Purification and Characterization of Flagella from Roseburia cecicola, an Obligately Anaerobic Bacterium

Martin, Joel; Savage, Dwayne C.

doi:10.1099/00221287-131-8-2075

Cited by 8 publications

(10 citation statements)

References 14 publications

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“…This perturbation might be either (i) in the higher-order structure of flagellins with identical primary sequences or (ii) a result of two flagellins with different primary sequences (resembling tubulins in the ot,4-heterodimeric structures of microtubuli; 24). This latter interpretation is supported by our finding of two structural genes that encode flagellin subunits with 87% (5,28,30,34,50). Reported exceptions are the filaments of Bdellovibrio bacteriovorus, which contain two subunits (45); those of Caulobacter crescentus, which contain three subunits (38); and those of Halobacterium halobium, which contain five subunits (9).…”

Section: Discussionsupporting

confidence: 70%

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Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti

Pleier

Schmitt

1989

J Bacteriol

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The genomic region that codes for the flagellin subunits of the complex flagellar filaments of Rhizobium meliloti was cloned and sequenced. Two structural genes, flaA and flaB, that encode 395-and 396-amino-acid polypeptides, respectively, were identified. These exhibit 87% sequence identity. The amino acid sequences of tryptic peptides suggest that both of these subunit proteins are represented in the flagellar fiatnents. The N-terminal methionine was absent from the mature flagellin subunits. Their derived primary structures show almost no relationship to flagellins from Escherichia coli, Salmonella typhimurium, or Bacillus subtilis but exhibit up to 60% similarity to the N-and C-terminal portions of flagellin from Caulobacter crescentus. It is suggested that the complex flagellar filaments of R. meliloti are unique in being assembled from heterodimers of two related flagellin subunits. The tandemly arranged flagellin genes were shown to be transcribed separately from unusual promoter sequences.Bacterial flagella consist of a helical filament, a proximal hook, and a basal body with the flagellar motor (31). The long helical filaments are polymers assembled from flagellin subunits, whose molecular weights range between 20,000 and 65,000, depending on the bacterial species (5,9,23,25,27,34,35,38).Two types of flagellar filaments, named plain and complex, have been distinguished by their electron microscopically determined surface structures (42). Plain filaments have a smooth surface structure with faint helical lines, whereas complex filaments exhibit a conspicuous helical pattern of alternating ridges and grooves (13,41,42,46). These helical undulations are considered to be responsible for the brittle and (by implication) rigid structure (46) that enables them to propel bacteria efficiently in viscous media (13). This may reflect environmental selection, since complex flagella have been observed in only three species of soil bacteria: Pseudomonas rhodos (42), Rhizobium lupini H13-3 (35, 41), and R. meliloti (13,27). Plain filaments have been observed in most well-studied swimming bacteria, such as Escherichia coli, Salmonella typhimurium, and Bacillus subtilis. Whereas flagella with plain filaments can alternate between clockwise and counterclockwise rotation (31, 43), all known flagella with complex filaments rotate only clockwise with intermittent stops (14).Measurements of mass per unit length and three-dimensional reconstruction from electron micrographs suggested that the complex filaments of R. lupini are composed of functional dimers, whereas the plain filaments of S. typhimurium consist of functional monomers (46,47). This is consistent with the present finding in R. meliloti of two flagellin genes, named flaA and flaB, that encode closely related proteins. We propose that the complex filaments are composed of heterodimeric subunits that require stoichiometric amounts of the flaA and flaB gene products. Curiously, the two tandemly arranged genes were shown to be transcribed individually from unusual pr...

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Section: Discussionsupporting

confidence: 70%

“…The long helical filaments are polymers assembled from flagellin subunits, whose molecular weights range between 20,000 and 65,000, depending on the bacterial species (5,9,23,25,27,34,35,38).…”

mentioning

confidence: 99%

Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti

Pleier

Schmitt

1989

J Bacteriol

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“…The flagella that we observed had a diameter of 16 nm, the diameter of typical flagella of eubacteria. In addition, the molecular masses of flagellins of Roseburia cecicola (18), Bacillus firmus (8), Rhizobium meliloti (14), and some species of genus Pseudomonas (28) range from 38 to 43 kDa. On the other hand, the molecular masses of pilins of Escherichia coli (12), Pseudomonas cepacia (21), Klebsiella (19) ranged from 16 to 21.5 kDa.…”

Section: Discussionmentioning

confidence: 99%

Sulfur-binding protein of flagella of Thiobacillus ferrooxidans

et al. 1996

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The sulfur-binding protein of Thiobacillus ferrooxidans ATCC 23270 was investigated. The protein composition of the bacterium's cell surface changed according to the culture substrate. Sulfur-grown cells showed greater adhesion to sulfur than iron-grown cells. The sulfur-grown cells synthesized a 40-kDa surface protein which was not synthesized by iron-grown cells. The 40-kDa protein had thiol groups and strongly adhered to elemental sulfur powder. This adhesion was not disturbed by Triton X-100, which can quench hydrophobic interactions. However, adhesion was disturbed by 2-mercaptoethanol, which broke the disulfide bond. The thiol groups of the 40-kDa protein formed a disulfide bond with elemental sulfur and mediated the strong adhesion between T. ferrooxidans cells and elemental sulfur. The 40-kDa protein was located on the flagella. The location of the protein would make it possible for cells to be in closer contact with the surface of elemental sulfur powder.

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“…Flagellin was purified as described previously (15 Cloning. After fragments of interest from each restriction enzyme digest were identified by Southern hybridization, another restriction digest and electrophoresis were performed under similar conditions.…”

Section: Methodsmentioning

confidence: 99%

Cloning, nucleotide sequence, and taxonomic implications of the flagellin gene of Roseburia cecicola

Martin

Savage

1988

J Bacteriol

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The gene coding for the flageliw protein of Roseburia cecicola, an oxygen-intolerant, gram-negative, anaerobic bacterium indigenous to the murine cecum, has been cloned and sequenced. NH2-terminal amino acid sequence data from the flagellin protein were used as a basis for the synthesis of two mixed-sequence deoxyoligonucleotides. The oligonucleotides were used to identify and clone the flagellin structural gene. DNA sequence analysis of M13mp8 and mp9 subclones revealed a protein with a length of 293 amino acids and a molecular weight of 31,370. Comparisons with the sequences of flagellins of other species revealed conserved regions and suggested that although R. cecicola has structural characteristics of a gram-negative bacterium, it may be most closely related to the gram-positive bacteria.Motility and chemotaxis may be ecologically important for many species of microorganisms colonizing specific habitats on mammalian cell surfaces (2, 19). In the oxygen-free environment of the murine gastrointestinal tract, especially in the cecum and colon, many species of bacteria are motile (6,17,23). Motility for some of these organisms is a propitious and possibly essential trait (25).One such bacterium is Roseburia cecicola, a motile, anaerobic, gram-negative rod in the Bacteroidaceae family that was originally isolated from cecal scrapings from a conventional laboratory mouse (24). This. organism has an unusual flagellar arrangement composed of a fascicle of 20 to 35 flagella anchored in the polar to subpolar region at one end of the cell (24). Even though motility is a metabolically expensive trait, it is essential for this strict anaerobe to be able to colonize the cecum and colon in animals with a competing indigenous microflora (25). We are using R. cecicola as a model organism to investigate the influence of motility and chemotaxis on the ability of motile bacteria to colonize the gastrointestinal tract.Despite considerable information on the flagellins of facultative anaerobes, little is known about these proteins in the many motile anaerobic species colonizing habitats in gastrointestinal ecosystems. In this report we describe the use of families of synthetic oligonucleotides to clone the R. cecicola flagellin structural gene.

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Purification and Characterization of Flagella from Roseburia cecicola, an Obligately Anaerobic Bacterium

Cited by 8 publications

References 14 publications

Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti

Identification and sequence analysis of two related flagellin genes in Rhizobium meliloti

Sulfur-binding protein of flagella of Thiobacillus ferrooxidans

Cloning, nucleotide sequence, and taxonomic implications of the flagellin gene of Roseburia cecicola

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