Versatile Rhodococcus equi?Escherichia coli shuttle vectors

Abstract

Rhodococcus equi is an intracellular pathogen of macrophages, causing disease in young foals, humans, and sporadically other animals. Although R. equi is easy to grow and manipulate, the analysis of virulence is hampered by a lack of molecular tools. This paper describes the development of a number of versatile plasmids for use in R. equi. Plasmids pREV2 and pREV5 use origins of replication derived from the Mycobacterium fortuitum plasmids pAL5000 and pMF1. These plasmids and their derivatives are compatible in R. equi, allowing their use for analysis of gene function in trans. The stability of these plasmids in R. equi in the absence of selection for the plasmid borne antibiotic resistance markers, and their integrity following passage through Escherichia coli and R. equi was determined.

The use of pAL5000 Replicon Vectors to Transform Rhodococcus and GordoniaSpecies

“…asteroides, N. nova, N. cyriacigergica, Dietzia sp. and R. equi (Chiba et al, 2007;Mangan et al, 2005;Szvetnik et al, 2010). This study has extended that host range to include Rhodococcus sp., Gordonia sp.…”

“…To ensure that pCCC2 was not rearranging upon transformation into Gram positive strains, we checked the structural stability of the vector as described by Mangan et al (2005). Briefly, this was evaluated by extracting the vector from their Gram positive hosts, retransforming them into E. coli, re-extracting the vector and performing digestions.…”

Site-specific integration ofStreptomycesΦC31 integrase-based vectors in the chromosome ofRhodococcus equi

“…A major hurdle to R. equi research has been the lack of molecular tools required for genetic analysis. Of late, great progress has been made in the areas of shuttle vector construction (Zheng et al, 1997;Sekizaki et al, 1998;Giguere et al, 1999;Mangan et al, 2005), transformation (Zheng et al, 1997), transposon-based random mutagenesis procedures (Mangan & Meijer, 2001;Ashour & Hondalus, 2003), and allelic exchange methodology (Navas et al, 2001;Jain et al, 2003). The study of mutant strains requires complementation analysis, which can be done using episomal shuttle vectors.…”

Advances in the Development of Genetic Tools for the Genus Rhodococcus

“…Data accumulated from basic studies on the characteristic features of rhodococci and the increasing knowledge obtained from the genome databases have enhanced the development of genetic manipulation techniques used for rhodococci analysis. Such techniques include the development of shuttle vectors using cryptic and/or antibiotic-resistant plasmids derived from Rhodococcus strains 10,19,20) , efficient transformation techniques using electroporation 21,22) , the expression vectors for protein production 23,24) , random mutagenesis methods using transposons or spontaneous illegitimate recombination 25,26) , and targeted gene disruption systems 11,27) . These techniques along with increasing genome information are enabling cell engineering in a manner such that there is an improvement in the useful properties of rhodococci such as their ability to act as biocatalysts; this improvement is due to the activation of catabolic pathways and/or depletion of the undesirable genes.…”