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

Construction and features of pHERD vectors.

Functional genetic analysis requires vectors capable of conditional expression. The P_BAD promoter has been used for gene expression extensively in E. coli and some in P. aeruginosa and Burkholderia spp. (12, 27, 31). We first constructed three shuttle vectors, pHERD20T, -28T, and -30T (Fig. 1), based on Escherichia-Pseudomonas shuttle vectors pUCP20T, pUCP28T, and pUCP30T (29) and the commercial expression vector pBAD/Thio-TOPO (Invitrogen). The 368-bp fragment of the pUCP vectors spanning two restriction sites, AflII and EcoRI, was replaced with the araC-P_BAD fragment (1.3 kb), produced via PCR with pBAD/Thio-TOPO as the template and primers pBAD-F and pBAD-R (Table 1). The PCR product was purified and directly digested with AflII and EcoRI, and the two fragments were ligated into the pUCP vectors, creating pHERD20T (Fig. 1). The EcoRI/AflII regions of these vectors were sequenced to confirm that no mutations were introduced during the cloning process. We next transferred the 2.4-kb AdhI-EcoRI fragment from pHERD20T to pUCP26, generating pHERD26T (Tet^r, 6,166 bp), which includes the araC-P_BAD cassette and the oriT sequence.

The pHERD vectors have the features of the pUCP vector family, including the pBR322 origin, four different antibiotic resistance markers, the oriT region for conjugation-mediated plasmid transfer (23), ori₁₆₀₀, and the rep gene encoding the replication-controlling protein (24, 29). However, the main advantage for cloning into the pHERD vectors is low expression, which occurs from the P_BAD promoter when it is not induced (Fig. 2). α complementation is inducible for blue-white screening, which facilitates the identification of 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 anneal to regions on both sides of the multiple cloning site, pHERD-SF 78 bp upstream of the EcoRI site and pHERD-SR 49 bp downstream of the HindIII site. If a gene is cloned in frame into the EcoRI site, a fusion protein with an additional seven NH₂-terminal amino acids (MGSDKNS) derived from thioredoxin of pBAD-TOPO/Thio will result. Thioredoxin acts as a translation leader to facilitate high-level expression and, in some cases, increase solubility in E. coli (9). These amino acids at the N terminus of the target protein may also serve as an epitope tag for protein analysis. pHERD vectors can be readily transferred from E. coli into Pseudomonas species and other bacteria via triparental conjugation (7) or by electroporation. It has been shown that the progenitor plasmid pRO1614 could replicate in a series of bacterial species, including P. aeruginosa, P. putida, P. fluorescens, Klebsiella pneumoniae (19), and Burkholderia spp. (5, 26). Therefore, the pHERD vectors are most likely functional in these bacteria. Another feature of the P_BAD promoter is catabolite repression of expression in the presence of glucose in the growth medium, which reduces intracellular cyclic AMP concentrations in E. coli cells, preventing the transcriptional activation of many genes by the cyclic AMP-binding protein (8).

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

We have observed that pHERD vectors can be used for the high-fidelity cloning and conditional expression of P_BAD transcription in the absence of l-arabinose (10). Initial attempts to clone the open reading frame of P. aeruginosa alternative sigma factor algU into pUCP20T were not successful. All of the algU alleles cloned were not functional, and sequence analysis showed that only mutant algU alleles were cloned into pUCP20T. This was consistent with the previous observations that algU/T cannot be cloned into the common expression vectors (16, 21). However, the algU gene was readily cloned into pHERD20T. Upon the expression of algU from P_BAD on pHERD20T, we observed dose-dependent alginate production or mucoidy in P. aeruginosa strain PAO1 in response to arabinose in the growth medium (Fig. 3).

Overexpression of the small peptide encoded by mucE activates AlgW, inducing alginate production (Fig. 4) in P. aeruginosa PAO1 and PA14 (20). Overexpression of mucE caused mucoidy in P. aeruginosa PAO1 and P. fluorescens Pf-5 (Table 2). The C-terminal WVF signal encoded by mucE is required for the activation of AlgW. The outer membrane protein OprF does not activate alginate production (Fig. 4); however, addition of the MucE WVF signal motif to the C terminus of OprF did cause alginate production (Table 2). Some genes are not highly expressed, and therefore expression in trans for complementation needs to be conditional. Expression of algW from P_BAD can complement an algW mutant back to 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 for complementation of the Δ(amrAB-oprA) efflux pump mutation in Burkholderia pseudomallei strain Bp50 (5). In this case, however, complementation was also observed in uninduced cells, presumably because of basal transcription from the P_BAD promoter, which could not be overcome by growing cells in the presence of 0.2% glucose and was not dependent on the growth medium used for the MIC assays (LB versus Mueller-Hinton broth) (data not shown).

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

Nucleotide sequence accession numbers.

The GenBank accession numbers for the nucleotide sequences of pHERD20T, -26T, -28T, and -30T are EU603324, EU603327, EU603325, and EU603326, respectively.