Phylogenetic diversity of numerically important arctic sea-ice bacteria cultured at subzero temperature

Heterotrophic bacteria in sea ice play a key role in carbon cycling, but little is known about the predominant players at the phylogenetic level. In a study of both algal bands and clear ice habitats within summertime Arctic pack ice from the Chukchi Sea, we determined the abundance of total bacteria and actively respiring cells in melted ice samples using epifluorescence microscopy and the stains 4', 6'-diamidino-2-phenylindole 2HCl (DAPI) and 5-cyano-2,3-ditolyl tetrazolium chloride (CTC), respectively. Organic-rich and -poor culturing media were used to determine culturable members by plating (at 0degreesC and 5degreesC) and most-probable-number (MPN) analyses (at -1degreesC). Total bacterial counts ranged from 5.44 x 10(4) ml(-1) in clear ice to 2.41 X 10(6) ml(-1) in algal-band ice samples, with 2-27% metabolically active by CTC stain. Plating and MPN results revealed a high degree of culturability in both types of media, but greater success in oligotrophic media (to 62% of total abundance) and from clear ice samples. The bacterial enumeration anomaly, commonly held to mean less than or equal to 0.01% cultured, was not demonstrated in any of our samples, Denaturing gradient get electrophoresis was used to check the purity of 44 isolates and select representatives for subsequent sequencing. Phylogenetic analyses based on 16S rRNA sequences indicated close relationships exclusively to known marine psychrophiles within two bacterial divisions, Proteobacteria (in the genera Alteromonas, Colwellia, Glaciecola, Octadecabacter, Pseudoaltermonas and Shewanella) and Cytophaga-Flexibacter-Bacteroides (Cytophaga, Flavobacterium, Gelidibacter and Polaribacter). All cultures from the clear ice sample with highest (62%) culturability were closely related to each other or to psychrophilic Cytophaga-Flexibacter-Bacteroides (94.9-99.6% sequence similarities). Overall, these findings suggest limited, heterotrophic bacterial diversity at cold temperatures and may provide insight into the recent evolution of psychrophilic bacteria.