vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (Tgt) of Escherichia coli K-12

Durand, Jérôme M. B.; Okada, Nobuhiko; Tobe, Toru; Watarai, Masahisa; Fukuda, I.; Suzuki, Toshihiko; Nakata, Noboru; Komatsu, Keiko; Yoshikawa, Masanosuke; Sasakawa, Chihiro

doi:10.1128/jb.176.15.4627-4634.1994

Cited by 131 publications

(126 citation statements)

References 43 publications

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“…Wild-type S. flexneri 2a YSH6000 (Sasakawa et al, 1986) was used together with the virulent strain 2457T (Formal et al, 1971). Strain N1436, the congenic tgt : : Tn5 derivative of YSH6000, has been described (Durand et al, 1994;Okada et al, 1991). Plasmid pHW848 (virF9-9lacZ) was provided by H. Watanabe (Nakayama & Watanabe, 1995).…”

Section: Methodsmentioning

confidence: 99%

Metabolic control through ornithine and uracil of epithelial cell invasion by Shigella flexneri

Durand

Björk

2009

Microbiology

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This paper shows that compounds in defined growth media strongly influence the expression of the effectors of virulence in the human invasive pathogen Shigella flexneri. Ornithine in conjunction with uracil reduces the haemolytic ability of wild-type cultures more than 20-fold and the expression of the type III secretion system more than 8-fold, as monitored by an mxiC : : lacZ transcriptional reporter. mxiC gene expression is further decreased by the presence of methionine or branched-chain amino acids (15-fold or 25-fold at least, respectively). Lysine and a few other aminated metabolites (cadaverine, homoserine and diaminopimelate) counteract the ornithinemediated inhibition of haemolytic activity and of the expression of a transcriptional activator virF reporter. The complete abolition of invasion of HeLa cells by wild-type bacteria by ornithine, uracil, methionine or branched-chain amino acids establishes that these metabolites are powerful effectors of virulence. These findings provide a direct connection between metabolism and virulence in S. flexneri. The inhibitory potential exhibited by the nutritional environment is stronger than temperature, the classical environmental effector of virulence. The implications and practical application of this finding in prophylaxis and treatment of shigellosis are discussed.

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Section: Methodsmentioning

confidence: 99%

Metabolic control through ornithine and uracil of epithelial cell invasion by Shigella flexneri

Durand

Björk

2009

Microbiology

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“…pBluescriptII SKϩ (pBS) (Stratagene, La Jolla, Calif.), pMW119Tp (12), pGEX-4T-1 (Amersham Pharmacia Biotech, Piscataway, N.J.), and pCACTUS-Tp r (45) were used for genetic engineering experiments. pTB101-mxiH, denoted previously for its construction, was used as a vector for the overexpression of MxiH in S. flexneri grown in the presence of 1 mM isopropyl-␤-D-thiogalactopyranoside (IPTG) (45).…”

Section: Methodsmentioning

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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“…Many eukaryotes require Q but attain it as the base queuine from gut flora or diet (29); its deficiency in germ-free mice compromises tyrosine production (30). In bacteria, Q elimination diminishes growth fitness in stationary phase (24), and can contribute to virulence loss (31). At present, the preQ 1 -III riboswitch has been identified in a handful of Ruminococcaceae with the preponderance of sequences originating from metagenomes (23).…”

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confidence: 99%

Structural analysis of a class III preQ ₁ riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

Liberman

Suddala

Aytenfisu

et al. 2015

Proc. Natl. Acad. Sci. U.S.A.

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PreQ 1 -III riboswitches are newly identified RNA elements that control bacterial genes in response to preQ 1 (7-aminomethyl-7-deazaguanine), a precursor to the essential hypermodified tRNA base queuosine. Although numerous riboswitches fold as H-type or HL out -type pseudoknots that integrate ligand-binding and regulatory sequences within a single folded domain, the preQ 1 -III riboswitch aptamer forms a HL out -type pseudoknot that does not appear to incorporate its ribosome-binding site (RBS). To understand how this unusual organization confers function, we determined the crystal structure of the class III preQ 1 riboswitch from Faecalibacterium prausnitzii at 2.75 Å resolution. PreQ 1 binds tightly (K D,app 6.5 ± 0.5 nM) between helices P1 and P2 of a three-way helical junction wherein the third helix, P4, projects orthogonally from the ligand-binding pocket, exposing its stem-loop to base pair with the 3′ RBS. Biochemical analysis, computational modeling, and single-molecule FRET imaging demonstrated that preQ 1 enhances P4 reorientation toward P1-P2, promoting a partially nested, H-type pseudoknot in which the RBS undergoes rapid docking (k dock ∼0.6 s −1 ) and undocking (k undock ∼1.1 s −1 ). Discovery of such dynamic conformational switching provides insight into how a riboswitch with bipartite architecture uses dynamics to modulate expression platform accessibility, thus expanding the known repertoire of gene control strategies used by regulatory RNAs. preQ 1 riboswitch | gene regulation | crystal structure | single-molecule FRET | molecular dynamics R iboswitches are structured RNA motifs that sense the cellular levels of small molecules to provide feedback regulation of genes (1). Although present in all domains of life, they are prominent in bacteria where they typically reside in the 5′-leader sequences of mRNA (2). Broad interest in riboswitches originates from the discovery that they can be targeted by antimicrobials (3-5), and the observation that they use complex scaffolds to achieve gene regulation without the need for protein partners. In the latter respect, riboswitches typically exhibit bipartite sequence organization comprising a conserved aptamer linked to a downstream expression platform (2). Aptamer binding to a cognate effector can induce conformational changes that alter the accessibility of expression platform sequences, such as those required for transcriptional read-through, or hybridization to the 16S rRNA as a preface to translation (2, 6).Numerous riboswitches fold as pseudoknots that conform to the H-type or closely related HL out -type topology, which have emerged as the most efficient RNA scaffolds to integrate aptamer and expression platform sequences (7). The preQ 1 -I, preQ 1 -II, S-adenosyl-L-methionine-II (SAM-II), and fluoride riboswitches are representative of this organizational strategy, and their analysis has contributed to a renaissance in our understanding of regulatory pseudoknot structure and dynamics (8-18). By contrast, pseudoknotted aptamers that do not integr...

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vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (Tgt) of Escherichia coli K-12

Cited by 131 publications

References 43 publications

Metabolic control through ornithine and uracil of epithelial cell invasion by Shigella flexneri

Metabolic control through ornithine and uracil of epithelial cell invasion by Shigella flexneri

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

Structural analysis of a class III preQ ₁ riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

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vacC, a virulence-associated chromosomal locus of Shigella flexneri, is homologous to tgt, a gene encoding tRNA-guanine transglycosylase (Tgt) of Escherichia coli K-12

Cited by 131 publications

References 43 publications

Metabolic control through ornithine and uracil of epithelial cell invasion by Shigella flexneri

Metabolic control through ornithine and uracil of epithelial cell invasion by Shigella flexneri

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

Structural analysis of a class III preQ 1 riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics

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Structural analysis of a class III preQ ₁ riboswitch reveals an aptamer distant from a ribosome-binding site regulated by fast dynamics