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Method Article
Here we describe a protocol using the mini-himar1 mariner transposon-mediated mutagenesis for generating a high-density insertion mutant library to screen, isolate and identify novel alginate regulators in the prototypic Pseudomonas aeruginosa strain PAO1.
Pseudomonas aeruginosa is a Gram-negative, environmental bacterium with versatile metabolic capabilities. P. aeruginosa is an opportunistic bacterial pathogen which establishes chronic pulmonary infections in patients with cystic fibrosis (CF). The overproduction of a capsular polysaccharide called alginate, also known as mucoidy, promotes the formation of mucoid biofilms which are more resistant than planktonic cells to antibiotic chemotherapy and host defenses. Additionally, the conversion from the nonmucoid to mucoid phenotype is a clinical marker for the onset of chronic infection in CF. Alginate overproduction by P. aeruginosa is an endergonic process which heavily taxes cellular energy. Therefore, alginate production is highly regulated in P. aeruginosa. To better understand alginate regulation, we describe a protocol using the mini-himar1 transposon mutagenesis for the identification of novel alginate regulators in a prototypic strain PAO1. The procedure consists of two basic steps. First, we transferred the mini-himar1 transposon (pFAC) from host E. coli SM10/λpir into recipient P. aeruginosa PAO1 via biparental conjugation to create a high-density insertion mutant library, which were selected on Pseudomonas isolation agar plates supplemented with gentamycin. Secondly, we screened and isolated the mucoid colonies to map the insertion site through inverse PCR using DNA primers pointing outward from the gentamycin cassette and DNA sequencing. Using this protocol, we have identified two novel alginate regulators, mucE (PA4033) and kinB (PA5484), in strain PAO1 with a wild-type mucA encoding the anti-sigma factor MucA for the master alginate regulator AlgU (AlgT, σ22). This high-throughput mutagenesis protocol can be modified for the identification of other virulence-related genes causing change in colony morphology.
The ability of the opportunistic, Gram-negative pathogen Pseudomonas aeruginosa to overproduce alginate is a major factor in its ability to establish a biofilm. The overproduction of alginate is a phenotype often referred to as mucoidy. The isolation of mucoid colonies from the sputa of individuals afflicted with cystic fibrosis (CF) is indicative of a chronic infection, and is directly associated with an overall decline in the patient's health1. Currently, it is understood that the regulation and production of alginate in P. aeruginosa primarily occurs at two operons. The first is the alginate biosynthetic operon, which contains 12 genes (algD-alg8-alg44-algK-algE-algG-algX-algL-algI-algJ-algF-algA) that are responsible for the synthesis and export of the alginate polymer across the periplasm to the extracellular environment2-5. The second operon is a cluster of genes beginning with the alternative sigma factor algU/T and continuing with mucA, mucB, and mucD. AlgU/T is a positive regulator, while mucAB-D are classified as negative regulators of alginate production6-8. Additionally, transcriptional regulators, such as AlgB, AlgQ, AlgR, and RpoN, as well as post-transcriptional and post-translational modification by catabolite repression control, kinase activity (KinB) and intramembrane proteolysis, have also been shown to be involved in alginate regulation9-14.
The mini-himar1 transposon vector known as pFAC was originally created in Dr. Mekalanos' lab at Harvard Medical School15. The pFAC plasmid consists of a transposable element flanked by two inverted repeats of 27 bps and a gentamycin resistance cassette in the middle (aacC1: 534 bp), a gene encoding the hyperactive mariner transposase16, and a gene encoding β-lactamase (bla) (Figure 1) . Sequence information for the transposable element of pFAC is available at the GenBank accession number: DQ36630013. In pFAC, there is a multiple cloning site (MCS) behind the aacC1 gene which is used for the identification of the chromosomal insertion site using inverse PCR. One major advantage with using the mini-himar1 transposon is no specific host factors are required for transposition (mutagenesis). Additionally, there is high abundance of the TA dinucleotide insertion sites found throughout the genome of P. aeruginosa. For example, TA dinucleotide insertion sites occurs 94,404 and 100,229 times in the PAO1 (6,264,404 bps, GenBank accession number NC_002516.2) and PA14 (6,537,648 bps; GenBank access number NC_008463) genomes, respectively. Because of the abundance of TA dinucleotide in the genome, mini-himar1 transposon can cause the high-density and random mutagenesis, which is particularly suitable for the analysis of virulence genes and the genes that are highly regulated. Theoretically, the mini-himar1 transposon can insert into any nonessential gene within the genome of P. aeruginosa. This provides approximately 18 TA insertion sites per open reading frame in the PAO1 genome.
Here, we describe a protocol using mini-himar1 transposon-mediated mutagenesis to identify novel regulators of mucoidy in P. aeruginosa. More specifically, we biparentally conjugated the pFAC vector containing a mini-himar1 transposon from E. coli SM10/λpir into the nonmucoid prototrophic strain PAO1. After the transposon is integrated in the genome, the recipient strain is cultured on Pseudomonas isolation agar (PIA) containing triclosan which inhibits the growth of E. coli. Thus, a library of mutants of P. aeruginosa can be selected by the growth on PIA plate supplemented with gentamycin and the presence of the mucoid phenotype. Note, a true transposon-mediated vs. an integration mutant will have a gentamycin resistant and carbenicillin-sensitive phenotype. In this study, approximately 80,000 insertion mutants of PAO1 were isolated through four separate conjugations. We then screened for mucoid isolates, and determined the site of insertion by restriction enzyme digestion, ligation and inverse PCR. We performed Southern blot analysis using the gentamycin resistance cassette as a probe to see the number of insertion per genome. We determined that more than 90% of mutants obtained per conjugation using this protocol had only a single copy of the himar1 in the genome and displayed the gentamycin resistant and carbenicillin-sensitive phenotype. A total of 32 mucoid isolates were identified, 22 of them were mapped to different loci of the P. aeruginosa PAO1 chromosome. This rate of insertion provides adequate coverage to identify several novel regulators of alginate overproduction.
1. Preparation of Bacterial Strains and Biparental Conjugation
2. Detection and Isolation of Mucoid Colonies
3. gDNA Restriction Digestion and Ligation
4. Inverse PCR (iPCR) and Sequence Analysis
As illustrated in Figure 1, the mini-himar1 mariner transposon vector, pFAC, contains two 27 bp inverted repeats with TA insertion sites flanking the aacC1 gentamycin resistance cassette, with its σ70-dependent promoter, and a multiple cloning site (MCS). Additionally, the pFAC vector contains genes encoding the highly active himar1 transposase, β-lactamase (bla), and the tra transfer operon. The TA insertion sites allow for an efficient i...
It is important to note that this method can be used in other Pseudomonas species with these alterations: incubate P. fluorescens and P. putida at 30 °C, and P. stutzeri at 42 °C; P. stuzeri should be cultured on LB plates supplemented with 150 µg/mL of gentamycin; on step 1.11, P. stutzeri cells should be transferred into 500 µl of LB instead of 1 ml. Additionally, there are two critical steps for this protocol. First, the recipient strain should ...
The author Hongwei D. Yu is the Chief Science Officer and Cofounder of Progenesis Technologies, LLC.
This work was supported by the National Aeronautics and Space Administration West Virginia Space Grant Consortium (NASA WVSGC), Cystic Fibrosis Foundation (CFF-YU11G0) and NIH P20RR016477 and P20GM103434 to the West Virginia IDeA Network for Biomedical Research Excellence. We thank Vonya M. Eisinger for the technical assistance with this work.
Name | Company | Catalog Number | Comments |
Luria Broth | Difco | 240230 | via Fisher Scientific |
Pseudomonas isolation agar | Difco | 292710 | via Fisher Scientific |
Small Plates (100 O.D. x 10 mm) | Fisher Scientific | 08-757-13 | |
Large Plates (150 O.D. x 15 mm) | Fisher Scientific | 08-757-14 | |
Glycerol | Fisher Scientific | BP229-4 | |
Benchtop Shaking Incubator | New Brunswick Scientific | Innova 4080 | shake at 200 rpm |
Cabinet Incubator | VWR | 1540 | |
Benchtop Microcentrifuge | Sorvall | 75-003-287 | via Fisher Scientific |
SmartSpec Plus Spectrophotometer | Bio-Rad | 170-2525 | or preferred method/vendor |
Diposable Inoculation Loops | Fisher Scientific | 22-363-597 | |
1.5 ml Microcentrifuge Tubes | Fisher Scientific | 05-408-129 | |
2.0 ml Cryogenic Vials | Corning | 430659 | via Fisher Scientific |
15 ml Tubes | Fisher Scientific | 05-539-12 | |
Skim Milk | Difco | DF0032-17-3 | via Fisher Scientific |
DNeasy Blood and Tissue (250) | Qiagen | 69506 | or preferred method/vendor |
QIAquick PCR Purification Kit (250) | Qiagen | 28106 | or preferred method/vendor |
QIAprep Spin Miniprep Kit (250) | Qiagen | 27106 | or preferred method/vendor |
FastLink II DNA Ligation Kit | Epicentre Technologies | LK6201H | via Fisher Scientific |
Accu block Digital Dry Bath | Labnet | NC0205808 | via Fisher Scientific |
Sal1, restriction endonuclease | New England BioLabs | R0138L | |
EasyStart Micro 50 | Molecular BioProducts | 6020 | via Fisher Scientific |
Taq DNA Polymerase | New England BioLabs | M0267L | |
iCycler, Thermocycler | Bio-Rad | 170-8740 | |
LE agarose | Genemate | 3120-500 | via Fisher Scientific |
Gentamycin Sulfate | Fisher Scientific | BP918-1 | |
2.0 ml Cryogenic Vials | Corning | 430659 | via Fisher Scientific |
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