Fructose utilization and pathogenicity of Spiroplasma citri: characterization of the fructose operon

Gaurivaud, Patrice; Laigret, Frédéric; Garnier, M.; Bové, Joseph M.

doi:10.1016/s0378-1119(00)00230-4

Cited by 47 publications

(42 citation statements)

References 32 publications

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“…In particular, it was shown that fructose utilization is a key factor of spiroplasmal pathogenicity (22,9). However, the interactions of S. citri with the host plant and with its insect vector are still poorly understood.…”

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confidence: 99%

Spiralin Is Not Essential for Helicity, Motility, or Pathogenicity but Is Required for Efficient Transmission of Spiroplasma citri by Its Leafhopper Vector Circulifer haematoceps

Duret

Berho

Danet

et al. 2003

Appl Environ Microbiol

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Spiralin is the most abundant protein at the surface of the plant pathogenic mollicute Spiroplasma citri and hence might play a role in the interactions of the spiroplasma with its host plant and/or its insect vector. To study spiralin function, mutants were produced by inactivating the spiralin gene through homologous recombination. A spiralin-green fluorescent protein (GFP) translational fusion was engineered and introduced into S. citri by using an oriC-based targeting vector. According to the strategy used, integration of the plasmid by a single-crossover recombination at the spiralin gene resulted in the expression of the spiralin-GFP fusion protein. Two distinct mutants were isolated. Western and colony immunoblot analyses showed that one mutant (GII3-9a5) did produce the spiralin-GFP fusion protein, which was found not to fluoresce, whereas the other (GII3-9a2) produced neither the fusion protein nor the wild-type spiralin. Both mutants displayed helical morphology and motility, similarly to the wild-type strain GII-3. Genomic DNA analyses revealed that GII3-9a5 was unstable and that GII3-9a2 was probably derived from GII3-9a5 by a double-crossover recombination between plasmid sequences integrated into the GII3-9a5 chromosome and free plasmid. When injected into the leafhopper vector Circulifer haematoceps, the spiralinless mutant GII3-9a2 multiplied to high titers in the insects (1.1 ؋ 10 6 to 2.8 ؋ 10 6 CFU/insect) but was transmitted to the host plant 100 times less efficiently than the wild-type strain. As a result, not all plants were infected, and symptom production in these plants was delayed for 2 to 4 weeks compared to that in the wild-type strain. In the infected plants however, the mutant multiplied to high titers (1.2 ؋ 10 6 to 1.4 ؋ 10 7 CFU/g of midribs) and produced the typical symptoms of the disease. These results indicate that spiralin is not essential for pathogenicity but is required for efficient transmission of S. citri by its insect vector.

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Spiralin Is Not Essential for Helicity, Motility, or Pathogenicity but Is Required for Efficient Transmission of Spiroplasma citri by Its Leafhopper Vector Circulifer haematoceps

Duret

Berho

Danet

et al. 2003

Appl Environ Microbiol

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“…In contrast, replicative plasmids have been obtained for the mollicute Spiroplasma citri by cloning the origin of replication of the chromosome (oriC) into artificial plasmids (34,41). The resulting oriC plasmids can be used to inactivate targeted genes by homologous recombination (8,41), to support heterologous gene expression in S. citri (35), and to functionally complement mutants obtained by transposon insertion (18).…”

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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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“…Transposon Tn4001 was originally isolated from Staphylococcus aureus (22) and has proved to be a useful tool for genetic analysis of several gram-positive bacteria, including Streptococcus gordonii (18), and mollicutes, such as the mycoplasmas (7,10), spiroplasmas (8,9), and acholeplasmas (41). This compound transposon is composed of the aacA-aphD genes, conferring resistance to gentamicin, kanamycin, and tobramycin, flanked by two copies of IS256 in inverted orientation ( Fig.…”

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Diversity of Tn4001Transposition Products: the Flanking IS256Elements Can Form Tandem Dimers and IS Circles

et al. 2002

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We show that both flanking IS256 elements carried by transposon Tn4001 are capable of generating head-to-tail tandem copies and free circular forms, implying that both are active. Our results suggest that the tandem structures arise from dimeric copies of the donor or vector plasmid present in the population by a mechanism in which an IS256 belonging to one Tn4001 copy attacks an IS256 end carried by the second Tn4001 copy. The resulting structures carry abutted left (inverted left repeat [IRL]) and right (inverted right repeat [IRR]) IS256 ends. Examination of the junction sequence suggested that it may form a relatively good promoter capable of driving transposase synthesis in Escherichia coli. This behavior resembles that of an increasing number of bacterial insertion sequences which generate integrative junctions as part of the transposition cycle. Sequence analysis of the IRL-IRR junctions demonstrated that attack of one end by the other is largely oriented (IRL attacks IRR). Our experiments also defined the functional tips of IS256 as the tips predicted from sequence alignments, confirming that the terminal 4 bp at each end are indeed different. The appearance of these multiple plasmid and transposon forms indicates that care should be exercised when Tn4001 is used in transposition mutagenesis. This is especially true when it is used with naturally transformable hosts, such as Streptococcus pneumoniae, in which reconstitution of the donor plasmid may select for higher-order multimers.Transposon Tn4001 was originally isolated from Staphylococcus aureus (22) and has proved to be a useful tool for genetic analysis of several gram-positive bacteria, including Streptococcus gordonii (18), and mollicutes, such as the mycoplasmas (7, 10), spiroplasmas (8, 9), and acholeplasmas (41). This compound transposon is composed of the aacA-aphD genes, conferring resistance to gentamicin, kanamycin, and tobramycin, flanked by two copies of IS256 in inverted orientation (Fig. 1A) (22).Although Tn4001 has been used to mutagenize various gram-positive bacteria, the initial evidence suggested that some of the transposition products might be complex (20). We also observed complex transposition behavior during Tn4001-mediated transposition mutagenesis of Streptococcus pneumoniae. These results prompted us to investigate transpositional recombination of Tn4001 in more detail.IS256 itself is the founding member of a family of insertion sequences which includes examples from a variety of grampositive and gram-negative bacterial species (4, 23). It is 1,324 bp long and carries 26-bp terminal imperfect inverted repeats (an inverted left repeat [IRL] and an inverted right repeat [IRR]); in the case of IS256, but not in the majority of family members, these inverted repeats diverge at the terminal dinucleotides. IS256 generally generates 8-bp direct flanking target repeats upon insertion and includes a single long open reading frame encoding the transposase. Very little is known about the transposition mechanism of this family of ele...

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Fructose utilization and pathogenicity of Spiroplasma citri: characterization of the fructose operon

Cited by 47 publications

References 32 publications

Spiralin Is Not Essential for Helicity, Motility, or Pathogenicity but Is Required for Efficient Transmission of Spiroplasma citri by Its Leafhopper Vector Circulifer haematoceps

Spiralin Is Not Essential for Helicity, Motility, or Pathogenicity but Is Required for Efficient Transmission of Spiroplasma citri by Its Leafhopper Vector Circulifer haematoceps

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

Diversity of Tn4001Transposition Products: the Flanking IS256Elements Can Form Tandem Dimers and IS Circles

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