Search

In Archaea, the preflagellin peptidase (a type IV prepilin-like peptidase designated FlaK in Methanococcus voltae and Methanococcus maripaludis) is the enzyme that cleaves the N-terminal signal peptide from preflagellins. In methanogens and several other archaeal species, the typical flagellin signal peptide length is 11 to 12 amino acids, while in other archaea preflagellins possess extremely short signal peptides. A systematic approach to address the signal peptide length requirement for preflagellin processing is presented in this study. M. voltae preflagellin FlaB2 proteins with signal peptides 3 to 12 amino acids in length were generated and used as a substrate in an in vitro assay utilizing M. voltae membranes as an enzyme source. Processing by FlaK was observed in FlaB2 proteins containing signal peptides shortened to 5 amino acids; signal peptides 4 or 3 amino acids in length were unprocessed. In the case of Sulfolobus solfataricus, where the preflagellin peptidase PibD has broader substrate specificity, some predicted substrates have predicted signal peptides as short as 3 amino acids. Interestingly, the shorter signal peptides of the various mutant FlaB2 proteins not processed by FIaK were processed by PibD, suggesting that some archaeal preflagellin peptidases are likely adapted toward cleaving shorter signal peptides. The functional complementation of signal peptidase activity by FlaK and PibD in an M. maripaludis [DELTA]flaK mutant indicated that processing of preflageilins was detected by complementation with either FlaK or PibD, yet only FlaK-complemented cells were flagellated. This suggested that a block in an assembly step subsequent to signal peptide removal occurred in the PibD complementation. doi: 10.1128/JB.00673-09

Salmonella flgG point mutations produce filamentous rod structures whose lengths are determined by FliK. FliK length variants produce rods with lengths proportional to the corresponding FliK molecular size, suggesting that FliK controls the length of not only the hook but also the rod by the same molecular mechanism. doi: 10.1128/JB.00509-09

The flagellar cytoplasmic ring (C ring), which consists of three proteins, FliG, FliM, and FliN, is located on the cytoplasmic side of the flagellum. The C ring is a multifunctional structure necessary for flagellar protein secretion, torque generation, and switching of the rotational direction of the motor. The deletion of any one of the fliG, fliM, and fliN genes results in a [Fla.sup.-] phenotype. Here, we show that the overproduction of the flagellum-specific ATPase FliI overcomes the inability of basal bodies with partial C-ring structures to produce complete flagella. Flagella made upon FliI overproduction were paralyzed, indicating that an intact C ring is essential for motor function. In FliN-or FliM-deficient mutants, flagellum production was about 10% of the wild-type level, while it was only a few percent in FliG-deficient mutants, suggesting that the size of partial C rings affects the extent of flagellation. For flagella made in C-ring mutants, the hook length varied considerably, with many being markedly shorter or longer than that of the wild type. The broad distribution of hook lengths suggests that defective C rings cannot control the hook length as tightly as the wild type even though FliK and FlhB are both intact. doi: 10.1128/JB.00601-09

Silicibacter sp. TM1040 is a member of the marine Roseobacter clade of Alphaproteobacteria that forms symbioses with unicellular eukaryotic phytoplankton, such as dinoflagellates. The symbiosis is complex and involves a series of steps that physiologically change highly motile bacteria into cells that readily form biofilms on the surface of the host. The initial phases of symbiosis require bacterial motility and chemotaxis that drive the swimming bacteria toward their planktonic host. Cells lacking wild-type motility fail to establish biofilms on host cells and do not produce effetive symbioses, emphasizing the importance of understanding the molecular mechanisms controlling flagellar biosynthesis and the biphasic 'swim-or-stick' switch. In the present study, we used a combination of bioinformatic and genetic approaches to identify the genes critical for swimming of Silicibacter sp. TM1040. More than 40 open reading frames with homology to known flagellar structural and regulatory genes were identified, most of which are organized into approximately eight operons comprising a 35.4-kb locus, with surprising similarity to the fla2 locus of Rhodobacter sphaeroides. The genome has homologs of CckA, CtrA, FlbT, and FlaF, proteins that in Caulobacter crescentus regulate flagellum biosynthesis. In addition, we uncovered three novel genes, flaB, flaC, and flaD, which encode flagellar regulatory proteins whose functions are likely to involve regulation of motor function (FlaD) and modulation of the swim-or-stick switch (FlaC). The data support the conclusion that Silicibacter sp. TM1040 uses components found in other Alphaproteobacteria, as well as novel molecular mechanisms, to regulate the expression of the genes required for motility and biofilm formation. These unique molecular mechanisms may enhance the symbiosis and survival of Roseobacter clade bacteria in the marine environment.

Upon completion of the flagellar hook-basal body (HBB) structure, the flagellar type III secretion system switches from secreting rod/hook-type to filament-type substrates. The secretion specificity switch has been reported to occur prematurely (prior to HBB completion) in flk-null mutants (P. Aldridge, J. E. Karlinsey, E. Becker, F. F. Chevance, and K. T. Hughes, Mol. Microbiol. 60:630-643, 2006) and in distal rod gene gain-of-function mutants (flgG* mutants) that produce filamentous rod structures (F. F. Chevance, N. Takahashi, J. E. Karlinsey, J. Gnerer, T. Hirano, R. Samudrala, S. Aizawa, and K. T. Hughes, Genes Dev. 21:2326-2335, 2007). A fusion of [beta]-lactamase (Bla) to the C terminus of the filament-type secretion substrate FlgM was used to select for mutants that would secrete FlgM-Bla into the periplasmic space and show ampicillin resistance ([Ap.sup.r]). [Ap.sup.r] resulted from null mutations in the flhE gene, C-terminal truncation mutations in the flhA gene, null and dominant mutations in the flk gene, and flgG* mutations. All mutant classes required the hook length control protein (FliK) and the rod cap protein (Fig J) for the secretion specificity switch to occur. However, neither the hook (FlgE) nor the hook cap (FlgD) protein was required for premature FlgM-Bla secretion in the flgG* and flk mutant strains, but it was in the flhE mutants. Unexpectedly, when deletions of either flgE or flgD were introduced into flgG* mutant strains, filaments were able to grow directly on the filamentous rod structures.

Glycosylation is a posttranslational modification utilized in all three domains of life. Compared to eukary. otic and bacterial systems, knowledge of the archaeal processes involved in glycosylation is limited. Recently, Methanococcus voltae flagellin proteins were found to have an N-linked trisaccharide necessary for proper flagellum assembly. Current analysis by mass spectrometry of Methanococcus maripaludis flagellin proteins also indicated the attachment of an N-glycan containing acetylated sugars. To identify genes involved in sugar biosynthesis in M. maripaludis, a putative acetyltransferase was targeted for in-frame deletion. Deletion of this gene (MMP0350) resulted in a flagellin molecular mass shift to a size comparable to that expected for underglycosylated or completely nonglycoslyated flagellins, as determined by immunoblotting. Assembled flagellar filaments were not observed by electron microscopy. Interestingly, the deletion also resulted in defective pilus anchoring. Mutant cells with a deletion of MMP0350 had very few, if any, pili attached to the cell surface compared to a nonflagellated but piliated strain. However, pili were obtained from culture supernatants of this strain, indicating that the defect was not in pilus assembly but in stable attachment to the cell surface. Complementation of MMP0350 on a plasmid restored pilus attachment, but it was unable to restore flagellation, likely because the mutant ceased to make detectable flagellin. These findings represent the first report of a biosynthetic gene involved in flagellin glycosylation in archaea. Also, it is the first gene to be associated with pili, linking flagellum and pilus structure and assembly through posttranslationai modifications.

Salmonella enterica serovar Typhimurium secretes virulence factors for invasion called Sip proteins or Sips into its hosts through a type III secretion system (T3SS). In the absence of a host, S. enterica induces Sip secretion in response to sucrose or simple salts, such as NaCl. We analyzed induction of host-independent Sip secretion by monitoring protein secretion by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), assembly of needle complexes by electron microscopy, and transcription of virulence regulatory genes by quantitative reverse transcriptase PCR (real-time PCR). SDS-PAGE showed that addition of sucrose or simple salts, such as NaCl, to the growth medium induced Sip secretion without altering flagellar protein secretion, which requires a distinct T3SS. Electron microscopy confirmed that the amount of secreted Sips increased as the number of assembled needle complexes increased. Real-time PCR revealed that added sucrose or NaCl enhanced transcription of hilA, hilC, and hilD, which encode known regulators of Salmonella virulence. However, epistasis analysis implicated HilD and HilA, but not HilC, in the direct pathway from the salt stimulus to the Sip secretion response. Further analyses showed that the BarA/SirA two-component signal transduction pathway, but not the two-component sensor kinase EnvZ, directly activated hilD and hiLA transcription and thus Sip secretion in response to either sucrose or NaCl. Finally, real-time PCR showed that salt does not influence transcription of the BarA/SirA-dependent csrB and csrC genes. A model is proposed for the major pathway in which sucrose or salt signals to enhance virulence gene expression.

Helicobacter pylori requires flagellar motility and chemotaxis to establish and maintain chronic infection of the human stomach. The pH gradient in the stomach mucus is essential for bacterial orientation and guides the bacterium toward a narrow layer of the mucus, suggesting that H. pylori is capable of energy sensing or taxis. In the present study, H. pylori wild-type behavior in a temporal swimming assay could be altered by electron transport inhibitors, indicating that a connection between metabolism and behavior exists. In order to elucidate mechanisms of behavioral responses of H. pylori related to energy sensing, we investigated the phenotypes of single and multiple mutants of the four proposed chemotaxis sensor proteins. All sensor mutants were motile, but they diverged in their behavior in media supporting different energy yields. One proposed intracellular sensor, TlpD, was crucial for behavioral responses of H. pylori in defined media which did not permit growth and led to reduced bacterial energy levels. Suboptimal energetic conditions and inhibition of electron transport induced an increased frequency of stops and direction changes in the wild type but not in tlpD mutants. Loss of metabolism-dependent behavior in tlpD mutants could be reversed by complementation but not by electron donors bypassing the activity of the electron transport chain, in contrast to the case for the wild type. TlpD, which apparently lacks transmembrane domains, was detected both in the bacterial cytoplasm and at the bacterial periphery. The proposed energy sensor TlpD was found to mediate a repellent tactic response away from conditions of reduced electron transport.

Borrelia burgdorferi is a flat-wave, motile spirochete that causes Lyme disease. Motility is provided by periplasmic flagella (PFs) located between the cell cylinder and an outer membrane sheath. The structure of these PFs, which are composed of a basal body, a hook, and a filament, is similar to the structure of flagella of other bacteria. To determine if hook formation influences flagellin gene transcription in B. burgdorferi, we inactivated the hook structural gene flgE by targeted mutagenesis. In many bacteria, completion of the hook structure serves as a checkpoint for transcriptional control of flagellum synthesis and other chemotaxis and motility genes. Specifically, the hook allows secretion of the anti-sigma factor FlgM and concomitant late gene transcription promoted by [[sigma].sup.28] However, the control of B. burgdorferi PF synthesis differs from the control of flagellum synthesis in other bacteria; the gene encoding [[sigma].sup.28] is not present in the genome of B. burgdorferi, nor are any [[sigma].sup.28] promoter recognition sequences associated with the motility genes. We found that B. burgdorferi flgE mutants lacked PFs, were rod shaped, and were nonmotile, which substantiates previous evidence that PFs are involved in both cell morphology and motility. Although most motility and chemotaxis gene products accumulated at wild-type levels in the absence of FlgE, mutant cells had markedly decreased levels of the flagellar filament proteins FlaA and FlaB. Further analyses showed that the reduction in the levels of flagellin proteins in the spirochetes lacking FlgE was mediated at the posttranscriptional level. Taken together, our results indicate that in B. burgdorferi, the completion of the hook does not serve as a checkpoint for transcriptional regulation of flagellum synthesis. In addition, we also present evidence that the hook protein in B. burgdorferi forms a high-molecular-weight complex and that formation of this complex occurs in the periplasmic space.

The role of flagellin glycosylation on motility was investigated in Pseudomonas syringae pv. tabaci. The swimming activity of glycosylation-defective mutants was prominently decreased in a highly viscous medium. The mutants showed differences in polymorphic transitions and in the bundle formation of flagella, indicating that glycosylation stabilizes the filament structure and lubricates the rotation of the bundle.

The ability of Escherichia coli cells to produce type 1 pili depends upon the orientation of the fimA promoter. The orientation depends upon the ratios of the FimB and FimE recombinases. Here, we report that the two-component response regulator RcsB influences the piliation state by controlling fimB and timE transcription.

Three flagellar proteins, FliG, FliM, and FliN (FliGMN), are the components of the C ring of the flagellar motor. The genes encoding these proteins are multifunctional; they show three different phenotypes ([Fla.sup.-], [Mot.sup.-], and [Che.sup.-]), depending on the sites and types of mutations. Some of the [Mot.sup.-] mutants previously characterized are found to be motile. Reexamination of all [Mot.sup.-] mutants in fliGMN genes so far studied revealed that many of them are actually temperature sensitive (TS); that is, they are motile at 20oC but nonmotile at 37[degrees]C. There were two types of TS mutants: one caused a loss of function that was not reversed by a return to the permissive temperature (rigid TS), and the other caused a loss that was reversed (hyper-TS). The rigid TS mutants showed an all-or-none phenotype; that is, once a structure was formed, the structure and function were stable against temperature shifts. All of fliM and fliN and most of the fliG TS mutants belong to this group. On the other hand, the hyper-TS mutants (three of the fliG mutants) showed a temporal swimming/ stop phenotype, responding to temporal temperature shifts when the structure was formed at a permissive temperature. Those hyper-TS mutation sites are localized in the C-terminal domain of the FliG molecules at sites that are different from the previously proposed functional sites. We discuss a role for this new region of FliG in the torque generation of the flagellar motor.

Bradyrhizobium japonicum is one of the soil bacteria that form nodules on soybean roots. The cell has two sets of flagellar systems, one thick flagellum and a few thin flagella, uniquely growing at subpolar positions. The thick flagellum appears to be semicoiled in morphology, and the thin flagella were in a tight-curly form as observed by dark-field microscopy. Flagellin genes were identified from the amino acid sequence of each flageflin. Flagellar genes for the thick flagellum are scattered into several clusters on the genome, while those genes for the thin flagellum are compactly organized in one cluster. Both types of flagella are powered by proton-driven motors. The swimming propulsion is supplied mainly by the thick flagellum. B. japonicum flagellar systems resemble the polar-lateral flagellar systems of Vibrio species but differ in several aspects.

Buchnera aphidicola is the endosymbiotic bacterium of the pea aphid. Due to its small genome size, Buchnera lacks many essential genes for autogenous life but obtains nutrients from the host. Although the Buchnera cell is nonmotile, it retains clusters of flagellar genes that lack the late genes necessary for motility, including the flagellin gene. In this study, we show that the flagellar genes are actually transcribed and translated and that the Buchnera cell surface is covered with hundreds of hook--basal-body (HBB) complexes. The abundance of HBB complexes suggests a role other than motility. We discuss the possibility that the HBB complex may serve as a protein transporter not only for the flagellar proteins but also for other proteins to maintain the symbiotic system.

The bacterium Vibrio fischeri requires bacterial motility to initiate colonization of the Hawaiian squid Euprymna scolopes. Once colonized, however, the bacterial population becomes largely unflagellated. To understand environmental influences on V. fischeri motility, we investigated migration of this organism in tryptone-based soft agar media supplemented with different salts. We round that optimal migration required divalent cations and, in particular, [Mg.sup.2+]. At concentrations naturally present in seawater, [Mg.sup.2+] improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that [Mg.sup.2+] addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein.

We report the complete genome sequence of the deep-sea [gamma]-proteobacterium, Idiomarina Ioihiensis, isolated recently from a hydrothermal vent at 1,300-m depth on the Loihi submarine volcano, Hawaii. The I. Ioihiensis genome comprises a single chromosome of 2,839,318 base pairs, encoding 2,640 proteins, four rRNA operons, and 56 tRNA genes. A comparison of I. Ioihiensis to the genomes of other [gamma]-proteobacteria reveals abundance of amino acid transport and degradation enzymes, but a loss of sugar transport systems and certain enzymes of sugar metabolism. This finding suggests that I. Ioihiensis relies primarily on amino acid catabolism, rather than on sugar fermentation, for carbon and energy. Enzymes for biosynthesis of purines, pyrimidines, the majority of amino acids, and coenzymes are encoded in the genome, but biosynthetic pathways for Leu, lle, Val, Thr, and Met are incomplete. Auxotrophy for Val and Thr was confirmed by in vivo experiments. The I. Ioihiensis genome contains a cluster of 32 genes encoding enzymes for exopolysaccharide and capsular polysaccharide synthesis. It also encodes diverse peptidases, a variety of peptide and amino acid uptake systems, and versatile signal transduction machinery. We propose that the source of amino acids for I. Ioihiensis growth are the proteinaceous particles present in the deep sea hydrothermal vent waters. I. Ioihiensis would colonize these particles by using the secreted exopolysaccharide, digest these proteins, and metabolize the resulting peptides and amino acids. In summary, the I. Ioihiensis genome reveals an integrated mechanism of metabolic adaptation to the constantly changing deep-sea hydrothermal ecosystem.

Research has been conducted on flagella type III secretion system. The conditions triggering expression of the locus of enterocyte effacement-encoded type III secretion system have been investigated, and the results are reported.

The bacterial flagellar export switching machinery consists of a ruler protein, FliK, and an export switch protein, FlhB and switches substrate specificity of the flagellar type III export apparatus upon completion of hook assembly. An interaction between the C-terminal domain of FliK (FliKC) and the C-terminal cytoplasmic domain of FlhB (FlhBC) is postulated to be responsible for this switch. FliKC has a compactly folded domain termed FliKT3S4 (residues 268-352) and an intrinsically disordered region composed of the last 53 residues, FliKCT (residues 353-405). Residues 301-350 of FliKT3S4 and the last five residues of FliKCT are critical for the switching function of FliK. FliKCT is postulated to regulate the interaction of FliKT3S4 with FlhBC, but it remains unknown how. Here we report the role of FliKCT in the export switching mechanism. Systematic deletion analyses of FliKCT revealed that residues of 351-370 are responsible for efficient switching of substrate specificity of the export apparatus. Suppressor mutant analyses showed that FliKCT coordinates FliKT3S4 action with the switching. Site-directed photo-cross-linking experiments showed that Val-302 and Ile-304 in the hydrophobic core of FliKT3S4 bind to FlhBC. We propose that FliKCT may induce conformational rearrangements of FliKT3S4 to bind to FlhBC. [ABSTRACT FROM AUTHOR]

BACKGROUND Astringent compounds contained in persimmon fruits have been widely used in Japan as food preservatives and thus as anti-bacterial and anti-fungi reagents. However, the molecular mechanism of the anti-microbial activity has been unclear. One of the virulence secretion systems in Salmonella enterica was used to test the anti-microbial activity of extracts from a persimmon ( Diospyros kaki Thunb 'Saijo'). RESULTS We found that the extract could inhibit the secretion of virulence proteins but did not affect cell growth and determined the critical concentrations of the extract to show the effect. Then, the effective fraction on the suppression of secretion of virulence proteins was purified from the crude extracts using solvent partition, absorption chromatography and gel filtration chromatography. The anti-bacterial fraction was analysed by HCl-butanol treatment and gel permeation chromatography followed by nuclear magnetic resonance and identified as the octamers of epigallocatechin and its gallate as major components. CONCLUSION Proanthocyanidins suppress the secretion of Salmonella pathogenicity island 1 virulence proteins. © 2015 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

In Methanococcus maripaludis S2, the swimming organelle, the archaellum, is composed of three archaellins, FlaB1S2, FlaB2S2 and FlaB3S2. All three are modified with an N-linked tetrasaccharide at multiple sites. Disruption of the N-linked glycosylation pathway is known to cause defects in archaella assembly or function. Here, we explored the potential requirement of N-glycosylation of archaellins on archaellation by investigating the effects of eliminating the 4 N-glycosylation sites in the wildtype FlaB2S2 protein in all possible combinations either by Asn to Glu (N to Q) substitution or Asn to Asp (N to D) substitutions of the N-glycosylation sequon asparagine. The ability of these mutant derivatives to complement a non-archaellated ΔflaB2S2 strain was examined by electron microscopy (for archaella assembly) and swarm plates (for analysis of swimming). Western blot results showed that all mutated FlaB2S2 proteins were expressed and of smaller apparent molecular mass compared to wildtype FlaB2S2, consistent with the loss of glycosylation sites. In the 8 single-site mutant complements, archaella were observed on the surface of Q2, D2 and D4 (numbers after N or Q refer to the 1st to 4th glycosylation site). Of the 6 double-site mutation complementations all were archaellated except D1,3. Of the 4 triple-site mutation complements, only D2,3,4 was archaellated. Elimination of all 4 N-glycosylation sites resulted in non-archaellated cells, indicating some minimum amount of archaellin glycosylation was necessary for their incorporation into stable archaella. All complementations that led to a return of archaella also resulted in motile cells with the exception of the D4 version. In addition, a series of FlaB2S2 scanning deletions each missing 10 amino acids was also generated and tested for their ability to complement the ΔflaB2S2 strain. While most variants were expressed, none of them restored archaellation, although FlaB2S2 harbouring a smaller 3-amino acid deletion was able to partially restore archaellation. [ABSTRACT FROM AUTHOR]

Methanococcus maripaludis is an archaeon with two studied surface appendages, archaella and type IV-like pili. Previously, the major structural pilin was identified as MMP1685 and three additional proteins were designated as minor pilins (EpdA, EpdB and EpdC). All of the proteins are likely processed by the pilin-specific prepilin peptidase EppA. Six other genes were identified earlier as likely encoding pilin proteins processed also by EppA. In this study, each of the six genes (mmp0528, mmp0600, mmp0601, mmp0709, mmp0903 and mmp1283) was deleted and the mutants examined by electron microscopy to determine their essentiality for pili formation. While mRNA transcripts of all genes were detected by RT-PCR, only the deletion of mmp1283 led to nonpiliated cells. This strain could be complemented back to a piliated state by supplying a wildtype copy of the mmp1283 gene in trans. This study adds to the complexity of the type IV pili system in M. maripaludis and raises questions about the functions of the remaining five pilin-like genes and whether M. maripaludis under other growth conditions may be able to assemble additional pili-like structures. [ABSTRACT FROM AUTHOR]