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Abstract

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 and have counterparts in all seven plasmids. Through successive deletions, various pSci2 derivatives were constructed and assessed for their ability to replicate by transformation of 44, a strain which has no plasmid. The smallest functional replicon was found to contain a single CDS () and its flanking intergenic regions. Shuttle (/) plasmids, in which CDS was disrupted, failed to replicate in , suggesting that PE is the replication protein of the plasmids. Successive propagations of pSci2-derived transformed spiroplasmas, in the absence of selection pressure, revealed that only pSci2 derivatives having an intact gene were stably maintained, indicating that the -encoded polypeptide is most likely involved in plasmid partitioning. Upon transformation, pSci2 derivatives, including shuttle (/) plasmids, were shown to replicate in all 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 and , the transformation of which had never, to our knowledge, been described before. These studies show that, besides their implications for the biology of , the pSci plasmids hold considerable promise as vectors of general use for genetic studies of plant-pathogenic spiroplasmas. As an example, a HA-tagged protein was expressed in . Detection of -hybridizing sequences in various group I spiroplasma species indicated that replicating plasmids were not restricted to the three plant-pathogenic spiroplasmas.

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2008-10-01
2019-10-21
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Supplements

vol. , part 10, pp. 3232 - 3244

The supplementary data file [ PDF, 165 kb] contains the following: Spiroplasma strains used in this study. Amino acid sequence alignments of spiroplasma PE proteins. Southern blot hybridization of cII-restricted DNAs from GII3-3X subclones with a mixture of scarp and p32 probes. Amino acid sequence alignment of Soj proteins. Phylogenetic relationships of ParA proteins from 91 different replicons. Phylogenetic relationships of ParA/Soj proteins from mollicutes and several other firmicutes.



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