MifS, a DctB family histidine kinase, is a specific regulator of α-ketoglutarate response in Pseudomonas aeruginosa PAO1

Abstract

The C5-dicarboxylate α-ketoglutarate (α-KG) is a preferred nutrient source for the opportunistic pathogen Pseudomonas aeruginosa . However, very little is known about how P. aeruginosa detects and responds to α-KG in the environment. Our laboratory has previously shown that the MifS/MifR two-component signal transduction system regulates α-KG assimilation in P. aeruginosa PAO1. In an effort to better understand how this bacterium detects α-KG, we characterized the MifS sensor histidine kinase. In this study we show that although MifS is a homologue of the C4-dicarboxylate sensor DctB, it specifically responds to the C5-dicarboxylate α-KG. MifS activity increased >10-fold in the presence of α-KG, while the related C5-dicarboxylate glutarate caused only a 2-fold increase in activity. All other dicarboxylates tested did not show any significant effect on MifS activity. Homology modelling of the MifS sensor domain revealed a substrate binding pocket for α-KG. Using protein modelling and mutational analysis, we identified nine residues that are important for α-KG response, including one residue that determines the substrate specificity of MifS. Further, we found that MifS has a novel cytoplasmic linker domain that is required for α-KG response and is probably involved in signal transduction from the sensor domain to the cytoplasmic transmitter domain. Until this study, DctB family histidine kinases were known to only respond to C4-dicarboxylates. Our work shows that MifS is a novel member of the DctB family histidine kinase that specifically responds to α-KG.

Transcriptional Regulators Controlling Virulence in Pseudomonas aeruginosa

“…Lastly, the MifSR TCS affects microcolony and cluster formation during the last stages of the biofilm maturation process. Upon sensing α-ketoglutarate, the HK MifS phosphorylates MifR, which acts as an EBP, assisting the σ 54 factor RpoN to bind to the promoter region of α-ketoglutarate transport genes [27,149]. α-ketoglutarate is a preferred carbon source for P. aeruginosa, and it is required for virulence in pathogenic bacteria [149].…”

“…Upon sensing α-ketoglutarate, the HK MifS phosphorylates MifR, which acts as an EBP, assisting the σ 54 factor RpoN to bind to the promoter region of α-ketoglutarate transport genes [27,149]. α-ketoglutarate is a preferred carbon source for P. aeruginosa, and it is required for virulence in pathogenic bacteria [149]. MifR has also been shown to alter the production of at least 18 proteins when mutated or overexpressed, many of which are involved in growth under anaerobic conditions, suggesting a link between biofilm formation and oxygen restriction [42].…”

Utilization of L-glutamate as a preferred or sole nutrient in Pseudomonas aeruginosa PAO1 depends on genes encoding for the enhancer-binding protein AauR, the sigma factor RpoN and the transporter complex AatJQMP

“…The MifSR TCS was originally identified for its role in microcolony formation and named accordingly [ 12 ]. It was later demonstrated that the MifSR TCS is required for the utilization of C 5 -dicarboxylates such as α-ketoglutarate (α-KG) in which MifR activates transcription of the PA5530 gene, encoding for a C 5 -dicarboxylate-proton symporter [ 13 – 15 ]. The utilization of C 4 - and C 5 -dicarboxylates as a sole carbon source in P. aeruginosa PAO1 is dependent on DctD and MifR, respectively [ 11 , 13 ].…”

Microbial Musings – September 2020

“…The sensor has two transmembrane domains, between which is a structured periplasmic domain that binds directly the molecule to be sensed, which results in the transduction of this signal through the membrane to the transmitter domain in the cytoplasm. Zaara Sarwar from The College of New Jersey, USA, working with the group of Christopher Nomura at the State University of New York, Syracuse, USA, has been looking at the function of MifS and has found that it is highly specific for α-KG and only recognizes the related C5-dicarboxylate glutarate [ 17 ]. There are structures of similar sensing domains such as the Vibrio cholerae DctB protein, which adopts α/β PDC fold [ 18 ], and the authors modelled the MifS sensing domain on these.…”