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Introduction Although Staphylococcus aureus is the leading cause of biofilm-related infections, the lipidomic distributions within these biofilms is poorly understood. Here, lipidomic mapping of S. aureus biofilm cross-sections was performed to investigate heterogeneity between horizontal biofilm layers. Methods S. aureus biofilms were grown statically, embedded in a mixture of carboxymethylcellulose/gelatin, and prepared for downstream matrix-assisted laser desorption/ionization imaging mass spectrometry (MALDI IMS). Trapped ion mobility spectrometry (TIMS) was also applied prior to mass analysis. Results Implementation of TIMS led to a ∼ threefold increase in the number of lipid species detected. Washing biofilm samples with ammonium formate (150 mM) increased signal intensity for some bacterial lipids by as much as tenfold, with minimal disruption of the biofilm structure. MALDI TIMS IMS revealed that most lipids localize primarily to a single biofilm layer, and species from the same lipid class such as cardiolipins CL(57:0) – CL(66:0) display starkly different localizations, exhibiting between 1.5 and 6.3-fold intensity differences between layers (n = 3, p < 0.03). No horizontal layers were observed within biofilms grown anaerobically, and lipids were distributed homogenously. Conclusions High spatial resolution analysis of S. aureus biofilm cross-sections by MALDI TIMS IMS revealed stark lipidomic heterogeneity between horizontal S. aureus biofilm layers demonstrating that each layer was molecularly distinct. Finally, this workflow uncovered an absence of layers in biofilms grown under anaerobic conditions, possibly indicating that oxygen contributes to the observed heterogeneity under aerobic conditions. Future applications of this workflow to study spatially localized molecular responses to antimicrobials could provide new therapeutic strategies. 

S. aureusUSA300 isolates utilize thecopBLandcopAZgene products to prevent Cu intoxication. We created and examined a ΔcopAZΔcopBLmutant strain (cop‐). Thecop‐ strain was sensitive to Cu and accumulated intracellular Cu. We screened a transposon (Tn) mutant library in thecop‐ background and isolated strains with Tn insertions in themntABCoperon that permitted growth in the presence of Cu. The mutations were inmntAand they were recessive. Under the growth conditions utilized, MntABC functioned in manganese (Mn) import. When cultured with Cu, strains containing amntA::Tnaccumulated less Cu than the parent strain. Mn(II) supplementation improved growth whencop‐ was cultured with Cu and this phenotype was dependent upon the presence of MntR, which is a repressor ofmntABCtranscription. A ΔmntRstrain had an increased Cu load and decreased growth in the presence of Cu, which was abrogated by the introduction ofmntA::Tn. Over‐expression ofmntABCincreased cellular Cu load and sensitivity to Cu. The presence of amntA::Tnmutation protected iron‐sulfur (FeS) enzymes from inactivation by Cu. The data presented are consistent with a model wherein defective MntABC results in decreased cellular Cu accumulation and protection to FeS enzymes from Cu poisoning.