Cryptic plasmid pSKU146 from the wall-less plant pathogen Spiroplasma kunkelii encodes an adhesin and components of a type IV translocation-related conjugation system

Abstract

A cryptic plasmid of the wall-less plant pathogenic mollicute, Spiroplasma kunkelii CR2-3X, was cloned and its sequence analyzed. The 14,615 bp plasmid, designated pSKU146, has a nucleotide content of 28 mol% G + C, and contains 18 potential protein-coding regions (open reading frames, ORFs), of which six encode proteins that exhibit similarity to virulence-associated proteins involved in cell-to-cell adhesion or conjugal DNA transfer. One ORF encodes a 96 kDa protein, SkARP1, that is highly similar to SARP1 adhesin involved in attachment of Spiroplasma citri to insect vector gut membrane. Five ORFs encode proteins similar to TraE and Mob in walled bacteria, and to ORFs found in the integrative, conjugative element (ICEF) of Mycoplasma fermentans, respectively. Presence of domains similar to proteins of the Type IV secretion system in pathogenic bacteria suggests that spiroplasma possesses a related translocation system. Plasmid pSKU146 also contains two identical oriT regions each containing a nick sequence characteristic of the IncP conjugative plasmid family, as well as a 58 bp palindromic sequence, palSK1. Features in pSKU146 suggest that the plasmid functions as a mobile genetic element in conjugative transmission of spiroplasma pathogenicity-related genes.

Introduction

Spiroplasmas are helical, motile, cell wall-less prokaryotes that are classified, along with members of genus Mycoplasma and other wall-less bacteria, in class Mollicutes. In their descent from the Gram-positive, low G + C Bacillus–Clostridium group of walled bacteria, spiroplasmas underwent massive evolutionary genome reduction, while acquiring capabilities for arthropod parasitism, and in some cases plant pathogenicity. First discovered in association with plant disease (Davis and Worley, 1973, Davis et al., 1972), spiroplasmas comprise broadly diverse species, most of which parasitize a variety of insects or ticks (Gasparich, 2002). As in other bacteria, plasmids presumably have played key roles in spiroplasma evolution, and contributed to the diversity of Spiroplasma species, through horizontal exchange of genes encoding virulence factors, niche adaptation factors, and factors conferring competitive advantages on the spiroplasma cell. In the present work, we characterized a plasmid from a plant pathogen, Spiroplasma kunkelii.

Spiroplasma kunkelii is the causative agent of corn stunt disease, a major factor limiting maize production in the Americas (Davis and Worley, 1973, Davis et al., 1972, Whitcomb and Williamson, 1975). This spiroplasma is transmitted between maize plants exclusively by phloem-feeding leafhopper insects, in which it multiplies. In maize plants, the spiroplasma resides specifically in sieve cells of the plant’s phloem tissue, and induces symptoms including general stunting, broad chlorotic stripes, poor filling of ears, sterility of tassels, and plant death. Although plasmids have been reported in S. kunkelii (Gasparich et al., 1993), none has previously been sequenced. Here we present the structural analysis of a previously unreported S. kunkelii plasmid, designated pSKU146, harboring regions potentially encoding proteins exhibiting significant homologies, respectively, with a spiroplasma adhesion-related protein and proteins involved in conjugal mating pair formation and DNA transfer.