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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

In silico analysis of mifS (PmifS) and PA5530 (PPA5530) promoter sequences.Motif search was done using the ensemble learning method SCOPE and GLAM2 (Gapped Local Alignment of Motifs) [113,114]. (A) Sequence analysis of the 81-bp (PmifS) (black) indicates a putative σ70-dependent -10 consensus (TATAAT). However, it lacks the -35 consensus (TTGACA) for σ70 promoter [80]. Arrows represent the long 17-bp direct and inverted repeats in PmifS with a consensus GGAt/cAGCGACATCGGCG. (B) The 315-bp promoter region of PA5530 showing strong -12 and -24 σ54-dependent promoter like element and the proposed transcription start site (+1). Dashed line (blue) depicts a common motif in PmifS and PPA5530 suggesting a common regulatory mechanism (A and B). The three pairs of direct repeats in PPA5530 are represented by green, blue and orange arrows. PPA5530 possess the signature sequence (AAc/uAAc/uAA) for catabolite repression control (Crc) protein (brown box) [90]. The uncharacterized small antisense RNA (asRNA) identified in the PPA5530 region [91] is indicated by marked line.
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pone.0129629.g013: In silico analysis of mifS (PmifS) and PA5530 (PPA5530) promoter sequences.Motif search was done using the ensemble learning method SCOPE and GLAM2 (Gapped Local Alignment of Motifs) [113,114]. (A) Sequence analysis of the 81-bp (PmifS) (black) indicates a putative σ70-dependent -10 consensus (TATAAT). However, it lacks the -35 consensus (TTGACA) for σ70 promoter [80]. Arrows represent the long 17-bp direct and inverted repeats in PmifS with a consensus GGAt/cAGCGACATCGGCG. (B) The 315-bp promoter region of PA5530 showing strong -12 and -24 σ54-dependent promoter like element and the proposed transcription start site (+1). Dashed line (blue) depicts a common motif in PmifS and PPA5530 suggesting a common regulatory mechanism (A and B). The three pairs of direct repeats in PPA5530 are represented by green, blue and orange arrows. PPA5530 possess the signature sequence (AAc/uAAc/uAA) for catabolite repression control (Crc) protein (brown box) [90]. The uncharacterized small antisense RNA (asRNA) identified in the PPA5530 region [91] is indicated by marked line.

Mentions: The small 81-bp mifSR promoter has no obvious RpoN sigma factor -12/-24 consensus sequence: 5’-TGGCACG-N4-TTGCW-3’ in which W stands for either A or T (Fig 13A) [53]. In fact, it appears to have a potential -10 (consensus: TATAAT) but lacked -35 (consensus: TTGACA) for sigma-70 promoter (Fig 13A) [54]. On the other hand, the promoter region of PA5530 is 315-bp long with strong -12 and -24 boxes upstream of the predicted transcription start site (Fig 13B). We hypothesized that the inability of rpoN mutant to utilize α-KG can be rescued by expressing PA5530 under a regulatable promoter PlacUV5. As expected, the growth of the rpoN mutant was restored when the plasmid harboring the transporter PA5530 was expressed in trans (Table 3). This suggests that expression of PA5530 requires both MifSR TCS and RpoN.


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)

In silico analysis of mifS (PmifS) and PA5530 (PPA5530) promoter sequences.Motif search was done using the ensemble learning method SCOPE and GLAM2 (Gapped Local Alignment of Motifs) [113,114]. (A) Sequence analysis of the 81-bp (PmifS) (black) indicates a putative σ70-dependent -10 consensus (TATAAT). However, it lacks the -35 consensus (TTGACA) for σ70 promoter [80]. Arrows represent the long 17-bp direct and inverted repeats in PmifS with a consensus GGAt/cAGCGACATCGGCG. (B) The 315-bp promoter region of PA5530 showing strong -12 and -24 σ54-dependent promoter like element and the proposed transcription start site (+1). Dashed line (blue) depicts a common motif in PmifS and PPA5530 suggesting a common regulatory mechanism (A and B). The three pairs of direct repeats in PPA5530 are represented by green, blue and orange arrows. PPA5530 possess the signature sequence (AAc/uAAc/uAA) for catabolite repression control (Crc) protein (brown box) [90]. The uncharacterized small antisense RNA (asRNA) identified in the PPA5530 region [91] is indicated by marked line.
© Copyright Policy
Related In: Results  -  Collection

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

pone.0129629.g013: In silico analysis of mifS (PmifS) and PA5530 (PPA5530) promoter sequences.Motif search was done using the ensemble learning method SCOPE and GLAM2 (Gapped Local Alignment of Motifs) [113,114]. (A) Sequence analysis of the 81-bp (PmifS) (black) indicates a putative σ70-dependent -10 consensus (TATAAT). However, it lacks the -35 consensus (TTGACA) for σ70 promoter [80]. Arrows represent the long 17-bp direct and inverted repeats in PmifS with a consensus GGAt/cAGCGACATCGGCG. (B) The 315-bp promoter region of PA5530 showing strong -12 and -24 σ54-dependent promoter like element and the proposed transcription start site (+1). Dashed line (blue) depicts a common motif in PmifS and PPA5530 suggesting a common regulatory mechanism (A and B). The three pairs of direct repeats in PPA5530 are represented by green, blue and orange arrows. PPA5530 possess the signature sequence (AAc/uAAc/uAA) for catabolite repression control (Crc) protein (brown box) [90]. The uncharacterized small antisense RNA (asRNA) identified in the PPA5530 region [91] is indicated by marked line.
Mentions: The small 81-bp mifSR promoter has no obvious RpoN sigma factor -12/-24 consensus sequence: 5’-TGGCACG-N4-TTGCW-3’ in which W stands for either A or T (Fig 13A) [53]. In fact, it appears to have a potential -10 (consensus: TATAAT) but lacked -35 (consensus: TTGACA) for sigma-70 promoter (Fig 13A) [54]. On the other hand, the promoter region of PA5530 is 315-bp long with strong -12 and -24 boxes upstream of the predicted transcription start site (Fig 13B). We hypothesized that the inability of rpoN mutant to utilize α-KG can be rescued by expressing PA5530 under a regulatable promoter PlacUV5. As expected, the growth of the rpoN mutant was restored when the plasmid harboring the transporter PA5530 was expressed in trans (Table 3). This suggests that expression of PA5530 requires both MifSR TCS and RpoN.

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