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We describe the construction of mini-Tn7-based broad-host-range vectors encoding lux genes as bioluminescent reporters. These constructs can be mobilized into the desired host(s) by conjugation for chromosomal mini-Tn7-lux integration and are useful for localization of bacteria during infections or for characterizing regulation of promoters of interest in Gram-negative bacteria.
The lux bioluminescent reporter genes are useful for quantifying and characterizing bacterial gene expression or pinpointing the location of bacteria within in vivo infection models (1). Expression of the luciferase genes (luxCDABE) from Photorhabdus luminescens, commonly referred to as the lux reporter, results in readily detectable bioluminescence (2). The light produced by luciferase is emitted at 490 to 500 nm and can be easily detected by several analytical platforms, such as luminometers, film exposure, or digital cameras. In this report, we describe the assembly of mobilizable lux reporter plasmid constructs capable of being transferred to the strain of interest by conjugation to integrate the mini-Tn7-lux elements into the chromosome of numerous Gram-negative bacteria. We provide experimental examples that highlight their utility and offer further suggestions for their application in future studies.
Mini-Tn7 vectors have been shown to be useful in many bacteria (3–9) for shuttling constructs to a highly conserved site on the chromosome downstream of the glmS gene(s) (4). A mini-Tn7 vector carrying lux driven by a constitutive promoter has been constructed and is known as pUC18-mini-Tn7T-lux-Gm (6). To permit conjugation of the plasmid into recipient cells, an oriT-containing fragment from the backbone of pUC18T-mini-Tn7T was released by digestion with NarI and StuI and ligated with a NarI-StuI fragment of pUC18-mini-Tn7T-Gm-lux to create the new vector pUC18T-mini-Tn7T-lux-Gm (see Fig. S1 in the supplemental material). Many bacterial mutant strains, such as those in the Pseudomonas aeruginosa PA14 nonredundant mutant library (10), were constructed with a gentamicin resistance marker. In order to make this vector compatible with a broader range of bacteria, we replaced the gentamicin resistance cassette (aacC1) with a trimethoprim resistance marker. To accomplish this, the XbaI-FRT-aacC1-FRT-XbaI fragment was deleted from pUC18T-mini-Tn7T-lux-Gm and replaced with the XbaI-FRT-dhfRIIb-FRT-XbaI fragment from pUC18T-mini-Tn7T-Tp, forming pUC18T-mini-Tn7T-lux-Tp (Fig. 1). In order to integrate the mini-Tn7-lux elements contained in this and related constructs into the chromosome, these delivery vectors can be transferred by conjugation into recipient strains along with helper plasmids (pRK2013 and pTNS3; Table 1), as previously described (11). Alternatively, the delivery plasmid can be (i) transformed into an Escherichia coli mobilizer strain with chromosomally integrated transfer functions (e.g., RHO3, S17-1, or SM10) and conjugally cotransferred into the target bacterium containing the pTNS3 helper plasmid (12) or (ii) codelivered into the target bacterium by coelectroporation of the mini-Tn7-lux delivery plasmid and pTNS3 (6, 13).
Both pUC18T-mini-Tn7T-lux-Tp and pUC18T-mini-Tn7T-lux-Gm contain the robust P1 integron promoter (14) which drives constitutive luxCDBAE gene expression. The P1 integron promoter region has two sigma70-dependent promoters; similar constructs with the P1 promoter have proven to be useful for bioluminescent localization studies (15). In addition, promoter lux fusions are useful for determining in vitro and in vivo promoter regulation, including the conditions that affect activity, and for basic characterization of promoter regions (2). To fuse a promoter of interest into the pUC18T-mini-Tn7T-lux vectors, BamHI, EcoRI, and PstI restriction sites can be used to remove the P1 integron promoter and replace it with a promoter of interest. We attempted to replace the P1 integron promoter region with a multiple cloning site but noticed instability of the plasmid during cloning and found it easier to delete the P1 promoter region by restriction enzyme digestion and replace it with the promoter of interest. The resulting construct was then able to be used to perform a variety of experiments aimed at characterizing factors governing gene expression from the cloned promoter region.
P. aeruginosa is a Gram-negative opportunistic pathogen that secretes an exopolysaccharide known as alginate to promote survival in the lungs of cystic fibrosis patients. AlgU is the master regulator of alginate biosynthesis and is an alternative sigma factor that is required for the expression of the algD alginate biosynthetic operon. PalgU and PalgD are important regulated promoters that have been extensively studied (16). DNA fragments containing the PalgU and PalgD promoters were amplified from strain PAO1 and directionally ligated into pUC18T-mini-Tn7T-lux-Tp to drive lux expression, and the respective mini-Tn7 elements were transferred to the chromosome of PAO1 as previously described (11). PAO1 derivatives with P1-lux, PalgU-lux, or PalgD-lux integrated into the chromosome were grown on Pseudomonas isolation agar (PIA) (Fig. 2A and andB).B). A Bio-Rad Chemidoc XRS+ Gel Doc was used to detect bioluminescence. Standard gel imaging systems with chemiluminescence detection capabilities can be utilized to take white light and bioluminescence images that can then be overlaid with an Adobe Photoshop action file (http://tinyurl.com/clzy83o). This action file can be used to generate overlays following the protocol described in Fig. S2 in the supplemental material to identify clones with higher or lower bioluminescence production in the environment being tested (Fig. 2A and andB).B). Using densitometry measurements (data not shown), each of the clones can be identified when they are mixed together and plated (Fig. 2B). Since PAO1 is nonmucoid on PIA, it was expected the PalgD-lux activity would be low on PIA (Fig. 2A). A PerkinElmer Victor3 luminescence plate reader was used to measure bioluminescence during growth. In addition to luminometer-based measurements, simple exposure to photographic film for a few seconds is sufficient to effectively visualize bioluminescence (data not shown). As shown in Fig. 2C, the activities of PAO1 with P1-lux, PalgU-lux, and PalgD-lux were measured in Pseudomonas isolation broth (PIB). Bioluminescence was the highest for the P1-lux reporter during log-phase growth (Fig. 2C). We would predict the PalgU promoter activity to be higher than that of PalgD in PIB because PIB does not cause alginate overproduction (Fig. 2C). PIA with ammonium metavanadate (PIAAMV) is a medium that induces alginate overproduction by P. aeruginosa and activates transcription from both PalgU and PalgD (17). The PalgU and PalgD reporter strains were grown on PIA and on PIAAMV, cells were collected in phosphate-buffered saline, samples were diluted to the same optical density, and then the bioluminescence was measured by a luminometer. As shown previously with promoter-lacZ fusions (17), compared to PIA results, growth on PIAAMV increases PalgU and PalgD promoter activity (Fig. 2D).
To illustrate how the lux constructs can be used for localization of bacteria and in vivo promoter analysis, PAO1, PAO1-P1-lux, PAO1-PalgU-lux, and PAO1-PalgD-lux strains were used to infect lettuce (18). P. aeruginosa was grown in PIB at 37°C for 18 h and washed with sterile 10 mM MgSO4. Fresh romaine lettuce leaves were washed with 0.1% (vol/vol) bleach and rinsed with sterile distilled water, and then the midrib was inoculated with 106 CFU of each strain. A Bio-Rad Chemidoc XRS+ Gel Doc was used to detect bioluminescence of P. aeruginosa strains 24 h after infection. While the diameters of the soft rot area at the site of inoculation for all the strains were nearly identical to the diameter at the site of the uninfected inoculation, the intensity of light emission for PAO1-P1-lux was the highest (Fig. 3A). PalgU and PalgD were also detected, albeit at lower levels, as expected. Comparable images were obtained with the Xenogen IVIS 100 imaging system (data not shown), but an imaging system of such ultrahigh sensitivity is not required for all applications. These promoter-lux constructs can also be used for in vivo imaging in other model infection systems, such as mice (Fig. 3B). Eight-week-old BALB/c mice were infected with 4 × 107 CFU of PAO1 P1-lux by intranasal administration. After 24 h, mice were anesthetized with isoflurane and images were captured by 10-s exposures with a Xenogen IVIS 100 imaging system (Fig. 3B). PAO1 P1-lux was clearly localized in the nares and lung of the mice shown in the representative image, indicating the utility of these constructs for in vivo bioluminescence imaging. All animal experiments were performed following protocols approved by the University of Virginia Animal Care and Use Committee.
In this report, we have described construction of two mobilizable mini-Tn7-based chromosomal integration vectors for luciferase tagging and promoter analysis using lux fusions. We showed several examples illustrating the utility of these vectors and imaging of the resultant bioluminescent bacteria. To discover novel regulators, a promoter of interest could be fused with the lux genes and then the strain could be submitted to transposon mutagenesis. Bioluminescent screening for resulting mutants with higher or lower promoter activity could identify novel regulators. Mini-Tn7-based vectors can be used in many Gram-negative bacteria, and we anticipate that these vectors will be of great utility for gene expression analyses in various model systems.
F.H.D. was supported by a postdoctoral fellowship from the Cystic Fibrosis Foundation (DAMRON10F0). M.B. was supported by a postdoctoral fellowship from the American Type Culture Collection. G.W.L. was supported in part by a University of Virginia Cancer Training Grant (5T32 CA 009109). H.P.S. was supported by NIAID grant U54 AI065357. J.B.G. was supported grants from the NIH (R01 AI068112) and the Cystic Fibrosis Foundation (GOLDBE10G0).
We thank Ian Glomski for critical review of the manuscript. We are grateful to Brian Hall for assistance in generating these constructs and methods of detection. We also thank the anonymous reviewers for their helpful comments.
Published ahead of print 12 April 2013
Supplemental material for this article may be found at http://dx.doi.org/10.1128/AEM.00640-13.