In Vitro Assay for Measuring Receptor-Kinase Activity in the Bacillus subtilis Chemotaxis Pathway

Walukiewicz, Hanna E.; Ordal, George; Rao, Christopher V.

doi:10.1007/978-1-4939-7577-8_10

Cited by 2 publications

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“…We next performed an in vitro receptor-coupled kinase assay to test whether ethanol is able to activate CheA kinase (32). This assay has been used to study how attractant binding to chemoreceptors modulates CheA kinase activity (22,32). Briefly, membranes expressing either McpB or HemAT were isolated.…”

Section: Resultsmentioning

confidence: 99%

“…The CheA, CheW, and CheD proteins used in the kinase assay were expressed from glutathione S ‐transferase (GST) fusion plasmids and purified from the E. coli BL21(DE3) strain as described previously ( 22 , 32 ). GSTrap columns (5 ml; GE Healthcare) were used with an Äkta Prime fast protein liquid chromatography (FPLC) system (GE Healthcare) for purification.…”

Section: Methodsmentioning

confidence: 99%

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The Unconventional Cytoplasmic Sensing Mechanism for Ethanol Chemotaxis in Bacillus subtilis

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Bodhankar

Pei

et al. 2020

mBio

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Motile bacteria sense chemical gradients using chemoreceptors, which consist of distinct sensing and signaling domains. The general model is that the sensing domain binds the chemical and the signaling domain induces the tactic response. Here, we investigated the unconventional sensing mechanism for ethanol taxis in Bacillus subtilis. Ethanol and other short-chain alcohols are attractants for B. subtilis. Two chemoreceptors, McpB and HemAT, sense these alcohols. In the case of McpB, the signaling domain directly binds ethanol. We were further able to identify a single amino acid residue, Ala431, on the cytoplasmic signaling domain of McpB that, when mutated to serine, reduces taxis to alcohols. Molecular dynamics simulations suggest that the conversion of Ala431 to serine increases coiled-coil packing within the signaling domain, thereby reducing the ability of ethanol to bind between the helices of the signaling domain. In the case of HemAT, the myoglobin-like sensing domain binds ethanol, likely between the helices encapsulating the heme group. Aside from being sensed by an unconventional mechanism, ethanol also differs from many other chemoattractants because it is not metabolized by B. subtilis and is toxic. We propose that B. subtilis uses ethanol and other short-chain alcohols to locate prey, namely, alcohol-producing microorganisms. IMPORTANCE Ethanol is a chemoattractant for Bacillus subtilis even though it is not metabolized and inhibits growth. B. subtilis likely uses ethanol to find ethanol-fermenting microorganisms to utilize as prey. Two chemoreceptors sense ethanol: HemAT and McpB. HemAT’s myoglobin-like sensing domain directly binds ethanol, but the heme group is not involved. McpB is a transmembrane receptor consisting of an extracellular sensing domain and a cytoplasmic signaling domain. While most attractants bind the extracellular sensing domain, we found that ethanol directly binds between intermonomer helices of the cytoplasmic signaling domain of McpB, using a mechanism akin to those identified in many mammalian ethanol-binding proteins. Our results indicate that the sensory repertoire of chemoreceptors extends beyond the sensing domain and can directly involve the signaling domain.

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Section: Resultsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

The Unconventional Cytoplasmic Sensing Mechanism for Ethanol Chemotaxis in Bacillus subtilis

Tohidifar

Bodhankar

Pei

et al. 2020

mBio

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The unconventional cytoplasmic sensing mechanism for ethanol chemotaxis inBacillus subtilis

Tohidifar

Bodhankar

Pei

et al. 2020

Preprint

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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 ...

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In Vitro Assay for Measuring Receptor-Kinase Activity in the Bacillus subtilis Chemotaxis Pathway

Cited by 2 publications

References 34 publications

The Unconventional Cytoplasmic Sensing Mechanism for Ethanol Chemotaxis in Bacillus subtilis

The Unconventional Cytoplasmic Sensing Mechanism for Ethanol Chemotaxis in Bacillus subtilis

The unconventional cytoplasmic sensing mechanism for ethanol chemotaxis inBacillus subtilis

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