Genetic variability of the stolbur phytoplasma vmp1 gene in grapevines, bindweeds and vegetables.

AIM: Evaluation of the genetic variability of stolbur phytoplasma infecting grapevines, bindweeds and vegetables, collected in different central and southern Italian regions. MATERIALS AND RESULTS: Phytoplasma isolates belonging to stolbur subgroup 16SrXII-A were subjected to molecular characterization by polymerase chain reaction/restriction fragment length polymorphism (PCR/RFLP), to investigate two different nonribosomal genes: tuf and vmp1. In grapevines, 32% of samples were infected by tuf-a type and 68% by tuf-b type, with different relative incidences in the regions surveyed. All herbaceous samples (bindweeds, tomato, tobacco, pepper, celery) were infected by tuf-b. The gene vmp1 showed higher polymorphism in grapevines (nine profiles) than herbaceous plants (six) by RFLP analysis, in agreement with nucleotide sequences' analysis and virtual digestions. CONCLUSIONS: The phylogenetic analysis of vmp1 gene sequences supports the RFLP data and demonstrates the accuracy of RFLP for preliminary assessments of genetic diversity of stolbur phytoplasmas and for screening different vmp types. SIGNIFICANCE AND IMPACT OF THE STUDY: Stolbur represents a serious phytosanitary problem in the areas under investigation, owing to heavy economic losses in infected grapevines and vegetables. Molecular information about the complex genotyping of the vmp1 gene provides useful data towards a better understanding of stolbur epidemiology. Moreover, this study clarifies some different vmp1 genotype classifications of stolbur, providing molecular data in comparison with previous investigations.

rRNA operons and genome size of 'Candidatus Liberibacter americanus', a bacterium associated with citrus huanglongbing in Brazil.

Huanglongbing is one of the most severe diseases of citrus worldwide and is associated with 'Candidatus (Ca.) Liberibacter africanus' in Africa, 'Ca. Liberibacter asiaticus' in Asia and the Americas (Brazil, USA and Cuba) and 'Ca. Liberibacter americanus' (Lam) in Brazil. In the absence of axenic cultures, genetic information on liberibacters is scarce. The sequences of the entire 23S rRNA and 5S rRNA genes from Lam have now been obtained, using a consensus primer designed on known tRNAMet sequences of rhizobia. The size of the Lam genome was determined by PFGE, using Lam-infected periwinkle plants for bacterial enrichment, and was found to be close to 1.31 Mbp. In order to determine the number of ribosomal operons on the Lam genome, probes designed to detect the 16S rRNA gene and the 3' end of the 23S rRNA gene were developed and used for Southern hybridization with I-CeuI-treated genomic DNA. Our results suggest that there are three ribosomal operons in a circular genome. Lam is the first liberibacter species for which such data are available.

Characterization of Bois noir isolates by restriction fragment length polymorphism of a Stolbur-specific putative membrane protein gene.

Bois noir phytoplasma (BNp), widespread in wine-producing areas of Europe and endemic in France and Italy, is classified in the 16SrXII-A subgroup, whose members are referred to as Stolbur phytoplasmas. The 16S rDNA gene of Stolbur phytoplasma shows low variability, and few non-ribosomal genes are available as markers to assess variation among isolates. We used the Stolbur-specific stol-1H10 gene, encoding a putative membrane-exposed protein, to investigate genetic diversity of French and Italian BNp isolates from plants and insects. Amplification of stol-1H10 from infected grapevines, weeds, and Hyalesthes obsoletus produced fragments of three sizes, and restriction fragment length polymorphism analysis divided these amplicons further into 12 profiles (V1 to V12). French BNp isolates were more variable than Italian ones, and different profiles were present in infected grapevines from France and Italy. Isolate V3, most abundant among Italian affected grapes but present among French ones, was found in one Urtica dioica sample and in all H. obsoletus collected on this species. Four Italian-specific profiles were represented among infected Convolvulus arvensis, the most frequent of which (V12) was also detected in H. obsoletus collected on this species. Most of the variability in the stol-1H10 sequence was associated with type II on the tuf gene.

Comparing the spatial genetic structures of the Flavescence dorée phytoplasma and its leafhopper vector Scaphoideus titanus.

The Nearctic leafhopper Scaphoideus titanus Ball is the vector of "Flavescence dorée" phytoplasma (FDp) in European vineyards. We studied the genetic diversity and structure of S. titanus populations in France and of the FDp they carried. A total of 621 S. titanus individuals, sampled in 24 FDp-infected and uninfected vineyards, were genotyped using seven polymorphic microsatellite loci. The mean observed heterozygosity in S. titanus populations was between 0.364 and 0.548. There was evidence of only a low level of population genetic differentiation (mean F(ST)=0.027) suggesting that there is long-distance gene flow between S. titanus populations. This may be a consequence of the high migration capacity of the vector associated with large effective population size and, at least in part, of passive dispersion over long distances by the transport of grapevine-planting material carrying eggs. For each insect, FDp was detected and typed by nested-PCR followed by RFLP and sequencing of a 674 bp fragment of the FDp map gene. Twelve of the 24 populations were found to be infected by FDp, with the percentage of infected individuals varying from 3% to 29%. FDp isolates were classified into two FDp genetic clusters (FD1 and FD2), which differed by 12-13 SNPs. FD1 genotypes were detected in the insect populations at two sites and the FD2 genotypes in the other ten populations. Both FD1 and FD2 genotypes were found to be transmitted by the insect. No significant relationship was found between the genetic structure of these French S. titanus populations and the distribution of the various FDp strain types they carried. Nevertheless, overall genetic differentiation between FDp-infected and healthy S. titanus "subsamples" was found to be significantly higher than zero. These results suggest that FDp-infected S. titanus individuals are more philopatric (disperse less) than healthy S. titanus.

First Report of a γ 3-Proteobacterium Associated with Diseased Strawberries in Italy.

During the fall seasons of 2005 and 2006, diseased strawberry plants (Fragaria × ananassa Duch.) were observed in nurseries and production fields in Ferrara, Forli-Cesena, and Ravenna provinces (Emilia-Romagna region, northern Italy). Symptoms consisted of a conspicuous plant stunting with a poor root system. Older leaves rolled upward and displayed a marked premature purplish discoloration, while young leaves were cupped, chlorotic, generally reduced in size, and had shortened petioles. This strawberry disorder was similar to "marginal chlorosis", an infectious disease occurring in France that can be induced by two different phloem-limited uncultured bacteria: the γ 3-proteobacterium 'Candidatus Phlomobacter fragariae' and the stolbur phytoplasma (16SrXII-A). In strawberry production fields, 'Ca. P. fragariae' is reported as being the prevalent agent of this disease (1). Sixty-seven diseased plants were collected from production fields and nurseries for testing for 'Ca. P. fragariae'. Leaf samples were analyzed by 4',6-diamidine-2-phenylindole staining and PCR. Forty samples showed fluorescent DNA in the phloem, whereas no fluorescence was observed in symptomless strawberries. When tested by PCR with primers Fra4/Fra5, which amplify a 550-bp fragment of the 16S rDNA region of 'Ca. P. fragariae' (1), 13 of 36 strawberries from production fields and 1 of 31 nursery plants gave a positive reaction. On the other hand, 21 samples from nurseries and 5 from production fields tested positive for stolbur phytoplasma (3). No amplification was obtained with DNA from symptomless or healthy strawberry plants. Sequencing Fra4/Fra5 amplicons from three samples (GenBank Accession Nos. DQ362916-DQ362918) showed a 98.1 to 98.6% and a 98.3 to 98.8% identity with the published sequences of the French isolate "LG2001" (GenBank Accession No. AM110766) and the Japanese isolate J-B (GenBank Accession No. AB246669) of 'Ca. P. fragariae', respectively. Higher homology (99.2 to 99.8%) was found with another bacterium-like organism (BLO) of the γ 3-proteobacteria subgroup (GenBank Accession No. AY057392) associated with the syndrome "basses richesses" of sugar beet (SBR). Furthermore, PCR assays performed with primers Pfr1/Pfr4, specific for spoT gene of 'Ca. P. fragariae', did not show any amplification with DNA from the 14 diseased strawberry plants tested. This is in agreement with the SBR BLO identification (2). To better characterize the Italian isolates, the full-length 16S rDNA gene was analyzed with primers fd1/Fra4 and Fra5/rp1, which amplify the 5' and 3' region of 16S rDNA gene of the proteobacteria, respectively (2). PCR products from eight isolates were sequenced, and the 16S rDNA sequences obtained (GenBank Accession Nos. DQ538372-DQ538379) showed a 96.4 to 97.3% identity with the known 'Ca. P. fragariae' isolates, while a higher homology (99.4 to 99.9%) was again found with the SBR BLO. To our knowledge, this is the first report of a γ 3-proteobacterium affecting strawberry in Italy. In the genome region analyzed, our isolates are more similar to the SBR BLO than to 'Ca. P. fragariae'. Further work is in progress to investigate incidence, geographical distribution, epidemiology, and host range of this pathogen in Italy. References: (1) J. L. Danet et al. Phytopathology 93:644, 2003. (2) O. Semetey et al. Phytopathology 97:72, 2007. (3) F. Terlizzi et al. Plant Dis. 90:831, 2006.

Asian prunus viruses: New related members of the family Flexiviridae in Prunus germplasm of Asian origin.

Serological reactivity to Plum pox virus (PPV) antisera has been described in several Prunus sources of Asian origin that are free of PPV infection. Using polyvalent or specific PCR assays, the presence of three closely related agents in two of these sources, Prunus mume cv. Bungo and P. persica cv. Ku Chu'a Hung, was demonstrated. Similarities in genome organization and sequence comparisons indicate that these agents should be regarded as members of the genus Foveavirus, their only singular trait being a very large (>800 nt) 3' non-coding region (NCR), as compared to the ca. 130-180 nt 3' NCR observed in other Foveaviruses. The three agents are very divergent from known Foveaviruses but are also significantly removed one from the others, with overall nucleotide sequence identity levels in the sequenced region of ca. 74-76% and of only 60.8-67.5% in their complete CP gene (61.9-71.3% amino acid sequence identity). Given the species discrimination criteria in the family Flexiviridae, these three agents should be regarded as three related yet distinct new viruses belonging to the Foveavirus genus, for which the names Asian prunus virus 1, 2 and 3 are proposed. Evidence is provided for the presence of variants of these new viruses in other Prunus germplasm of Asian origin.

First Report of a 16SrII Group Phytoplasma Associated with Shoot Proliferation of a Cactus (Opuntia monacantha) in Lebanon.

In October 2003, during a survey to evaluate the incidence of phytoplasma diseases in Lebanon, symptoms suggestive of phytoplasma infection in Opuntia monacantha (Haworth) were observed in Saghbine, Bekaa Valley. Symptoms were excessive stem and shoot proliferation. Three symptomatic and as well as symptomless plants were collected and analyzed for the presence of phytoplasmas. Nucleic acids were extracted from 0.5 g of shoot tissue and tested using polymerase chain reaction (PCR) with universal phytoplasma primers (fU5rU3) for partial amplification of the ribosomal 16SrDNA (4). PCR resulted in amplification of an expected 881-bp rDNA fragment from the symptomatic but not from symptomless samples. For characterization, sequence of the amplified DNA was determined (Genbank Accession No. AY939815). The sequence showed a high similarity with several isolates of the 16srII group of phytoplasmas. The highest similarity has been oserved with 16S rDNA of two isolates of cactus witches'-broom phytoplasma found in China (1) and Mexico (3) (Genbank Accession Nos. AJ293216 and AF320575, respectively) (99.8%) as well as faba bean phyllody phytoplasma (Genbank Accession No. X83432) (99.7%) and "Candidatus Phytoplasma aurantifolia" (Genbank Accession No. U15442) (99.3%). The presence of phytoplasmas was confirmed using nested-PCR with primers R16mF2/R1 and R16F2n/R2 (2). The Tru9I digestion pattern of the amplified product R16F2n/F16R2 detected in O. monacantha was identical to the digestion pattern obtained from periwinkle infected by "Ca. P. aurantifolia" (subgroup 16SrII-B) and soybean phyllody phytoplasma (subgroup 16SrII-C), but different from the Tru9I digestion pattern observed for cleome phyllody phytoplasma (subgroup 16SrII-A) and tomato big bud phytoplasma (subgroup 16SrII-E). To our knowledge, this is the first report of an infection with a phytoplasma belonging to16SrII group in Lebanon. References: (1) H. Cai et al. Plant Pathol. 51:394, 2002. (2) D. E. Gundersen and I. M. Lee. Phytopathol. Mediterr. 35:144, 1996. (3) N. E. Leyva-Lopez et al. Phytopathology. (Abstr.) 89(suppl):S45, 1999. (4) B. Schneider et al. Pages 369-380 in: Molecular and Diagnostic Procedures in Mycoplasmology. Academic Press, NY, 1995.

Putative protein digestion in a sap-sucking homopteran plant pest (rice brown plant hopper; Nilaparvata lugens: Delphacidae)--identification of trypsin-like and cathepsin B-like proteases.

Sap-sucking phytophagous insect species of the order Hemiptera have been assumed not to carry out digestive proteolysis, but instead to rely on free amino acids in the phloem and xylem saps for their nutritional requirements. Extracts prepared from isolated guts of rice brown planthopper (Nilaparvata lugens), a homopteran crop pest, were shown to contain protease activity, with hydrolysis of both protein and synthetic peptide substrates being observed. Assays with specific inhibitors suggested that a trypsin-like serine protease was responsible for most of hydrolytic activity against synthetic substrates. A cDNA library was prepared from RNA extracted from N. lugens gut tissue, and screened for protease-encoding sequences. cDNAs for a cathepsin B-like protease and a trypsin-like protease were isolated and fully characterised; the latter exhibits a novel C-terminal region and an unusual activation mechanism, and represents a small gene family. Soya bean Kunitz trypsin inhibitor (SKTI) is an effective inhibitor of protein hydrolysis by N. lugens gut extracts in vitro, explaining why transgenic rice plants expressing this protein are partially resistant to the insect (Mol. Breed. 5 (1999) 1). It is suggested that digestive proteolysis may be widespread in sap-sucking homoptera, and can make a significant contribution to nutrition.

Molecular characterization of foveaviruses associated with the cherry necrotic mottle leaf disease and complete sequencing of an European isolate of Cherry green ring mottle virus.

Analysis of the P1C124 source (associated with the cherry necrotic mottle leaf (CNML) disease) revealed the presence of two different viral agents. The complete nucleotide sequence of one of these agents, P1A, had an overall nucleotide sequence similarity of 83% with a previously sequenced North American isolate of Cherry green ring mottle virus (CGRMV) and should therefore be regarded as an European isolate of CGRMV. Approximately 2 kb of the 5' end of the genome of the second agent, P1B, were also sequenced and were shown to be 82% homologous with Cherry necrotic rusty mottle virus (CNRMV), another member of the Foveavirus genus. The possible involvement of CGRMV-P1A and of CNRMV-P1B in the etiology of the CNML disease is discussed.

Characterization of two different apricot latent virus variants associated with peach asteroid spot and peach sooty ringspot diseases.

Peach asteroid spot (PAS) and peach sooty ringspot (PSRS) are two diseases of stone fruit trees of unknown aetiology. The use of a cRNA probe of the newly described Apricot latent virus (ApLV), a tentative member of the Foveavirus genus, indicated the presence of cross-hybridizing agents in PAS isolate LA2 and in PSRS isolates Caserta 12 and Clava J4. Analysis of dsRNA patterns revealed in each case the presence of a major dsRNA band of about 9.6 kbp. The purified dsRNAs were used to obtain cDNA clones for isolates LA2 and Caserta 12. Sequence analysis of a 1.1 kbp cDNA clone from isolate LA2 showed very high homology with the known ApLV sequence, indicating that this isolate represents a closely related variant of ApLV. Sequence analysis of a 3.06 kbp Caserta 12 cDNA clone representing the 3' region of the genome revealed a genomic organization similar to that reported for other members of the Foveavirus genus, including the triple gene block and a large, 43.6 kDa coat protein. Sequence comparison with the CP gene of ApLV, the only sequenced region so far for this virus, showed an overall homology of 78%. These results indicate that the foveavirus represented by the Caserta 12 isolate of PSRS disease may be regarded as a distant variant of ApLV. The present results indicate that the viral agents associated with peach asteroid spot and peach sooty ringspot diseases might be variants of the recently described ApLV.

Mycoplasmas, plants, insect vectors: a matrimonial triangle.

Plant pathogenic mycoplasmas were discovered by electron microscopy, in 1967, long after the discovery and culture in 1898 of the first pathogenic mycoplasma of animal origin, Mycoplasma mycoides. Mycoplasmas are Eubacteria of the class Mollicutes, a group of organisms phylogenetically related to Gram-positive bacteria. Their more characteristic features reside in the small size of their genomes, the low guanine (G) plus cytosine (C) content of their genomic DNA and the lack of a cell wall. Plant pathogenic mycoplasmas are responsible for several hundred diseases and belong to two groups: the phytoplasmas and the spiroplasmas. The phytoplasmas (previously called MLOs, for mycoplasma like organisms) were discovered first; they are pleiomorphic, and have so far resisted in vitro cultivation. Phytoplasmas represent the largest group of plant pathogenic Mollicutes. Only three plant pathogenic spiroplasmas are known today. Spiroplasma citri, the agent of citrus stubborn was discovered and cultured in 1970 and shown to be helical and motile. S. kunkelii is the causal agent of corn stunt. S. phoeniceum, responsible for periwinkle yellows, was discovered in Syria. There are many other spiroplasmas associated with insects and ticks. Plant pathogenic mycoplasmas are restricted to the phloem sieve tubes in which circulates the photosynthetically-enriched sap, the food for many phloem-feeding insects (aphids, leafhoppers, psyllids, etc.). Interestingly, phytopathogenic mycoplasmas are very specifically transmitted by leafhoppers or psyllid species. In this paper, the most recent knowledge on phytopathogenic mycoplasmas in relation with their insect and plant habitats is presented as well as the experiments carried out to control plant mycoplasma diseases, by expression of mycoplasma-directed-antibodies in plants (plantibodies).

Characterisation of two citrus apscaviroids isolated in Spain.

Sequence variability in the PCR amplified cDNAs from two citrus apscaviroid isolates CVd-Ia and CVd-IIId from Spain, was analysed. CVd-IIId sequence was shown to be identical to previously described CVd-III sequences and no important variability was encountered within the viroid population. Conversely, CVd-Ia displayed population heterogeneity as shown by SSCP analysis, Sal I restriction site polymorphism and sequences of 27 CVd-Ia cloned DNAs. The CVd-Ia genomic heterogeneity is characterised by two major subpopulations with the most divergent sequences, and by the presence of individual variants, making a sequence continuum between the two major groups. Most sequence variations are clustered in the left part of the viroid molecule.

Cloning of the spoT gene of "Candidatus Phlomobacter fragariae" and development of a PCR-restriction fragment length polymorphism assay for detection of the bacterium in insects.

Marginal chlorosis is a new disease of strawberry in which the uncultured phloem-restricted proteobacterium "Candidatus Phlomobacter fragariae" is involved. In order to identify the insect(s) vector(s) of this bacterium, homopteran insects have been captured. Because a PCR test based on the 16S rRNA gene (rDNA) applied to these insects was unable to discriminate between "P. fragariae" and other insect-associated proteobacteria, isolation of "P. fragariae" genes other than 16S rDNA was undertaken. Using comparative randomly amplified polymorphic DNAs, an amplicon was specifically amplified from "P. fragariae"-infected strawberry plants. It encodes part of a "P. fragariae" open reading frame sharing appreciable homology with the spoT gene from other proteobacteria. A spoT-based PCR test combined with restriction fragment length polymorphisms was developed and was able to distinguish "P. fragariae" from other insect bacteria. None of the many leafhoppers and psyllids captured during several years in and around infected strawberry fields was found to carry "P. fragariae." Interestingly however, the "P. fragariae" spoT sequence could be easily detected in whiteflies proliferating on "P. fragariae"-infected strawberry plants under confined greenhouse conditions but not on control whiteflies, indicating that these insects can become infected with the bacterium.

Ferritin acts as the most abundant binding protein for snowdrop lectin in the midgut of rice brown planthoppers (Nilaparvata lugens).

The mannose-specific snowdrop lectin [Galanthus nivalis agglutinin (GNA)] displays toxicity to the rice brown planthopper Nilaparvata lugens. A 26kDa GNA-binding polypeptide from N. lugens midgut was identified by lectin blotting and affinity chromatography, and characterized by N-terminal sequencing. This polypeptide is the most abundant binding protein for GNA in the N. lugens midgut. A cDNA (fersub2) encoding this protein was isolated from an N. lugens cDNA library. The deduced amino acid sequence shows significant homology to ferritin subunits from Manduca sexta and other arthropods, plants and vertebrates, and contains a putative N-glycosylation site. Native ferritin was purified from whole insects as a protein of more than 400kDa in size and characterized biochemically. Three subunits of 20, 26 and 27kDa were released from the native complex. The 26kDa subunit binds GNA, and its N-terminal sequence was identical to that of fersub2. A second cDNA (fersub1), exhibiting strong homology with dipteran ferritin, was identified as an abundant cDNA in an N. lugens midgut-specific cDNA library, and could encode the larger ferritin subunit. The fersub1 cDNA carries a stem-loop structure (iron-responsive element) upstream from the start codon, similar to structures that have been shown to play a role in the control of ferritin synthesis in other insects.

Resistance to green leafhopper (Nephotettix virescens) and brown planthopper (Nilaparvata lugens) in transgenic rice expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA).

Transgenic rice plants expressing snowdrop lectin (Galanthus nivalis agglutinin; GNA) were screened for resistance to green leafhopper (Nephotettix virescens; GLH), a major homopteran pest of rice. Survival was reduced by 29% and 53% (P<0.05) respectively, on plants where GNA expression was tissue-specific (phloem and epidermal layer) or constitutive. Similar levels of resistance in GNA-expressing transgenic rice were previously reported for rice brown planthopper (Nilaparvata lugens; BPH). GNA binding to glycoproteins in gut tissues showed that BPH contained more "receptors" than GLH, and that the binding affinity was stronger, particularly in the midgut. Subsequent toxicity of GNA is thus unlikely to be directly related to the amount of lectin bound. GNA was not detected in the honeydew of either insect species when they were fed on GNA-expressing plants, in contrast to results from artificial diet studies. This result suggests that GNA is not being delivered to the insect efficiently. When offered a free choice vs control plants, BPH nymphs tended to avoid plants expressing GNA; avoidance was less pronounced and took longer to develop on plants where GNA expression was tissue-specific, In contrast to BPH, GLH nymphs were attracted to plants expressing GNA, whether constitutively or in a tissue-specific manner.

Sequence analysis of Spiroplasma phoeniceum and Spiroplasma kunkelii spiralin genes and comparison with other spiralin genes.

The spiralin genes from two phytopathogenic spiroplasmas, Spiroplasma phoeniceum and Spiroplasma kunkelii, were amplified by PCR, cloned, and sequenced. Comparison of the amino acid sequences of the five spiralins analyzed to date confirm that the spiralins have a general amphiphilic character and possess a conserved lipoprotein signal peptide. It also shows that a conserved central region and an amino acid repetition, including a VTKXE consensus sequence, are present in all spiralins analyzed.

Random insertion of transposon Tn4001 in the genome of Spiroplasma citri strain GII3.

Electroporation of Spiroplasma citri strain GII3 with plasmid pMUT containing the Staphylococcus aureus transposon Tn4001 resulted in random insertion of Tn4001 into the spiroplasmal genome. Transformation frequencies reached 10(-8) per colony-forming unit (CFU) when 100 microg of plasmid DNA and 3 x 10(9) S. citri CFU were used. Three other strains of S. citri failed to be transformed under the same conditions. In most cases Tn4001 was randomly inserted in the genome of S. citri strain GII3, without insertion of the carrier plasmid. For most transformed spiroplasmas, Tn4001 was stably maintained in the absence of antibiotic selection for at least 80 bacterial generations, making Tn4001 a potential tool for S. citri mutagenesis.

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

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 [3H]palmitic acid, at least 10 proteins, including spiralin, became labeled. In the presence of globomycin, a lipoprotein signal peptidase inhibitor in eubacteria, apparently unprocessed spiralin could be detected. Formic acid hydrolysis of the [3H]palmitic acid-labeled spiralins of four representative S. citri strains yielded two peptide fragments for each spiralin, as expected from the gene sequence. On fragment was [3H]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 (delta Da approximately equal to 800 Da); however, these were much smaller than those of the whole spiralins before formic acid hydrolysis (delta Da approximately equal to 8,000 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.

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.