Pseudomonas aeruginosa MifS-Mif R Two-Component System Is Specific for alpha-Ketoglutarate Utilization

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 (σ). In this study we demonstrate that themifS andmifR genes form a two-gene operon. The close proximity ofmifSR 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 PAOΔmifS, PAOΔmifR and PAOΔmifSR. The loss ofmifSR had no effect on the antibiotic resistance profile. Phenotypic microarray (BioLOG) analyses of PAOΔmifS and PAOΔmifR 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 bymifR ormifSR expressed in trans. ThesemifSRmutants 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 themifSRmutants 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 thatP. aeruginosaMifSR TCS is involved in sensing α-KG and regulating its transport and subsequent metabolism. PLOS ONE | DOI:10.1371/journal.pone. 0129629 June 26, 2015 1 / 31 OPEN ACCESS Citation: Tatke G, Kumari H, Silva-Herzog E, Ramirez L, Mathee K (2015) Pseudomonas aeruginosa MifS-MifR Two-Component System Is Specific for α-Ketoglutarate Utilization. PLoS ONE 10 (6): e0129629. doi:10.1371/journal.pone. 0129629 Academic Editor: Min Wu, University of North Dakota, UNITED STATES Received: February 24, 2015 Accepted: May 10, 2015 Published: June 26, 2015 Copyright: © 2015 Tatke et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. Data Availability Statement: All relevant data are within the paper. Funding: This study was supported in part by the National Institutes of Health – Minority Biomedical Research Support SCORE (SC1AI081376; to KM), National Science Foundation IIP-1237818 [PFI-AIR: CREST-I/UCRC-Industry Ecosystem to Pipeline Research] (to KM), Biomedical Research Initiative (BRI) student research award (NIH/NIGMS R25 GM061347; to GT), Florida International University (FIU) teaching assistantship (Biological Sciences; to GT), FIU research assistantship (Herbert Wertheim College of Medicine; to GT), MBRS-RISE fellowship