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

Abstract

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.

PelN Is a New Pectate Lyase of Dickeya dadantii with Unusual Characteristics

“…PecS is a negative regulator controlling a large set of genes involved in the symptomatic phase of infection (37,39). GacA is a positive regulator involved in the control of virulence genes during plant infection (40,41). In culture media, the pelN expression was weakly induced in the presence of polygalacturonate (Table 3), but it was not affected by the addition of other molecular inducers of pectate lyase synthesis, such as plant extracts or ferulic acid (1,42).…”

The Role of Secretion Systems and Small Molecules in Soft-Rot Enterobacteriaceae Pathogenicity

“…Despite this, there has been significant progress in modeling gene regulation in SRE, including mathematical models of virulence (75) and examination of gene expression at the single cell level (87,158,159,166),).…”

“…These two compounds upregulate the expression of the small regulatory RNA rsmB and the T3SS regulator hrpL, suggesting that OCA and TCA signaling occurs via the GacA/S two component system (159). This finding led to the discovery that another plant phenolic compound, p-coumaric acid (PCA) represses the expression of T3SS genes (87).…”

SlyA Regulates Type III Secretion System (T3SS) Genes in Parallel with the T3SS Master Regulator HrpL in Dickeya dadantii 3937

“…HrpS, an NtrC family enhancer protein, initiates the transcription of hrpL in an RpoN-dependent manner (27,41). In addition to the transcriptional regulation, the amount of hrpL mRNA is controlled by the regulator of a secondary metabolism (Rsm) system through the GacS-GacA-rsmB-RsmA pathway (35,43). The Rsm system in D. dadantii is composed of RsmA and rsmB.…”