Nucleotide sequence of the Spiroplasma citri fibril protein gene

Williamson, David L.; Renaudin, Joël; Bové, J.M.

doi:10.1128/jb.173.14.4353-4362.1991

Cited by 65 publications

(56 citation statements)

References 30 publications

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“…1), which harbors the tetM gene (25) from the transposon Tn916 inserted into the pBS plasmid (Stratagene, La Jolla, Calif.). In pSRT2, the tetM gene is under the control of the S. citri spiralin gene promoter (40). The 1.9-kbp oriC region of M. pulmonis, containing the dnaA gene with its upstream and downstream intergenic regions ( Fig.…”

Section: Methodsmentioning

confidence: 99%

Identification of the Origin of Replication of the Mycoplasma pulmonis Chromosome and Its Use in oriC Replicative Plasmids

Córdova

Lartigue²,

Sirand‐Pugnet³

et al. 2002

J Bacteriol

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Mycoplasma pulmonis is a natural rodent pathogen, considered a privileged model for studying respiratory mycoplasmosis. The complete genome of this bacterium, which belongs to the class Mollicutes, has recently been sequenced, but studying the role of specific genes requires improved genetic tools. In silico comparative analysis of sequenced mollicute genomes indicated the lack of conservation of gene order in the region containing the predicted origin of replication (oriC) and the existence, in most of the mollicute genomes examined, of putative DnaA boxes lying upstream and downstream from the dnaA gene. The predicted M. pulmonis oriC region was shown to be functional after cloning it into an artificial plasmid and after transformation of the mycoplasma, which was obtained with a frequency of 3 ؋ 10 ؊6 transformants/CFU/g of plasmid DNA. However, after a few in vitro passages, this plasmid integrated into the chromosomal oriC region. Reduction of this oriC region by subcloning experiments to the region either upstream or downstream from dnaA resulted in plasmids that failed to replicate in M. pulmonis, except when these two intergenic regions were cloned with the tetM determinant as a spacer in between them. An internal fragment of the M. pulmonis hemolysin A gene (hlyA) was cloned into this oriC plasmid, and the resulting construct was used to transform M. pulmonis. Targeted integration of this genetic element into the chromosomal hlyA by a single crossing over, which results in the disruption of the gene, could be documented. These mycoplasmal oriC plasmids may therefore become valuable tools for investigating the roles of specific genes, including those potentially implicated in pathogenesis.The mollicutes are the smallest free-living microorganisms capable of autoreplication (33). They are related to grampositive eubacteria from which they evolved by a drastic reduction of genome size, being thus considered the best representatives of the concept of a minimal cell. (964 kbp [5]). Postgenomic approaches aiming at deciphering the role of specific genes discovered by in silico analysis now require efficient strategies to genetically manipulate these organisms. The transposon Tn916 or Tn4001 can transpose in all the mollicute genomes, at least for the species amenable to transformation, such as M. genitalium, M. pneumoniae, and M. pulmonis. In M. genitalium and M. pneumoniae, a global approach of gene inactivation with the transposon Tn4001 was used to define the genes that are essential for the life of these bacteria (22). Derivatives of this transposon, obtained by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant, were also shown to transpose in M. pulmonis (12). The use of these genetic elements for gene inactivation, which relies on the random insertion of the transposon in the genome of the organism, does not allow specific targeting of a gene of interest. In addition, the complementation of mutants would require the cloning and expression of genes in...

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Section: Methodsmentioning

confidence: 99%

Identification of the Origin of Replication of the Mycoplasma pulmonis Chromosome and Its Use in oriC Replicative Plasmids

Córdova

Lartigue²,

Sirand‐Pugnet³

et al. 2002

J Bacteriol

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“…Large-scale and small-scale preparations of plasmids and M13 DNA amplified in E. coli were carried out according to standard procedures (37). Isolation of spiroplasmal genomic DNA has been described elsewhere (51).…”

Section: Methodsmentioning

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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“…Sequence determination of several spiroplasmal genes has shown that both UGG and UGA are used as tryptophan codons (5,7,28,38). In the present study, we analyzed the tRNATrP situation in S. citri.…”

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

et al. 1992

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From the total tRNAs of Spiroplasma citri, we isolated and purified two tRNA(Trp) species by using chromatography on an RPC-5 column followed by denaturing polyacrylamide gel electrophoresis. The sequence of the two tRNAs, as well as the sequences of the corresponding genes, were determined. One of the two tRNA(Trp) species has a CCA anticodon and is able to pair with the universal UGG tryptophan codon, while the second has a U*CA (U* is a modified uridine) anticodon and is able to pair with UGA but also with UGG in accordance with the "U:N wobble" rule. Thus, in S. citri, UGA is not a stop codon but codes for tryptophan. The two tRNA(Trp) genes, together with a third tRNA gene, tRNA(Ser) (CGA), belong to a single transcription unit. The nucleotide sequences of the two tRNA(Trp) species show 82.9% similarity. The two spiroplasmal tRNA(Trp) species can be aminoacylated by using an aminoacyl-tRNA synthetase fraction from S. citri. In contrast, the enzyme fraction from Escherichia coli aminoacylates tRNA(Trp) (CCA) but not tRNA(Trp) (U*CA).

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Nucleotide sequence of the Spiroplasma citri fibril protein gene

Cited by 65 publications

References 30 publications

Identification of the Origin of Replication of the Mycoplasma pulmonis Chromosome and Its Use in oriC Replicative Plasmids

Identification of the Origin of Replication of the Mycoplasma pulmonis Chromosome and Its Use in oriC Replicative Plasmids

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

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

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