• We are sorry, but NCBI web applications do not support your browser and may not function properly. More information
Logo of aemPermissionsJournals.ASM.orgJournalAEM ArticleJournal InfoAuthorsReviewers
Appl Environ Microbiol. Dec 2008; 74(23): 7422–7426.
Published online Oct 10, 2008. doi:  10.1128/AEM.01369-08
PMCID: PMC2592904

PBAD-Based Shuttle Vectors for Functional Analysis of Toxic and Highly Regulated Genes in Pseudomonas and Burkholderia spp. and Other Bacteria[down-pointing small open triangle]

Abstract

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

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

Construction and features of pHERD vectors.

Functional genetic analysis requires vectors capable of conditional expression. The PBAD 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. (Fig.1),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-PBAD fragment (1.3 kb), produced via PCR with pBAD/Thio-TOPO as the template and primers pBAD-F and pBAD-R (Table (Table1).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. (Fig.1).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 (Tetr, 6,166 bp), which includes the araC-PBAD cassette and the oriT sequence.

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 ...
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 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 PBAD promoter when it is not induced (Fig. (Fig.2).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 PBAD promoter responds in a dose-dependent manner (Fig. (Fig.2).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 PBAD 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).

FIG. 2.
Arabinose-regulated lacZα 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 ...

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 PBAD 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 PBAD on pHERD20T, we observed dose-dependent alginate production or mucoidy in P. aeruginosa strain PAO1 in response to arabinose in the growth medium (Fig. (Fig.33).

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% ...

Overexpression of the small peptide encoded by mucE activates AlgW, inducing alginate production (Fig. (Fig.4)4) in P. aeruginosa PAO1 and PA14 (20). Overexpression of mucE caused mucoidy in P. aeruginosa PAO1 and P. fluorescens Pf-5 (Table (Table2).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. (Fig.4);4); however, addition of the MucE WVF signal motif to the C terminus of OprF did cause alginate production (Table (Table2).2). Some genes are not highly expressed, and therefore expression in trans for complementation needs to be conditional. Expression of algW from PBAD can complement an algW mutant back to alginate production due to titratable expression (Table (Table2).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 PBAD 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).

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 ...
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-Pseudomonas shuttle vectors with the E. coli araC and PBAD 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.

Acknowledgments

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

This work was supported by a research grant (NNA04CC74G) from the National Aeronautics and Space Administration (NASA) and research grants from the NASA West Virginia Space Grant Consortium to H.D.Y. H.P.S.'s Burkholderia research was supported by NIH grant 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 algU alleles in pUCP20T, V. M. Eisinger for the generation of the VE mutants, and K. D. Dillon for technical assistance with the alginate assay.

Footnotes

[down-pointing small open triangle]Published ahead of print on 10 October 2008.

REFERENCES

1. Baynham, P. J., D. M. Ramsey, B. V. Gvozdyev, E. M. Cordonnier, and D. J. Wozniak. 2006. The Pseudomonas aeruginosa ribbon-helix-helix DNA-binding protein AlgZ (AmrZ) controls twitching motility and biogenesis of type IV pili. J. Bacteriol. 188:132-140. [PMC free article] [PubMed]
2. Ben-Samoun, K., G. Leblon, and O. Reyes. 1999. Positively regulated expression of the Escherichia coli araBAD promoter in Corynebacterium glutamicum. FEMS Microbiol. Lett. 174:125-130. [PubMed]
3. Boles, B. R., M. Thoendel, and P. K. Singh. 2005. Rhamnolipids mediate detachment of Pseudomonas aeruginosa from biofilms. Mol. Microbiol. 57:1210-1223. [PubMed]
4. Brosius, J., M. Erfle, and J. Storella. 1985. Spacing of the −10 and −35 regions in the tac promoter. Effect on its in vivo activity. J. Biol. Chem. 260:3539-3541. [PubMed]
5. Choi, K.-H., T. Mima, Y. Casart, D. Rholl, A. Kumar, I. R. Beacham, and H. P. Schweizer. 2008. Genetic tools for select agent compliant manipulation of Burkholderia pseudomallei. Appl. Environ. Microbiol. 74:1064-1075. [PMC free article] [PubMed]
6. de Boer, H. A., L. J. Comstock, and M. Vasser. 1983. The tac promoter: a functional hybrid derived from the trp and lac promoters. Proc. Natl. Acad. Sci. USA 80:21-25. [PMC free article] [PubMed]
7. Figurski, D. H., and D. R. Helinski. 1979. Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans. Proc. Natl. Acad. Sci. USA 76:1648-1652. [PMC free article] [PubMed]
8. Guzman, L.-M., D. Belin, M. J. Carson, and J. Beckwith. 1995. Tight regulation, modulation, and high-level expression by vectors containing the arabinose PBAD promoter. J. Bacteriol. 177:4121-4130. [PMC free article] [PubMed]
9. LaVallie, E. R., E. A. DiBlasio, S. Kovacic, K. L. Grant, P. F. Schendel, and J. M. McCoy. 1993. A thioredoxin gene fusion expression system that circumvents inclusion body formation in the E. coli cytoplasm. Bio/Technology 11:187-193. [PubMed]
10. Lee, N., C. Francklyn, and E. P. Hamilton. 1987. Arabinose-induced binding of AraC protein to araI2 activates the araBAD operon promoter. Proc. Natl. Acad. Sci. USA 84:8814-8818. [PMC free article] [PubMed]
11. Lee, N. L., W. O. Gielow, and R. G. Wallace. 1981. Mechanism of araC autoregulation and the domains of two overlapping promoters, Pc and PBAD, in the l-arabinose regulatory region of Escherichia coli. Proc. Natl. Acad. Sci. USA 78:752-756. [PMC free article] [PubMed]
12. Lefebre, M. D., and M. A. Valvano. 2002. Construction and evaluation of plasmid vectors optimized for constitutive and regulated gene expression in Burkholderia cepacia complex isolates. Appl. Environ. Microbiol. 68:5956-5964. [PMC free article] [PubMed]
13. Ma, L., K. D. Jackson, R. M. Landry, M. R. Parsek, and D. J. Wozniak. 2006. Analysis of Pseudomonas aeruginosa conditional psl variants reveals roles for the psl polysaccharide in adhesion and maintaining biofilm structure postattachment. J. Bacteriol. 188:8213-8221. [PMC free article] [PubMed]
14. Martin, D. W., B. W. Holloway, and V. Deretic. 1993. Characterization of a locus determining the mucoid status of Pseudomonas aeruginosa: AlgU shows sequence similarities with a Bacillus sigma factor. J. Bacteriol. 175:1153-1164. [PMC free article] [PubMed]
15. Martin, D. W., M. J. Schurr, M. H. Mudd, J. R. Govan, B. W. Holloway, and V. Deretic. 1993. Mechanism of conversion to mucoidy in Pseudomonas aeruginosa infecting cystic fibrosis patients. Proc. Natl. Acad. Sci. USA 90:8377-8381. [PMC free article] [PubMed]
16. Mathee, K., C. J. McPherson, and D. E. Ohman. 1997. Posttranslational control of the algT (algU)-encoded σ22 for expression of the alginate regulon in Pseudomonas aeruginosa and localization of its antagonist proteins MucA and MucB (AlgN). J. Bacteriol. 179:3711-3720. [PMC free article] [PubMed]
17. Mdluli, K. E., P. R. Witte, T. Kline, A. W. Barb, A. L. Erwin, B. E. Mansfield, A. L. McClerren, M. C. Pirrung, L. N. Tumey, P. Warrener, C. R. Raetz, and C. K. Stover. 2006. Molecular validation of LpxC as an antibacterial drug target in Pseudomonas aeruginosa. Antimicrob. Agents Chemother. 50:2178-2184. [PMC free article] [PubMed]
18. Newman, J. R., and C. Fuqua. 1999. Broad-host-range expression vectors that carry the l-arabinose-inducible Escherichia coli araBAD promoter and the araC regulator. Gene 227:197-203. [PubMed]
19. Olsen, R. H., G. DeBusscher, and W. R. McCombie. 1982. Development of broad-host-range vectors and gene banks: self-cloning of the Pseudomonas aeruginosa PAO chromosome. J. Bacteriol. 150:60-69. [PMC free article] [PubMed]
20. Qiu, D., V. M. Eisinger, D. W. Rowen, and H. D. Yu. 2007. Regulated proteolysis controls mucoid conversion in Pseudomonas aeruginosa. Proc. Natl. Acad. Sci. USA 104:8107-8112. [PMC free article] [PubMed]
21. Schurr, M. J., D. W. Martin, M. H. Mudd, and V. Deretic. 1994. Gene cluster controlling conversion to alginate-overproducing phenotype in Pseudomonas aeruginosa: functional analysis in a heterologous host and role in the instability of mucoidy. J. Bacteriol. 176:3375-3382. [PMC free article] [PubMed]
22. Schurr, M. J., H. Yu, J. M. Martinez-Salazar, J. C. Boucher, and V. Deretic. 1996. Control of AlgU, a member of the sigma E-like family of stress sigma factors, by the negative regulators MucA and MucB and Pseudomonas aeruginosa conversion to mucoidy in cystic fibrosis. J. Bacteriol. 178:4997-5004. [PMC free article] [PubMed]
23. Schweizer, H. P. 1992. Allelic exchange in Pseudomonas aeruginosa using novel ColE1-type vectors and a family of cassettes containing a portable oriT and the counter-selectable Bacillus subtilis sacB marker. Mol. Microbiol. 6:1195-1204. [PubMed]
24. Schweizer, H. P. 1991. Escherichia-Pseudomonas shuttle vectors derived from pUC18/19. Gene 97:109-121. [PubMed]
25. Schweizer, H. P. 2001. Vectors to express foreign genes and techniques to monitor gene expression in pseudomonads. Curr. Opin. Biotechnol. 12:439-445. [PubMed]
26. Schweizer, H. P., T. R. Klassen, and T. Hoang. 1996. Improved methods for gene analysis and expression in Pseudomonas, p. 229-237. In T. Nakazawa, K. Furukawa, D. Haas, and S. Silver (ed.), Molecular biology of pseudomonads. ASM Press, Washington, DC.
27. Suh, S. J., L. A. Silo-Suh, and D. E. Ohman. 2004. Development of tools for the genetic manipulation of Pseudomonas aeruginosa. J. Microbiol. Methods 58:203-212. [PubMed]
28. Tabor, S., and C. C. Richardson. 1985. A bacteriophage T7 RNA polymerase/promoter system for controlled exclusive expression of specific genes. Proc. Natl. Acad. Sci. USA 82:1074-1078. [PMC free article] [PubMed]
29. West, S. E., H. P. Schweizer, C. Dall, A. K. Sample, and L. J. Runyen-Janecky. 1994. Construction of improved Escherichia-Pseudomonas shuttle vectors derived from pUC18/19 and sequence of the region required for their replication in Pseudomonas aeruginosa. Gene 148:81-86. [PubMed]
30. Woolwine, S. C., A. B. Sprinkle, and D. J. Wozniak. 2001. Loss of Pseudomonas aeruginosa PhpA aminopeptidase activity results in increased algD transcription. J. Bacteriol. 183:4674-4679. [PMC free article] [PubMed]
31. Wyckoff, T. J., B. Thomas, D. J. Hassett, and D. J. Wozniak. 2002. Static growth of mucoid Pseudomonas aeruginosa selects for nonmucoid variants that have acquired flagellum-dependent motility. Microbiology 148:3423-3430. [PubMed]
32. Yanisch-Perron, C., J. Vieira, and J. Messing. 1985. Improved M13 phage cloning vectors and host strains: nucleotide sequences of the M13mp18 and pUC19 vectors. Gene 33:103-119. [PubMed]

Articles from Applied and Environmental Microbiology are provided here courtesy of American Society for Microbiology (ASM)
PubReader format: click here to try

Formats:

Related citations in PubMed

See reviews...See all...

Cited by other articles in PMC

See all...

Links

Recent Activity

Your browsing activity is empty.

Activity recording is turned off.

Turn recording back on

See more...