18The bacterial plasma membrane is a key interface during pathogen-host interactions, and 19 membrane composition enhances resistance against host antimicrobial defenses. Branched chain 20 fatty acids (BCFAs) are the major plasma membrane component in the intracellular Gram-21 positive pathogen Listeria monocytogenes (Lm) and BCFA metabolism is essential for Lm 22 growth and virulence. BCFA synthesis requires branched chain amino acids (BCAAs), and the 23 BCAA Isoleucine (Ile) is a necessary substrate for the predominant membrane anteiso-BCFAs 24 (ai-BCFAs) as well as an environmental signal for virulence regulation in Lm. In this study, we 25 explored how two proteins that metabolize or sense Ile contribute to Lm growth, BCFA 26 metabolism, and virulence. The IlvE aminotransferase incorporates Ile into ai-BCFAs, while 27CodY is an Ile-sensing regulator that coordinates BCAA synthesis and virulence gene 28 expression. Analysis of deletion mutants lacking IlvE (ilvE) or CodY (codY) revealed a major 29 role for IlvE under nutrient restriction and stress conditions. Cultures of the ilvE mutant 30 contained proportionally less ai-BCFAs relative to wild type, while of the codY mutant had a 31 lower proportion of ai-BCFAs in stationary phase, despite containing more cell-associated Ile. 32

The Forkhead box O transcription factor DAF-16 is an important downstream transcription factor in the well-conserved insulin signalling (IIS) pathway in Caenorhabditis elegans. The nuclear localization of DAF-16 can activate a series of genes that mediate oxidative stress, heat shock, xenobiotic responses, innate immunity, metabolism and autophagy. In this study, a Bxdaf-16-2b gene encoding 510 amino acids was