Broad-Host-Range Shuttle Vectors for Screening of Regulated Promoter Activity in Viridans Group Streptococci: Isolation of a pH-Regulated Promoter

Viridans group streptococci are major constituents of the normal human oral flora and are also identified as the predominant pathogenic bacteria in native valve infective endocarditis. Little information is available regarding the regulation of gene expression in viridans group streptococci, either in response to changes in the oral environment or during development of endocarditis. We therefore constructed a set of broad-host-range vectors for the isolation of promoters from viridans group streptococci that are activated by specific environmental stimuli in vitro or in vivo. A genomic library of Streptococcus gordonii strain CH1 was constructed in one of the new vectors, and this library was introduced into a homologous bacterium by using an optimized electroporation protocol for viridans group streptococci. Because viridans group streptococci entering the bloodstream from the oral cavity encounter an increase in pH, we selected promoters upregulated by this specific stimulus. One of the selected promoter sequences showed homology to the promoter region of the hydA gene from Clostridium acetobutylicum, the expression of which is known to be regulated by the environmental pH. The isolation of this pH-regulated promoter shows that S. gordonii can sense an increase in the environmental pH, which serves as a signal for bacterial gene activation. Furthermore, this demonstrates the usefulness of these new selection vectors in research on adaptive gene expression of viridans group streptococci and possibly also of other gram-positive bacteria.

草绿色链球菌群（Viridans group streptococci）是人体正常口腔菌群的主要组成成员，同时也是自体瓣膜感染性心内膜炎中占主导地位的病原菌。目前关于草绿色链球菌群的基因表达调控的研究仍较为匮乏，无论是针对口腔环境变化的响应，还是在感染性心内膜炎发生过程中的调控机制均鲜有报道。为此，我们构建了一套宽宿主范围载体（broad-host-range vectors），用于分离可在体外或体内被特定环境刺激激活的草绿色链球菌群启动子。我们以其中一款新型载体构建了戈登链球菌（Streptococcus gordonii）CH1菌株的基因组文库，并通过针对草绿色链球菌群优化的电转化方案（electroporation protocol）将该文库导入同源宿主菌中。由于从口腔进入血液循环的草绿色链球菌群会遭遇环境pH升高的应激条件，我们筛选了受该特定刺激上调的启动子。其中一条筛选得到的启动子序列与丙酮丁醇梭菌（Clostridium acetobutylicum）hydA基因的启动子区域具有同源性，而该基因的表达已知受环境pH调控。该受pH调控的启动子的成功分离，证实戈登链球菌能够感知环境pH升高这一信号，并以此触发细菌基因的激活。此外，本研究证明了这些新型筛选载体在草绿色链球菌群（乃至其他革兰氏阳性菌）的适应性基因表达研究中具有良好的应用价值。