A system for transposon mutagenesis of Bartonella bacilliformis

Abstract

Bartonella bacilliformis is the etiologic agent of Carrión's disease in South America. Lack of a system for random mutagenesis has significantly hampered research on the pathogen's molecular biology. Here, we describe a transposon (Tn)-based mutagenesis strategy for B. bacilliformis using pSAM_Rl; a Tn-mariner delivery vector originally constructed for members of the Rhizobiaceae family. Following electroporation of the vector, five candidate mutant strains were selected based on aberrant colony morphologies, and four mutations confirmed and identified using arbitrarily-primed PCR coupled with Sanger sequencing. One mutant strain, 4B2, was found to have a disrupted flgI gene, encoding the P-ring component of the flagellar motor. We therefore investigated the flgI strain's motility phenotype in a novel motility medium and found that insertional mutagenesis produced a non-motile mutant. Taken as a whole, the results show that: 1) pSAM_R1 is a practical Tn delivery vector for B. bacilliformis, 2) the plasmid can be used to create random Tn mariner mutants, 3) arbitrarily-primed PCR coupled with Sanger sequencing is a rapid and simple method for identifying and locating mutations generated by this Tn, and 4) in silico-predicted mutant phenotypes can be verified in vitro following mutagenesis. This system of Tn mutagenesis and mutation identification provides a novel and straightforward approach to investigate the molecular biology of B. bacilliformis.

Keywords: Bartonella bacilliformis; Genetic manipulation; Random mutagenesis; Tn mariner; Transposon.