We investigated pH taxis in Bacillus subtilis. This bacterium was found to perform bidirectional taxis in response to external pH gradients, enabling it to preferentially migrate to neutral environments. We next investigated the chemoreceptors involved in sensing pH gradients. We identified four chemoreceptors involved in sensing pH: McpA and TlpA for sensing acidic environments and McpB and TlpB for sensing alkaline ones. In addition, TlpA was found to also weakly sense alkaline environments. By analyzing chimeras between McpA and TlpB, the principal acid- and base-sensing chemoreceptors, we identified four critical amino acid residues—Thr199, Gln200, His273, and Glu274 on McpA and Lys199, Glu200, Gln273, and Asp274 on TlpB—involved in sensing pH. Swapping these four residues between McpA and TlpB converted the former into a base receptor and the latter into an acid receptor. Based on the results, we propose that disruption of hydrogen bonding between the adjacent residues upon pH changes induces signaling. Collectively, our results further our understanding of chemotaxis in B. subtilis and provide a new model for pH sensing in bacteria. IMPORTANCE Many bacteria can sense the pH in their environment and then use this information to direct their movement toward more favorable locations. In this study, we investigated the pH sensing mechanism in Bacillus subtilis. This bacterium preferentially migrates to neutral environments. It employs four chemoreceptors to sense pH. Two are involved in sensing acidic environments, and two are involved in sensing alkaline ones. To identify the mechanism for pH sensing, we constructed receptor chimeras of acid- and base-sensing chemoreceptors. By analyzing the responses of these chimeric receptors, we were able to identify four critical amino acid residues involved in pH sensing and propose a model for the pH sensing mechanism in B. subtilis.

SummaryThe Bacillus subtilis chemotaxis pathway employs three systems for sensory adaptation: the methylation system, the CheC/CheD/CheYp system, and the CheV system. Little is known in general about how these three adaptation systems contribute to chemotaxis in B. subtilis and whether they interact with one another. To further understand these three adaptation systems, we employed a quantitative in vitro receptor-kinase assay. Using this assay, we were able to determine how CheD and CheV affect receptor-kinase activity as a function of the receptor modification state. CheD was found to increase receptor-kinase activity, where the magnitude of the increase depends on the modification state of the receptor. The principal new findings concern CheV. Little was known about this protein before now. Our data suggest that this protein has two roles depending on the modification state of the receptor, one for sensory adaptation when the receptors are modified (methylated) and the other for signal amplification when they are unmodified (unmethylated). In addition, our data suggest that methylation of site 630 tunes the strength of the CheV adaptation system. Collectively, our results provide new insight regarding the integrated function of the three adaptation systems in B. subtilis.

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.

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 ...

Bacillus subtilis employs ten chemoreceptors to move in response to chemicals in its environment. While the sensing mechanisms have been determined for many attractants, little is known about the sensing mechanisms for repellents. In this work, we investigated phenol chemotaxis in B. subtilis . Phenol is an attractant at low, micromolar concentrations, and a repellent at high, millimolar concentrations. McpA was found to be the principal chemoreceptor governing the repellent response to phenol and other related aromatic compounds. In addition, the chemoreceptors McpC and HemAT were found to govern the attractant response to phenol and related compounds. Using chemoreceptor chimeras, McpA was found to sense phenol using its signaling domain rather than its sensing domain. These observations were substantiated in vitro, where direct binding of phenol to the signaling domain of McpA was observed using saturation-transfer difference nuclear magnetic resonance. These results further advance our understanding of B. subtilis chemotaxis and further demonstrate that the signaling domain of B. subtilis chemoreceptors can directly sense chemoeffectors. IMPORTANCE Bacterial chemotaxis is commonly thought to employ a sensing mechanism involving the extracellular sensing domain of chemoreceptors. Some ligands, however, appear to be sensed by the signaling domain. Phenolic compounds, commonly found in soil and root exudates, provide environmental cues for soil microbes like Bacillus subtilis . We show that phenol is sensed both as an attractant and a repellent. While the mechanism for sensing phenol as an attractant is still unknown, we found that phenol is sensed as a repellent by the signaling domain of the chemoreceptor McpA. This study furthers our understanding of the unconventional sensing mechanisms employed by the B. subtilis chemotaxis pathway.

16We investigated pH taxis in Bacillus subtilis. This bacterium was found to perform 17 bidirectional taxis in response to external pH gradients, enabling it to preferentially 18 migrate to neutral environments. We next investigated the chemoreceptors involved in 19 sensing pH gradients. We found that four chemoreceptors are involved in sensing pH: 20McpA and TlpA for sensing acidic environments and McpB and TlpB for alkaline ones. 21In addition, TlpA was found to also weakly sense alkaline environments. By analyzing 22 IMPORTANCE 32Many bacteria can sense the pH in their environment and then use this information to 33 direct their movement towards more favorable locations. In this study, we investigated 34 the pH sensing mechanism in Bacillus subtilis. This bacterium preferentially migrates to 35 neutral environments. It employs four chemoreceptors to sense pH. Two are involved in 36 sensing acidic environments and two are involved in sensing alkaline ones. To identify 37 the mechanism for pH sensing, we constructed receptor chimeras of acid and base 38 sensing chemoreceptors. By analyzing the response of these chimeric receptors, we 39were able to identify four critical amino-acid residues involved in pH sensing and 40 propose a model for the pH sensing mechanism in B. subtilis. 41 42 RESULTS AND DISCUSSION 99B. subtilis exhibits bidirectional taxis to external pH gradients. To determine 100 whether B. subtilis performs chemotaxis in response to external pH gradients, we 101 employed the capillary assay (26). Briefly, cells suspended in buffer at different pH's 102 (6.0-8.5) were incubated with capillaries filled with buffer at pH 7.0 and then the number 103 of cells that entered the capillaries after 1 h were counted. The resulting data show that 104 B. subtilis exhibits bidirectional taxis to pH gradients in manner similar to what is 105 observed in E. coli (Figure 1A). In particular, we found that B. subtilis preferentially 106 migrates to neutral (pH 7) environments when the cells were initially suspended in either 107 acidic or alkaline buffer (pH 6-8). Outside of this pH range, however, the cells were less 108 motile (data not shown) and, consequently, taxis was reduced. 109 110The methylation system is required for pH taxis. B. subtilis employs three 111 adaptions systems for sensing chemical gradients (11). Of the three adaptation 112 systems, the methylation system is the dominant one for sensing amino-acid gradients. 113To determine whether the methylation system is also required for pH taxis, we tested 114 whether a cheRcheB mutant was capable of pH taxis (Figure 1B). This mutant lacks 115 the requisite methyltransferase (CheR) and methylesterase (CheB). It was unable to 116 perform pH taxis, indicating that the methylation system is necessary for sensing pH 117 gradients. 118 119 Four chemoreceptors are involved in sensing pH gradients. B. subtilis 120 possesses ten chemoreceptors (4). To determine which chemoreceptors are involved in 595

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Bacillus subtilis employs ten chemoreceptors to move in response to chemicals in its environment. While the sensing mechanisms have been determined for many attractants, little is known about the sensing mechanisms for repellents. In this work, we investigated phenol chemotaxis in B. subtilis. Phenol is an attractant at low, micromolar concentrations, and a repellent at high, millimolar concentrations. McpA was found to be the principal chemoreceptor governing the repellent response to phenol and other related aromatic compounds. In addition, the chemoreceptors McpC and HemAT were found to the govern the attractant response to phenol and related compounds. Using receptor chimeras, McpA was found to sense phenol using its signaling domain rather than its sensing domain. These observations were substantiated in vitro, where direct binding of phenol to the signaling domain of McpA was observed using saturation-transfer difference nuclear magnetic resonance. These results further advance our understanding of B. subtilis chemotaxis and demonstrate that the signaling domain of B. subtilis chemoreceptors can directly sense chemoeffectors.