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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

Growth curve analysis in the presence to methionine.Growth curves of P. aeruginosa wild-type PAO1 and mifSR mutants in M9 minimal media supplemented with glucose (30 mM) and methionine (5 mM) as carbon and nitrogen source.
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pone.0129629.g004: Growth curve analysis in the presence to methionine.Growth curves of P. aeruginosa wild-type PAO1 and mifSR mutants in M9 minimal media supplemented with glucose (30 mM) and methionine (5 mM) as carbon and nitrogen source.

Mentions: The PAO∆mifS, PAO∆mifR and PAO∆mifSR mutants exhibited no discernible phenotype compared to the parent PAO1 when tested for growth, swimming, swarming, twitching motility (LB media), pyocyanin production (LB & King’s A media), pyoverdine production (LB & King’s B Media), congo red binding assay (CR media) and antibiotic resistance (MH media) (Data not shown). Hence, a comparative phenotypic microarray analysis was performed with the wild-type PAO1, PAO∆mifR and PAO∆mifS mutants (BioLOG Inc.). Out of approximately 2000 metabolic and chemical sensitivity assays tested, PAO∆mifR exhibited four gain-of-function and 29 loss-of-function phenotypes whereas PAO∆mifS exhibited two gain-of-function and 23 loss-of-function phenotypes (Fig 3A). A single gain of function phenotype shared between PAO∆mifS and PAO∆mifR, was the ability to utilize L-methionine. When metabolism and chemical sensitivity were compared, the mutants appear more sensitive to various antibiotics (Fig 3B). However, none of these were reproducible in the lab in the MH media. The loss of mifS and mifR resulted in differential phenotype in the presence of six metabolites, amongst which, two were common to both mifS and mifR mutants (Fig 3B). The shared metabolic phenotypes involved the utilization of L-methionine and α-KG (Fig 3C). Compared to the parent PAO1, the mutants did not exhibit any growth increase when provided with L-methionine (Fig 4). This could be simply due to the difference in culture conditions and BioLOG proprietary media.


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)

Growth curve analysis in the presence to methionine.Growth curves of P. aeruginosa wild-type PAO1 and mifSR mutants in M9 minimal media supplemented with glucose (30 mM) and methionine (5 mM) as carbon and nitrogen source.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129629.g004: Growth curve analysis in the presence to methionine.Growth curves of P. aeruginosa wild-type PAO1 and mifSR mutants in M9 minimal media supplemented with glucose (30 mM) and methionine (5 mM) as carbon and nitrogen source.
Mentions: The PAO∆mifS, PAO∆mifR and PAO∆mifSR mutants exhibited no discernible phenotype compared to the parent PAO1 when tested for growth, swimming, swarming, twitching motility (LB media), pyocyanin production (LB & King’s A media), pyoverdine production (LB & King’s B Media), congo red binding assay (CR media) and antibiotic resistance (MH media) (Data not shown). Hence, a comparative phenotypic microarray analysis was performed with the wild-type PAO1, PAO∆mifR and PAO∆mifS mutants (BioLOG Inc.). Out of approximately 2000 metabolic and chemical sensitivity assays tested, PAO∆mifR exhibited four gain-of-function and 29 loss-of-function phenotypes whereas PAO∆mifS exhibited two gain-of-function and 23 loss-of-function phenotypes (Fig 3A). A single gain of function phenotype shared between PAO∆mifS and PAO∆mifR, was the ability to utilize L-methionine. When metabolism and chemical sensitivity were compared, the mutants appear more sensitive to various antibiotics (Fig 3B). However, none of these were reproducible in the lab in the MH media. The loss of mifS and mifR resulted in differential phenotype in the presence of six metabolites, amongst which, two were common to both mifS and mifR mutants (Fig 3B). The shared metabolic phenotypes involved the utilization of L-methionine and α-KG (Fig 3C). Compared to the parent PAO1, the mutants did not exhibit any growth increase when provided with L-methionine (Fig 4). This could be simply due to the difference in culture conditions and BioLOG proprietary media.

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