Regulation of urease gene expression by Streptococcus salivarius growing in biofilms.
Regulation of urease gene expression by Streptococcus salivarius growing in biofilms.
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DOI：
10.1046/j.1462-2920.2000.00088.x
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发表时间：
2000-04
影响因子：
5.1
通讯作者：
Y. Li;Y. Y. Chen-Y.;R. Burne
中科院分区：
文献类型：
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作者：
Y. Li;Y. Y. Chen-Y.;R. Burne
口腔细菌的尿素酶对尿素的代谢深刻地影响口腔生物膜pH稳态和口腔微生物生态。本研究的目的是深入了解低pH值诱导的尿素酶基因在唾液链球菌在体外生长的生物膜中的群体中的表达的调节，并探讨尿素酶调节或尿素酶表达水平在生物膜细胞中是否与非生物膜细胞显著不同。两株S.使用含有与氯霉素乙酰转移酶（cat）基因的尿素酶启动子融合体的唾液腺细菌：PurelCAT，含有与全长pH敏感启动子的融合体;或Pureldelta 100CAT，尿素酶基因启动子的组成型去阻遏缺失衍生物。菌株在Rototorge生物膜反应器中在胰蛋白胨-酵母提取物-蔗糖培养基中生长，有或没有pH控制。CAT和尿素酶活性在生物膜中进行了测量，在“准稳态”和25 mM葡萄糖脉冲后。结果表明，PurelCAT中CAT的表达在相对中性的pH值下被抑制，并且在碳水化合物攻击后，该表达可以被酸性pH诱导。在低pH下生长的PurelCAT的生物膜，没有缓冲，具有约20倍高的CAT水平，并且只有适度的进一步诱导可以引起碳水化合物脉冲。在缓冲培养基中生长的PurelCAT的生物膜中的CAT水平略高于在pH 7.0下培养的增殖细胞所报道的那些，并且在低pH下生长或诱导后生长的Purel-CAT中的CAT水平与完全诱导的增殖细胞所报道的那些相似。在Pureldelta 100CAT中的CAT活性是组成性高的，无论生长条件如何。有趣的是，在亲株S. salivarius 57.1，可能比在类似条件下生长的流体恒化器培养物所报道的高130倍。生物膜中较高水平的脲酶活性可能是由生物膜细胞内稳定的脲酶的积累、低pH微环境和生物膜中细胞群体的生长阶段引起的。S.唾液生物膜细胞响应于pH梯度而上调尿素酶表达以及当在生物膜中生长时积累更大量的尿素酶可能对口腔生物膜pH稳态和体内微生物生态学具有显著影响。此外，S.将pH敏感型尿素酶基因启动子与合适的报告基因融合，构建了一种可用于原位检测生物膜pH的生物探针。
The metabolism of urea by urease enzymes of oral bacteria profoundly influences oral biofilm pH homeostasis and oral microbial ecology. The purpose of this study was to gain insight into the regulation of expression of the low pH-inducible urease genes in populations of Streptococcus salivarius growing in vitro in biofilms and to explore whether urease regulation or the levels of urease expression in biofilm cells differed significantly from planktonic cells. Two strains of S. salivarius harbouring urease promoter fusions to a chloramphenicol acetyltransferase (cat) gene were used: PurelCAT, containing a fusion to the full-length, pH-sensitive promoter; or Pureldelta100CAT, a constitutively derepressed deletion derivative of the urease gene promoter. The strains were grown in a Rototorque biofilm reactor in a tryptone-yeast extract-sucrose medium with or without pH control. Both CAT and urease activities in biofilms were measured at 'quasi-steady state' and after a 25mM glucose pulse. The results showed that CAT expression in PurelCAT was repressed at relatively neutral pH values, and that expression could be induced by acidic pH after carbohydrate challenge. Biofilms of PurelCAT grown at low pH, without buffering, had about 20-fold higher CAT levels, and only a modest further induction could be elicited with carbohydrate pulsing. The levels of CAT in biofilms of PurelCAT grown in buffered medium were slightly higher than those reported for planktonic cells cultured at pH 7.0, and the levels of CAT in Purel-CAT growing at low pH or after induction were similar to those reported for fully induced planktonic cells. CAT activity in Pureldelta100CAT was constitutively high, regardless of growth conditions. Interestingly, urease activity detected in biofilms of the parent strain, S. salivarius 57.1, could be as much as 130-fold higher than that reported for fluid chemostat cultures grown under similar conditions. The higher level of urease activity in biofilms was probably caused by the accumulation of the stable urease enzyme within biofilm cells, low pH microenvironments and the growth phase of populations of cells in the biofilm. The ability of S. salivarius biofilm cells to upregulate urease expression in response to pH gradients and to accumulate greater quantities of urease enzyme when growing in biofilms may have a significant impact on oral biofilm pH homeostasis and microbial ecology in vivo. Additionally, S. salivarius carrying the pH-sensitive urease gene promoter fused to an appropriate reporter gene may be a useful biological probe for sensing biofilm pH in situ.