Lemos José A C, Brown Thomas A, Burne Robert A
Department of Oral Biology, University of Florida College of Dentistry, Gainesville, Florida 32610, USA.
Infect Immun. 2004 Mar;72(3):1431-40. doi: 10.1128/IAI.72.3.1431-1440.2004.
Streptococcus mutans is a biofilm-forming bacterium that is adapted to tolerate rapid and dramatic fluctuations in nutrient availability, carbohydrate source, and pH in its natural environment, the human oral cavity. Dissecting the pathways used to form stable biofilms and to tolerate environmental stress is central to understanding the virulence of this organism. Here, we investigated the role of the S. mutans relA gene, which codes for a guanosine tetraphosphate and guanosine pentaphosphate [(p)ppGpp] synthetase/hydrolase, in biofilm formation and acid tolerance. Two mutants in which relA was insertionally inactivated or replaced by an antibiotic resistance determinant were constructed. Under normal growth and stress conditions, the mutants grew slower than the wild-type strain, although the final yields were similar. The mutants, which were still able to accumulate (p)ppGpp after the induction of a stringent response, showed significant reductions in biofilm formation on microtiter plates or hydroxylapatite disks. There was no difference in the sensitivities to acid killing of the parent and relA strains grown in planktonic cultures. However, when cells were grown in biofilms, the mutants became more acid resistant and could lower the pH through glycolysis faster and to a greater extent than the wild-type strain. Differences in acid resistance were not correlated with increases in F-ATPase activity, although bacterial sugar:phosphotransferase activity was elevated in the mutants. Expression of the luxS gene was increased as much as fivefold in the relA mutants, suggesting a link between AI-2 quorum sensing and the stringent response.
变形链球菌是一种形成生物膜的细菌，它能够适应其天然生存环境——人类口腔中营养物质可用性、碳水化合物来源以及pH值的快速而显著的波动。剖析用于形成稳定生物膜以及耐受环境压力的途径对于理解这种生物体的毒力至关重要。在此，我们研究了变形链球菌relA基因在生物膜形成和耐酸性中的作用，该基因编码一种鸟苷四磷酸和鸟苷五磷酸[（p）ppGpp]合成酶/水解酶。构建了两个relA基因被插入失活或被抗生素抗性决定簇取代的突变体。在正常生长和应激条件下，突变体的生长速度比野生型菌株慢，尽管最终产量相似。在严紧反应诱导后仍能积累（p）ppGpp的突变体，在微量滴定板或羟基磷灰石圆盘上的生物膜形成显著减少。在浮游培养中生长的亲本菌株和relA菌株对酸杀伤的敏感性没有差异。然而，当细胞在生物膜中生长时，突变体变得更耐酸，并且通过糖酵解降低pH值的速度比野生型菌株更快、程度更大。耐酸性的差异与F-ATPase活性的增加无关，尽管突变体中的细菌糖：磷酸转移酶活性有所升高。relA突变体中luxS基因的表达增加了多达五倍，这表明自诱导物-2群体感应与严紧反应之间存在联系。
