The formation of the Salmonella extracellular matrix is a multicellular behavior important for environmental persistence. It is comprised of uniquely but ill-defined assembled thin aggregative fimbriae (Tafi), cellulose and uncharacterized polysaccharides. Consequently, investigations were launched into further clarifying Tafi assembly and the polysaccharide constituents of the extracellular matrix. In the Salmonella agfBAC Tafi operon, the transcription and role of agfC has been elusive. In this study using the clinical isolate, Salmonella Enteritidis 27655-3b, agfBAC transcripts were detected using a reverse transcriptase and transcription was not enhanced by replacement of a stem-loop structure immediately preceding agfC. AgfChis was purified, localized to the periplasm, and found to specifically bind noncrystalline cellulose suggesting an association with the extracellular matrix. An inframe ΔagfC mutant displayed an abundance of 20 nm fibers, which could be complemented with agfC in trans, in addition to Tafi and an increase in cell hydrophobicity. Depolymerization of purified 20 nm fibers required exceptionally stringent conditions to release what proved to be AgfA subunits revealing the 20 nm fibers as AgfA assemblages of unique morphology. The role of AgfC in Tafi assembly was investigated further via a novel, quantitative antibody-capture assay of in-frame agf mutants. A soluble antibody-accessible form of AgfA was captured in wt, ΔagfB and ΔagfF strains in support of the extracellular nucleation-precipitation pathway of Tafi assembly, but not in ΔagfC or ΔagfE mutants. These results suggest that AgfC and AgfE are required for AgfA’s extracellular assembly and thus may act as atypical AgfAspecific chaperones which facilitate Tafi assembly. The implications of these results are presented in an assembly model for Tafi. Additional investigations revealed that Salmonella produces an O-Antigen capsule co-regulated with the extracellular matrix. Structural analysis of purified extracellular polysaccharides (EPS) yielded a repeating oligosaccharide unit similar to iv that of lipopolysaccharide O-Antigen with modifications. Putative carbohydrate transport and regulatory operons important for capsule expression, designated emcA-H and emcIJ, were identified by screening a random transposon library with immune serum generated to the capsule. The absence of capsule was confirmed by generating various in-frame Δemc mutants where emcG and emcE were shown to be important in capsule assembly and translocation. Luciferase-based expression studies showed that, AgfD differentially regulated the emc operons in coordination with extracellular matrix genes. Survival assays demonstrated the capsule is important for desiccation tolerance. The emc genes were found to be conserved in Salmonellae and thus, the O-Antigen extracellular matrix capsule may be a conserved survival strategy important for environmental persistence. Finally, a compositionally unique acidic EPS was found associated with the extracellular matrix. In-frame ΔbcsA, ΔemcG and ΔagfA mutants but neither ΔagfAΔbcsA nor ΔagfD mutants bound calcofluor, a β-glucan binding fluorescent agent, suggesting that multicellular behavior itself and not necessarily AgfD alone was influencing EPS expression. A transposon library was screened by ELISA using serum generated against purified EPS. This identified mutations inactivating genes involved in quorum sensing AI-2 degradation, flagella repression and Tafi and TolA expression. All mutations resulted in the loss of multicellular behavior and immunologically decreased levels of Tafi. This is the first report that implicates quorum sensing AI-2 degradation and flagella repression as part of the regulatory circuit for Tafi expression. Together, the results reveal Tafi uses assembly factors to facilitate extracellular polymerization which likely assists the formation of a network of branched, amorphous fimbriae. Tafi together with EPS form the extracellular matrix: Tafi stabilizes the EPS on the microbial communities; EPS imparts it with physical properties such as hydration, charge and diffusion barriers that protect it from adverse environmental conditions such as desiccation and antimicrobials. This probably contributes to Salmonella survival in the environment and facilitates its cyclic lifestyle.

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