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PubMed1985

Excretion of unassembled hook-associated proteins by Salmonella typhimurium.

Hook-associated proteins (HAPs) were excreted into the culture medium of the Fla+ strain as well as into the growth medium of the filamentless mutants of Salmonella typhimurium. This indicates that the bacteria synthesize HAPs excessively, beyond the amount required for construction of flagella. The extra HAPs are shed into the culture medium after a definite amount of each HAP has been assembled into the flagellar structure.

Bacterial Proteins↗

PubMed1986

Subdivision of flagellar genes of Salmonella typhimurium into regions responsible for assembly, rotation, and switching.

Three flagellar genes of Salmonella typhimurium (flaAII.2, flaQ, and flaN) were found to be multifunctional, each being associated with four distinct mutant phenotypes: nonflagellate (Fla-), paralyzed (Mot-), nonchemotactic (Che-) with clockwise motor bias, and nonchemotactic (Che-) with counterclockwise motor bias. The distribution of Fla, Mot, and Che mutational sites within each gene was examined. Fla sites were fairly broadly distributed, whereas Mot and Che sites were more narrowly defined. Local subregions rich in sites of one type were not generally rich in sites of another type. Among Che sites, there was little overlap between those corresponding to a clockwise bias and those corresponding to a counterclockwise bias. Our results suggest that within the corresponding gene products there are specialized subregions for flagellar structure, motor rotation, and control of the sense of rotation.

Alleles↗

PubMed1994

Characterization of the fliL gene in the flagellar regulon of Escherichia coli and Salmonella typhimurium.

filL is a small gene of unknown function that lies within the beginning of a large flagellar operon of Salmonella typhimurium and Escherichia coli. A spontaneous fliL mutant of S. typhimurium, containing a frameshift mutation about 40% from the 3' end of the gene, was moderately motile but swarmed poorly, suggesting that FliL might be a component of the flagellar motor or switch. However, in-frame deletions of the E. coli gene, including an essentially total deletion, had little or no effect on motility or chemotaxis. Thus, FliL does not appear to have a major role in flagellar structure or function and is therefore unlikely to be a component of the motor or switch; the effect on motility caused by truncation of the gene is probably an indirect one.

Bacterial Proteins↗

PubMed1995

A mutation that uncouples flagellum assembly from transcription alters the temporal pattern of flagellar gene expression in Caulobacter crescentus.

The transcription of flagellar genes in Caulobacter crescentus is regulated by cell cycle events that culminate in the synthesis of a new flagellum once every cell division. Early flagellar gene products regulate the expression of late flagellar genes at two distinct stages of the flagellar trans-acting hierarchy. Here we investigate the coupling of early flagellar biogenesis with middle and late flagellar gene expression. We have isolated mutants (bfa) that do not require early class II flagellar gene products for the transcription of middle or late flagellar genes. bfa mutant strains are apparently defective in a negative regulatory pathway that couples early flagellar biogenesis to late flagellar gene expression. The bfa regulatory pathway functions solely at the level of transcription. Although flagellin promoters are transcribed in class II/bfa double mutants, there is no detectable flagellin protein on immunoblots prepared from mutant cell extracts. This finding suggests that early flagellar biogenesis is coupled to gene expression by two distinct mechanisms: one that negatively regulates transcription, mediated by bfa, and another that functions posttranscriptionally. To determine whether bfa affects the temporal pattern of late flagellar gene expression, cell cycle experiments were performed in bfa mutant strains. In a bfa mutant strain, flagellin expression fails to shut off at its normal time in the cell division cycle. This experimental result indicates that bfa may function as a regulator of flagellar gene transcription late in the cell cycle, after early flagellar structures have been assembled.

Caulobacter↗

PubMed2003

FlrA, a sigma54-dependent transcriptional activator in Vibrio fischeri, is required for motility and symbiotic light-organ colonization.

Flagellum-mediated motility of Vibrio fischeri is an essential factor in the bacterium's ability to colonize its host, the Hawaiian squid Euprymna scolopes. To begin characterizing the nature of the flagellar regulon, we have cloned a gene, designated flrA, from V. fischeri that encodes a putative sigma(54)-dependent transcriptional activator. Genetic arrangement of the flrA locus in V. fischeri is similar to motility master-regulator operons of Vibrio cholerae and Vibrio parahaemolyticus. In addition, examination of regulatory regions of a number of flagellar operons in V. fischeri revealed apparent sigma(54) recognition motifs, suggesting that the flagellar regulatory hierarchy is controlled by a similar mechanism to that described in V. cholerae. However, in contrast to its closest known relatives, flrA mutant strains of V. fischeri ES114 were completely abolished in swimming capability. Although flrA provided in trans restored motility to the flrA mutant, the complemented strain was unable to reach wild-type levels of symbiotic colonization in juvenile squid, suggesting a possible role for the proper expression of FlrA in regulating symbiotic colonization factors in addition to those required for motility. Comparative RNA arbitrarily primed PCR analysis of the flrA mutant and its wild-type parent revealed several differentially expressed transcripts. These results define a regulon that includes both flagellar structural genes and other genes apparently not involved in flagellum elaboration or function. Thus, the transcriptional activator FlrA plays an essential role in regulating motility, and apparently in modulating other symbiotic functions, in V. fischeri.

Amino Acid Sequence↗

PubMed1965

Electron microscopy of bundled flagella of the curly mutant of Salmonella abortivoequina.

Mitani, Michiko (National Institute of Genetics, Mishima, Japan), and Tetsuo Iino. Electron microscopy of bundled flagella of the curly mutant of Salmonella abortivoequina. J. Bacteriol. 90:1096-1101. 1965.-The arrangement of flagella was observed by dark-field and electron microscopy in three strains of Salmonella abortivoequina, namely, normal flagellar, curly flagellar, and paralyzed curly flagellar strains. With dark-field microscopy, bundled flagella could be seen in 5 to 10% of actively moving normal or curly mutant cells. Under the electron microscope, a great many bundled flagella were observed in the curly mutant strain, but in the normal strain most of the flagella were dissociated or the bundles were rather loose and irregular. Normal flagella seem to separate easily during the process of preparation, but not the curly ones. Single flagella were found to run parallel with each other and to form a bundle consisting of five or more flagella; the bundle was spirally gyrating, with the characteristic flagellar wave. It is thought that the bundle observed with the electron microscope corresponds to that observed under the dark-field microscope. Further, the marked decrease of bundle formation in the paralyzed curly mutant cells suggests that bundle formation is not caused by curly flagellar structure per se, but corresponds to the mode of locomotion of peritrichously flagellated bacteria.

Flagella↗

PubMed2001

CHLAMYDOMONAS AS A MODEL ORGANISM.

The unicellular green alga Chlamydomonas offers a simple life cycle, easy isolation of mutants, and a growing array of tools and techniques for molecular genetic studies. Among the principal areas of current investigation using this model system are flagellar structure and function, genetics of basal bodies (centrioles), chloroplast biogenesis, photosynthesis, light perception, cell-cell recognition, and cell cycle control. A genome project has begun with compilation of expressed sequence tag data and gene expression studies and will lead to a complete genome sequence. Resources available to the research community include wild-type and mutant strains, plasmid constructs for transformation studies, and a comprehensive on-line database.

Journal Article↗

PubMed2001

Extraction of IgE-binding components of Helicobacter pylori by immunoblotting analysis in chronic urticaria patients.

BACKGROUND: An association between Helicobacter pylori and chronic urticaria has been suspected previously. An IgE-mediated pathway might be a possible link between H. pylori infection and chronic urticaria, and therefore we wanted to prepare an optimal H. pylori antigen to detect H. pylori-specific IgE antibodies in chronic urticaria patients. METHODS: H. pylori antigen extracts were prepared in different ways to find the optimal antigen extract to be used in the assays. Immunoblotting was used to detect IgE-binding bands. The results were applied in an H. pylori RAST assay for specific H. pylori IgE antibodies in patient sera. RESULTS: In immunoblotting, the largest number of IgE-stained bands were visualized in the washing fluids and sonicated extracts, while strong heating and denaturing treatments destroyed the epitopes for IgE binding, suggesting that they belonged to the flagellar structures of H. pylori. However, in H. pylori-specific RAST analysis, specific IgE was found only in 1 of 25 H. pylori-infected patients. CONCLUSIONS: Our findings suggest that although IgE-binding epitopes were found in H. pylori, H. pylori-specific IgE antibodies are not common in chronic urticaria, and the clinical significance of the IgE response is unclear.

Adult↗

PubMed2001

Diverse virulence traits underlying different clinical outcomes of Salmonella infection.

Salmonella strains have evolved to infect a wide variety of reptiles, birds, and mammals resulting in many different syndromes ranging from colonization and chronic carriage to acute fatal disease. Adaptation to a large number of different evolutionary niches has undoubtedly driven the high degree of phenotypic and genotypic diversity in Salmonella strains. Differences in LPS and flagellar structure generate the antigenic variation that is reflected in the more than 2,000 known serotypes. Moreover, variations of LPS structure affect the virulence of the strain. The differential expression of various fimbriae by Salmonella is likely to be due to the wide variety of mucosal surfaces that are encountered by various strains, and the host immune response may select for a different expression pattern. As with these surface structures, a variety of other important virulence determinants show a variable distribution in Salmonella strains and also serve to delineate the divergence of the Salmonella lineage from E. coli. The acquisition of the SPI-1 region may have represented the defining genetic event in the separation of the Salmonella and E. coli lineages. The SPI-1 cell invasion function allowed Salmonella to establish a separate niche in epithelial cells. The mgtC locus on SPI-3 is also present in all lineages and facilitates the adaptation of the bacteria to the low Mg2+, low pH environment of the endosome that results from SPI-1-mediated invasion. Subsequent acquisition of SPI-2 allowed Salmonella to manipulate the sorting of the endosome or phagosome, altering the intracellular environment and facilitating bacterial growth within infected cells. The ability to disseminate from the bowel and establish extraintestinal niches is promoted by the spv locus. Since Salmonella proliferates within macrophages and must avoid phagocytosis by neutrophils to establish a systemic infection, the spv genes appear to promote the macrophage phase of the disease process. Here the polymorphism of the spv locus is clearly demonstrated, since the serovars that cause most cases of nontyphoid bacteremia contain the spv genes. The absence of the spv genes from S. typhi is particularly puzzling and is a strong indication that the pathogenesis of typhoid fever is fundamentally different from that of bacteremia due to nontyphoid Salmonella. There is currently no genetic explanation for the phenotype of host adaptation or for the finding that only a few serovars cause the majority of human infections. Based on recent findings that multiple individual virulence genes have a variable distribution in Salmonella, it is unlikely that a single locus will be found to be responsible for these complex biological traits. Instead, a complicated combination of genes are likely to contribute to the overall virulence phenotype.

Animals↗

PubMed2003

Kinesin II and regulated intraflagellar transport of Chlamydomonas aurora protein kinase.

The assembly and functioning of cilia and flagella depend on a complex system of traffic between the organelles and the cell body. Two types of transport into these organelles have been identified. The best characterized is constitutive: in a process termed intraflagellar transport (IFT), flagellar structural components are continuously carried into cilia and flagella on transport complexes termed IFT particles via the microtubule motor protein kinesin II. Previous studies have shown that the flagella of the unicellular green alga Chlamydomonas exhibit a second type of protein import that is regulated. During fertilization, the Chlamydomonas aurora protein kinase CALK undergoes regulated translocation from the cell body into the flagella. The motor that powers this second, regulated type of movement is unknown. Here, we have examined the cellular properties of the CALK in Chlamydomonas and used a kinesin II mutant to test the idea that the motor protein is essential for regulated translocation of proteins into flagella. We found that the CALK that is transported into flagella of wild-type gametes becomes part of a membrane-associated complex, that kinesin II is essential for the normal localization of this Chlamydomonas aurora protein kinase in unactivated gametes and that the cAMP-induced translocation of the protein kinase into flagella is disrupted in the fla10 mutants. Our results indicate that, in addition to its role in the constitutive transport of IFT particles and their cargo, kinesin II is essential for regulated translocation of proteins into flagella.

Algal Proteins↗

PubMed2004

Tyrosine phosphorylation activates surface chaperones facilitating sperm-zona recognition.

Mammalian spermatozoa undergo a series of molecular and biochemical changes collectively termed capacitation prior to acquiring the ability to fertilise the oocyte. Although phosphorylation of sperm proteins on tyrosine residues has been recognised as an important component of this process, the precise relationship between the phosphorylation status of mammalian spermatozoa and their capacity for fertilisation has remained unclear. In this study we demonstrate a causal relationship between tyrosine phosphorylation in spermatozoa and sperm-zona interaction. The phosphotyrosine expression associated with sperm capacitation localised to internal flagellar structures in permeabilised cells but could also be detected on the exterior surface of the sperm head in live cells. Importantly, almost all spermatozoa bound to the zona pellucida demonstrated this pattern of phosphoprotein localisation, compared to fewer than 15% of the free-swimming population. These data suggest that tyrosine phosphorylation plays a significant role in remodelling the sperm surface, so that these cells are able to recognise the zona pellucida. Phosphoproteome analysis yielded the first evidence of molecular chaperones, endoplasmin (erp99) and heat shock protein 60 (hsp60), as targets for phosphorylation on the surface of mouse spermatozoa, whereas immunofluorescence localised these proteins to the precise region of the sperm head that participates in zona recognition. Based on these results, we propose a novel mechanism for mammalian gamete interaction whereby the activation of sperm-surface chaperones by tyrosine phosphorylation during capacitation may trigger conformational changes facilitating the formation of a functional zona pellucida receptor complex on the surface of mammalian spermatozoa.

Animals↗

PubMed2004

Absence of Dp71 in mdx3cv mouse spermatozoa alters flagellar morphology and the distribution of ion channels and nNOS.

In muscle, the absence of dystrophin alters the dystrophin-associated protein complex (DAPC), which is involved in the clustering and anchoring of signaling proteins and ion and water channels. Here we show that mice spermatozoa express only dystrophin Dp71 and utrophin Up71. The purpose of this study was to explore the effect of the absence of Dp71 on the morphology and membrane distribution of members of the DAPC, ion channels and signaling proteins of spermatozoa obtained from dystrophic mutant mdx3cv mice. Our work indicates that although the absence of Dp71 results in a dramatic decrease in beta-dystroglycan, it induces membrane redistribution and an increase in the total level of alpha-syntrophin, voltage-dependent Na+ (micro1) and K+ (Kv1.1) channels and neural nitric oxide synthase (nNOS). The short utrophin (Up71) was upregulated and redistributed in the spermatozoa of mdx3cv mice. A significant increase in abnormal flagella morphology was observed in the absence of Dp71, which was partially corrected when the plasma membrane was eliminated by detergent treatment. Our observations point to a new phenotype associated with the absence of Dp71. Abnormal flagellar structure and altered distribution of ion channels and signaling proteins may be responsible for the fertility problems of mdx3cv mice.

Animals↗

PubMed2005

The counterbend phenomenon in dynein-disabled rat sperm flagella and what it reveals about the interdoublet elasticity.

Rat sperm that have been rendered passive by disabling the dynein motors with 50 muM sodium metavanadate and 0.1 mM ATP exhibit an interesting response to imposed bending. When the proximal flagellum is bent with a microprobe, the portion of the flagellum distal to the probe contact point develops a bend in the direction opposite the imposed bend. This "counterbend" is not compatible with a simple elastic beam. It can be satisfactorily explained by the sliding tubule model of flagellar structure but only if there are permanent elastic connections between the outer doublets of the axoneme. The elastic component that contributes the bending torque for the counterbend does not reset to a new equilibrium position after an imposed bend but returns the flagellum to a nearly straight or slightly curved final position after release from the probe. This suggests it is based on fixed, rather than mobile, attachments. It is also disrupted by elastase or trypsin digestion, confirming that it is dependent on a protein linkage. Adopting the assumption that the elasticity is attributed to the nexin links that repeat at 96 nm intervals, we find an apparent elasticity for each link that ranges from 1.6 to 10 x 10(-5) N/m. However, the elasticity is nonlinear and does not follow Hooke's law but appears to decrease with increased stretch. In addition, the responsible elastic elements must be able to stretch to more than 10 times their resting length without breakage to account for the observed counterbend formation. Elasticity created by some type of protein unfolding may be the only viable explanation consistent with both the extreme capacity for extension and the nonlinear character of the restoring force that is observed.

Animals↗

PubMed1991

Guidelines for drug treatment of male infertility.

The prerequisite for rational therapy of male fertility disorders is an exact diagnosis. While the possibilities of influencing disturbances of spermiogenesis are limited, male adnexal diseases can be successfully treated in many cases. Drugs for the treatment of fertility disorders must be applied with this in mind, and empiric therapy is often performed in addition to causal treatment which, however, may be quite rationally determined. The therapeutic spectrum in andrology includes antibiotic and antiphlogistic agents, mast cell blockers, zinc, vitamins, and immunosuppressive drugs (corticosteroids). These agents are used for the treatment of inflammatory diseases of the testes and the accessory glands or for suppression of antispermatozoal antibodies. Hormonal disturbances are infrequently encountered by the andrologist, but they can be treated, with proven efficacy, with gonadotrophins, gonadotrophin-releasing hormone (GnRH) or androgens. In certain cases that are not hormonally related, the use of antiestrogens (clomifene, tamoxifen) as stimulating agents may be successful. Furthermore, tissue hormone releasing proteases (kallikrein) can be used both therapeutically (especially in motility disturbances that are not due to structural flagellar defects) and diagnostically (in order to distinguish between inflammatory and noninflammatory testicular damage). Anticholinergics and alpha-sympathomimetics are applied to ameliorate ejaculation or emission failure. In addition to a review of these treatment forms, the development of new concepts, e.g. angiotensin converting enzyme (ACE) inhibitors, is discussed.

Adrenal Cortex Hormones↗

PubMed1979

Ultrastructure of spermiogenesis in men with congenital absence of the vasa deferentia.

Spermiogenesis has been investigated in four cases of agenesia of vasa deferentia. During acrosome formation various anomalies gave rise to late spermatids with deformed heads. Chromatin condensation proceeded normally, but completion of this process appeared to be delayed. Redundant nuclear membranes frequently persisted at the basal region of the nuclei in mature spermatozoa, which occupied niches within Sertoli cells, as the tubules had no lumen. Flagellar structure was normal. These findings support the view that the altered local milieu and variations in acrosome formation may induce the observed anomalies.

Acrosome↗

PubMed2003

Flagellin is the major proinflammatory determinant of enteropathogenic Salmonella.

The gastroenteritis-causing pathogen Salmonella typhimurium induces profound transcriptional changes in intestinal epithelia resulting in the recruitment of neutrophils whose presence is the histopathologic hallmark of salmonellosis. Here we used cDNA microarray expression profiling to define the molecular determinants that mediate such changes in model intestinal epithelia. Enteropathogenic Salmonella induced a classical proinflammatory gene expression program similar to that activated by the canonical proinflammatory agonist TNF-alpha. Nonproinflammatory bacteria, both commensals (Escherichia coli) and systemic pathogens (S. typhi), did not activate this expression profile. While S. typhimurium strains lacking the SPI-1-encoded type III system were fully proinflammatory, strains lacking the genes for the flagellar structural component flagellin were nearly devoid of proinflammatory signaling. Lastly, the epithelial proinflammatory response could be largely recapitulated by basolateral addition of purified flagellin. Thus, S. typhimurium flagellin is the major molecular trigger by which this pathogen activates gut epithelial proinflammatory gene expression.

Cell Line↗

PubMed2000

A multiplex polymerase chain reaction assay for rapid detection and identification of Escherichia coli O157:H7 in foods and bovine feces.

A multiplex polymerase chain reaction (PCR) assay was designed to simplify detection of Escherichia coli O157:H7 and to identify the H serogroup and the type of Shiga toxin produced by this bacterium. Primers for a plasmid-encoded hemolysin gene (hly933), and chromosomal flagella (fliCh7; flagellar structural gene of H7 serogroup), Shiga toxins (stx1, stx2), and attaching and effacing (eaeA) genes were used in a multiplex PCR for coamplification of the corresponding DNA sequences from enterohemorrhagic E. coli (EHEC) O157:H7. Enrichment cultures of ground beef, blue cheese, mussels, alfalfa sprouts, and bovine feces, artificially inoculated with various levels of E. coli O157:H7 strain 933, were subjected to a simple DNA extraction step prior to the PCR, and the resulting amplification products were analyzed by agarose gel electrophoresis. Sensitivity of the assay was < or = 1 CFU/g of food or bovine feces (initial inoculum level), and results could be obtained within 24 h. Similar detection levels were obtained with ground beef samples that underwent enrichment culturing immediately after inoculation and samples that were frozen or refrigerated prior to enrichment. The multiplex PCR facilitates detection of E. coli O157:H7 and can reduce the time required for confirmation of isolates by up to 3 to 4 days.

Animals↗

PubMed1995

Structural effects of mutations in Salmonella typhimurium flagellar switch complex.

Mutations in Salmonella typhimurium fliG, fliM and fliN give rise either to non-flagellate, non-motile or non-chemotactic mutant bacteria. The FliG, FliM and FliN proteins form part of recently characterized extended flagellar basal structures, and have been postulated to form a mutually interacting structural complex. We have examined basal body preparations from non-motile or non-chemotactic fliG, fliM and fliN mutant strains by electron microscopy and immunoblot gel analysis. Most flagellar preparations isolated from the non-motile mutants lacked FliM, but contained FliG. The basal bodies lacked the belled morphology characteristic of the wild-type structures, but had protrusions which could be labelled with anti-FliG. Non-motile mutant preparations severely depleted of FliG but containing FliM were also obtained. These preparations contained extended, belled flagellar structures that were labelled with anti-FliM. Thus, FliM is part of the shell of the extended structures responsible for the belled morphology, while FliG may be part of the inner substructure. The extended basal structures from a FliG temperature-sensitive mutant strain rapidly lost FliM, as well as FliG, upon a shift to a non-permissive temperature, implying interaction between the FliG- and FliM-containing substructures. In dramatic contrast to non-motile mutants, extended basal structures isolated from non-chemotactic mutants were indistinguishable from wild-type structures. This difference may reflect the energetics of the different protein-protein interactions operative during torque generation and the switching of rotation sense.

Antibodies, Bacterial↗