Lipoylation is a post-translational modification in which lipoic acid is attached to specific apoproteins of enzyme complexes, like E2 subunits of dehydrogenases or GcvH of the glycine cleavage system. A defining feature of organisms with a lipoyl-relay system is the presence of amidotransferase activity, which enables the transfer of lipoyl groups attached to intermediary proteins to the E2 subunits. In this study, we characterized the lipoate metabolism of Capsaspora owczarzaki and Plasmodium falciparum. Both organisms utilize amidotransferases in their lipoylation pathways, with the filasterian enzyme playing a key role in lipoate synthesis, while the apicomplexan counterpart, previously considered a lipoyltransferase, is essential in its lipoate salvage pathway. We also discovered that specific structural features and certain conserved domains in eukaryotic amidotransferases can significantly influence their mechanism of action and susceptibility to the lipoate analog bromooctanoate. Overall, this study highlights the metabolic strategies of C. owczarzaki and emphasizes the critical role of amidotransferases as ancestral enzymes in the evolution of lipoate metabolism, suggesting that the lipoyl relay may represent a universal pathway across diverse clades.

Two-component systems (TCSs) are vital signal transduction pathways ubiquitous among bacteria, facilitating their responses to diverse environmental stimuli. In Bacillus subtilis, the DesK histidine kinase thermosensor, together with the response regulator DesR, constitute a TCS dedicated to membrane lipid homeostasis maintenance. This TCS orchestrates the transcriptional regulation of the des gene, encoding the sole desaturase in these bacteria, Δ5-Des. Additionally, B. subtilis possesses a paralog TCS, YvfT/YvfU, with unknown target gene(s). In this work we show that YvfT/YvfU controls the expression of the yvfRS operon that codes for an ABC transporter. Interestingly, we found that this regulation also involves the action of DesK/DesR. Notably, opposite to des, yvfRS transcription is induced at 37°C and not at 25°C. Our in vivo and in vitro experiments demonstrate that both YvfU and DesR directly bind to the operon promoter region, with DesR exerting its control over yvfRS expression in its unphosphorylated state. Our study uncovers an intriguing case of cross-regulation where two homologous TCSs interact closely to finely tune gene expression in response to environmental cues. These findings shed light on the complexity of bacterial signal transduction systems and their critical role in bacterial adaptability.