Biofilm, ice recrystallization inhibition and freeze-thaw protection in an epiphyte community

Microbial communities found on the surface of overwintering plants may be exposed to low temperatures as well as multiple freeze-thaw events. To explore the adaptive mechanisms of these epiphytes, with the objective of identifying products for freeze-protection, enrichment libraries were made from frost-exposed leaves. Of 15 identified bacteria from 60 individual clones, approximately half had ice-association activities, with the great majority showing high freeze-thaw resistance. Isolates with ice nucleation activity and ice recrystallization inhibition activity were recovered. Of the latter, two (Erwinia billingiae J10, and Sphingobacterium kitahiroshimense Y2) showed culture and electron microscopic evidence of motility and/or biofilm production. Mass spectrometric characterization of the E. billingiae extracellular polymeric substance (EPS) identified the major proteins as 35 kDa outer membrane protein A and F, supporting its biofilm character. The addition of the EPS preparation increased the freeze-thaw survival of the more susceptible bacteria 1000-10000 times, and protection was at least partially dependent on the protein component.