Identification of NpdA as the protein forming the surface layer in and evidence of its occurrence as a surface layer protein in diverse genera of the Betaproteobacteria and Gammaproteobacteria.

The phytopathogen possesses an ortholog of a newly identified surface layer protein (SLP) termed NpdA but has not been reported to produce a surface layer (S-layer). This study had two objectives. First, to determine if formed an NpdA-based S-layer and, if so, assess the effects of S-layer formation on virulence, production of nanostructures termed nanopods, and other phenotypes. Second, to establish the distribution of orthologs throughout the Pseudomonadota and examine selected candidate cultures for physical evidence of S-layer formation. Formation of an NpdA-based S-layer by AAC00-1 was confirmed by gene deletion mutagenesis (Δ), proteomics, and cryo-electron microscopy. There were no significant differences between the wild-type and mutant in virulence assays with detached watermelon fruit. Nanopods contiguous with S-layers of multiple biofilm cells were visualized by transmission electron microscopy. Orthologs of were identified in 62 Betaproteobacteria species and 49 Gammaproteobacteria species. In phylogenetic analyses, NpdA orthologs largely segregated into distinct groups. Cryo-electron microscopy imaging revealed an NpdA-like S-layer in all but one of the 16 additional cultures examined. We conclude that NpdA represents a new family of SLP, forming an S-layer in and other Pseudomonadota. While the S-layer did not contribute to virulence in watermelon fruit, a potential role of the S-layer in another dimension of pathogenesis cannot be ruled out. Lastly, formation of cell-bridging nanopods in biofilms is a new property of S-layers; it remains to be determined if nanopods can mediate intercellular movement of materials.