Skip to main page content
U.S. flag

An official website of the United States government

Dot gov

The .gov means it’s official.
Federal government websites often end in .gov or .mil. Before sharing sensitive information, make sure you’re on a federal government site.

Https

The site is secure.
The https:// ensures that you are connecting to the official website and that any information you provide is encrypted and transmitted securely.

Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
. 2006 Feb 21;103(8):2833-8.
doi: 10.1073/pnas.0511100103. Epub 2006 Feb 13.

An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants

Nicole T Liberati  1 , Jonathan M Urbach, Sachiko Miyata, Daniel G Lee, Eliana Drenkard, Gang Wu, Jacinto Villanueva, Tao Wei, Frederick M Ausubel
Affiliations

An ordered, nonredundant library of Pseudomonas aeruginosa strain PA14 transposon insertion mutants

Nicole T Liberati et al. Proc Natl Acad Sci U S A. .

Erratum in

  • Proc Natl Acad Sci U S A. 2006 Dec 26;103(52):19931

Abstract

Random transposon insertion libraries have proven invaluable in studying bacterial genomes. Libraries that approach saturation must be large, with multiple insertions per gene, making comprehensive genome-wide scanning difficult. To facilitate genome-scale study of the opportunistic human pathogen Pseudomonas aeruginosa strain PA14, we constructed a nonredundant library of PA14 transposon mutants (the PA14NR Set) in which nonessential PA14 genes are represented by a single transposon insertion chosen from a comprehensive library of insertion mutants. The parental library of PA14 transposon insertion mutants was generated by using MAR2xT7, a transposon compatible with transposon-site hybridization and based on mariner. The transposon-site hybridization genetic footprinting feature broadens the utility of the library by allowing pooled MAR2xT7 mutants to be individually tracked under different experimental conditions. A public, internet-accessible database (the PA14 Transposon Insertion Mutant Database, http://ausubellab.mgh.harvard.edu/cgi-bin/pa14/home.cgi) was developed to facilitate construction, distribution, and use of the PA14NR Set. The usefulness of the PA14NR Set in genome-wide scanning for phenotypic mutants was validated in a screen for attachment to abiotic surfaces. Comparison of the genes disrupted in the PA14 transposon insertion library with an independently constructed insertion library in P. aeruginosa strain PAO1 provides an estimate of the number of P. aeruginosa essential genes.

PubMed Disclaimer

Conflict of interest statement

Conflict of interest statement: No conflicts declared.

Figures

Fig. 1.
Fig. 1.
The MAR2xT7 transposon is TraSH-compatible. (A) Schematic of MAR2xT7 showing the gentamicin resistance cassette, two outwardly directed T7 promoter sites at both ends of the tranposon, and short, 28-bp inverted repeats. The top strand of MAR2xT7 as it is pictured represents Strand A, and the bottom strand represents Strand B. Primers that anneal to Strand B were used for sequencing and identification of mutants. (B) T7 polymerase-generated RNA created from five different MAR2xT7 mutants. Genomic DNA from each mutant was HinP1-digested, and Y-linkers were ligated to the ends as described in ref. . PCR-amplified DNA products were used as template for T7 RNA polymerase. The average size of the RNA products is ≈500 bp.
Fig. 2.
Fig. 2.
The PA14 MAR2xT7 mutant library is approaching saturation. The number of insertions mapped is shown on the left y axis (solid line), and the number of genes disrupted is shown on the right y axis (dashed curve).
Fig. 3.
Fig. 3.
MAR2xT7 insertion-site distribution. The number of insertion sites in every 10 kb of PA14 genomic sequence. The upper set of data points represents insertions in which MAR2xT7 Strand B runs 5′ to 3′ with the top strand of the PA14 chromosome. The lower set of data points denotes MAR2xT7 insertions oriented in the opposite direction. Mb, megabase.
Fig. 4.
Fig. 4.
Frequency of insertions within genes. The left y axis shows the number of genes disrupted once, twice, etc. (solid trace). The right y axis shows the average length of the genes in kilobases at each insertion frequency (dashed trace).
Fig. 5.
Fig. 5.
Insertion site distribution relative to MAR2xT7 insertion position within each gene. (A) The fraction of all mutants (black bar) and the fraction of mutants carrying an insertion in a gene that was disrupted only once (hatched bar) are shown for each insertion site position as a percentage of gene length in base pairs. Fractions are based on either the sum of all mutants or the sum of all mutants with insertions in genes disrupted only one time. The fraction of total or single mutants containing an insertion in the first 5% of the gene length is represented by the 5% category. (B) The fraction of mutants carrying an insertion in a gene that was disrupted only once in the library at each gene position (black bars), the fraction of mutants with single gene insertions in which MAR2xT7 Strand A is oriented in the same direction as the coding sequence (hatched bars), and the fraction of mutants with single gene insertions with MAR2xT7 Strand B oriented in the same direction as the coding sequence (gray bars) are shown. Fractions are based on the sum of all mutants, the sum of all mutants with Strand A insertions in genes disrupted only once, or the sum of all mutants with Strand B insertions in genes disrupted only once.
Fig. 6.
Fig. 6.
Length in kilobases of gaps between transposon insertion sites. Actual gap sizes for the PA14 transposon insertion library (black trace, ■) and predicted gap sizes based on a Monte Carlo random distribution model of insertion sites (dashed trace, ♦) are shown.
Fig. 7.
Fig. 7.
PA14NR Set mutants show PVC-attachment deficiencies. Shown are absorbance measurements at 550 nm of crystal violet stain extracted from PA14NR Set mutant cells attached to PVC. The flgK mutant is a previously characterized PA14 strain carrying a transposon insertion in the flgK gene that was included as a control (31). Error bars represent SDs of two replicates.
See this image and copyright information in PMC

Similar articles

See all similar articles

Cited by

See all "Cited by" articles

References

    1. Riley M. Nucleic Acids Res. 1997;25:51–52. - PMC - PubMed
    1. Kang Y., Durfee T., Glasner J. D., Qiu Y., Frisch D., Winterberg K. M., Blattner F. R. J. Bacteriol. 2004;186:4921–4930. - PMC - PubMed
    1. Kobayashi K., Ehrlich S. D., Albertini A., Amati G., Andersen K. K., Arnaud M., Asai K., Ashikaga S., Aymerich S., Bessieres P., et al. Proc. Natl. Acad. Sci. USA. 2003;100:4678–4683. - PMC - PubMed
    1. Giaever G., Chu A. M., Ni L., Connelly C., Riles L., Veronneau S., Dow S., Lucau-Danila A., Anderson K., Andre B., et al. Nature. 2002;418:387–391. - PubMed
    1. Alonso J. M., Stepanova A. N., Leisse T. J., Kim C. J., Chen H., Shinn P., Stevenson D. K., Zimmerman J., Barajas P., Cheuk R., et al. Science. 2003;301:653–657. - PubMed

Publication types

Substances

LinkOut - more resources

-