Redundancy in citrate and cis-aconitate transport in Pseudomonas aeruginosa

Abstract

Tricarboxylates such as citrate are the preferred carbon sources for Pseudomonas aeruginosa, an opportunistic pathogen that causes chronic human infections. However, the membrane transport process for the TCA cycle intermediates citrate and cis-aconitate is poorly characterized. Transport is thought to be controlled by the TctDE two-component system, which mediates transcription of the putative major transporter OpdH. Loss of tctDE has been associated with sensitization to aminoglycosides, possibly linking tricarboxylate transport to enhanced antimicrobial resistance. In this work, we search for previously unidentified transporters of citrate and cis-aconitate using both protein homology and RNA sequencing approaches. We uncover new transporters and show that OpdH is not the major citrate porin; instead, citrate transport primarily relies on the tripartite TctCBA system, which is encoded in the opdH operon. Deletion of tctA causes a growth lag on citrate and loss of growth on cis-aconitate. Combinatorial deletion of newly discovered transporters can fully block citrate utilization. We then characterize transcriptional control of the opdH operon in tctDE mutants and show that loss of tctD blocks citrate utilization due to its inability to express opdH-tctCBA. However, tctE and tctDE mutants evolve heritable adaptations that restore growth on citrate as the sole carbon source. Author SummaryPseudomonas aeruginosa is a bacterium that infects hospitalized patients and is often highly resistant to antibiotic treatment. It preferentially uses small organic acids called tricarboxylates rather than sugars as a source of carbon for growth. The transport of many of these molecules from outside the cell to the interior occurs through unknown channels. In some cases, there may be links between antibiotic uptake and the transport of metabolic molecules, making cross-membrane transport medically important. In this work we examined how the tricarboxylates citrate and cis-aconitate are transported in P. aeruginosa. We then sought to understand how production of proteins that permit citrate and cis-aconitate transport is regulated by a signaling system called TctDE. We identified new transporters for these molecules, clarified the function of a known transport system, and directly tied transporter expression to the presence of an intact TctDE system.