Identification of Novel Genes Associated with Alginate Production in Pseudomonas aeruginosa Using Mini-himar1 Mariner Transposon-mediated Mutagenesis

Summary

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.

Abstract

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.

Introduction

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 ge....

Protocol

1. Preparation of Bacterial Strains and Biparental Conjugation

1. Inoculate E. coli SM10/λpir/pFAC in 5 ml of Luria Broth (LB) supplemented with 15 µg/ml of gentamycin and place in a shaking incubator overnight at 37 °C.
2. Inoculate P. aeruginosa strain PAO1 in 5 ml of LB, and place in a shaking incubator overnight at 42 °C.
3. Measure OD₆₀₀ of overnight cultures and mix equal amounts of PAO1 and E. coli pFAC such that the final volume is between 1-1.4 ml.
4. Centrifuge mixture at 6,000 x g for 5 min.
5. Meanwhile, dry LB plates for conjugation: leave plates open in incubat....

Results

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 integration.......

Discussion

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 .......

Materials

List of materials used in this article

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