The diguanylate cyclase GcpA inhibits the production of pectate lyases via the H‐NS protein and RsmB regulatory RNA in Dickeya dadantii

Abstract

Dickeya dadantii 3937 secretes pectate lyases (Pels) to degrade plant cell walls. Previously, we have demonstrated that EGcpB and EcpC function as bis-(3',5')-cyclic dimeric guanosine monophosphate (c-di-GMP)-specific phosphodiesterases (PDEs) to positively regulate Pel production. However, the diguanylate cyclase (DGC) responsible for the synthesis of c-di-GMP and the dichotomous regulation of Pel has remained a mystery. Here, we identified GcpA as the dominant DGC to negatively regulate Pel production by the specific repression of pelD gene expression. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) assays revealed that the expression levels of histone-like, nucleoid-structuring protein encoding gene hns and post-transcriptional regulator encoding genes rsmA and rsmB were significantly affected by GcpA. Deletion of hns or rsmB in the gcpA site-directed mutant restored its Pel production and pelD expression, demonstrating that H-NS and RsmB contribute to the GcpA-dependent regulation of Pel in D. dadantii. In addition, RsmB expression was subject to positive regulation by H-NS. Thus, we propose a novel pathway consisting of GcpA-H-NS-RsmB-RsmA-pelD that controls Pel production in D. dadantii. Furthermore, we showed that H-NS and RsmB are responsible for the GcpA-dependent regulation of motility and type III secretion system (T3SS) gene expression, respectively. Of the two PDEs involved in the regulation of Pels, only EGcpB regulates pelD expression through the same pathway as GcpA.

A feed‐forward signalling circuit controls bacterial virulence through linking cyclic di‐GMP and two mechanistically distinct sRNAs, ArcZ and RsmB

“…As, our results (Fig. B and C) showed that Hfq controls T3SS and Pel through additional pathways independent of PecT, and c‐di‐GMP negatively regulates T3SS, Pel and swimming motility in D. dadantii (Yi et al ., ; Yuan et al ., ; Yuan et al ., ), we further assessed whether Hfq controls T3SS and Pel through c‐di‐GMP signalling. We first compared the intracellular c‐di‐GMP levels between wild‐type and Δ hfq using ultra performance liquid chromatography coupled with tandem mass spectrometry (UPLC‐MS–MS).…”

“…Additionally, we explored the contributions of GcpA and GcpL to T3SS and Pel in wild‐type, the hfq mutant, and the hfq / pecT double mutant. Unlike the gcpA deletion that increases both the hrpA expression and Pel production (Yuan et al ., ), deletion of gcpL in the wild‐type background only slightly increased Pel production (Yuan et al ., ) and had no effect on the expression of hrpA (data not shown). Deletion of either of gcpA or gcpL in the hfq mutant background did not alter T3SS or Pel (Supporting Information Fig.…”

“…A similar phenotype has been reported in the plague pathogen Yersinia pestis , in which Hfq modulates the abundance of a DGC and a PDE (Bellows et al ., ). D. dadantii encodes 18 GGDEF and/or EAL domain encoding genes in the genome (Yuan et al ., ). After individually inactivating each one of these genes, we found that GcpA, EGcpB and EcpC modulate T3SS, Pel and swimming motility (Yi et al ., ; Yuan et al ., ), whereas GcpL seems to be involved only in swimming regulation, suggesting a functional specificity in different c‐di‐GMP signalling pathways.…”

“…D. dadantii encodes 18 GGDEF and/or EAL domain encoding genes in the genome (Yuan et al ., ). After individually inactivating each one of these genes, we found that GcpA, EGcpB and EcpC modulate T3SS, Pel and swimming motility (Yi et al ., ; Yuan et al ., ), whereas GcpL seems to be involved only in swimming regulation, suggesting a functional specificity in different c‐di‐GMP signalling pathways. Surprisingly, Hfq selectively repressed the abundance of two DGCs, GcpA and GcpL, and that both c‐di‐GMP signalling pathways were required for the positive effects of Hfq on swimming motility, T3SS and Pel (Fig.…”

“…Why does D. dadantii require Hfq to control two DGCs that affect the same biological function? Structurally, GcpA is a predicted cytoplasmic protein that contains two types of N‐terminal sensory domains, GAF (c G MP phosphodiesterase, A denyl cyclase, F hlA domain) and PAS (Per/Arnt/Sim), whereas GcpL contains two N‐terminal transmembrane domains and a predicted periplasmic calcium channel chemotaxis receptor (CACHE) domain (Yuan et al ., ; Supporting Information Fig. S13).…”