Gundlach, J. | Search bioRxiv

bioRxiv2021

MspA Porin as a Local Nanopore Probe for Membrane-bound Proteins

Nanopore sensing is based on detection and analysis of nanopore transient conductance changes induced by analyte capture. We have recently shown that -Synuclein (Syn), an intrinsically disordered, membrane-active, neuronal protein implicated in Parkinson disease, can be reversibly captured by the VDAC nanopore. The capture process is a highly voltage dependent complexation of the two proteins where transmembrane potential drives the polyanionic C-terminal domain of Syn into VDAC--exactly the mechanism by which generic nanopore-based interrogation of proteins and polynucleotides proceeds. The complex formation, and the motion of Syn in the nanopore, thus may be expected to be only indirectly dependent on the pore identity. Here, we confirm this prediction by demonstrating that when VDAC is replaced with a different transmembrane pore, the engineered mycobacterial porin M2MspA, all the qualitative features of the Syn/nanopore interaction are preserved. The rate of Syn capture by M2MspA rises exponentially with the applied field, while the residence time displays a crossover behavior, indicating that at voltages >50 mV M2MspA-bound Syn largely undergoes translocation to the other side of the membrane. The translocation is directly confirmed using the selectivity tag method, in which the polyanionic C-terminal and neutral N-terminal regions of Syn alter the selectivity of the M2MspA channel differently, allowing direct discrimination of translocation vs retraction for single Syn molecules. We thus prove that the physical model of the motion of disordered protein chains in the nanopore confinement and the selectivity tag technique are not limited to VDAC but are broadly applicable to nanopore-based protein detection, analysis, and separation technologies.

biophysics↗

bioRxiv2020

Essentiality of c-di-AMP in Bacillus subtilis: Bypassing mutations converge in potassium and glutamate homeostasis

In order to adjust to changing environmental conditions, bacteria use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Cyclic di-adenosine monophosphate (c-di-AMP) is the only known essential nucleotide second messenger. In addition to the well-established role of this second messenger in the control of potassium homeostasis, we observed that glutamate is as toxic as potassium for a c-di-AMP-free strain of the Gram-positive model bacterium Bacillus subtilis. In this work, we isolated suppressor mutants that allow growth of a c-di-AMP-free strain under these toxic conditions. Characterization of glutamate resistant suppressors revealed that they contain pairs of mutations, in most cases affecting glutamate and potassium homeostasis. Among these mutations, several independent mutations affected a novel glutamate transporter, AimA (Amino acid importer A, formerly YbeC). This protein is the major transporter for glutamate and serine in B. subtilis. Unexpectedly, some of the isolated suppressor mutants could suppress glutamate toxicity by a combination of mutations that affect phospholipid biosynthesis and a specific gain-of-function mutation of a mechanosensitive channel of small conductance (YfkC) suggesting the acquisition of a device for glutamate export. Cultivation of the c-di-AMP-free strain on complex medium was an even greater challenge because the amounts of potassium, glutamate, and other osmolytes are substantially higher than in minimal mediu. Suppressor mutants viable on complex medium could only be isolated under anaerobic conditions if one of the two c-di-AMP receptor proteins, DarA or DarB, was absent. Also on complex medium, potassium and osmolyte toxicity are the major bottlenecks for the growth of B. subtilis in the absence of c-di-AMP. Our results indicate that the essentiality of c-di-AMP in B. subtilis is caused by the global impact of the second messenger nucleotide on different aspects of cellular physiology. AUTHOR SUMMARYBacteria are exposed to constantly changing environmental conditions. In order to respond to these changes, they use nucleotide second messengers to transduce external signals and translate them into a specific cellular response. Among the repertoire of bacterial second messenger nucleotides, cyclic di-AMP (c-di-AMP) stands out as it is the only second messenger that is essential for the bacteria that produce it, including the Gram-positive model organism Bacillus subtilis. C-di-AMP plays a major role in the control of potassium homeostasis, and we found that glutamate is toxic to a B. subtilis strain lacking c-di-AMP to the same extent as potassium. These toxic conditions were the starting point for an extensive suppressor analysis, which led to the identification of a novel glutamate transporter (AimA). If the B. subtilis strain lacking c-di-AMP was cultivated on complex medium, the isolation of suppressor mutants was only possible under anaerobic conditions and if either of the two c-di-AMP-binding signal transduction proteins was absent. This suggests that these proteins are a major burden for the cell on complex medium in their c-di-AMP free state. Our result underline the complexity of c-di-AMP signaling and propose new directions for research.

microbiology↗