New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy

Khan, Shahid; Khan, Imran; Reese, T. R.

doi:10.1128/jb.173.9.2888-2896.1991

Cited by 63 publications

(41 citation statements)

References 66 publications

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“…These results suggest that the S. flexneri needle and Salmonella hook are both still controlled even in the absence of Spa32 and FliK, respectively. Recent studies have led to the hypothesis that the C-ring compartment formed in the cytoplasm beneath the MS ring is involved in determining the length of the hook on the flagellar basal body (13,21,27,30). The C-ring is predicted to function as a compartment for the storage of a fixed amount of the flagellar hook component FlgE proteins, although there is no direct evidence yet that FlgE fills the C-ring, and thus the mechanism of length control of the hook is still largely speculative (30).…”

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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“…Recently, gentler isolation procedures have allowed purification of flagella that contain large, cytoplasmically located structures not observed previously (9,16,18). These ring- (9) or bell-shaped (16,18) structures extend approximately 17 nm into the cytoplasm and have a maximum diameter of about 45 nm (9).…”

Section: Discussionmentioning

confidence: 99%

“…These ring- (9) or bell-shaped (16,18) structures extend approximately 17 nm into the cytoplasm and have a maximum diameter of about 45 nm (9). This structure probably contains the FliM and FliN proteins, because basal bodies that have it can be decorated with antibodies against FliM or FliN whereas those that lack it cannot (9).…”

Section: Discussionmentioning

confidence: 99%

Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN

et al. 1996

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Among the many proteins needed for assembly and function of bacterial flagella, FliG, FliM, and FliN have attracted special attention because mutant phenotypes suggest that they are needed not only for flagellar assembly but also for torque generation and for controlling the direction of motor rotation. A role for these proteins in torque generation is suggested by the existence of mutations in each of them that produce the Mot ؊ (or paralyzed) phenotype, in which flagella are assembled and appear normal but do not rotate. The presumption is that Mot ؊ defects cause paralysis by specifically disrupting functions essential for torque generation, while preserving the features of a protein needed for flagellar assembly. Here, we present evidence that the reported mot mutations in fliM and fliN do not disrupt torque-generating functions specifically but, instead, affect the incorporation of proteins into the flagellum. The fliM and fliN mutants are immotile at normal expression levels but become motile when the mutant proteins and/or other, evidently interacting flagellar proteins are overexpressed. In contrast, many of the reported fliG mot mutations abolish motility at all expression levels, while permitting flagellar assembly, and thus appear to disrupt torque generation specifically. These mutations are clustered in a segment of about 100 residues at the carboxyl terminus of FliG. A slightly larger carboxyl-terminal segment of 126 residues accumulates in the cells when expressed alone and thus probably constitutes a stable, independently folded domain. We suggest that the carboxyl-terminal domain of FliG functions specifically in torque generation, forming the rotor portion of the site of energy transduction in the flagellar motor.Many motile bacteria are propelled by thin helical filaments that are each driven at the base by a rotary motor embedded in the cell membrane (reviewed in references 1, 20, 25). The filament/motor organelle is called a flagellum. In contrast to eukaryotic molecular motors, which use nucleoside triphosphates as an energy source, bacterial rotary motors are powered by the transmembrane gradient of protons (19,21) or, in some species, sodium ions (14). The mechanism by which bacterial flagellar motors convert the chemical energy of the ion gradient into the mechanical energy of rotation is not yet understood.Approximately 50 genes are needed for the assembly and operation of the flagella of Escherichia coli or Salmonella typhimurium (20). Among these, only a few encode products thought to be directly involved in the process of torque generation. Genes whose products might participate in torque generation have been identified in extensive mutant screens as those giving the Mot Ϫ phenotype, in which flagella are assembled but do not rotate. The presumption has been that mot mutations affect torque-generating activities specifically, while preserving the features of a protein needed for flagellar assembly. By using this criterion, five proteins that could have direct roles in torque generati...

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“…The rapid-freeze method described previously was applied to mycoplasma with slight modifications (11,12). M. mobile strain 163K (ATCC 43663) and its mutants (23) were grown as described previously (28).…”

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confidence: 99%

Spike Structure at the Interface between GlidingMycoplasma mobileCells and Glass Surfaces Visualized by Rapid-Freeze-and-Fracture Electron Microscopy

Miyata

Petersen

2004

J Bacteriol

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Mycoplasma mobile is a flask-shaped bacteria that binds to a substrate and glides towards its tapered end, the so-called "head-like protrusion," by an unknown mechanism. To search for cellular structures underlying this motility, the cell-substrate interface of actively gliding cells was visualized by rapid-freeze-and-freezefracture rotary-shadow electron microscopy. Novel structures, called "spikes," were observed to protrude from the cell membrane and attach to the glass surface at their distal end. The spikes were on average 50 nm in length and 4 nm in diameter, most abundant around the head, and not observed in a nonbinding mutant. The spikes may be involved in the mechanism of binding, gliding, or both.Mycoplasma gliding. Mycoplasmas are bacteria that lack a cell wall and are parasitic or commensal to many kinds of hosts, including humans, animals, and plants (25). Several mycoplasma species have a membrane protrusion at one end, called the "head-like protrusion," which creates the organism's characteristic flask-shaped cell morphology. These bacteria are capable of gliding motility, enabling them to translocate smoothly on solid surfaces, always in the direction of the headlike protrusion (3, 13). Although gliding motility of mycoplasmas is believed to be involved in pathogenicity, the mechanism of gliding motility has not been well investigated. Mycoplasmas do not have any homologs of genes that encode pili, flagella, genes related to other bacterial motility, or motor proteins that are common in eukaryotic motility, such as myosins (5,7,9,19). These facts suggest that mycoplasmas glide by an entirely unknown mechanism, but this mechanism has been difficult to isolate because many species of mycoplasma travel at low speed and with interrupted motility.The species Mycoplasma mobile provides an opportunity to study mycoplasma gliding motility, because this species is the fastest gliding and, unlike other species, glides without interruption (13-15, 20, 21, 27). At all stages of growth, M. mobile glides smoothly and continuously on glass at an average speed of 2.0 to 4.5 m/s, or about 3 to 7 times the length of the cell per s (27), exerting a force of up to 27 piconewtons (pN) (20,21). These distinct characteristics enabled detailed analyses of gliding (6,(20)(21)(22)(26)(27)(28) and isolation of gliding mutants that were characterized by reduced or deficient gliding or enhanced speed (23,28).

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New structural features of the flagellar base in Salmonella typhimurium revealed by rapid-freeze electron microscopy

Cited by 63 publications

References 66 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

Torque generation in the flagellar motor of Escherichia coli: evidence of a direct role for FliG but not for FliM or FliN

Spike Structure at the Interface between GlidingMycoplasma mobileCells and Glass Surfaces Visualized by Rapid-Freeze-and-Fracture Electron Microscopy

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