA1802) gene 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. Difference in the expression levels of genes is not statistically significant at p-value < 0.05.
Mentions: The absence of growth in the presence of exogenous α-KG could be due to either failure to enter the cells or loss of the mutants’ ability to convert α-KG to succinate. The latter is likely if the mutants failed to express a functional α-KG dehydrogenase complex. The ability of mifSR mutants to grow effectively in the presence of citrate and succinate suggests that these mutants are likely to harbor a functional α-KG dehydrogenase complex, unless the mutants bypass it using the glyoxylate shunt (Fig 8). The former is likely as qPCR analysis of genes encoding isocitrate dehydrogenase (idh, icd) and α-KG dehydrogenase complex (sucA, sucB, lpd3) revealed no difference in the expression levels in the wild-type PAO1 and mifSR mutants (Fig 10).

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