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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]

Escherichia coli cultures can be fractionated into more than 20 cell populations, each having a different bouyant density and apparently representing a specific stage of cell differentiation from exponential growth to stationary phase (H. Makinoshima, A. Nishimura, and A. Ishihama, Mol. Microbiol. 43:269-279, 2002). The density increase was found to be impaired at an early step for a mutant E. coli with the disrupted rpoS gene, which encodes the RNA polymerase RpoS (sigma-S) for stationary-phase gene transcription. This finding suggests that RpoS is need for the entire process of cell density increase. In the absence of RpoF sigma factor, the flagella are not formed as observed by electron microscopy, but the growth phase-coupled density increase takes place as in wild-type E. coli, confirming that the alteration in cell density is not directly correlated with the presence or absence of flagella. In the stationary-phase cells, accumulation of electron-dense areas was observed by electron microscopic observation of bacterial thin sections. By chemical determination, the increase in glycogen (or polysaccharides) was suggested to be one component, which contributes to the increase in weight-to-volume ratio of stationary-phase E. coli cells. [ABSTRACT FROM AUTHOR]

We investigated the supramolecular structure of the Shigella type III secretion machinery including its major components. Our results indicated that the machinery was composed of needle and basal parts with respective lengths of 45.4 ± 3.3 and 31.6 ± 0.3 nm, and contained MxiD, MxiG, MxiJ and MxiH. spa47, encoding a putative F1-type ATPase, was required for the secretion of effector proteins via the type III system and was involved in the formation of the needle. The spa47 mutant produced a defective, needle-less type III structure, which contained MxiD, MxiG and MxiJ but not MxiH. The mxiH mutant produced a defective type III structure lacking the needle and failed to secrete cffector proteins. Upon ovcrcxprcssion of MxiH in the mxiH mutant, the bacteria produced type III structures with protruding dramatically long needles, and showed a remarkable increase in invasiveness. Our results suggest that MxiH is the major needle component of the type III machinery and is essential for delivery of the effector proteins, and that the level of MxiH affects the length of the needle. [ABSTRACT FROM AUTHOR]

Summarizes information presented at the Fifth Biennial Conference on Bacterial Locomotion and Signal Transduction on January 16-21, 1999. Two-component regulatory systems that control chemotactic behavior and gene expression in response to environmental signals; Quorum sensing; Behavior on surfaces; Flagellar structure and assembly; Diversity of taxis processes.

The peritrichous flagella of Salmonella are synthesized and function through many cell generations. There are two different aspects in the relationship between flagellar biogenesis and cell division. Filament growth is independent from the cell cycle and the length of filaments appear to be locally controlled at each flagellar base, whereas the number of filaments (or flagellar basal bodies) is dependent on cell cycle. We present a model to explain how the number of filaments is maintained through generations. We will also introduce a new direction for research that might directly connect flagellation and cell division; the global communication between flagellar genes and external factors of a complex regulatory network in a cell. [ABSTRACT FROM AUTHOR]

Examines the flagellar filament structure and cell motility of Salmonella typhimurium mutants lacking a part of the outer domain of flagellin. Isolation of mutants which can swim in the presence of antifilament antibodies; Morphology of flagellins isolated from the mutant types; Deletions in the flagellin structure; Role of the flagellin in cell motility.

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]

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 notproduce effective 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. [ABSTRACT FROM AUTHOR]

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 β-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 (Apr). Apr 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 (FlgJ) 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. [ABSTRACT FROM AUTHOR]

Abstract: The bacterial flagellum transforms its shape into several distinguishable helical shapes (polymorphs) under various environmental conditions. Polymorphs of each type of flagellum stay on a circle in the pitch-diameter (P versus πD) plot, indicating that they all belong to one family. Previously, we showed that the flagellar family of a marine bacterium Idiomarina loihiensis (Family II) differed from the conventional flagellar family of Salmonella typhimurium (Family I). The pitch and diameter of Family II flagella are half those of Family I flagella. We have suggested that Family I encompasses peritrichous flagella, while Family II forms a polar flagellum. In this study, we have surveyed the polymorphs of flagella from 18 other species and categorized their family types. Previous observations were confirmed; Family I form peritrichous flagella and Family II form polar flagella. Furthermore, we found that lateral flagella had helical parameters much smaller than those of the other two Families and thus belong to a new family (Family III). [Copyright &y& Elsevier]

As part of our attempt to map the impact of acetyl phosphate (acetyl∼P) on the entire network of two-component signal transduction pathways in Escherichia coli, we asked whether the influence of acetyl∼P on capsular biosynthesis and flagellar biogenesis depends on the Rcs phosphorelay. To do so, we performed a series of epistasis experiments: mutations in the components of the pathway that controls acetyl∼P levels were combined with mutations in components of the Rcs phosphorelay. Cells that did not synthesize acetyl∼P produced no capsule under normally permissive conditions, while those that accumulated acetyl∼P synthesized capsule under conditions previously considered to be non-permissive. Acetyl∼P-dependent capsular biosynthesis required both RcsB and RcsA, while the lack of RcsC restored capsular biosynthesis to acetyl∼P-deficient cells. Similarly, acetyl∼P-sensitive repression of flagellar biogenesis was suppressed by the loss of RcsB (but not of RcsA), while it was enhanced by the lack of RcsC. Taken together, these results show that both acetyl∼P-sensitive activation of capsular biosynthesis and acetyl∼P-sensitive repression of flagellar biogenesis require the Rcs phosphorelay. Moreover, they provide strong genetic support for the hypothesis that RcsC can function as either a kinase or a phosphatase dependent on environmental conditions. Finally, we learned that RcsB and RcsC inversely regulated the timing of flagellar biogenesis: rcsB mutants elaborated flagella prematurely, while rcsC mutants delayed their display of flagella. Temporal control of flagella biogenesis implicates the Rcs phosphorelay (and, by extension, acetyl∼P) in the transition of motile, planktonic individuals into sessile biofilm communities. [ABSTRACT FROM AUTHOR]

The helical filaments of the bacterial flagella so far studied seem to be universal in the bacterial kingdom. Despite the variation in flagellin molecular masses, which range from 24kDa to 62kDa in different species, there are only two forms: either the so-called Normal (left-handed) or the Curly (right-handed). The Normal and Curly helical forms are asymmetric; the two characteristic helical parameters, which are the pitch and diameter, of Normal filaments are twice those of Curly filaments. Both the universality of these two helical forms and their asymmetry are biological puzzles. We found that the marine bacteria Idiomarina loihiensis have flagella with left-handed Curly-like filaments. Analysis of the polymorphic forms under different pH conditions showed that the Curly-like filaments are actually Normal filaments having a smaller pitch and diameter than those of Salmonella typhimurium. A minor modification of Calladine''s model for a filament lattice can explain the variant helical forms. Pseudomonas aeruginosa filaments also belong to the family of I.loihiensis filaments. Thus, there are at least two families of flagella filaments. [Copyright &y& Elsevier]

Abstract: To clarify the functions of flagella for pathogenesis of Erwinia carotovora subsp. carotovora EC1, non-polar in-frame deletion mutants of fliC and motA homologues were constructed. Both the ΔfliC and the ΔmotA mutants were confirmed to be non-motile on semisolid agar medium. The ΔmotA mutant was peritrichously flagellated like its parental strain under electron microscope, whereas the ΔfliC mutant was defective in flagellin production and aflagellated. We demonstrated that both the ΔfliC and the ΔmotA mutants reduced their ability to elicit the soft-rot symptoms on Chinese cabbage without any deleterious effect on the production of major extracellular enzymes. They retained their ability to induce hypersensitive reaction (HR) upon inoculation into tobacco leaves. These results suggest that motility but not flagellar component (s) is (are) involved in pathogenesis of E. carotovora subsp. carotovora. [Copyright &y& Elsevier]

The classic controversy of whether genotoxic chemicals induce cancers with or without a certain low-dose limit, i.e., the threshold, is revisited because of a number of current publications available addressing the plausibility of “practical” thresholds even for genotoxic carcinogens, the mechanism of which may be hypothesized to be due, in part, to a repair system composed of ordinarily available various defense mechanisms under the steady-state DNA damage. The question of whether an absolute nonthreshold or a relative nonthreshold, i.e., a “practical” threshold specifically in the low-dose level, is present may not be answered even with the use of a prohibitively large number of wild-type mice. Could the excessive incidence of tumorigenesis in p53-deficient mice contribute to our understanding of the threshold vs nonthreshold issue in genotoxic carcinogenesis? This is considered because an exaggeration of tumorigenesis in p53-deficient mice is hypothesized to reduce or eliminate the range of threshold due to the p53-deficiency-mediated reduction of DNA repair and apoptosis. The present study of chemical leukemogenesis in p53-deficient mice by transplantation assay was designed to answer this question. Briefly, 218 C3H/He mice were lethally irradiated and repopulated with bone marrow cells from wild-type, heterozygous p53-deficient, and homozygous p53-deficient C3H/He mice. This was followed by treatment with a single and graded dose of methyl nitrosourea at 6.6, 14.8, 33.3, 50.0, and 75.0 mg/kg body wt, with the vehicle-treated control groups treated with zero dose for each genotype. Whereas mice repopulated with p53-deficient bone marrow cells showed a marked reduction of the threshold for leukemogenicity, mice repopulated with wild-type bone marrow cells did not exhibit leukemia at a dose of 33.3 mg/kg body wt and showed a curve with a high probability for the linear regression model with a positive dose intercept, predicting a threshold by the likelihood ratio test. Thus, the failure of wild-type mice to show an increase in incidence of leukemogenesis at low doses of genotoxic carcinogens may be due not to a statistical rarity, but to various p53-related pharmacophysiological functions, possibly including DNA repair and apoptosis that may account for a threshold. [Copyright &y& Elsevier]

The flagellar apparatus consists of components that function as a type III secretion system (TTSS). Enteropathogenic and enterohaemorrhagic E. coli (EPEC and EHEC, respectively) produce an additional TTSS, which is involved in virulence via the translocation of effector proteins into infected host cells. This system is encoded by the locus of enterocyte effacement (LEE). The authors observed that EPEC and EHEC grown in Dulbecco's modified Eagle's medium to the mid- and late-exponential growth phase at 37 °C are non-motile. At the same time these conditions trigger the expression of the LEE-encoded TTSS. Furthermore, it was found that EPEC with an inactivated ihfA, which encodes the IHFα subunit of the integration host factor (IHF), becomes hyperflagellated and motile. Similar hypermotility was seen upon inactivation of the ihfA of EHEC strains. IHF-mediated repression of the EPEC flagella involves down-regulation of flhDC, which encodes a positive regulator of the flagellar regulon. IHF indirectly mediates flhDC repression, via a putative EPEC-unique regulator which is not encoded by LEE. [ABSTRACT FROM AUTHOR]

Compares the Type III secretion systems and bacterial flagella. Genetic structure; Regulation; Function.

In wild-type Salmonella, the length of the flagellar hook, a structure consisting of subunits of the hook protein FlgE, is fairly tightly controlled at ≈ 55 nm. Because fliK mutants produce abnormally elongated hook structures that lack the filament structure, FliK appears to be involved in both the termination of hook elongation and the initiation of filament formation. FliK, a soluble protein, is believed to function together with a membrane protein, FlhB, of the export apparatus to mediate the switching of export substrate specificity (from hook protein to flagellin) upon completion of hook assembly. We have examined the location of FliK during flagellar morphogenesis. FliK was found in the culture supernatants from the wild-type strain and from flgD (hook capping protein), flgE (hook protein) and flgK (hook-filament junction protein) mutants, but not in that from a flgB (rod protein) mutant. The amount of FliK in the culture supernatant from the flgE mutant was much higher than in that from the flgK mutant, indicating that FliK is most efficiently exported prior to the completion of hook assembly. Export was impaired by deletions within the N-terminal region of FliK, but not by C-terminal truncations. A decrease in the level of exported FliK resulted in elongated hook structures, sometimes with filaments attached. Our results suggest that the export of FliK during hook assembly is important for hook-length control and the switching of export substrate specificity. [ABSTRACT FROM AUTHOR]

The bacterium Staphylococcus aureus, which colonizes healthy human skin, may cause diseases, such as atopic dermatitis (AD). Treatment for such AD cases involves antibiotic use; however, alternate treatments are preferred owing to the development of antimicrobial resistance. This study aimed to characterize the novel bacteriophage SaGU1 as a potential agent for phage therapy to treat S. aureus infections. SaGU1 that infects S. aureus strains previously isolated from the skin of patients with AD was screened from sewage samples in Gifu, Japan. Its genome was sequenced and analyzed using bioinformatics tools, and the morphology, lytic activity, stability, and host range of the phage were determined. The SaGU1 genome was 140,909 bp with an average GC content of 30.2%. The viral chromosome contained 225 putative protein-coding genes and four tRNA genes, carrying neither toxic nor antibiotic resistance genes. Electron microscopy analysis revealed that SaGU1 belongs to the Myoviridae family. Stability tests showed that SaGU1 was heat-stable under physiological and acidic conditions. Host range testing revealed that SaGU1 can infect a broad range of S. aureus clinical isolates present on the skin of AD patients, whereas it did not kill strains of Staphylococcus epidermidis, which are symbiotic resident bacteria on human skin. Hence, our data suggest that SaGU1 is a potential candidate for developing a phage therapy to treat AD caused by pathogenic S. aureus. [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]

Methanococcus maripaludis has two surface appendages, archaella and type IV pili, which are composed of glycoprotein subunits. Archaellins are modified with an N-linked tetrasaccharide with the structure Sug-1,4-β-ManNAc3NAmA6Thr-1,4-β- GlcNAc3NAcA-1,3-β-GalNAc, where Sug is (5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L-erythro-hexos-5-ulo-1,5-pyranose. The pilin glycan has an additional hexose attached to GalNAc. In this study, genes located in two adjacent, divergently transcribed operons (mmp0350-mmp0354 and mmp0359-mmp0355) were targeted for study based on annotations suggesting their involvement in biosynthesis of N-glycan sugars. Mutants carrying deletions in mmp0350, mmp0351, mmp0352, or mmp0353 were nonarchaellated and synthesized archaellins modified with a 1-sugar glycan, as estimated from Western blots. Mass spectroscopy analysis of pili purified from the Δmmp0352 strain confirmed a glycan with only GalNAc, suggesting mmp0350 to mmp0353 were all involved in biosynthesis of the second sugar (GlcNAc3NAcA). The Δmmp0357 mutant was archaellated and had archaellins with a 2-sugar glycan, as confirmed by mass spectroscopy of purified archaella, indicating a role for MMP0357 in biosynthesis of the third sugar (ManNAc3NAmA6Thr). M. maripaludis mmp0350, mmp0351, mmp0352, mmp0353, and mmp0357 are proposed to be functionally equivalent to Pseudomonas aeruginosa wbpABEDI, involved in converting UDP-N-acetylglucosamine to UDP-2,3-diacetamido-2,3-dideoxy-D-mannuronic acid, an O5-specific antigen sugar. Cross-domain complementation of the final step of the P. aeruginosa pathway with mmp0357 supports this hypothesis. [ABSTRACT FROM AUTHOR]

The bacterial flagellar hook is a short and uniquely curved tube that connects the basal body to the filament. Hook length is controlled at 55 nm on average by a soluble protein FliK in Salmonella enterica serovar Typhimurium. The N-terminal segment of FliK responsible for measuring the hook length is considered to be intrinsically disordered. Here, we show by high-speed atomic force microscopy that a FliK molecule in solution takes on a shape of two balls linked by a flexible string; the larger ball corresponds to the N-terminal region and the smaller one corresponds to the C-terminal region. The N-terminal domain is stable but the C-terminal domain fluctuates in shape. Based on these and other features of FliK, we propose that the folding of the N-terminal segment at the tip of the growing hook plays a major role in determining the minimal length of the hook. [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]

Bdellovibrio are predatory bacteria that have evolved to invade virtually all Gram-negative bacteria, including many prominent pathogens. Upon invasion, prey bacteria become rounded up into an osmotically stable niche for the Bdellovibrio, preventing further superinfection and allowing Bdellovibrio to replicate inside without competition, killing the prey bacterium and degrading its contents. Historically, prey rounding was hypothesized to be associated with peptidoglycan (PG) metabolism; we found two Bdellovibrio genes, bd0816 and bd3459, expressed at prey entry and encoding proteins with limited homologies to conventional dacB/PBP4 DD-endo/carboxypeptidases (responsible for peptidoglycan maintenance during growth and division). We tested possible links between Bd0816/3459 activity and predation. Bd3459, but not an active site serine mutant protein, bound β-lactam, exhibited DD-endo/carboxypeptidase activity against purified peptidoglycan and, importantly, rounded up E. coli cells upon periplasmic expression. A ΔBd0816 ΔBd3459 double mutant invaded prey more slowly than the wild type (with negligible prey cell rounding) and double invasions of single prey by more than one Bdellovibrio became more frequent. We solved the crystal structure of Bd3459 to 1.45 Å and this revealed predation-associated domain differences to conventional PBP4 housekeeping enzymes (loss of the regulatory domain III, alteration of domain II and a more exposed active site). The Bd3459 active site (and by similarity the Bd0816 active site) can thus accommodate and remodel the various bacterial PGs that Bdellovibrio may encounter across its diverse prey range, compared to the more closed active site that "regular" PBP4s have for self cell wall maintenance. Therefore, during evolution, Bdellovibrio peptidoglycan endopeptidases have adapted into secreted predation-specific proteins, preventing wasteful double invasion, and allowing activity upon the diverse prey peptidoglycan structures to sculpt the prey cell into a stable intracellular niche for replication. [ABSTRACT FROM AUTHOR]

Bdellovibrio bacteriovorus is a Delta-proteobacterium that oscillates between free-living growth and predation on Gram-negative bacteria including important pathogens of man, animals and plants. After entering the prey periplasm, killing the prey and replicating inside the prey bdelloplast, several motile B. bacteriovorus progeny cells emerge. The B. bacteriovorus HD100 genome encodes numerous proteins predicted to be involved in signalling via the secondary messenger cyclic di-GMP (c-di-GMP), which is known to affect bacterial lifestyle choices. We investigated the role of c-di-GMP signalling in B. bacteriovorus, focussing on the five GGDEF domain proteins that are predicted to function as diguanylyl cyclases initiating c-di-GMP signalling cascades. Inactivation of individual GGDEF domain genes resulted in remarkably distinct phenotypes. Deletion of dgcB (Bd0742) resulted in a predation impaired, obligately axenic mutant, while deletion of dgcC (Bd1434) resulted in the opposite, obligately predatory mutant. Deletion of dgcA (Bd0367) abolished gliding motility, producing bacteria capable of predatory invasion but unable to leave the exhausted prey. Complementation was achieved with wild type dgc genes, but not with GGAAF versions. Deletion of cdgA (Bd3125) substantially slowed predation; this was restored by wild type complementation. Deletion of dgcD (Bd3766) had no observable phenotype. In vitro assays showed that DgcA, DgcB, and DgcC were diguanylyl cyclases. CdgA lacks enzymatic activity but functions as a c-di-GMP receptor apparently in the DgcB pathway. Activity of DgcD was not detected. Deletion of DgcA strongly decreased the extractable c-di-GMP content of axenic Bdellovibrio cells. We show that c-di-GMP signalling pathways are essential for both the free-living and predatory lifestyles of B. bacteriovorus and that obligately predatory dgcC- can be made lacking a propensity to survive without predation of bacterial pathogens and thus possibly useful in anti-pathogen applications. In contrast to many studies in other bacteria, Bdellovibrio shows specificity and lack of overlap in c-di-GMP signalling pathways. [ABSTRACT FROM AUTHOR]

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 fimE transcription. [ABSTRACT FROM AUTHOR]

Abstract: The flagellar cytoplasmic protein FliK controls hook elongation by two successive events: by determining hook length and by stopping the supply of hook protein. These two distinct roles are assigned to different parts of FliK: the N-terminal half (FliKN) determines length and the C-terminal half (FliKC) switches secretion from the hook protein to the filament protein. The interaction of FliKC with FlhB, the switchable secretion gate, triggers the switch. By NMR spectroscopy, we demonstrated that FliK is largely unstructured and determined the structure of a compact domain in FliKC. The compact domain, denoted the FliKC core domain, consists of two α-helices, a β-sheet with two parallel and two antiparallel strands, and several exposed loops. Based on the functional data obtained by a series of deletion mutants of the FliKC core domain, we constructed a model of the complex between the FliKC core domain and FlhBC. The model suggested that one of the FliKC loops has a high probability of interacting with the C-terminal domain of FlhB (FlhBC) as the FliK molecule enters the secretion gate. We suggest that the autocleaved NPTH sequence in FlhB contacts loop 2 of FliKC to trigger the switching event. This contact is sterically prevented when NPTH is not cleaved. Thus, the structure of FliK provides insight into the mechanism by which this bifunctional protein triggers a switch in the export of substrates. [Copyright &y& Elsevier]

Selenomonas ruminantium produces a tuft of flagella near the midpoint of the cell body and swims by rotating the cell body along the cell's long axis. The flagellum is composed of a single kind of flagellin, which is heavily glycosylated. The hook length of S. ruminantium is almost double that of Salmonella. [ABSTRACT FROM AUTHOR]

Actinoplanes missouriensis spores swim with a tuft of flagella. Flagella of newborn spores are wrapped with a membranous sheath. When the sheath is unwrapped, spores start swimming. Flagellar length is kept short, at around 1.9 μm, which covers half the circumference of the spore. [ABSTRACT FROM AUTHOR]

The structure of pili from the archaeon Methanococcus maripaludis is unlike that of any bacterial pili. However, genetic analysis of the genes involved in the formation of these pili has been lacking until this study. Pili were isolated from a nonflagellated (AflaK) mutant and shown by sodium dodecyl sulfate-polyacrylamide gel electrophoresis to consist primarily of subunits with an apparent molecular mass of 17 kDa. In-frame deletions were created in three genes, MMP0233, MMP0236, and MMP0237, which encode proteins with bacterial type IV pilin-like signal peptides previously identified by in silico methodology as likely candidates for pilus structural proteins. Deletion of MMP0236 or MMP0237 resulted in mutant cells completely devoid of pili on the cell surface, while deletion of the third pilin-like gene, MMP0233, resulted in cells greatly reduced in the number of pili on the surface. Complementation with the deleted gene in each case returned the cells to a piliated state. Surprisingly, mass spectrometry analysis of purified pili identified the major structural pilin as another type IV pilin-like protein, MMP1685, whose gene is located outside the first pilus locus. This protein was found to be glycosylated with an N-linked branched pentasaccharide glycan. Deletion and complementation analysis confirmed that MMP1685 is required for piliation. [ABSTRACT FROM AUTHOR]

A molecular ruler, FliK, controls the length of the flagellar hook. FliK measures hook length and catalyses the secretion-substrate specificity switch from rod-hook substrate specificity to late substrate secretion, which includes the filament subunits. Here, we show normal hook-length control and filament assembly in the complete absence of the C-ring thus refuting the previous ‘cup’ model for hook-length control. Mutants of C-ring components, which are reported to produce short hooks, show a reduced rate of hook–basal body assembly thereby allowing for a premature secretion-substrate specificity switch. Unlike fliK null mutants, hook-length control in an autocleavage-defective mutant of flhB, the protein responsible for the switch to late substrate secretion, is completely abolished. FliK deletion variants that retain the ability to measure hook length are secreted thus demonstrating that FliK directly measures rod-hook length during the secretion process. Finally, we present a unifying model accounting for all published data on hook-length control in which FliK acts as a molecular ruler that takes measurements of rod-hook length while being intermittently secreted during the assembly process of the hook–basal body complex. [ABSTRACT FROM AUTHOR]

A novel Acinetobacter strain, Ud-4, possessing a strong capacity to degrade edible, lubricating, and heavy oil was isolated from seawater in a fishing port located in Toyama, Japan. It was identified by morphological and physiological analyses and 16S rDNA sequencing. This strain could utilize five types of edible oils (canola oil, olive oil, sesame oil, soybean oil, and lard), lubricating oil, and C-heavy oil as the sole carbon source for growth in M9 medium. The strain grew well and heavily degraded edible oils in Luria–Bertani medium during a 7-day culture at 25°C; it also degraded all kinds of oils in artificial seawater medium for marine bacteria. Furthermore, this strain was capable of degrading almost all C10–C25 n-alkanes in C-heavy oil during a 4-week culture. Oligonucleotide primers specific to two catabolic genes involved in the degradation of n-alkanes ( Acinetobacter sp. alkM) and triglyceride ( Acinetobacter sp. lipA) allowed amplification of these genes in strain Ud-4. To our knowledge, this is the first report on the isolation of a bacterium that can efficiently degrade both edible and mineral oils. [ABSTRACT FROM AUTHOR]

Abstract: Bdellovibrio bacteriovorus cells have a single polar flagellum whose helical pitch and diameter characteristically change near the midpoint, resulting in a tapered wave. There are six flagellin genes in the genome: fliC1 to fliC6. Accordingly, the flagellar filament is composed of several similar flagellin species. We have used knockout mutants of each gene and analyzed the mutational effects on the filament length and on the composition and localization of each flagellin species in the filament by electron microscopy and one- and two-dimensional polyacrylamide gel electrophoresis. The location and amounts of flagellins in a filament were determined to be as follows: a small amount of FliC3 at the proximal end, followed by a large amount of FliC5, a large amount of FliC1, a small amount of FliC2 in this order, and a large amount of FliC6 at the distal end. FliC4 was present at a low level, but the location was not determined. Filament lengths of newly born progeny cells increased during prolonged incubation in nutrient-deficient buffer. The newly formed part of the elongated filament was composed of mainly FliC6. Reverse transcription PCR analysis of flagellar gene expression over 5 days in buffer showed that fliC gene expression tailed off over 5 days in the wild-type cells, but in the fliC5 mutant, expression of the fliC2, fliC4, and fliC6 genes was elevated on day 5, suggesting that they may be expressed to compensate for the absence of a major component, FliC5. [Copyright &y& Elsevier]

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 - 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. [ABSTRACT FROM AUTHOR]

Recently, the flagellin proteins of Methanococcus maripaludis were found to harbour an N-linked tetrasaccharide composed of N-acetylgalactosamine, di-acetylated glucuronic acid, an acetylated and acetamidino-modified mannuronic acid linked to threonine, and a novel terminal sugar [( 5S)-2-acetamido-2,4-dideoxy-5-O-methyl-α-L- erythro-hexos-5-ulo-1,5-pyranose]. To identify genes involved in the assembly and attachment of this glycan, in-frame deletions were constructed in putative glycan assembly genes. Successful deletion of genes encoding three glycosyltransferases and an oligosaccharyltransferase (Stt3p homologue) resulted in flagellins of decreased molecular masses as evidenced by immunoblotting, indicating partial or completely absent glycan structures. Deletion of the oligosaccharyltransferase or the glycosyltransferase responsible for the transfer of the second sugar in the chain resulted in flagellins that were not assembled into flagella filaments, as evidenced by electron microscopy. Deletions of the glycosyltransferases responsible for the addition of the third and terminal sugars in the glycan were confirmed by mass spectrometry analysis of purified flagellins from these mutants. Although flagellated, these mutants had decreased motility as evidenced by semi-swarm plate analysis with the presence of each additional sugar improving movement capabilities. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Abstract: A bacterial cell tethered through a flagellum on a glass slide rotates its cell body in either counter-clockwise or clockwise at around 10 Hz. To analyze the detailed manner of rotation, we have constructed and expressed yellow fluorescent protein (YFP)-FliN fusion protein in a fliN deletion mutant, resulting in the recovery of motility of the Fla- mutant cells. The tethered cells that incorporated the fusion protein in the flagellar motor rotate around one of the fluorescent spots. Tracing the center spot of a rotating cell, we have found that the rotating circles of the tethered cells were often distorted, and that the cell has seldom rotated smoothly but gyrated around the center point. The radii of the gyrating circles were 100–200 nm for the wild-type cells, and 50 nm for the cells carrying short hooks, suggesting that the flexibility of the hook is responsible for asymmetrical rotation. These observations indicate that tethered cells almost always interact with the glass surface in one cycle of rotation, where the length and flexibility of the hook have an important role. [Copyright &y& Elsevier]

The archaeal flagellum is a unique motility apparatus in the prokaryotic domain, distinct from the bacterial flagellum. Most of the currently recognized archaeal flagella-associated genes fall into a single fla operon that contains the genes for the flagellin proteins (two or more genes designated as flaA or flaB), some variation of a set of conserved proteins of unknown function ( flaC, flaD, flaE, flaF, flaG and flaH), an ATPase ( flaI) and a membrane protein ( flaJ). In addition, the flaD gene has been demonstrated to encode two proteins: a full-length gene product and a truncated product derived from an alternate, internal start site. A systematic deletion approach was taken using the methanogen Methanococcus maripaludis to investigate the requirement and a possible role for these proposed flagella-associated genes. Markerless in-frame deletion strains were created for most of the genes in the M. maripaludis fla operon. In addition, a strain lacking the truncated FlaD protein [FlaD M(191)I] was also created. DNA sequencing and Southern blot analysis confirmed each mutant strain, and the integrity of the remaining operon was confirmed by immunoblot. With the exception of the ΔFlaB3 and FlaD M(191)I strains, all mutants were non-motile by light microscopy and non-flagellated by electron microscopy. A detailed examination of the ΔFlaB3 mutant flagella revealed that these structures had no hook region, while the FlaD M(191)I strain appeared identical to wild type. Each deletion strain was complemented, and motility and flagellation was restored. Collectively, these results demonstrate for first time that these fla operon genes are directly involved and critically required for proper archaeal flagella assembly and function. [ABSTRACT FROM AUTHOR]

Haemophilus influenzae transits between niches within the human host that are predicted to differ in oxygen levels. The ArcAB two-component signal transduction system controls gene expression in response to respiratory conditions of growth and has been implicated in bacterial pathogenesis, yet the mechanism is not understood. We undertook a genome-scale study to identify genes of the H. influenzae ArcA regulon. Deletion of arcA resulted in increased anaerobic expression of genes of the respiratory chain and of H. influenzae's partial tricarboxylic acid cycle, and decreased anaerobic expression levels of genes of polyamine metabolism, and iron sequestration. Deletion of arcA also conferred a susceptibility to transient exposure to hydrogen peroxide that was greater following anaerobic growth than after aerobic growth. Array data revealed that the dps gene, not previously assigned to the ArcA modulon in bacteria, exhibited decreased expression in the arcA mutant. Deletion of dps resulted in hydrogen peroxide sensitivity and complementation restored resistance, providing insight into the previously uncharacterized mechanism of arcA-mediated H2O2 resistance. The results indicate a role for H. influenzae arcA and dps in pre-emptive defence against transitions from growth in low oxygen environments to aerobic exposure to hydrogen peroxide, an antibacterial oxidant produced by phagocytes during infection. [ABSTRACT FROM AUTHOR]

The predatory bacterium Bdellovibrio bacteriovorus swims rapidly by rotation of a single, polar flagellum comprised of a helical filament of flagellin monomers, contained within a membrane sheath and powered by a basal motor complex. Bdellovibrio collides with, enters and replicates within bacterial prey, a process previously suggested to firstly require flagellar motility and then flagellar shedding upon prey entry. Here we show that flagella are not always shed upon prey entry and we study the six fliC flagellin genes of B. bacteriovorus, finding them all conserved and expressed in genome strain HD100 and the widely studied lab strain 109J. Individual inactivation of five of the fliC genes gave mutant Bdellovibrio that still made flagella, and which were motile and predatory. Inactivation of the sixth fliC gene abolished normal flagellar synthesis and motility, but a disordered flagellar sheath was still seen. We find that this non-motile mutant was still able to predate when directly applied to lawns of YFP-labelled prey bacteria, showing that flagellar motility is not essential for prey entry but important for efficient encounters with prey in liquid environments. [ABSTRACT FROM AUTHOR]

The length of the flagellar hook is regulated; it is 55 ± 6 nm long inSalmonella. Five genes involved in hook-length regulation arefliK,flhB,fliG,fliMandfliN. The last four genes encode structural components of the protein export apparatus in the flagellar base, whereas FliK is soluble and secreted during flagellar assembly. The role of FliK, however, remains ambiguous. We constructed two kinds of FliK variants: N-terminally truncated FliK protein and FliK N-terminally fused with cyan fluorescent protein (CFP-FliK). Both N-terminally truncated FliK missing the first 99 amino acids (aa) and CFP-FliK fusion variants partially complemented afliKnull (polyhook) mutant to produce cells with filaments, allowing cells to swim; the hooks, however, were not normal but were polyhooks. When the N-terminally defective FliK variants were expressed at high levels, the average polyhook length was shortened coming close to the length of the wild-type hook, independently of the sizes of the FliK variants. These FliK variants were not secreted. CFP-FliK fusion proteins were observed to homogeneously distribute in the cytoplasm. We conclude that FliK does not need to be exported to control hook length and is unlikely to be a ruler; instead, we conclude that FliK controls hook length by the timely switching of secretion modes of the flagellar type III secretion system by the FliK C-terminal domain, and that the N-terminal region is dispensable for hook length control. [ABSTRACT FROM AUTHOR]

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 found that optimal migration required divalent cations and, in particular, Me2+. At concentrations naturally present in seawater, Mg2+ improved migration without altering the growth rate of the cells. Transmission electron microscopy and Western blot experiments suggested that Mg2+ addition enhanced flagellation, at least in part through an effect on the steady-state levels of flagellin protein. [ABSTRACT FROM AUTHOR]

We report the complete genome sequence of the deep-sea γ-- proteobacterium, idiomarina Ioihiensis, isolated recently from a hydrothermal vent at 1,300-m depth on the Löihi 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. loihiensis to the genomes of other γ-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. loihiensis 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, Ile, Val, Thr, and Met are incomplete. Auxotrophy for Val and Thr was confirmed by in vivo experiments. The I loihiensis genome contains a cluster of 32 genes encoding enzymes for exopolysaccharide and capsular polysaccharide synthesis. lt 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. loihiensis growth are the proteinaceous particles present in the deep sea hydrothermal vent waters. I. loihiensis would colonize these particles by using the secreted exopolysaccharide, digest these proteins, and metabolize the resulting peptides and amino acids. In summary, the I. loihiensis genome reveals an integrated mechanism of metabolic adaptation to the constantly changing deep-sea hydrothermal ecosystem. [ABSTRACT FROM AUTHOR]

Helicobacter pylori is a flagellated chronic pathogen, which colonizes the gastric mucus and mucosal cell surfaces. Flagella and motility are essential for the survival of this bacterium in the stomach environment. Flagellins of several bacterial species are potent activators of the human innate immune system by binding to TOLL-like receptor 5 (TLR5). The possible role of the two H. pylori flagellins FlaA and FlaB in stimulation of the innate immune system and induction of IL-8 release by human gastric epithelial cells was investigated in this study. Transcription and expression of TLR5 in three different human gastric epithelial cell lines was demonstrated. Salmonella enterica serovar Typhimurium FliC flagellin was able to activate human gastric epithelial cells. TLR5 transcription was modulated by H. pylori infection. However, both H. pylori flagellins appeared to possess no immunostimulatory potential on human gastric cells via TLR5, despite their extensive amino acid homology to stimulating flagellins of other bacterial species. The evolutionary development of such unique flagellins of low activating potential is proposed to be a novel mechanism of H. pylori to preserve the essential function of its flagella during chronic colonization of the stomach and to evade the deleterious host immune responses. [Copyright &y& Elsevier]

Focuses on the localization of flagellin genes of Methanococcus voltae. Electron microscopy examination of flagella; Role of the bacterial hook protein; Use of FlaA-specific antibodies.

Enteropathogenic Escherichia coli (EPEC) uses a type III secretion system (TTSS) to inject effector proteins into the plasma membrane and cytosol of infected cells. To translocate proteins, EPEC, like Salmonella and Shigella, is believed to assemble a macromolecular complex (type III secreton) that spans both bacterial membranes and has a short needle-like projection. However, there is a special interest in studying the EPEC TTSS owing to the fact that one of the secreted proteins, EspA, is assembled into a unique filamentous structure also required for protein translocation. In this report we present electron micrographs of EspA filaments which reveal a regular segmented substructure. Recently we have shown that deletion of the putative structural needle protein, EscF, abolished protein secretion and formation of EspA filaments. Moreover, we demonstrated that EspA can bind directly to EscF, suggesting that EspA filaments are physically linked to the EPEC needle complex. In this paper we provide direct evidence for the association between an EPEC bacterial membrane needle complex and EspA filaments, defining a new class of filamentous TTSS. [ABSTRACT FROM AUTHOR]

Describes the polymorphic forms of the flagellar filament of Rhodobacter sphaeroides induced in vitro by changes in pH and ionic strength. Sequence analysis and expression studies on the fliC gene, which encodes the flagellin protein of R. sphaeroides; Control of the fliC gene by a sigma[sup 28]-dependent promoter.

The flhDC operon of Salmonella typhimurium is the master control operon required for the expression of the entire flagellar regulon. The flagellar master operon was placed under the tetracycline-inducible promoter PtetA using the T-POP transposon. Cells containing this construct are motile in the presence of tetracycline and non-motile without inducer present. No flagella were visible under the electron microscope when cells were grown without inducer. The class 1, class 2 and class 3 promoters of the flagellar regulon are temporally regulated. After addition of tetracycline, the class 1 flhDC operon was transcribed immediately. Transcription of flgM (which is transcribed from both class 2 and class 3 promoters) began 15 min after induction. At 20 min after induction, the class 2 fliA promoter became active and intracellular FliA protein levels increased; at 30 min after induction, the class 3 fliC promoter was activated. Induction of fliC gene expression coincides with the appearance of FlgM anti-sigma factor in the growth medium. This also coincides with the completion of hook–basal body structures. Rolling cells first appeared 35 min after induction, and excess hook protein (FlgE) was also found in the growth medium at this time. At 45 min after induction, nascent flagellar filaments became visible in electron micrographs and over 40% of the cells exhibited some swimming behaviour. Multiple flagella assemble and grow on individual cells after induction of the master operon. These results confirm that the flagellar regulatory hierarchy of S. typhimurium is temporally regulated after induction. Both FlgM secretion and class 3 gene expression occur upon completion of the hook–basal body structure. [ABSTRACT FROM AUTHOR]

We analysed all major proteins secreted into culture media from Salmonella typhimurium . Proteins in culture supernatants were collected by trichloroacetic acid precipitation, separated in SDS–polyacrylamide gels and analysed by amino acid sequencing. Wild-type strain SJW1103 cells typically gave rise to nine bands in SDS gels: 89, 67, 58, 52, 50, 42, 40, 35 and (sometimes) 28 kDa. A search of the sequences in the available databases revealed that they were either flagellar proteins or virulence factors. Six of them were flagella specific: FlgK or HAP1 (58 kDa), FliC or flagellin (52 kDa), FliD or HAP2 (50 kDa), FlgE or hook protein (42 kDa), FlgL or HAP3 (35 kDa) and FlgD or hook-cap protein (28 kDa). The other four bands were specific for virulence factors: SipA (89 kDa), SipB (67 kDa), SipC (42 kDa) and InvJ (40 kDa). The 42 kDa band was a mixture of FlgE and SipC. We also analysed secreted proteins from more than 30 flagellar mutants, and they were categorized into four groups according to their band patterns: wild type, mot type, polyhook type and master gene type. Virulence factors were constantly secreted at a higher level in all flagellar mutants except a Δmot (motAB deletion) mutant, in which the amounts were greatly reduced. A new morphological pathway of flagellar biogenesis including protein secretion is presented. [ABSTRACT FROM AUTHOR]