Development of a novel plasmid as a shuttle vector for heterologous gene expression in Mycoplasma yeatsii

• Published in: Journal of microbiological methods 2012-10, Vol.91 (1), p.121-127
• Main Authors: Kent, Bethany N., Foecking, Mark F., Calcutt, Michael J.
• Format: Article
• Language: English
• Subjects: Bacteriological methods and techniques used in bacteriology; Bacteriology; Biological and medical sciences; Electroporation; Escherichia coli; Escherichia coli - genetics; Fundamental and applied biological sciences. Psychology; Gene Expression; genes; genetic engineering; Genetic transformation; Genetic Vectors; Genetics, Microbial - methods; heterologous gene expression; Microbiology; Molecular Biology - methods; Mycoplasma; Mycoplasma - genetics; Mycoplasma fermentans; Mycoplasma yeatsii; open reading frames; Plasmid; Plasmids; proteins; replicon; sequence homology; Shuttle vector; Transfection; Transformation, Genetic
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2012.07.018

A circular plasmid, pMyBK1, was detected in Mycoplasma yeatsii strain GIHT. Analysis of the sequence of the 3432-bp replicon identified two predicted open reading frames (ORFs), one with sequence similarity to multiple plasmid mobilization proteins and one that matches only to hypothetical ORFs encoded by integrated chromosomal elements in the sequenced genomes of two Mycoplasma species. Shuttle vectors were constructed in Escherichia coli which could be introduced into M. yeatsii at high efficiency (104–105 per μg DNA) by electroporation. Independent deletion analysis of the two ORFs disclosed that whereas mob was dispensable, orf2 was necessary for plasmid replication or maintenance. The absence of plasmid-encoded database matches for ORF2 indicates that pMyBK1 represents a novel plasmid family. One shuttle vector was used to demonstrate heterologous expression of the Mycoplasma fermentans malp gene and was stable during multiple passages. The host–plasmid system described has potential application for genetic manipulation in a genus for which few replicative vectors are available. ► A novel plasmid, pMyBK1, has been identified in Mycoplasma yeatsii Type strain GIH. ► pMyBK1-based shuttle vectors transform M. yeatsii at high efficiency. ► Heterologous protein expression has been demonstrated for MALP-404. ► ORF2, dissimilar to known plasmid-encoded proteins, is essential for replication.