Inactivation of the Stress- and Starvation-Inducible gls24 Operon Has a Pleiotrophic Effect on Cell Morphology, Stress Sensitivity, and Gene Expression in Enterococcus faecalis

Enterococcus faecalis induces the synthesis of at least 42 proteins during 24 h of glucose starvation. Because of its induction during carbohydrate and complete starvation (incubation in tap water) and CdCl(2) and bile salts stresses, one of these proteins (Gls24) was qualified as a “general stress protein” and was analyzed at the molecular level. Its corresponding gene, gls24, seems to be the penultimate gene of an operon composed, altogether, of six open reading frames (ORFs). The ORF preceding gls24 (orf4) showed very strong identity with gls24. The deduced polypeptides of these two genes showed similarity with a 20-kDa hypothetical protein from Lactococcus lactis and an alkaline stress protein from Staphylococcus aureus with no previously known biological significance. Data from the operon sequence and Northern analysis led to the conclusions that (i) gls24 possesses its own promoter which is especially induced at the onset of starvation and (ii) the operon promoter is stress inducible in exponential-phase cells. A mutation in the gls24 gene led to a severe reduction of growth rate and reduction of survival against 0.3% bile salts in the 24-h-starved cells compared to the wild-type strain. Moreover, the chain length of the mutant is significantly reduced during growth. These results argue strongly for a role of the protein Gls24 and/or GlsB in morphological changes and in stress tolerance in E. faecalis. Comparison of two-dimensional protein gels from wild-type cells with those from gls24 mutant cells revealed a pleiotropic effect of the mutation on gene expression. At least nine proteins were present in larger amounts in the mutant. For six of them, the corresponding N-terminal microsequence has been obtained. Three of these sequences map in genes coding for l-lactate dehydrogenase, lipoamide dehydrogenase, and pyruvate decarboxylase, all involved in pyruvate metabolism.

粪肠球菌（Enterococcus faecalis）在葡萄糖饥饿24小时期间可诱导至少42种蛋白质的合成。由于该类蛋白质在碳水化合物饥饿、完全饥饿（于自来水中孵育）以及氯化镉（CdCl₂）和胆汁盐胁迫下均会被诱导，其中一种名为Gls24的蛋白质被归类为“通用胁迫蛋白”，并在分子水平上得到了深入分析。其对应的基因gls24似乎是一个由6个开放阅读框（open reading frames, ORFs）组成的操纵子的倒数第二个基因。位于gls24上游的ORF（orf4）与gls24具有极高的序列同源性。这两个基因推导得到的多肽与乳酸乳球菌（Lactococcus lactis）的20 kDa假定蛋白以及金黄色葡萄球菌（Staphylococcus aureus）的碱性胁迫蛋白具有序列相似性，此前二者的生物学功能均未被阐明。通过操纵子序列分析和Northern印迹分析（Northern analysis），研究得出以下结论：（1）gls24拥有自身的启动子，该启动子在饥饿起始阶段被显著诱导；（2）该操纵子的启动子可在指数生长期细胞中被胁迫诱导。与野生型菌株相比，gls24基因发生突变后，饥饿24小时的细胞的生长速率大幅下降，且在0.3%胆汁盐胁迫下的存活率显著降低。此外，突变株在生长过程中的细胞链长明显缩短。上述结果强烈表明，Gls24和/或GlsB蛋白在粪肠球菌的形态变化及胁迫耐受中发挥关键作用。将野生型细胞与gls24突变株的二维蛋白质凝胶（two-dimensional protein gels）进行对比分析，发现该突变对基因表达具有多效性效应。至少有9种蛋白质在突变株中的表达丰度更高，其中6种蛋白质的N端微序列（N-terminal microsequence）已被成功获取。其中3个序列定位在分别编码L-乳酸脱氢酶（L-lactate dehydrogenase）、硫辛酰胺脱氢酶（lipoamide dehydrogenase）和丙酮酸脱羧酶（pyruvate decarboxylase）的基因中，这三种酶均参与丙酮酸代谢（pyruvate metabolism）。