Gitton Christophe, Meyrand Mickael, Wang Juhui, Caron Christophe, Trubuil Alain, Guillot Alain, Mistou Michel-Yves
Unité Biochimie et Structure des Protéines, INRA, Jouy-en-Josas, France.
Appl Environ Microbiol. 2005 Nov;71(11):7152-63. doi: 10.1128/AEM.71.11.7152-7163.2005.
We have compared the proteomic profiles of L. lactis subsp. cremoris NCDO763 growing in the synthetic medium M17Lac, skim milk microfiltrate (SMM), and skim milk. SMM was used as a simple model medium to reproduce the initial phase of growth of L. lactis in milk. To widen the analysis of the cytoplasmic proteome, we used two different gel systems (pH ranges of 4 to 7 and 4.5 to 5.5), and the proteins associated with the cell envelopes were also studied by two-dimensional electrophoresis. In the course of the study, we analyzed about 800 spots and identified 330 proteins by mass spectrometry. We observed that the levels of more than 50 and 30 proteins were significantly increased upon growth in SMM and milk, respectively. The large redeployment of protein synthesis was essentially associated with an activation of pathways involved in the metabolism of nitrogenous compounds: peptidolytic and peptide transport systems, amino acid biosynthesis and interconversion, and de novo biosynthesis of purines. We also showed that enzymes involved in reactions feeding the purine biosynthetic pathway in one-carbon units and amino acids have an increased level in SMM and milk. The analysis of the proteomic data suggested that the glutamine synthetase (GS) would play a pivotal role in the adaptation to SMM and milk. The analysis of glnA expression during growth in milk and the construction of a glnA-defective mutant confirmed that GS is an essential enzyme for the development of L. lactis in dairy media. This analysis thus provides a proteomic signature of L. lactis, a model lactic acid bacterium, growing in its technological environment.
我们比较了乳酸乳球菌亚种cremoris NCDO763在合成培养基M17Lac、脱脂乳微滤液（SMM）和脱脂乳中生长时的蛋白质组图谱。SMM被用作一种简单的模型培养基，以重现乳酸乳球菌在牛奶中生长的初始阶段。为了扩大对细胞质蛋白质组的分析，我们使用了两种不同的凝胶系统（pH范围为4至7和4.5至5.5），并且还通过二维电泳研究了与细胞包膜相关的蛋白质。在研究过程中，我们分析了约800个斑点，并通过质谱鉴定了330种蛋白质。我们观察到，分别在SMM和牛奶中生长时，超过50种和30种蛋白质的水平显著增加。蛋白质合成的大量重新部署基本上与含氮化合物代谢相关途径的激活有关：肽水解和肽转运系统、氨基酸生物合成和相互转化以及嘌呤的从头生物合成。我们还表明，参与以一碳单位和氨基酸为嘌呤生物合成途径提供原料的反应的酶在SMM和牛奶中的水平有所增加。蛋白质组数据分析表明，谷氨酰胺合成酶（GS）在适应SMM和牛奶方面将发挥关键作用。对牛奶中生长期间glnA表达的分析以及glnA缺陷突变体的构建证实，GS是乳酸乳球菌在乳制品培养基中生长所必需的酶。因此，该分析提供了乳酸乳球菌（一种典型的乳酸菌）在其技术环境中生长的蛋白质组特征。
