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1.
The uropathogenic Gram-negative bacterium Proteus mirabilis exhibits a form of multicellular behaviour termed swarming, which involves cyclical differentiation of typical vegetative cells into filamentous, multinucleate, hyperflagellate swarm cells capable of rapid and co-ordinated population migration across surfaces. We observed that differentiation into swarm cells was accompanied by substantial increases in the activities of intracellular urease and extracellular haemolysin and metalloprotease, which are believed to be central to the pathogenicity of P. mirabilis. In addition, the ability of P. mirabilis to invade human urothelial cells in vitro was primarily a characteristic of differentiated swarm cells, not vegetative cells. These virulence factor activities fell back as the cells underwent cyclical reversion to the vegetative form (consolidation), in parallel with the diagnostic modulation of flagellin levels on the cell surface. Control cellular alkaline phosphatase activities did not increase during differentiation or consolidation. Non-flagellated, nonmotile transposon insertion mutants were unable to invade urothelial cells and they generated only low-level activities of haemolysin, urease and protease (0-10% of wild type). Motile mutants unable to differentiate into swarm cells were comparably reduced in their haemolytic, ureolytic and invasive phenotypes and generated threefold less protease activity. Mutants that were able to form swarm cells but exhibited various aberrant patterns of swarming migration produced wild-type activities of haemolysin, urease and protease, but their ability to enter urothelial cells was three- to 10-fold lower.(ABSTRACT TRUNCATED AT 250 WORDS)  相似文献   

2.
The pathogenic bacterium Proteus mirabilis exhibits a form of multicellular behaviour called swarming migration. This involves the differentiation of vegetative cells at the colony margin into swarm cells which are long, aseptate, multinucleate, hyper-flagellated filaments able to undergo repeated cycles of co-ordinated population migration and consolidation (reversion to vegetative cells). Transposon mutagenesis of uropathogenic P. mirabilis strain U6450 with Tn5 generated 4860 chromosomal insertions and, of these, 75 (1.6%) caused visibly abnormal swarming behaviour, indicating that at least 45 genes are involved in directing motility, cell differentiation and multicellular behaviour. While about one fifth of the swarm-defective mutants lacked flagella and were non-motile non-swarming (NMNS) the majority were normally flagellated and motile but were unable to form swarm cells (motile non-swarming, MNS), or were motile and able to form swarm cells but displayed aberrant patterns of multicellular migration (dendritic swarming, DS) or consolidation (frequent and infrequent consolidation, FC and IC). Restriction enzyme mapping of representative mutant DNAs by Southern hybridization with transposon DNA probes identified eight different mutated genetic loci within the five phenotypic classes. Subsequent Southern analysis of large restriction fragments separated by pulsed-field electrophoresis showed that these eight mutated loci required for motility, cell differentiation and multicellular migration were clustered on a region of DNA spanning approximately 8% of the 4.2 mbp P. mirabilis chromosome. Further linkage analysis showed that the DS locus involved in the ordered migration of the swarm cell population mapped separately from two main clusters of swarm loci, one cluster containing, within 112 kbp, genetic determinants of motility (NMNS) and also differentiation into swarm cells (MNS1, MNS2), and a second within a neighbouring 95 kbp DNA sequence containing three loci involved in the control of consolidation (FC, IC1, IC2).  相似文献   

3.
Proteus mirabilis is a urinary tract pathogen that differentiates from a short swimmer cell to an elongated, highly flagellated swarmer cell. Swarmer cell differentiation parallels an increased expression of several virulence factors, suggesting that both processes are controlled by the same signal. The molecular nature of this signal is not known but is hypothesized to involve the inhibition of flagellar rotation. In this study, data are presented supporting the idea that conditions inhibiting flagellar rotation induce swarmer cell differentiation and implicating a rotating flagellar filament as critical to the sensing mechanism. Mutations in three genes, fliL, fliF, and fliG, encoding components of the flagellar basal body, result in the inappropriate development of swarmer cells in noninducing liquid media or hyperelongated swarmer cells on agar media. The fliL mutation was studied in detail. FliL- mutants are nonmotile and fail to synthesize flagellin, while complementation of fliL restores wild-type cell elongation but not motility. Overexpression of fliL+ in wild-type cells prevents swarmer cell differentiation and motility, a result also observed when P. mirabilis fliL+ was expressed in Escherichia coli. These results suggest that FliL plays a role in swarmer cell differentiation and implicate FliL as critical to transduction of the signal inducing swarmer cell differentiation and virulence gene expression. In concert with this idea, defects in fliL up-regulate the expression of two virulence genes, zapA and hpmB. These results support the hypothesis that P. mirabilis ascertains its location in the environment or host by assessing the status of its flagellar motors, which in turn control swarmer cell gene expression.  相似文献   

4.
To study the assembly of the Caulobacter crescentus flagellar filament, we have devised a fractionation protocol that separates the cellular flagellin into three compartments: soluble, membrane, and assembled. Radioactive labeling in pulse-chase and pulse-labeling experiments has demonstrated for the first time that both soluble and membrane-associated flagellin pools are precursors in the assembly of the flagellar filament. The results of these experiments also indicate that flagellar filament assembly occurs via the translocation of newly synthesized flagellins from the soluble pool to the membrane pool to the assembled flagellar filaments. It is not possible to conclude whether the soluble flagellin fraction is synthesized cytoplasmically or as a loosely associated membrane intermediate which is released during lysis. It is clear, however, that the soluble and membrane flagellins are in physically and functionally distinct pools. The implications of these findings for the study of protein secretion from cells and the invariant targeting of flagellar proteins to the stalk-distal pole of the dividing cell during flagellum morphogenesis are discussed.  相似文献   

5.
Swarming in Proteus mirabilis is characterized by the coordinated surface migration of multicellular rafts of highly elongated, hyperflagellated swarm cells. We describe a transposon mutant, MNS185, that was unable to swarm even though vegetative cells retained normal motility and the ability to differentiate into swarm cells. However, these elongated cells were irregularly curved and had variable diameters, suggesting that the migration defect results from the inability of these deformed swarm cells to align into multicellular rafts. The transposon was inserted at codon 196 of a 228-codon gene that lacks recognizable homologs. Multiple copies of the wild-type gene, called ccmA, for curved cell morphology, restored swarming to the mutant. The 25-kDa CcmA protein is predicted to span the inner membrane twice, with its C-terminal major domain being present in the cytoplasm. Membrane localization was confirmed both by immunoblotting and by electron microscopy of immunogold-labelled sections. Two forms of CcmA were identified for wild-type P. mirabilis; they were full-length integral membrane CcmA1 and N-terminally truncated peripheral membrane CcmA2, both present at approximately 20-fold higher concentrations in swarm cells. Differentiated MNS185 mutant cells contained wild-type levels of the C-terminally truncated versions of both proteins. Elongated cells of a ccmA null mutant were less misshapen than those of MNS185 and were able to swarm, albeit more slowly than wild-type cells. The truncated CcmA proteins may therefore interfere with normal morphogenesis, while the wild-type proteins, which are not essential for swarming, may enhance migration by maintaining the linearity of highly elongated cells. Consistent with this view, overexpression of the ccmA gene caused cells of both Escherichia coli and P. mirabilis to become enlarged and ellipsoidal.  相似文献   

6.
7.
Swarming by Proteus mirabilis is characterized by cycles of rapid and coordinated population migration across surfaces following differentiation of vegetative cells into elongated hyperflagellated swarm cells. It has been shown that surface colony expansion by the swarm cell population is facilitated by a colony migration factor (Cmf), a capsular polysaccharide (CPS) that also contributes to the uropathogenicity of P. mirabilis (Gygi, D., Rahman, M. M., Lai, H.-C., Carlson, R., Guard-Petter, J., and Hughes, C. (1995) Mol. Microbiol. 17, 1167-1175). In this report, the Cmf-CPS was extracted with hot water, precipitated with ethanol, and further purified by gel permeation chromatography. Its structure was established by glycosyl composition and linkage analyses, and by one- and two-dimensional NMR spectroscopy. The Cmf-CPS is composed of the following tetrasaccharide repeating unit. [see text]  相似文献   

8.
In Caulobacter crescentus biogenesis of the flagellar organelle occurs during one stage of its complex life cycle. Thus in synchronous cultures it is possible to assay the sequential synthesis and assembly of the flagellum and hook in vivo with a combination of biochemical and radioimmunological techniques. The periodicity of synthesis and the subcellular compartmentation of the basal hook and filament subunits were determined by radioimmune assay procedures. Unassembled 27,000-dalton (27K) flagellin was preferentially located in isolated membrane fractions, whereas the 25K flagellin was distributed between the membrane and cytoplasm. The synthesis of hook began before that of flagellin, although appreciable overlap of the two processes occurred. Initiation of filament assembly coincided with the association of newly synthesized hook and flagellin subunits. Caulobacter flagella are unusual in that they contain two different flagellin subunits. Data are presented which suggest that the ratio of the two flagellin subunits changes along the length of the filament. Only the newly synthesized 25K flagellin subunit is detected in filaments assembled during the swarmer cell stage. By monitoring the appearance of flagellar hooks in the culture medium, the time at which flagella are released was determined.  相似文献   

9.
To determine the ultrastructural and biochemical bases for flagellar adhesiveness in the mating reaction in Chlamydomonas, gametic and vegetative flagella and flagellar membranes were studied by use of electron microscope and electrophoretic procedures. Negative staining with uranyl acetate revealed no differences in gametic and vegetative flagellar surfaces; both had flagellar membranes, flagellar sheaths, and similar numbers and distributions of mastigonemes. Freezecleave procedures suggested that there may be a greater density of intramembranous particles on the B faces of gametic flagellar membranes than on the B faces of vegetative flagellar membranes. Gamone, the adhesive material that gametes release into their medium, was demonstrated, on the basis of ultrastructural and biochemical analyses, to be composed of flagellar surface components, i.e., membrane vesicles and mastigonemes. Comparison of vegetative (nonadhesive) and gametic (adhesive) "gamones" by use of SDS polyacrylamide gel electrophoresis showed both preparations to be composed of membrane, mastigoneme, and some microtubule proteins, as well as several unidentified protein and carbohydrate-staining components. However, there was an additional protein of approximately 70,000 mol wt in gametic gamone which was not present in vegetative gamone. When gametic gamone was separated into a membrane and a mastigoneme fraction on CSCl gradients, only the membrane fraction had isoagglutinating activity; the mastigoneme fraction was inactive, suggesting that mastigonemes are not involved in adhesion.  相似文献   

10.
Swarming motility   总被引:10,自引:0,他引:10  
Swarming involves differentiation of vegetative cells into hyperflagellated swarm cells that undergo rapid and coordinated population migration across solid surfaces. Cell density, surface contact, and physiological signals all provide critical stimuli, and close cell alignment and the production of secreted migration factors facilitate mass translocation. Flagella biogenesis is central to swarming, and the flhDC flagellar master operon is the focal point of a regulatory network governing differentiation and migration.  相似文献   

11.
12.
The aglU gene of Myxococcus xanthus encodes a protein similar to Het-E1 (vegetative incompatibility) from Podospora anserina, acylaminoacyl-peptidase from Bacillus subtilis, and TolB from Escherichia coli. These proteins all have evenly spaced SPDG repeats that are characteristic of a larger motif called the WD-repeat. The WD-repeat is predicted to form a beta-propeller structure that mediates the assembly of heteromeric protein complexes. AglU has a consensus lipoprotein attachment motif that includes a type II signal sequence followed by a cysteine residue. This suggests that AglU is matured, then attached to the outer membrane via fatty acid acylation at this Cys. Cells carrying a mutation in aglU are blocked in adventurous gliding and can swarm only if cells are in contact with one another. When starved of nutrients, the aglU mutant aggregates and forms multicellular fruiting bodies like the wild-type strain, but is unable to produce heat-resistant spores. This suggests that adventurous gliding motility, per se, is not required for development, but that AglU is essential for a terminal step of spore differentiation.  相似文献   

13.
The ability to rapidly translocate polystyrene microspheres attached to the surface of a plasma membrane domain reflects a unique form of cellular force transduction occurring in association with the plasma membrane of microtubule based cell extensions. This unusual form of cell motility can be utilized by protistan organisms for whole cell locomotion, the early events in mating, and transport of food organisms along the cell surface, and possibly intracellular transport of certain organelles. Since surface motility is observed in association with cilia and flagella of algae, sea urchin embryos and cultured mammalian cells, it is likely that it serves an additional role beyond those already cited; this is likely to be the transport of precursors for the assembly and turnover of ciliary and flagellar membranes and axonemes. In the case of the Chlamydomonas flagellum, where surface motility has been most extensively studied, it appears that cross-linking of flagellar surface exposed proteins induces a transmembrane signaling pathway that activates machinery for moving flagellar membrane proteins in the plane of the flagellar membrane. This signaling pathway in vegetative Chlamydomonas reinhardtii appears to involve an influx of calcium, a rise in intraflagellar free calcium concentration and a change in the level of phosphorylation of specific membrane-matrix proteins. It is hypothesized that flagellar surface contact with a solid substrate (during gliding), a polystyrene microsphere or another flagellum (during mating) will all activate a signaling pathway similar to the one artificially activated by the use of monoclonal antibodies to flagellar membrane glycoproteins. A somewhat different signaling pathway, involving a transient rise in intracellular cAMP level, may be associated with the mating of Chlamydomonas gametes, which is initiated by flagellum-flagellum contact. The hypothesis that the widespread observation of microsphere movements on various ciliary and flagellar surfaces may reflect a mechanism normally utilized to transport axonemal and membrane subunits along the internal surface of the organelle membrane presents a paradox in that one would expect this to be a constitutive mechanism, not one necessarily activated by a signaling pathway.  相似文献   

14.
Radioactive labeling studies demonstrate a continuous incorporation of newly synthesized proteins and glycoproteins into the intact flagella of Chlamydomonas. This apparent turnover is preferentially occurring for membrane components. In particular, two classes of flagellar membrane components, one a high molecular weight (HMW) group of closely migrating glycoproteins and the other a protein with a MW around 65 kD, are continuously turning over in the vegetative cell. This selective protein turnover may explain the ability of Chlamydomonas to rapidly recover from proteolytic modification of the flagellar surface and to change its flagellar surface properties during the early events in mating.  相似文献   

15.
The developmental cytology of the vegetative filament of Vaucheria has been investigated with light and electron microscopes. The vegetative filament consists of three distinct zones: the apical zone, the sub-apical zone and the zone of vacuolation. Organelle distribution and associations in these zones have a primary role in controlling morphogenetic events which influence growth and induce differentiation of sexual and asexual organs. Cell elongation in the polarised vegetative filament occurs by vesicular addition in the apical zone. Two types of active cyclosis are found exclusively in the zone of vacuolation. Nuclear cyclosis involves microtubular bands which associate with persistent centrioles found adjacent to the outer nuclear membrane. A specialised mitochondrial-dictyosome association is transported by a microfilament-like system of anastomosing strands. This latter system is extremely labile to conventional fixatives.  相似文献   

16.
The IgA-degrading metalloprotease, ZapA, of the urinary tract pathogen Proteus mirabilis is co-ordinately expressed along with other proteins and virulence factors during swarmer cell differentiation. In this communication, we have used zapA to monitor IgA protease expression during the differentiation of vegetative swimmer cells to fully differentiated swarmer cells. Northern blot analysis of wild-type cells and beta-galactosidase measurements using a zapA:lacZ fusion strain indicate that zapA is fully expressed only in differentiated swarmer cells. Moreover, the expression of zapA on nutrient agar medium is co-ordinately regulated in concert with the cycles of cellular differentiation, swarm migration and consolidation that produce the bull's-eye colonies typically associated with P. mirabilis. ZapA activity is not required for swarmer cell differentiation or swarming behaviour, as ZapA- strains produce wild-type colony patterns. ZapA- strains fail to degrade IgA and show decreased survival compared with the wild-type cells during infection in a mouse model of ascending urinary tract infection (UTI). These data underscore the importance of the P. mirabilis IgA-degrading metalloprotease in UTI. Analysis of the nucleotide sequences adjacent to zapA reveals four additional genes, zapE, zapB, zapC and zapD, which appear to possess functions required for ZapA activity and IgA proteolysis. Based on homology to other known proteins, these genes encode a second metalloprotease, ZapE, as well as a ZapA-specific ABC transporter system (ZapB, ZapC and ZapD). A model describing the function and interaction of each of these five proteins in the degradation of host IgA during UTI is presented.  相似文献   

17.
Bacteria secrete flagella subunits and deliver virulence effectors via type III export systems. During flagellar filament assembly, a chaperone escort mechanism has been proposed to enhance the export of early, minor flagellar filament components by selectively binding and cycling their chaperones. Here we identify virulence orthologues of the flagellar chaperone escort FliJ and show that the orthologues Salmonella InvI and Yersinia YscO are, like FliJ, essential for their type III export pathway and similarly, do not bind export substrates. Like FliJ, they recognize a subset of export chaperones, in particular those of the host membrane translocon components required for subsequent effector delivery.  相似文献   

18.
Many types of bacteria propel themselves using elongated structures known as flagella. The bacterial flagellar filament is a relatively simple and well-studied macromolecular assembly, which assumes different helical shapes when rotated in different directions. This polymorphism enables a bacterium to switch between running and tumbling modes; however, the mechanism governing the filament polymorphism is not completely understood. Here we report a study of the bacterial flagellar filament using numerical simulations that employ a novel coarse-grained molecular dynamics method. The simulations reveal the dynamics of a half-micrometer-long flagellum segment on a timescale of tens of microseconds. Depending on the rotation direction, specific modes of filament coiling and arrangement of monomers are observed, in qualitative agreement with experimental observations of flagellar polymorphism. We find that solvent-protein interactions are likely to contribute to the polymorphic helical shapes of the filament.  相似文献   

19.
Campylobacter jejuni, a gram-negative motile bacterium, secretes a set of proteins termed the Campylobacter invasion antigens (Cia proteins). The purpose of this study was to determine whether the flagellar apparatus serves as the export apparatus for the Cia proteins. Mutations were generated in five genes encoding three structural components of the flagella, the flagellar basal body (flgB and flgC), hook (flgE2), and filament (flaA and flaB) genes, as well as in genes whose products are essential for flagellar protein export (flhB and fliI). While mutations that affected filament assembly were found to be nonmotile (Mot-) and did not secrete Cia proteins (S-), a flaA (flaB+) filament mutant was found to be nonmotile but Cia protein secretion competent (Mot-, S+). Complementation of a flaA flaB double mutant with a shuttle plasmid harboring either the flaA or flaB gene restored Cia protein secretion, suggesting that Cia export requires at least one of the two filament proteins. Infection of INT 407 human intestinal cells with the C. jejuni mutants revealed that maximal invasion of the epithelial cells required motile bacteria that are secretion competent. Collectively, these data suggest that the C. jejuni Cia proteins are secreted from the flagellar export apparatus.  相似文献   

20.
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