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.

Genetic determinants of biofilm development of opaque and translucent Vibrio parahaemolyticus

“…It appeared that strong promoters were required to confer sufficient levels of chloramphenicol resistance. Subsequently, methods for using Larsen and Metcalf's Tn 5 -RL27 transposon (Larsen et al ., 2002) in V. parahaemolyticus were developed (Stewart and McCarter, 2003) and used to isolate pellicle mutants in both OP (bank II) and TR backgrounds (bank III).…”

“…Three derivatives of Tn5 were employed for transposon mutagenesis: Tn5cat-TS (this work), Tn5-RL27 (Larsen et al, 2002) and Tn5lux-RL27 (Stewart and McCarter, 2003). Tn5cat-TS contains a promoterless reporter gene encoding chloramphenicol resistance, a kanamycin-resistance gene, and an ori6K origin of replication; it was constructed by introducing a promoterless chloramphenicol acetyl transferase gene (derived from pUCM; Schweizer, 1990) into the KpnI site of pTnMod-RKm' (Dennis and Zylstra, 1998) to make pLM2274.…”

Disruption of MiaA provides insights into the regulation of phenazine biosynthesis under suboptimal growth conditions in Pseudomonas chlororaphis 30-84

“…Isolation of the miaA mutant P. chlororaphis 30-84 was mutagenized using the pRL27 :: Tn5Km R plasposon, as described previously [39]. Briefly, overnight cultures of the E. coli donor containing pRL27 :: Tn5Km R and recipient 30-84 were grown to mid-logarithmic phase.…”

“…Briefly, overnight cultures of the E. coli donor containing pRL27 :: Tn5Km R and recipient 30-84 were grown to mid-logarithmic phase. Cells were collected by centrifugation and spotted onto sterile nitrocellulose membranes on LB agar and incubated at The plasposon and adjacent chromosomal regions of 30-84 :: 19-30 were isolated as a plasmid as described previously [39]. Using the outwardly directed sequencing primers (tpnRL17 and tpnRL13, Table S1), the sequence adjacent to the insertion was determined.…”

Verminephrobacter eiseniae type IV pili and flagella are required to colonize earthworm nephridia

“…Conjugation of EF05-2r was performed as above and mutants were screened for marker insertion. A mini-Tn5 transposon conferring kanamycin resistance was introduced into V. eiseniae EF05-2r from E. coli S17-1 (pRL27) (Larsen et al, 2002) through biparental mating, at a 1:10 ratio, O/N at 28 1C and subsequent selection on ACM with appropriate antibiotics to generate RTn5.1 (Random Tn5 clone1) and RTn5.2. All resulting mutants (VEflgKÀ, VEflgKLÀ and VEpilBCÀ) screened for resistance marker were confirmed using PCR to contain the appropriate insertion.…”