Copper resistance genes of Burkholderia cenocepacia H111 identified by transposon sequencing

Abstract

Summary Copper is an essential element but in excess is highly toxic and therefore cytoplasmic levels must be tightly controlled. Member of the genus Burkholderia are highly resistant to various heavy metals and are often isolated from acidic soils where copper bioavailability is high. In this study, we employed transposon sequencing (Tn‐Seq) to identify copper resistance genes in Burkholderia cenocepacia H111. We identified a copper efflux system that shares similarities with the plasmid‐based copper detoxification systems found in Escherichia coli and Pseudomonas syringae. We also found that several of the identified resistance determinants are involved in maintaining the integrity of the cell envelope, suggesting that proteins located in the outer membrane and periplasmic space are particularly sensitive to copper stress. Given that several of the resistance genes are required for the repair and turnover of misfolded proteins, we suggest that copper toxicity is caused by protein damage rather than by oxidative stress.

Identification of genes required for gold and silver tolerance in Burkholderia cenocepacia H111 by transposon sequencing

“…The functional categories 'translation/ribosomal structure and biogenesis' and 'transcription' were overrepresented in the silver and gold datasets respectively. The copper dataset was found to be enriched for genes of the categories 'post translational modification' and 'intracellular trafficking/secretion transport, inorganic ion transport/ metabolism', possibly reflecting the fact that copper detoxification depends on a specific copper efflux system (Higgins et al, 2020). We also noticed that genes of the categories 'lipid transport/metabolism' and 'replication/ recombination repair' were absent in the copper dataset.…”

“…Two fitness determinants are required for tolerance against all three group IB transition metals A comparison of the silver and gold data sets with the list of genes required for copper resistance of B. cenocepacia H111 (Higgins et al, 2020) revealed that only two genes, BCAL3204 and BCAL2395, are required for tolerance to all three group IB transition metals. BCAL3204 encodes an outer-membrane protein of the OmpA family.…”

“…We decided to use heavy metal concentrations that reduced growth by approximately 30%. We passaged the library two times with increasing concentrations of AgNO 3 (10 μM and 20 μM) or AuCl 3 (200 μM and 400 μM) following the protocol previously used to identify copper resistance genes in B. cenocepacia H111 (Higgins et al, 2020). The first growth cycle was initiated by inoculating the selective medium with 2-4 × 10 6 Tn5 mutants per ml, which avoided bottlenecks, as the entire mutant collection consisting of approximately 0.8 × 10 6 is represented in the culture.…”

“…Since membrane proteins are important targets of heavy metal toxicity, many genes identified in our screen are involved in membrane integrity (Jung et al, 2008;Mijnendonckx et al, 2013;Tambosi et al, 2018). Gene BCAL3204 was found to be essential for growth in the presence of silver and gold (Tables 2 and 3) as well as copper ions (Higgins et al, 2020). This gene encodes a component of the Tol multiprotein complex, which is required for outer membrane integrity (Lazzaroni et al, 1999;Llamas et al, 2000).…”

A type IVB secretion system adapted for bacterial killing, biofilm invasion and biocontrol

“…DNA extraction was done using the Bacterial Genomic DNA Kit (Sigma-Aldrich). All sequencing steps were performed using the circle method described by Gallager et al, 2011 95 with several modifications described in Higgins et al, 2020 96 .…”