Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium

Williams, A W; Yamaguchi, Shigeru; Togashi, F; Aizawa, Shin‐Ichi; Kawagishi, Ikuro; Macnab, Robert M.

doi:10.1128/jb.178.10.2960-2970.1996

Cited by 175 publications

(302 citation statements)

References 27 publications

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“…However, the placements of spa32, invJ, fliK, and yscP in the respective operons were all similar, and each of the gene products was secreted (6,32,36,40,42; this study). Furthermore, the production of each protein was required for a functional type III secretion system (Spa32, InvJ, and YscP) (7, 36, 42; this study) or flagellar export system (FliK) (20,33,48). Consequently, we wondered whether Spa32 and its putative cognates would share similar functions.…”

Section: Resultsmentioning

confidence: 99%

Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length

et al. 2002

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The Shigella type III secretion machinery is responsible for delivering to host cells the set of effectors required for invasion. The type III secretion complex comprises a needle composed of MxiH and MxiI and a basal body made up of MxiD, MxiG, and MxiJ. In S. flexneri, the needle length has a narrow range, with a mean of approximately 45 nm, suggesting that it is strictly regulated. Here we show that Spa32, encoded by one of the spa genes, is an essential protein translocated via the type III secretion system and is involved in the control of needle length as well as type III secretion activity. When the spa32 gene was mutated, the type III secretion complexes possessed needles of various lengths, ranging from 40 to 1,150 nm. Upon introduction of a cloned spa32 into the spa32 mutant, the bacteria produced needles of wild-type length. The spa32 mutant overexpressing MxiH produced extremely long (>5 m) needles. Spa32 was secreted into the medium via the type III secretion system, but secretion did not depend on activation of the system. The spa32 mutant and the mutant overexpressing MxiH did not secrete effectors such as Ipa proteins into the medium or invade HeLa cells. Upon introduction of Salmonella invJ, encoding InvJ, which has 15.4% amino acid identity with Spa32, into the spa32 mutant, the bacteria produced type III needles of wild-type length and efficiently entered HeLa cells. These findings suggest that Spa32 is an essential secreted protein for a functional type III secretion system in Shigella spp. and is involved in the control of needle length. Furthermore, its function is interchangeable with that of Salmonella InvJ.The type III protein secretion system is found among various gram-negative animal-and plant-pathogenic bacteria, in which the subsets of effector proteins that are secreted via the system into host cells have crucial roles in the bacterial infection process (8,9,16,38). Genetic and functional studies indicate that the type III secretion system requires proteins encoded by more than 20 genes, which contain the structural components of the secretion complex, secreted proteins, chaperones, and regulators (10,11,19,37,43). In Shigella spp., the system is encoded by approximately 20 genes located in the mxi and spa operons on a large 230-kb plasmid (5, 19). The proteins of the type III secretion system show considerable amino acid homology among different pathogens, and some of them also show amino acid homology with the components of the bacterial flagellar export system (26). Although the function of the type III secretion system in bacterial infection is distinct from that of the flagellar export system, the type III secretion system is believed to have evolved from the flagellar export system and diverged in each pathogen to become involved in the infection process (15).Recent studies indicate that the type III secretion complexes of Shigella flexneri and Salmonella spp. share structural similarities; the complexes are composed of two distinct parts, a basal body and a needle. The basal...

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Section: Resultsmentioning

confidence: 99%

Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length

et al. 2002

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“…Orthologues of HP1575 occur in several other bacteria and are referred to as FlhX proteins (Pallen et al, 2005; Wand et al, 2006). Wand and co-workers reported that FlhX can functionally substitute for FlhB CC in H. pylori (Wand et al, 2006), suggesting that in the absence of FlhB CC , FlhX can associate with the N-terminal subdomain of FlhB C (FlhB CN ).As observed in S. typhimurium, disruption of H. pylori fliK (hp0906) results in reduced motility and formation of polyhook structures due to a delay in the switch between export states (Ryan et al, 2005;Williams et al, 1996). Inactivation of fliK in H. pylori and C. jejuni stimulates expression of RpoN-dependent flagellar genes (Kamal et al, 2007;Ryan et al, 2005).…”

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confidence: 95%

Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression

2009

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Regulation of the Helicobacter pylori flagellar gene cascade involves the transcription factors s 54 (RpoN), employed for expression of genes required midway through flagellar assembly, and s 28 (FliA), required for expression of late genes. Previous studies revealed that mutations in genes encoding components of the flagellar protein export apparatus block expression of the H. pylori RpoN and FliA regulons. FlhB is a membrane-bound component of the export apparatus that possesses a large cytoplasmic domain (FlhB C ). The hook length control protein FliK interacts with FlhB C to modulate the substrate specificity of the export apparatus. FlhB C undergoes autocleavage as part of the switch in substrate specificity. Consistent with previous reports, deletion of flhB in H. pylori interfered with expression of RpoN-dependent reporter genes, while deletion of fliK stimulated expression of these reporter genes. In the DflhB mutant, disrupting fliK did not restore expression of RpoN-dependent reporter genes, suggesting that the inhibitory effect of the DflhB mutation is not due to the inability to export FliK. Amino acid substitutions (N265A and P266G) at the putative autocleavage site of H. pylori FlhB prevented processing of FlhB and export of filament-type substrates. The FlhB variants supported wild-type expression of RpoN-and FliA-dependent reporter genes. In the strain producing FlhB N265A , expression of RpoN-and FliA-dependent reporter genes was inhibited when fliK was disrupted. In contrast, expression of these reporter genes was unaffected or slightly stimulated when fliK was disrupted in the strain producing FlhB P266G. H. pylori HP1575 (FlhX) shares homology with the C-terminal portion of FlhB C (FlhB CC ) and can substitute for FlhB CC in flagellar assembly. Disrupting flhX inhibited expression of a flaB reporter gene in the wild-type but not in the DfliK mutant or strains producing FlhB variants, suggesting a role for FlhX or FlhB CC in normal expression of the RpoN regulon. Taken together, these data indicate that the mechanism by which the flagellar protein export apparatus exerts control over the H. pylori RpoN regulon is complex and involves more than simply switching substrate specificity of the flagellar protein export apparatus. INTRODUCTIONBacterial flagellar biosynthesis involves transcriptional hierarchies that coordinate flagellar gene expression with assembly. Flagellar gene hierarchies are well characterized in a few bacteria, including Salmonella enterica serovar Typhimurium (S. typhimurium) and Caulobacter crescentus (Chilcott & Hughes, 2000;Wu & Newton, 1997). Flagellar gene regulation in e-Proteobacteria, such as Helicobacter pylori and Campylobacter jejuni, shares some similarities with these established paradigms but also differs significantly. As in S. typhimurium, H. pylori flagellar genes required late in assembly, such as the major flagellin (FlaA) and filament cap protein, are transcribed by s 28 (FliA)-RNA polymerase holoenzyme (Kim et al., 1999;Leying et al., 1992), and, as in C....

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“…These results are in line with earlier findings showing that the 33 last amino acids of FlhB are redundant for hook-basal body assembly. Interestingly this domain of FlhB has an inhibitory effect on the substrate specificity switch suggesting that the C-terminal region of FlhB is involved in the temporal regulation of the flagellum assembly (33,34). In fact it cannot be excluded that YscU via its C-terminal secretion signal possesses a negative activity similarly to FlhB, that affects the switch from secretion of early to late substrates.…”

Section: Discussionmentioning

confidence: 98%

“…Several studies have demonstrated the involvement of the YscU/FlhB family of protein in the substrate specificity switch (11,13,14,34). YscU is anchored in the inner membrane via four transmembrane segments.…”

Section: Discussionmentioning

confidence: 99%

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YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal

Wolf‐Watz

2015

Journal of Biological Chemistry

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Background: After auto-proteolysis and dissociation, YscU CC is secreted by the T3SS of Yersinia pseudotuberculosis. Results: YscU CC harbors a specific C-terminal T3S signal and its deletion triggers an increase of YscF secretion without affecting Yops secretion. Conclusion: C-terminal end of YscU participate in YscF secretion regulation but not in the substrate specificity switch. Significance: This is the first report of a C-terminal T3S signal.

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Mutations in fliK and flhB affecting flagellar hook and filament assembly in Salmonella typhimurium

Cited by 175 publications

References 27 publications

Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length

Shigella Spa32 Is an Essential Secretory Protein for Functional Type III Secretion Machinery and Uniformity of Its Needle Length

Helicobacter pylori FlhB processing-deficient variants affect flagellar assembly but not flagellar gene expression

YscU/FlhB of Yersinia pseudotuberculosis Harbors a C-terminal Type III Secretion Signal

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