Formation of bacterial flagella. I. Demonstration of a functional flagellin pool in spirillum serpens and bacillus subtilis

Martinez, R. J. (University of California, Los Angeles), and E. Z. Gordee. Formation of bacterial flagella. I. Demonstration of a functional flagellin pool in Spirillum serpens and Bacillus subtilis. J. Bacteriol. 91:870-875. 1966-Exponentially growing cultures of Spirillum serpens and Bacillus subtilis regained motility and flagella within one generation after mechanical deflagellation. Regeneration of flagella occurred in both cultures in the presence of chloramphenicol at concentrations shown to inhibit flagellin synthesis. Cells labeled with C(14)-amino acids regenerated radioactive flagella in the presence of chloramphenicol. A conditional mutant of S. serpens (T-45) was isolated. This strain did not produce flagella when grown at 45 C, but formed the organelles upon temperature shift to 30 C, even in the presence of chloramphenicol. A reduction of intracellular antibody-precipitable flagellin counts in labeled S. serpens T-45 occurred concomitant with the generation of flagella at 30 C. The data suggest that the flagella of S. serpens and B. subtilis are formed from a pool of intracellular flagellin proteins.     

pH-dependent flagella formation by facultative alkaliphilic Bacillus sp. C-125.

A facultative alkaliphilic strain of Bacillus sp. C-125 grown at alkaline pH had many sinuous peritrichous flagella and was highly motile. However, most of the cells grown initially at pH 7 were non-motile and possessed few straight flagella. The amount of flagellin was low when the organism was grown at pH 7, suggesting that non-motility is due to poor synthesis of flagellin. The molecular mass of the flagellin was 37 kDa and the isoelectric point was pH 5.0. The amino acid composition of the flagellin was similar to that found in the flagellin from neutrophilic Bacillus subtilis 168.     

Surface-associated flagellum formation and swarming differentiation in Bacillus subtilis are controlled by the ifm locus

Knowledge of the highly regulated processes governing the production of flagella in Bacillus subtilis is the result of several observations obtained from growing this microorganism in liquid cultures. No information is available regarding the regulation of flagellar formation in B. subtilis in response to contact with a solid surface. One of the best-characterized responses of flagellated eubacteria to surfaces is swarming motility, a coordinate cell differentiation process that allows collective movement of bacteria over solid substrates. This study describes the swarming ability of a B. subtilis hypermotile mutant harboring a mutation in the ifm locus that has long been known to affect the degree of flagellation and motility in liquid media. On solid media, the mutant produces elongated and hyperflagellated cells displaying a 10-fold increase in extracellular flagellin. In contrast to the mutant, the parental strain, as well as other laboratory strains carrying a wild-type ifm locus, fails to activate a swarm response. Furthermore, it stops to produce flagella when transferred from liquid to solid medium. Evidence is provided that the absence of flagella is due to the lack of flagellin gene expression. However, restoration of flagellin synthesis in cells overexpressing sigma(D) or carrying a deletion of flgM does not recover the ability to assemble flagella. Thus, the ifm gene plays a determinantal role in the ability of B. subtilis to contact with solid surfaces.     

Identification of flagellin associated with the Bacillus subtilis folded chromosome.

We have analyzed the nature and contents of a major protein, P36, in the nucleoid of the Bacillus subtilis wild type and an isogenic mutant devoid of flagella. It appears that deoxyribonucleic acid-P36 complex is flagellin present as membrane-associated flagella.     

Pleiotropic phenomena in autolytic enzyme(s) content, flagellation, and simultaneous hyperproduction of extracellular alpha-amylase and protease in a Bacillus subtilis mutant.

A mutant of Bacillus subtilis 6160 that had been isolated by its hyperproduction of alpha-amylase and protease lacked flagella and motility, and its content of autolytic enzyme(s) was reduced to one-third to one-fourth that of the parent. These phenotypic differences were completely co-transferred by the deoxyribonucleic acid (DNA) of the mutant when five DNA recipient strains of B. subtilis were transformed. The revertants, isolated by motility with a frequency of approximately 10(-7), recovered a normal level of autolytic activity and showed reduced productivity of alpha-amylase and protease. This point mutation allowed normal flagellin synthesis, spore formation, and rate of growth. The comparison of cell envelope of the mutant with that of the parent indicated that there was no significant difference except loss of flagella. Therefore the association at the cell surface of a group of extracellular proteins consisting of alpha-amylase, proteases, flagellin, and autolytic enzymes(s) seem to be coordinately regulated by the gene or seem to be affected coordinately by certain undetected alterations of the cell envelope.     

Establishment of monoclonal antibodies specific for Bacillus subtilis DB9011

Bacillus subtilis DB9011 is a strain with useful functions for agriculture. To establish a method for the discrimination of this strain from others, monoclonal antibodies (MAbs) were prepared. Although two established MAbs (MAb9B6 and MAb14D2) cross-react with some other Bacillus strains in ELISA, only B. subtilis DB9011 vegetative cells are recognized by both MAbs. MAb14D2 recognizes flagellin, a 34-kDa unit protein of flagella. The two MAbs established will provide powerful tools with which detailed analysis of this bacterial strain can be obtained under environmental conditions.     

Variation in the primary structure of Bacillus subtilis flagellins

The flagella derived from 18 strains Bacillus subtilis were tested for their reaction with antiflagellar filament antibody and antiflagellin antibody. On the basis of their reactivity, at least five serologically distinct classes could be identified. Peptide map analysis of tryptic digests of the subunit proteins were consistent with the immunochemical analysis. Large differences in sequence existed among proteins of the different classes; proteins within an antigenic group differed by only a few peptides. Furthermore, 9 of the 27 tryptic peptides resolved were common to flagellin proteins from all the classes examined. The relationship between antigenic specificity, variability in peptide pattern, and the conformation of the flagellin protein are discussed.     

Comparison of the flagellins from different flagellar morphotypes of Escherichia coli.

The molecular weights of the flagellins of 13 strains of Escherichia coli, each with a different H antigen, were estimated using polyacrylamide gel electrophoresis. In each case only one major polypeptide was demonstrated, although some strains possessed apparently sheathed flagella. Considerable differences in the molecular weight of flagellin accompanied the previously described structural differences between flagella from strains with different H antigens. The relationship between flagellar diameter and the molecular weight of the corresponding flagellins was similar for both unsheathed and apparently sheathed flagella. Crosss-polymerization occurred between seed consisting of fragment of unsheathed flagella and flagellin solution from apparently sheathed flagella and vice versa. Co-polymerization of flagellin from unsheathed flagella and flagellin from apparently sheathed flagella was also demonstrated. These polymerization experiments indicate that the assembly pattern of flagellin molecules is probably the same in all E. coli flagella. The above and other evidence suggests that there is no true sheath, but that the differences in flagellar surface structure between different E. coli flagella are the result of differences in the superficial parts of the flagellin molecules.     

Sequential polymerization of flagellin A and flagellin B into Caulobacter flagella

The mode of polymerization of two species of flagellins, flagellin A and flagellin B, in polar flagella of Caulobacter crescentus was examined. By immunological staining we found that 1 to 1.2 μm of the portion of the flagellar filament proximal to the cell was composed of flagellin B, whereas about 5 μm of the distal portion was composed of flagellin A. This result, together with the previous observation that a flagellin B-less mutant cannot form normal flagella but instead forms stubs in spite of their high level of flagellin A synthesis, indicates that flagellin B is very important for the formation of complete flagella and/or for the initiation of filament formation from the hook.     

Reconstitution and purification of flagellar filaments from Caulobacter crescentus.

Filaments from isolated flagella of Caulobacter crescentus have been purified by successive dissociation and reconstitution. After the second and third reconstitutions from subunits in 0.8 M sodium citrate, filament preparations contained only two proteins, flagellin A (26,000 daltons) and flagellin B (28,000 daltons). There was some enrichment for flagellin A during reconstitution by this procedure, since isolated flagella contained flagellin A and flagellin B in a ratio of approximately 3.8:1 and filaments after the third reconstitution contained the two proteins in a ratio of 5.0:1.     

A method for the purification of bacterial flagellin that allows simple upscaling

There is a growing interest in enterobacterial flagellins that may result in a demand to produce flagellin on an industrial scale for possible applications as an adjuvant, immunomodulatory agent or vaccine antigen. Traditionally, small-scale production of flagellin has occurred in the laboratory by flagellar shearing of bacterial surfaces and subsequent ultracentrifugation. The main drawback of this method is the need to use low-agitation cultures to avoid the loss of flagella due to shearing during culture. In the present work, we describe a scalable protocol for the production of flagellin with higher yields than traditional laboratory-scale protocols. The use of cross-flow filtration to concentrate bacterial cultures combines extensive shearing of flagella with a reduction in volume, greatly simplifying downstream processing. This technique also allows the use of highly-agitated culture conditions because any sheared flagella are retained in the bacterial concentrate. Flagella obtained with this procedure showed in vivo and in vitro innate activating capacities similar to those of flagella produced at laboratory scale. This procedure is flexible, allowing an increase in production scale, an enhancement of flagellin yield and no requirement for expensive equipment.     

"Cap" on the tip of Salmonella flagella

Flagellar filaments isolated intact from a Salmonella short-flagella mutant are unable to serve as nuclei for flagellin polymerization in vitro, whereas the filaments reconstructed in vitro from the mutant flagellin are able to do so. The inability of intact flagella to nucleate flagellin polymerization appears to be common to wild-type bacteria and thus suggests that the tip of intact flagella are generally inactivated or capped in vivo. Careful observations of the tips of intact flagella and reconstructed flagellar filaments of a wild-type species have revealed marked difference between them: the intact flagella usually have blunt ends, whereas reconstructed filaments have concave, "fish-tail" ends. Moreover, a thin structure is often observed attaching to the very end of the intact flagella. We suspect that this "capping" structure is essential to the elongation mechanism of flagellar filaments.     

Purification and antigenic analysis of flagella of Campylobacter jejuni

The flagella of Campylobacter jejuni strain FUM158432 were purified and a flagellin preparation consisting of only a single peptide of 63,000 daltons was obtained. The peptide of 92,000 daltons usually associated with a flagellar preparation was shown to be a peptide derived from the hook region. Antiserum was prepared by immunizing a rabbit with the flagellin preparation. The reaction of the antiserum was found to be highly specific for the flagellar filament by immunoelectron microscopy and for flagellin peptide by the immunoblotting method. Seventeen of 23 clinically isolated strains of C. jejuni reacted with this antiserum but the other six strains did not, indicating the existence of antigenic variation of the flagella of C. jejuni. The flagella of a few strains of C. coli also reacted with this antiserum.     

Antigenic Difference Between Polar Monotrichous and Peritrichous Flagella of Vibrio parahaemolyticus

Polar monotrichous and peritrichous flagella of Vibrio parahaemolyticus were isolated and purified separately. On hydroxylapatite column chromatography, the flagellins of polar monotrichous flagella were eluted with a higher concentration of phosphate than those of peritrichous flagella. Gel diffusion tests showed an antigenic difference between the flagellins of polar monotrichous and peritrichous flagella. Electron microscope observations on cells stained with ferritin-conjugated antibodies demonstrated that polar monotrichous and peritrichous flagella reacted specifically with antimonotrichous flagellin and antiperitrichous flagellin antisera, respectively.     

Effect of flagella expression on adhesion of Achromobacter piechaudii to chalk surfaces

Aims: To examine flagella role and cell motility in adhesion of Achromobacter piechaudii to chalk. Methods and Results: Transmission electron microscopy revealed that stationary cells have thicker and longer flagella than logarithmic cells. SDS‐PAGE analysis showed that flagellin was more abundant in stationary cells than logarithmic ones. Sonication or inhibition of flagellin synthesis caused a 30% reduction in adhesion to chalk. Preincubation of chalk with flagella extracts reduced adhesion, by 50%. Three motility mutants were isolated. Mutants 94 and 153 were nonmotile, expressed normal levels of flagellin, have regular flagella and exhibited reduced adhesion. Mutant 208 expressed low levels of flagellin, no flagella and a spherical cell shape but with normal adhesion capacity. Conclusions: Multiple cell surface factors affect the adhesion efficiency to chalk. Flagella per se through physical interaction and through cell motility contribute to the adhesion process. The adhesion behaviour of mutant 208 suggests that cell shape can compensate for flagellar removal and motility. Significance and Impact of the Study: Physiological status affects bacterial cell surface properties and hence adhesion efficiency to chalk. This interaction is essential to sustain biodegradation activities and thus, remediation of contaminated chalk aquifers.     

Synthesis and Structure of Caulobacter crescentus Flagella

During the normal cell cycle of Caulobacter crescentus, flagella are released into the culture fluid as swarmer cells differentiate into stalked cells. The released flagellum is composed of a filament, hook, and rod. The molecular weight of purified flagellin (subunit of flagella filament) is 25,000 as determined by sodium dodecyl sulfate-polyacrylamide gel electrophoresis. The formation of a flagellum opposite the stalk has been observed by microscope during the differentiation of a stalked cell in preparation for cell division. By pulsing synchronized cultures with (14)C-amino acids it has been demonstrated that the synthesis of flagellin occurs approximately 30 to 40 min before cell division. Flagellin, therefore, is synthesized at a discrete time in the cell cycle and is assembled into flagella at a specific site on the cell. A mutant of C. crescentus that fails to synthesize flagellin has been isolated.     

Rhamnolipid-induced shedding of flagellin from Pseudomonas aeruginosa provokes hBD-2 and IL-8 response in human keratinocytes

Several 'pathogen-associated molecular pattern' (PAMP) of the opportunistic pathogen Pseudomonas aeruginosa activate the innate immune system in epithelial cells. Particularly the production of antimicrobial peptides such as the human beta-defensin-2 (hBD-2) and proinflammatory cytokines as the interleukin (IL)-8 is boosted. In the present study culture supernatants of static grown P. aeruginosa were found to be potent hBD-2 and IL-8 inducers, indicating a soluble or shedded PAMP, comparable to that of heat-killed bacterial supernatants. In subsequent analyses this PAMP was identified as flagellin, the major structural protein of the flagella. Flagellin is known to be an immunostimulatory potent factor, but the mechanisms by which P. aeruginosa is able to remove flagellin from the flagella remain unknown. Here we provide evidence for the presence of a factor responsible for release of flagellin from the flagella. Purification of this factor and subsequent mass spectrometry analyses identified rhamnolipids as responsible agents. Our findings indicate that maybe upon adhesion to surfaces P. aeruginosa alters the outer membrane composition in a rhamnolipid-depending manner, thereby shedding flagellin from the flagella. In turn epithelial cells recognize flagellin and cause the synthesis of antimicrobial peptides as well as recruitment of inflammatory cells by induction of proinflammatory cytokines.