Essential genome of Pseudomonas aeruginosa in cystic fibrosis sputum

Abstract

Defining the essential genome of bacterial pathogens is central to developing an understanding of the biological processes controlling disease. This has proven elusive for Pseudomonas aeruginosa during chronic infection of the cystic fibrosis (CF) lung. In this paper, using a Monte Carlo simulation-based method to analyze high-throughput transposon sequencing data, we establish the P. aeruginosa essential genome with statistical precision in laboratory media and CF sputum. Reconstruction of the global requirements for growth in CF sputum compared with defined growth conditions shows that the latter requires several cofactors including biotin, riboflavin, and pantothenate. Comparison of P. aeruginosa strains PAO1 and PA14 demonstrates that essential genes are primarily restricted to the core genome; however, some orthologous genes in these strains exhibit differential essentiality. These results indicate that genes with similar molecular functions may have distinct genetic roles in different P. aeruginosa strains during growth in CF sputum. We also show that growth in a defined growth medium developed to mimic CF sputum yielded virtually identical fitness requirements to CF sputum, providing support for this medium as a relevant in vitro model for CF microbiology studies.

Defining Genetic Fitness Determinants and Creating Genomic Resources for an Oral Pathogen

“…We then compared the expected to the observed data and observed that the fold changes in mutant abundance per gene follow typical (41,48) bimodal distributions (Fig. 1A).…”

“…1A). These numbers are [41] and 358 in Haemophilus influenzae [42]). The core A. actinomycetemcomitans essential genome.…”

“…We next used a Monte Carlo-based approach (41) to define the essential genomes of VT1169 and 624 with statistical rigor. In this approach, the actual mariner insertions identified in the genome (observed data) are compared to "expected" simulated data, where each gene is assumed to be fitness neutral (equally capable of being disrupted by a transposon).…”

“…The lower mode in these distributions corresponds to genes that cannot sustain insertions. As described previously (41), we determined a gene as essential if (i) it contained significantly fewer insertions than expected and (ii) clustered significantly with the lower of the two modes. Using these criteria, we identified 413 (22% of total) coding genes as essential in VT1169 and 386 (18% of total) coding genes as essential in strain 624 (Fig.…”

The Limitations of In Vitro Experimentation in Understanding Biofilms and Chronic Infection

“…A follow up study rectified these issues, incorporating these components into SCFM. Using Tn-seq (which is a more precise way to measure fitness, compared to RNA-seq), it was shown that near identical selection pressures exist between synthetic and expectorated CF sputum [128]. In a separate study, the use of an artificial sputum media was shown to increase diversity within a population, something not observed in Lysogeny Broth (LB) [108].…”