Genetic analysis of pigment biosynthesis in Xanthobacter autotrophicus Py2 using a new, highly efficient transposon mutagenesis system that is functional in a wide variety of bacteria.

Abstract:

:A highly efficient method of transposon mutagenesis was developed for genetic analysis of Xanthobacter autotrophicus Py2. The method makes use of a transposon delivery vector that encodes a hyperactive Tn 5 transposase that is 1,000-fold more active than the wild-type transposase. In this construct, the transposase is expressed from the promoter of the tetA gene of plasmid RP4, which is functional in a wide variety of organisms. The transposon itself contains a kanamycin resistance gene as a selectable marker and the origin of replication from plasmid R6K to facilitate subsequent cloning of the resulting insertion site. To test the effectiveness of this method, mutants unable to produce the characteristic yellow pigment (zeaxanthin dirhamnoside) of X. autotrophicus Py2 were isolated and analyzed. Transposon insertions were obtained at high frequency: approximately 1 x 10(-3) per recipient cell. Among these, pigment mutants were observed at a frequency of approximately 10(-3). Such mutants were found to have transposon insertions in genes homologous to known carotenoid biosynthetic genes previously characterized in other pigmented bacteria. Mutants were also isolated in Pseudomonas stutzeri and in an Alcaligenes faecalis, demonstrating the effectiveness of the method in diverse Proteobacteria. Preliminary results from other laboratories have confirmed the effectiveness of this method in additional phylogenetically diverse species.

journal_name

Arch Microbiol

journal_title

Archives of microbiology

authors

Larsen RA,Wilson MM,Guss AM,Metcalf WW

doi

10.1007/s00203-002-0442-2

keywords:

subject

Has Abstract

pub_date

2002-09-01 00:00:00

pages

193-201

issue

3

eissn

0302-8933

issn

1432-072X

journal_volume

178

pub_type

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