Dynamic Regulation of GacA in Type III Secretion, Pectinase Gene Expression, Pellicle Formation, and Pathogenicity of Dickeya dadantii (Erwinia chrysanthemi 3937)

摘要

Dickeya dadantii (Erwinia chrysanthemi 3937) secretes exoenzymes, including pectin-degrading enzymes, leading to the loss of structural integrity of plant cell walls. A type III secretion system (T3SS) is essential for full virulence of this bacterium within plant hosts. The GacS/GacA two-component signal transduction system participates in important biological roles in several gram-negative bacteria. In this study, a gacA deletion mutant (Ech137) of D. dadantii was constructed to investigate the effect of this mutation on pathogenesis and other phenotypes. Compared with wild-type D. dadantii, Ech137 had a delayed biofilm-pellicle formation. The production of pectate lyase (Pel), protease, and cellulase was diminished in Ech137 compared with the wild-type cells. Reduced transcription of two endo-Pel genes, pelD and pelL, was found in Ech137 using a green fluorescence protein-based fluorescence-activated cell sorter promoter activity assay. In addition, the transcription of T3SS genes dspE (an effector), hrpA (a structural protein of the T3SS pilus), and hrpN (a T3SS harpin) was reduced in Ech137. A lower amount of rsmB regulatory RNA was found in gacA mutant Ech137 compared with the wild-type bacterium by quantitative reverse-transcription polymerase chain reaction. Compared with wild-type D. dadantii, a lower amount of hrpL mRNA was observed in Ech137 at 12 h grown in medium. Although the role of RsmA, rsmB, and RsmC in D. dadantii is not clear, from the regulatory pathway revealed in E. carotovora, the lower expression of dspE, hrpA, and hrpN in Ech137 may be due to a post-transcriptional regulation of hrpL through the Gac-Rsm regulatory pathway. Consequently, the reduced exoenzyme production and Pel gene expression in the mutant may be sue partially to the regulatory role of rsmB-RsmA on exoenzyme expression. Similar to in vitro results, a lower expression of T3SS and pectinase genes of Ech137 also was observed in bacterial cells inoculated into Saintpaulia ionantha leaves, perhaps accounting for the observed reduction in local maceration. Interestingly, compared with the wild-type D. dadantii, although a lower concentration of Ech137 was observed at day 3 and 4 postinoculation, there is no significant difference in bacterial concentration between the wild-type bacterium and Ech137 in the early stage of infection. Finally, the nearly abolished systemic invasion ability of Ech137 suggests that GacA of D. dadantii is essential for the pathogenicity and systemic movement of the bacterium in S. ionantha.