Jungnitz H, West N P, Walker M J, Chhatwal G S, Guzmán C A
Division of Microbiology, GBF-National Research Centre for Biotechnology, 38124 Braunschweig, Germany.
Infect Immun. 1998 Oct;66(10):4640-50. doi: 10.1128/IAI.66.10.4640-4650.1998.
Random minitransposon mutagenesis was used to identify genes involved in the survival of Bordetella bronchiseptica within eukaryotic cells. One of the mutants which exhibited a reduced ability to survive intracellularly harbored a minitransposon insertion in a locus (ris) which displays a high degree of homology to two-component regulatory systems. This system exhibited less than 25% amino acid sequence homology to the only other two-component regulatory system described in Bordetella spp., the bvg locus. A risA'-'lacZ translational fusion was constructed and integrated into the chromosome of B. bronchiseptica. Determination of beta-galactosidase activity under different environmental conditions suggested that ris is regulated independently of bvg and is optimally expressed at 37 degrees C, in the absence of Mg2+, and when bacteria are in the intracellular niche. This novel regulatory locus, present in all Bordetella spp., is required for the expression of acid phosphatase by B. bronchiseptica. Although catalase and superoxide dismutase production were unaffected, the ris mutant was more sensitive to oxidative stress than the wild-type strain. Complementation of bvg-positive and bvg-negative ris mutants with the intact ris operon incorporated as a single copy into the chromosome resulted in the reestablishment of the ability of the bacterium to produce acid phosphatase and to resist oxidative stress. Mouse colonization studies demonstrated that the ris mutant is cleared by the host much earlier than the wild-type strain, suggesting that ris-regulated products play a significant role in natural infections. The identification of a second two-component system in B. bronchiseptica highlights the complexity of the regulatory network needed for organisms with a life cycle requiring adaptation to both the external environment and a mammalian host.
随机微型转座子诱变被用于鉴定支气管败血波氏杆菌在真核细胞内存活所涉及的基因。其中一个在细胞内存活能力降低的突变体，其微型转座子插入到一个与双组分调节系统具有高度同源性的位点（ris）中。该系统与波氏杆菌属中描述的唯一另一个双组分调节系统bvg位点的氨基酸序列同源性不到25%。构建了一个risA'-'lacZ翻译融合体，并将其整合到支气管败血波氏杆菌的染色体中。在不同环境条件下测定β-半乳糖苷酶活性表明，ris的调节独立于bvg，并且在37摄氏度、不存在Mg2+以及细菌处于细胞内生态位时最佳表达。这个存在于所有波氏杆菌属中的新型调节位点是支气管败血波氏杆菌表达酸性磷酸酶所必需的。虽然过氧化氢酶和超氧化物歧化酶的产生不受影响，但ris突变体比野生型菌株对氧化应激更敏感。用完整的ris操纵子作为单拷贝整合到染色体中对bvg阳性和bvg阴性ris突变体进行互补，导致细菌重新获得产生酸性磷酸酶和抵抗氧化应激的能力。小鼠定殖研究表明，ris突变体比野生型菌株更早被宿主清除，这表明ris调节的产物在自然感染中起重要作用。支气管败血波氏杆菌中第二个双组分系统的鉴定突出了对于具有需要适应外部环境和哺乳动物宿主的生命周期的生物体而言所需调节网络的复杂性。
