Cryopreservation of Cyanobacteria and Eukaryotic Microalgae Using Exopolysaccharide Extracted from a Glacier Bacterium.

Exopolysaccharide (EPS) has been known to be a good cryoprotective agent for bacteria, but it has not been tested for cyanobacteria and eukaryotic microalgae. In this study, we used EPS extracted from a glacier bacterium as a cryoprotective agent for the cryopreservation of three unicellular cyanobacteria and two eukaryotic microalgae. Different concentrations of EPS (10%, 15%, and 20%) were tested, and the highest concentration (20%) of EPS yielded the best growth recovery for the algal strains we tested. We also compared EPS with 5% dimethyl sulfoxide (DMSO) and 10% glycerol for the cryopreservation recovery. The growth recovery for the microalgal strains after nine months of cryopreservation was better than 5% DMSO, a well-known cryoprotectant for microalgae. A poor recovery was recorded for all the tested strains with 10% glycerol as a cryoprotective agent. The patterns of growth recovery for most of these strains were similar after 5 days, 15 days, and 9 months of cryopreservation. Unlike common cryopreservants such as DMSO or methanol, which are hazardous materials, EPS is safe to handle. We demonstrate that the EPS from a psychrotrophic bacterium helped in the long-term cryopreservation of cyanobacteria and microalgae, and it has the potential to be used as natural cryoprotective agent for other cells.

Bacterial community characterization of Batura Glacier in the Karakoram Range of Pakistan.

High-altitude cold habitats of the Karakoram are rarely explored for their bacterial community characterization and metabolite productions. In the present study, bacterial communities in ice, water, and sediments of Batura Glacier were investigated using culture-dependent and culture-independent methods. Twenty-seven cold-adapted bacterial strains (mostly psychrotrophic) were isolated using R2A, Tryptic Soy Agar (TSA), and Luria-Bertani (LB) media, at 4 °C and 15 °C. Most of the isolates exhibited growth at a wide range of temperature (4-35 °C), pH (5-12), and salinity (1-6%). Among the bacterial isolates, 52% were identified as Gram-positive and the remaining 48% represented as Gram-negative. The results of phylogenetic analysis indicated that all the culturable bacteria belonged to 3 major phylogenetic groups, i.e., Actinobacteria (48%), Bacteroidetes (26%), and Proteobacteria (22%), while Flavobacterium (26%), Arthrobacter (22%), and Pseudomonas (19%) were represented as the dominant genera. Similarly, Illumina amplicon sequencing of 16S rRNA genes after PCR amplification of DNA from the whole community revealed dominance of the same phylogenetic groups, Proteobacteria, Actinobacteria, and Bacteroidetes, while Arthrobacter, Mycoplana, Ochrobactrum, Kaistobacter, Janthinobacterium, and Flavobacterium were found as the dominant genera. Among the culturable isolates, 70% demonstrated activity for cellulases, 48% lipases, 41% proteases, 41% DNases, and only 7% for amylases. Most of the glacial isolates demonstrated antimicrobial activity against other microorganisms including the multiple-drug-resistant strains of Candida albicans, Klebsiella pneumoniae, Acinetobacter sp., and Bacillus sp. 67% of Gram-negative while 46% of Gram-positive glacial bacteria were resistant to trimethoprim/sulfamethoxazole. Resistance against methicillin and vancomycin among the Gram-positive isolates was 23% and 15%, respectively, while 11% of the Gram-negative isolates exhibited resistance against both colistin sulfate and nalidixic acid.

Draft Genome Sequence of a Cold-Adapted Pseudomonas sp. Strain, BGI-2, Isolated from the Ice of Batura Glacier, Pakistan.

Pseudomonas sp. strain BGI-2 is a psychrotrophic bacterium isolated from the ice of Batura Glacier in the Karakoram mountain range. This strain produces a high yield of exopolysaccharide (EPS) at low temperatures and exhibits high freeze-thaw tolerance. The BGI-2 genome contains 11 EPS-producing genes, which are not found in the closely related Pseudomonas strains.

A Glacier Bacterium Produces High Yield of Cryoprotective Exopolysaccharide.

Pseudomonas sp. BGI-2 is a psychrotrophic bacterium isolated from the ice sample collected from Batura glacier, Pakistan. This strain produces highly viscous colonies on agar media supplemented with glucose. In this study, we have optimized growth and production of exopolysaccharide (EPS) by the cold-adapted Pseudomonas sp. BGI-2 using different nutritional and environmental conditions. Pseudomonas sp. BGI-2 is able to grow in a wide range of temperatures (4-35°C), pH (5-11), and salt concentrations (1-5%). Carbon utilization for growth and EPS production was extensively studied and we found that glucose, galactose, mannose, mannitol, and glycerol are the preferable carbon sources. The strain is also able to use sugar waste molasses as a growth substrate, an alternative for the relatively expensive sugars for large scale EPS production. Maximum EPS production was observed at 15°C, pH 6, NaCl (10 g L-1), glucose as carbon source (100 g L-1), yeast extract as nitrogen source (10 g L-1), and glucose/yeast extract ratio (10/1). Under optimized conditions, EPS production was 2.01 g L-1, which is relatively high for a Pseudomonas species compared to previous studies using the same method for quantification. High-performance anion-exchange chromatography with pulsed amperometric detection (HPAEC-PAD) analysis of EPS revealed glucose, galactose, and glucosamine as the main sugar monomers. Membrane protection assay using human RBCs revealed significant reduction in cell lysis (∼50%) in the presence of EPS, suggesting its role in membrane protection. The EPS (5%) also conferred significant cryoprotection for a mesophilic Escherichia coli k12 which was comparable to glycerol (20%). Also, improvement in lipid peroxidation inhibition (in vitro) resulted when lipids from the E. coli was pretreated with EPS. Increased EPS production at low temperatures, freeze thaw tolerance of the EPS producing strain, and increased survivability of E. coli in the presence of EPS as cryoprotective agent supports the hypothesis that EPS production is a strategy for survival in extremely cold environments such as the glacier ice.

Allelic dropout can cause false-positive results for Prader-Willi and Angelman syndrome testing.

The diagnosis of many genetic disorders relies on a combination of clinical suspicion and confirmatory genetic testing. Our laboratory uses a standard methylation-sensitive PCR (MSP) to target the differentially methylated SNRPN gene to test for Prader-Willi syndrome (PWS) and Angelman syndrome. One patient, a 27-month-old female, who lacked the classical clinical features of PWS, but had a molecular diagnosis of PWS by MSP by another laboratory, had repeat testing in our laboratory. Testing by MSP in our laboratory also identified an apparent loss of the unmethylated paternal allele, consistent with a diagnosis of PWS. Confirmatory testing using Southern blot analysis with a methylation-sensitive restriction enzyme showed a normal pattern of methylation, detecting both the methylated maternal and unmethylated paternal alleles. To investigate these discrepant results, we amplified and sequenced the SNRPN locus in this patient and identified a single nucleotide change within the binding site for the unmethylated DNA-specific primer. These results indicate this nucleotide change led to allelic dropout in the MSP analysis, yielding the false-positive result. Subsequently, MSP analysis using an alternate primer set that was developed by our laboratory detected both methylated and unmethylated alleles. These findings illustrate that allelic dropout due to the presence of rare polymorphisms can cause false-positive results in commonly used MSP assays and lead to molecular misdiagnosis.