Identification of <i>iso</i> -diabolic acid-based tetraester and mixed ether/ester membrane-spanning lipids in members of the Bacillota provides insight into the biosynthesis of bacterial branched glycerol dialkyl glycerol tetraethers

ABSTRACT Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane-spanning lipids (MSLs) resembling archaeal MSLs, as they form monolayers and are linked to glycerol backbones via ether bonds. Ubiquitous in soils, sediments, and aquatic environments, their distributions are widely applied as paleoclimate proxies for reconstructing past temperature and pH. Despite this, the understanding of their biological origins and functional roles in cells remains incomplete. While some Acidobacteria are known producers of brGDGTs, genomic evidence and environmental surveys indicate additional bacterial contributors. In this study, we report the first detection of structural analogs of brGDGT in Bacillota that could reflect biosynthetic intermediates in brGDGT biosynthesis. Ultra-high pressure liquid chromatography high resolution multi-stage mass spectrometry (UHPLC-HRMS n ) revealed membrane-spanning diglycerol lipids containing iso -diabolic acid (13,16-dimethyl octacosanedioic acid)-derived alkyl chains. These diglycerol lipids displayed diverse structures, including tetraesters, mixed ester/ether combinations, and often contained vinyl ether bonds. Additionally, “open” MSL, structurally analogous to branched glycerol trialkyl glycerol tetraether (brGTGTs), was also identified. Notably, all brGDGT and brGTGT structural analogs were also detected with a phosphatidylglycerol head group. Experiments showed that the two Bacillota strains, which produce these brGDGT biosynthetic intermediates, responded differently to changes in temperature and oxygen availability, suggesting that environmental regulation of brGDGT-related lipids is taxonomically dependent. Based on these findings, we propose a biosynthetic pathway for brGDGT formation and highlight the physiological implications for interpreting brGDGT-based paleoclimate proxies. This work also expands the known diversity of bacterial sources of brGDGTs and provides new insights into the ecological and evolutionary significance of these lipids. IMPORTANCE Branched glycerol dialkyl glycerol tetraethers (brGDGTs) are bacterial membrane-spanning lipids forming monolayers and linked via ether bonds to a glycerol backbone, traits more commonly found in archaeal membranes organized in monolayers. BrGDGTs are commonly used in paleoclimate proxies to assess past temperature and pH, but their predictive power is hampered by the lack of information regarding their biological producers. Branched GDGTs have been detected in just a few species of the Acidobacteria, but there are strong indications that other bacterial phyla also contribute to the pool of brGDGTs in the environment. Here, we report for the first time the production of structural analogs of brGDGTs in Bacillota species, which we speculate could be intermediates leading to their synthesis. This study demonstrates that brGDGTs are probably much more widespread in the bacterial domain than previously thought and opens a new chapter in the determination of potential brGDGT sources in the environment and the potential application in paleoclimatology.