Purification and characterization of the flagellar hook-basal body complex of Salmonella typhimurium

The Yersinia Ysc apparatus is made of more than 20 proteins, 11 of which have homologues in many type III systems. Here, we characterize YscP from Yersinia enterocolitica. This 515‐residue protein has a high proline content, a large tandem repetition and a slow migration in SDS–PAGE. Unlike the products of neighbouring genes, it has a counterpart only in Pseudomonas aeruginosa and it varies even between Yersinia Ysc machineries. An yscPΔ97−465 mutant was unable to secrete any Yop, even under conditions overcoming feedback inhibition of Yop synthesis. Interestingly, a cloned yscPΔ57−324 from Yersinia pestis introduced in the yscPΔ97−465 mutant can sustain a significant Yop secretion and thus partially complemented the mutation. This explains the leaky phenotype observed with the yscP mutant of Y. pestis. In accordance with this secretion deficiency, YscP is required for the delivery of Yop effectors into macrophages. Mechanical shearing, immunolabelling and electron microscopy experiments showed that YscP is exposed at the bacterial surface when bacteria are incubated at 37°C in the presence of Ca2+ and thus do not secrete Yops. At 37°C, when Ca2+ ions are chelated, YscP is released like a Yop protein. We conclude that YscP is a part of the Ysc injectisome which is localized at the bacterial surface and is destabilized by Ca2+ chelation.

Section: Shearing Of Bacteriamentioning

confidence: 99%

YscP, a Yersinia protein required for Yop secretion that is surface exposed, and released in low Ca²⁺

Stainier

Bleves

Josenhans

et al. 2000

“…Intact flagella of B. subtilis were purified by modifying the original protocol for S. typhimurium (Aizawa et al, 1985). Major modifications are as follows: (i) EDTA was not added when making sphaeroplasts, because lysozyme freely penetrates the cell wall of B. subtilis.…”

Section: Purification Of the Hbb Complexmentioning

confidence: 99%

“…The HBB complex of B. subtilis was purified by a modified version of the original protocol used for S. typhimurium (Aizawa et al, 1985). Modified procedures only are described, in contrast with the original protocol.…”

Section: Purification Of the Hbb Complexmentioning

confidence: 99%

“…Biochemical analysis of the structure of the basal body had not been accomplished until a new protocol for purifying the hook-basal body (HBB) complex from Salmonella typhimurium was developed in 1985 (Aizawa et al, 1985). The analysis of the basal-body structure of S. typhimurium was almost finished by 1991 when the S ring, one of the inner rings and a long-time-elusive component, were identified as a part of a multidomain complex (now called the MS ring complex) consisting of a single protein, FliF (Ueno et al, 1992;.…”

Section: Introductionmentioning

confidence: 99%

See 1 more Smart Citation

Purification and characterization of the flagellar hook–basal body complex of Bacillus subtilis

Kubori

Okumura

Kobayashi

et al. 1997

SummaryThe flagellar hook-basal body (HBB) complex of the Gram-positive bacterium Bacillus subtilis was purified and analysed by electron microscopy, gel electrophoresis, and amino acid sequencing of the major component proteins. The purified HBB complex consisted of the inner (M and S) rings, a rod and a hook. There were no outer (P and L) rings that are found in Gram-negative bacteria. The hook was 15 nm in thickness and 70 nm in length, which is thinner and longer than the hook of Salmonella typhimurium. The hook protein had an apparent molecular mass of 29 kDa, and its N-terminal sequence was identical to that of B. subtilis FlgG

“…The hook-basal body complexes were isolated from SJW3122 and purified according to Aizawa et al (1985). Osmotically shocked cells of SJW3122 were prepared according to Kubori et al (1992). Measurements of the cellular level of flagellin and the amounts of cellular and secreted FlgM SJW3122 cells were grown in 20 ml of Luria-Bertani (LB) medium containing various concentrations of NaCl to late log phase.…”

Section: Purification Of Hook-basal Body Complexes From Sjw3122mentioning

confidence: 99%

Flagellar filament elongation can be impaired by mutations in the hook protein FlgE of Salmonella typhimurium: a possible role of the hook as a passage for the anti‐sigma factor FlgM

Saitō

Ueno

Kubori

et al. 1998

SummaryAmong motile revertants isolated from flagellar hookdeficient (flgE ) mutants of Salmonella typhimurium, one produced only short flagellar filaments in L broth, despite the fact that flagellin itself has the ability to polymerize into long filaments in vitro. This pseudorevertant has an intragenic suppressor, resulting in a two-amino-acid substitution (Asp-Gln→Ala-Arg) in the C-terminal region of the hook protein, FlgE. The flagellation of the pseudorevertant was greatly affected by the concentration of NaCl in the culture media: we observed no filaments in the absence of NaCl, short filaments in 1% NaCl and full-length filaments in 2% NaCl. Electron microscopy of osmotically shocked cells showed that the number of hook-basal bodies on cells was constant under various NaCl conditions. Furthermore, we found that the mutant hook was straight rather than curved. We monitored the cellular flagellin level of this pseudorevertant under various NaCl concentrations by immunoblotting. It was revealed that little flagellin was present under NaCl-free conditions in contrast with the ordinary amounts of flagellin present in 2% NaCl. As the expression of flagellin is regulated by competitive interaction of a sigma factor, FliA, and a corresponding anti-sigma factor, FlgM, we also observed the effect of NaCl on the secretion of FlgM. FlgM was secreted into the media in more than 1% NaCl but accumulated inside the cells in the absence of NaCl, indicating that the failure of secretion of FlgM in the absence of salt was the cause of the impaired elongation of filaments.