Microorganisms enhance fitness by prioritizing catabolism of available carbon sources using a process known as carbon catabolite repression (CCR). Planktonically grown Pseudomonas aeruginosa is known to prioritize the consumption of organic acids including lactic acid over catabolism of glucose using a CCR strategy termed “reverse diauxie.” P. aeruginosa is an opportunistic pathogen with well-documented biofilm phenotypes that are distinct from its planktonic phenotypes. Reverse diauxie has been described in planktonic cultures, but it has not been documented explicitly in P. aeruginosa biofilms. Here a combination of exometabolomics and label-free proteomics was used to analyze planktonic and biofilm phenotypes for reverse diauxie. P. aeruginosa biofilm cultures preferentially consumed lactic acid over glucose, and in addition, the cultures catabolized the substrates completely and did not exhibit the acetate secreting “overflow” metabolism that is typical of many model microorganisms. The biofilm phenotype was enabled by changes in protein abundances, including lactate dehydrogenase, fumarate hydratase, GTP cyclohydrolase, L-ornithine N(5)-monooxygenase, and superoxide dismutase. These results are noteworthy because reverse diauxie-mediated catabolism of organic acids necessitates a terminal electron acceptor like O₂, which is typically in low supply in biofilms due to diffusion limitation. Label-free proteomics identified dozens of proteins associated with biofilm formation including 16 that have not been previously reported, highlighting both the advantages of the methodology utilized here and the complexity of the proteomic adaptation for P. aeruginosa biofilms. Documenting the reverse diauxic phenotype in P. aeruginosa biofilms is foundational for understanding cellular nutrient and energy fluxes, which ultimately control growth and virulence.

微生物通过使用称为碳分解代谢物抑制（CCR）的过程优先分解可用碳源来增强适应性。众所周知，浮游生长的铜绿假单胞菌会优先消耗包括乳酸在内的有机酸，而不是使用称为“反向二氧化”的 CCR 策略分解代谢葡萄糖。铜绿假单胞菌是一种机会性病原体，具有与其浮游表型不同的生物膜表型。浮游培养物中已描述了反向二氧化性，但在铜绿假单胞菌生物膜中尚未明确记录。这里结合外代谢组学和无标记蛋白质组学来分析反向二元的浮游和生物膜表型。铜绿假单胞菌生物膜培养物优先消耗乳酸而不是葡萄糖，此外，培养物完全分解代谢底物并且不表现出许多模型微生物典型的分泌乙酸盐的“溢出”代谢。生物膜表型是通过蛋白质丰度的变化实现的，包括乳酸脱氢酶、富马酸水合酶、GTP环水解酶、L-鸟氨酸N(5)-单加氧酶和超氧化物歧化酶。这些结果值得注意，因为有机酸的反向双辅助介导的分解代谢需要末端电子受体，如 O ₂，​​由于扩散限制，其在生物膜中通常供应不足。无标记蛋白质组学鉴定了数十种与生物膜形成相关的蛋白质，其中包括 16 种以前未报道过的蛋白质，突出了此处使用的方法的优点以及铜绿假单胞菌生物膜的蛋白质组适应的复杂性。记录铜绿假单胞菌生物膜中的反向双峰表型是了解细胞营养和能量通量的基础，最终控制生长和毒力。