ComX activity of Streptococcus mutans growing in biofilms
ComX activity of Streptococcus mutans growing in biofilms
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DOI：
10.1016/j.femsle.2004.07.032
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发表时间：
2004-09-01
影响因子：
2.1
通讯作者：
Cvitkovitch, DG
中科院分区：
文献类型：
--
作者：
Aspiras, MB;Ellen, RP;Cvitkovitch, DG
在许多链球菌中，包括变形链球菌，遗传能力由群体感应系统调节，该系统由 comC 基因编码的能力刺激肽 (CSP) 信息素介导。在肺炎链球菌中，该系统的核心组成部分是 ComX，它作为替代 sigma 因子来激活参与 DNA 摄取和加工的能力基因。负责变异链球菌遗传能力诱导的群体感应系统与生物膜形成和耐酸反应有关。为了检测变形链球菌中 comX 对 CSP 的反应，构建了 comX 启动子 (pcomX) 与 lacZ 的转录融合，以生成报告载体 pcomx::pALH122（复制载体），并转化到不能产生内源 CSP 的变形链球菌 UA159 comC(-) 中。添加 CSP 并检查 pcomX::lacZ 相对表达指数 (REI)，结果显示添加 CSP 5 分钟和 10 分钟后最大 β-gal 活性增加了 2 倍。还通过测量生长为生物膜的细胞中的 REI 来检查内源 CSP 对 pcomX::lacZ 表达的影响； 3 小时时观察到峰值 pcomX 活性。为了确定转化频率的时间模式，将pMA2（一种Sp(r)穿梭载体）转化到生物膜生长的细胞中，在3小时时观察到最大转化频率。使用类似构建的绿色荧光蛋白报告载体 pcomX::gfp 在 4 小时生物膜中进行共聚焦显微镜检查 pcomX 活性，揭示了生物膜群体内高细胞密度区域中的活性 pcomX 活性。这些结果表明 pcomX 活性、自然转化和生物膜能力发展之间呈正相关。 (C) 2004 年由 Elsevier B.V. 代表欧洲微生物学会联合会出版。
In many streptococci, including Streptococcus mutans, genetic competence is regulated by a quorum sensing system mediated by a competence stimulating peptide (CSP) pheromone, encoded by the comC gene. In Streptococcus pneumoniae, a central component of this system is ComX, which acts as an alternative sigma factor to activate competence genes involved in DNA uptake and processing. The quorum sensing system responsible for genetic competence induction in S. mutans has been linked to biofilm formation and the acid tolerance response. To examine the response of comX to CSP in S. mutans, a transcriptional fusion of the comX promoter (pcomX) with lacZ was constructed to generate reporter vector pcomx::pALH122 (replicative vector) and transformed into S. mutans UA159 comC(-), which is unable to produce endogenous CSP. CSP was added and pcomX::lacZ relative expression index (REI) examined, revealing a 2-fold increase in maximal beta-gal activity 5 and 10 min after CSP addition. The effect of endogenous CSP on pcomX::lacZ expression was also examined by measuring REI in cells grown as a biofilm; peak pcomX activity was observed at 3 h. To determine the temporal pattern of transformation frequency, pMA2, a Sp(r) shuttle vector, was transformed into biofilm-grown cells, with maximal transformation frequency observed at 3 h. Confocal microscopy was performed to examine pcomX activity using a similarly constructed green fluorescent protein reporter vector, pcomX::gfp, in a 4-h biofilm, revealing active pcomX activity in high cell density areas within the biofilm population. These results demonstrated a positive correlation between pcomX activity, natural transformation and competence development in biofilms. (C) 2004 Published by Elsevier B.V. on behalf of the Federation of European Microbiological Societies.