Pseudomonas aeruginosa is a nosocomial pathogen with a prevalence in immunocompromised individuals and is particularly abundant in the lung microbiome of cystic fibrosis patients. A clinically important adaptation for bacterial pathogens during infection is their ability to survive and proliferate under phosphorus-limited growth conditions. Here, we demonstrate that P. aeruginosa adapts to P-limitation by substituting membrane glycerophospholipids with sugar-containing glycolipids through a lipid renovation pathway involving a phospholipase and two glycosyltransferases. Combining bacterial genetics and multi-omics (proteomics, lipidomics and metatranscriptomic analyses), we show that the surrogate glycolipids monoglucosyldiacylglycerol and glucuronic acid-diacylglycerol are synthesised through the action of a new phospholipase (PA3219) and two glycosyltransferases (PA3218 and PA0842). Comparative genomic analyses revealed that this pathway is strictly conserved in all P. aeruginosa strains isolated from a range of clinical and environmental settings and actively expressed in the metatranscriptome of cystic fibrosis patients. Importantly, this phospholipid-to-glycolipid transition comes with significant ecophysiological consequence in terms of antibiotic sensitivity. Mutants defective in glycolipid synthesis survive poorly when challenged with polymyxin B, a last-resort antibiotic for treating multi-drug resistant P. aeruginosa. Thus, we demonstrate an intriguing link between adaptation to environmental stress (nutrient availability) and antibiotic resistance, mediated through membrane lipid renovation that is an important new facet in our understanding of the ecophysiology of this bacterium in the lung microbiome of cystic fibrosis patients.

The Burkholderia cepacia complex is a group of Burkholderia species that are opportunistic pathogens causing high mortality rates in patients with cystic fibrosis. An environmental stress often encountered by these soil-dwelling and pathogenic bacteria is phosphorus limitation, an essential element for cellular processes. Here, we describe cellular and extracellular proteins differentially regulated between phosphate-deplete (0 mM, no added phosphate) and phosphate-replete (1 mM) growth conditions using a comparative proteomics (LC-MS/MS) approach. We observed a total of 128 and 65 unique proteins were downregulated and upregulated respectively, in the B. cenocepacia proteome. Of those downregulated proteins, many have functions in amino acid transport/ metabolism. We have identified 24 upregulated proteins that are directly/indirectly involved in inorganic phosphate or organic phosphorus acquisition. Also, proteins involved in virulence and antimicrobial resistance were differentially regulated, suggesting B. cenocepacia experiences a dramatic shift in metabolism under these stress conditions. Overall, this study provides a baseline for further research into the biology of Burkholderia in response to phosphorus stress.

How double-membraned Gram-negative bacteria overcome lipid peroxidation is virtually unknown. Bactericidal antibiotics and superoxide ion stress stimulate the transcription of the Burkholderia cenocepacia bcnA gene that encodes a secreted lipocalin. bcnA gene orthologs are conserved in bacteria and generally linked to a conserved upstream gene encoding a cytochrome b561 membrane protein (herein named lcoA, lipocalin-associated cytochrome oxidase gene). Mutants in bcnA, lcoA, and in a gene encoding a conserved cytoplasmic aldehyde reductase (peroxidative stress-associated aldehyde reductase gene, psrA) display enhanced membrane lipid peroxidation. Compared to wild type, the levels of the peroxidation biomarker malondialdehyde (MDA) increase in the mutants upon exposure to sublethal concentrations of the bactericidal antibiotics polymyxin B and norfloxacin. Microscopy with lipid peroxidation–sensitive fluorescent probes shows that lipid peroxyl radicals accumulate at the bacterial cell poles and septum and peroxidation is associated with a redistribution of anionic phospholipids and reduced antimicrobial resistance in the mutants. We conclude that BcnA, LcoA, and PsrA are components of an evolutionary conserved, hitherto unrecognized peroxidation detoxification system that protects the bacterial cell envelope from lipid peroxyl radicals.