Spiralin Diversity Within Iranian Strains of Spiroplasma citri

The first-cultured and most-studied spiroplasma is Spiroplasma citri, the causal agent of citrus stubborn disease, one of the three plant-pathogenic, sieve-tube-restricted, and leafhopper vector-transmitted mollicutes. In Iranian Fars province, S. citri cultures were obtained from stubborn affected citrus trees, sesame and safflower plants, and from the leafhopper vector Circulifer haematoceps. Spiralin gene sequences from different S. citri isolates were amplified by PCR, cloned, and sequenced. Phylogenetic trees based on spiralin gene sequence showed diversity and indicated the presence of three clusters among the S. citri strains. Comparison of the amino acid sequences of eleven spiralins from Iranian strains and those from the reference S. citri strain GII-3 (241 aa), Palmyre strain (242 aa), Spiroplasma kunkelii (240 aa), and Spiroplasma phoeniceum (237 aa) confirmed the conservation of general features of the protein. However, the spiralin of an S. citri isolate named Shiraz I comprised 346 amino acids and showed a large duplication of the region comprised between two short repeats previously identified in S. citri spiralins. We report in this paper the spiralin diversity in Spiroplasma strains from southern Iran and for the first time a partial internal duplication of the spiralin gene.     

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 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 x 10(6) to 2.8 x 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 x 10(6) to 1.4 x 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.     

Morphology and ultrastructure of helical and nonhelical strains of Spiroplasma citri.

Cells of the nonhelical strain of Spiroplasma citri underwent changes of morphology comparable to those which occurred in the normal helical strain. Cells of the nonhelical strain had the same ultrastructural features as helical cells and released long flexible fibrils similar to those seen in other spiroplasmas. Nonhelical organisms showed an increased tendency to aggregate, forming cell clusters of an unusual annular form. The cytoplasmic membrane of the nonhelical strain lacked a single protein present in all helical strains. Loss of helicity associated with the senescence of spiroplasma cells was not accompanied by the disappearance of this protein. Differences in colony morphology were shown to be a consequence of motility, and a technique was developed which facilitated the identification of nonmotile organisms.     

Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri.

The replication region (oriC) of the Spiroplasma citri chromosome has been recently sequenced, and a 2-kbp DNA fragment was characterized as an autonomously replicating sequence (F. Ye, J. Renaudin, J. M. Bové, and F. Laigret, Curr. Microbiol. 29:23-29, 1994). In the present studies, we have combined this DNA fragment, containing the dnaA gene and the flanking dnaA boxes, with a ColE1-derived Escherichia coli replicon and the Tet M determinant, which confers resistance to tetracycline. The recombinant plasmid, named pBOT1, was introduced into S. citri cells, in which it replicated. Plasmid pBOT1 was shuttled from E. coli to S. citri and back to E. coli. In S. citri, replication of pBOT1 did not require the presence of a functional dnaA gene on the plasmid. However, the dnaA box region downstream of the dnaA gene was essential. Upon passaging of the S. citri transformants, the plasmid integrated into the spiroplasmal host chromosome by recombination at the replication origin. The integration process led to duplication of the oriC sequences. In contrast to the integrative pBOT1, plasmid pOT1, which does not contain the E. coli replicon, was stably maintained as a free extrachromosomal element. Plasmid pOT1 was used as a vector to introduce into S. citri the G fragment of the cytadhesin P1 gene of Mycoplasma pneumoniae and the spiralin gene of Spiroplasma phoeniceum. The recombinant plasmids, pOTPG with the G fragment and pOTPS with the spiralin gene, were stably maintained in spiroplasmal transformants. Expression of the heterologous S. phoeniceum spiralin in S. citri was demonstrated by Western immunoblotting.     

Antigenic relatedness between the spiralins of Spiroplasma citri and Spiroplasma melliferum.

Four spiralins were compared by rocket immunoelectrophoresis, quantitative immunoblotting techniques, and the spiroplasma deformation test with the use of antispiralin (polyclonal) monospecific antibodies. This investigation revealed that the spiralins of Spiroplasma citri and S. melliferum are antigenically related and that probably no more than two epitopes simultaneously saturable with antibodies are shared by the two proteins. One at least of these epitopes is accessible to antibodies on the spiroplasma cell surface.     

Characterization of the recA gene regions of Spiroplasma citri and Spiroplasma melliferum.

In previous studies (A. Marais, J. M. Bove, and J. Renaudin, J. Bacteriol. 178:862-870, 1996), we have shown that the recA gene of Spiroplasma citri R8A2 was restricted to the first 390 nucleotides of the N-terminal part. PCR amplification and sequencing studies of five additional strains of S. citri have revealed that these strains had the same organization at the recA region as the R8A2 strain. In contrast to S. citri, Spiroplasma melliferum was found to contain a full-length recA gene. However, in all five S. melliferum strains tested, a TAA stop codon was found within the N-terminal region of the recA reading frame. Our results suggest that S. melliferum, as well as S. citri, is RecA deficient. In agreement with the recA mutant genotype of S. citri and S. melliferum, we have shown that these organisms are highly sensitive to UV irradiation.     

Comparison of the membrane composition of Spiroplasma citri and the corn stunt Spiroplasma.

Components of membranes isolated from Spiroplasma citri and corn stunt spiroplasma grown at 28 degrees C were analyzed. On a protein basis, lipid phosphorus was lower and cholesterol was higher in S. citri. Only minor differences between the two species were found in fatty acid composition, reduced nicotinamide adenine dinucleotide diaphorase, and adenosine triphosphatase.     

Lipid composition and lipid metabolism of Spiroplasma citri.

In a horse serum-based medium containing a full complement of fatty acids, cells of Spiroplasma citri were seen to preferentially incorporate palmitic acid. In the same medium, which had a steryl ester-to-sterol ratio of 3.64, a steryl ester-to-sterol ratio of 0.23 was seen in the cells, cholesterol being preferentially incorporated over cholesteryl ester. Like most other mycoplasmas, S. citri was shown to be unable to synthesize fatty acids or esterify cholesterol. The neutral lipids of S. citri grown in a medium containing horse serum consisted of free cholesterol, cholesteryl ester, free fatty acids, triglycerides and diglycerides. All polar lipids were phospholipids, with no glycolipids detected. These phospholipids, which are characteristic of many mycoplasmas, are phosphatidyl glycerol, diphosphatidyl glycerol, and their lyso derivatives. Sphingomyelin was also incorporated when cells were grown on horse serum. A sterol requirement for the growth of S. citri was confirmed using a serum-free medium supplemented with bovine serum albumin, palmitic acid, and various concentrations of sterols dissolved in Tween 80. The addition of palmitic acid stimulated growth but was not essential for growth. S citri was shown to grow best on cholesterol and beta-sitosterol and was able to grow on stigmasterol and ergosterol to a lesser degree. No growth was obtained using mevalonate, deoxycholate, or taurodeoxycholate as an alternative to sterol. S. citri was also able to grow when palmitic acid was replaced with oleic acid, linoleic acid, or linolenic acid. Alterations in the lipid composition of the growth medium and hence in the lipid composition of S. citri induced changes in the characteristic helical morphology of the cells, concurrent with loss of cell viability. Culture, age, and pH were also factors in determining cell morphology and viability.     

Composition and enzyme activities of Spiroplasma citri membranes.

Spiroplasma citri was cultured in three different media that supplied cholesterol and fatty acids from: (i) horse serum, (ii) pleuropneumonia-like organism (PPLO) serum fraction, or (iii) bovine serum albumin-fatty acid-cholesterol. The ability of PPLO serum fraction to support growth varied by lot number. Neither PPLO serum fraction nor the bovine serum albumin medium supported growth as well as the horse serum medium. Analysis of cholesterol, lipid phosphorus, and membrane protein showed the horse serum- and PPLO-grown cells to be indistinguishable, but the bovine serum albumin-grown cells were deficient in lipid phosphorus. The three cultures did not show markedly different fatty acid compositions, but, in all cases, the cultures preferentially incorporated palmitic acid and discriminated against linoleic acid. Cultures grown for different times from logarithmic growth through a degenerative phase showed relatively constant ratios of cholesterol/protein and lipid phosphorus/protein. Fatty acid composition was also relatively constant at the different stages. Adenosine triphosphatase and p-nitrophenyl phosphatase were mainly associated with the membrane, whereas reduced nicotinamide adenine dinucleotide oxidase was either readily removed or not associated with the membrane. The reduced nicotinamide adenine dinucleotide oxidase was inactivated at temperatures above 35 degrees C.     

An in situ Freeze-Fracture Study of Spiroplasma citri and the Corn Stunt Spiroplasma

Spiroplasma citri and the corn stunt spiroplasma in sieve cells of Catharanthus roseus were investigated using freeze -fracture electron microscopy. Only the particle studded fracture faces of the plasmalemma could be exposed and not the surfaces of both the extraplasmatic and the plasmatic leaflet. The extraplasmatic fracture face (EF) shows a lower particle density than the plasmatic fracture face (PF). On the PF particle free areas could be observed, which are helically arranged in helical filaments. We suppose that the cytoplasmic fibrils, probably involved in motility processes and in maintaining the helical shape, underly the particle free area only.     

A physical and genetic map of the Spiroplasma citri genome.

A physical and genetic map of the Spiroplasma citri genome has been constructed using several restriction enzymes and pulsed field gel electrophoresis. A number of genes were subsequently localized on the map by the use of appropriate probes. The genome size of the spiroplasma estimated from restriction fragments is close to 1780 kbp, the largest of all Mollicutes studied so far. It contains multisite insertions of Spiroplasma virus 1 (SpV1) sequences. The physical and genetic map of the S. citri genome shares several features with that of other Mollicutes, especially those in the Mycoplasma mycoides cluster. This supports the finding that S. citri and these Mycoplasma spp. are phylogenetically related.     

Comparison of lipids from Spiroplasma citri and corn stunt spiroplasma.

The qualitative lipid composition of Spiroplasma citri and corn stunt spiroplasma is identical. Small amounts of acylated glucose and steryl glucoside were found.     

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 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 × 10⁶ to 2.8 × 10⁶ 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 × 10⁶ to 1.4 × 10⁷ 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.     

Lethal and mutation frequency responses of Spiroplasma citri cells to UV irradiation

The effect of UV irradiation on viability and mutant colony frequency in the Mollicute Spiroplasma citri was investigated at 3 phases of growth. The first UV-induced mutants obtained in Mollicutes were selected: xylitol-resistant (XylR) and arsenic acid-resistant mutants (ArsR). Lethal and mutation frequency responses of S. citri cells increase with the age of the cell cultures. In all UV-irradiated populations, light exposure slightly increases the number of survivors and decreases the induced mutation frequency; liquid holding conditions increase the number of both survivors and mutant colonies. This suggests that, in UV-irradiated S. citri cells maintained under liquid holding conditions, there is no dark reactivation but induction of an error-prone repair system of the SOS type. In S. citri, the error-free light and dark repair systems are inefficient. Results allow the development of a method to select UV-induced mutations usable as markers in genetic studies of Spiroplasma cells.     

Growth and division of Spiroplasma citri: elongation of elementary helices.

The smallest viable cell of Spiroplasma citri is a two-turn helix (elementary helix). This elementary helix grows into longer parental cells, which then divide by constriction. The helical morphology is conserved during this process. The growth pattern of S. citri membranes has been investigated by different methods of membrane labeling. When labeling is done with specific antibodies, a diffuse growth of the membrane is observed. On the contrary, pulse-labeling of the membrane with tritiated amino acids reveals a polar growth of the organism. Finally, labeling of oxydo reduction sites with potassium tellurite also indicates a polarity in the organism. These results are discussed, and a scheme for spiroplasma growth is proposed.     

Effect of external environmental factors on the morphology of Spiroplasma citri.

Spiroplasma citri was examined by electron microscopy for morphological changes when maintained under a variety of conditions. PPLO serum fraction maintained spiral and helical morphology of S. citri at pH values of 8.0, 7.5, and 7.0, but only partially at pH 6.0 and 5.0. The absence of PPLO serum fraction resulted in round, deteriorated cells at all pH values tested. Bovine serum albumin (BSA), Phytone, soluble starch, potato starch, spermine, lipid-extracted PPLO serum fraction, and lipid-extracted BSA could substitute for PPLO serum fraction in maintaining spiral and helical morphology at pH 7.5. At pH 5.0, only BSA, lipid-extracted BSA, and lipid-extracted PPLO serum fraction were effective. Only BSA supported growth of S. citri for more than two transfers, whereas all other substitutes could not support growth longer than two transfers.     

Involvement of a Spiroplasma citri plasmid in the erythromycin-resistance transfer

An erythromycin-resistant strain (M4 Er-1) was selected from Spiroplasma citri M4+. The transfer by transformation of the erythromycin-resistance character to the erythromycin-sensitive S. citri strain R8A2+ was studied. Transfer became effective and reproducible when cells were treated with alkali cations plus polyethylene glycol. Comparison of the efficiency of transformation of the erythromycin-sensitive strain S. citri R8A2+ by total and extrachromosomal DNA purified from the erythromycin-resistant strain M4 Er-1 showed that the plasmid pM42 was able to transfer the erythromycin-resistance. pM42 was mapped with restriction endonucleases and found to be related to the pMH1 plasmid previously isolated from S. citri MH. Hybridization analysis of DNA from sensitive and resistant strains has shown that a sequence from pM42, analogous to a sequence from pMH1, was integrated at a specific locus in the chromosome of the erythromycin-resistant cells, i.e., of the transformed R8A2 cells and of the spontaneous mutant M4 Er-1 strain.