Many bacteria move using a complex, self-assembling nanomachine, the bacterial flagellum. Biosynthesis of the flagellum depends on a flagellar-specific type III secretion system (T3SS), a protein export machine homologous to the export machinery of the virulence-associated injectisome. Six cytoplasmic (FliH/I/J/G/M/N) and seven integral-membrane proteins (FlhA/B FliF/O/P/Q/R) form the flagellar basal body and are involved in the transport of flagellar building blocks across the inner membrane in a proton motive force-dependent manner. However, how the large, multi-component transmembrane export gate complex assembles in a coordinated manner remains enigmatic. Specific for most flagellar T3SSs is the presence of FliO, a small bitopic membrane protein with a large cytoplasmic domain. The function of FliO is unknown, but homologs of FliO are found in >80% of all flagellated bacteria. Here, we demonstrate that FliO protects FliP from proteolytic degradation and promotes the formation of a stable FliP–FliR complex required for the assembly of a functional core export apparatus. We further reveal the subcellular localization of FliO by super-resolution microscopy and show that FliO is not part of the assembled flagellar basal body. In summary, our results suggest that FliO functions as a novel, flagellar T3SS-specific chaperone, which facilitates quality control and productive assembly of the core T3SS export machinery.

Many bacteria use the bacterial flagellum for directed movement in various environments. The assembly and function of the bacterial flagellum and the related virulence-associated injectisome relies on protein export via a conserved type III secretion system (T3SS). The multicomponent transmembrane core export apparatus of the flagellar T3SS consists of FlhA/B and FliP/Q/R and must assemble in a highly coordinated manner. In the present study, we determined the role of the transmembrane protein FliO in the maturation of the flagellar core protein export apparatus. We show that FliO functions as a flagellum-specific chaperone during the initial step of export apparatus assembly. FliO facilitates the efficient formation of a stable FliP–FliR core complex and is thus required for quality management and productive assembly of the flagellar export apparatus. Our results suggest a coordinated assembly process of the flagellar core export apparatus that nucleates with the FliO-dependent formation of a FliP–FliR complex. Subsequent incorporation of FliQ, FlhB, and FlhA leads to the assembly of a secretion-competent flagellar T3SS.

Funding: Deutsche Forschungsgemeinschaft (grant number SFB 944 projects P4 and Z). Received by M.H.. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Deutsche Forschungsgemeinschaft (grant number SFB 766 project B14). Received by S.W.. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Helmholtz Association (grant number VH-GS-202). Received by F.D.F.. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Alexander von Humboldt Foundation. Received by T.T.R.. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Europeans Unions FP7 (grant number 334030). Received by M.E.. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Deutsche Forschungsgemeinschaft (grant number ER 778/2-1). Received by M.E.. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. Helmholtz Association (grant number VH-NG-932). Received by M.E.. The funder had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

Copyright: © 2017 Fabiani et al. This is an open access article distributed under the terms of the Creative Commons Attribution License , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

Here, we determined the molecular function of FliO in maturation of the flagellar type III secretion system (T3SS). We propose that FliO functions as a flagellum-specific, integral membrane chaperone that stabilizes FliP protein and facilitates the formation of a stable FliP–FliR core complex, which is essential for the productive assembly of a functional flagellar basal body.

Six cytoplasmic and seven membrane proteins are essential for the export of the majority of the extra-cytoplasmic building blocks of the flagellum. The MS ring is made of 26 subunits of FliF and likely assembles after the completion of the core integral membrane export apparatus, similar to the injectisome system [ 10 – 13 ]. Tightly associated with the MS ring is a cytoplasmic ring (C ring) made of FliG/M/N. The C ring functions as a rotor/switch complex and serves also as a docking platform for cargo [ 14 ] and the FliH/I/J ATPase complex [ 15 , 16 ], which facilitates export via ATP hydrolysis [ 7 , 17 ]. Within the MS ring, the integral membrane proteins FlhB/A and FliO/P/Q/R are thought to form the export gate. FlhA was reported to form a nonameric ring and presumably energizes export using the pmf [ 11 , 18 – 20 ]. FlhB is involved in the switch of secretion specificity between late and early substrates [ 21 ]. FliO (17.5 kDa), FliP (25 kDa), FliQ (9 kDa), and FliR (26 kDa) are integral membrane proteins and essential for the export of flagellar substrates [ 22 ]. FliP/Q/R are highly conserved in all fT3SS and have homologs in the vT3SS [ 23 ]. Interestingly, FliO homologs are apparently absent in the vT3SS, suggesting an important fT3SS-specific role [ 24 ]. FliO is a bitopic membrane protein with a large cytosolic C-terminal domain. While FliO is required for flagella formation and motility under physiological conditions, upregulation of flagellar secretion substrates and a secondary-site suppressor mutation in FliP restored partial motility of a ΔfliO strain, indicating a functional link between FliO and FliP [ 25 – 27 ].

The ability of many bacteria to move on surfaces and swim through liquid environments depends on the function of a rotating nanomachine, the bacterial flagellum. The flagellum is highly conserved among bacterial species and is best characterized in Salmonella enterica serovar Typhimurium. The structure of the flagellum can be divided into 3 main parts: a basal body, a flexible hook, and a long rigid filament [ 1 , 2 ]. The basal body itself is composed of several substructures located in the cytosol or spanning the bacterial cell envelope. The inner membrane MS ring, the periplasmic P ring, and the outer membrane L ring assemble using the Sec-pathway. The self-assembly of all other axial parts of the flagellum is dependent on protein export through the flagellar-specific type III secretion system (fT3SS) [ 3 ]. The core integral-membrane components of the flagellar export apparatus (FlhA/B and FliP/Q/R) are closely related to the virulence-associated T3SS (vT3SS) of the injectisome device used by many gram-negative bacteria to inject toxins into host cells [ 4 ]. Protein export via both fT3SS and vT3SS is primarily dependent on the proton motive force (pmf) [ 5 – 9 ]. How the bacterial cell coordinates the self-assembly of the multicomponent export apparatus complex in the inner membrane remains elusive.

Results

Phylogenetic distribution of FliO reveals widespread conservation Homologs of FliO are absent in vT3SS of the injectisome and were also thought to be absent in many flagellated species. However, Pallen et al. [28] suggested that FliO homologs are misannotated as FliZ in some bacterial species and identified several potential FliO homologs through PSI-BLAST searches: Cj0352 in Campylobacter jejuni, LA2612 in Leptospira interrogans, RB9276 in Rhodopirellula baltica, and TP0719 in Treponema pallidum. This suggested that FliO homologs are more widespread than previously thought, and we thus performed a detailed phylogenetic analysis of the distribution of FliO proteins across different bacterial phyla (Fig 1). We retrieved the full collection of representative genomes from the refseq NCBI repository (n = 4771) and queried the genomes for the presence of flagellin, FliO, FliP, FliQ, and FliR homologs using regular annotations. We found that FliO homologs in particular were poorly annotated and in fact sometimes misannotated as FliZ, as previously noted by Pallen et al. [28]. This suggested that a large proportion of FliO homologs are missed by the automated annotation algorithms. PPT PowerPoint slide

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larger image TIFF original image Download: Fig 1. Computational prediction and phylogenetic distribution of FliO, FliP, FliQ, FliR, and flagellin across all bacterial phyla. The distribution of FliO, FliP, FliQ, FliR, and flagellin in all The National Center for Biotechnology Information (NCBI) reference genomes (n = 4771) was analyzed according to NCBI annotation and de novo prediction using a Hidden Markov Model (HMM) based on curated Pfam (protein family) database models. (A) Phylogenetic tree based on NCBI taxonomy—outer rings indicate the presence/absence of flagellin, FliO, FliP, FliQ, and FliR. Each colored branch highlights a bacterial phylum. The number of retrieved genomes is indicated for the major phyla; C = Cyanobacteria (119), T = Tenericutes (116), S = Spirochaetes (60), D = Deinococcus-Thermus (39), A = Acidobacteria (24). (B) Gene set representation of de novo predicted FliO, FliP, FliQ, FliR, and flagellin. Left bars show the total number of predicted proteins including previously annotated (dark color) and newly predicted ones (light color) across the NCBI reference genomes. The top bars represent the number of genomes for each combination of predicted FliO, FliP, FliQ, FliR, and flagellin. The pie chart highlights the improved annotation of FliO through the HMM de novo prediction. https://doi.org/10.1371/journal.pbio.2002267.g001 We next performed a de novo prediction of flagellin, FliO, FliP, FliQ, and FliR homologs using Hidden Markov Models (HMMs) based on curated Pfam (protein family) database models (Fig 1, S1 Fig). We screened all genomes using the HMM and identified a large number of FliO homologs in genomes in which also FliP and other flagella components were predicted. The majority of predicted FliO hits corresponded to hypothetical proteins, and we identified possible FliO homologs in >80% of flagellated bacteria across most bacterial phyla (Fig 1).