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Cytophaga hutchinsonii SprA and SprT Are Essential Components of the Type IX Secretion System Required for Ca²⁺ Acquisition, Cellulose Degradation, and Cell Motility.

ABSTRACT: The type IX secretion system (T9SS) is a novel protein secretion system, which is found in and confined to the phylum Bacteroidetes. T9SS is involved in the secretion of virulence factors, cell surface adhesins, and complex biopolymer degrading enzymes to the cell surface or extracellular medium. Cytophaga hutchinsonii is a widely distributed bacterium, which is able to efficiently digest cellulose and rapidly glide along the solid surfaces. C. hutchinsonii has a full set of orthologs of T9SS components. However, the functions of most homologous proteins have not been verified. In C. hutchinsonii, CHU_0029 and CHU_2709 are similar in sequence to Flavobacterium johnsoniae T9SS components SprA and SprT, respectively. In this study, the single deletion mutants of chu_0029 (sprA) and chu_2709 (sprT) were obtained using a complex medium with the addition of Ca²⁺ and Mg²⁺. Single deletion of sprA or sprT resulted in defects in cellulose utilization and gliding motility. Moreover, the ?sprA and ?sprT mutants showed growth defects in Ca²⁺- and Mg²⁺-deficient media. The results of ICP-MS test showed that both the whole cell and intracellular concentrations of Ca²⁺ were dramatically reduced in the ?sprA and ?sprT mutants, indicating that SprA and SprT are both important for the assimilation of trace amount of Ca²⁺. While the assimilation of Mg²⁺ was not obviously influenced in the ?sprA and ?sprT mutants. Through proteomics analysis of the cell surface proteins of the wild type and mutants, we found that the ?sprA and ?sprT mutants were defective in secretion of the majority of T9SS substrates. Together, these results indicate that SprA and SprT are both essential components of C. hutchinsonii T9SS, which is required for protein secretion, Ca²⁺ acquisition, cellulose degradation, and gliding motility in C. hutchinsonii. Our study shed more light on the functions of SprA and SprT in T9SS, and further proved the link between the T9SS and Ca²⁺ uptake system.

SUBMITTER: Gao L

PROVIDER: S-EPMC7906972 | biostudies-literature | 2021

REPOSITORIES: biostudies-literature

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Cytophaga hutchinsonii SprA and SprT Are Essential Components of the Type IX Secretion System Required for Ca2+ Acquisition, Cellulose Degradation, and Cell Motility.

Gao Lijuan L Tan Yahong Y Zhang Weican W Qi Qingsheng Q Lu Xuemei X

Frontiers in microbiology 20210212

The type IX secretion system (T9SS) is a novel protein secretion system, which is found in and confined to the phylum Bacteroidetes. T9SS is involved in the secretion of virulence factors, cell surface adhesins, and complex biopolymer degrading enzymes to the cell surface or extracellular medium. Cytophaga hutchinsonii is a widely distributed bacterium, which is able to efficiently digest cellulose and rapidly glide along the solid surfaces. C. hutchinsonii has a full set of ...[more]

PMID: 33643255

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Project description: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 BacteroidetesCytophaga 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. hutchinsoniiIMPORTANCE 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.

Project description:Cytophaga hutchinsonii cells can bind to the surface of insoluble cellulose and degrade it by utilizing a novel cell contact-dependent mechanism, in which the outer membrane proteins may play important roles. In this study, the deletion of a gene locus, chu_1165, which encodes a hypothetical protein with 32% identity with TlpB, a disulfide oxidoreductase in Flavobacterium psychrophilum, caused a complete cellulolytic defect in C. hutchinsonii Further study showed that cells of the Δ1165 strain could not bind to cellulose, and the levels of many outer membrane proteins that can bind to cellulose were significantly decreased. The N-terminal region of CHU_1165 is anchored to the cytoplasmic membrane with five predicted transmembrane helices, and the C-terminal region is predicted to stretch to the periplasm and has a similar thioredoxin (Trx) fold containing a Cys-X-X-Cys motif that is conserved in disulfide oxidoreductases. Recombinant CHU_1165His containing the Cys-X-X-Cys motif was able to reduce the disulfide bonds of insulin in vitro Site-directed mutation showed that the cysteines in the Cys-X-X-Cys motif and at residues 106 and 108 were indispensable for the function of CHU_1165. Western blotting showed that CHU_1165 was in an oxidized state in vivo, suggesting that it may act as an oxidase to catalyze disulfide bond formation. However, many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of the cysteine in these proteins did not affect cellulose degradation, indicating that CHU_1165 may have an indirect or pleiotropic effect on the function of these outer membrane proteins.IMPORTANCECytophaga hutchinsonii can rapidly digest cellulose in a contact-dependent manner, in which the outer membrane proteins may play important roles. In this study, a hypothetical protein, CHU_1165, characterized as a disulfide oxidoreductase, is essential for cellulose degradation by affecting the cellulose binding ability of many outer membrane proteins in C. hutchinsonii Disulfide oxidoreductases are involved in disulfide bond formation. However, our studies show that many of the decreased outer membrane proteins that were essential for cellulose degradation contained no or one cysteine, and mutation of cysteine did not affect their function, indicating that CHU_1165 did not facilitate the formation of a disulfide bond in these proteins. It may have an indirect or pleiotropic effect on the function of these outer membrane proteins. Our study provides an orientation for exploring the proteins that assist in the appropriate conformation of many outer membrane proteins essential for cellulose degradation, which is important for exploring the novel mechanism of cellulose degradation in C. hutchinsonii.

Project description:The soil bacterium Cytophaga hutchinsonii actively digests crystalline cellulose by a poorly understood mechanism. Genome analyses identified nine genes predicted to encode endoglucanases with roles in this process. No predicted cellobiohydrolases, which are usually involved in the utilization of crystalline cellulose, were identified. Chromosomal deletions were performed in eight of the endoglucanase-encoding genes: cel5A, cel5B, cel5C, cel9A, cel9B, cel9C, cel9E, and cel9F Each mutant retained the ability to digest crystalline cellulose, although the deletion of cel9C caused a modest decrease in cellulose utilization. Strains with multiple deletions were constructed to identify the critical cellulases. Cells of a mutant lacking both cel5B and cel9C were completely deficient in growth on cellulose. Cell fractionation and biochemical analyses indicate that Cel5B and Cel9C are periplasmic nonprocessive endoglucanases. The requirement of periplasmic endoglucanases for cellulose utilization suggests that cellodextrins are transported across the outer membrane during this process. Bioinformatic analyses predict that Cel5A, Cel9A, Cel9B, Cel9D, and Cel9E are secreted across the outer membrane by the type IX secretion system, which has been linked to cellulose utilization. These secreted endoglucanases may perform the initial digestion within amorphous regions on the cellulose fibers, releasing oligomers that are transported into the periplasm for further digestion by Cel5B and Cel9C. The results suggest that both cell surface and periplasmic endoglucanases are required for the growth of C. hutchinsonii on cellulose and that novel cell surface proteins may solubilize and transport cellodextrins across the outer membrane.The bacterium Cytophaga hutchinsonii digests crystalline cellulose by an unknown mechanism. It lacks processive cellobiohydrolases that are often involved in cellulose digestion. Critical cellulolytic enzymes were identified by genetic analyses. Intracellular (periplasmic) nonprocessive endoglucanases performed an important role in cellulose utilization. The results suggest a model involving partial digestion at the cell surface, solubilization and uptake of cellodextrins across the outer membrane by an unknown mechanism, and further digestion within the periplasm. The ability to sequester cellodextrins and digest them intracellularly may limit losses of soluble cellobiose to other organisms. C. hutchinsonii uses an unusual approach to digest cellulose and is a potential source of novel proteins to increase the efficiency of conversion of cellulose into soluble sugars and biofuels.

Dataset Information

Cytophaga hutchinsonii SprA and SprT Are Essential Components of the Type IX Secretion System Required for Ca²⁺ Acquisition, Cellulose Degradation, and Cell Motility.

Publications

<i>Cytophaga hutchinsonii</i> SprA and SprT Are Essential Components of the Type IX Secretion System Required for Ca<sup>2+</sup> Acquisition, Cellulose Degradation, and Cell Motility.

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