Many bacteria use the flagellum for locomotion and chemotaxis. Its bi-directional rotation is driven by the membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport and how these changes power rotation of the flagellum, remain unknown. Here we present ~3 Å-resolution cryo-electron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a mechanistic model for motor function and switching of rotational direction.

cInfection of intestinal epithelial cells is dependent on the Salmonella enterica serovar Typhimurium pathogenicity island 1 (Spi1)-encoded type III injectisome system and flagellar motility. Thus, the expression of virulence and flagellar genes is subject to tight regulatory control mechanisms in order to ensure the correct spatiotemporal production of the respective gene products. In this work, we reveal a new level of cross-regulation between the Spi1 and flagellar regulatory systems. Transposon mutagenesis identified a class of mutants that prevented flhDC autorepression by overexpressing HilD. HilD, HilC, RtsA, and HilA comprise a positive regulatory circuit for the expression of the Spi1 genes. Here, we report a novel transcriptional cross talk between the Spi1 and flagellar regulons where HilD transcriptionally activates flhDC gene expression by binding to nucleotides ؊68 to ؊24 upstream from the P5 transcriptional start site. We additionally show that, in contrast to the results of a previous report, HilA does not affect flagellar gene expression. Finally, we discuss a model of the cross-regulation network between Spi1 and the flagellar system and propose a regulatory mechanism via the Spi1 master regulator HilD that would prime flagellar genes for rapid reactivation during host infection.

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The long external filament of bacterial flagella is composed of several thousand copies of a single protein, flagellin. Here, we explore the role played by lysine methylation of flagellin in Salmonella, which requires the methylase FliB. We show that both flagellins of Salmonella enterica serovar Typhimurium, FliC and FljB, are methylated at surface-exposed lysine residues by FliB. A Salmonella Typhimurium mutant deficient in flagellin methylation is outcompeted for gut colonization in a gastroenteritis mouse model, and methylation of flagellin promotes bacterial invasion of epithelial cells in vitro. Lysine methylation increases the surface hydrophobicity of flagellin, and enhances flagella-dependent adhesion of Salmonella to phosphatidylcholine vesicles and epithelial cells. Therefore, posttranslational methylation of flagellin facilitates adhesion of Salmonella Typhimurium to hydrophobic host cell surfaces, and contributes to efficient gut colonization and host infection.

Salmonella enterica utilizes flagellar motility to swim through liquid environments and on surfaces. The biosynthesis of the flagellum is regulated on various levels, including transcriptional and posttranscriptional mechanisms. Here, we investigated the motility phenotype of 24 selected single gene deletions that were previously described to display swimming and swarming motility effects. Mutations in flgE, fliH, ydiV, rfaG, yjcC, STM1267 and STM3363 showed an altered motility phenotype. Deletions of flgE and fliH displayed a non-motile phenotype in both swimming and swarming motility assays as expected. The deletions of STM1267, STM3363, ydiV, rfaG and yjcC were further analyzed in detail for flagellar and fimbrial gene expression and filament formation. A ΔydiV mutant showed increased swimming motility, but a decrease in swarming motility, which coincided with derepression of curli fimbriae. A deletion of yjcC, encoding for an EAL domain-containing protein, increased swimming motility independent on flagellar gene expression. A ΔSTM1267 mutant displayed a hypermotile phenotype on swarm agar plates and was found to have increased numbers of flagella. In contrast, a knockout of STM3363 did also display an increase in swarming motility, but did not alter flagella numbers. Finally, a deletion of the LPS biosynthesis-related protein RfaG reduced swimming and swarming motility, associated with a decrease in transcription from flagellar class II and class III promoters and a lack of flagellar filaments.

The bacterial flagellum enables directed movement of Salmonella enterica towards favorable conditions in liquid environments. Regulation of flagellar synthesis is tightly controlled by various environmental signals at transcriptional and post-transcriptional levels. The flagellar master regulator FlhD4 C2 resides on top of the flagellar transcriptional hierarchy and is under autogenous control by FlhD4 C2 -dependent activation of the repressor rflM. The inhibitory activity of RflM depends on the presence of RcsB, the response regulator of the RcsCDB phosphorelay system. In this study, we elucidated the molecular mechanism of RflM-dependent repression of flhDC. We show that RcsB and RflM form a heterodimer that coordinately represses flhDC transcription independent of RcsB phosphorylation. RcsB-RflM complex binds to a RcsB box downstream the P1 transcriptional start site of the flhDC promoter with increased affinity compared to RcsB in the absence of RflM. We propose that RflM stabilizes binding of unphosphorylated RcsB to the flhDC promoter in absence of environmental cues. Thus, RflM is a novel auxiliary regulatory protein that mediates target specificity of RcsB for flhDC repression. The cooperative action of the RcsB-RflM repressor complex allows Salmonella to fine-tune initiation of flagellar gene expression and adds another level to the complex regulation of flagellar synthesis.

Many bacteria use the flagellum for locomotion and chemotaxis. Its bi-directional rotation is driven by the membrane-embedded motor, which uses energy from the transmembrane ion gradient to generate torque at the interface between stator units and rotor. The structural organization of the stator unit (MotAB), its conformational changes upon ion transport and how 15 these changes power rotation of the flagellum, remain unknown. Here we present ~3 Å-resolution cryo-electron microscopy reconstructions of the stator unit in different functional states. We show that the stator unit consists of a dimer of MotB surrounded by a pentamer of MotA. Combining structural data with mutagenesis and functional studies, we identify key residues involved in torque generation and present a mechanistic model for motor function and switching of rotational 20 direction.One Sentence Summary: Structural basis of torque generation in the bidirectional bacterial flagellar motor

35The flagellum is the motility device of many bacteria and the long external filament is 36 made of several thousand copies of a single protein, flagellin. While posttranslational 37 modifications of flagellin are common among bacterial pathogens, the role of lysine 38 methylation remained unknown. Here, we show that both flagellins of Salmonella 39 enterica, FliC and FljB, are methylated at surface-exposed lysine residues. A 40 Salmonella mutant deficient in flagellin methylation was outcompeted for gut 41 colonization in a gastroenteritis mouse model. In support, methylation of flagellin 42 promoted invasion of epithelial cells in vitro. Lysine methylation increased the surface 43 hydrophobicity of flagellin and enhanced flagella-dependent adhesion of Salmonella to 44 phosphatidylcholine vesicles and epithelial cells. In summary, posttranslational flagellin 45 methylation constitutes a novel mechanism how flagellated bacteria facilitate adhesion 46 to hydrophobic host cell surfaces and thereby contributes to efficient gut colonization 47 and successful infection of the host. 48 49 50 51 52 53 54 Introduction: 57 The Gram-negative enteropathogen Salmonella enterica uses a variety of strategies to 58 successfully enter and replicate within a host. In this respect, bacterial motility enables 59 the directed movement of the bacteria towards nutrients or the target site of infection. A 60 rotary nanomachine, the flagellum, mediates motility of many bacteria, including 61 Salmonella enterica 1 . Flagella also play a central role in other infection processes, 62 involving biofilm formation, immune system modulation and adhesion 2-4 . 63 The eukaryotic plasma membrane plays an important role in the interaction of 64 flagellated bacteria with host cells during the early stages of infection 5 . The flagella of S. 65 enterica, Escherichia coli and Pseudomonas aeruginosa can function as adhesion 66 molecules 6-8 mediating the contact to various lipidic plasma membrane components, 67 including cholesterol, phospholipids, sulfolipids and the gangliosides GM1 and aGM1 9-68 12 . 69 Structurally, the flagellum consists out of three main parts: the basal body embedded 70 within the inner and outer membranes of the bacterium, a flexible linking structure -the 71 hook, and the long, external filament, which functions as the propeller of the motility 72 device 13 . The filament is formed by more than 20,000 subunits of a single protein, 73 flagellin. Many S. enterica serovars express either of two distinct flagellins, FliC or FljB, 74 in a process called flagellar phase variation 14,15 . FliC-expressing bacteria display a 75 distinct motility behavior on host cell surfaces and a competitive advantage in 76 colonization of the intestinal epithelia compared to FljB-expressing bacteria 16 . However, 77while the structure of FliC has been determined previously 17 , the structure of FljB 78 remained unknown. 130We next aligned the amino acid sequences of FljB and FliC up-and downstream of the 131 identified ɛ-N-methyl-lysine residues (...