O'Hara M B, Hageman J H
Department of Chemistry, New Mexico State University, Las Cruces 88003.
J Bacteriol. 1990 Aug;172(8):4161-70. doi: 10.1128/jb.172.8.4161-4170.1990.
Bacterial cells degrade intracellular proteins at elevated rates during starvation and can selectively degrade proteins by energy-dependent processes. Sporulating bacteria can degrade protein with apparent first-order rate constants of over 0.20 h-1. We have shown, with an optimized [14C]leucine-labeling and chasing procedure, in a chemically defined sporulation medium, that intracellular protein degradation in sporulating cells of Bacillus subtilis 168 (trpC2) is apparently energy dependent. Sodium arsenate, sodium azide, carbonyl cyanide m-chlorophenylhydrozone, and N,N'-dicyclohexylcarbodiimide, at levels which did not induce appreciable lysis (less than or equal to 10%) over 10-h periods of sporulation, inhibited intracellular proteolysis by 13 to 93%. Exponentially growing cells acquired arsenate resistance. In contrast to earlier reports, we found that chloramphenicol (100 micrograms/ml) strongly inhibited proteolysis (68%) even when added 6 h into the sporulation process. Restricting the calcium ion concentration (less than 2 microM) in the medium had no effect on rates or extent of vegetative growth, strongly inhibited sporulation (98%), and inhibited rates of proteolysis by 60% or more. Inhibitors of energy metabolism, at the same levels which inhibited proteolysis, did not affect the rate or degree of uptake of Ca2+ by cells, which suggested that the Ca2+ and metabolic energy requirements of proteolysis were independent. Restricting the Ca2+ concentration in the medium reduced by threefold the specific activity in cells of the major intracellular serine proteinase after 12 h of sporulation. Finally, cells of a mutant of B. subtilis bearing an insertionally inactivated gene for the Ca2(+)-dependent intracellular proteinase-1 degraded protein in chemically defined sporulation medium at a rate indistinguishable from that of the wild-type cells for periods of 8 h.
在饥饿期间，细菌细胞会以较高的速率降解细胞内蛋白质，并且能够通过能量依赖过程选择性地降解蛋白质。形成芽孢的细菌能够以超过0.20 h⁻¹的表观一级速率常数降解蛋白质。我们采用优化的[¹⁴C]亮氨酸标记和追踪程序，在化学限定的芽孢形成培养基中表明，枯草芽孢杆菌168（trpC2）芽孢形成细胞中的细胞内蛋白质降解明显依赖能量。在芽孢形成的10小时期间，砷酸钠、叠氮化钠、羰基氰化物间氯苯腙和N，N'-二环己基碳二亚胺在不引起明显裂解（小于或等于10%）的水平下，抑制细胞内蛋白水解达13%至93%。指数生长期的细胞获得了砷酸盐抗性。与早期报告相反，我们发现氯霉素（100微克/毫升）即使在芽孢形成过程6小时后添加，也能强烈抑制蛋白水解（68%）。限制培养基中的钙离子浓度（小于2微摩尔）对营养生长的速率或程度没有影响，但强烈抑制芽孢形成（98%），并抑制蛋白水解速率达60%或更高。能量代谢抑制剂在抑制蛋白水解的相同水平下，不影响细胞对Ca²⁺的摄取速率或程度，这表明蛋白水解对Ca²⁺和代谢能量的需求是独立的。在芽孢形成12小时后，限制培养基中的Ca²⁺浓度使主要细胞内丝氨酸蛋白酶在细胞中的比活性降低了三倍。最后，枯草芽孢杆菌的一个突变体，其Ca²⁺依赖性细胞内蛋白酶-1基因插入失活，在化学限定的芽孢形成培养基中降解蛋白质的速率在8小时内与野生型细胞的速率无明显差异。
