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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

P. aeruginosa MifR domain organization and sequence alignment.(A) MifR domain organization determined using the Simple Modular Architecture Research Tool (SMART) [51]. MifR is a sigma-54 dependent transcriptional activator [57]. There are three functional domains, N-terminal receiver with the conserved aspartate residue at position 53 (Asp-53) (Purple), central AAA+ ATPase, characteristic of sigma-54 dependent activation proteins (Green), and the C-terminal helix-turn-helix (HTH) DNA binding (Red) domains. (B) Sequence alignment of MifR with P. aeruginosa DctD (PA5166), NtrC (PA5125) and R. meliloti DctD. Vertical bars indicate conserved residues, asterisk (*) indicate residues are identical at that position. Key residues of the central AAA+ domain (C1 to C7) are well conserved amongst sigma-54 dependent transcriptional activators. The horizontal arrow bars indicate HTH domain. Asp-53 indicates the conserved phosphorylation site of P. aeruginosa MifR. The alignment was generated using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
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pone.0129629.g012: P. aeruginosa MifR domain organization and sequence alignment.(A) MifR domain organization determined using the Simple Modular Architecture Research Tool (SMART) [51]. MifR is a sigma-54 dependent transcriptional activator [57]. There are three functional domains, N-terminal receiver with the conserved aspartate residue at position 53 (Asp-53) (Purple), central AAA+ ATPase, characteristic of sigma-54 dependent activation proteins (Green), and the C-terminal helix-turn-helix (HTH) DNA binding (Red) domains. (B) Sequence alignment of MifR with P. aeruginosa DctD (PA5166), NtrC (PA5125) and R. meliloti DctD. Vertical bars indicate conserved residues, asterisk (*) indicate residues are identical at that position. Key residues of the central AAA+ domain (C1 to C7) are well conserved amongst sigma-54 dependent transcriptional activators. The horizontal arrow bars indicate HTH domain. Asp-53 indicates the conserved phosphorylation site of P. aeruginosa MifR. The alignment was generated using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).

Mentions: The closest P. aeruginosa MifS and MifR homologs are R. meliloti DctB and DctD [40]. In fact, MifR is 69% similar to R. meliloti DctD that belongs to the Sigma 54 (σ54) dependent NtrC family of transcriptional regulators [39,40]. Thus, it is likely that MifR has the conserved domains found among NtrC family of regulators, an N-terminal regulatory, a central σ54 activation and a C-terminal DNA binding domains [49,50]. MifR analysis using the simple modular architecture research tool (SMART) [51] and InterPro [52] revealed the presence of three domains: CheY-homologous receiver/regulatory, a central AAA+ region required for σ54 activation, and the DNA binding helix-turn-helix domains (Fig 12A). The central AAA+ domain contains seven conserved regions designated C1 to C7 [50] that are characteristic of σ54- dependent transcriptional regulators. Sequence analysis of MifR revealed the presence of all the seven conserved regions in the AAA+ domain between amino acid residues 144 to 373 (Fig 12B).


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)

P. aeruginosa MifR domain organization and sequence alignment.(A) MifR domain organization determined using the Simple Modular Architecture Research Tool (SMART) [51]. MifR is a sigma-54 dependent transcriptional activator [57]. There are three functional domains, N-terminal receiver with the conserved aspartate residue at position 53 (Asp-53) (Purple), central AAA+ ATPase, characteristic of sigma-54 dependent activation proteins (Green), and the C-terminal helix-turn-helix (HTH) DNA binding (Red) domains. (B) Sequence alignment of MifR with P. aeruginosa DctD (PA5166), NtrC (PA5125) and R. meliloti DctD. Vertical bars indicate conserved residues, asterisk (*) indicate residues are identical at that position. Key residues of the central AAA+ domain (C1 to C7) are well conserved amongst sigma-54 dependent transcriptional activators. The horizontal arrow bars indicate HTH domain. Asp-53 indicates the conserved phosphorylation site of P. aeruginosa MifR. The alignment was generated using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129629.g012: P. aeruginosa MifR domain organization and sequence alignment.(A) MifR domain organization determined using the Simple Modular Architecture Research Tool (SMART) [51]. MifR is a sigma-54 dependent transcriptional activator [57]. There are three functional domains, N-terminal receiver with the conserved aspartate residue at position 53 (Asp-53) (Purple), central AAA+ ATPase, characteristic of sigma-54 dependent activation proteins (Green), and the C-terminal helix-turn-helix (HTH) DNA binding (Red) domains. (B) Sequence alignment of MifR with P. aeruginosa DctD (PA5166), NtrC (PA5125) and R. meliloti DctD. Vertical bars indicate conserved residues, asterisk (*) indicate residues are identical at that position. Key residues of the central AAA+ domain (C1 to C7) are well conserved amongst sigma-54 dependent transcriptional activators. The horizontal arrow bars indicate HTH domain. Asp-53 indicates the conserved phosphorylation site of P. aeruginosa MifR. The alignment was generated using ClustalW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/).
Mentions: The closest P. aeruginosa MifS and MifR homologs are R. meliloti DctB and DctD [40]. In fact, MifR is 69% similar to R. meliloti DctD that belongs to the Sigma 54 (σ54) dependent NtrC family of transcriptional regulators [39,40]. Thus, it is likely that MifR has the conserved domains found among NtrC family of regulators, an N-terminal regulatory, a central σ54 activation and a C-terminal DNA binding domains [49,50]. MifR analysis using the simple modular architecture research tool (SMART) [51] and InterPro [52] revealed the presence of three domains: CheY-homologous receiver/regulatory, a central AAA+ region required for σ54 activation, and the DNA binding helix-turn-helix domains (Fig 12A). The central AAA+ domain contains seven conserved regions designated C1 to C7 [50] that are characteristic of σ54- dependent transcriptional regulators. Sequence analysis of MifR revealed the presence of all the seven conserved regions in the AAA+ domain between amino acid residues 144 to 373 (Fig 12B).

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