Sanchez-Salas J L, Setlow P
Department of Biochemistry, University of Connecticut Health Center, Farmington 06030-3305.
J Bacteriol. 1993 May;175(9):2568-77. doi: 10.1128/jb.175.9.2568-2577.1993.
Degradation of small, acid-soluble spore proteins during germination of Bacillus subtilis spores is initiated by a sequence-specific protease called GPR. Western blot (immunoblot) analysis of either Bacillus megaterium or B. subtilis GPR expressed in B. subtilis showed that GPR is synthesized at about the third hour of sporulation in a precursor form and is processed to an approximately 2- to 5-kDa-smaller species 2 to 3 h later, at or slightly before the time of accumulation of dipicolinic acid by the forespore. This was found with both normal levels of expression of B. subtilis and B. megaterium GPR in B. subtilis, as well as when either protein was overexpressed up to 100-fold. The sporulation-specific processing of GPR was blocked in all spoIII, -IV, and -V mutants tested (none of which accumulated dipicolinic acid), but not in a spoVI mutant which accumulated dipicolinic acid. The amino-terminal sequences of the B. megaterium and B. subtilis GPR initially synthesized in sporulation were identical to those predicted from the coding genes' sequences. However, the processed form generated in sporulation lacked 15 (B. megaterium) or 16 (B. subtilis) amino-terminal residues. The amino acid sequence surrounding this proteolytic cleavage site was very homologous to the consensus sequence recognized and cleaved by GPR in its small, acid-soluble spore protein substrates. This observation, plus the efficient processing of overproduced GPR during sporulation, suggests that the GPR precursor may autoproteolyze itself during sporulation. During spore germination, the GPR from either species expressed in B. subtilis was further processed by removal of one additional amino-terminal amino acid (leucine), generating the mature protease which acts during spore germination.
枯草芽孢杆菌芽孢萌发过程中，小的酸溶性芽孢蛋白的降解由一种名为GPR的序列特异性蛋白酶启动。对在枯草芽孢杆菌中表达的巨大芽孢杆菌或枯草芽孢杆菌GPR进行的蛋白质免疫印迹（免疫印迹）分析表明，GPR在前体形式下于芽孢形成约3小时时合成，并在2至3小时后，即前芽孢积累吡啶二羧酸之时或稍前，加工成一种分子量约小2至5 kDa的形式。在枯草芽孢杆菌中正常表达水平的枯草芽孢杆菌和巨大芽孢杆菌GPR以及两种蛋白均过表达高达100倍时，均发现了这一现象。在所有测试的spoIII、-IV和-V突变体（均不积累吡啶二羧酸）中，GPR的芽孢形成特异性加工均被阻断，但在积累吡啶二羧酸的spoVI突变体中未被阻断。在芽孢形成过程中最初合成的巨大芽孢杆菌和枯草芽孢杆菌GPR的氨基末端序列与从编码基因序列预测的序列相同。然而，在芽孢形成过程中产生的加工形式缺少15个（巨大芽孢杆菌）或16个（枯草芽孢杆菌）氨基末端残基。该蛋白水解切割位点周围的氨基酸序列与GPR在其小的酸溶性芽孢蛋白底物中识别并切割的共有序列非常同源。这一观察结果，加上芽孢形成过程中过量产生的GPR的有效加工，表明GPR前体可能在芽孢形成过程中自我自催化。在芽孢萌发过程中，在枯草芽孢杆菌中表达的任一物种的GPR通过去除另外一个氨基末端氨基酸（亮氨酸）进一步加工，产生在芽孢萌发过程中起作用的成熟蛋白酶。
