The unconventional cytoplasmic sensing mechanism for ethanol chemotaxis in Bacillus 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 a serine, reduces taxis to ethanol. Molecular dynamics simulations suggest ethanol binds McpB near residue Ala431 and mutation of this residue 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

Non-canonical sensing mechanisms in bacteria

Bacteria continuously experience changing environments. Their ability to sense their local habitat and respond appropriately is essential for their survival. For example, bacteria form biofilms to share nutrients and protect themselves from harmful factors. Or they synchronize their gene expression patterns within the community for optimal performance in response to fluctuations in population density through quorum sensing. Additionally, bacteria have evolved chemosensory navigation machineries to sense chemicals around them and move towards nutrient-rich regions or away from toxins in the process known as chemotaxis. Chemotaxis has been extensively investigated in number of bacterial species, such as Escherichia coli, Salmonella typhimurium, Pseudomonas aeruginosa, Helicobacter pylori, and Bacillus subtilis. In the past five decades the study of bacterial chemotaxis has been mainly focused on simple molecules, including amino acids and sugars, which are essential for cells growth and survival. Although the details of the underlying molecular basis in chemotaxis vary among bacterial species, they all share a canonical mechanism to sense and respond to these simple molecules. Little is known about chemotaxis to other molecules and the governing sensing mechanisms. In this work, we studied chemotaxis to unconventional molecules in the B. subtilis bacterium and demonstrated the potential molecular mechanisms for sensing these compounds. Many biological processes are influenced by pH. Therefore, cells have to sense and respond to intracellular and extracellular pH. Chemotaxis to pH has been studied in number of bacterial species. We found that B. subtilis also exhibits chemotaxis to pH. Interestingly, pH chemotaxis is bidirectional in B. subtilis. McpB and its three paralogs, namely McpA, TlpA and TlpB are responsible for pH sensing. We investigated the molecular basis for bipolar pH sensing. Modified capillary assay was used to measure responses to opposite pH gradients. Through in vivo chimeric receptor and site-directed mutagenesis studies, we found that the lower regions of the extracellular ligand binding domains of the chemoreceptors are involved in pH sensing. In particular, we identified number of key amino acid residues that define the polarity of pH sensing. We recently found that B. subtilis performs chemotaxis to DNA. While DNA can serve as a nutrient for B. subtilis, our data suggest that the chemotaxis response is not to the DNA itself but rather to the information encoded within the DNA. Our evidence comes from experiments showing that B. subtilis prefers the DNA of more closely related species than the DNA of more distantly related ones. These results suggest that B. subtilis responds to particular DNA sequences that are enriched within the genomes of closely related bacteria. We employed the in vivo capillary assay to measure chemotaxis to DNA from different organisms. We then used SELEX-Seq to identify the specific sequences of DNA that B. subtilis responds to. The binding properties of these sequences were then evaluated using isothermal titration calorimetry (ITC) and the in vitro receptor-kinase assay. Chemotaxis to DNA is dose-dependent. Among the organisms tested, Bacilli are the preferred sources of DNA. McpC is the sole chemoreceptor for DNA. Using SELEX-Seq, we identified a number of chemotactic DNA motifs. The abundance of these motifs partially explains the organismal preference of DNA chemotaxis. While the physiological role of DNA chemotaxis is unknown, its selectivity suggests that it may be involved in horizontal gene transfer or kin selection. Alcohols are known for their antibacterial activity. E. coli, for example, performs chemotaxis away from straight and branched alcohols. Unexpectedly, we found that B. subtilis can exhibit chemotaxis towards short-chain alcohols. Among ten chemoreceptors of B. subtilis, HemAT and McpB were found to sense alcohols. In this study, we investigated the mechanism for sensing these alcohols. In vivo chemotaxis responses were measured using the capillary assay. In vitro chemotaxis responses were measured using the kinase assay. We found that the alcohol response is dose dependent, and the kinase assays indicated that alcohol may directly interacts with chemoreceptors. Analysis of chimeric chemoreceptors revealed that the cytoplasmic domain of McpB is involved in sensing alcohols. In addition, the sensing domain of HemAT was analyzed using UV spectroscopy. UV spectroscopy suggests that alcohols do not directly bind or interact with the heme group within the HemAT sensor domain. However, Isothermal Titration Calorimetry analysis demonstrated that the cytoplasmic signaling regions of both McpB and HemAT can directly bind ethanol. Interestingly, B. subtilis does not consume alcohol. We speculate that B. subtilis may follow alcohol gradients to colonize plants or attack yeast.U of I OnlyAuthor requested U of Illinois access only (OA after 2yrs) in Vireo ETD syste