Sphingolipids are defined by their sphingoid bases, also called long chain bases (LCBs), which are aliphatic amino alcohols. When an LCB is N-acylated with a fatty acid, the resulting compound is a ceramide. Ceramides are nearly ubiquitous in eukaryotic cell membranes and structures, but their production in bacteria has only been reported in a handful of taxa. Until recently, bacterial sphingolipids were thought to be exclusive to the Sphingomonadaceae, but over the past decade they have been found in members of the Bacteroidetes, Chlorobi, and other Proteobacteria. Despite this growing list, the physiological role of sphingolipids in bacterial membranes outside of pathogenicity remains largely unexplored, and bacterial contributions to environmental sphingolipid pools are poorly understood.

The research team, led by Toby A. Halamka of the University of Colorado Boulder, grew S. usitatus strain Ellin6076 — a bacterium originally isolated from a temperate Australian pasture soil — in triplicate under a carefully controlled matrix of environmentally relevant conditions: temperatures ranging from 15°C to 30°C, pH values of 5.5 and 6.5, and oxygen levels from fully aerobic (21% O₂) down to suboxic (5% O₂). Using a modified DSMZ 1266 medium buffered with MES and adjusted with sodium hydroxide, the researchers harvested cells in stationary phase, freeze-dried the biomass, and extracted intact polar lipids using a CTAB freeze-thaw pre-treatment followed by a modified Bligh-Dyer extraction protocol. The total lipid extracts were then analyzed on a high-performance liquid chromatography mass spectrometer consisting of a Dionex UltiMate 3000 UHPLC coupled to a Bruker maXis qTOF mass spectrometer operated in positive ion mode, with intact polar lipids identified based on data-dependent MS-MS fragmentation patterns.

What the team found was striking. Two varieties of core ceramides were identified in the polar lipid profile of S. usitatus. The most abundant was ceramide NDS — non-hydroxy fatty acid dihydrosphingosine ceramides — the same configuration as the bacterial dihydroceramide structure. The second variety, referred to as Cer-HHDS, contains a hydroxylated fatty acid component and a hydroxylated dihydrosphingosine LCB, though the exact positions of the additional hydroxylations could not be confirmed from the available mass spectra. The overall intact polar lipid composition of S. usitatus comprised four major polar headgroups: phosphohexose, phosphatidylethanolamine (PE), phosphatidylcholine, and di-phosphatidylglycerol. Of these, only PE headgroups were detected in association with NDS ceramides, while no polar headgroups were detected for Cer-HHDS.

Having confirmed the presence of sphingolipids, the researchers turned to the genome to identify the biosynthetic machinery. Homologs for the key genes involved in eukaryotic ceramide biosynthesis were not detected, with the exception of serine palmitoyltransferase (Spt), the only shared step between the eukaryotic and bacterial pathways. The bacterial pathway, recently resolved in the Proteobacterium Caulobacter crescentus, consists of three essential steps: the condensation of L-serine and fatty-acyl CoA by Spt, the addition of a second acyl chain by bacterial ceramide synthase (bCerS), and the reduction of the LCB by bacterial ceramide reductase (CerR). In S. usitatus, the proposed homologs of spt (gene ACID_5448), bcerS (gene ACID_5449), and cerR (gene ACID_5451) occur in the same predicted biosynthetic operon, further suggesting a shared functional relationship. Label-free quantitative proteomics confirmed that all three proteins were expressed at average to above-average levels across the culturing experiments, while a fourth gene, ACID_2641, predicted to encode a fatty acid desaturase similar to the bacterial ceramide hydroxylase CerH, was not detected in the proteome.

The dominant intact ceramide detected across all analyzed temperature, pH, and oxygen conditions was PE-Cer NDS d17:0/15:0, with a mass-to-charge ratio of 635.51. When the researchers normalized the response units of this compound by the amount of biomass extracted, clear trends emerged. The highest production of PE-Cer m/z 635 across all tested conditions was at 30°C, pH 5.5, 21% O₂, grown in the base medium. At the same pH, oxygen, and media conditions, PE-Cer m/z 635 production positively correlated with increasing culturing temperatures. Efforts to culture S. usitatus below 15°C and above 30°C resulted in little or zero cell growth, suggesting that the observed temperature trend applies across the growth range of this strain and that increased ceramide production is a temperature adaptation mechanism. Evidence of bacterial ceramide production in response to elevated temperature stress has previously been reported for the acetic acid bacterium Acetobacter malorum and for C. crescentus, and the same study of A. malorum identified low pH stress as a trigger for ceramide production. Although only two pH conditions were tested in this study, a decrease in the production of PE-Cer m/z 635 was found when culturing pH was increased, suggesting higher production of ceramides at lower pH values.

The oxygen-limited cultures presented a more nuanced picture. Cellular clumping and an excess of extracellular materials are often observed in liquid cultures of S. usitatus grown at suboxic conditions, which suggests that additional cellular components not observed in fully oxygenated cultures could be contributing to the amount of biomass generated in the suboxic experiments. Thus, the reported decrease in counts per milligram biomass of PE-Cer m/z 635 at 10% and 5% O₂ may be reflective of an increase in cellular components other than ceramides rather than a physiological shift in ceramide production itself. Interestingly, the lowest production of PE-Cer m/z 635 was observed in the experiment given excess phosphate. Phosphate starvation was previously found to trigger the production of a novel glycosphingolipid in C. crescentus, attributed to a lack of available phosphate for the biosynthesis of phospho-glycerol or sphingolipids. Data from S. usitatus suggests that a reduction in phosphate availability correlates to an increase in the production of PE ceramides, though neither of the experimental phosphate conditions would be considered phosphate-limited for this organism, and the 10 mM phosphate concentration represents a bioavailable phosphorus quantity not representative of most soils.

To assess the broader significance of these findings, the team searched available Acidobacteria genomes from the Joint Genome Institute and NCBI databases for bCerS homologs, using the S. usitatus bCerS gene as the BLAST query. The results identified 359 bCerS sequences belonging to various classes of Acidobacteria, including Blastocatellia (n = 36), Holophagae (n = 17), Terriglobia (n = 72), Thermoanaerobaculia (n = 79), Vicinamibacteria (n = 37), and 118 unclassified Acidobacteria. Acidobacteria represent on average 20% of the microbial communities of global soils and are metabolically diverse heterotrophs, making them the third most abundant bacterial phylum in global soils. The extent of ceramide biosynthetic potential across five different classes of the phylum — coupled to the abundance of their genes in soil metagenomes — suggests that soil sphingolipids should not be solely attributed to eukaryotic production.

The environmental implications are profound. Soil metagenomes from JGI were queried for bCerS sequences, yielding a total of 16,477 sequences from grasslands (n = 4,593), tundras (n = 447), and peats (n = 11,437). A gene phylogeny constructed from the NCBI non-redundant protein database, using bCerS sequences from both non-Acidobacteria (n = 2,656) and Acidobacteria (n = 319), revealed that 2,963 bCerS genes from the soil metagenomes clustered within Acidobacterial bCerS clades: 74 from tundra, 187 from grasslands, and strikingly, 2,702 from peats. When grouped by source environment, only 4% of the bCerS sequences from grassland soils clustered with Acidobacteria, whereas 16.5% and 23.6% of the sequences from tundra and peat, respectively, clustered within Acidobacterial bCerS gene clades. This suggests that while Acidobacteria may not be significant drivers of bacterial sphingolipid production in grassland soils, they represent nearly a quarter of the genetic potential for bacterial ceramide biosynthesis in peats. Notably, 84% of the environmental peat bCerS sequences associated with Acidobacteria were found in the same clade as the bCerS gene from S. usitatus, suggesting that investigations of ceramide biosynthesis and physiology in this single cultured strain may be particularly relevant for understanding bacterial sphingolipid dynamics in peat soil environments.

The findings also dovetail with recent work investigating shifts in the environmental lipidomes of grassland soils during wetting and drying cycles, in which a close association was found between increases in amplicon sequence variants of Acidobacteria and the increased occurrence of sphingolipids in dry soils. Without previous evidence of ceramide biosynthesis in Acidobacteria, that study’s authors discussed the underexplored potential of bacterial ceramide production and cautioned against the assumption that soil sphingolipids are eukaryotic biomarkers. The phylum Bacteroidetes, which contains multiple sphingolipid-producing genera including the soil-associated genus Sphingobacterium, undoubtedly also contributes to soil sphingolipid pools. But the work presented here, combining the first insights into the physiological role of ceramides in a cultured soil bacterium with a broad investigation of how the phylum Acidobacteria may influence environmental sphingolipid pools, opens an entirely new chapter in our understanding of who makes these important lipids in the world’s soils — and why. Additional physiology-based studies of Acidobacterial sphingolipid producers should be conducted to further constrain the role of ceramides in Acidobacteria and their subsequent role in soil ecosystems, but the message is already clear: the bacterial domain of life is a far more significant source of soil sphingolipids than anyone previously imagined.

Subject of Research: Occurrence of ceramides in the Acidobacterium Solibacter usitatus: implications for bacterial physiology and sphingolipids in soils

Article Title: Occurrence of ceramides in the Acidobacterium Solibacter usitatus: implications for bacterial physiology and sphingolipids in soils

Article References: Halamka, T. A., Garcia, A., Evans, T. W., Schubert, S., Younkin, A., Hinrichs, K.-U., & Kopf, S. (2024). Occurrence of ceramides in the Acidobacterium Solibacter usitatus: implications for bacterial physiology and sphingolipids in soils. Frontiers in Geochemistry, 2, Article 1400278. https://doi.org/10.3389/fgeoc.2024.1400278

Image Credits: AI Generated

DOI: 10.3389/fgeoc.2024.1400278

Keywords: ceramides, acidobacteria, sphingolipids, soil bacteria, biosynthesis, microbiology, geochemistry, Occurrence, Acidobacterium, Solibacter, usitatus, implications