Microbial abundance in surface ice on the Greenland Ice Sheet

Measuring microbial abundance in glacier ice and identifying its controls is essential for a better understanding and quantification of biogeochemical processes in glacial ecosystems. However, cell enumeration of glacier ice samples is challenging due to typically low cell numbers and the presence of interfering mineral particles. We quantified for the first time the abundance of microbial cells in surface ice from geographically distinct sites on the Greenland Ice Sheet (GrIS), using three enumeration methods: epifluorescence microscopy (EFM), flow cytometry (FCM), and quantitative polymerase chain reaction (qPCR). In addition, we reviewed published data on microbial abundance in glacier ice and tested the three methods on artificial ice samples of realistic cell (10(2)–10(7) cells ml(−1)) and mineral particle (0.1–100 mg ml(−1)) concentrations, simulating a range of glacial ice types, from clean subsurface ice to surface ice to sediment-laden basal ice. We then used multivariate statistical analysis to identify factors responsible for the variation in microbial abundance on the ice sheet. EFM gave the most accurate and reproducible results of the tested methodologies, and was therefore selected as the most suitable technique for cell enumeration of ice containing dust. Cell numbers in surface ice samples, determined by EFM, ranged from ~ 2 × 10(3) to ~ 2 × 10(6) cells ml(−1) while dust concentrations ranged from 0.01 to 2 mg ml(−1). The lowest abundances were found in ice sampled from the accumulation area of the ice sheet and in samples affected by fresh snow; these samples may be considered as a reference point of the cell abundance of precipitants that are deposited on the ice sheet surface. Dust content was the most significant variable to explain the variation in the abundance data, which suggests a direct association between deposited dust particles and cells and/or by their provision of limited nutrients to microbial communities on the GrIS

Meltwater export of prokaryotic cells from the Greenland ice sheet

Microorganisms are flushed from the Greenland Ice Sheet (GrIS) where they may contribute towards the nutrient cycling and community compositions of downstream ecosystems. We investigate meltwater microbial assemblages as they exit the GrIS from a large outlet glacier, and as they enter a downstream river delta during the record melt year of 2012. Prokaryotic abundance, flux and community composition was studied, and factors affecting community structures were statistically considered. The mean concentration of cells exiting the ice sheet was 8.30 × 104 cells mL−1 and we estimate that ∼1.02 × 1021 cells were transported to the downstream fjord in 2012, equivalent to 30.95 Mg of carbon. Prokaryotic microbial assemblages were dominated by Proteobacteria, Bacteroidetes, and Actinobacteria. Cell concentrations and community compositions were stable throughout the sample period, and were statistically similar at both sample sites. Based on our observations, we argue that the subglacial environment is the primary source of the river-transported microbiota, and that cell export from the GrIS is dependent on discharge. We hypothesise that the release of subglacial microbiota to downstream ecosystems will increase as freshwater flux from the GrIS rises in a warming world

Supraglacial bacterial community structures vary across the Greenland ice sheet

The composition and spatial variability of microbial communities that reside within the extensive (>200 000 km2) biologically active area encompassing the Greenland ice sheet (GrIS) is hypothesized to be variable. We examined bacterial communities from cryoconite debris and surface ice across the GrIS, using sequence analysis and quantitative PCR of 16S rRNA genes from co-extracted DNA and RNA. Communities were found to differ across the ice sheet, with 82.8% of the total calculated variation attributed to spatial distribution on a scale of tens of kilometers separation. Amplicons related to Sphingobacteriaceae, Pseudanabaenaceae and WPS-2 accounted for the greatest portion of calculated dissimilarities. The bacterial communities of ice and cryoconite were moderately similar (global R = 0.360, P = 0.002) and the sampled surface type (ice versus cryoconite) did not contribute heavily towards community dissimilarities (2.3% of total variability calculated). The majority of dissimilarities found between cryoconite 16S rRNA gene amplicons from DNA and RNA was calculated to be the result of changes in three taxa, Pseudanabaenaceae, Sphingobacteriaceae and WPS-2, which together contributed towards 80.8 ± 12.6% of dissimilarities between samples. Bacterial communities across the GrIS are spatially variable active communities that are likely influenced by localized biological inputs and physicochemical conditions

Primers used for TD-PCR.

<p>Primers used for TD-PCR.</p

Novel Insight into the Genetic Context of the <i>cadAB</i> Genes from a 4-chloro-2-methylphenoxyacetic Acid-Degrading <i>Sphingomonas</i>

<div><p>The 2-methyl-4-chlorophenoxyacetic (MCPA) acid-degrader <i>Sphingomonas</i> sp. ERG5 has recently been isolated from MCPA-degrading bacterial communities. Using Illumina-sequencing, the 5.7 Mb genome of this isolate was sequenced in this study, revealing the 138 kbp plasmid pCADAB1 harboring the 32.5 kbp composite transposon Tn6228 which contains genes encoding proteins for the removal of 2,4-dichlorophenoxyacetic acid (2,4-D) and MCPA, as well as the regulation of this pathway. Transposon Tn6228 was confirmed by PCR to be situated on the plasmid and also exist in a circular intermediate state - typical of IS3 elements. The canonical <i>tfdAα</i>-gene of group III 2,4-D degraders, encoding the first step in degradation of 2,4-D and related compounds, was not present in the chromosomal contigs. However, the alternative <i>cadAB</i> genes, also providing the initial degradation step, were found in Tn6228, along with the 2,4-D-degradation-associated genes <i>tfdBCDEFKR</i> and <i>cadR</i>. Putative reductase and ferredoxin genes <i>cadCD</i> of Rieske non-heme iron oxygenases were also present in close proximity to <i>cadAB</i>, suggesting that these might have an unknown role in the initial degradation reaction. Parts of the composite transposon contain sequence displaying high similarity to previously analyzed 2,4-D degradation genes, suggesting rapid dissemination and high conservation of the chlorinated-phenoxyacetic acid (PAA)-degradation genotype among the sphingomonads.</p></div

Genetic map of plasmid pCADAB1 and composite transposon Tn6228.

<p>The outer ring shows the functional regions of the plasmid: conjugative transfer (blue), plasmid stability and maintenance (yellow) and region containing multiple IS-elements (red). The composite transposon Tn6228 is displayed on the outside of the plasmid backbone (black line) Primers 74F, 74R, 251F and 251R for PCR have been marked on the outer ring. Inside of the backbone, predicted ORFs on either the positive strand (top blocks) or negative strand (bottom blocks) are displayed. Genes involved in degradation of MCPA are highlighted (green). Also highlighted are IS-elements (red), genes related to the type 4 secretion system (blue) and genes involved in plasmid maintenance and stability (yellow). The middle circle shows similarity to other sequences: Grey bars indicate collinear blocks of similarity to A) <i>Sphingomonas</i> sp. tfd44 (acc. no. AY598949.1) and B) <i>Sphingomonas</i> sp. 58-1 (acc. no. AB353895.1), while bright green bars indicate collinearity with plasmid pNL1 (acc. no. CP000676.1) and dark green with plasmid pCAR3 (acc. no. AB270530.1). The minimum nucleotide similarity in collinear blocks is 72%. The inner circle displays the G+C content (window size  = 1000, step size  = 10).</p

Combination of primers for TD-PCR and tested hypotheses.

<p>Combination of primers for TD-PCR and tested hypotheses.</p

Dendrogram representing the phylogenetic relationship of representative CadA sequences.

<p>Representative sequences for alignment were identified from a BLASTX search using the derived amino acid sequence of CadA from <i>Sphingomonas</i> sp. ERG5 as query. BLASTX hits were picked for comparison with a cutoff value of 50% for amino acid identities. The support of each branch, as determined from 100 bootstrap samples, is indicated as a percentage at each node. Sequences were aligned with T-Coffee (v6.85) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083346#pone.0083346-Notredame1" target="_blank">[30]</a> and the alignment was curated with Gblocks (v0.91b) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083346#pone.0083346-Castresana1" target="_blank">[31]</a>. A phylogenetic tree was constructed using a maximum likelihood approach and the JTT substitution model <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083346#pone.0083346-Jones1" target="_blank">[32]</a> in the PhyML program (v3.0 aLRT) <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083346#pone.0083346-Guindon1" target="_blank">[33]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083346#pone.0083346-Anisimova1" target="_blank">[34]</a>. The resulting unrooted phylogenetic tree was visualized in MEGA4.0.2<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0083346#pone.0083346-Tamura1" target="_blank">[35]</a>.</p

Comparing Metabolic Functionalities, Community Structures, and Dynamics of Herbicide-Degrading Communities Cultivated with Different Substrate Concentrations

Two 4-chloro-2-methylphenoxyacetic acid (MCPA)-degrading enrichment cultures selected from an aquifer on low (0.1 mg liter(-1)) or high (25 mg liter(-1)) MCPA concentrations were compared in terms of metabolic activity, community composition, population growth, and single cell physiology. Different community compositions and major shifts in community structure following exposure to different MCPA concentrations were observed using both 16S rRNA gene denaturing gradient gel electrophoresis fingerprinting and pyrosequencing. The communities also differed in their MCPA-mineralizing activities. The enrichments selected on low concentrations mineralized MCPA with shorter lag phases than those selected on high concentrations. Flow cytometry measurements revealed that mineralization led to cell growth. The presence of low-nucleic acid-content bacteria (LNA bacteria) was correlated with mineralization activity in cultures selected on low herbicide concentrations. This suggests that LNA bacteria may play a role in degradation of low herbicide concentrations in aquifers impacted by agriculture. This study shows that subpopulations of herbicide-degrading bacteria that are adapted to different pesticide concentrations can coexist in the same environment and that using a low herbicide concentration enables enrichment of apparently oligotrophic subpopulations