Limits...
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

Proposed model for α-KG utilization in P. aeruginosa.HK-MifS senses the extracellular α-KG to undergo phosphorylation. The phosphate is transferred to the RR-MifR. The phosphorylated MifR in coordination with RpoN (σ54) activates the expression of α-KG specific transporter gene PA5530. PA5530 thus enables the influx of α-KG to meet the metabolic and energy demands of the cells. PA5530 promoter (PPA5530) region has a Crc binding site (Fig 13), suggesting that it is under the catabolite repression control by Crc/CrcZ. The PPA5530 also shows the presence of another uncharacterized small non-coding asRNA indicating a multilayered and complex regulation of the α-KG transport system.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4482717&req=5

pone.0129629.g014: Proposed model for α-KG utilization in P. aeruginosa.HK-MifS senses the extracellular α-KG to undergo phosphorylation. The phosphate is transferred to the RR-MifR. The phosphorylated MifR in coordination with RpoN (σ54) activates the expression of α-KG specific transporter gene PA5530. PA5530 thus enables the influx of α-KG to meet the metabolic and energy demands of the cells. PA5530 promoter (PPA5530) region has a Crc binding site (Fig 13), suggesting that it is under the catabolite repression control by Crc/CrcZ. The PPA5530 also shows the presence of another uncharacterized small non-coding asRNA indicating a multilayered and complex regulation of the α-KG transport system.

Mentions: In eukaryotic cells, the mitochondria serve as a hub and reservoir of the TCA cycle and its intermediates, respectively. Bacterial pathogens can be highly virulent intruders of the host tissue, causing significant damage leading to cellular aberrations and injury. Mitochondrial dysfunction, a consequence of cell injury, results in efflux of TCA cycle intermediates leading to an increase in their extracellular concentrations [92]. It is known that TCA cycle intermediates (C4, C5, and C6 dicarboxylates) are present at micromolar (μM) concentrations in blood that increase with tissues damaged [26,92]. α-KG can also act as a reactive oxygen species scavenger, especially for hydrogen peroxide, protecting both host and pathogen [93]. For pathogenic bacteria such as P. aeruginosa, efficient uptake of TCA intermediates from the host is crucial for its survival, especially when it is bombarded with host reactive oxygen species, and requires the activity of bacterial carboxylate transport proteins. The transport proteins could be specific for C4, C5, and C6 intermediates and may use a cognate TCS. This study suggests a complex regulatory cascade in modulating P. aeruginosa C5-dicarboxylate, α-KG uptake involving the PA5530 transporter, the MifS/MifR TCS and the sigma factor RpoN (Fig 14). It appears that MifS senses the presence of α-KG and signals MifR. The activated MifR in concert with RpoN initiates the transcription of α-KG-specific transporter gene PA5530. Analyses of the published data suggests that the PA5530 promoter is under several layers of regulation including catabolite repression mediated by Crc/CrcZ [90] and the small non-coding asRNA [91]. Though the asRNA has been identified [91], it has not been characterized. It is not surprising that the PA5530 expression is potentially regulated by Crc, as it would allow control of transporter(s) in response to the presence of carbon sources in the environment.


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)

Proposed model for α-KG utilization in P. aeruginosa.HK-MifS senses the extracellular α-KG to undergo phosphorylation. The phosphate is transferred to the RR-MifR. The phosphorylated MifR in coordination with RpoN (σ54) activates the expression of α-KG specific transporter gene PA5530. PA5530 thus enables the influx of α-KG to meet the metabolic and energy demands of the cells. PA5530 promoter (PPA5530) region has a Crc binding site (Fig 13), suggesting that it is under the catabolite repression control by Crc/CrcZ. The PPA5530 also shows the presence of another uncharacterized small non-coding asRNA indicating a multilayered and complex regulation of the α-KG transport system.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129629.g014: Proposed model for α-KG utilization in P. aeruginosa.HK-MifS senses the extracellular α-KG to undergo phosphorylation. The phosphate is transferred to the RR-MifR. The phosphorylated MifR in coordination with RpoN (σ54) activates the expression of α-KG specific transporter gene PA5530. PA5530 thus enables the influx of α-KG to meet the metabolic and energy demands of the cells. PA5530 promoter (PPA5530) region has a Crc binding site (Fig 13), suggesting that it is under the catabolite repression control by Crc/CrcZ. The PPA5530 also shows the presence of another uncharacterized small non-coding asRNA indicating a multilayered and complex regulation of the α-KG transport system.
Mentions: In eukaryotic cells, the mitochondria serve as a hub and reservoir of the TCA cycle and its intermediates, respectively. Bacterial pathogens can be highly virulent intruders of the host tissue, causing significant damage leading to cellular aberrations and injury. Mitochondrial dysfunction, a consequence of cell injury, results in efflux of TCA cycle intermediates leading to an increase in their extracellular concentrations [92]. It is known that TCA cycle intermediates (C4, C5, and C6 dicarboxylates) are present at micromolar (μM) concentrations in blood that increase with tissues damaged [26,92]. α-KG can also act as a reactive oxygen species scavenger, especially for hydrogen peroxide, protecting both host and pathogen [93]. For pathogenic bacteria such as P. aeruginosa, efficient uptake of TCA intermediates from the host is crucial for its survival, especially when it is bombarded with host reactive oxygen species, and requires the activity of bacterial carboxylate transport proteins. The transport proteins could be specific for C4, C5, and C6 intermediates and may use a cognate TCS. This study suggests a complex regulatory cascade in modulating P. aeruginosa C5-dicarboxylate, α-KG uptake involving the PA5530 transporter, the MifS/MifR TCS and the sigma factor RpoN (Fig 14). It appears that MifS senses the presence of α-KG and signals MifR. The activated MifR in concert with RpoN initiates the transcription of α-KG-specific transporter gene PA5530. Analyses of the published data suggests that the PA5530 promoter is under several layers of regulation including catabolite repression mediated by Crc/CrcZ [90] and the small non-coding asRNA [91]. Though the asRNA has been identified [91], it has not been characterized. It is not surprising that the PA5530 expression is potentially regulated by Crc, as it would allow control of transporter(s) in response to the presence of carbon sources in the environment.

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