Summary One common mechanism of resistance against antimicrobial peptides in Gram‐negative bacteria is the addition of 4‐amino‐4‐deoxy‐l‐arabinose (l‐Ara4N) to the lipopolysaccharide (LPS) molecule. Burkholderia cenocepacia exhibits extraordinary intrinsic resistance to antimicrobial peptides and other antibiotics. We have previously discovered that unlike other bacteria, B. cenocepacia requires l‐Ara4N for viability. Here, we describe the isolation of B. cenocepacia suppressor mutants that remain viable despite the deletion of genes required for l‐Ara4N synthesis and transfer to the LPS. The absence of l‐Ara4N is the only structural difference in the LPS of the mutants compared with that of the parental strain. The mutants also become highly sensitive to polymyxin B and melittin, two different classes of antimicrobial peptides. The suppressor phenotype resulted from a single amino acid replacement (aspartic acid to histidine) at position 31 of LptG, a protein component of the multi‐protein pathway responsible for the export of the LPS molecule from the inner to the outer membrane. We propose that l‐Ara4N modification of LPS provides a molecular signature required for LPS export and proper assembly at the outer membrane of B. cenocepacia, and is the most critical determinant for the intrinsic resistance of this bacterium to antimicrobial peptides.

Burkholderia cenocepacia is a multidrug-resistant opportunistic pathogen that infects the airways of patients with cystic fibrosis (CF) and can survive intracellularly in macrophages and epithelial cells. The gentamicin protection assay, which relies on the poor ability of gentamicin or other aminoglycosides to permeate eukaryotic cell membranes, is traditionally employed to quantify intracellular bacteria. However, the high resistance of these bacteria to aminoglycosides hampers the use of the gentamicin protection assay to investigate intracellular infection by B. cenocepacia. Here, we report the construction of gentamicin-sensitive strains of B. cenocepacia carrying a deletion of the BCAL1674, BCAL1675, and BCAL1676 genes that form an operon encoding an AmrAB-OprA-like efflux pump. We show that bacteria carrying this deletion are hypersensitive to gentamicin and also delay phagolysosomal fusion upon infection of RAW 264.7 murine macrophages, as previously demonstrated for the parental strain. We also demonstrate for the first time that low concentrations of gentamicin can be used to effectively kill extracellular bacteria and reliably quantify the intracellular infection by B. cenocepacia, which can replicate in RAW 264.7 macrophages.

This is the pre-peer reviewed version of the following article: Schmerk, C. L., Welander, P. V., Hamad, M. A., Bain, K. L., Bernards, M. A., Summons, R. E. and Valvano, M. A. (2015), Elucidation of the Burkholderia cenocepacia hopanoid biosynthesis pathway uncovers functions for conserved proteins in hopanoid-producing bacteria. Environmental Microbiology, 17: 735-750, which has been published in final form at http://onlinelibrary.wiley.com/wol1/doi/10.1111/1462-2920.12509/abstract. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving. General rightsCopyright for the publications made accessible via the Queen's University Belfast Research Portal is retained by the author(s) and / or other copyright owners and it is a condition of accessing these publications that users recognise and abide by the legal requirements associated with these rights.Take down policy The Research Portal is Queen's institutional repository that provides access to Queen's research output. Every effort has been made to ensure that content in the Research Portal does not infringe any person's rights, or applicable UK laws. If you discover content in the Research Portal that you believe breaches copyright or violates any law, please contact openaccess@qub.ac.uk. grow under any stress condition tested, whereas ΔhpnI, ΔhpnJ, and ΔhpnK displayed wild type 42 growth rates when exposed to detergent, but varying levels of sensitivity to low pH and 43 polymyxin B. This study not only elucidates the biosynthetic pathway of hopanoids in B. 44 cenocepacia, but also uncovers a biosynthetic role for the conserved proteins HpnI, HpnJ, and 45

Genetic manipulation of multidrug-resistant bacteria is often difficult and hinders progress in understanding their physiology and pathogenesis. This book chapter highlights advances in genetic manipulation of Burkholderia cenocepacia, which are also applicable to other members of the Burkholderia cepacia complex and multidrug-resistant gram-negative bacteria of other genera. The method detailed here is based on the I-SceI homing endonuclease system, which can be efficiently used for chromosomal integration, deletion, and genetic replacement. This system creates markerless mutations and insertions without leaving a genetic scar and thus can be reused successively to generate multiple modifications in the same strain.

Background:The Burkholderia cenocepacia lipid A is hypoacylated. Results: Aminoarabinose residues in lipid A contribute to Burkholderia lipid A binding to the TLR4⅐MD-2 complex. Conclusion: A novel mode of Burkholderia lipopolysaccharide-TLR4⅐MD-2 interactions promotes inflammation. Significance: Modifications of the lipid A structure enhance proinflammatory responses of hypoacylated lipopolysaccharide.