RESUMEN
Like amino acids, the sugars glucose and the nonmetabolizable 2-deoxyglucose caused a turnover of methyl groups on the methyl-accepting chemotaxis proteins. These sugars also caused methanol formation on addition. Thus, in contrast to chemotaxis in Escherichia coli, taxis to phosphotransferase sugars by Bacillus subtilis utilizes the methyl-accepting chemotaxis proteins.
Asunto(s)
Bacillus subtilis/fisiología , Proteínas Bacterianas , Factores Quimiotácticos/metabolismo , Quimiotaxis , Desoxiazúcares/farmacología , Desoxiglucosa/farmacología , Glucosa/farmacología , Proteínas de la Membrana/metabolismo , Bacillus subtilis/efectos de los fármacos , Metanol/metabolismo , Proteínas Quimiotácticas Aceptoras de MetiloRESUMEN
The addition of attractant to Bacillus subtilis briefly exposed to radioactive methionine causes an increase of labeling of the methyl-accepting chemotaxis proteins. The addition of attractant to cells radiolabeled for longer times shows no change in the extent of methylation. Therefore, the increase in labeling for the briefly labeled cells is due to an increased turnover of methyl groups caused by attractant. All amino acids gave enhanced turnover. This turnover lasted for a prolonged time, probably spanning the period of smooth swimming caused by the attractant addition. Repellent did not affect the turnover when added alone or simultaneously with attractant. Thus, for amino acid attractants, the turnover is probably the excitatory signal, which is seen to extend long into or throughout the adaptation period, not just at the start of it.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas , Factores Quimiotácticos/metabolismo , Quimiotaxis , Proteínas de la Membrana/metabolismo , Aminoácidos/farmacología , Bacillus subtilis/efectos de los fármacos , Cinética , Metionina/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , Metilación , Técnica de Dilución de Radioisótopos , TritioRESUMEN
If Bacillus subtilis is incubated in radioactive methionine in the absence of protein synthesis, the methyl-accepting chemotaxis proteins (MCPs) become radioactively methylated. If the bacteria are further incubated in excess nonradioactive methionine ("cold-chased") and then given the attractant aspartate, the MCPs lose about half of their radioactivity due to turnover, in which lower specific activity methyl groups from S-adenosylmethionine (AdoMet) replace higher specific activity ones. Due to the cold-chase, the specific activity of the AdoMet pool is reduced at least 2-fold. If, later, the attractant is removed, higher specific activity methyl groups return to the MCPs. Thus, there must exist an unidentified methyl carrier that can "reversibly" receive methyl groups from the MCPs. In a similar experiment, labeled cells were transferred to a flow cell and exposed to addition and removal of attractant and of repellent. All four kinds of stimuli were found to cause methanol production. Bacteria with maximally labeled MCPs were exposed to many cycles of addition and removal of attractant; the maximum amount of radioactive methanol was evolved on the third, not the first, cycle. This result suggests that there is a precursor-product relationship between methyl groups on the MCPs and on the unidentified carrier, which might be the direct source of methanol. However, since no methanol was produced when a methyltransferase mutant, whose MCPs were unmethylated, was exposed to addition and removal of attractant or repellent, the methanol must ultimately derive from methylated MCPs.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas , Factores Quimiotácticos/metabolismo , Quimiotaxis , Proteínas de la Membrana/metabolismo , Metionina/metabolismo , Bacillus subtilis/fisiología , Cinética , Metanol/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , MetilaciónRESUMEN
In Bacillus subtilis, addition of chemotactic attractant causes an immediate change in distribution of methyl groups on methyl-accepting chemotaxis proteins (MCPs), whereas in Escherichia coli, it causes changes that occur throughout the adaptation period. Thus, methylation changes in B. subtilis are probably related to excitation, not adaptation. If labeled cells are exposed to excess nonradioactive methionine, then attractant causes immediate 50% delabeling of the MCPs, suggesting that a flux of methyl groups through the MCPs occurs. Methanol is given off at a high rate during the adaptation period and probably reflects demethylation of some substance to bring about adaptation. The fact that many radioactive methyl groups are lost immediately from the MCPs but only slowly arise as methanol is consistent with the hypothesis that they are transferred from the MCPs to a carrier from which methanol arises. Demethylation of this carrier may cause adaptation.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas , Factores Quimiotácticos/farmacología , Proteínas de la Membrana/metabolismo , Adaptación Fisiológica , Ácido Aspártico/farmacología , Bacillus subtilis/efectos de los fármacos , Metanol/metabolismo , Proteínas Quimiotácticas Aceptoras de Metilo , MetilaciónRESUMEN
We present evidence for methyl (as methyl or methoxy) transfer from the methyl-accepting chemotaxis proteins H1 and possibly H3 of Bacillus subtilis to the methyl-accepting chemotaxis protein H2. This methyl transfer, which has been observed in vitro (D. J. Goldman and G. W. Ordal, Biochemistry 23:2600-2606, 1984), was strongly stimulated by the chemoattractant aspartate and thus may play an important role in the sensory processing system of this organism. Although radiolabeling of H1 and H3 began at once after the addition of [3H]methionine, radiolabeling of H2 showed a lag. Furthermore, the addition of excess nonradioactive methionine caused immediate exponential delabeling of H1 and H3 while labeling of H2 continued to increase. Methylation of H2 required the chemotactic methyltransferase, probably to first methylate H1 and H3. Aspartate caused increased labeling of H2 and strongly decreased labeling of H1 and H3 after the addition of nonradioactive methionine. Without the addition of nonradioactive methionine, aspartate caused demethylation of H1 and to a lesser extent H3, with an approximately equal increase of methylation of H2.
Asunto(s)
Bacillus subtilis/metabolismo , Proteínas Bacterianas , Factores Quimiotácticos/metabolismo , Proteínas de la Membrana/metabolismo , Electroforesis en Gel de Poliacrilamida , Proteínas Quimiotácticas Aceptoras de Metilo , MetilaciónRESUMEN
Bacillus subtilis responds to chemotactic attractants by demethylating certain membrane-bound proteins, termed methyl-accepting chemotaxis proteins (MCPs) and by augmenting the evolution of methanol. We propose that the methanol comes from a methylated intermediate rather than directly from the MCPs themselves. First, repellent blocks attractant-induced smooth swimming and methanol formation, but not MCP demethylation. Second, prior treatment of cells with much attractant to reduce radiolabeling of MCPs and increase that of the putative intermediate caused increased, rather than decreased, production of methanol upon addition and then removal of the repellent. Third, such cells also produced much, rather than little, methanol upon addition of less attractant than during the pretreatment. We speculate that unmethylated intermediate causes tumbling; attractant causes its methylation and hence absence of tumbling (smooth swimming). Its demethylation during the period of smooth swimming affords adaptation.