Integrative and free Spiroplasma citri oriC plasmids: expression of the Spiroplasma phoeniceum spiralin in Spiroplasma citri

Abstract

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.Mollicutes are a class of eubacteria phylogenetically related to gram-positive bacteria with low GϩC contents (30). They are characterized by the lack of a cell wall, a small genome size, a small number of rRNA operons, and relatively few tRNA genes, and their metabolic activities are limited. The molecular features of mollicutes have been recently reviewed (1). The high adenine-thymine content has resulted in a particular codon usage. For instance, in mycoplasmas, ureaplasmas, mesoplasmas, entomoplasmas, and spiroplasmas, UGA is read as a tryptophan codon and not as a termination signal. As a result, expression of mollicute genes by cloning in Escherichia coli has been limited. To circumvent these difficulties, an opal suppressor strain of E. coli has been used to fully express the CpG DNA methylase of Spiroplasma monobiae (22) and, more recently, to screen Mycoplasma hyopneumoniae gene libraries (17). However, gene function can only be ascertained by mutational analysis. A classical approach consists of isolating mutants and, subsequently, identifying the DNA sequences from the wild-type species that are able to complement the particular mutations. Complementation of gene mutations requires that genes be transferred between isolates. In mollicutes, genetic analyses have long been hampered by the lack of methods for transformation and of suitable gene vectors. Recently, several approaches have been examined...

Identification of the Origin of Replication of the Mycoplasma pulmonis Chromosome and Its Use in oriC Replicative Plasmids

“…In contrast, replicative plasmids have been obtained for the mollicute Spiroplasma citri by cloning the origin of replication of the chromosome (oriC) into artificial plasmids (34,41). The resulting oriC plasmids can be used to inactivate targeted genes by homologous recombination (8,41), to support heterologous gene expression in S. citri (35), and to functionally complement mutants obtained by transposon insertion (18).…”

Fructose operon mutants of Spiroplasma citri The GenBank accession number for the sequence reported in this paper is AF202665.

“…Plasmid pBS was purchased from Stratagene. Plasmid pBOT carrying the S. citri oriC and tetracycline-resistance gene (tetM) was described elsewhere (Renaudin et al, 1995). Transformation of S. citri by electroporation with pBOT and its derivatives was as previously described (Stamburski et al, 1991).…”

Molecular biology of mycoplasmas: from the minimum cell concept to the artificial cell

“…and maintain stability as an extrachromosomal element (Renaudin et al 1995). Vectors derived from this plasmid show themselves to be capable of inactivating specific S. citri genes (Duret et al 1999).…”