Ito Akinobu, May Thithiwat, Taniuchi Asami, Kawata Koji, Okabe Satoshi
Department of Urban and Environmental Engineering, Graduate School of Engineering, Hokkaido University, North-13, West-8, Kita-ku, Sapporo 060-8628, Japan.
Biotechnol Bioeng. 2009 Aug 1;103(5):975-83. doi: 10.1002/bit.22305.
Although importance of the rpoS gene on biofilm formation by Escherichia coli has been suggested, there has not been any report showing where the rpoS is expressed during biofilm formation process. Since physiological state of the cells in the biofilms is considerably heterogeneous, the expression of the rpoS gene must be heterogeneous. In this study, in situ spatial expression of the rpoS gene during biofilm formation was investigated with an rpoS-gfp transcriptional fusion mutant strain. A ribosomal binding site and a gene encoding a green fluorescent protein were introduced into the downstream of the rpoS gene, which enabled us to observe the in situ spatial expression of the rpoS gene during biofilm formation processes without any disturbance of the rpoS expression. In the early stages of the biofilm formation process, the rpoS gene was expressed in the most of the cells. On the other hand, the rpoS expression was observed only at the outside of the biofilms during the late stages of the biofilm formation process. The in situ spatial expression of the rpoS gene in the biofilm was verified by quantifying the expression levels of the rpoS at the outside and the inside of the biofilms with the real time RT-PCR. In addition, global gene expression analysis was performed with DNA microarray to investigate physiological difference between the outside and the inside of the biofilms. This heterogeneous rpoS expression profile suggested that the cells at the outside of the biofilm need to express the rpoS to shift the physiological state to the stationary growth mode such as induction of various stress responses and suppression of the motility.
尽管已有研究表明rpoS基因对大肠杆菌生物膜形成具有重要作用，但尚无任何报告显示在生物膜形成过程中rpoS基因是在哪里表达的。由于生物膜中细胞的生理状态存在很大异质性，rpoS基因的表达也必然是异质性的。在本研究中，利用rpoS - gfp转录融合突变菌株研究了生物膜形成过程中rpoS基因的原位空间表达。在rpoS基因下游引入核糖体结合位点和编码绿色荧光蛋白的基因，这使我们能够在不干扰rpoS表达的情况下观察生物膜形成过程中rpoS基因的原位空间表达。在生物膜形成过程的早期阶段，大多数细胞中rpoS基因都有表达。另一方面，在生物膜形成过程的后期阶段，仅在生物膜外部观察到rpoS的表达。通过实时RT - PCR定量生物膜外部和内部rpoS的表达水平，验证了生物膜中rpoS基因的原位空间表达。此外，利用DNA微阵列进行全局基因表达分析，以研究生物膜外部和内部的生理差异。这种异质性的rpoS表达谱表明，生物膜外部的细胞需要表达rpoS来将生理状态转变为静止生长模式，如诱导各种应激反应和抑制运动性。
