Pucciarelli M Graciela, Prieto Ana I, Casadesús Josep, Garcı A-Del Portillo Francisco
Departamento de Biotecnologı́a Microbiana, Centro Nacional de Biotecnologı́a, Consejo Superior de Investigaciones Cientı́ficas (CSIC), Campus de Cantoblanco, 28049 Madrid, Spain1.
Departamento de Genética, Facultad de Biologı́a, Universidad de Sevilla, 41080 Sevilla, Spain2.
Microbiology (Reading). 2002 Apr;148(Pt 4):1171-1182. doi: 10.1099/00221287-148-4-1171.
Mutants of Salmonella enterica serovar Typhimurium lacking DNA adenine (Dam) methylase show reduced secretion of invasion effectors encoded in the Salmonella-pathogenicity island 1 (SPI-1). Concomitant with this alteration, a high number and quantity of extracellular proteins are detected in cultures of Dam(-) mutants. This study shows by subcellular fractionation analysis that the presence of numerous extracellular proteins in cultures of Dam(-) mutants is linked to an exacerbated release of membrane particulate material. The membrane 'leaky' phenotype and the impaired functionality of type III secretion systems were, however, unrelated since exacerbated release of proteins to the medium was evident in Dam(-) strains carrying mutations in either SPI-1 (invA, invJ) or flagellar (flhD) genes. This result supports the view that Dam methylation controls a plethora of cellular processes. Electron microscopy analysis demonstrated that the accumulation of membrane particulate material occurs preferentially as vesicles in stationary cultures of Dam(-) strains. In addition, a reduction in the relative amount of peptidoglycan-associated lipoprotein (PAL), TolB, OmpA and murein lipoprotein (Lpp) bound to peptidoglycan was observed in actively growing Dam(-) mutants. The existence of an envelope defect was further confirmed by the increased sensitivity to deoxycholate exhibited by Dam(-) mutants, mostly during exponential growth. Unexpectedly, lack of Dam methylation neither increased envelope instability nor impaired the association of PAL-Tol-Lpp proteins to the peptidoglycan in Escherichia coli. Accordingly, E. coli Dam(-) mutants did not show sensitivity to deoxycholate. Altogether, these results indicate that, besides its role in modulating the secretion of effectors by the SPI-1-encoded type III apparatus, Dam methylation controls cell envelope integrity in S. enterica.
缺乏DNA腺嘌呤（Dam）甲基化酶的鼠伤寒沙门氏菌血清型鼠伤寒变种显示，沙门氏菌致病岛1（SPI-1）中编码的侵袭效应蛋白的分泌减少。伴随着这种变化，在Dam（-）突变体培养物中检测到大量且数量较多的细胞外蛋白质。本研究通过亚细胞分级分离分析表明，Dam（-）突变体培养物中大量细胞外蛋白质的存在与膜颗粒物质的加剧释放有关。然而，膜“渗漏”表型和III型分泌系统的功能受损并无关联，因为在携带SPI-1（invA、invJ）或鞭毛（flhD）基因突变的Dam（-）菌株中，蛋白质向培养基中的加剧释放很明显。这一结果支持了Dam甲基化控制大量细胞过程的观点。电子显微镜分析表明，膜颗粒物质的积累在Dam（-）菌株的静止培养物中优先以囊泡形式出现。此外，在活跃生长的Dam（-）突变体中，观察到与肽聚糖结合的肽聚糖相关脂蛋白（PAL）、TolB、OmpA和胞壁质脂蛋白（Lpp）的相对量减少。Dam（-）突变体对脱氧胆酸盐的敏感性增加进一步证实了包膜缺陷的存在，这主要发生在指数生长期。出乎意料的是，缺乏Dam甲基化既没有增加包膜不稳定性，也没有损害大肠杆菌中PAL-Tol-Lpp蛋白与肽聚糖的结合。因此，大肠杆菌Dam（-）突变体对脱氧胆酸盐不敏感。总之，这些结果表明，除了在调节SPI-1编码的III型装置效应蛋白分泌中的作用外，Dam甲基化还控制肠炎沙门氏菌的细胞包膜完整性。
