Comparative analysis of the Burkholderia cenocepacia K56-2 essential genome reveals cell envelope functions that are uniquely required for survival in species of the genus Burkholderia

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.

A general protein O-glycosylation machinery conserved in Burkholderia species improves bacterial fitness and elicits glycan immunogenicity in humans

“…The inability to generate DogcA suggests that absence of this gene may be deleterious for bacterial cell viability. This interpretation is consistent with previous Tn-Seq analysis showing that insertions are unrepresented within the B. cenocepacia ogcA (40) and with a report indicating the B. pseudomallei K96243 ogcA homologue (BPSL2668) is essential for survival (41). It is likely that without the terminal Gal the incomplete O-linked disaccharide glycan cannot be either effectively translocated across the membrane or processed by PglL, leading in both cases to the accumulation of Und-PP that cannot be recycled thus becoming growth limiting.…”

Genetic Determinants Associated With in Vivo Survival of Burkholderia cenocepacia in the Caenorhabditis elegans Model

“…As O -antigen production is thought to be not required for B. cenocepacia viability, Hanuszkiewicz et al (2014) raised the possibility that RmlB is involved in the biosynthesis of other nucleotide sugars that may play an essential role in a yet unknown metabolic pathway in strain K56-2. The essentiality of rmlB in strain K56-2 is further supported by a recent Tn-seq study ( Gislason et al, 2017 ), which also identified distinct sets of genes that are uniquely essential in K56-2 and J2315. Nonetheless, while disruption of J2315 rmlB in our study did not affect mutant accumulation in the worm in a single-strain infection experiment, abolishment of RmlB led to reduced killing of C. elegans .…”

Competitive Fitness of Essential Gene Knockdowns Reveals a Broad-Spectrum Antibacterial Inhibitor of the Cell Division Protein FtsZ

“…Previously, we developed a high-density transposon mutant (HDTM) library in B. cenocepacia K56-2 by delivering a transposon element containing an outward rhamnose-inducible promoter ( P r haB ) into the genome ( 14 ). Using an enrichment process to isolate mutants in which P r haB is driving the expression of essential genes, we built a redundant knockdown library of 830 clones.…”