Flagellar Assembly Process Research Articles

Adaptation of the Marine Bacterium Shewanella baltica to Low Temperature Stress.

Marine bacteria display significant versatility in adaptation to variations in the environment and stress conditions, including temperature shifts. Shewanella baltica plays a major role in denitrification and bioremediation in the marine environment, but is also identified to be responsible for spoilage of ice-stored seafood. We aimed to characterize transcriptional response of S. baltica to cold stress in order to achieve a better insight into mechanisms governing its adaptation. We exposed bacterial cells to 8 °C for 90 and 180 min, and assessed changes in the bacterial transcriptome with RNA sequencing validated with the RT-qPCR method. We found that S. baltica general response to cold stress is associated with massive downregulation of gene expression, which covered about 70% of differentially expressed genes. Enrichment analysis revealed upregulation of only few pathways, including aminoacyl-tRNA biosynthesis, sulfur metabolism and the flagellar assembly process. Downregulation was observed for fatty acid degradation, amino acid metabolism and a bacterial secretion system. We found that the entire type II secretion system was transcriptionally shut down at low temperatures. We also observed transcriptional reprogramming through the induction of RpoE and repression of RpoD sigma factors to mediate the cold stress response. Our study revealed how diverse and complex the cold stress response in S. baltica is.

FliK-Driven Conformational Rearrangements of FlhA and FlhB Are Required for Export Switching of the Flagellar Protein Export Apparatus.

FlhA and FlhB are transmembrane proteins of the flagellar type III protein export apparatus, and their C-terminal cytoplasmic domains (FlhAC and FlhBC) coordinate flagellar protein export with assembly. FlhBC undergoes autocleavage between Asn-269 and Pro-270 in a well-conserved NPTH loop located between FlhBCN and FlhBCC polypeptides and interacts with the C-terminal domain of the FliK ruler when the length of the hook has reached about 55 nm in Salmonella As a result, the flagellar protein export apparatus switches its substrate specificity, thereby terminating hook assembly and initiating filament assembly. The mechanism of export switching remains unclear. Here, we report the role of FlhBC cleavage in the switching mechanism. Photo-cross-linking experiments revealed that the flhB(N269A) and flhB(P270A) mutations did not affect the binding affinity of FlhBC for FliK. Genetic analysis of the flhB(P270A) mutant revealed that the P270A mutation affects a FliK-dependent conformational change of FlhBC, thereby inhibiting the substrate specificity switching. The flhA(A489E) mutation in FlhAC suppressed the flhB(P270A) mutation, suggesting that an interaction between FlhBC and FlhAC is critical for the export switching. We propose that the interaction between FliKC and a cleaved form of FlhBC promotes a conformational change in FlhBC responsible for the termination of hook-type protein export and a structural remodeling of the FlhAC ring responsible for the initiation of filament-type protein export.IMPORTANCE The flagellar type III protein export apparatus coordinates protein export with assembly, which allows the flagellum to be efficiently built at the cell surface. Hook completion is an important morphological checkpoint for the sequential flagellar assembly process. The protein export apparatus switches its substrate specificity from the hook protein to the filament protein upon hook completion. FliK, FlhB, and FlhA are involved in the export-switching process, but the mechanism remains a mystery. By analyzing a slow-cleaving flhB(P270A) mutant, we provide evidence that an interaction between FliK and FlhB induces conformational rearrangements in FlhB, followed by a structural remodeling of the FlhA ring structure that terminates hook assembly and initiates filament formation.

In situ imaging of the bacterial flagellar motor disassembly and assembly processes.

The self-assembly of cellular macromolecular machines such as the bacterial flagellar motor requires the spatio-temporal synchronization of gene expression with proper protein localization and association of dozens of protein components. In Salmonella and Escherichia coli, a sequential, outward assembly mechanism has been proposed for the flagellar motor starting from the inner membrane, with the addition of each new component stabilizing the previous one. However, very little is known about flagellar disassembly. Here, using electron cryo-tomography and sub-tomogram averaging of intact Legionella pneumophila, Pseudomonas aeruginosa, and Shewanella oneidensis cells, we study flagellar motor disassembly and assembly insitu. We first show that motor disassembly results in stable outer membrane-embedded sub-complexes. These sub-complexes consist of the periplasmic embellished P- and L-rings, and bend the membrane inward while it remains apparently sealed. Additionally, we also observe various intermediates of the assembly process including an inner-membrane sub-complex consisting of the C-ring, MS-ring, and export apparatus. Finally, we show that the L-ring is responsible for reshaping the outer membrane, a crucial step in the flagellar assembly process.

Hierarchical protein export mechanism of the bacterial flagellar type III protein export apparatus.

The bacterial flagellum is supramolecular motility machinery consisting of the basal body, the hook and the filament. Flagellar proteins are translocated across the cytoplasmic membrane via a type III protein export apparatus, diffuse down the central channel of the growing structure and assemble at the distal end. Flagellar assembly begins with the basal body, followed by the hook and finally the filament. The completion of hook assembly is the most important morphological checkpoint of the sequential flagellar assembly process. When the hook reaches its mature length of about 55 nm in Salmonella enterica, the type III protein export apparatus switches export specificity from proteins required for the structure and assembly of the hook to those responsible for filament assembly, thereby terminating hook assembly and initiating filament assembly. Three flagellar proteins, namely FliK, FlhB and FlhA, are responsible for this substrate specificity switching. Upon completion of the switching event, interactions among FlhA, the cytoplasmic ATPase complex and flagellar type III export chaperones establish the assembly order of the filament at the hook tip. Here, we describe our current understanding of a hierarchical protein export mechanism used in flagellar type III protein export.

Comprehensive mapping of the Escherichia coli flagellar regulatory network.

Flagellar synthesis is a highly regulated process in all motile bacteria. In Escherichia coli and related species, the transcription factor FlhDC is the master regulator of a multi-tiered transcription network. FlhDC activates transcription of a number of genes, including some flagellar genes and the gene encoding the alternative Sigma factor FliA. Genes whose expression is required late in flagellar assembly are primarily transcribed by FliA, imparting temporal regulation of transcription and coupling expression to flagellar assembly. In this study, we use ChIP-seq and RNA-seq to comprehensively map the E. coli FlhDC and FliA regulons. We define a surprisingly restricted FlhDC regulon, including two novel regulated targets and two binding sites not associated with detectable regulation of surrounding genes. In contrast, we greatly expand the known FliA regulon. Surprisingly, 30 of the 52 FliA binding sites are located inside genes. Two of these intragenic promoters are associated with detectable noncoding RNAs, while the others either produce highly unstable RNAs or are inactive under these conditions. Together, our data redefine the E. coli flagellar regulatory network, and provide new insight into the temporal orchestration of gene expression that coordinates the flagellar assembly process.

Structural insight into the regulatory mechanisms of interactions of the flagellar type III chaperone FliT with its binding partners

For self-assembly of the bacterial flagellum, most of the flagellar component proteins synthesized in the cytoplasm are exported by the flagellar type III export apparatus to the growing, distal end. Flagellar protein export is highly organized and well controlled in every step of the flagellar assembly process. Flagellar-specific chaperones not only facilitate the export of their cognate proteins, as well as prevent their premature aggregation in the cytoplasm, but also play a role in fine-tuning flagellar gene expression to be coupled with the flagellar assembly process. FliT is a flagellar-specific chaperone responsible for the export of the filament-capping protein FliD and for negative control of flagellar gene expression by binding to the FlhDC complex. Here we report the crystal structure of Salmonella FliT at 3.2-A resolution. The structural and biochemical analyses clearly reveal that the C-terminal segment of FliT regulates its interactions with the FlhDC complex, FliI ATPase, and FliJ (subunits of the export apparatus), and that its conformational change is responsible for the switch in its binding partners during flagellar protein export.

Mechanisms of type III protein export for bacterial flagellar assembly

Flagellar type III protein export is highly organized and well controlled in a timely manner by dynamic, specific and cooperative interactions among components of the export apparatus, allowing the huge and complex macromolecular assembly to be built efficiently. The bacterial flagellum, which is required for motility, consists of a rotary motor, a universal joint and a helical propeller. Most of the flagellar components are translocated to the distal, growing end of the flagellum for assembly through the central channel of the flagellum itself by the flagellar type III protein export apparatus, which is postulated to be located on the cytoplasmic side of the flagellar basal body. The export specificity switching machinery, which consists of at least two proteins that function as a molecular ruler and an export switch, respectively, monitors the state of hook-basal body assembly in the cell exterior and switches export specificity, thereby coupling sequential flagellar gene expression with the flagellar assembly process. The export ATPase complex composed of an ATPase and its regulator acts as a pilot to deliver its export substrate to the export gate and helps initial entry of the substrate N-terminal chain into a narrow pore of the export gate. The energy of ATP hydrolysis appears to be used to disassemble and release the ATPase complex from the protein about to be exported, and the rest of the successive unfolding/translocation process of the long polypeptide chain is driven solely by proton motive force (PMF), perhaps through biased one-dimensional Brownian diffusion. Interestingly, the subunits of the ATPase complex have significant sequence similarities to subunits of F(0)F(1)-ATP synthase, a rotary motor that drives the chemical reaction of ATP synthesis using PMF, and the entire crystal structure of the export ATPase is extremely similar to the alpha/beta subunits of F(0)F(1)-ATP synthase, suggesting that the flagellar export apparatus and F(0)F(1)-ATP synthase share the mechanism for their two distinct functions.

Evolution: Reducible Complexity — The Case for Bacterial Flagella

A recent paper, which will surely figure centrally in the debate between evolutionists and Intelligent Design creationists, proposes a (perhaps too simple) scheme for the evolution of bacterial flagella.

Chlamydomonas flagella

Chlamydomonas reinhardtii Dangard has been widely adopted as a model system for studies of eukaryotic cilia and flagella. Here I review recent progress in understanding flagellar ultrastructure, the mechanisms that generate and regulate flagellar beating and gliding motility, the flagellar assembly process, basal body structure and function, and adhesion‐based signaling, all advanced by work with this single‐celled organism.

Identification of a Caulobacter basal body structural gene and a cis-acting site required for activation of transcription

The genes that encode the components and regulatory proteins of the Caulobacter crescentus flagellum are transcribed at specific times in the cell cycle. One of these genes, flbN, is required early in the flagellar assembly process. The flbN gene was cloned and sequenced, and the time of transcription activation was determined. The derived amino acid sequence indicates that fibN encodes a 25-kilodalton protein with a cleavable leader peptide. The flbN-encoded protein has 30.8% identity with the protein encoded by the Salmonella typhimurium basal body L-ring gene, flgH. Site-directed mutagenesis and gel mobility shift assays identified a binding site at -100 from the transcription start site for a trans-acting protein, RF-2, that functions to partially activate flbN transcription at a defined time in the cell cycle. The RF-2 binding region is similar to a NifA binding site normally used in the activation of some sigma 54 promoters involved in nitrogen fixation in other bacteria. Transcription of a flbN-reporter gene fusion in an Escherichia coli background was dependent on the presence of a NifA transcription factor supplied by a plasmid-borne Rhizobium meliloti gene encoding NifA. A deletion or base changes in the RF-2 binding region eliminated expression of the flbN gene in E. coli even when a NifA protein was provided in trans, suggesting that a sigma 54 promoter with an upstream activator element is used by the C. crescentus flbN gene. A consensus sequence for a sigma 54 promoter was found at the appropriate distance 5' to one of two identified transcription start sites. Site-directed mutagenesis confirmed that a conserved nucleotide in this sigma 54 promoter consensus sequence was required for transcription. Deletion of the region 5' to the apparent sigma 54 promoter caused a complete loss of transcription activation. Transcription activation of flbN in C. crescentus involves the combination of several elements: the NifA-like site is required for full activation, and other sequence elements 5' to the promoter and 3' to the transcription start site are necessary for the correct time of transcription initiation.

Altered Flagellar Size‐Control in shf‐1 Short‐Flagella Mutants of Chlamydomonas reinhardtii

ABSTRACTTwo allelic Mendelian mutations which confer a short flagella phenotype were used to explore flagellar size control in Chlamydomonas reinhardtii. When mutant/wild type quadriflagellate dikaryon cells were constructed, their two short flagella rapidly grew out to near wild type length. The kinetics of elongation suggest that the flagellar assembly process is not intrinsically self‐limiting as a number of otherwise attractive models for size control require. Instead, we suggest that there exists a cellular machinery dedicated to flagellar size control and that the short‐flagella mutations alter the machinery in some as yet unknown way. One of the mutants shows temperature‐sensitive flagellar assembly, and both are flagellaless in acetate media. Genetic analysis indicates that the temperaturesensitive, acetate‐sensitive, and short‐flagella phenotypes have a common genetic basis. The responsible gene has been named shf‐1, and it has been mapped to chromosome VI, approximately 5 map units from the centromere.