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
In the Gram-positive soil bacterium Bacillus subtilis, the chemoreceptors are coupled to the central two-component kinase CheA via two proteins, CheW and CheV. CheV is a two-domain protein with an N-terminal CheW-like domain and a C-terminal two-component receiver domain. In this study, we show that CheV is phosphorylated in vitro on a conserved aspartate in the presence of phosphorylated CheA (CheA-P). This reaction is slower compared with the phospho-transfer reaction between CheA-P and one other response regulator of the system, CheB. CheV-P is also highly stable in comparison with CheB-P. Both of these properties are more pronounced in the full-length protein compared with a truncated form composed only of the receiver domain, that is, deletion of the CheW-like domain results in increase in the rate of the phospho-transfer reaction and decrease in stability of the phosphorylated protein. Phosphorylation of CheV is required for adaptation to the addition of the chemoattractant asparagine. In tethered-cell assays, strains expressing an unphosphorylatable point mutant of cheV or a truncated mutant lacking the entire receiver domain are severely impaired in adaptation to the addition of asparagine. Both of these strains, however, show near normal counterclockwise biases, suggesting that in the absence of the attractant the chemoreceptors are efficiently coupled to CheA kinase by the mutant CheV proteins. Inability of the CheW-like domain of CheV to support complete adaptation to the addition of asparagine also suggests that unlike CheW, this domain by itself may lead to the formation of signaling complexes that stay overactive in the presence of the attractant. A possible structural basis for this feature is discussed.
Asunto(s)
Adaptación Fisiológica/fisiología , Bacillus subtilis/fisiología , Proteínas Bacterianas , Factores Quimiotácticos/metabolismo , Quimiotaxis , Secuencia de Aminoácidos , Bacillus subtilis/metabolismo , Factores Quimiotácticos/química , Datos de Secuencia Molecular , Fosforilación , Homología de Secuencia de AminoácidoRESUMEN
For the Gram-positive organism Bacillus subtilis, chemotaxis to the attractant asparagine is mediated by the chemoreceptor McpB. In this study, we show that rapid net demethylation of B. subtilis McpB results in the immediate production of methanol, presumably due to the action of CheB. We also show that net demethylation of McpB occurs upon both addition and removal of asparagine. After each demethylation event, McpB is remethylated to nearly prestimulus levels. Both remethylation events are attributable to CheR using S-adenosylmethionine as a substrate. Therefore, no methyl transfer to an intermediate carrier need be postulated to occur during chemotaxis in B. subtilis as was previously suggested. Furthermore, we show that the remethylation of asparagine-bound McpB requires the response regulator, CheY-P, suggesting that CheY-P acts in a feedback mechanism to facilitate adaptation to positive stimuli during chemotaxis in B. subtilis. This hypothesis is supported by two observations: a cheRBCD mutant is capable of transient excitation and subsequent oscillations that bring the flagellar rotational bias below the prestimulus value in the tethered cell assay, and the cheRBCD mutant is capable of swarming in a Tryptone swarm plate.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas/metabolismo , Células Quimiorreceptoras/metabolismo , Quimiotaxis , Proteínas de la Membrana/metabolismo , Adaptación Fisiológica , Asparagina/metabolismo , Bacillus subtilis/fisiología , Proteínas de la Membrana/genética , Metanol/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , Metilación , MutagénesisRESUMEN
As space missions become longer in duration, the need to recycle waste into useful compounds rises dramatically. This problem can be addressed by the development of Controlled Ecological Life Support Systems (CELSS) (i.e., Engineered Closed/Controlled Eco-Systems (ECCES)), consisting of human and plant modules. One of the waste streams leaving the human module is urine. In addition to the reclamation of water from urine, recovery of the nitrogen is important because it is an essential nutrient for the plant module. A 3-step biological process for the recycling of nitrogenous waste (urea) is proposed. A packed-bed bioreactor system for this purpose was modeled, and the issues of reaction step segregation, reactor type and volume, support particle size, and pressure drop were addressed. Based on minimization of volume, a bioreactor system consisting of a plug flow immobilized urease reactor, a completely mixed flow immobilized cell reactor to convert ammonia to nitrite, and a plug flow immobilized cell reactor to produce nitrate from nitrite is recommended. It is apparent that this 3-step bioprocess meets the requirements for space applications.