Genetic footprinting with mariner -based transposition in Pseudomonas aeruginosa

Abstract

The complete DNA sequence of Pseudomonas aeruginosa provides an opportunity to apply functional genomics to a major human pathogen. A comparative genomics approach combined with genetic footprinting was used as a strategy to identify genes required for viability in P. aeruginosa. Use of a highly efficient in vivo mariner transposition system in P. aeruginosa facilitated the analysis of candidate genes of this class. We have developed a rapid and efficient allelic exchange system by using the I-SceI homing endonuclease in conjunction with in vitro mariner mutagenesis to generate mutants within targeted regions of the P. aeruginosa chromosome for genetic footprinting analyses. This technique for generating transposon insertion mutants should be widely applicable to other organisms that are not naturally transformable or may lack well developed in vivo transposition systems. We tested this system with three genes in P. aeruginosa that have putative essential homologs in Haemophilus influenzae. We show that one of three H. influenzae essential gene homologs is needed for growth in P. aeruginosa, validating the practicality of this comparative genomics strategy to identify essential genes in P. aeruginosa.transposon ͉ SCE jumping

A genome-scale analysis for identification of genes required for growth or survival of Haemophilus influenzae

“…Genes essential for viability under a defined growth condition (in this case, the ability to form colonies on BHIs agar medium) sustain a markedly reduced frequency of transposon insertions as detected by genetic footprinting. Results of this approach correlate well with independent methods for assigning essentiality (9,17) and, therefore, genes that cannot sustain transposon insertions are designated as putatively essential. Adjacent nonessential genes or intergenic regions in each DNA segment serve as internal controls that can sustain insertions.…”

“…For example, a kinetic approach to monitoring loss of mutants from a pool directly after in vitro transposon mutagenesis can provide a rapid means of initial verification of essentiality (5,32). In addition, insertions in specific DNA regions can be more precisely mapped by PAGE to examine genes that were not evaluated (17,33). This study integrates genomic sequence data with genome-scale mutagenesis.…”

“…Table 2 notes roles in viability for several conserved genes (homologs of HI0004, HI0767, HI1282, HI1608, and HI1654) that have been examined in B. subtilis (ref. 4; http:͞͞locus.jouy.inra.fr͞cgi-bin͞ genmic͞madbasehome.pl), E. coli (27), M. genitalium (6), or Pseudomonas aeruginosa (17). Both of the putatively essential H. influenzae genes in the list that are conserved in M. genitalium are potentially essential in M. genitalium.…”

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

“…First, the recipient strain should be cultured at the appropriate temperature. For example, PAO1 should be incubated at 42 °C to increase the frequency of recombination 15 . The efficiency of the mutant library was significantly reduced if the recipient strain if not cultured at the correct temperature.…”

“…The mini-himar1 transposon vector known as pFAC was originally created in Dr. Mekalanos' lab at Harvard Medical School 15 . 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 transposase 16 , and a gene encoding β-lactamase (bla) (Figure 1) .…”