Cryle, M. J. | bioRxiv Science

bioRxiv2020

Structural basis for control of antibiotic production by bacterial hormones

Actinobacteria produce numerous antibiotics and other specialised metabolites with important applications in medicine and agriculture. Diffusible hormones frequently control the production of such metabolites by binding TetR family transcriptional repressors (TFTRs), but the molecular basis for this remains unclear. The production of methylenomycin antibiotics in Streptomyces coelicolor A3(2) is initiated by binding of 2-alkyl-4-hydroxymethylfuran-3-carboxylic acid (AHFCA) hormones to the TFTR MmfR. Here, we report the X-ray crystal structure of an MmfR-AHFCA complex, establishing the structural basis for hormone recognition. We also elucidate the mechanism for DNA release upon hormone binding by single particle cryo-electron microscopy of an MmfR-operator complex. Electrophoretic mobility shift assays with MmfR mutants and synthetic AHFCA analogues illuminate the role played by individual amino acid residues and hormone functional groups in ligand recognition and DNA release. These findings will facilitate the exploitation of Actinobacterial hormones and their associated TFTRs in synthetic biology and novel antibiotic discovery.

biochemistry↗

bioRxiv2019

Two uptake hydrogenases differentially interact with the aerobic respiratory chain during mycobacterial growth and persistence

Aerobic soil bacteria metabolize atmospheric hydrogen (H2) to persist when nutrient sources are limited. This process is the primary sink in the global H2 cycle and supports the productivity of microbes in oligotrophic environments. To mediate this function, bacteria possess [NiFe]-hydrogenases capable of oxidising H2 to subatmospheric concentrations. The soil saprophyte Mycobacterium smegmatis has two such [NiFe]-hydrogenases, designated Huc and Hhy, which belong to different phylogenetic subgroups. Huc and Hhy exhibit similar characteristics: both are oxygen-tolerant, oxidise H2 to subatmospheric concentrations, and enhance survival during hypoxia and carbon limitation. These shared characteristics pose the question: Why does M. smegmatis require two hydrogenases mediating a seemingly similar function? In this work we resolve this question by showing that Huc and Hhy are differentially expressed, localised, and integrated into the respiratory chain. Huc is active in late exponential and early stationary phase, supporting energy conservation during mixotrophic growth and the transition into dormancy. In contrast, Hhy is most active during long-term persistence, providing energy for maintenance processes when carbon sources are depleted. We show that Huc and Hhy are obligately linked to the aerobic respiratory chain via the menaquinone pool and are differentially affected by respiratory uncouplers. Consistent with their distinct expression profiles, Huc and Hhy interact differentially with the terminal oxidases of the respiratory chain. Huc exclusively donates electrons to, and possibly physically associates with, the proton pumping cytochrome bcc-aa3 supercomplex. In contrast, the more promiscuous Hhy can also provide electrons to the cytochrome bd oxidase complex. These data demonstrate that, despite their similar characteristics, Huc and Hhy perform distinct functions during mycobacterial growth and survival.

microbiology↗