Structural studies of the FliG protein of the bacterial flagellar rotor

Flagella are complex multiprotein structures that not only enable bacteria to move through the environment, but are also key virulence factors for pathogens. The flagellar rotor that drives the entire flagellum, with rotation that can happen in both directions, is composed of the C-ring (built of proteins FliG, FliM and FliN) and MS-ring (built of the FliF protein). Interaction between FliG and FliF proteins and their higher-order complex formation is central to the bacterial flagellum biosynthesis. This is because FliF and its co-folding partners are among the earliest structures to assemble during the flagella building process, and because they physically link all the flagellar associated structures in the cytoplasm with those in the periplasmic space (in Gram negative bacteria), outer-membrane and finally the filament that resides outside the cell. Meanwhile, FliG functions to transfer the torque from the membrane anchored stators, transducing the energy from ion flow to rotation of the rotor and thus the entire flagellar structure. To expand our mechanistic understanding of this molecular machine, a combination of methods was employed here. Molecular dynamics simulations were used to study the structure of the FliG, revealing that the linker between the FliG N-terminal and middle domains likely adopts an extended conformation in vivo, in contrast with crystallographic data. An integrative modelling approach was then taken, encompassing homology modelling and molecular dynamics flexible fitting approaches, to create full length viable FliG models in a C-ring assembly. Furthermore, no high resolution structure of the FliG ring has been solved to date; thus, two cryo-electron tomography datasets were collected to visualize motor reconstructions from C. jejuni (a pathogen and a flagella research model organism), and preliminary reconstructions were obtained. Construction of a ΔfliF strain and a set of complementation strains was created to study a function of previously identified pseudorevertant mutations in FliF that restored motility to non-motile C. jejuni. Whole genome sequencing and negative stain electron microscopy highlighted the importance of the two genes coding FliG and FliF to be co-transcribed and co-translated for an efficient motor assembly. Sequence analysis also identified a novel mutation in FliF associated with increased motility. Collectively, these methods expanded the knowledge of the bacterial flagellar rotor, as well as providing models to generate new hypotheses that lay groundwork for future experiments

Bridging the N-terminal and middle domains in FliG of the flagellar rotor

Flagella are necessary for bacterial movement and contribute to various aspects of virulence. They are complex cylindrical structures built of multiple molecular rings with self-assembly properties. The flagellar rotor is composed of the MS-ring and the C-ring. The FliG protein of the C-ring is central to flagellar assembly and function due to its roles in linking the C-ring with the MS-ring and in torque transmission from stator to rotor. No high-resolution structure of an assembled C-ring has been resolved to date, and the conformation adopted by FliG within the ring is unclear due to variations in available crystallographic data. Here, we use molecular dynamics (MD) simulations to study the conformation and dynamics of FliG in different states of assembly, including both in physiologically relevant and crystallographic lattice environments. We conclude that the linker between the FliG N-terminal and middle domain likely adopts an extended helical conformation in vivo, in contrast with the contracted conformation observed in some previous X-ray studies. We further support our findings with integrative model building of full-length FliG and a FliG ring model that is compatible with cryo-electron tomography (cryo-ET) and electron microscopy (EM) densities of the C-ring. Collectively, our study contributes to a better mechanistic understanding of the flagellar rotor assembly and its function

Transcriptome analysis of barley (Hordeum vulgare L.) mutant nec1

nec1 ir miežu nekrotiskais mutants, kuram ir mutācija ciklisko nukleotīdu regulētā jonu kanāla 4 (CNGC4) gēnā. nec1 ir raksturīgs HR līdzīgs fenotips, palielināts auksīna un salicilskābes daudzums, pastiprināta PR-1 un β-glukanāzes gēnu ekspresija, izmainīta koleoptiļu un atvārsnīšu atbilde uz auksīnu. nec1 transkriptoma analīze izmantojot Affymetrix miežu gēnu čipu parādīja, ka nec1 ir pastiprināta triptofāna un auksīna biosintēze, par kuru visticamāk atbildīgs indol-3-acetamīda biosintēzes ceļš, kā arī pastiprināts salicilskābes metabolisms un biosintēze. nec1 transkriptoma analīze norāda arī uz citu fitohormonu – etilēna un jasmonāta sintēzes izmaiņām. Par nec1 fenotipu atbildīgā mutācija izraisījusi arī jonu homeostāzes izmaiņas un izmaiņas sekundāro metabolītu, kas saistīti ar auga aizsargreakcijām – fenilpropanoīdu, lignīnu un flavonoīdu – biosintēzē. Dažas no nec1 transkriptoma analīzē redzamajām metabolisma izmaiņām varētu būt raksturīgas arī citiem nekrotiskajiem mutantiem.nec1 is a barley necrotic mutant with a mutation in a gene encoding cyclic nucletotide regulated ion channel 4 (CNGC4). nec1 is characterised by hypersensitive response like phenotype, elevated levels of auxin and salicylic acid, overexpression of PR-1 and β-glucanase genes, differential coleoptile and stomatal response to exogenous auxin. Trascriptome analysis of nec1 using Affymetrix Barley GeneChip showed an enhanced tryptophan and auxin biosythesis that most likely occurs via indole-3-actetamide pathway and enhanced biosynthesis and metabolism of salycilic acid. Transcriptome analysis indicates changes in synthesis of other phytohormones – ethylene and jasmonic acid. Mutation responsible for nec1 phenotype has also altered ion homeostasis and biosynthesis of defence related secondary metabolites – phenylpropanoids, lignins and flavonoids. Some of metabolism changes revealed by nec1 transcriptome analysis may be characteristic to other necrotic mutants as well