Department of Environmental Protection, Estación Experimental del Zaidín, Consejo Superior de Investigaciones Científicas, Profesor Albareda 1, Granada 18008, Spain.
Environ Microbiol. 2011 Jul;13(7):1745-66. doi: 10.1111/j.1462-2920.2011.02499.x. Epub 2011 May 9.
GGDEF and EAL/HD-GYP protein domains are responsible for the synthesis and hydrolysis of the bacterial secondary messenger cyclic diguanylate (c-di-GMP) through their diguanylate cyclase and phosphodiesterase activities, respectively. Forty-three genes in Pseudomonas putida KT2440 are putatively involved in the turnover of c-di-GMP. Of them only rup4959 (locus PP4959) encodes a GGDEF/EAL response regulator, which was identified in a genome wide analysis as preferentially induced while this bacterium colonizes roots and adjacent soil areas (the rhizosphere). By using fusions to reporter genes it was confirmed that the rup4959 promoter is active in the rhizosphere and inducible by corn plant root exudates and microaerobiosis. Transcription of rup4959 was strictly dependent on the alternative transcriptional factor σ(S) . The inactivation of the rup4959-4957 operon altered the expression of 22 genes in the rhizosphere and had a negative effect upon oligopeptide utilization and biofilm formation. In multicopy or when overexpressed, rup4959 enhanced adhesin LapA-dependent biofilm formation, the development of wrinkly colony morphology, and increased Calcofluor stainable exopolysaccharides (EPS). Under these conditions the inhibition of swarming motility was total and plant root tip colonization considerably less efficient, whereas swimming was partially diminished. This pleiotropic phenotype, which correlated with an increase in the global level of c-di-GMP, was not acquired with increased levels of Rup4959 catalytic mutant at GGDEF as a proof of this response regulator exhibiting diguanylate cyclase activity. A screen for mutants in putative targets of c-di-GMP led to the identification of a surface polysaccharide specific to KT2440, which is encoded by the genes cluster PP3133-PP3141, as essential for phenotypes associated with increased c-di-GMP. Cellulose and alginate were discarded as the overproduced EPS, and lipopolysaccharide (LPS) core and O-antigen were found to be essential for the development of wrinkly colony morphology.
GGDEF 和 EAL/HD-GYP 蛋白结构域通过其环二鸟苷酸 (c-di-GMP) 合酶和磷酸二酯酶活性分别负责细菌二级信使 c-di-GMP 的合成和水解。恶臭假单胞菌 KT2440 中的 43 个基因被认为参与 c-di-GMP 的周转。其中只有 rup4959（PP4959 基因座）编码 GGDEF/EAL 反应调节剂，该调节剂在全基因组分析中被鉴定为优先诱导，而该细菌定殖于根和相邻土壤区域（根际）。通过与报告基因融合，证实 rup4959 启动子在根际中具有活性，并可被玉米植物根分泌物和微需氧诱导。 rup4959 的转录严格依赖于替代转录因子 σ(S)。 rup4959-4957 操纵子的失活改变了根际中 22 个基因的表达，并对寡肽利用和生物膜形成产生负面影响。在多拷贝或过表达时，rup4959 增强了黏附素 LapA 依赖性生物膜形成、褶皱菌落形态的发育和 Calcofluor 可染色胞外多糖 (EPS) 的增加。在这些条件下，群体运动的抑制是完全的，植物根尖定殖效率大大降低，而游泳部分减少。这种多效表型与 c-di-GMP 整体水平的增加相关，这是通过增加 GGDEF 上 rup4959 催化突变体的水平来证明该反应调节剂具有环二鸟苷酸合酶活性而获得的。对 c-di-GMP 潜在靶标的突变体进行筛选，导致鉴定出一种特定于 KT2440 的表面多糖，该多糖由基因簇 PP3133-PP3141 编码，是与增加的 c-di-GMP 相关表型相关的必需基因。纤维素和藻酸盐被排除在产生的 EPS 之外，发现脂多糖 (LPS) 核心和 O-抗原对于褶皱菌落形态的发育是必需的。
