Sequence-Verified Two-Allele Transposon Mutant Library for Pseudomonas aeruginosa PAO1

Abstract

Mutant hunts using comprehensive sequence-defined libraries make it possible to identify virtually all of the nonessential functions required for different bacterial processes. However, the success of such screening depends on the accuracy of mutant identification in the mutant library used. To provide a high-quality library for Pseudomonas aeruginosa PAO1, we created a sequence-verified collection of 9,437 transposon mutants that provides genome coverage and includes two mutants for most genes. Mutants were cherry-picked from a larger library, colony-purified, and resequenced both individually using Sanger sequencing and in a pool using Tn-seq. About 8% of the insertion assignments were corrected, and in the final library nearly 93% of the transposon locations were confirmed by at least one of the resequencing procedures. The extensive sequence verification and inclusion of more than one mutant for most genes should help minimize missed or erroneous genotype-phenotype assignments in studies using the new library. Comprehensive sequence-defined mutant libraries have facilitated the genetic dissection of complex processes in several bacterial species (7). In principle, such libraries can be screened directly for mutants exhibiting a given phenotype to provide relatively complete identification of nonessential functions responsible for the trait. In practice, however, such screens can fail to achieve completeness and may even suggest incorrect genotypephenotype associations. There are several potential contributing factors that arise from how mutant libraries are created and quality control tested.Most defined mutant libraries have been generated by largescale transposon mutagenesis and sequencing. Relatively complete genome coverage requires that an average of 5 to 10 unique insertions per gene be identified (7). Such large primary libraries serve as a source of mutants for smaller secondary libraries that retain genome coverage and facilitate phenotype screening (1-3, 6). Secondary libraries are usually made up of one or more colonypurified mutants per gene, with insertions situated toward the centers of coding regions to help ensure inactivation. A limitation of most secondary libraries is that mutant identities have not been verified by resequencing. In rare cases in which they have been checked, many assignments (typically on the order of 10%) have been found to be incorrect (3). An additional limitation of secondary libraries consisting of only one mutant per gene is that screens using them are at risk of missing genotype-phenotype associations due to mutant cross-contamination and insertion alleles that fail to inactivate. Libraries with more than one mutant per gene provide more than one chance to identify an association.This report describes a Pseudomonas aeruginosa PAO1 secondary transposon mutant library with two mutants for most genes in which mutant identities have been confirmed by multiple resequencings. The redundancy and verification of mutant identities should make the library particularly useful...

Enterobacteria secrete an inhibitor of Pseudomonas virulence during clinical bacteriuria

“…Although pyochelin enhances P. aeruginosa growth during infections (53,54), it is functionally redundant with pyoverdin, a second siderophore, during in vitro growth in standard culture media. Escherichelin addition to a defined, iron-restricted minimal medium caused a dose-dependent reduction in growth of a pyoverdin-deficient P. aeruginosa mutant strain, PW5011 ( Figure 3H) (55)(56)(57). These results are consistent with the escherichelin-mediated inhibition of iron uptake in P. aeruginosa noted by Mislin et al (25).…”

“…Escherichelin activity assay on P. aeruginosa. PW5011 (pvdA-E02::ISlacZ/hah) (55,56) was grown in LB broth overnight at 37°C under shaking conditions and then back-diluted into succinate medium (25) at 10 8 CFU/ml. The growth experiments were performed in a final volume of 100 μl in 96-well plates.…”

Resources for Genetic and Genomic Analysis of Emerging Pathogen Acinetobacter baumannii

“…Such defined mutant libraries have proved useful for genome-scale studies of other pathogens (71)(72)(73). The A. baumannii library provides redundant, multiple-allele coverage of most nonessential genes, with most mutant identities verified by two independent sequencings.…”

The Stringent Response Controls Catalases in Pseudomonas aeruginosa and Is Required for Hydrogen Peroxide and Antibiotic Tolerance

“…The P. aeruginosa ⌬relA ⌬spoT (⌬SR) strain and complemented mutant (ϩSR) strain were obtained from a previous study (33). The katA, katB, and rpoS transposon mutants were obtained from the PAO1 two-allele transposon mutant library (41). To create the ⌬SR rpoS triple mutant, the rpoS-B03 ISlacZ/hah allele was moved from strain PW7151 into the ⌬SR mutant by transformation of the electrocompetent ⌬SR mutant with PW7151 genomic DNA as previously described (42).…”