Pseudomonas aeruginosa MifS-MifR Two-Component System Is Specific for α-Ketoglutarate Utilization

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.

Suppressor mutations reveal an NtrC-like response regulator, NmpR, for modulation of Type-IV Pili-dependent motility in Myxococcus xanthus

“…The NtrC-like RR family has been extensively studied and has served as a model protein for intramolecular structure-function dynamics [ 18 , 19 , 23 , 47 – 50 ]. NtrC-like RRs also continue to be discovered as important transcriptional regulators controlling diverse biological processes in diverse bacterial species [ 51 – 54 ]. For example, NtrC-like RRs and σ 54 play critical roles in processes exterior to the bacterial cell, such as lipopolysaccharide production, EPS production, and motility [ 24 ].…”

Identification of a Chemoreceptor in Pseudomonas aeruginosa That Specifically Mediates Chemotaxis Toward α-Ketoglutarate

“…The activity of this TCS is regulated by the small protein PII that senses αKG as carbon signal and glutamine as nitrogen signals (Ninfa and Jiang, 2005). In addition, a model was proposed in which αKG regulates in P. aeruginosa the activity of another TCS, namely the MifS/MifR system, which regulates genes that are involved in αKG transport and subsequent metabolism (Tatke et al, 2015). …”

The Complex Relationship between Virulence and Antibiotic Resistance

“…Communication amongst bacteria is essential for many cellular functions, such as biofilm formation, sporulation, competence, antibiotic resistance, and regulation of a variety of other virulence factors [ 182 , 183 , 184 ]. Bacteria communicate either through (1) quorum sensing and (2) two component systems, which are directed and deliberate forms of communication of bacteria.…”