General and condition-specific essential functions of Pseudomonas aeruginosa

Abstract

The essential functions of a bacterial pathogen reflect the most basic processes required for its viability and growth, and represent potential therapeutic targets. Most screens for essential genes have assayed a single condition-growth in a rich undefined medium-and thus have not distinguished genes that are generally essential from those that are specific to this particular condition. To help define these classes for Pseudomonas aeruginosa, we identified genes required for growth on six different media, including a medium made from cystic fibrosis patient sputum. The analysis used the Tn-seq circle method to achieve high genome coverage and analyzed more than 1,000,000 unique insertion positions (an average of one insertion every 6.0 bp). We identified 352 general and 199 condition-specific essential genes. A subset of assignments was verified in individual strains with regulated expression alleles. The profile of essential genes revealed that, compared with Escherichia coli, P. aeruginosa is highly vulnerable to mutations disrupting central carbon-energy metabolism and reactive oxygen defenses. These vulnerabilities may arise from the stripped-down architecture of the organism's carbohydrate utilization pathways and its reliance on respiration for energy generation. The essential function profile thus provides fundamental insights into P. aeruginosa physiology as well as identifying candidate targets for new antibacterial agents.Tn-seq | ESKAPE | cystic fibrosis | antibiotic target | sputum

Loving the poison: the methylcitrate cycle and bacterial pathogenesis

“…Such observations indicate that the methylcitrate cycle can fulfil atypical functionalities when required [49]. The metabolic redundancy of PrpC may also explain why the citrate synthase of Pseudomonas aeruginosa (GltA) is not generally essential for growth [50].…”

High molecular weight DNA assembly in vivo for synthetic biology applications

“…Furthermore, a number of essential genes are hypothetical open reading frames with unknown function. [86][87][88][89][90][91][92][93][94] The unknown genes constitute 9.8% of general Pseudomonas aeruginosa essential gene set required for growth in three primary growth conditions (minimal medium, sputum, LB medium). Furthermore, unknown genes constitute 13.4% of highly confirmed P. aeruginosa essential genes identified as essential in four genome-wide analyses.…”

Essential Genes Predicted in the Genome of Rubrivivax gelatinosus

“…Mutants are pooled, and high-throughput sequencing is used to map hundreds of thousands of unique insertions; these data are then used to find genes that are essential under mutagenesis conditions by identifying genes that cannot tolerate disruptive insertions. This technique has been used on a number of model bacterial systems (13)(14)(15)(16)(17). Here, TnSeq was applied to R. gelatinosus, not only to investigate its metabolism but also to reveal other unusual features of the organism.…”

“…In those three categories, B. subvibrioides has 35 essential genes and A. tumefaciens has 39 essential genes (15). C. crescentus TnSeq found 49 essential genes of unknown function (14), and P. aeruginosa has 72 essential genes listed as hypothetical alone (16). While this unknown essential gene content may represent biology yet to be discovered, the number of genes in these categories is likely also dependent on the age or effectiveness of genome annotation.…”

“…There were very few essential genes from any of the glycolytic pathways. This result is not unusual; few glycolytic genes are essential in other organisms (13)(14)(15)(16)(17). TnSeq is usually performed on rich media containing combinations of tryptone, peptone, and yeast extract; therefore, the principal carbon and energy sources in these media are amino acids and small peptides.…”