Functional Insights Into Flagella Biogenesis Genes In A Non-Flagellated Biocontrol Bacterium,Lysobacter Enzymogenes OH11

Lysobacter enzymogenes is a soil,Gram-negative bacterium with antifungal activities against numerous crop fungal pathogens.This non-flagellated biocontrol bacterium utilizes type Ⅳ pilus(T4P)-driven twitching motility to prey upon nearby fungi for food by secreting abundant lytic enzymes and an antifungal antibiotic,known as heat-stable antifungal factor(HSAF).Interestingly,L.enzymogenes does not own surface-attached flagellum,but its model strain OH 11 contains several homologous genes coding for the flagellum biogenesis components,including the flagellar type Ⅲ secretion system(FT3SS).The reason for this unique phenomenon is unclear.TheFT3SS system in flagellated bacteria is composed of a proton motive force(PMF)-driven transmembrane export gate complex comprising FlhA,FlhB,FliP.FliQ.and FliR,as well as a cytoplasmic ATPase ring complex comprising of FliH,FliI,and FliJ.The cooperation between the transmembrane export gate and the ATPase ring complex drives the transport of flagellar gene products from the cytoplasm to the distal end of the growing flagellar structure.After carrying out a systematic genomic survey using the FT3SS components from the taxonomy-related Xanthomonas oryzae pv.oryzae PXO99A as queries,we found that such FT3SS genes are present in the model strain OH11 of L.enzymogenes.The gene products of flhA.flhB,fliP,fliQ,and fliR.which form a PMF-driven export gate complex inside the basal body MS ring,were all detected on the genome of strain OH11.However,only fliI gene encoding a component of the cytoplasmic ATPase ring complex of the FT3SS was present.Further,it is possible that filH and fliJ homologues are present in strain OH11 although their sequence similarities are too low to allow clear identification.We also identified the presence of other flagellar genes responsible for flagellar assembly in OH11 such as the flagellar hook gene(flgE).basal-body rod genes(flgC and flgG),L-ring gene(flgH).P-ring gene(flgI).MSring gene(fliF).C-ring gene(fliN),hook cap gene(flgD).and hook-length control gene(fliK).missing multiple flagellar assembly genes could explain a loss of surface-attached flagellum for OH 11 during the evolutionary process.Previous studies show that this non-flagellated strain moves in the environment through the formation of type Ⅳ pili,it is possible that the genes involved in FT3SS formation might be involved in other functions.For this purpose,the present work investigated the roles of the FT3SS genes in the formation of T4P-driven twitching motility in OH11.We found that the non-flagellated OH 11 employed the homologous FT3SS genes to involve the export of natively-retained flagellar proteins.Blocking the FT3SS-like system in OH11 by the in-frame deletion mutation of either flhA,flhB,fliP,fliQ,fliR,or fliI gene abolished the secretion of two flagellar proteins,FliK and FlgD.Although FT3SS is still functional in mediating the export of retained flagellar proteins,four of its gene products(FlhA,FlhB,FliI,and FliR)are reprogrammed to affect twitching motility that is commonly governed by a canonical T4P system in both flagellated and nonflagellated bacteria.We further found that the flagellar FliI ATPase,but not FlhA,FlhB and FliR,is required for the export of major pilus assembly protein PiIA,whose secretion is generally controlled by the pilus ATPase PilB.Interestingly,the involvement of FT3SS genes in regulating twitching motility in L.enzymogenes could be functionally replaced by other canonical FT3SS counterparts from X.oryzae pv.oryzae PXO99A,a flagellated,taxonomyrelated species of L.enzymogenes.Our results thus provide an intriguing case demonstrating that a non-flagellated bacterium can preserve FT3SS genes for reprogramming their native functions to twitching motility for better ecological adaption.Besides the above interesting observations,the present study also investigated the role of the FT3SS system in the antagonistic interaction between strain OH 11 and fungi.We chose the fliI mutant of OH11,because the flagellar FliI is a well-known FT3SS-specific ATPase required for the export of flagellar proteins in flagellated bacterium.As expected,we found that the OH11 FliI-His fusion protein exhibited an ATPase activity similar to the FliIxoo-His fusion protein deriving from X.oryzae pv.oryzae PXO99A.Using L.enzymogenesSaccharomyces cerevisiae antagonism as a bacterial-fungal interaction system,we report that FliI of L.enzymogenes OH 11 is required for the secretion of several antifungal toxins.Wecarried out a comparative proteomic assay between the wild-type OH 11 and the ΔfliI mutant,and we found that the abundance of several secreted non-flagellar proteins toxic to the model fungus S.cerevisiae was diminished remarkably in the mutant relative to the wildtype OH11.In agreement,while the wild-type OH11 inhibited the growth of S.cerevisiae in a contact-dependent manner,the ΔfliI mutant failed to do so.We then generated two heterologous toxins by individually fusing an N-terminal segment(133-aa length)of the flagellin FliC fromX.oryzae pv.oryzae PXO99A with two antibacterial peptides from garlic.Interestingly,both recombinant toxins generated in OH11 were found to be successfully secreted into the culture media via the FT3S-like system,enabling OH11 to inhibit the growth of the bacterial pathogen,Ralstonia solanacearum.Overall,we discovered an evolved FT3SS-like system for mediating the export of antimicrobial toxins in a non-flagellated,biocontrol bacterium.In conclusion,the present study provides experimental evidence showing the functional divergence or evolution of the FT3SS genes in a non-flagellated biocontrol bacterium,L.enzymogenes OH11,in which the remaining FT3SS genes seem to play a coordinated role in promoting the T4P-driven twitching motility to move towards nearby fungal pathogens and killing them through secreting several antifungal toxins via the FT3SS.We also transferred these basic findings to applied microbiology and crop protection,where we developed a strategy to allow strain OH 11 to export heterologous antibacterial toxins inhibiting the growth of bacterial crop pathogen.The discovery of a functional FT3SS in a non-flagellated bacterium provides a platform for future studies on mechanistic insights as far us retention of these flagellar genes is concerned.