The unconventional cytoplasmic sensing mechanism for ethanol chemotaxis inBacillus subtilis

Abstract

205 words; Importance: 121 words Main Text: 4732 23 2 Abstract 24Motile bacteria sense chemical gradients using chemoreceptors, which consist of 25 distinct sensing and signaling domains. The general model is that the sensing domain 26 binds the chemical and the signaling domain induces the tactic response. Here, we 27 investigated the unconventional sensing mechanism for ethanol taxis in Bacillus subtilis. 28 Ethanol and other short-chain alcohols are attractants for B. subtilis. Two 29 chemoreceptors, McpB and HemAT, sense these alcohols. In the case of McpB, the 30 signaling domain directly binds ethanol. We were further able to identify a single amino-31 acid residue Ala 431 on the cytoplasmic signaling domain of McpB, that when mutated to 32 a serine, reduces taxis to ethanol. Molecular dynamics simulations suggest ethanol 33 binds McpB near residue Ala 431 and mutation of this residue to serine increases coiled-34 coil packing within the signaling domain, thereby reducing the ability of ethanol to bind 35between the helices of the signaling domain. In the case of HemAT, the myoglobin-like 36 sensing domain binds ethanol, likely between the helices encapsulating the heme 37 group. Aside from being sensed by an unconventional mechanism, ethanol also differs 38 from many other chemoattractants because it is not metabolized by B. subtilis and is 39 toxic. We propose that B. subtilis uses ethanol and other short-chain alcohols to locate 40 prey, namely alcohol-producing microorganisms. 41 42 3 Importance 43 Ethanol is a chemoattractant for Bacillus subtilis even though it is not metabolized and 44 inhibits growth. B. subtilis likely uses ethanol to find ethanol-fermenting microorganisms 45 for prey. Two chemoreceptors sense ethanol: HemAT and McpB. HemAT's myoglobin-46 like sensing domain directly binds ethanol, but the heme group is not involved. McpB is 47 48 cytoplasmic signaling domain. While most attractants bind the extracellular sensing 49 domain, we found that ethanol directly binds between inter-monomer helices of the 50 cytoplasmic signaling domain of McpB, using a mechanism akin to those identified in 51 many mammalian ethanol-binding proteins. Our results indicate that the sensory 52 repertoire of chemoreceptors extends beyond the sensing domain and can directly 53 involve the signaling domain.54 55 56Many bacteria move in response to external chemical gradients through a process 57 known as chemotaxis (1). Typically, bacteria migrate up gradients of chemicals that 58 support their growth and down ones that inhibit it. These chemicals are commonly 59 sensed using transmembrane chemoreceptors, which consist of an extracellular 60 sensing domain and a cytoplasmic signaling domain along with a cytoplasmic HAMP 61 domain that couples the two domains. While a number of sensing mechanisms exist, 62 the best understood one involves direct binding of the chemical to the extracellular 63 sensing domain (2). In flagellated bacteria such as Bacillus subtilis and Escherichia coli, 64 this binding event ...

PyLipID: A Python package for analysis of protein-lipid interactions from MD simulations

“…To demonstrate the application of PyLipID to small-molecule/fragment binding, we analyzed the interactions of ethanol with a bacterial chemoreceptor, McpB, for which ethanol is a known attractant. The analyses were carried out on previously conducted atomistic simulations (3 × 600 ns) of an McpB cytoplasmic homodimer with 165 ethanol molecules (0.316 M) included to reproduce experimental conditions 40 . As anticipated, ethanol molecules showed transient interactions with the receptor due to their small size and simple structure.…”

“…It is suggested that these may facilitate conformational changes induced by ethanol binding to be transmitted to other parts of the receptor, thereby enabling the signaling response. To test the sensitivity of PyLipID to minor changes in protein sequence, we additionally analyzed atomistic simulations (3 × 600 ns) of McpB carrying the A431S mutation, which is known to considerably reduce taxis to alcohols 40 . While the 51 ethanol binding sites identified by PyLipID largely overlap with those on wild-type McpB, ethanol binding to the sidechain of residue 431 was no longer observed (SI Fig S16).…”

“…Subsequently, we present two cases of the application of PyLipID to interactions of membrane proteins with phospholipids illustrating the potential application of PyLipID to assist the interpretation of lipid-like densities in cryo-EM maps. Finally, we demonstrate the application of PyLipID to non-lipid molecules, using it characterize ethanol binding to the cytoplasmic domain of the B. subtilis McpB chemoreceptor as seen in atomistic simulations 40 .…”

Characterization of opposing responses to phenol by Bacillus subtilis chemoreceptors

“…2D). McpC was previously found to sense amino acids and HemAT to sense oxygen and alcohols (23)(24)(25). Functional redundancy of chemoreceptors for sensing aromatic compounds is also observed in other bacteria (26).…”

Ethanol pre-exposure enhances alcohol-seeking behavior at cellular level by chemoattraction and exhibits bleb-driven cellular stress response in uniform ethanol concentration

“…subtilis (Tohidifar et al, 2020). However, ethanol as a chemotactic agent has not been studied in eukaryotic cells to the best of the authors' knowledge.…”