Construction of the first shuttle vectors for gene cloning and homologous recombination in Mycoplasma agalactiae

Abstract

Mycoplasma agalactiae is a worldwide ruminant pathogen that causes significant economic losses by inflicting contagious agalactia in sheep and goats. The development of efficient control strategies requires a better understanding of the mycoplasma factors that promote successful infection. However, lack of genetic tools has been a major impediment in studying the pathogenic mechanisms of M. agalactiae. This study describes the identification and cloning of the M. agalactiae origin of replication (oriC) in order to construct the first shuttle vectors for targeted gene disruption, gene complementation and expression studies. Additionally, this report provides the first evidence of the occurrence of homologous recombination and the functionality of heterologous tetM determinant in this pathogen.

Simultaneous Identification of Potential Pathogenicity Factors of Mycoplasma agalactiae in the Natural Ovine Host by Negative Selection

“…However, there is a complete lack of studies on the systematic analysis of M. agalactiae genetic factors involved in the colonization of the udder and subsequent systemic spread and persistence leading to chronic infections. M. agalactiae genome sequencing, together with the development of tools for its genetic manipulation, has opened new dimensions in understanding its pathogenesis (2,(11)(12)(13).…”

Critical Role of Dispensable Genes inMycoplasma agalactiaeInteraction with Mammalian Cells

“…However, the genetic information necessary to develop interactions of mycoplasma with its animal host is likely to differ from the minimal set of essential genes required for laboratory growth. New opportunities to investigate factors involved in mycoplasma-host interaction have emerged through the development of genomic tools that facilitate the manipulation of animal mycoplasmas including M. agalactiae (8,9). However, in vivo screening of mutant libraries of ruminant mycoplasmas involves difficulties inherent in experiments with infections in large animals.…”

Development of oriC -Based Plasmids for Mesoplasma florum

“…Many Mollicutes phylogenetically related to M. florum, including M. mycoides, M. capricolum, and Spiroplasma citri, have been successfully transformed with artificial plasmids containing a chromosomal origin of replication (oriC) (18)(19)(20)(21)(22)(23)(24)(25)(26). oriC-based plasmids have multiple uses, such as expression of exogenous genes, inactivation of target genes by recombination, or complementation of chromosomal mutations.…”