Spiroplasma citri UGG and UGA tryptophan codons: sequence of the two tryptophanyl-tRNAs and organization of the corresponding genes

Citti, Christine; Maréchal-Drouard, Laurence; Saillard, Colette; Weil, Jacques-Henry; Bové, J.M.

doi:10.1128/jb.174.20.6471-6478.1992

Cited by 39 publications

(23 citation statements)

References 42 publications

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“…Taking into account the fact that, in spiroplasmas, UGA codes for tryptophan (7,36), we found this sequence to contain three ORFs (ORFs 1, 2, and 3), each starting at an ATG initiation codon located downstream of a possible ribosome binding site. The nucleotide sequence and the deduced amino acid sequences of the putative translation products are presented in Fig.…”

Section: Resultsmentioning

confidence: 99%

Isolation, characterization, and complementation of a motility mutant of Spiroplasma citri

Jacob¹,

Nouzières²,

Duret³

et al. 1997

J Bacteriol

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The helical mollicute Spiroplasma citri, when growing on low-agar medium, forms fuzzy colonies with occasional surrounding satellite colonies due to the ability of the spiroplasmal cells to move through the agar matrix. In liquid medium, these helical organisms flex, twist, and rotate rapidly. By using Tn4001 insertion mutagenesis, a motility mutant was isolated on the basis of its nondiffuse, sharp-edged colonies. Dark-field microscopy observations revealed that the organism flexed at a low frequency and had lost the ability to rotate about the helix axis. In this mutant, the transposon was shown to be inserted into an open reading frame encoding a putative polypeptide of 409 amino acids for which no significant homology with known proteins was found. The corresponding gene, named scm1, was recovered from the wild-type strain and introduced into the motility mutant by using the S. citri oriC plasmid pBOT1 as the vector. The appearance of fuzzy colonies and the observation that spiroplasma cells displayed rotatory and flexional movements showed the motile phenotype to be restored in the spiroplasmal transformants. The functional complementation of the motility mutant proves the scm1 gene product to be involved in the motility mechanism of S. citri.Spiroplasmas are helical, wall-free bacteria belonging to the class Mollicutes, a group of microorganisms phylogenetically related to gram-positive bacteria with low GϩC contents (48). Besides helical morphology, motility is the most distinctive property of spiroplasmas, especially since these organisms possess no flagella, no periplasmic fibrils, and no axial filaments. Motility of spiroplasmas was first described by Davis and Worley (12) for the corn stunt spiroplasma in extracts of infected plants and was confirmed by Cole and coworkers (9) with cultured cells of Spiroplasma citri, the causal agent of citrus subborn disease. Spiroplasma cells display both rotatory and flexional movements which do not produce translational motility in liquid media of low viscosity. Translational motility could be observed only when viscosity was increased by incorporating agar or methylcellulose in the medium (11). As a result of the ability of spiroplasma cells to move through the agar matrix, spiroplasmas, and in particular S. citri, form diffuse colonies when plated on low-agar medium. Aspects of motility, chemotaxis, and viscotaxis in spiroplasmas have been further documented by Daniels and coworkers (11). On the basis of their work, these authors suggest that translational motility is driven by rotation about the helix axis, whereas flexing of the cell body causes changes of direction. Also from these studies, it is known that motility of spiroplasmas is energy dependent. However, the mechanism of motility has not been elucidated, and the cellular elements involved in motility are still unknown. In order to identify such cellular components, Cohen and coworkers (8) tried to obtain nonmotile mutants of Spiroplasma melliferum BC3. In those studies, nitrous acid was used as the mut...

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Section: Resultsmentioning

confidence: 99%

Isolation, characterization, and complementation of a motility mutant of Spiroplasma citri

Jacob¹,

Nouzières²,

Duret³

et al. 1997

J Bacteriol

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“…S. citri uses the UGA opal codon to incorporate tryptophan rather than a stop codon as in the universal genetic code (16). For PGK expression in Escherichia coli, the two opal codons contained in the gene were changed by site-directed mutagenesis by using overlap extension PCR (23).…”

Section: Methodsmentioning

confidence: 99%

Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin

Labroussaa

Arricau‐Bouvery

Dubrana

et al. 2010

Appl Environ Microbiol

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Transmission of the phytopathogenic mollicutes, spiroplasmas, and phytoplasmas by their insect vectors mainly depends on their ability to pass through gut cells, to multiply in various tissues, and to traverse the salivary gland cells. The passage of these different barriers suggests molecular interactions between the plant mollicute and the insect vector that regulate transmission. In the present study, we focused on the interaction between Spiroplasma citri and its leafhopper vector, Circulifer haematoceps. An in vitro protein overlay assay identified five significant binding activities between S. citri proteins and insect host proteins from salivary glands. One insect protein involved in one binding activity was identified by liquid chromatography-tandem mass spectrometry (LC-MS/MS) as actin. Confocal microscopy observations of infected salivary glands revealed that spiroplasmas colocated with the host actin filaments. An S. citri actin-binding protein of 44 kDa was isolated by affinity chromatography and identified by LC-MS/MS as phosphoglycerate kinase (PGK). To investigate the role of the PGK-actin interaction, we performed competitive binding and internalization assays on leafhopper cultured cell lines (Ciha-1) in which His 6 -tagged PGK from S. citri or purified PGK from Saccharomyces cerevisiae was added prior to the addition of S. citri inoculum. The results suggested that exogenous PGK has no effect on spiroplasmal attachment to leafhopper cell surfaces but inhibits S. citri internalization, demonstrating that the process leading to internalization of S. citri in eukaryotic cells requires the presence of PGK. PGK, regardless of origin, reduced the entry of spiroplasmas into Ciha-1 cells in a dose-dependent manner.

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“…All of these mollicutes are associated with arthropods, mainly insects. The spiroplasmas, like the mycoplasmas, ureaplasmas, mesoplasmas, and entomoplasmas, but unlike the acholeplasmas, use the triplet UGA, usually a stop codon, as a tryptophan codon (8,21).Spiralin is the most abundant membrane protein in S. citri (36). Its amphiphilic and antigenic properties (34, 38) and its membrane location and arrangement (30, 35) have been extensively studied.…”

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Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation

et al. 1996

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Spiralin is defined as the major membrane protein of the helical mollicute Spiroplasma citri. According to the S. citri strain used, spiralin shows polymorphism in its electrophoretic mobility. The spiralin gene sequences of eight S. citri strains were determined by direct sequencing of the PCR-amplified genes. All spiralins were found to be 241 amino acids long, except for the spiralin of strain Palmyre, which is 242 amino acids long. The molecular masses calculated from these sequences did not explain the differences observed in the electrophoretic mobilities. In all of the spiralins examined, the first 24 N-terminal amino acids were conserved, including a cysteine at position 24, and had the features of typical signal peptides of procaryotic lipoproteins. When S. citri strains were grown in the presence of [ ]palmitic acid labeled, and it had almost the same electrophoretic mobility irrespective of the spiralins used. Samples of the unlabeled peptide fragments from the four representative strains had slightly different electrophoretic mobilities (⌬DaХ800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (⌬DaХ8,000 Da). These results suggest that spiralin polymorphism in S. citri is not due to differences in posttranslational modification by palmitic acid and is certainly a structural property of the whole protein or could result from an unidentified posttranslational modification of spiralin.Among the mollicutes (i.e., wall-less eubacteria with low GϩC contents and small genomes), spiroplasmas are characterized by their helical morphology and motility. Spiroplasma citri, the first spiroplasma to have been cultured, is the causal agent of ''Stubborn'' disease of citrus (24) and affects many other plant species. S. citri also multiplies in various leafhoppers, which act as insect vectors. The spiroplasmas belong to the recently established order Entomoplasmatales, which also contains Entomoplasma and Mesoplasma species (31). All of these mollicutes are associated with arthropods, mainly insects. The spiroplasmas, like the mycoplasmas, ureaplasmas, mesoplasmas, and entomoplasmas, but unlike the acholeplasmas, use the triplet UGA, usually a stop codon, as a tryptophan codon (8,21).Spiralin is the most abundant membrane protein in S. citri (36). Its amphiphilic and antigenic properties (34, 38) and its membrane location and arrangement (30, 35) have been extensively studied. The spiralin gene has been identified, cloned, and expressed in Escherichia coli cells (19). Several forms of spiralin were produced in E. coli, including a major form with a slightly larger molecular mass than that of spiralin in S. citri. S. citri spiralin is free of tryptophan, and its gene has no UGA codon (6). This feature explains why the gene could be efficiently translated in E. coli at a time when it was not yet known that in spiroplasmas, UGA is not read as a stop codon but instead is read as a tryptophan codon. However, the function of spiralin in the spiroplasma cell remains undet...

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Spiroplasma citri UGG and UGA tryptophan codons: sequence of the two tryptophanyl-tRNAs and organization of the corresponding genes

Cited by 39 publications

References 42 publications

Isolation, characterization, and complementation of a motility mutant of Spiroplasma citri

Isolation, characterization, and complementation of a motility mutant of Spiroplasma citri

Entry of Spiroplasma citri into Circulifer haematoceps Cells Involves Interaction between Spiroplasma Phosphoglycerate Kinase and Leafhopper Actin

Spiralin polymorphism in strains of Spiroplasma citri is not due to differences in posttranslational palmitoylation

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