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Cytophaga hutchinsonii can efficiently degrade cellulose and rapidly glide over surfaces, but the underlying mechanisms remain unclear. The type IX secretion system (T9SS) is involved in protein secretion and gliding motility, which is unique to the phylum Bacteroidetes. In this study, we deleted a homologous gene of PorV (chu_3238), a shuttle protein in the T9SS. The Δ3238 mutant caused cellulolytic and gliding defects, while the porV deletion mutants in other Bacteroidetes could glide normally. Adding Ca2+ and K+ improved growth in the PY6 medium, suggesting a potential role of chu_3238 in ion uptake. A proteomic analysis showed an increase in the number of extracellular proteins in the Δ3238 mutant and a decrease in the outer membrane proteins compared to the wild type (WT). Endoglucanase activity in the Δ3238 intact cells was reduced by approximately 70% compared to that of the WT. These results indicate that the secreted proteins could not attach to the cell surface but were released into the extracellular space in the Δ3238 mutant. However, the cargo proteins accumulated in the periplasm of other reported porV deletion mutants. In addition, the homologs of the translocon SprA and a Plug protein were pulled down by co-immunoprecipitation in the 3238-FLAG strain, which are involved in protein transport in the T9SS of Flavobacterium johnsoniae. The integrity of the lipopolysaccharide (LPS) was also affected in the Δ3238 mutant, which may be the reason for the sensitivity of the cell to toxic reagents. The functional diversity of CHU_3238 suggests its important role in the T9SS of C. hutchinsonii and highlights the functional differences of PorV in the T9SS among the Bacteroidetes. [ABSTRACT FROM AUTHOR]

Cytophaga hutchinsonii is a gliding Gram-negative bacterium in the phylum Bacteroidetes with the capability to digest crystalline cellulose rapidly, but the mechanism is unclear. In this study, deletion of chu_0125, encoding a homolog of the peptidoglycan-associated lipoprotein (Pal), was determined to prevent degradation of the crystalline region of cellulose. We found that the chu_0125 deletion mutant grew normally in regenerated amorphous cellulose medium but displayed defective growth in crystalline cellulose medium and increased the degree of crystallinity of Avicel. The endoglucanase and β-glucosidase activities on the cell surface were reduced by 60 and 30% without chu_0125, respectively. Moreover, compared with the wild type, the chu_0125 deletion mutant was found to be more sensitive to some harmful compounds and to release sixfold more outer membrane vesicles (OMVs) whose protein varieties were dramatically increased. These results indicated that CHU_0125 played a critical role in maintaining the integrity of the outer membrane. Further study showed that the amounts of some outer membrane proteins were remarkably decreased in the chu_0125 deletion mutant. Western blotting revealed that CHU_3220, the only reported outer membrane protein that was necessary and specialized for degradation of the crystalline region of cellulose, was largely leaked from the outer membrane and packaged into OMVs. We concluded that the deletion of chu_0125 affected the integrity of outer membrane and thus influenced the localization of some outer membrane proteins including CHU_3220. This might be the reason why deletion of chu_0125 prevented degradation of the crystalline region of cellulose. [ABSTRACT FROM AUTHOR]

• Chu DegQ harbored both proteolytic activity and chaperone-like activity. • Trimers of Chu DegQ possessed robust proteolytic activity as cage-like polyhedrons. • Trimers suffered serious self-degradation in degrading substrates while cage-like polyhedrons were stable. • Chaperone activity of cage-like polyhedrons was much higher than that of trimers. DegQ is a member of highly conserved htrA family proteins, acting as a chaperone-protease facilitating refolding or degradation of periplasmic misfolded proteins. In the current regulatory model of DegQ, the resting state was inactive hexamers and they would reassemble into cage-like polyhedrons to refold or degrade substrates. Here, the characteristics of DegQ from Cytophaga hutchinsonii were reported. The resting state of Chu DegQ was trimers and no hexamers were detected, suggesting a different regulatory mechanism. Cage-like polyhedrons were formed depending on the concentration of substrates, which were able to degrade unfolded BSA and prevent the aggregation of unfolded lysozyme, suggesting that Chu DegQ harbored both proteolytic and chaperone-like activity. Further study showed that trimers possessed robust proteolytic activity as cage-like polyhedrons. However, when degrading unfolded substrates, trimers suffered serious self-degradation while cage-like polyhedrons were stable. Moreover, trimers were able to prevent the aggregation of unfolded lysozyme and refold chemically denatured MalS, indicating that trimers had a certain chaperone activity. While the chaperone activity of cage-like polyhedrons was much higher than that of trimers. These results suggested that the formation of cage-like polyhedrons was important for both stable protease activity and robust chaperone activity. This study enriched our knowledge of both the proteolytic activity and chaperone activity of the trimeric DegQ and shed light on the reason for the cage formation of DegQ. [ABSTRACT FROM AUTHOR]