RESUMEN
Chemotaxis by Bacillus subtilis requires the inter-acting chemotaxis proteins CheC and CheD. In this study, we show that CheD is absolutely required for a behavioural response to proline mediated by McpC but is not required for the response to asparagine mediated by McpB. We also show that CheC is not required for the excitation response to asparagine stimulation but is required for adaptation while asparagine remains complexed with the McpB chemoreceptor. CheC displayed an interaction with the histidine kinase CheA as well as with McpB in the yeast two-hybrid assay, suggesting that the mechanism by which CheC affects adaptation may result from an interaction with the receptor-CheA complex. Furthermore, CheC was found to be related to the family of flagellar switch proteins comprising FliM and FliY but is not present in many proteobacterial genomes in which CheD homologues exist. The distinct physiological roles for CheC and CheD during B. subtilis chemotaxis and the observation that CheD is present in bacterial genomes that lack CheC indicate that these proteins can function independently and may define unique pathways during chemotactic signal transduction. We speculate that CheC interacts with flagellar switch components and dissociates upon CheY-P binding and subsequently interacts with the receptor complex to facilitate adaptation.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas/genética , Proteínas Bacterianas/metabolismo , Quimiotaxis/fisiología , Regulación Bacteriana de la Expresión Génica , Secuencia de Aminoácidos , Asparagina/farmacología , Bacillus subtilis/genética , Proteínas Bacterianas/química , Quimiotaxis/genética , Datos de Secuencia Molecular , Mutación , Prolina/farmacología , Alineación de Secuencia , Técnicas del Sistema de Dos HíbridosRESUMEN
The phosphoenolpyruvate-dependent phosphotransferase system (PTS) plays a major role in the ability of Escherichia coli to migrate toward PTS carbohydrates. The present study establishes that chemotaxis toward PTS substrates in Bacillus subtilis is mediated by the PTS as well as by a methyl-accepting chemotaxis protein (MCP). As for E. coli, a B. subtilis ptsH null mutant is severely deficient in chemotaxis toward most PTS carbohydrates. Tethering analysis revealed that this mutant does respond normally to the stepwise addition of a PTS substrate (positive stimulus) but fails to respond normally to the stepwise removal of such a substrate (negative stimulus). An mcpC null mutant showed no response to the stepwise addition or removal of D-glucose or D-mannitol, both of which are PTS substrates. Therefore, in contrast to E. coli PTS carbohydrate chemotaxis, B. subtilis PTS carbohydrate chemotaxis is mediated by both MCPs and the PTS; the response to positive stimulus is primarily McpC mediated, while the duration or magnitude of the response to negative PTS carbohydrate stimulus is greatly influenced by components of the PTS and McpC. In the case of the PTS substrate D-glucose, the response to negative stimulus is also partially mediated by McpA. Finally, we show that B. subtilis EnzymeI-P has the ability to inhibit B. subtilis CheA autophosphorylation in vitro. We hypothesize that chemotaxis in the spatial gradient of the capillary assay may result from a combination of a transient increase in the intracellular concentration of EnzymeI-P and a decrease in the concentration of carbohydrate-associated McpC as the cell moves down the carbohydrate concentration gradient. Both events appear to contribute to inhibition of CheA activity that increases the tendency of the bacteria to tumble. In the case of D-glucose, a decrease in D-glucose-associated McpA may also contribute to the inhibition of CheA. This bias on the otherwise random walk allows net migration, or chemotaxis, to occur.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas , Quimiotaxis , Glucosa/metabolismo , Proteínas de la Membrana/metabolismo , Sistema de Fosfotransferasa de Azúcar del Fosfoenolpiruvato/metabolismo , Bacillus subtilis/enzimología , Proteínas de Escherichia coli , Histidina Quinasa , Proteínas de la Membrana/aislamiento & purificación , Proteínas Quimiotácticas Aceptoras de Metilo , FosforilaciónRESUMEN
Virtually all organisms have means of monitoring their environment and making use of information gained to aid their survival. Many organisms, from bacteria to animals, move from place to place and can alter their movements. Chemotaxis is a signal transduction system found in motile bacteria that allows them to sense changes in the concentrations of various extracellular compounds and change their swimming behavior in a way that moves them toward more favorable environments. Chemotaxis is the most ancient sensory-motor process in nature. For years, studies of enteric bacteria, such as Escherichia coli and Salmonella typhimurium, have served as the paradigm for understanding this process on a molecular level. Recent studies on the gram-positive bacterium, Bacillus subtilis, and other bacteria, suggest that a slightly more complex system may be ancestral to that of the more extensively studied enterics. Aspects of chemotaxis that are unique to B. subtilis include a more complex adaptation system, with protein-protein methyl group transfer, chemotaxis proteins having no counterparts in E. coli, and a very extensive repertoire of repellents that are sensed at very low concentrations by receptors.
Asunto(s)
Bacillus subtilis/fisiología , Quimiotaxis/fisiología , Escherichia coli/fisiologíaRESUMEN
The role of indoleamine 2,3-dioxygenase (IDO) in IFN-gamma-mediated inhibition of intracellular parasite growth has been examined previously, although earlier work has been largely correlative. In this study, we defined more completely the role of IDO in the IFN-antimicrobial response. Two mutant cell lines, derived from ME180 cells and exhibiting reduced IDO activity (IR3B6A, IR3B6B) were characterized to determine if they retained the capacity to inhibit intracellular Chlamydia and Toxoplasma growth. Mutant cells treated with IFN-gamma exhibited reduced capacity to suppress pathogen growth. The expression of several IFN-regulated genes also was measured to confirm that the inability to inhibit pathogen growth was because of the lack of IDO. The expression of class II MHC, intracellular adhesion molecule-1, MxA, and P68 kinase genes was induced in the IFN-gamma-treated wild type ME180 cells, but was variable in the mutant cell lines, supporting the hypothesis that IFN-gamma-induced production of IDO is a key IFN-gamma-mediated antimicrobial mechanism.