Department of Microbiology, University of Texas Southwestern Medical Center, Dallas, TX 75390-9048, USA.
J Bacteriol. 2010 Apr;192(7):1965-74. doi: 10.1128/JB.01616-09. Epub 2010 Jan 29.
Decorin-binding proteins B and A (DbpB and DbpA) are thought to play important roles in Borrelia burgdorferi pathogenesis by serving as adhesins for the extracellular matrix. It has been established that the expression of DbpBA is governed by the Rrp2-RpoN-RpoS regulatory pathway. However, the precise mechanism underlying the control of DbpBA expression has been unclear. In particular, it has been unknown whether RpoS influences DbpBA expression directly or indirectly (through an additional regulatory molecule[s]). Here, employing a wild-type B. burgdorferi strain and a dbpBA-deficient mutant, we analyzed the 5' genetic elements of the dbpBA operon using deletion analysis, coupled with luciferase reporter assays, quantitative reverse transcription PCR, and immunoblot analyses. A minimal promoter, encompassed within 70 bp upstream of the ATG start codon of dbpBA, was identified and found to be necessary and sufficient to initiate dbpBA transcription. The minimal dbpBA promoter was responsive to environmental stimuli such as temperature, pH, and whole blood. Two in silico-identified inverted repeat elements were not involved in the response of dbpBA expression to in vitro stimulation by environmental factors. The expression of dbpBA from the minimal promoter was abolished when rpoS was inactivated. In addition, the targeted mutagenesis of a C at position -14 within the extended -10 region of dbpBA, which has been postulated to be strategic for Esigma(S) binding in Escherichia coli, abolished dbpBA expression in B. burgdorferi. These combined data suggest that the Rrp2-RpoN-RpoS pathway controls dbpBA expression by the direct binding of RpoS to an RpoS-dependent promoter. However, given that there remains a distinct difference between the expression of DbpBA and other genes under the direct control of RpoS (e.g., OspC), our findings do not preclude the existence of another layer of gene regulation that may contribute to the modulation of DbpBA expression via an as-yet unknown mechanism.
卷曲相关蛋白结合蛋白 B 和 A（DbpB 和 DbpA）被认为通过作为细胞外基质的黏附素在伯氏疏螺旋体的发病机制中发挥重要作用。已经证实 DbpBA 的表达受 Rrp2-RpoN-RpoS 调控途径调控。然而，DbpBA 表达调控的确切机制尚不清楚。特别是，尚不清楚 RpoS 是否直接或间接（通过额外的调节分子）影响 DbpBA 的表达。在这里，我们使用野生型伯氏疏螺旋体菌株和 dbpBA 缺陷突变体，通过缺失分析，结合荧光素酶报告基因检测、定量逆转录 PCR 和免疫印迹分析，分析了 dbpBA 操纵子的 5' 遗传元件。鉴定出一个包含在 dbpBA ATG 起始密码子上游 70bp 内的最小启动子，并发现该启动子对于起始 dbpBA 转录是必需且充分的。最小的 dbpBA 启动子对温度、pH 值和全血等环境刺激有反应。两个在计算机上鉴定的反向重复元件不参与 dbpBA 表达对环境因素体外刺激的反应。当 rpoS 失活时，最小 dbpBA 启动子的 dbpBA 表达被废除。此外，在 dbpBA 延长 -10 区的位置 -14 处靶向突变 C，该突变被假设为大肠杆菌中 Esigma(S) 结合的关键，在伯氏疏螺旋体中也废除了 dbpBA 的表达。这些综合数据表明，Rrp2-RpoN-RpoS 途径通过 RpoS 直接结合到依赖 RpoS 的启动子来控制 dbpBA 的表达。然而，鉴于 DbpBA 的表达与其他直接受 RpoS 调控的基因（例如 OspC）之间仍然存在明显差异，我们的发现并不排除存在另一种基因调控层，该调控层可能通过未知机制对 DbpBA 表达进行调节。
