PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria - PubMed (original) (raw)

PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria

Dongru Qiu et al. Appl Environ Microbiol. 2008 Dec.

Abstract

We report the construction of a series of Escherichia-Pseudomonas broad-host-range expression vectors utilizing the P(BAD) promoter and the araC regulator for routine cloning, conditional expression, and analysis of tightly controlled and/or toxic genes in pseudomonads.

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Figures

FIG. 1.

Construction of an _Escherichia_-Pseudomonas shuttle vector, pHERD20T, an arabinose-inducible expression vector. pHERD20T is a pUCP20T-based, conjugatable vector with pBR322- and pRO1600-derived replicons which support replication in E. coli, P. aeruginosa, and other bacteria, respectively. The PBAD promoter was derived from the expression vector pBAD/Thio-TOPO (Invitrogen). The Plac promoter in pUCP20T was replaced with the PBAD promoter-containing segment with an EcoRI-AflII fragment generated via PCR containing the araC gene and PBAD. Black arrows indicate the region transferred from pBAD/Thio-TOPO into pUCP20T. pHERD20T contains a multiple cloning site within _lacZ_α encoding the β-galactosidase α peptide.

FIG. 2.

Arabinose-regulated _lacZ_α expression in B. pseudomallei. RNA was extracted from log-phase B. pseudomallei Bp50 cells harboring pHERD30T that either had no arabinose added (None) or were induced for 2 h by the addition of the indicated amounts of

-arabinose. Quantitative real-time PCR was performed by using _lacZ_α-specific primers. Data were normalized by using the 23S rRNA gene as the housekeeping control.

FIG. 3.

Arabinose-dependent induction of alginate production in P. aeruginosa PAO1 carrying pHERD20T-algU. PAO1 with pHERD20T-algU was grown at 37°C for 24 h on Pseudomonas isolation agar and LB plates supplemented with carbenicillin and 0, 0.1, and 1.0% arabinose, respectively. The empty pHERD20T vector was used as the control (open box). Bars indicate means with standard errors. PAO1/pHERD20T with 0.1 and 1.0% arabinose does not increase alginate production (data not shown). OD, optical density.

FIG. 4.

Regulated alginate production in P. aeruginosa. Regulation of alginate production in P. aeruginosa involves many genes coding for products with many different functions. Mucoidy or alginate production is directed by the alternative σ22 factor AlgU (14). MucA is the cognate anti-sigma factor that negatively regulates AlgU activity by sequestering AlgU to the inner membrane (IM) (22). Sequestering of AlgU by MucA can be relieved by either mutation of mucA (15) or proteolytic degradation of MucA by the intramembrane protease AlgW (20). Derepression of MucA causes AlgU activation and alginate production. OM, outer membrane.

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PBAD-based shuttle vectors for functional analysis of toxic and highly regulated genes in Pseudomonas and Burkholderia spp. and other bacteria - PubMed (original) (raw)