Temperature-Sensitive Lipids Reveal Intraspecific Diversity in Bacteria Isolated from an Ancient Antarctic Microbial Mat

Abstract

Ancient Antarctic microbial mats harbour viable bacteria that have been exposed to extreme cold and arid conditions for hundreds of years. To delve into the molecular mechanisms underlaying their survival, we isolated 12 bacterial strains from a 1,000-year-old desiccated microbial mat from the McMurdo Ice Shelf and studied their lipid composition as a function of temperature. Six of the bacterial strains were classified as Paenisporosarcina macmurdoensis, and the other six as Arthrobacter sp., according to 16S rRNA gene analyses. Two strains of each taxon were incubated at their minimum and maximum growth temperatures, and the changes in their lipid profiles were analyzed. The proportion of major fatty acids (saturated, unsaturated, and iso- and anteiso-) remained relatively constant across temperature in the four strains, but the composition of hydrocarbons and alcohols differed between taxa (e.g., presence of unsaturated alkenes in Arthrobacter sp., or unidentified isoprenoid alcohols in P. macmurdoensis). This highlights the diagnostic value of non-fatty acid lipids and revealed a taxon-dependent lipid composition. Despite the taxon-associated lipid profile, incubation temperature also influenced lipid composition in both taxa, with higher temperature correlating with greater lipid richness. Interestingly, the two P. macmurdoensis strains showed distinct lipid profiles at 20°C, suggesting that intraspecific lipid diversity reflects within-species physiological variability with potential relevance for adaptation to temperature fluctuations in the mat. Therefore, assessing the influence of temperature on bacterial lipids is crucial for understanding their adaptation and survival in extreme environments, as well as for expanding species lipid inventories for biological interpretations of ancient samples.

Keywords: Antarctica; Cold adaptation; Lipid biomarkers; Polar ecology; Psychrophiles; Psychrotrophs.

Fig. 1

Methodological scheme to assess the influence of temperature on the lipid composition of extremophilic bacteria isolated from a 1,000-year-old microbial mat from Antarctica. 1. Bacterial growth on plates and colony selection; 2. Bacterial isolation of 12 strains (B1-B12, based on distinct colour and morphology) on liquid and solid R2A or LB media; 3. Taxonomic identification of the 12 bacterial strains by 16S rRNA gene analysis; 4. Identification of fast-growing bacteria by incubating the 12 isolates at 15 ºC, 20 ºC and 25 ºC for two months on liquid R2A or LB media; 5. Selection of fast-growing bacterial strains of different species (i.e., Paenisporosarcina macmurdoensis B1 and B2, and Arthrobacter sp. B7 and B8); 6. Growth curves of the selected strains at distinct incubation temperatures (from 0 ºC to 25 ºC); 7. Lipid extraction of the selected strains incubated at their maximum and minimum growth temperatures, and 8. Multivariate statistical analysis. Figure made using BioRender software

Fig. 2

Taxonomic affiliations of the isolated bacterial strains (B1-B12). Maximum likelihood tree based on the 16S rRNA gene showing in bold the bacterial strains isolated in this study from the ancient microbial mat from the McMurdo Ice Shelf (Antarctica). Numbers near the nodes indicate bootstrap values greater than or equal to 70, as a percentage of 1000 replicates resulting from the phylogenetic analysis. The bar indicates 0.12 substitutions per nucleotide position. Clusters I, II and III include bacterial isolates B7-B12 and are identified with the genus Arthrobacter, while cluster IV comprises bacterial isolates B1-B6 and are identified with the species Paenisporosarcina macmurdoensis. GenBank accession numbers for the sequences used in the phylogenetic tree are provided in brackets

Fig. 3

Growth of bacterial isolates as a function of incubation temperature. Growth curves of Paenisporosarcina macmurdoensis strains B1 and B2, and Arthrobacter sp. strains B7 and B8 under low (0 ºC, 5 ºC and 10 ºC) and warm (20 ºC and 25 ºC) temperatures up to 21 days of incubation under shaking and dark conditions. Coloured arrows indicate the optical density of each bacterial culture at the time of lipid extraction, corresponding to their early/mid exponential growth phase. Bars indicate standard deviation of triplicates. Media controls without bacteria showed no growth (Fig. S2)

Fig. 4

Lipid composition of bacterial isolates as a function of incubation temperature. A) Lipid concentration relative to dry weight (mg · g⁻¹ dry weight) and B) composition and relative abundance of each type of lipid within hydrocarbons, fatty acids and alcohol families in the Paenisporosarcina macmurdoensis strains B1 and B2 and Arthrobacter sp. strains B7 and B8 after incubation at their maximum and minimum temperatures for growth. For strains B1 and B2, an additional temperature for each strain (5 ºC for B1 and 10 ºC for B2) was included to assure enough biomass for lipid extraction

Fig. 5

Specific lipid composition and concentration of bacterial isolates as a function of incubation temperature. Composition and concentration (µg · g⁻¹ dry weight) of specific lipid compounds in Paenisporosarcina macmurdoensis strains B1 and B2 and Arthrobacter sp. strains B7 and B8 after incubation at their maximum and minimum temperatures for growth (Fig. 3). For strains B1 and B2, an additional temperature for each strain (5 ºC for B1 and 10 ºC for B2) was included to assure enough biomass for lipid detection. The negative control of the experiment (phosphate buffered saline medium without bacteria) is also included (i.e., “Neg”). In A), lipid compounds from the acidic fraction or fatty acids; in B), from the apolar fraction or hydrocarbons, and in C), from the polar fraction or alcohols. “B&U” means branched and unsaturated fatty acids or alkanols, “B&S” stands for branched and saturated fatty acids, and “DiMe” or “Mme” indicate dimethylated or monomethylated monounsaturated fatty acids. The position of the double bond in the “C_14:1” and “C_16:1” fatty acids is unknown. Roman numbers in brackets refer to different isomers of the same compound

Fig. 6

Similarities/dissimilarities in the lipid profile of bacterial isolates as a function of taxonomy and incubation temperature. Principal Coordinates Analysis (PCoA) based on the Bray-Curtis dissimilarity distances using the concentrations (µg · g⁻¹ dw) of the specific lipid compounds produced by Paenisporosarcina macmurdoensis strains B1 and B2, and Arthrobacter sp. strains B7 and B8 incubated at their maximum and minimum temperatures for growth (Fig. 5)