The L-lactate dehydrogenases of <i>Pseudomonas aeruginosa</i> are conditionally regulated but both contribute to survival during macrophage infection

<i>Pseudomonas aeruginosa</i> is an opportunistic pathogen that thrives in environments associated with human activity, including soil and water altered by agriculture or pollution. Because L-lactate is a significant product of plant and animal metabolism, it can serve as a carbon source for <i>P. aeruginosa</i> in the diverse settings that it inhabits. In this study, we evaluate the production and use of two redundant <i>P. aeruginosa</i> L-lactate dehydrogenases, termed LldD and LldA. We confirm that the protein LldR represses <i>lldD</i> and identify a new transcription factor, called LldS, that activates <i>lldA</i>; these distinct regulators and the genomic contexts of <i>lldD</i> and <i>lldA</i> contribute to their differential expression. We demonstrate that the <i>lldD</i> and <i>lldA</i> genes are conditionally controlled in response to lactate isomers as well as to glycolate and ɑ-hydroxybutyrate, which, like lactate, are ɑ-hydroxycarboxylates. We also show that <i>lldA</i> is induced when iron availability is low. Our examination of <i>lldD</i> and <i>lldA</i> expression across depth in biofilms indicates a complex pattern that is consistent with the effects of glycolate production, iron availability, and cross-regulation on enzyme preference. Finally, macrophage infection assays reveal that both <i>lldD</i> and <i>lldA</i> contribute to persistence within host cells, underscoring the potential role of L-lactate as a carbon source during <i>P. aeruginosa-</i>eukaryote interactions. Together, these findings help us understand the metabolism of a key resource that may promote <i>P. aeruginosa</i>'s success as a resident of contaminated environments and animal hosts.IMPORTANCE<i>Pseudomonas aeruginosa</i> is a major cause of lung infections in people with cystic fibrosis, of hospital-acquired infections, and of wound infections. It consumes L-lactate, which is found at substantial levels in human blood and tissues. In this study, we investigated the spatial regulation of two redundant enzymes, called LldD and LldA, which enable L-lactate metabolism in <i>P. aeruginosa</i> biofilms. We uncovered mechanisms and identified compounds that control the preference of <i>P. aeruginosa</i> for LldD versus LldA. We also showed that both enzymes contribute to its ability to survive within macrophages, a behavior that is thought to augment the chronicity and recalcitrance of infections. Our findings shed light on a key metabolic strategy used by <i>P. aeruginosa</i> and have the potential to inform the development of therapies targeting bacterial metabolism during infection.