Cytophaga hutchinsonii gldN , Encoding a Core Component of the Type IX Secretion System, Is Essential for Ion Assimilation, Cellulose Degradation, and Cell Motility

Abstract

The type IX secretion system (T9SS), which is involved in pathogenicity, motility, and utilization of complex biopolymers, is a novel protein secretion system confined to the phylum Bacteroidetes. Cytophaga hutchinsonii, a common cellulolytic soil bacterium belonging to the phylum Bacteroidetes, can rapidly digest crystalline cellulose using a novel strategy. In this study, the deletion mutant of chu_0174 (gldN) was obtained using PY6 medium supplemented with Stanier salts. GldN was verified to be a core component of C. hutchinsonii T9SS, and is indispensable for cellulose degradation, motility, and secretion of C-terminal domain (CTD) proteins. Notably, the ΔgldN mutant showed significant growth defects in Ca2+- and Mg2+-deficient media. These growth defects could be relieved by the addition of Ca2+ or Mg2+. The intracellular concentrations of Ca2+ and Mg2+ were markedly reduced in ΔgldN. These results demonstrated that GldN is essential for the acquisition of trace amounts of Ca2+ and Mg2+, especially for Ca2+. Moreover, an outer membrane efflux protein, CHU_2807, which was decreased in abundance on the outer membrane of ΔgldN, is essential for normal growth in PY6 medium. The reduced intracellular accumulation of Ca2+ and Mg2+ in the Δ2807 mutant indicated that CHU_2807 is involved in the uptake of trace amounts of Ca2+ and Mg2+. This study provides insights into the role of T9SS in metal ion assimilation in C. hutchinsonii. IMPORTANCE The widespread Gram-negative bacterium Cytophaga hutchinsonii uses a novel but poorly understood strategy to utilize crystalline cellulose. Recent studies showed that a T9SS exists in C. hutchinsonii and is involved in cellulose degradation and motility. However, the main components of the C. hutchinsonii T9SS and their functions are still unclear. Our study characterized the function of GldN, which is a core component of the T9SS. GldN was proved to play vital roles in cellulose degradation and cell motility. Notably, GldN is essential for the acquisition of Ca2+ and Mg2+ ions under Ca2+- and Mg2+-deficient conditions, revealing a link between the T9SS and the metal ion transport system. The outer membrane abundance of CHU_2807, which is essential for Ca2+ and Mg2+ uptake in PY6 medium, was affected by the deletion of GldN. This study demonstrated that the C. hutchinsonii T9SS has extensive functions, including cellulose degradation, motility, and metal ion assimilation, and contributes to further understanding of the function of the T9SS in the phylum Bacteroidetes.

Identification of the Type IX Secretion System Component, PorV (CHU_3238), Involved in Secretion and Localization of Proteins in Cytophaga hutchinsonii

“…These results suggest that Ca 2+ and K + might be important for Δ3238 mutant growth. Studies from our group have shown that chu_0174 ( gldN ), chu_0029 ( sprA ), and chu_2709 ( sprT ) deletion mutants in C. hutchinsonii had growth defects in Ca 2+ - and Mg 2+ -deficient media ( Gao et al, 2020 , 2021 ). chu_3238 may also play a role in ion assimilation of Ca 2+ and K + , but not in the assimilation of Mg 2+ .…”

“…Moreover, outer membrane proteins are necessary for C. hutchinsonii to bind and degrade cellulose, and many of these proteins are the components or substrates of the T9SS ( Ji et al, 2012 , 2014 ; Wang et al, 2017 , 2019 ; Zhao et al, 2020 ). The components of C. hutchinsonii , including CHU_0170 (SprP), CHU_3237 (PorU), CHU_0174 (GldN), CHU_0029 (SprA), and CHU_2709 (SprT), were found to affect the gliding motility and cellulose utilization ( Wang et al, 2014 ; Zhu and McBride, 2014 ; Gao et al, 2020 ; Gao et al, 2021 ). In addition, CHU_0174, CHU_0029, and CHU_2709 were found to be essential for the assimilation of Ca 2+ and Mg 2+ ( Gao et al, 2020 , 2021 ).…”

“…The components of C. hutchinsonii , including CHU_0170 (SprP), CHU_3237 (PorU), CHU_0174 (GldN), CHU_0029 (SprA), and CHU_2709 (SprT), were found to affect the gliding motility and cellulose utilization ( Wang et al, 2014 ; Zhu and McBride, 2014 ; Gao et al, 2020 ; Gao et al, 2021 ). In addition, CHU_0174, CHU_0029, and CHU_2709 were found to be essential for the assimilation of Ca 2+ and Mg 2+ ( Gao et al, 2020 , 2021 ). Ion channels are required for most nutrients’ passive transportation into the periplasm, which is beneficial for bacterial growth.…”

The Type IX Secretion System: Advances in Structure, Function and Organisation

“…The Bacteroidetes are an important group of bacteria found in the digestive tract of animals and humans and are also widespread in the environment. Many members of this phylum utilise the type IX secretion system (T9SS) to secrete various effector proteins, such as proteases, adhesins, cellulases, chitin and surface layer proteins [ 1 , 2 , 3 , 4 , 5 , 6 ]. The T9SS is important in the periodontal pathogen, Porphyromonas gingivalis , for the secretion of at least 30 proteins, including its major virulence factors called gingipains (Kgp, RgpA, RgpB) [ 7 , 8 , 9 ].…”

Biofilm Spreading by the Adhesin-Dependent Gliding Motility of Flavobacterium johnsoniae. 1. Internal Structure of the Biofilm

“…Cryo-electron tomography revealed filaments and outer membrane-associated patches near the base of the outer membrane of WT cells, but these were not observed when the nonmotile gldF mutant cells were similarly examined [ 8 ]. SprB is secreted and translocated to the cell surface by the Bacteroidetes-specific type IX secretion system (T9SS), which is intertwined with the gliding motility machinery [ 9 , 10 , 11 , 12 , 13 , 14 , 15 ]. Therefore, T9SS mutants influenced gliding and caused nonspreading colonies on both agar surfaces.…”