Abstract
Burkholderia cenocepacia K56-2 belongs to the Burkholderia cepacia complex, a group of Gram-negative opportunistic pathogens that have large and dynamic genomes. In this work, we identified the essential genome of B. cenocepacia K56-2 using high-density transposon mutagenesis and insertion site sequencing (Tn-seq circle). We constructed a library of one million transposon mutants and identified the transposon insertions at an average of one insertion per 27 bp. The probability of gene essentiality was determined by comparing of the insertion density per gene with the variance of neutral datasets generated by Monte Carlo simulations. Five hundred and eight genes were not significantly disrupted, suggesting that these genes are essential for survival in rich, undefined medium. Comparison of the B. cenocepacia K56-2 essential genome with that of the closely related B. cenocepacia J2315 revealed partial overlapping, suggesting that some essential genes are strain-specific. Furthermore, 158 essential genes were conserved in B. cenocepacia and two species belonging to the Burkholderia pseudomallei complex, B. pseudomallei K96243 and Burkholderia thailandensis E264. Porins, including OpcC, a lysophospholipid transporter, LplT, and a protein involved in the modification of lipid A with aminoarabinose were found to be essential in Burkholderia genomes but not in other bacterial essential genomes identified so far. Our results highlight the existence of cell envelope processes that are uniquely essential in species of the genus Burkholderia for which the essential genomes have been identified by Tn-seq.
Highlights
Determining the essential genomes of bacteria has furthered our understanding about the fundamental processes required for survival [1,2,3,4,5,6] and provided a first step in identifying putative targets for developing antibacterial therapies [7,8,9]
With the goal of identifying the essential genome of B. cenocepacia K56-2, we used high-density transposon mutant (HDTM) followed by Illumina sequencing of the transposon insertion sites
The total reads from PCR-amplification of the HDTM library with the iTaq DNA polymerase were more evenly distributed over the insertion sites, whereas the use of the KAPA DNA polymerase resulted in many insertions with a low read count and a large proportion of reads mapping to a small number of insertion sites (Fig. S4)
Summary
Determining the essential genomes of bacteria has furthered our understanding about the fundamental processes required for survival [1,2,3,4,5,6] and provided a first step in identifying putative targets for developing antibacterial therapies [7,8,9]. The identification of essential genes is challenging due to the lethal phenotype that mutagenesis of essential genes cause. Identification of essential genes in laboratory conditions can be achieved by recovering mutants growing in rich, undefined medium after concerted disruption of non-essential genes [10,11,12,13]. Generation sequencing has facilitated the use of saturated transposon mutagenesis to identify the essential genomes of many bacteria. A recently developed data analysis pipeline compares the abundance of each transposon mutant in the library with the variance of a neutral dataset generated by Monte Carlo simulations to determine the probability that a gene is essential [16], increasing the confidence of the essential gene prediction
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