, 2004). It has been shown that under antibiotic stress, E. coli CspD causes arrest in growth and reduced viability under the direct regulation of a motility and quorum-sensing regulator toxin–antitoxin pair, MqsR/MqsA, that possesses RNase find more activity and influence cell physiology (e.g. biofilm formation and motility) leading to the establishment of the persister population (Kim et al., 2010; Kim & Wood, 2010). It is apparent from the above findings that although all Csps contain highly conserved CSD, their physiological roles may vary depending upon the organisms, proteins and environment. Most structural and functional analyses were performed on Csps from mesophiles,

but not from psychrophiles or psychrotolerant bacteria. Among all the classes of phylum Proteobacteria, the Csps from bacteria belonging to class Betaproteobacteria have not been characterized yet. Therefore, the major objective of this study was to elucidate the structure and physiological role of a cold-shock protein, CspD from a psychrotolerant Janthinobacterium sp. Ant5-2 isolated from Antarctica that has to survive extreme cold environment. Janthinobacterium sp. strain Ant5-2 BYL719 (ATCC BAA-2154)

was isolated from Lake Podprudnoye a small proglacial lake located in central Schirmacher Oasis, East Antarctica (latitude −70.766758°, longitude 11.610249°). Samples were collected aseptically in November 2008 from shallow water near the shoreline when ice-cover was absent and transported to the lab frozen and processed shortly after arrival. For all experiments in this study, Ant5-2 was grown in 1 : 2 (v/v) trypticase soy broth (TSB) (BD, Franklin, NJ). Escherichia coli BL21(DE3)pLysS (F – ompT hsdSB(rB -, mB -) galdcm (DE3) pLysS (CamR) (Novagen, WI) was grown at 37 °C in Luria–Bertini (LB) medium. For growth and cold tolerance, 250 mL of 1 : 2 (v/v) TSB medium was inoculated with 1 mL of overnight culture of Ant5-2 and incubated at 22 °C until the OD450 nm

reached 0.2. Then, 50 mL aliquots were transferred to incubator shakers set at 37, 22, 15, 4 and −1 °C. The OD was determined PAK5 at various time intervals using a Lambda 2 spectrophotometer (Perkin Elmer, Norwalk, CT). To determine freeze tolerance, Ant5-2 culture was grown to exponential phase and frozen at −20 °C. The frozen culture was then subjected to one freeze–thaw cycle and viable plate count was determined before and after freezing as described previously (Panicker et al., 2002). Total cellular proteins of Ant 5-2 cultures were radiolabeled using EasyTag™ Express 35S methionine Protein Labeling Mix (11 μCi μL−1) (Perkin Elmer) and transferred immediately to −1, 4, 15 and 22 °C, respectively, and incubated for 1.5 h as described previously (Panicker et al., 2002).