Characterizing the replication and stability regions of Spiroplasma citri plasmids identifies a novel replication protein and expands the genetic toolbox for plant-pathogenic spiroplasmas

Abstract

Spiroplasma citri strain GII3 contains seven plasmids, pSciA and pSci1-6, that share extensive regions of sequence homology and display a mosaic gene organization. Plasmid pSci2 comprises 12 coding sequences (CDS), three of which encode polypeptides homologous to proteins Soj/ ParA, involved in chromosome partitioning, and TrsE and Mob/TraG, implicated in the type IV secretion pathway. One CDS encodes the adhesin-like protein ScARP3d whereas the other eight encode polypeptides with no homology to known proteins. The pSci2 CDS pE and soj have counterparts in all seven plasmids. Through successive deletions, various pSci2 derivatives were constructed and assessed for their ability to replicate by transformation of S. citri 44, a strain which has no plasmid. The smallest functional replicon was found to contain a single CDS (pE) and its flanking intergenic regions. Shuttle (S. citri/Escherichia coli) plasmids, in which CDS pE was disrupted, failed to replicate in S. citri, suggesting that PE is the replication protein of the S. citri plasmids. Successive propagations of pSci2-derived transformed spiroplasmas, in the absence of selection pressure, revealed that only pSci2 derivatives having an intact soj gene were stably maintained, indicating that the soj-encoded polypeptide is most likely involved in plasmid partitioning. Upon transformation, pSci2 derivatives, including shuttle (S. citri/E. coli) plasmids, were shown to replicate in all S. citri strains tested regardless of whether the strain possesses endogenous plasmids, such as strain GII3, or not, such as strain R8A2. In addition, the pSci replicons were introduced efficiently into the plant-pathogenic spiroplasmas Spiroplasma kunkelii and Spiroplasma phoeniceum, the transformation of which had never, to our knowledge, been described before. These studies show that, besides their implications for the biology of S. citri, the pSci plasmids hold considerable promise as vectors of general use for genetic studies of plantpathogenic spiroplasmas. As an example, a HA-tagged S. citri protein was expressed in S. kunkelii. Detection of pE-hybridizing sequences in various group I spiroplasma species indicated that pE replicating plasmids were not restricted to the three plant-pathogenic spiroplasmas. INTRODUCTIONSpiroplasmas are a group of helical, cell-wall-free bacteria belonging to the class Mollicutes. Three spiroplasma species, Spiroplasma citri (Saglio et al., 1973), Spiroplasma kunkelii (Whitcomb et al., 1986) and Spiroplasma phoeniceum (Saillard et al., 1987), have been associated with plant diseases. S. citri is the aetiological agent of the citrus stubborn and horseradish brittle root diseases and was the first plant mollicute to be cultured in vitro (Fletcher et al., 1981;Saglio et al., 1971). Since then, S. citri has been studied extensively (Bové et al., 1989(Bové et al., , 2003. The availability of both an experimental transmission assay via the leafhopper vector (Foissac et al., 1996) and gene transfer systems (Foissac et al., 1997;Renaudin, 2002;Renaudi...

Mobility of Plasmids

“…In other clades, the smallest VirB4-containing plasmid was pSci1, with only 13 kb, found in Spiroplasma citri GII3. This strain harbors several plasmids for which transmission has been demonstrated, but it is unclear if the plasmid codes for a selfsufficient T4SS (17). Mollicutes contain small conjugative elements measuring much less than 30 kb (23, 98, 107).…”

Sequences Essential for Transmission of Spiroplasma citri by Its Leafhopper Vector, Circulifer haematoceps , Revealed by Plasmid Curing and Replacement Based on Incompatibility

“…Plasmids pSci6PT, pSci21NT, pFL4, and pFL5 have been described previously (4,8). Plasmid pSci6B resulted from self-ligation of the 18,487-bp BstBI restriction fragment of pSci6PT (Fig.…”

“…As a result, transformation of S. citri GII3 by the pSci2 derivatives yielded a large set of spiroplasmal mutants differing in their plasmid contents. We have previously established that, due to defective partitioning, pSci2 derivatives lacking soj are lost during propagation of the spiroplasmal transformants in the absence of selection (8). Indeed, further propagation of pFL4 or pFL5 transformants in nonselective medium resulted in the loss of these tetM-marked plasmids, yielding spiroplasmas with native plasmids, exclusively.…”

“…1). This region comprises 12 CDSs, including two truncated copies of CDS E (proved to be the replication protein) (8), two copies of the CDS cluster K traG M, CDS N, two hypothetical CDSs, and a truncated copy of CDS F. In pSci6BA, the 10.4-kbp NciI fragment was further reduced to a 4.6-kbp region, in which the traG and M CDSs were the only CDSs specifically encoded by pSci6 (Fig. 1).…”

“…Previously, we characterized the replication and stability regions of the S. citri GII3 plasmids and showed that each plasmid has its own partition system involving the Soj protein (8).…”