I-SceI-mediated plasmid deletion and intra-molecular recombination in Spiroplasma citri

S. citri wild-type strain GII3 carries six plasmids (pSci1 to -6) that are thought to encode determinants involved in the transmission of the spiroplasma by its leafhopper vector. In this study we report the use of meganuclease I-SceI for plasmid deletion in S. citri. Plasmids pSci1NT-I and pSci6PT-I, pSci1 and pSci6 derivatives that contain the tetM selection marker and a unique I-SceI recognition site were first introduced into S. citri strains 44 (having no plasmid) and GII3 (carrying pSci1-6), respectively. Due to incompatibility of homologous replication regions, propagation of the S. citri GII3 transformant in selective medium resulted in the replacement of the natural pSci6 by pSci6PT-I. The spiroplasmal transformants were further transformed by an oriC plasmid carrying the I-SceI gene under the control of the spiralin gene promoter. In the S. citri 44 transformant, expression of I-SceI resulted in rapid loss of pSciNT-I showing that expression of I-SceI can be used as a counter-selection tool in spiroplasmas. In the case of the S. citri GII3 transformant carrying pSci6PT-I, expression of I-SceI resulted in the deletion of plasmid fragments comprising the I-SceI site and the tetM marker. Delineating the I-SceI generated deletions proved they had occurred though recombination between homologous sequences. To our knowledge this is the first report of I-SceI mediated intra-molecular recombination in mollicutes.     

Involvement of a minimal actin-binding region of Spiroplasma citri phosphoglycerate kinase in spiroplasma transmission by its leafhopper vector

Background

Spiroplasma citri is a wall-less bacterium that colonizes phloem vessels of a large number of host plants. Leafhopper vectors transmit S. citri in a propagative and circulative manner, involving colonization and multiplication of bacteria in various insect organs. Previously we reported that phosphoglycerate kinase (PGK), the well-known glycolytic enzyme, bound to leafhopper actin and was unexpectedly implicated in the internalization process of S. citri into Circulifer haematoceps cells.

Methodology/Principal Findings

In an attempt to identify the actin-interacting regions of PGK, several overlapping PGK truncations were generated. Binding assays, using the truncations as probes on insect protein blots, revealed that the actin-binding region of PGK was located on the truncated peptide designated PGK-FL5 containing amino acids 49–154. To investigate the role of PGK-FL5-actin interaction, competitive spiroplasma attachment and internalization assays, in which His₆-tagged PGK-FL5 was added to Ciha-1 cells prior to infection with S. citri, were performed. No effect on the efficiency of attachment of S. citri to leafhopper cells was observed while internalization was drastically reduced. The in vivo effect of PGK-FL5 was confirmed by competitive experimental transmission assays as injection of PGK-FL5 into S. citri infected leafhoppers significantly affected spiroplasmal transmission.

Conclusion

These results suggest that S. citri transmission by its insect vector is correlated to PGK ability to bind actin.     

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

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 His₆-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.Phloem-feeding leafhoppers transmit plant pathogenic mollicutes, spiroplasmas, and phytoplasmas from plant to plant in a persistent propagative manner (26, 43). These phytopathogenic mollicutes are restricted to phloem and to certain vector tissues; thus, their vectors are phloem sap-feeding specialists. After being ingested from plant phloem by their insect vectors, they traverse the insect gut wall, move into the hemolymph, where they multiply, and invade the salivary glands (20, 33, 34, 36). During their movements in the insect vector until its transmission to a new host plant, spiroplasmas and phytoplasmas must traverse two major physical barriers, namely, the insect intestine and the salivary gland (35, 53). Until now, little was known about the molecular and cellular interactions contributing to the crossing of these physical barriers. Several lines of evidence suggest that host-pathogen interactions could be a prerequisite for invasion and colonization of insect vector organs (2, 48, 53). For human and animal pathogenic mollicutes, it is well established that successful colonization of the host cells requires adhesion as the first step. This event is mediated by surface proteins, and among these proteins adhesins play an important role (8, 44). Recently, it was reported that an antigenic membrane protein (Amp) of onion yellow phytoplasma interacts with the insect microfilament complex and that interaction plays an important role in determining the insect vector specificity (48). Several other immunodominant membrane proteins from various phytoplasmas have been mentioned in the literature as candidates for involvement in host-phytoplasma interactions (29, 30).Spiroplasma citri, the first phytopathogenic mollicute available in culture (45), has emerged as an outstanding model for studying spiroplasma interactions with its two hosts: the periwinkle plant and the insect vector Circulifer haematoceps. Following observations of membrane-bound cytoplasmic vesicles of midgut epithelium and salivary gland cells, S. citri was hypothesized to cross these physical barriers by receptor-mediated cell endocytosis (3, 33, 39). Several S. citri protein candidates have been identified as involved in transmission and, for a few of them, in an interaction with leafhopper vector proteins. Spiralin, the most abundant membrane protein, was suspected to be involved in the transmission for two reasons: (i) a S. citri spiralinless mutant was less effective in its transmissibility (19); (ii) spiralin acted in vitro as a lectin able to bind to glycoproteins of insect vectors and therefore might function as a ligand able to interact with leafhopper receptors (32). In addition, the ability of S. citri to be transmitted by C. haematoceps is clearly affected by disruption of a gene predicted to encode a lipoprotein with homology to a solute-binding protein of an ABC transporter (14). The proteome of nontransmissible S. citri strains specifically lacks adhesion-related proteins (ScARPs) and the membrane-associated protein P32 present in the proteome of transmissible strains (12, 13, 31). These proteins are encoded by plasmids pSci1 to -6 (46), which are present only in transmissible strains, and ScARPs share strong similarities with the adhesion-related protein SARP1 of S. citri strain BR3, in which the presence has been correlated to the ability for the spiroplasma to adhere to insect cells in vitro (9, 55). The specific interactions of S. citri with eukaryotic cells remain to be elucidated, but a combination of the effects of several proteins or a complex would be necessary to explain the invasion of a variety of host cell types by S. citri (33).Nevertheless, in the last sequence of events involved in insect vector transmission, the first contact and recognition for the efficient penetration of the salivary gland cells represents an essential step. In the present study, confocal images of infected salivary glands show the localization of S. citri cells along the actin filaments. We report the results of the first attempt to decipher the role of the spiroplasma''s phosphoglycerate kinase (PGK) in the internalization of S. citri in its insect vector''s cells.     

Partial Chromosome Sequence of Spiroplasma citri Reveals Extensive Viral Invasion and Important Gene Decay

The assembly of 20,000 sequencing reads obtained from shotgun and chromosome-specific libraries of the Spiroplasma citri genome yielded 77 chromosomal contigs totaling 1,674 kbp (92%) of the 1,820-kbp chromosome. The largest chromosomal contigs were positioned on the physical and genetic maps constructed from pulsed-field gel electrophoresis and Southern blot hybridizations. Thirty-eight contigs were annotated, resulting in 1,908 predicted coding sequences (CDS) representing an overall coding density of only 74%. Cellular processes, cell metabolism, and structural-element CDS account for 29% of the coding capacity, CDS of external origin such as viruses and mobile elements account for 24% of the coding capacity, and CDS of unknown function account for 47% of the coding capacity. Among these, 21% of the CDS group into 63 paralog families. The organization of these paralogs into conserved blocks suggests that they represent potential mobile units. Phage-related sequences were particularly abundant and include plectrovirus SpV1 and SVGII3 and lambda-like SpV2 sequences. Sixty-nine copies of transposases belonging to four insertion sequence (IS) families (IS30, IS481, IS3, and ISNCY) were detected. Similarity analyses showed that 21% of chromosomal CDS were truncated compared to their bacterial orthologs. Transmembrane domains, including signal peptides, were predicted for 599 CDS, of which 58 were putative lipoproteins. S. citri has a Sec-dependent protein export pathway. Eighty-four CDS were assigned to transport, such as phosphoenolpyruvate phosphotransferase systems (PTS), the ATP binding cassette (ABC), and other transporters. Besides glycolytic and ATP synthesis pathways, it is noteworthy that S. citri possesses a nearly complete pathway for the biosynthesis of a terpenoid.Spiroplasmas are arthropod-associated bacteria belonging to the class Mollicutes, a group of wall-less microorganisms phylogenetically related to low-G+C-content, Gram-positive bacteria (51). Spiroplasma citri is a helical plant-pathogenic mollicute responsible for the “stubborn” disease of citrus (39). It inhabits the phloem sap of infected plants to which it is transmitted by sap-sucking hemipteran insect in a circulative and propagative manner (31, 32). S. citri can infect a wide range of plant species, including crop and wild plants, as it is transmitted by polyphagous leafhoppers (13). Spiroplasmas are available in pure culture, and their study has therefore benefited from the use of molecular genetics. In particular, the relationships of spiroplasmas with their two hosts, the plant and the leafhopper vector, have been extensively studied (11, 22). In S. citri, the inactivation of genes and functional complementation of mutants have shown that (i) fructose consumption by the spiroplasma is a major cause for symptom production in plants, (ii) the solute binding protein of a putative ABC-type transporter is involved in the insect transmission process, and (iii) spiralin, the major membrane protein, is not essential for helicity, motility, and pathogenicity but is required for efficient transmission by the leafhopper vector (10, 19, 23, 24, 28). To characterize other spiroplasma genes potentially involved in insect transmission and pathogenicity, the genome of S. citri strain GII3-3X is currently being deciphered.The S. citri genome is characterized by an abundance of extrachromosomal elements, including seven plasmids, pSciA and pSci1 to pSci6, present as 10 to 14 copies per cell. These plasmids are vertically inherited, but some of them could also be horizontally transferred, as they encode proteins involved in partitioning and the cell-to-cell transfer of DNA molecules (12, 40). Plasmids pSci1 to pSci5 encode surface proteins of the S. citri adhesion-related protein (ScARP) family, and pSci6 was previously shown to confer insect transmissibility (9). Therefore, it is likely that the abundance and diversity of plasmids could provide S. citri strain GII3-3X with the ability to quickly adapt to various vector insects and, hence, to be transmitted to diverse host plants. However, chromosome-encoded determinants are also expected to play a role in spiroplasma biology. In S. citri, the chromosome sizes vary from 1.6 to 1.9 Mbp among strains (53, 54), and part of the size variation is thought to result from different amounts of prophage sequences (35). Many S. citri strains are infected by single-stranded DNA-containing filamentous phages (Plectrovirus), whose sequences also occur as partial or full-length prophages integrated into the spiroplasma chromosome (7, 35, 38). Here we report the partial chromosome sequence of S. citri strain GII3-3X and the functional assignment of the predicted coding sequences.     

Invasion of insect cells by Spiroplasma citri involves spiralin relocalization and lectin/glycoconjugate‐type interactions

Spiroplamas are helical, cell wall‐less bacteria belonging to the Class Mollicutes, a group of microorganisms phylogenetically related to low G+C, Gram‐positive bacteria. Spiroplasma species are all found associated with arthropods and a few, including Spiroplasma citri are pathogenic to plant. Thus S. citri has the ability to colonize cells of two very distinct hosts, the plant and the insect vector. While spiroplasmal factors involved in transmission by the leafhopper Circulifer haematoceps have been identified, their specific contribution to invasion of insect cells is poorly understood. In this study we provide evidence that the lipoprotein spiralin plays a major role in the very early step of cell invasion. Confocal laser scanning immunomicroscopy revealed a relocalization of spiralin at the contact zone of adhering spiroplasmas. The implication of a role for spiralin in adhesion to insect cells was further supported by adhesion assays showing that a spiralin‐less mutant was impaired in adhesion and that recombinant spiralin triggered adhesion of latex beads. We also showed that cytochalasin D induced changes in the surface‐exposed glycoconjugates, as inferred from the lectin binding patterns, and specifically improved adhesion of S. citri wild‐type but not of the spiralin‐less mutant. These results indicate that cytochalasin D exposes insect cell receptors of spiralin that are masked in untreated cells. In addition, competitive adhesion assays with lectins strongly suggest spiralin to exhibit glycoconjugate binding properties similar to that of the Vicia villosa agglutinin (VVA) lectin.     

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.     

Disruption of a Gene Predicted To Encode a Solute Binding Protein of an ABC Transporter Reduces Transmission of Spiroplasma citri by the Leafhopper Circulifer haematoceps

Spiroplasma citri is transmitted from plant to plant by phloem-feeding leafhoppers. In an attempt to identify mechanisms involved in transmission, mutants of S. citri affected in their transmission must be available. For this purpose, transposon (Tn4001) mutagenesis was used to produce mutants which have been screened for their ability to be transmitted by the leafhopper vector Circulifer haematoceps to periwinkle plants. With one mutant (G76) which multiplied in leafhoppers as efficiently as S. citri wild-type (wt) strain GII-3, the plants showed symptoms 4 to 5 weeks later than those infected with wt GII-3. Thirty to fifty percent of plants exposed to leafhoppers injected with G76 remained symptomless, whereas for wt GII-3, all plants exposed to the transmission showed severe symptoms. This suggests that the mutant G76 was injected into plants by the leafhoppers less efficiently than wt GII-3. To check this possibility, the number of spiroplasma cells injected by a leafhopper through a Parafilm membrane into SP4 medium was determined. Thirty times less mutant G76 than wt GII-3 was transmitted through the membrane. These results suggest that mutant G76 was affected either in its capacity to penetrate the salivary glands and/or to multiply within them. In mutant G76, transposon Tn4001 was shown to be inserted into a gene encoding a putative lipoprotein (Sc76) In the ABCdb database Sc76 protein was noted as a solute binding protein of an ABC transporter of the family S1_b. Functional complementation of the G76 mutant with the Sc76 gene restored the wild phenotype, showing that Sc76 protein is involved in S. citri transmission by the leafhopper vector C. haematoceps.     

Spiralin: Major membrane protein specific for subgroup I-1 Spiroplasmas

Preparations of spiralin from membranes ofSpiroplasma citri, strain C189, purified by sequential solubilization with detergents followed by agarose-suspension electrophoresis induced rabbit antibodies that were largely specific forSpiroplasma citri Group I-1 spiroplasmas, as demonstrated by metabolic inhibition (MI), growth inhibition (GI), and deformation (DF) tests. By contrast, antibodies againstS. citri whole-membrane protein preparations reacted broadly with representative type cultures of seven subgroups of theS. citri complex. Neither antimembrane nor antispiralin sera reacted withS. floricola, S. mirum, or Group IV, (VI), (VII), or (VIII) spiroplasmas. Minor cross-reactions in MI and DF tests between antispiralin serum and Subgroup I-2 and I-3 antigens may have represented shared epitopes in a set of homologous membrane proteins of the three spiroplasmas, or antibodies against highly antigenic traces of other common membrane proteins in the purified spiralin preparations. The unique antigenic properties of spiralin, the most abundant protein in theS. citri membrane, explain in part the unique profiles shown by this spiroplasma species in comparative taxonomic serological tests.     

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.     

Chemically Defined Medium for Cultivation of Several Epiphytic and Phytopathogenic Spiroplasmas

A chemically defined medium, LD82, was formulated for in vitro cultivation of spiroplasmas. Medium LD82 supported good growth for four epiphytic and insect-pathogenic spiroplasmas, Spiroplasma floricola 23-6^T, Spiroplasma sp. strain SR3, Spiroplasma sp. strain brevi, and Spiroplasma sp. strain AS576, and of the phytopathogenic spiroplasmas Spiroplasma citri Maroc R8A2^T and PC1. Titers of all six strains grown in defined medium LD82 reached 2.0 × 10⁹ to 6.0 × 10⁹ CFU/ml of culture. All spiroplasma strains tested formed colonies readily on agar medium LD82. None of the spiroplasmas formed typical fried-egg colonies. All formed diffuse colonies, but the forms of colonies differed somewhat among the spiroplasma strains. In preliminary studies of nutritional requirements, phospholipids slightly enhanced the growth of the epiphytic and insect-pathogenic strains in medium LD82 and were found essential for good growth of S. citri.     

Effects of infection with a spiroplasma virus on the symptoms produced by Spiroplasma citri

Multiplication of a virus resembling spiroplasma-virus citri type 3 was observed in Spiroplasma citri cells within an infected Madagascar periwinkle plant showing unusually mild symptoms. Sap filtrates from this plant contained virus particles which gave rise to plaques on lawns of the lethal SP-A strain of S. citri. Transmission of spiroplasmas containing virus to plants already infected with strain SP-A resulted in suppression of symptoms and a reduction in the number of viable spiroplasmas.     

Effect of Polyclonal, Monoclonal, and Recombinant (Single-Chain Variable Fragment) Antibodies on In Vitro Morphology, Growth, and Metabolism of the Phytopathogenic Mollicute Spiroplasma citri

Antibodies are known to affect the morphology, growth, and metabolism of mollicutes and thus may serve as candidate molecules for a plantibody-based control strategy for plant-pathogenic spiroplasmas and phytoplasmas. Recombinant single-chain variable fragment (scFv) antibodies are easy to engineer and express in plants, but their inhibitory effects on mollicutes have never been evaluated and compared with those of polyclonal and monoclonal antibodies. We describe the morphology, growth, and glucose metabolism of Spiroplasma citri in the presence of polyclonal, monoclonal, and recombinant antibodies directed against the immunodominant membrane protein spiralin. We showed that the scFv antibodies had no effect on S. citri glucose metabolism but were as efficient as polyclonal antibodies in inhibiting S. citri growth in liquid medium. Inhibition of motility was also observed.     

Identification of surface proteins ofSpiroplasma citri with protease and antibody probes

Spiroplasma citri, a helical, wall-less prokaryote, is an insect-borne phytopathogen. Though proteins having domains on the surface ofS. citri cells may be important in pathogenicity or transmissibility, only one surface protein, spiralin (29 KDa), has previously been identified. Intact cells of strain BR3 were treated with chymotrypsin, proteinase K, or trypsin, and the surviving proteins were analyzed by SDS-PAGE. Seven proteins, in addition to spiralin, were degraded, indicative of surface exposure of those polypeptides. Surface immunoprecipitation (SIP) was used to test accessibility of the proteins to anti-S. citri membrane serum, another indication of surface exposure. With unlabeled cells, five such proteins were identified. Four of these have sizes that correspond to those seen with protease treatments. When¹²⁵I surfacelabeled spiroplasmas were used for SIP, twelve surface proteins were detected, eight of which correspond to bands identified by the other methods. A protein of 89 KDa in strain BR3 was not universally detected in otherS. citri strains and spiroplasma species.     

The spiroplasma deformation test,a new serological method

The deformation test is a simple and highly sensitive technique capable of demonstrating significant antigenic differences among helical, wall-less prokaryotes (spiroplasmas). Specific identified. Quantitative relationships among various antisera are determined by examining, under dark-field microscopy, samples containing serum dilutions and a measured number of organisms which is held constant in each test. Antisera dilutions of 1:2,000 to 1;16,000 deformed spiroplasmas in homologous tests involvingSpiroplasma citri and the corn stunt and suckling mouse cataract spiroplasmas. With the exception of some heterologous cross-reactions in the deformation test betweenS. citri and corn stunt spiroplasmas, antisera and preimmunization sera failed to deform heterologous spiroplasmas at dilutions higher than 1:16.     

Growth inhibition of phytopathogenic spiroplasmas by membrane-interactive antimicrobial peptides Novispirin T7 and Caerin 1.1

Spiroplasma kunkelii and Spiroplasma citri, both helical-shaped cell wall-less bacteria, are the causative agents of corn stunt disease and citrus stubborn disease, respectively. Plants exhibiting natural resistance to these phytopathogenic spiroplasmas are currently lacking. Engineering artificial plant resistance using antimicrobial peptides (AMPs) has been conceived as a new approach to control the agronomically important spiroplasmal diseases. In preparation for such task, the present study focused on screening of AMPs that have potentials to curb the growth of S. kunkelii and S. citri. Four AMPs, including Novispirin T7, Caerin 1.1, Tricholongin and Dhvar4, were selected for in vitro growth inhibition test. A liquid assay method was developed for quick qualitative and quantitative evaluations of the AMPs. Our results demonstrated that Novispirin T7 and Caerin 1.1 were able to inhibit the growth of both phytopathogenic spiroplasmas with the efficacy comparable to that of tetracycline. Cell deformations were observed in spiroplasma cultures treated with these two peptides, indicating interactions of the AMPs with the spiroplasma cell membranes. The minimum inhibitory concentrations (MICs) of the AMPs against S. kunkelii and S. citri were determined.     

Xylella fastidiosa Afimbrial Adhesins Mediate Cell Transmission to Plants by Leafhopper Vectors

The interactions between the economically important plant-pathogenic bacterium Xylella fastidiosa and its leafhopper vectors are poorly characterized. We used different approaches to determine how X. fastidiosa cells interact with the cuticular surface of the foreguts of vectors. We demonstrate that X. fastidiosa binds to different polysaccharides with various affinities and that these interactions are mediated by cell surface carbohydrate-binding proteins. In addition, competition assays showed that N-acetylglucosamine inhibits bacterial adhesion to vector foregut extracts and intact wings, demonstrating that attachment to leafhopper surfaces is affected in the presence of specific polysaccharides. In vitro experiments with several X. fastidiosa knockout mutants indicated that hemagglutinin-like proteins are associated with cell adhesion to polysaccharides. These results were confirmed with biological experiments in which hemagglutinin-like protein mutants were transmitted to plants by vectors at lower rates than that of the wild type. Furthermore, although these mutants were defective in adhesion to the cuticle of vectors, their growth rate once attached to leafhoppers was similar to that of the wild type, suggesting that these proteins are important for initial adhesion of X. fastidiosa to leafhoppers. We propose that X. fastidiosa colonization of leafhopper vectors is a complex, stepwise process similar to the formation of biofilms on surfaces.     

Characterization of Spiroplasmas by one- and two-dimensional protein analysis on polyacrylamide slab gels

Differences betweenSpiroplasma citri isolates were detected by one-dimensional electrophoresis of proteins on gradient polyacrylamide slab gels. Two-dimensional protein maps (electrofocusing followed by electrophoresis) showed a highly characteristic pattern for allS. citri isolates examined. Coanalysis of mixed protein samples from pairs ofS. citri strains revealed more than 150 comigrating proteins common to allS. citri isolates, but also a number of noncomigrating proteins. Some noncomigrating proteins were present in one isolate but not in another, while other proteins whose migrational properties were only slightly different from one isolate to the other (homologous proteins), were present in more than one isolate.S. citri isolates had many common and only a few homologous proteins. Comparisons ofS. citri with the corn stunt spiroplasma revealed few common proteins and a large number of homologous proteins. When comparingS. citri and the suckling mouse cataract spiroplasma, few common and homologous proteins were apparent. However, several of these common proteins were also shared by the corn stunt spiroplasma, suggesting that they may well represent genus-specific proteins. The data also offer additional evidence that the suckling mouse cataract spiroplasma differs significantly fromS. citri and corn stunt spiroplasmas and probably deserves a separate species designation.     

Powder puff spiroplasma: A new epiphytic mycoplasma

A spiroplasma (strain PPS1) isolated from healthy flowers ofCalliandra haematocephala in Florida has been found to be a member of a serogroup of the Spiroplasmataceae. It is distinct fromSpiroplasma citri and from other described spiroplasmas as determined by growth inhibition, fluorescent antibody, and ELISA serological tests. PPS1 was also distinguished fromS. citri and several other spiroplasmas by the guanine + cytosine content of its DNA. PPS1 requires sterol for growth, is inhibited by digitonin, grows at 20–30°C, and does not hydrolyze arginine or urea. The ready isolation of this and similar organisms from surfaces of healthy plants emphasizes that caution should be exercised in attempts to isolate cell wall-less prokaryotes from the interior of diseased plants. Although some strains of spiroplasmas are known as insect pathogens in nature, the ecological role(s) of the flower-inhabiting spiroplasmas has yet to be fully determined.     

Streptococcus pyogenes CAMP factor promotes bacterial adhesion and invasion in pharyngeal epithelial cells without serum via PI3K/Akt signaling pathway

Streptococcus pyogenes is a bacterium that causes systemic diseases, such as pharyngitis and toxic shock syndrome, via oral- or nasal-cavity infection. S. pyogenes produces various molecules known to function with serum components that lead to bacterial adhesion and invasion in human tissues. In this study, we identified a novel S. pyogenes adhesin/invasin. Our results revealed that CAMP factor promoted streptococcal adhesion and invasion in pharyngeal epithelial Detroit562 cells without serum. Recombinant CAMP factor initially localized on the membranes of cells and then became internalized in the cytosol following S. pyogenes infection. Additionally, CAMP factor phosphorylated phosphoinositide 3-kinase and serine–threonine kinase in the cells. ELISA results demonstrate that CAMP factor affected the amount of phosphorylated phosphoinositide 3-kinase and serine–threonine kinase in Detroit562 cells. Furthermore, CAMP factor did not reverse the effect of phosphoinositide 3-kinase knockdown by small interfering RNA in reducing the level of adhesion and invasion of S. pyogenes isogenic cfa-deficient mutant. These results suggested that S. pyogenes CAMP factor activated the phosphoinositide 3-kinase/serine–threonine kinase signaling pathway, promoting S. pyogenes invasion of Detroit562 cells without serum. Our findings suggested that CAMP factor played an important role on adhesion and invasion in pharyngeal epithelial cells.     

Identity of cactus and lettuce spiroplasmas withSpiroplasma citri as determined by DNA-DNA hybridization

The isolation of spiroplasma strains from the cactusOpuntia tuna monstrosa and from aster yellows-diseased lettuce is described. DNA from these strains (ATCC 29594 and ATCC 29747) is compared with DNA fromSpiroplasma citri, and from the corn stunt and suckling mouse cataract spiroplasmas. The cactus and the lettuce isolates are found to be identical withS. citri by this method.