Selection and characterization of Spiroplasma citri mutants by random transposome mutagenesis.

Abstract:

:Phytopathogenic spiroplasmas can multiply in vascular plants and insects. A deeper understanding of this dual-host life could be furthered through the identification by random mutagenesis of spiroplasma genes required. The ability of the EZ::TN™ Tnp transposome™ system to create random insertional mutations in the genome of Spiroplasma citri was evaluated. The efficiency of electroporation-mediated transformation of S. citri BR3-3X averaged 28.8 CFUs/ng transposome for 10(9) spiroplasma cells. Many transformants appearing on the selection plates were growth impaired when transferred to broth. Altering broth composition in various ways did not improve their growth. However, placing colonies into a small broth volume resulted in robust growth and successful subsequent passages of a subset of transformants. PCR using primers for the dihydrofolate reductase gene confirmed the transposon's presence in the genomes of selected transformants. Southern blot hybridization and nucleotide sequencing suggested that insertion was random within the chromosome and usually at single sites. The insertions were stable. Growth rates of all transformants were lower than that of the wild-type S. citri, but none lost the ability to adhere to a Circulifer tenellus (CT-1) cell line. The EZ::TN™ Tnp transposome™ system represents an additional tool for genetic manipulation of the fastidious spiroplasmas.

journal_name

Can J Microbiol

authors

Mutaqin K,Comer JL,Wayadande AC,Melcher U,Fletcher J

doi

10.1139/w11-026

subject

Has Abstract

pub_date

2011-06-01 00:00:00

pages

525-32

issue

6

eissn

0008-4166

issn

1480-3275

journal_volume

57

pub_type

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