Phylogenetic Diversity of Numerically Important Arctic Sea-Ice Bacteria Cultured at Subzero Temperature

Abstract

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 0 degrees C and 5 degrees C) and most-probable-number (MPN) analyses (at -1 degrees C). 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 <or= 0.01% cultured, was not demonstrated in any of our samples. Denaturing gradient gel 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.

Hydrocarbon biodegradation by Arctic sea-ice and sub-ice microbial communities during microcosm experiments, Northwest Passage (Nunavut, Canada)

“…Gammaproteobacteria is typically the predominant group in Arctic sea ice (Junge et al 2002;Brinkmeyer et al 2003;Bowman et al 2012) followed by Bacteroidetes (Deming 2010), whereas Alphaproteobacteria dominates in Arctic surface waters (Garneau et al 2006;Alonso-Sáez et al 2008). Accordingly, our results showed a dominance of Gammaproteobacteria and Bacteroidetes in the marine communities on the underside of landfast ice but no Alphaproteobacteria were detected (Fig.…”

Abundance, size distribution and bacterial colonization of exopolymer particles in Antarctic sea ice (Bellingshausen Sea)

“…In the pelagic realm, EP and larger aggregates can serve bacteria as special microhabitats, and have been recognized as sites of increased microbial activity (Smith et al 1992, Sherr et al 1999, Ayo et al 2001, Grossart et al 2003. Junge et al (2002) report that a remarkably high fraction of the total number of respiring sea ice bacteria is associated with particles. In the present study, the total number of bacteria attached to individual EP ranged between 1 and 220 bacteria EP -1 and was within the range reported in pelagic studies , Mari & Kiørboe 1996, Simon et al 2002.…”

Characterization of a cold-active bacteriophage on two psychrophilic marine hosts

“…The phage, 9A, was isolated from an Arctic nepheloid layer (a particle-rich region of the water column) against 34H, itself originally cultured from Arctic shelf sediments (depth of 305 m; Huston 2003). Although little is known about the quantitative ecology of Colwellia, they are routinely isolated from cold, particle-or surface-rich environments, including flounder eggs (D'Aoust & Kushner 1972), sea ice (Junge et al 2002, Borriss et al 2003, Brinkmeyer et al 2003 and nepheloid layers (Wells 2006); association with particles or surfaces is also supported by molecular approaches (DeLong et al 1993, Gillan et al 1998, Eilers et al 2000, Bowman & McCuaig 2003. With a growth temperature optimum among the coldest of cultivated psychrophiles (8 to 9°C; Huston 2003) and its genome sequenced (Methé et al 2005), 34H is an attractive organism with which to explore host and viral features enabling cold activity and their ecological consequences.…”

“…There the water was gravity-filtered through a 0.2 µm HT Tuffryn membrane capsule filter (effective filtration area of 500 cm 2 ; Pall Gelman) to remove bacteria but not viruses. After filtration, which took ~36 h due to the large volume, high particle load and small filter area, the water was divided into two carboys and enriched with 4 to 8 ml each of turbid cultures of 34H, Colwellia psychrerythraea NRC 1004, and sea-ice isolates 6M3 and 11B5 (putative Colwellia and Shewanella, respectively; Junge et al 2002). Multiple hosts were used for enrichment in an attempt to avoid bias toward phage monovalence (Jensen et al 1998).…”