PBAD-Based Shuttle Vectors for Functional Analysis of Toxic and Highly Regulated Genes in Pseudomonas and Burkholderia spp. and Other Bacteria

We report the construction of a series of Escherichia-Pseudomonasbroad-host-range expression vectors utilizing the P_BAD promoterand the araC regulator for routine cloning, conditional expression,and analysis of tightly controlled and/or toxic genes in pseudomonads.

INTRODUCTION

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INTRODUCTION

Gene cloning, disruption, deletion, complementation analysis,and allelic exchange are central to prokaryotic molecular genetics.In Pseudomonas aeruginosa, Schweizer and colleagues developedthe pUCP family of general-purpose vectors for cloning and geneexpression (24, 29) based on the well-characterized pUC18/19vectors (32) and the cryptic mini-plasmid pRO1614 (19). Otherpromoters are also in routine use, such as the tac (4, 6), T7(28), and araBAD promoter-based (8, 11) vectors for regulatedexpression in Escherichia coli and many other bacterial species(e.g., see references 2, 18, and 25). In E. coli, AraC repressesthe araBAD promoter (P_BAD) and the expression of a cloned geneis induced by the addition of L-arabinose. Pseudomonas researchershave used the inducible properties of the araC regulator andthe P_BAD promoter cassette for the controlled gene expressionby integrating the araC-P_BAD-specific transcription fusion intothe chromosome by using a suicide vector or an integration-proficientvector (1, 3, 13, 17, 30, 31). In the present study, we modifiedthe existing Escherichia-Pseudomonas shuttle vectors pUCP20T,-26, -28T, and -30T by replacing the lac promoter with the araC-P_BADcassette to allow conditional expression in pseudomonads andother bacteria, e.g., Burkholderia spp.

Construction and features of pHERD vectors.

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Construction and features of...

Functional genetic analysis requires vectors capable of conditionalexpression. The P_BAD promoter has been used for gene expressionextensively in E. coli and some in P. aeruginosa and Burkholderiaspp. (12, 27, 31). We first constructed three shuttle vectors,pHERD20T, -28T, and -30T (Fig. 1), based on Escherichia-Pseudomonasshuttle vectors pUCP20T, pUCP28T, and pUCP30T (29) and the commercialexpression vector pBAD/Thio-TOPO (Invitrogen). The 368-bp fragmentof the pUCP vectors spanning two restriction sites, AflII andEcoRI, was replaced with the araC-P_BAD fragment (1.3 kb), producedvia PCR with pBAD/Thio-TOPO as the template and primers pBAD-Fand pBAD-R (Table 1). The PCR product was purified and directlydigested with AflII and EcoRI, and the two fragments were ligatedinto the pUCP vectors, creating pHERD20T (Fig. 1). The EcoRI/AflIIregions of these vectors were sequenced to confirm that no mutationswere introduced during the cloning process. We next transferredthe 2.4-kb AdhI-EcoRI fragment from pHERD20T to pUCP26, generatingpHERD26T (Tet^r, 6,166 bp), which includes the araC-P_BAD cassetteand the oriT sequence.

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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 P_BAD promoter was derived from the expression vector pBAD/Thio-TOPO (Invitrogen). The P_lac promoter in pUCP20T was replaced with the P_BAD promoter-containing segment with an EcoRI-AflII fragment generated via PCR containing the araC gene and P_BAD. Black arrows indicate the region transferred from pBAD/Thio-TOPO into pUCP20T. pHERD20T contains a multiple cloning site within lacZ ${alpha}$ encoding the β-galactosidase ${alpha}$ peptide.

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TABLE 1. Bacterial strains and plasmids used in this study

The pHERD vectors have the features of the pUCP vector family,including the pBR322 origin, four different antibiotic resistancemarkers, the oriT region for conjugation-mediated plasmid transfer(23), ori₁₆₀₀, and the rep gene encoding the replication-controllingprotein (24, 29). However, the main advantage for cloning intothe pHERD vectors is low expression, which occurs from the P_BADpromoter when it is not induced (Fig. 2). ${alpha}$ complementation isinducible for blue-white screening, which facilitates the identificationof recombinants on a 5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside(X-Gal)-containing plate supplemented with arabinose (0.01%).The P_BAD promoter responds in a dose-dependent manner (Fig.2). Two sequencing and PCR primers were designed that annealto regions on both sides of the multiple cloning site, pHERD-SF78 bp upstream of the EcoRI site and pHERD-SR 49 bp downstreamof the HindIII site. If a gene is cloned in frame into the EcoRIsite, a fusion protein with an additional seven NH₂-terminalamino acids (MGSDKNS) derived from thioredoxin of pBAD-TOPO/Thiowill result. Thioredoxin acts as a translation leader to facilitatehigh-level expression and, in some cases, increase solubilityin E. coli (9). These amino acids at the N terminus of the targetprotein may also serve as an epitope tag for protein analysis.pHERD vectors can be readily transferred from E. coli into Pseudomonasspecies and other bacteria via triparental conjugation (7) orby electroporation. It has been shown that the progenitor plasmidpRO1614 could replicate in a series of bacterial species, includingP. aeruginosa, P. putida, P. fluorescens, Klebsiella pneumoniae(19), and Burkholderia spp. (5, 26). Therefore, the pHERD vectorsare most likely functional in these bacteria. Another featureof the P_BAD promoter is catabolite repression of expressionin the presence of glucose in the growth medium, which reducesintracellular cyclic AMP concentrations in E. coli cells, preventingthe transcriptional activation of many genes by the cyclic AMP-bindingprotein (8).

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FIG. 2. Arabinose-regulated lacZ ${alpha}$ 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 L-arabinose. Quantitative real-time PCR was performed by using lacZ ${alpha}$ -specific primers. Data were normalized by using the 23S rRNA gene as the housekeeping control.

Validation of pHERD20T in P. aeruginosa by modulating alginate production.

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Validation of pHERD20T in...

We have observed that pHERD vectors can be used for the high-fidelitycloning and conditional expression of P_BAD transcription inthe absence of L-arabinose (10). Initial attempts to clone theopen reading frame of P. aeruginosa alternative sigma factoralgU into pUCP20T were not successful. All of the algU allelescloned were not functional, and sequence analysis showed thatonly mutant algU alleles were cloned into pUCP20T. This wasconsistent with the previous observations that algU/T cannotbe cloned into the common expression vectors (16, 21). However,the algU gene was readily cloned into pHERD20T. Upon the expressionof algU from P_BAD on pHERD20T, we observed dose-dependent alginateproduction or mucoidy in P. aeruginosa strain PAO1 in responseto arabinose in the growth medium (Fig. 3).

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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.

Overexpression of the small peptide encoded by mucE activatesAlgW, inducing alginate production (Fig. 4) in P. aeruginosaPAO1 and PA14 (20). Overexpression of mucE caused mucoidy inP. aeruginosa PAO1 and P. fluorescens Pf-5 (Table 2). The C-terminalWVF signal encoded by mucE is required for the activation ofAlgW. The outer membrane protein OprF does not activate alginateproduction (Fig. 4); however, addition of the MucE WVF signalmotif to the C terminus of OprF did cause alginate production(Table 2). Some genes are not highly expressed, and thereforeexpression in trans for complementation needs to be conditional.Expression of algW from P_BAD can complement an algW mutant backto alginate production due to titratable expression (Table 2).In addition to PAO1, we have used the pHERD vectors in PA14,CF149, environmental P. fluorescens isolates, and P. putida(data not shown). We have successfully employed pHERD30T forcomplementation of the ${Delta}$ (amrAB-oprA) efflux pump mutation inBurkholderia pseudomallei strain Bp50 (5). In this case, however,complementation was also observed in uninduced cells, presumablybecause of basal transcription from the P_BAD promoter, whichcould not be overcome by growing cells in the presence of 0.2%glucose and was not dependent on the growth medium used forthe MIC assays (LB versus Mueller-Hinton broth) (data not shown).

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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 ${sigma}$ ²² 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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TABLE 2. Modulation of mucoidy in P. aeruginosa and P. fluorescens by pHERD20T-borne alginate regulators

In summary, we constructed a series of small Escherichia-Pseudomonasshuttle vectors with the E. coli araC and P_BAD promoter forhighly regulated expression of cloned genes in Pseudomonas speciesand other bacteria and confirmed their utility by modulationof alginate production. Results presented here demonstrate thatpHERD vectors are useful tools for bacterial physiological researchand gene function studies with pseudomonads, as well as otherbacteria, including medically significant Burkholderia spp.

Nucleotide sequence accession numbers.

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Nucleotide sequence accession...

The GenBank accession numbers for the nucleotide sequences ofpHERD20T, -26T, -28T, and -30T are EU603324, EU603327, EU603325,and EU603326, respectively.

ACKNOWLEDGMENTS

The pHERD vectors described here are dedicated to the memoryof the 1970 Marshall University Thundering Herd football teamas depicted in the 2006 Warner Bros. film We Are Marshall.

This work was supported by a research grant (NNA04CC74G) fromthe National Aeronautics and Space Administration (NASA) andresearch grants from the NASA West Virginia Space Grant Consortiumto H.D.Y. H.P.S.'s Burkholderia research was supported by NIHgrant AI065357. F.H.D. was supported by a training grant (NNX06AH20H)from the NASA Graduate Student Researchers Program (GSRP).

We thank N. E. Head for the initial analysis of the mutant algUalleles in pUCP20T, V. M. Eisinger for the generation of theVE mutants, and K. D. Dillon for technical assistance with thealginate assay.

FOOTNOTES

* Corresponding author. Mailing address: Robert C. Byrd Biotechnology Science Center, 1 John Marshall Drive, Huntington, WV 25755-9320. Phone: (304) 696-7356. Fax: (304) 696-7207. E-mail: yuh{at}marshall.edu

Published ahead of print on 10 October 2008.

D.Q. and F.H.D. contributed equally to this publication.

REFERENCES

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