Control of pellicle biogenesis involves the diguanylate cyclases PdgA and PdgB, the c‐di‐GMP binding protein MxdA and the chemotaxis response regulator CheY3 in Shewanella oneidensis

Abstract

Summary Shewanella oneidensis is an aquatic proteobacterium with remarkable respiratory and chemotactic abilities. It is also capable of forming biofilms either associated to surfaces (SSA‐biofilm) or at the air–liquid interface (pellicle). We have previously shown that pellicle biogenesis in S. oneidensis requires the flagellum and the chemotaxis regulatory system including CheA3 kinase and CheY3 response regulator. Here we searched for additional factors involved in pellicle development. Using a multicopy library of S. oneidensis chromosomal fragments, we identified two genes encoding putative diguanylate cyclases (pdgA and pdgB) and allowing pellicle formation in the non‐pellicle‐forming cheY3‐deleted mutant. A mutant deleted of both pdgA and pdgB is affected during pellicle development. By overexpressing phosphodiesterase encoding genes, we confirmed the key role of c‐di‐GMP in pellicle biogenesis. The mxd operon, previously proposed to encode proteins involved in exopolysaccharide biosynthesis, is also essential for pellicle formation. In addition, we showed that the MxdA protein, containing a degenerate GGDEF motif, binds c‐di‐GMP and interacts with both CheY3 and PdgA. Therefore, we propose that pellicle biogenesis in S. oneidensis is controlled by a complex pathway that involves the chemotaxis response regulator CheY3, the two putative diguanylate cyclases PdgA and PdgB, and the c‐di‐GMP binding protein MxdA.

The phosphorylated regulator of chemotaxis is crucial throughout biofilm biogenesis in Shewanella oneidensis

“…These results strongly suggest that the CheY3 active form allowing SSA-biofilm formation is the phosphorylated one. In contrast, for pellicle formation, neither the cheY3D56A nor the cheY3D56E allele was able to complement the ΔcheY3 strain 32,40 .…”

“…Given the crucial role played by CheY3 in SSA-biofilm biogenesis, we hypothesized that other DGCs could interact with CheY3 and be involved in SSA-biofilm formation. Therefore we applied the same strategy that was previously used in the pellicle context for SSA-biofilm recovery 40 . Briefly, we incubated the ΔcheY3 strain containing the genomic plasmid-borne library in LM medium containing lactate for 3 or 4 days.…”

“…Like CheY3, CheY EC and CheY VC interact with the c-di-GMP effector MxdA It was previously shown that the Mxd machinery, which is probably involved in the synthesis and secretion of an exopolysaccharide, was required for SSA-biofilm development 34,37 . As it was recently demonstrated that CheY3 interacts with the c-di-GMP effector MxdA 40 , which probably activates the Mxd machinery, we wanted to determine whether the orthologous CheY proteins could bind MxdA. We therefore performed bacterial two-hybrid assays using MxdA fused to the T25 domain of adenylate cyclase and the CheY proteins fused to the T18 domain of adenylate cyclase.…”

“…To construct the ΔcheA3ΔcheY3 mutant strain, the cheY3 (SO3209) gene was deleted from the ΔcheA3 genome 32 . The Δ4DGC quadruple mutant strain was constructed by sequential deletion of the SO4207 and SO4407 genes, starting with the ΔpdgAΔpdgB double mutant strain 40 . To construct the Δ4DGCΔcheY3 mutant strain, the cheY3 gene was deleted from the Δ4DGC genome.…”

“…Recently, we identified two DGCs implicated in pellicle formation, PdgA, and PdgB, and showed that they interact with CheY3. Interestingly, PdgA also interacts with MxdA that proved to be a cdi-GMP-binding protein 40 . This partnership DGC-effector protein strengthens the c-di-GMP local signaling model.…”