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Home / Equine Research Bank / J Bacteriol / Phenotypic mutants of the intracellular actinomycete Rhodoco...
Journal of bacteriology2003; 185(8); 2644-2652; doi: 10.1128/JB.185.8.2644-2652.2003

Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis.

Abstract: Rhodococcus equi is a facultative intracellular opportunistic pathogen of immunocompromised people and a major cause of pneumonia in young horses. An effective live attenuated vaccine would be extremely useful in the prevention of R. equi disease in horses. Toward that end, we have developed an efficient transposon mutagenesis system that makes use of a Himar1 minitransposon delivered by a conditionally replicating plasmid for construction of R. equi mutants. We show that Himar1 transposition in R. equi is random and needs no apparent consensus sequence beyond the required TA dinucleotide. The diversity of the transposon library was demonstrated by the ease with which we were able to screen for auxotrophs and mutants with pigmentation and capsular phenotypes. One of the pigmentation mutants contained an insertion in a gene encoding phytoene desaturase, an enzyme of carotenoid biosynthesis, the pathway necessary for production of the characteristic salmon color of R. equi. We identified an auxotrophic mutant with a transposon insertion in the gene encoding a putative dual-functioning GTP cyclohydrolase II-3,4-dihydroxy-2-butanone-4-phosphate synthase, an enzyme essential for riboflavin biosynthesis. This mutant cannot grow in minimal medium in the absence of riboflavin supplementation. Experimental murine infection studies showed that, in contrast to wild-type R. equi, the riboflavin-requiring mutant is attenuated because it is unable to replicate in vivo. The mutagenesis methodology we have developed will allow the characterization of R. equi virulence mechanisms and the creation of other attenuated strains with vaccine potential.
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Publication Date: 2003-04-03 PubMed ID: 12670990PubMed Central: PMC152612DOI: 10.1128/JB.185.8.2644-2652.2003Google Scholar: Lookup
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  • Journal Article
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  • Non-U.S. Gov't

Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

The research article outlines a study on the construction of Rhodococcus equi mutants in an attempt to understand the virulence mechanisms of this intracellular pathogen and develop an effective live vaccine to prevent R. equi disease in horses.

Introduction and Objective

  • The aim of this study was to develop a mutagenesis system for the intracellular pathogen Rhodococcus equi, which is an opportunistic pathogen known to cause pneumonia in young horses. The researchers were seeking to create an effective live attenuated vaccine to counter this disease.

Methodology

  • The authors employed a Himar1 minitransposon, delivered via a conditionally replicating plasmid, to create R. equi mutants.
  • The transposition of Himar1 within R. equi was shown to be random, with no required consensus sequence beyond a TA dinucleotide.
  • The authors also highlighted that the diversity of the mutants created by this transposon mutagenesis was demonstrated by how easily mutants with auxotrophs, pigmentation, and capsular phenotypes could be identified and separated.

Findings

  • Notably, one pigmentation mutant had a transposon insertion in a gene encoding phytoene desaturase, an essential enzyme for the production of the salmon color characteristic of R. equi.
  • Another identified mutant had an insertion in the gene encoding an enzyme necessary for riboflavin biosynthesis. This mutant was auxotrophic, meaning it could not grow without riboflavin supplementation.
  • Murine infection studies demonstrated that this riboflavin-requiring mutant was attenuated or less virulent because it couldn’t replicate in vivo.

Conclusions and Implications

  • The authors concluded that the mutagenesis methods used will assist in the understanding of R. equi’s virulence mechanisms, potentially leading to the development of additional attenuated strains with the potential for a vaccine.

Cite This Article

APA
Ashour J, Hondalus MK. (2003). Phenotypic mutants of the intracellular actinomycete Rhodococcus equi created by in vivo Himar1 transposon mutagenesis. J Bacteriol, 185(8), 2644-2652. https://doi.org/10.1128/JB.185.8.2644-2652.2003

Publication

Journal of bacteriology
ISSN: 0021-9193
NlmUniqueID: 2985120R
Country: United States
Language: English
Volume: 185
Issue: 8
Pages: 2644-2652

Researcher Affiliations

Ashour, Joseph
  • Department of Immunology and Infectious Diseases, Harvard School of Public Health, Boston, Massachusetts 02115, USA.
Hondalus, Mary K

    MeSH Terms

    • Actinomycetales Infections / microbiology
    • Actinomycetales Infections / veterinary
    • Animals
    • DNA Transposable Elements / physiology
    • Disease Models, Animal
    • Female
    • GTP Cyclohydrolase / genetics
    • Mice
    • Mice, Inbred BALB C
    • Mutagenesis, Insertional
    • Oxidoreductases / genetics
    • Plasmids
    • Rhodococcus equi / genetics
    • Rhodococcus equi / metabolism
    • Rhodococcus equi / pathogenicity
    • Riboflavin / biosynthesis
    • Sugar Phosphates / biosynthesis
    • Virulence / genetics

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