Biogenic Quorum-Sensing Amides from <i>Streptomyces</i> sp. NP10

Volatile organic compounds produced by microbes are increasingly recognized as modulators of microbial interactions and mediators of both intra- and inter-kingdom communication. This study explored the possible ecophysiological roles of nine amides from <i>Streptomyces</i> sp. NP10 in quorum sensing (QS) and biofilm formation in <i>Pseudomonas aeruginosa</i> PAO1. GC-MS profiling, synthesis, spectral validation, and co-injection experiments confirmed compound identities. Notably, <i>N</i>-(3-methyl-2-butenyl)acetamide is reported as a new natural product and <i>N</i>-(2-methylbutyl)acetamide as a new <i>Streptomyces</i>-produced metabolite. At subinhibitory concentrations (250 μg/mL), most of the amides enhanced <i>P. aeruginosa</i> biofilm formation, with <i>N</i>-(2-methylbutyl)acetamide, <i>N</i>-(3-methyl-2-butenyl)acetamide, and 2-phenylacetamide showing the strongest effects. Simultaneously, these compounds suppressed QS by reducing the production of <i>N</i>-acyl homoserine lactones (AHLs) and 2-alkyl-4-quinolones (AHQs). Aliphatic acetamides preferentially inhibited short-chain AHLs, while <i>N</i>-acetyltyramine and 2-phenylacetamide mainly affected quinolone signaling. These opposing effects on QS and biofilm are consistent with the involvement of alternative regulatory circuits. Motility assays showed biofilm stimulation was not correlated with altered swarming or twitching. Cross-species assays revealed limited QS inhibition, with only <i>N</i>-acetyltryptamine reducing violacein production in <i>Chromobacterium violaceum</i> CV026. Most of the amides were non-cytotoxic at 100 μM (10.5–20.2 μg/mL), except for 2-phenylacetamide. Overall, these amides likely serve as microbial signals influencing QS and biofilm formation, offering leads for anti-virulence strategies.