Revisiting the functions of periplasmic chaperones in the quality control of the autotransporter Ag43 using a phenotypically homogeneous Escherichia coli strain.

Antigen 43 is a surface-displayed autotransporter protein that mediates bacterial self-association and pathogenicity. The quality control factors that facilitate Ag43 crossing the periplasm and inserting into the outer membrane remain enigmatic, mostly because Ag43 is phase variable and associated with heterologous phenotypes, which obscures the mutational effects of potential quality control factors. Here, we describe a screening method that allowed us to isolate a subpopulation of Escherichia coli that consistently displays an Ag43-mediated autoaggregation phenotype. Based on this subpopulation, we analyzed how disruptions of known periplasmic chaperones affect Ag43 biogenesis. We found that only the disruption of surA reduced Ag43 levels and abolished the autoaggregation phenotype of cells, but surA disruption did not affect the phase-variable expression of agn43. Using purified proteins, we showed that SurA effectively protected the ß-barrel domain of Ag43 from aggregation. In contrast, the previously reported Ag43 biogenesis factor OsmY showed weak chaperoning effects on Ag43 only in the absence of SurA. Our results shed light on the roles of different periplasmic chaperones in Ag43 biogenesis and provide a methodology applicable to the study of other phase-variable proteins.

Evaluation of substrate clogging in a full-scale horizontal subsurface flow treatment wetland using electrical resistivity tomography with an optimized electrode configuration.

This study investigated the spatial distribution of clogging matter in a full-scale horizontal subsurface flow treatment wetland (HSSF TW) based on an electrical resistivity tomography (ERT) method, comparing the performance of two different electrode configurations (i.e., Schlumberger and Wenner arrays). The results indicated that during the draining phase, the substrate apparent resistivities of the full-scale HSSF TWs were negatively correlated with the clogging matter fraction (v/v), and a functional relationship between the two parameters was established using a first-order k-C* model. The detected clogging matter fraction (v/v) based on the Schlumberger array showed higher accuracy (linear slope = 0.900, R-squared = 0.902) than the Wenner array (linear slope = 0.685, R-squared = 0.685). Most of the severe substrate clogging in the full-scale HSSF TW occurred within a 10-m flow distance, and the distribution of the clogging matter showed different characteristics at different substrate depths. From a cross section positioned 1 m from the inlet, the average clogging matter fraction (v/v) at a 0-0.30 m depth (23.1 ± 14.9%) was significantly higher than that at a 0.30-0.80 m depth (5.0 ± 2.1%). The clogging matter at a 5-m flow distance was evenly distributed at different substrate depths. Only a few localized clogging zones were observed in the cross section at a 10-m flow distance. This study provided an accurate and feasible method for investigating the volume fraction of clogging matters containing different organic contents and demonstrates the spatial heterogeneity of clogging matter in HSSF TWs.

Chaperone Spy Protects Outer Membrane Proteins from Folding Stress via Dynamic Complex Formation.

Gram-negative bacteria have a multicomponent and constitutively active periplasmic chaperone system to ensure the quality control of their outer membrane proteins (OMPs). Recently, OMPs have been identified as a new class of vulnerable targets for antibiotic development, and therefore a comprehensive understanding of OMP quality control network components will be critical for discovering antimicrobials. Here, we demonstrate that the periplasmic chaperone Spy protects certain OMPs against protein-unfolding stress and can functionally compensate for other periplasmic chaperones, namely Skp and FkpA, in the Escherichia coli K-12 MG1655 strain. After extensive in vivo genetic experiments for functional characterization of Spy, we use nuclear magnetic resonance and circular dichroism spectroscopy to elucidate the mechanism by which Spy binds and folds two different OMPs. Along with holding OMP substrates in a dynamic conformational ensemble, Spy binding enables OmpX to form a partially folded ß-strand secondary structure. The bound OMP experiences temperature-dependent conformational exchange within the chaperone, pointing to a multitude of local dynamics. Our findings thus deepen the understanding of functional compensation among periplasmic chaperones during OMP biogenesis and will promote the development of innovative antimicrobials against pathogenic Gram-negative bacteria. IMPORTANCE Outer membrane proteins (OMPs) play critical roles in bacterial pathogenicity and provide a new niche for antibiotic development. A comprehensive understanding of the OMP quality control network will strongly impact antimicrobial discovery. Here, we systematically demonstrate that the periplasmic chaperone Spy has a role in maintaining the homeostasis of certain OMPs. Remarkably, Spy utilizes a unique chaperone mechanism to bind OmpX and allows it to form a partially folded ß-strand secondary structure in a dynamic exchange of conformations. This mechanism differs from that of other E. coli periplasmic chaperones such as Skp and SurA, both of which maintain OMPs in disordered conformations. Our study thus deepens the understanding of the complex OMP quality control system and highlights the differences in the mechanisms of ATP-independent chaperones.