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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

Genome organization of the mifSR gene locus.In P. aeruginosa PAO1 the mifR (PA5511) ORF has a translation start codon (ATG) overlapping the mifS (PA5512) termination codon (TGA), denoted in red (B), suggesting that the mifS and mifR genes are physically linked. The cDNA amplification of the intergenic region spanning the mifS and mifR genes using GDT_cotrans F1-R1 and GDT_cotrans F2-R2 primers (Table 1) confirm that the two genes mifS and mifR are co-transcribed and form an operon (C).
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pone.0129629.g001: Genome organization of the mifSR gene locus.In P. aeruginosa PAO1 the mifR (PA5511) ORF has a translation start codon (ATG) overlapping the mifS (PA5512) termination codon (TGA), denoted in red (B), suggesting that the mifS and mifR genes are physically linked. The cDNA amplification of the intergenic region spanning the mifS and mifR genes using GDT_cotrans F1-R1 and GDT_cotrans F2-R2 primers (Table 1) confirm that the two genes mifS and mifR are co-transcribed and form an operon (C).

Mentions: In eubacteria, the genes that encode a HK and its cognate RR are often linked and are co-transcribed [30]. Our sequence analysis of P. aeruginosa PAO1 genome revealed that mifS (PA5512) and mifR (PA5511) are adjacent to each other, in the same orientation. The predicted translation start site of mifR ORF overlaps with mifS translation termination codon indicating that they are cotranscribed (Fig 1A and 1B). To determine if these two genes form an operon, cDNA across the intergenic regions spanning mifS and mifR was amplified using GDT_cotransF1-R1 and GDT_cotransF2-R2 primers (see Materials and Methods). As expected, 200 bp and 100 bp products were detected when using primers that span the overlapping region (Fig 1C, Lane 3 and Lane 4). These results confirm that mifS and mifR are a part of a two-gene operon. As controls, the mifSR genes were also amplified (Fig 1C, Lane 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)

Genome organization of the mifSR gene locus.In P. aeruginosa PAO1 the mifR (PA5511) ORF has a translation start codon (ATG) overlapping the mifS (PA5512) termination codon (TGA), denoted in red (B), suggesting that the mifS and mifR genes are physically linked. The cDNA amplification of the intergenic region spanning the mifS and mifR genes using GDT_cotrans F1-R1 and GDT_cotrans F2-R2 primers (Table 1) confirm that the two genes mifS and mifR are co-transcribed and form an operon (C).
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129629.g001: Genome organization of the mifSR gene locus.In P. aeruginosa PAO1 the mifR (PA5511) ORF has a translation start codon (ATG) overlapping the mifS (PA5512) termination codon (TGA), denoted in red (B), suggesting that the mifS and mifR genes are physically linked. The cDNA amplification of the intergenic region spanning the mifS and mifR genes using GDT_cotrans F1-R1 and GDT_cotrans F2-R2 primers (Table 1) confirm that the two genes mifS and mifR are co-transcribed and form an operon (C).
Mentions: In eubacteria, the genes that encode a HK and its cognate RR are often linked and are co-transcribed [30]. Our sequence analysis of P. aeruginosa PAO1 genome revealed that mifS (PA5512) and mifR (PA5511) are adjacent to each other, in the same orientation. The predicted translation start site of mifR ORF overlaps with mifS translation termination codon indicating that they are cotranscribed (Fig 1A and 1B). To determine if these two genes form an operon, cDNA across the intergenic regions spanning mifS and mifR was amplified using GDT_cotransF1-R1 and GDT_cotransF2-R2 primers (see Materials and Methods). As expected, 200 bp and 100 bp products were detected when using primers that span the overlapping region (Fig 1C, Lane 3 and Lane 4). These results confirm that mifS and mifR are a part of a two-gene operon. As controls, the mifSR genes were also amplified (Fig 1C, Lane 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