Biology subjects

Unden, G.

Publications and source records attributed to Unden, G..

3 recordsLinked to original sources

bioRxiv2023

Mechanism of sensor kinase CitA transmembrane signaling

Membrane bound histidine kinases (HKs) are ubiquitous sensors of extracellular stimuli in bacteria. Here, we used solid-state NMR in conjunction with crystallography, solution NMR and distance measurements to investigate the transmembrane signaling mechanism of a paradigmatic citrate sensing membrane embedded HK, CitA. Citrate binding in the sensory extracytoplasmic PAS domain (PASp) causes the linker to transmembrane helix 2 (TM2) to adopt a helical conformation. This triggers a piston-like pulling of TM2 and a quaternary structure rearrangement in the cytosolic PAS domain (PASc). Crystal structures of PASc reveal both anti-parallel and parallel dimer conformations. An anti-parallel to parallel transition upon citrate binding agrees with interdimer distances measured in the lipid embedded protein using a site-specific 19F label in PASc. These data show how Angstrom scale structural changes in the sensor domain are transmitted across the membrane to be converted and amplified into a nm scale shift in the linker to the phosphorylation subdomain of the kinase. One-Sentence SummaryTransmembrane signal transduction of a PAS-domain containing histidine kinase occurs via a piston-like pulling of a transmembrane helix, and amplification by cytoplasmic PAS domain dimer rearrangement.

molecular biology↗

bioRxiv2021

Regulation of dctA and DctA by cAMP-CRP and EIIAGlc at the transcriptional and post-translational levels in E. coli: Consequences for aerobic uptake and metabolism of C4-dicarboxylates

The expression of dctA, encoding the aerobic C4-dicarboxylate (C4-DC) transporter DctA of Escherichia coli, and its use in the presence of alternative carbon sources was characterized. dctA is regulated by cAMP-CRP and substrates that control cAMP levels, either through the phosphotransferase system (PTS), or through their metabolic link to PEP synthesis. The data indicates that phosphorylation of the regulator EIIAGlc of the glucose-specific PTS represents the mediator for regulation. The dctA promotor region contains a class I CRP-binding site (position -81.5) and a DcuR-binding site (position -105.5). The response regulator DcuR of the C4-DC-activated DcuS-DcuR two-component system is known to stimulate expression of dctA, and cAMP-CRP is known to stimulate expression of dcuS-dcuR. Thus, activation of dctA expression by cAMP-CRP and DcuR is organized in a coherent feed-forward loop (FFL) where cAMP-CRP positively regulates the expression of dctA by direct stimulation and by stimulating the expression of dcuR. Stimulation by DcuR is presumed to require DNA bending by cAMP-CRP. In this way, CRP-FFL integrates carbon catabolite control and C4-DC-specific regulation. Moreover, EIIAGlc of the glucose-specific PTS strongly interacts with DctA, which could lead to substrate exclusion of C4-DCs when preferred carbon substrates such as sugars are present. Since C4-DCs are perceived in the periplasmic space by the sensor DcuS, the substrate exclusion is not linked to inducer exclusion, contrasting classical inducer exclusion known for the lactose permease LacY. Thus, aerobic C4-DC metabolism is tightly regulated at the transcriptional and post-translational levels, whereas uptake of L-aspartate by DcuA is essentially unaffected. Overall, transcriptional and post-translational regulation of dctA expression and DctA function efficiently fine-tunes C4-DC catabolism in response to other preferred carbon sources.

microbiology↗

bioRxiv2021

C4-dicarboxylate metabolons: Interaction of C4-dicarboxylate transporters of Escherichia coli with cytosolic enzymes and regulators

Metabolons represent the structural organization of proteins for metabolic or regulatory pathways. Here the interaction of enzymes fumarase FumB and aspartase AspA with the C4-DC transporters DcuA and DcuB of Escherichia coli was tested by a bacterial two-hybrid (BACTH) assay in situ, or by co-chromatography (mSPINE). DcuB interacted strongly with FumB and AspA, and DcuA with AspA. The fumB-dcuB and the dcuA-aspA genes encoding the respective proteins are known for their colocalization on the genome and the production of co-transcripts. The data consistently suggest the formation of DcuB/FumB, DcuB/AspA and DcuA/AspA metabolons in fumarate respiration for the uptake of L-malate, or L-aspartate, conversion to fumarate and excretion of succinate after reduction. The DcuA/AspA metabolon catalyzes L-Asp uptake and fumarate excretion in concerted action also to provide ammonia for nitrogen assimilation.

microbiology↗