Kunji E R, Fang G, Jeronimus-Stratingh C M, Bruins A P, Poolman B, Konings W N
Department of Microbiology, Groningen Biomolecular Sciences and Biotechnology Institute, University of Groningen, Haren, The Netherlands.
Mol Microbiol. 1998 Mar;27(6):1107-18. doi: 10.1046/j.1365-2958.1998.00769.x.
Amino acid auxotrophous bacteria such as Lactococcus lactis use proteins as a source of amino acids. For this process, they possess a complex proteolytic system to degrade the protein(s) and to transport the degradation products into the cell. We have been able to dissect the various steps of the pathway by deleting one or more genes encoding key enzymes/components of the system and using mass spectrometry to analyse the complex peptide mixtures. This approach revealed in detail how L. lactis liberates the required amino acids from beta-casein, the major component of the lactococcal diet. Mutants containing the extracellular proteinase PrtP, but lacking the oligopeptide transport system Opp and the autolysin AcmA, were used to determine the proteinase specificity in vivo. To identify the substrates of Opp present in the casein hydrolysate, the PrtP-generated peptide pool was offered to mutants lacking the proteinase, but containing Opp, and the disappearance of peptides from the medium as well as the intracellular accumulation of amino acids and peptides was monitored in peptidase-proficient and fivefold peptidase-deficient genetic backgrounds. The results are unambiguous and firmly establish that (i) the carboxyl-terminal end of beta-casein is degraded preferentially despite the broad specificity of the proteinase; (ii) peptides smaller than five residues are not formed in vivo; (iii) use of oligopeptides of 5-10 residues becomes only possible after uptake via Opp; (iv) only a few (10-14) of the peptides generated by PrtP are actually used, even though the system facilitates the transport of oligopeptides up to at least 10 residues. The technology described here allows us to monitor the fate of individual peptides in complex mixtures and is applicable to other proteolytic systems.
诸如乳酸乳球菌之类的氨基酸营养缺陷型细菌利用蛋白质作为氨基酸来源。对于这一过程，它们拥有一套复杂的蛋白水解系统来降解蛋白质并将降解产物转运到细胞内。我们通过删除编码该系统关键酶/组分的一个或多个基因，并使用质谱分析法分析复杂的肽混合物，得以剖析该途径的各个步骤。这种方法详细揭示了乳酸乳球菌如何从β-酪蛋白（乳酸乳球菌饮食的主要成分）中释放所需的氨基酸。含有细胞外蛋白酶PrtP但缺乏寡肽转运系统Opp和自溶素AcmA的突变体被用于确定体内蛋白酶的特异性。为了鉴定酪蛋白水解物中存在的Opp底物，将PrtP产生的肽库提供给缺乏蛋白酶但含有Opp的突变体，并在肽酶水平正常和肽酶水平为正常五分之一的遗传背景下监测培养基中肽的消失以及氨基酸和肽在细胞内的积累情况。结果明确无误地证实：（i）尽管蛋白酶具有广泛的特异性，但β-酪蛋白的羧基末端优先被降解；（ii）体内不会形成小于五个残基的肽；（iii）只有通过Opp摄取后，才能利用5至10个残基的寡肽；（iv）尽管该系统有助于至少10个残基的寡肽的转运，但PrtP产生的肽中实际上只有少数（10至14个）被利用。这里描述的技术使我们能够监测复杂混合物中单个肽的去向，并且适用于其他蛋白水解系统。
