Functional Studies On The Components Of The Type Ⅸ Secretion System In Cytophaga Hutchinsonii

Nine types of protein secretion systems have been identified in bacteria,which exert crucial physiological functions,including nutrition acquisition,attachment to various surfaces,and pathogenicity.In 2010,a novel protein secretion system,the type IX secretion system（T9SS）,was first discovered in Porphyromonas gingivalis and Flavobacterium johnsoniae,which is composed of a series of Por,Gld,and Spr proteins.T9SS is widely distributed in and confined to the phylum Bacteroidete,which is involved in pathogenicity,motility,and the degradation of complex biopolymers.There are N-terminal signal peptides and conserved C-terminal domains（CTDs）in proteins secreted by T9SS.These proteins are first transported across the cytoplasmic membrane via the Sec transport system,and then are translocated across the outer membrane by T9SS directed by the CTDs to the cell surface or extracellular.As reported,most metal ions can pass through the outer membrane by passive diffusion through porins However,when the concentration of extracellular metal ion is low,diffusion is not effective.Bacteria have developed many effective means to obtain scarce metal ions in an energy-dependent manner（Such as the transport of zinc in Burkholderia thailandensis）.Cytophaga hutchinsonii is a widely distributed Gram-negative soil bacterium,which belongs to the phylum Bacteroidetes.C.hutchinsonii can efficiently digest crystalline cellulose using a novel strategy,which is different from the free soluble cellulase system and multiprotein cellulosome.Wilson proposed that C.hutchinsonii used a third mechanism to digest cellulose.Another feature of C.hutchinsonii is that it can rapidly glide along solid surfaces without of flagella and Ⅳpilus.The motility mechanism of C.hutchinsonii is still mysterious.Recently,it was found that outer membrane proteins play crucial roles in cellulose degradation and motility.Orthologs of all of the core genes of T9SS were identified in the genome of C.hutchinsonii.It was reported that PorU and SprP are components of C.hutchinsonii T9SS,which are involved in cellulose utilization and motility.The functions of other components（especially the core components）of C.hutchinsonii T9SS are still unknown.This study optimized the culture medium of C.hutchinsonii,using which we obtained the deletion mutants of gldN（encoding a core component of T9SS）,sprA and sprT（encoding the important components of T9SS）.In addition,we identified two hypothetical protein to be the components of C.hutchinsonii T9SS.Through investigating the functions of these T9SS components,we first found that T9SS is involved in the acquisition of trace amount of metal ions.The specific contents and main results are as follows:Research on the function of GldN,a core component of C.hutchinsonii T9SS:Among the identified genes encoding components of the T9SS,gldK,gldL,gldM,and gldN exist consecutively in the genome of at least 37 members of the phylum Bacteroidete,and are transcribed in the same direction.GldK,GldL,GldM,and GldN may form a large secretion apparatus that spans the cell envelope,so they are considered to be the core components of the T9SS.In order to investigate the function of GldN,we improved the screening medium through the addition of 0.9 mM calcium and 0.8 mM magnesium to the complete medium（PY6）,which is designated PYT medium.Using PYT medium and homologous recombination,we successfully deleted gldN,and obtained the △gldN mutant.Our study found that GldN is indispensable for cellulose utilization,motility,and the secretion of CTD proteins.△gldN completely lost the ability to utilize cellulose,including crystalline and amorphous cellulose.Moreover,deletion of gldN resulted in complete defects in motility.△gldN lost the ability to spread on hard and soft agar,and the individual cells of △gldN had decreased adhesin ability to the glass surface and the adhered cells lost the ability to glide.△gldN was defective in secretion of CHU₀344,which is the major extracellular protein with conserved CTD.Moreover,cell surface proteomics identified 38 CTD proteins on the cell surface of wild type（WT）.△gldN lost 35 of them on the cell surface,including CHU₃220,which is indispensable for the degradation of crystalline cellulose.Western blot using the antibody against CHU₃220 confirmed that the secretion defect of CHU₃220 in the △gldN mutant,and the accumulation of CHU₃220 in periplasmic space of the△gldN mutant.Notably,our study found that GldN is involved the assimilation of trace amount of Ca2+ and Mg2+ across the outer membrane.△gldN showed significant growth defects in Ca2+-and Mg2+-deficient media,and the intracellular concentrations of Ca2+and Mg2+were dramatically decreased in △gldN.The outer membrane location of CHU 2807,which is annotated as the outer membrane efflux protein,was dependent on GldN.Deletion of chu 2807 also caused significant growth defect in PY6 medium and decreased intracellular concentration of Ca2+and Mg2+.So far,limited studies have reported the transport mechanism of cations across the outer membrane of bacteria,especially the transport mechanism of Ca2+ and Mg2+.Our study first found that T9SS was involved in uptake of Ca2+and Mg2+,indicating the relationship between protein secretion system and ion acquisition system.Our study demonstrated that GldN is a core component protein of C.hutchinsonii T9SS,which is crucial for the secretion of the majority of CTD proteins,ion assimilation,cellulose degradation,and motilityResearch on the functions of SprA and SprT,important components of C.hutchinsonii T9SS:SprT（PorT）was found as the first component of T9SS,which are essential for the secretion of gingipains in P.gingivalis and SprB,Rem A,ChiA in F.johnsoniae.Sov（SprA）was subsequently found to be involved in the secretion of gingipains.Recently,Lauber et al.reported that SprA is the outer membrane translocon of the T9SS.SprA forms an extremely large（36-strand）single-polypeptide transmembrane barrel,and the pore,at 70 A in diameter,is large enough to permit the passage of the folded large substrates of the T9SS.In order to identify the homologous proteins of SprA and SprT,and study the functions of them in C.hutchinsonii,we deleted homologous genes of sprA（chu₀029）and sprT（chu₂709）.The deletion mutants of sprA and sprT could not be obtained using PY6 medium,whereas could be obtained using PYT medium.Further study indicated that SprA and SprT are involved in assimilation of trace amount of Ca2+ and Mg2+ across the outer membrane,especially SprT.△sprT showed significant growth defects in PY6,Stanier without the addition of CaCl2,Stanier with reduced MgSO4（0.1 mM）.However,△sprT could grew well in PYT and Stanier,which contain rich Ca2+and Mg2+.These results demonstrated that SprT played crucial roles in transportation of Ca2+ and Mg2+.Deletion of sprA and sprT resulted in defects in utilization of crystalline and amorphous cellulose.Moreover,cells of △sprA and △sprT could not arrange regularly along the filter paper fiber.Meanwhile,cells of the △sprA and △sprT mutants lost colony spreading ability and individual cell gliding ability.These results suggested that SprA and SprT are essential for the secretion of cellulose degradation related proteins,cellulose adhesion proteins,and adhesins.The secretion defects of CHU₀344（the dominant extracellular protein）and Ce19A（a cell surface endoglucanase）in the △sprA and △sprT mutants demonstrated that SprA and SprT are components of C.hutchinsonii T9SS.Cell surface proteomic found that SprA and SprT were required for the secretion of 32 and 27 CTD proteins（38 CTD proteins were identified on the cell surface of the WT）.Our study demonstrated that SprA and SprT are important components of C.hutchinsonii T9SS,which are indispensable for the secretion of the majority of CTD proteins,ion assimilation,cellulose degradation,and cell motility.Functions of other T9SS components and the regulatory system of T9SS in C.hutchinsonii:To further investigate the composition of C.hutchinsonii T9SS,we identified two hypothetical proteins CHU₂991 and CHU₃41 0,which participated the secretion of CHU₀344,to be the components of C.hutchinsonii T9SS.CHU₂991 exerted vital role in cellulose utilization.However,cells of △2991 exhibited partial defect in colony spreading,indicating that CHU₂991 is involved in the secretion of cellulose degradation related proteins and partial motility related proteins.CHU₂991 is the first T9SS component found to be partially involved in colony spreading,indicating that CHU₂991 is not a core component of C.hutchinsonii T9SS.CHU₃410 was found as a novel component of C.hutchinsonii T9SS,which played crucial roles in cellulose utilization and cell motility.Notably,CHU₃410 is essential for acquisition of Ca2+,and △3410 could only grow in media with rich Ca2+.Further study on the function of CHU 3410 would help to uncover the transport mechanism of Ca2+ in C.hutchinsonii.Function of PorX-PorY,which is the regulatory system of T9SS,was investigated in C.hutchinsonii.It was found that deletion of the encoding genes of porX orporY caused partial defects in cellulose utilization.The △porX and △porY could spread normally on soft agar.Moreover,deletion of porX or porY did not resulted in significant growth defects under Ca2+-and Mg2+-deficient condition.These results demonstrated that PorX-PorY does not participate in multiple physiological processes in C.hutchinsoniiDeletion of the encoding genes of T9SS caused growth defect in PY6 medium,which may be the reason for the difficulty in getting gldN,sprA,and sprT deleted.Our study optimized the medium of C.hutchinsonii and successfully obtained the T9SS mutants using PYT medium with rich Ca2+ and Mg2+.It is the first time to find that C.hutchinsonii T9SS is not only involved in cellulose degradation and cell motility,but also participates in assimilation of trace amounts of cations through the outer membrane,revealing a link between the T9SS and metal ion transport system.Our study also demonstrated that different components of C.hutchinsonii T9SS play different roles in protein secretion and ion assimilation.This study revealed the multifunction of T9SS and provided a strategy to investigate the function of T9SS in other bacteria of the phylum Bacteroidetes.