Non-flagellar Proteins Research Articles

FliW controls growth-phase expression of Campylobacter jejuni flagellar and non-flagellar proteins via the post-transcriptional regulator CsrA.

Campylobacter jejuni is an important human pathogen that causes 96 million cases of acute diarrheal disease worldwide each year. We have shown that C. jejuni CsrA is involved in the post-transcriptional regulation of more than 100 proteins, and altered expression of these proteins is presumably involved in the altered virulence-related phenotypes of a csrA mutant. Mutation of fliW results in C. jejuni cells that have greatly truncated flagella, are less motile, less able to form biofilms, and exhibit a reduced ability to colonize chicks. The loss of FliW results in the altered expression of 153 flagellar and non-flagellar proteins, the majority of which are members of the CsrA regulon. The number of proteins dysregulated in the fliW mutant was greater at mid-log phase (120 proteins) than at stationary phase (85 proteins); 52 proteins showed altered expression at both growth phases. Loss of FliW altered the growth-phase- and CsrA-mediated regulation of FlaA flagellin. FliW exerts these effects by binding to both FlaA and to CsrA, as evidenced by pull-down assays, protein-protein cross-linking, and size-exclusion chromatography. Taken together, these results show that CsrA-mediated regulation of both flagellar and non-flagellar proteins is modulated by direct binding of CsrA to the flagellar chaperone FliW. Changing FliW:CsrA stoichiometries at different growth phases allow C. jejuni to couple the expression of flagellar motility to metabolic and virulence characteristics.

A quantitative proteomic screen of the Campylobacter jejuni flagellar-dependent secretome

Campylobacter jejuni is the leading cause of bacterial gastroenteritis in the world. A number of factors are believed to contribute to the ability of C. jejuni to cause disease within the human host including the secretion of non-flagellar proteins via the flagellar type III secretion system (FT3SS). Here for the first time we have utilised quantitative proteomics using stable isotope labelling by amino acids in cell culture (SILAC), and label-free liquid chromatography-mass spectrometry (LC/MS), to compare supernatant samples from C. jejuni M1 wild type and flagella-deficient (flgG mutant) strains to identify putative novel proteins secreted via the FT3SS. Genes encoding proteins that were candidates for flagellar secretion, derived from the LC/MS and SILAC datasets, were deleted. Infection of human CACO-2 tissue culture cells using these mutants resulted in the identification of novel genes required for interactions with these cells. This work has shown for the first time that both CJM1_0791 and CJM1_0395 are dependent on the flagellum for their presence in supernatants from C. jejuni stains M1 and 81–176. Biological significanceThis study provides the most complete description of the Campylobac er jejuni secretome to date. SILAC and label-free proteomics comparing mutants with or without flagella have resulted in the identification of two C. jejuni proteins that are dependent on flagella for their export from the bacterial cell.

FliZ Is a Global Regulatory Protein Affecting the Expression of Flagellar and Virulence Genes in Individual Xenorhabdus nematophila Bacterial Cells

Heterogeneity in the expression of various bacterial genes has been shown to result in the presence of individuals with different phenotypes within clonal bacterial populations. The genes specifying motility and flagellar functions are coordinately regulated and form a complex regulon, the flagellar regulon. Complex interplay has recently been demonstrated in the regulation of flagellar and virulence gene expression in many bacterial pathogens. We show here that FliZ, a DNA-binding protein, plays a key role in the insect pathogen, Xenorhabdus nematophila, affecting not only hemolysin production and virulence in insects, but efficient swimming motility. RNA-Seq analysis identified FliZ as a global regulatory protein controlling the expression of 278 Xenorhabdus genes either directly or indirectly. FliZ is required for the efficient expression of all flagellar genes, probably through its positive feedback loop, which controls expression of the flhDC operon, the master regulator of the flagellar circuit. FliZ also up- or downregulates the expression of numerous genes encoding non-flagellar proteins potentially involved in key steps of the Xenorhabdus lifecycle. Single-cell analysis revealed the bimodal expression of six identified markers of the FliZ regulon during exponential growth of the bacterial population. In addition, a combination of fluorescence-activated cell sorting and RT-qPCR quantification showed that this bimodality generated a mixed population of cells either expressing (“ON state”) or not expressing (“OFF state”) FliZ-dependent genes. Moreover, studies of a bacterial population exposed to a graded series of FliZ concentrations showed that FliZ functioned as a rheostat, controlling the rate of transition between the “OFF” and “ON” states in individuals. FliZ thus plays a key role in cell fate decisions, by transiently creating individuals with different potentials for motility and host interactions.

Are “Power-Packs” Associated with the Switch-Motor Complex of Bacterial Flagella?

Bacterial flagella are rotated by a motor embedded in the cytoplasmic membrane, the direction of rotation being dictated by a switch at the base of the motor. In the case of bacteria such as E.coli and Salmonella, the motor is driven by the proton-motive force (PMF) across the membrane. The PMF is also used, inter alia, to synthesize ATP by means of the reversible membrane enzyme F0F1ATPase, whose mechanism involves the rotary action of a spindle. Recently, the switch-motor complex of bacterial flagella was found to be associated with a number of non-flagellar proteins, which, in spite of not belonging to the chemotaxis system, affect the function of the flagella. The observation that one of these proteins, fumarate reductase, is involved in electron transport under anaerobic conditions raised the question of whether other energy-linked enzymes are associated with the switch-motor complex as well. Here, we identified two additional such enzymes in E.coli. Employing Forster resonance energy transfer in vivo and pull-down assays in vitro, we provided evidence for the interaction of F0F1ATP synthase via its β subunit with the flagellar switch protein FliG and for the interaction of NADH-ubiquinone oxidoreductase with FliG, FliM, and possibly FliN. Furthermore, we measured higher rates of ATP synthesis, ATP hydrolysis, and electron transport from NADH to oxygen in membrane areas adjacent to the flagellar motor than in other membrane areas. All these observations suggest the association of energy complexes with the flagellar switch-motor complex. Finding that deletion of the β subunit in vivo affected the direction of flagellar rotation and switching frequency further implied that the interaction of F0F1ATP synthase with FliG is important for the function of the switch of bacterial flagella.

Selective Purification of Recombinant Neuroactive Peptides Using the Flagellar Type III Secretion System

ABSTRACTThe structure, assembly, and function of the bacterial flagellum involves about 60 different proteins, many of which are selectively secreted via a specific type III secretion system (T3SS) (J. Frye et al., J. Bacteriol. 188:2233–2243, 2006). The T3SS is reported to secrete proteins at rates of up to 10,000 amino acid residues per second. In this work, we showed that the flagellar T3SS of Salmonella enterica serovar Typhimurium could be manipulated to export recombinant nonflagellar proteins through the flagellum and into the surrounding medium. We translationally fused various neuroactive peptides and proteins from snails, spiders, snakes, sea anemone, and bacteria to the flagellar secretion substrate FlgM. We found that all tested peptides of various sizes were secreted via the bacterial flagellar T3SS. We subsequently purified the recombinant μ-conotoxin SIIIA (rSIIIA) from Conus striatus by affinity chromatography and confirmed that T3SS-derived rSIIIA inhibited mammalian voltage-gated sodium channel NaV1.2 comparably to chemically synthesized SIIIA.

Energy Complexes Are Apparently Associated with the Switch–Motor Complex of Bacterial Flagella

Recently, the switch–motor complex of bacterial flagella was found to be associated with a number of non-flagellar proteins, which, in spite of not being known as belonging to the chemotaxis system, affect the function of the flagella. The observation that one of these proteins, fumarate reductase, is essentially involved in electron transport under anaerobic conditions raised the question of whether other energy-linked enzymes are associated with the switch–motor complex as well. Here, we identified two additional such enzymes in Escherichia coli. Employing fluorescence resonance energy transfer in vivo and pull-down assays invitro, we provided evidence for the interaction of F0F1 ATP synthase via its β subunit with the flagellar switch protein FliG and for the interaction of NADH-ubiquinone oxidoreductase with FliG, FliM, and possibly FliN. Furthermore, we measured higher rates of ATP synthesis, ATP hydrolysis, and electron transport from NADH to oxygen in membrane areas adjacent to the flagellar motor than in other membrane areas. All these observations suggest the association of energy complexes with the flagellar switch–motor complex. Finding that deletion of the β subunit in vivo affected the direction of flagellar rotation and switching frequency further implied that the interaction of F0F1 ATP synthase with FliG is important for the function of the switch of bacterial flagella.

Distribution of flagella secreted protein and integral membrane protein among Campylobacter jejuni isolated from Thailand

BackgroundCampylobacter jejuni, a gram-negative bacterium, is a frequent cause of gastrointestinal food-borne illness in humans throughout the world. There are several reports that the virulence of C. jejuni might be modulated by non-flagellar proteins that are secreted through the filament. Recently, FspA (Flagella secreted proteins) have been described. Two alleles of fspA (fspA1 and fspA2) based on sequence analysis were previously reported and only the fspA2 allele was found in Thai isolates. The aim of this study is to analyze the deduced amino acid sequences fspA and the adjacent putative integral membrane protein from 103 Thai C. jejuni isolates.ResultsA total of 103 representative C. jejuni isolates were amplified by PCR for the fspA gene and the adjacent integral membrane protein gene. Two PCR product sizes were amplified using the same primers, an approximately 1600-bp PCR product from 19 strains that contained fspA and integral membrane protein genes and an approximately 800-bp PCR product from 84 strains that contained only the fspA gene. DNA sequencing was performed on the amplified products. The deduced amino acid sequences of both genes were analyzed separately using CLC Free Workbench 4 software. The analysis revealed three groups of FspA. Only FspA group 1 sequences (19/103) (corresponding to fspA1) consisting of 5 subgroups were associated with the adjacent gene encoding the integral membrane protein. FspA group 2 was the largest group (67/103) consisting of 9 subgroups. FspA group 2p (17/103) consisting of 7 subgroups was found to contain stop codons at a position before the terminal 142 position.ConclusionsThis study reveals greater heterogeneity of FspA (group 1, 2 and 2p) among Thai C. jejuni isolates than previously reported. Furthermore, the subgroups of FspA groups 1 were associated with groups of integral membrane protein. The significance of these different FspA variants to virulence requires further study.

Identification of a Campylobacter jejuni‐secreted protein required for maximal invasion of host cells

The food-borne pathogen Campylobacter jejuni is dependent on a functional flagellum for motility and the export of virulence proteins that promote maximal host cell invasion. Both the flagellar and non-flagellar proteins exported via the flagellar type III secretion system contain a sequence within the amino-terminus that directs their export from the bacterial cell. Accordingly, we developed a genetic screen to identify C. jejuni genes that encode a type III secretion amino-terminal sequence that utilizes the flagellar type III secretion system of Yersinia enterocolitica and a phospholipase reporter (yplA). We screened a library of 321 C. jejuni genes and identified proteins with putative type III secretion amino-terminal sequences. One gene identified by the screen was Cj1242. We generated a mutation in Cj1242, and performed growth rate, motility, secretion and INT 407 cell adherence and internalization assays. The C. jejuni Cj1242 mutant was not altered in growth rate or motility when compared with the wild-type strain, but displayed an altered secretion profile and a reduction in host cell internalization. Based on the phenotype of the C. jejuni Cj1242 mutant, we designated the protein Campylobacter invasion antigen C (CiaC). Collectively, our findings indicate that CiaC is a potentially important virulence factor.

Identification of non-flagellar genes involved in swarm cell differentiation using a Bacillus thuringiensis mini-Tn10 mutant library

Swarming is a social phenomenon that enables motile bacteria to move co-ordinately over solid surfaces. The molecular basis regulating this process is not completely known and may vary among species. Insertional mutagenesis of a swarming-proficient Bacillus thuringiensis strain was performed, by use of the transposon mini-Tn10, to identify novel genetic determinants of swarming that are dispensable for flagellation, swimming motility, chemotaxis and active growth. Among the 67 non-swarming mutants obtained, six were selected that showed no defect in flagellar assembly and function, chemotaxis or growth rate. Sequence analysis of DNA flanking the transposon insertion led to the identification of previously uncharacterized genes that are involved in the development of swarming colonies by B. thuringiensis and that are highly conserved in all members of the Bacillus cereus sensu lato group. These genes encode non-flagellar proteins with putative activity as sarcosine oxidase, catalase-2, amino acid permease, ATP-binding cassette transporter, dGTP triphosphohydrolase and acetyltransferase. Functional analysis of two of the isolated mutants demonstrated that swarming differentiation depends on the intracellular levels of the osmoprotectant glycine betaine and on the quantity of synthesized phenazine secondary metabolites. The finding that proteins involved in diverse physiological processes have a role in swarming motility underlines the complexity of the molecular mechanisms governing this behaviour in B. thuringiensis.

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.

Type III secretion: a secretory pathway serving both motility and virulence (Review)

'Type III secretion' (T3S) refers to a secretion pathway that is common to the flagellae of eubacteria and the injectisomes of some gram-negative bacteria. Flagellae are rotary nanomachines allowing motility but they contain a built-in secretion apparatus that exports their own distal components to the distal end of the growing structure where they polymerize. In some cases they have been shown to export non-flagellar proteins. Injectisomes are transkingdom communication apparatuses allowing bacteria docked at the surface of a eukaryotic cell membrane to inject effector proteins across the two bacterial membranes and the eukaryotic cell membrane. Both nanomachines share a similar basal body embedded in the two bacterial membranes, topped either by a hook and a filament or by a stiff short needle. Both appear to be assembled in the same fashion. They recognize their substrate by a loose N-terminal peptide signal and the help of individual chaperones of a new type.

Requirement of flhA for swarming differentiation, flagellin export, and secretion of virulence-associated proteins in Bacillus thuringiensis.

Bacillus thuringiensis is being used worldwide as a biopesticide, although increasing evidence suggests that it is emerging as an opportunistic human pathogen. While phospholipases, hemolysins, and enterotoxins are claimed to be responsible for B. thuringiensis virulence, there is no direct evidence to indicate that the flagellum-driven motility plays a role in parasite-host interactions. This report describes the characterization of a mini-Tn10 mutant of B. thuringiensis that is defective in flagellum filament assembly and in swimming and swarming motility as well as in the production of hemolysin BL and phosphatidylcholine-preferring phospholipase C. The mutant strain was determined to carry the transposon insertion in flhA, a flagellar class II gene encoding a protein of the flagellar type III export apparatus. Interestingly, the flhA mutant of B. thuringiensis synthesized flagellin but was impaired in flagellin export. Moreover, a protein similar to the anti-sigma factor FlgM that acts in regulating flagellar class III gene transcription was not detectable in B. thuringiensis, thus suggesting that the flagellar gene expression hierarchy of B. thuringiensis differs from that described for Bacillus subtilis. The flhA mutant of B. thuringiensis was also defective in the secretion of hemolysin BL and phosphatidylcholine-preferring phospholipase C, although both of these virulence factors were synthesized by the mutant. Since complementation of the mutant with a plasmid harboring the flhA gene restored swimming and swarming motility as well as secretion of toxins, the overall results indicate that motility and virulence in B. thuringiensis may be coordinately regulated by flhA, which appears to play a crucial role in the export of flagellar as well as nonflagellar proteins.

Measuring antibody to non-flagellar proteins of Salmonella typhi for the diagnosis of typhoid fever

Measuring antibody to non-flagellar proteins of Salmonella typhi for the diagnosis of typhoid fever

Increased levels of mRNAs for tubulin and other flagellar proteins after amputation or shortening of Chlamydomonas Flagella

A dramatic stimulation of synthesis of flagellar proteins occurs in Chlamydomonas following flagellar removal or experimentally induced resorption of the flagella into the cell. In this report we show that this stimulation involves an increase in the levels of mRNAs for tubulin and many other flagellar proteins. Total RNA and poly(A) RNA were isolated from cells after deflagellation or flagellar resorption, and were then translated in the reticulocyte lysate system. Two-dimensional gel analysis of the translation products demonstrates that the RNA-directed in vitro synthesis of α and β tubulins, and a number of other flagellar proteins, increases after deflagellation or flagellar resorption. Surprisingly, the α-tubulin synthesized in vitro does not co-migrate on two-dimensional gels with mature flagellar α-tubulin. Moreover, in vivo labeling experiments show that the major α-tubulin synthesized in the cell after deflagellation co-migrates with the major α-tubulin made in vitro, not with the major α-tubulin present in the flagella. These results suggest that flagellar α-tubulin is synthesized as a precursor, and undergoes post-translational modification before assembly into the flagella. In addition, we report that the synthesis of tubulin and other flagellar proteins can be specifically inhibited, as well as stimulated. Treatment of cells with IBMX, which induces flagellar resorption, causes a marked decrease in the levels of translatable mRNAs for tubulin and other flagellar proteins, without affecting levels of mRNAs for nonflagellar proteins.