Structural and physiological adaptations of soil microorganisms to freezing revealed by position-specific labeling and compound-specific 13C analysis

Psychrotolerant microbes are crucial for carbon cycling and biotechnological applications. Nonetheless, the mechanisms enabling their survival and functioning in frozen environments remain unclear. To elucidate adaptations of microbial cell membranes to freezing, we incubated soils with position-specific C-13 labeled glucose at +5 (control), -5 and -20 degrees C and quantified C-13 in CO2 and phospholipid fatty acids. High oxidation of glucose C-1 at +5 degrees C revealed a transformation via the pentose phosphate pathway. At subzero temperatures, however, the preferential oxidation of C-4 position suggested a switch to glycolysis. The threefold increase of Gram-negative phospholipid fatty acids in soil incubated at -5 degrees C was accompanied by a twofold increase in C-13 incorporation. This unequal increase of phospholipid fatty acids and incorporated C-13 can be explained by simultaneous desaturation of existing fatty acid chains and the de novo synthesis of monounsaturated fatty acids, which indicates microbial growth. In contrast, Gram-positive bacteria incorporated 2 times higher C-13 into their phospholipid fatty acids at -20 degrees C than at -5 and +5 degrees C without a significant increase in their fatty acid contents. This reflects intensive repair of membranes damaged at -20 degrees C without microbial growth. The fungal/bacterial ratio was 1.5 times lower at subzero temperatures than at +5 degrees C, reflecting a shift in microbial community structure towards bacteria. Accordingly, soil microorganisms adapted to freezing by (1) switching their metabolic pathway from the pentose phosphate pathway to glycolysis, (2) modifying phospholipid fatty acids by desaturation and, (3) shifting microbial community structure towards Gram-negative bacteria by reducing the fungal population.