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[Preprint]. 2023 Nov 22:2023.11.22.568322. [Version 1] doi: 10.1101/2023.11.22.568322

Figure 1: Simplified model of two-step Type 1 secretion system (T1SS).

Figure 1:

a) T1SS in Pseudomonas fluorescens Pf0-1. The large adhesin LapA is localized to the outer membrane (OM) via a retention module, allowing the bacterium to form a biofilm. The localization of LapA to the OM is regulated by a periplasmic protease LapG and a cyclic di-GMP (cdG) effector protein LapD. At high intracellular cdG levels, the catalytically inactive EAL domain of LapD binds cdG leading to a conformational change whereby the LapD periplasmic domain sequesters LapG, thereby retaining LapA on the OM. When c-di-GMP levels are low, LapD is in its autoinhibited conformation, which releases LapG to cleave LapA at dialanine motif (shown in red), resulting in the release of LapA to the extracellular environment and loss of biofilm formation. b) Proposed model of T1SS in Desulfovibrio vulgaris Hildenborough. DvhA is a LapA-like adhesin localized to the OM. DvhG is a LapG-like protease that can cleave DvhA at the dialanine site (shown in red), however, unlike the periplasmic-localized LapG, DvhG is hypothesized to be inner membrane (IM) bound. DvhD is a structurally different but functionally analogous LapD-like protein containing a catalytically inactive HD-GYP domain that can bind c-di-GMP and thus regulate DvhA localization and biofilm formation in D. vulgaris Hildenborough. c) Simplified protein architecture representing different domains and the dialanine site of Pf0-1 adhesin LapA, DvH adhesin DvhA and fusion proteins DvhA-RM-swap and DvhA-hlx-swap (RM - retention module; polyG – polyglycine linker; SD – C-terminal secretion domain). Protein domains are not to scale. d) Biofilm formed in K10T medium at 24 hours by strains expressing the adhesins DvhA-RM-swap and DvhA-hlx-swap compared to the native adhesin LapA in Pf0-1, all in the ΔlapGΔlapD background strain. e) Quantification of cell surface levels of HA-tagged adhesins at 24 hours in K10T medium. f) Western blots indicating total cellular levels of the different adhesins. Statistical analysis was performed using one way ANOVA multiple comparisons (ns, p > 0.05; *, p ≤ 0.05; **, p ≤ 0.01; ***, p ≤ 0.001; ****, p ≤ 0.0001); ****, P<0.0001).