Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG

Roman, S J; Frantz, Betsy; Matsumura, Philip

doi:10.1016/0378-1119(93)90232-r

Cited by 20 publications

(18 citation statements)

References 19 publications

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“…E. coli CheY and FliG proteins were purified as previously described (19,23). S. typhimurium FliG and FliM proteins were purified following protocols detailed in references 39 and 21, respectively.…”

Section: Methodsmentioning

confidence: 99%

“…Antibodies to S. typhimurium FlgE and FliM proteins (39) and to E. coli FliG (23) and CheY proteins were used in this study. The FlgE and FliM antisera were initially purified by preadsorption against the vesicular high-speed fraction (39).…”

Section: Methodsmentioning

confidence: 99%

“…The FlgE and FliM antisera were initially purified by preadsorption against the vesicular high-speed fraction (39). The FliG and CheY antisera were affinity purified against the purified proteins immobilized on Sepharose columns (23).…”

Section: Methodsmentioning

confidence: 99%

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FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies

et al. 1996

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Salmonella typhimurium FliG and FliM are two of three proteins known to be necessary for flagellar morphogenesis as well as energization and switching of flagellar rotation. We have determined FliG and FliM levels in cellular fractions and in extended flagellar basal bodies, using antibodies raised against the purified proteins. Both proteins were found predominantly in the detergent-solubilized particulate fraction containing flagellar structures. Basal flagellar fragments could be separated from partially constructed basal bodies by gel filtration chromatography. FliG and FliM were present in an approximately equimolar ratio in all gel-filtered fractions. FliG and FliM copy numbers, estimated relative to that of the hook protein from the early fractions containing long, basal, flagellar fragments, were (means ؎ standard errors) 41 ؎ 10 and 37 ؎ 13 per flagellum, respectively. Extended structures were present in the earliest identifiable basal bodies. Immunoelectron microscopy and immunoblot gel analysis suggested that the FliG and, to a less certain degree, the FliM contents of these structures were the same as those for the complete basal bodies. These facts are consistent with the postulate that FliG and FliM affect flagellar morphogenesis as part of the extended basal structure, formation of which is necessary for assembly of more-distal components of the flagellum. The determined stoichiometries will provide important constraints to modelling energization and switching of flagellar rotation.The propulsion of swimming bacteria is driven by the rotation of motile organelles, the flagella. Each flagellum consists of an external, rigid, helical filament contiguous with an elaborate basal body. The flagellar basal body traverses the cell wall and cytoplasmic membrane, protruding into the cytoplasm. It harbors machinery for energization and switching of flagellar rotation. The molecular motors that drive flagellar rotation are distinct from other biological molecular motors studied thus far in that they are capable of bidirectional force generation and are energized by transmembrane ion gradients instead of ATP (26).Proteins specifically involved in motility have been identified on the basis of the analysis of paralyzed mutants (mot) in the enteric bacteria Escherichia coli and Salmonella typhimurium. Five proteins have thus been identified from a total of 50 or so proteins required for flagellation and chemotaxis (18). Two, MotA and MotB, are integral membrane proteins whose expression determines the assembly of the intramembrane particle ring structures of the flagellar basal body (13). The other three, FliG, FliM, and FliN, have been localized to recently described cytoplasmic extensions of the flagellar basal body by immunoelectron microscopy (7, 39). Mutations that give rise to nonflagellate (fla) or nonchemotactic (che) bacteria have also been isolated in each of these proteins (37). To account for these and additional suppressor mutation data, it has been postulated that these proteins form a macromolecul...

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Section: Methodsmentioning

confidence: 99%

Section: Methodsmentioning

confidence: 99%

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FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies

et al. 1996

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“…Our sequenced clone was derived from the original sequenced clone, and upon side-by-side comparison on sequencing gels, both had the same differences from the reported sequence. Comparison of the translated E. coli (16) and Salmonella (11) fliG sequences revealed that the two predicted proteins were virtually identical except for a region of 12 amino acids in the middle. Our DNA sequence when translated was in closer agreement with the predicted Salmonella protein.…”

Section: Methodsmentioning

confidence: 99%

A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli

Marykwas

Berg

1996

J Bacteriol

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An Escherichia coli bacterium swims by a series of runs and tumbles that allows it to locate attractants and avoid repellents, a behavioral phenomenon called chemotaxis. Runs are episodes of steady forward movement that occur when flagellar rotary motors rotate counterclockwise (CCW), causing multiple flagellar filaments to coalesce into a coherent helical bundle that generates thrust. Tumbles are episodes of little net displacement that occur when the motors rotate clockwise (CW), causing the flagellar filaments to separate and move independently. Runs are resumed, but in a new direction chosen at random, when the motors rotate CCW once again. Regulation of motor activity at the level of CW-CCW switching allows the cell to prolong runs when conditions improve and thus to drift towards favorable environments (1).

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“…Loci of fli genes were then confirmed by complementation with E. coli fli mutants after being subcloned. Some loci (fliZY, fliST, fliG, and fliO) were determined only by comparing restriction sites with the sequence data (25,34,40,45) because corresponding E. coli mutants were not available for complementation tests.…”

Section: Methodsmentioning

confidence: 99%

Cloning and characterization of the region III flagellar operons of the four Shigella subgroups: genetic defects that cause loss of flagella of Shigella boydii and Shigella sonnei

1997

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To detect genetic defects that might have caused loss of flagella in Shigella boydii and Shigella sonnei, the region III flagellar (fli) operons were cloned from certain strains and analyzed with reference to the restriction maps and genetic maps of Escherichia coli fli operons. S. boydii NCTC9733 (strain C5 in this paper) had the 988-bp internal deletion in the fliF gene that encodes a large substructural protein of the basal body. Two strains (C1 and C8) had deletions of the entire fliF operon, and the remaining three (C3, C4, and C9) differed in the size of the restriction fragments carrying the fliF and fliL operons. Loss of flagella in S. boydii appears to originate in some defect in the fliF operon. S. sonnei IID969 lacked the fliD gene and, in place of it, carried two IS600 elements as inverted repeats. Genes downstream from fliD were not detected in the cloned fragment despite its large size but did appear elsewhere in the chromosome. The fliD gene encodes a cap protein of the flagellar filament, and its deletion results in overexpression of class 3 operons by the increased amount of FliA ( F ) caused by the excess export of the anti-sigma factor FlgM. Three other strains also had the fliD deletion, and two of them had another deletion in the fliF-fliG-fliH region. The fliD deletion might be the primary cause of loss of flagella in S. sonnei. The lack of FliF or FliD in each subgroup is discussed in connection with the maintenance of virulence and bacterial growth. We also discuss the process of loss of flagella in relation to transposition of IS elements and alterations of the noncoding region, which were found to be common to at least three subgroups.The genus Shigella is divided into four subgroups or species: Shigella dysenteriae, Shigella flexneri, Shigella boydii, and Shigella sonnei (46). All are pathogens which cause diarrhea and dysentery in humans and certain other primates (37). Shigellae have been regarded as nonmotile organisms lacking flagella (15), and this characteristic has been used as one of the taxonomic and diagnostic criteria to distinguish shigellae from other enteric bacteria. However, it has been reported that some prototypical strains of the four subgroups and two fresh clinical isolates of S. sonnei are sparsely flagellate (19). We have not yet detected any motile strain among the four subgroups in the more than 20 laboratory strains we have examined so far; instead we have detected defective flagellar genes in some strains of these subgroups (4, 56). Therefore, we have been studying loss of motility by shigellae from the point of view that almost all shigellae are nonmotile because of a lack of flagellation.Approximately 50 genes, composing at least 13 operons, are involved in the biogenesis and functioning of a flagellum of Escherichia coli and Salmonella typhimurium (33). These operons are clustered into three different regions on the chromosome. Region I operons mainly contain flagellar structural genes designated flg; region II contains a mixture of flagellar (flh) and mot...

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Gene sequence, overproduction, purification and determination of the wild-type level of the Escherichia coli flagellar switch protein FliG

Cited by 20 publications

References 19 publications

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies

FliG and FliM distribution in the Salmonella typhimurium cell and flagellar basal bodies

A mutational analysis of the interaction between FliG and FliM, two components of the flagellar motor of Escherichia coli

Cloning and characterization of the region III flagellar operons of the four Shigella subgroups: genetic defects that cause loss of flagella of Shigella boydii and Shigella sonnei

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