Microbiomes attached to fresh perennial ryegrass are temporally resilient and adapt to changing ecological niches.

BACKGROUND: Gut microbiomes, such as the rumen, greatly influence host nutrition due to their feed energy-harvesting capacity. We investigated temporal ecological interactions facilitating energy harvesting at the fresh perennial ryegrass (PRG)-biofilm interface in the rumen using an in sacco approach and prokaryotic metatranscriptomic profiling. RESULTS: Network analysis identified two distinct sub-microbiomes primarily representing primary (≤ 4 h) and secondary (≥ 4 h) colonisation phases and the most transcriptionally active bacterial families (i.e Fibrobacteriaceae, Selemondaceae and Methanobacteriaceae) did not interact with either sub-microbiome, indicating non-cooperative behaviour. Conversely, Prevotellaceae had most transcriptional activity within the primary sub-microbiome (focussed on protein metabolism) and Lachnospiraceae within the secondary sub-microbiome (focussed on carbohydrate degradation). Putative keystone taxa, with low transcriptional activity, were identified within both sub-microbiomes, highlighting the important synergistic role of minor bacterial families; however, we hypothesise that they may be 'cheating' in order to capitalise on the energy-harvesting capacity of other microbes. In terms of chemical cues underlying transition from primary to secondary colonisation phases, we suggest that AI-2-based quorum sensing plays a role, based on LuxS gene expression data, coupled with changes in PRG chemistry. CONCLUSIONS: In summary, we show that fresh PRG-attached prokaryotes are resilient and adapt quickly to changing niches. This study provides the first major insight into the complex temporal ecological interactions occurring at the plant-biofilm interface within the rumen. The study also provides valuable insights into potential plant breeding strategies for development of the utopian plant, allowing optimal sustainable production of ruminants. Video Abstract.

The Isolation and Genome Sequencing of Five Novel Bacteriophages From the Rumen Active Against Butyrivibrio fibrisolvens.

Although the prokaryotic communities of the rumen microbiome are being uncovered through genome sequencing, little is known about the resident viral populations. Whilst temperate phages can be predicted as integrated prophages when analyzing bacterial and archaeal genomes, the genetics underpinning lytic phages remain poorly characterized. To the five genomes of bacteriophages isolated from rumen-associated samples sequenced and analyzed previously, this study adds a further five novel genomes and predictions gleaned from them to further the understanding of the rumen phage population. Lytic bacteriophages isolated from fresh ovine and bovine fecal and rumen fluid samples were active against the predominant fibrolytic ruminal bacterium Butyrivibrio fibrisolvens. The double stranded DNA genomes were sequenced and reconstructed into single circular complete contigs. Based on sequence similarity and genome distances, the five phages represent four species from three separate genera, consisting of: (1) Butyrivibrio phages Arian and Bo-Finn; (2) Butyrivibrio phages Idris and Arawn; and (3) Butyrivibrio phage Ceridwen. They were predicted to all belong to the Siphoviridae family, based on evidence in the genomes such as size, the presence of the tail morphogenesis module, genes that share similarity to those in other siphovirus isolates and phylogenetic analysis using phage proteomes. Yet, phylogenomic analysis and sequence similarity of the entire phage genomes revealed that these five phages are unique and novel. These phages have only been observed undergoing the lytic lifecycle, but there is evidence in the genomes of phages Arawn and Idris for the potential to be temperate. However, there is no evidence in the genome of the bacterial host Butyrivibrio fibrisolvens of prophage genes or genes that share similarity with the phage genomes.

Domestication of Campylobacter jejuni NCTC 11168.

Reference and type strains of well-known bacteria have been a cornerstone of microbiology research for decades. The sharing of well-characterized isolates among laboratories has run in parallel with research efforts and enhanced the reproducibility of experiments, leading to a wealth of knowledge about trait variation in different species and the underlying genetics. Campylobacter jejuni strain NCTC 11168, deposited at the National Collection of Type Cultures in 1977, has been adopted widely as a reference strain by researchers worldwide and was the first Campylobacter for which the complete genome was published (in 2000). In this study, we collected 23 C. jejuni NCTC 11168 reference isolates from laboratories across the UK and compared variation in simple laboratory phenotypes with genetic variation in sequenced genomes. Putatively identical isolates, identified previously to have aberrant phenotypes, varied by up to 281 SNPs (in 15 genes) compared to the most recent reference strain. Isolates also display considerable phenotype variation in motility, morphology, growth at 37 °C, invasion of chicken and human cell lines, and susceptibility to ampicillin. This study provides evidence of ongoing evolutionary change among C. jejuni isolates as they are cultured in different laboratories and highlights the need for careful consideration of genetic variation within laboratory reference strains. This article contains data hosted by Microreact.

Evolutionary genomics of anthroponosis in Cryptosporidium.

Human cryptosporidiosis is the leading protozoan cause of diarrhoeal mortality worldwide, and a preponderance of infections is caused by Cryptosporidium hominis and C. parvum. Both species consist of several subtypes with distinct geographical distributions and host preferences (that is, generalist zoonotic and specialist anthroponotic subtypes). The evolutionary processes that drive the adaptation to the human host and the population structures of Cryptosporidium remain unknown. In this study, we analyse 21 whole-genome sequences to elucidate the evolution of anthroponosis. We show that Cryptosporidium parvum splits into two subclades and that the specialist anthroponotic subtype IIc-a shares a subset of loci with C. hominis that is undergoing rapid convergent evolution driven by positive selection. C. parvum subtype IIc-a also has an elevated level of insertion and deletion mutations in the peri-telomeric genes, which is also a characteristic of other specialist subtypes. Genetic exchange between Cryptosporidium subtypes plays a prominent role throughout the evolution of the genus. Interestingly, recombinant regions are enriched for positively selected genes and potential virulence factors, which indicates adaptive introgression. Analysis of 467 gp60 sequences collected from locations across the world shows that the population genetic structure differs markedly between the main zoonotic subtype (isolation-by-distance) and the anthroponotic subtype (admixed population structure). We also show that introgression between the four anthroponotic Cryptosporidium subtypes and species included in this study has occurred recently, probably within the past millennium.

Genomic analysis of bacteria in the Acute Oak Decline pathobiome.

The UK's native oak is under serious threat from Acute Oak Decline (AOD). Stem tissue necrosis is a primary symptom of AOD and several bacteria are associated with necrotic lesions. Two members of the lesion pathobiome, Brenneria goodwinii and Gibbsiella quercinecans, have been identified as causative agents of tissue necrosis. However, additional bacteria including Lonsdalea britannica and Rahnella species have been detected in the lesion microbiome, but their role in tissue degradation is unclear. Consequently, information on potential genome-encoded mechanisms for tissue necrosis is critical to understand the role and mechanisms used by bacterial members of the lesion pathobiome in the aetiology of AOD. Here, the whole genomes of bacteria isolated from AOD-affected trees were sequenced, annotated and compared against canonical bacterial phytopathogens and non-pathogenic symbionts. Using orthologous gene inference methods, shared virulence genes that retain the same function were identified. Furthermore, functional annotation of phytopathogenic virulence genes demonstrated that all studied members of the AOD lesion microbiota possessed genes associated with phytopathogens. However, the genome of B. goodwinii was the most characteristic of a necrogenic phytopathogen, corroborating previous pathological and metatranscriptomic studies that implicate it as the key causal agent of AOD lesions. Furthermore, we investigated the genome sequences of other AOD lesion microbiota to understand the potential ability of microbes to cause disease or contribute to pathogenic potential of organisms isolated from this complex pathobiome. The role of these members remains uncertain but some such as G. quercinecans may contribute to tissue necrosis through the release of necrotizing enzymes and may help more dangerous pathogens activate and realize their pathogenic potential or they may contribute as secondary/opportunistic pathogens with the potential to act as accessory species for B. goodwinii. We demonstrate that in combination with ecological data, whole genome sequencing provides key insights into the pathogenic potential of bacterial species whether they be phytopathogens, part-contributors or stimulators of the pathobiome.

A pilot study using metagenomic sequencing of the sputum microbiome suggests potential bacterial biomarkers for lung cancer.

Lung cancer (LC) is the most prevalent cancer worldwide, and responsible for over 1.3 million deaths each year. Currently, LC has a low five year survival rates relative to other cancers, and thus, novel methods to screen for and diagnose malignancies are necessary to improve patient outcomes. Here, we report on a pilot-sized study to evaluate the potential of the sputum microbiome as a source of non-invasive bacterial biomarkers for lung cancer status and stage. Spontaneous sputum samples were collected from ten patients referred with possible LC, of which four were eventually diagnosed with LC (LC+), and six had no LC after one year (LC-). Of the seven bacterial species found in all samples, Streptococcus viridans was significantly higher in LC+ samples. Seven further bacterial species were found only in LC-, and 16 were found only in samples from LC+. Additional taxonomic differences were identified in regards to significant fold changes between LC+ and LC-cases, with five species having significantly higher abundances in LC+, with Granulicatella adiacens showing the highest level of abundance change. Functional differences, evident through significant fold changes, included polyamine metabolism and iron siderophore receptors. G. adiacens abundance was correlated with six other bacterial species, namely Enterococcus sp. 130, Streptococcus intermedius, Escherichia coli, S. viridans, Acinetobacter junii, and Streptococcus sp. 6, in LC+ samples only, which could also be related to LC stage. Spontaneous sputum appears to be a viable source of bacterial biomarkers which may have utility as biomarkers for LC status and stage.

Terminator Operon Reporter: combining a transcription termination switch with reporter technology for improved gene synthesis and synthetic biology applications.

Synthetic biology is characterized by the development of novel and powerful DNA fabrication methods and by the application of engineering principles to biology. The current study describes Terminator Operon Reporter (TOR), a new gene assembly technology based on the conditional activation of a reporter gene in response to sequence errors occurring at the assembly stage of the synthetic element. These errors are monitored by a transcription terminator that is placed between the synthetic gene and reporter gene. Switching of this terminator between active and inactive states dictates the transcription status of the downstream reporter gene to provide a rapid and facile readout of the accuracy of synthetic assembly. Designed specifically and uniquely for the synthesis of protein coding genes in bacteria, TOR allows the rapid and cost-effective fabrication of synthetic constructs by employing oligonucleotides at the most basic purification level (desalted) and without the need for costly and time-consuming post-synthesis correction methods. Thus, TOR streamlines gene assembly approaches, which are central to the future development of synthetic biology.

Seasonal immunoregulation in a naturally-occurring vertebrate.

BACKGROUND: Fishes show seasonal patterns of immunity, but such phenomena are imperfectly understood in vertebrates generally, even in humans and mice. As these seasonal patterns may link to infectious disease risk and individual condition, the nature of their control has real practical implications. Here we characterize seasonal dynamics in the expression of conserved vertebrate immunity genes in a naturally-occurring piscine model, the three-spined stickleback. RESULTS: We made genome-wide measurements (RNAseq) of whole-fish mRNA pools (n = 36) at the end of summer and winter in contrasting habitats (riverine and lacustrine) and focussed on common trends to filter habitat-specific from overarching temporal responses. We corroborated this analysis with targeted year-round whole-fish gene expression (Q-PCR) studies in a different year (n = 478). We also considered seasonal tissue-specific expression (6 tissues) (n = 15) at a third contrasting (euryhaline) locality by Q-PCR, further validating the generality of the patterns seen in whole fish analyses. Extremes of season were the dominant predictor of immune expression (compared to sex, ontogeny or habitat). Signatures of adaptive immunity were elevated in late summer. In contrast, late winter was accompanied by signatures of innate immunity (including IL-1 signalling and non-classical complement activity) and modulated toll-like receptor signalling. Negative regulators of T-cell activity were prominent amongst winter-biased genes, suggesting that adaptive immunity is actively down-regulated during winter rather than passively tracking ambient temperature. Network analyses identified a small set of immune genes that might lie close to a regulatory axis. These genes acted as hubs linking summer-biased adaptive pathways, winter-biased innate pathways and other organismal processes, including growth, metabolic dynamics and responses to stress and temperature. Seasonal change was most pronounced in the gill, which contains a considerable concentration of T-cell activity in the stickleback. CONCLUSIONS: Our results suggest major and predictable seasonal re-adjustments of immunity. Further consideration should be given to the effects of such responses in seasonally-occurring disease.

Metagenomic Sequencing of the Chronic Obstructive Pulmonary Disease Upper Bronchial Tract Microbiome Reveals Functional Changes Associated with Disease Severity.

Chronic Obstructive Pulmonary Disease (COPD) is a major source of mortality and morbidity worldwide. The microbiome associated with this disease may be an important component of the disease, though studies to date have been based on sequencing of the 16S rRNA gene, and have revealed unequivocal results. Here, we employed metagenomic sequencing of the upper bronchial tract (UBT) microbiome to allow for greater elucidation of its taxonomic composition, and revealing functional changes associated with the disease. The bacterial metagenomes within sputum samples from eight COPD patients and ten 'healthy' smokers (Controls) were sequenced, and suggested significant changes in the abundance of bacterial species, particularly within the Streptococcus genus. The functional capacity of the COPD UBT microbiome indicated an increased capacity for bacterial growth, which could be an important feature in bacterial-associated acute exacerbations. Regression analyses correlated COPD severity (FEV1% of predicted) with differences in the abundance of Streptococcus pneumoniae and functional classifications related to a reduced capacity for bacterial sialic acid metabolism. This study suggests that the COPD UBT microbiome could be used in patient risk stratification and in identifying novel monitoring and treatment methods, but study of a longitudinal cohort will be required to unequivocally relate these features of the microbiome with COPD severity.

Draft Genome Sequence of Probiotic Enterococcus faecium Strain L-3.

We report here the draft genome sequence of the bacteriocin producer Enterococcus faecium strain L-3, isolated from a probiotic preparation, Laminolact, which is widely used in the Russian Federation. The draft genome sequence is composed of 74 contigs for a total of 2,643,001 bp, with 2,646 coding genes. Five clusters for bacteriocin production were found.

Temporal dynamics of the metabolically active rumen bacteria colonizing fresh perennial ryegrass.

This study investigated successional colonization of fresh perennial ryegrass (PRG) by the rumen microbiota over time. Fresh PRG was incubated in sacco in the rumens of three Holstein × Friesian cows over a period of 8 h, with samples recovered at various times. The diversity of attached bacteria was assessed using 454 pyrosequencing of 16S rRNA (cDNA). Results showed that plant epiphytic communities either decreased to low relative abundances or disappeared following rumen incubation, and that temporal colonization of the PRG by the rumen bacteria was biphasic with primary (1 and 2 h) and secondary (4-8 h) events evident with the transition period being with 2-4 h. A decrease in sequence reads pertaining to Succinivibrio spp. and increases in Pseudobutyrivibrio, Roseburia and Ruminococcus spp. (the latter all order Clostridiales) were evident during secondary colonization. Irrespective of temporal changes, the continually high abundances of Butyrivibrio, Fibrobacter, Olsenella and Prevotella suggest that they play a major role in the degradation of the plant. It is clear that a temporal understanding of the functional roles of these microbiota within the rumen is now required to unravel the role of these bacteria in the ruminal degradation of fresh PRG.

C4 Photosynthesis in the Rice Paddy: Insights from the Noxious Weed Echinochloa glabrescens.

The C4 pathway is a highly complex trait that increases photosynthetic efficiency in more than 60 plant lineages. Although the majority of C4 plants occupy disturbed, arid, and nutrient-poor habitats, some grow in high-nutrient, waterlogged conditions. One such example is Echinochloa glabrescens, which is an aggressive weed of rice paddies. We generated comprehensive transcriptome datasets for C4 E. glabrescens and C3 rice to identify genes associated with adaption to waterlogged, nutrient-replete conditions, but also used the data to better understand how C4 photosynthesis operates in these conditions. Leaves of E. glabrescens exhibited classical Kranz anatomy with lightly lobed mesophyll cells having low chloroplast coverage. As with rice and other hygrophytic C3 species, leaves of E. glabrescens accumulated a chloroplastic phosphoenolpyruvate carboxylase protein, albeit at reduced amounts relative to rice. The arid-grown species Setaria italica (C4) and Brachypodium distachyon (C3) were also found to accumulate chloroplastic phosphoenolpyruvate carboxylase. We identified a molecular signature associated with C4 photosynthesis in nutrient-replete, waterlogged conditions that is highly similar to those previously reported from C4 plants that grow in more arid conditions. We also identified a cohort of genes that have been subjected to a selective sweep associated with growth in paddy conditions. Overall, this approach highlights the value of using wild species such as weeds to identify adaptions to specific conditions associated with high-yielding crops in agriculture.

Generation of whole genome sequences of new Cryptosporidium hominis and Cryptosporidium parvum isolates directly from stool samples.

BACKGROUND: Whole genome sequencing (WGS) of Cryptosporidium spp. has previously relied on propagation of the parasite in animals to generate enough oocysts from which to extract DNA of sufficient quantity and purity for analysis. We have developed and validated a method for preparation of genomic Cryptosporidium DNA suitable for WGS directly from human stool samples and used it to generate 10 high-quality whole Cryptosporidium genome assemblies. Our method uses a combination of salt flotation, immunomagnetic separation (IMS), and surface sterilisation of oocysts prior to DNA extraction, with subsequent use of the transposome-based Nextera XT kit to generate libraries for sequencing on Illumina platforms. IMS was found to be superior to caesium chloride density centrifugation for purification of oocysts from small volume stool samples and for reducing levels of contaminant DNA. RESULTS: The IMS-based method was used initially to sequence whole genomes of Cryptosporidium hominis gp60 subtype IbA10G2 and Cryptosporidium parvum gp60 subtype IIaA19G1R2 from small amounts of stool left over from diagnostic testing of clinical cases of cryptosporidiosis. The C. parvum isolate was sequenced to a mean depth of 51.8X with reads covering 100 % of the bases of the C. parvum Iowa II reference genome (Bioproject PRJNA 15586), while the C. hominis isolate was sequenced to a mean depth of 34.7X with reads covering 98 % of the bases of the C. hominis TU502 v1 reference genome (Bioproject PRJNA 15585). The method was then applied to a further 17 stools, successfully generating another eight new whole genome sequences, of which two were C. hominis (gp60 subtypes IbA10G2 and IaA14R3) and six C. parvum (gp60 subtypes IIaA15G2R1 from three samples, and one each of IIaA17G1R1, IIaA18G2R1, and IIdA22G1), demonstrating the utility of this method to sequence Cryptosporidium genomes directly from clinical samples. This development is especially important as it reduces the requirement to propagate Cryptosporidium oocysts in animal models prior to genome sequencing. CONCLUSION: This represents the first report of high-quality whole genome sequencing of Cryptosporidium isolates prepared directly from human stool samples.

Draft Genomes, Phylogenetic Reconstruction, and Comparative Genomics of Two Novel Cohabiting Bacterial Symbionts Isolated from Frankliniella occidentalis.

Obligate bacterial symbionts are widespread in many invertebrates, where they are often confined to specialized host cells and are transmitted directly from mother to progeny. Increasing numbers of these bacteria are being characterized but questions remain about their population structure and evolution. Here we take a comparative genomics approach to investigate two prominent bacterial symbionts (BFo1 and BFo2) isolated from geographically separated populations of western flower thrips, Frankliniella occidentalis. Our multifaceted approach to classifying these symbionts includes concatenated multilocus sequence analysis (MLSA) phylogenies, ribosomal multilocus sequence typing (rMLST), construction of whole-genome phylogenies, and in-depth genomic comparisons. We showed that the BFo1 genome clusters more closely to species in the genus Erwinia, and is a putative close relative to Erwinia aphidicola. BFo1 is also likely to have shared a common ancestor with Erwinia pyrifoliae/Erwinia amylovora and the nonpathogenic Erwinia tasmaniensis and genetic traits similar to Erwinia billingiae. The BFo1 genome contained virulence factors found in the genus Erwinia but represented a divergent lineage. In contrast, we showed that BFo2 belongs within the Enterobacteriales but does not group closely with any currently known bacterial species. Concatenated MLSA phylogenies indicate that it may have shared a common ancestor to the Erwinia and Pantoea genera, and based on the clustering of rMLST genes, it was most closely related to Pantoea ananatis but represented a divergent lineage. We reconstructed a core genome of a putative common ancestor of Erwinia and Pantoea and compared this with the genomes of BFo bacteria. BFo2 possessed none of the virulence determinants that were omnipresent in the Erwinia and Pantoea genera. Taken together, these data are consistent with BFo2 representing a highly novel species that maybe related to known Pantoea.

RapGene: a fast and accurate strategy for synthetic gene assembly in Escherichia coli.

The ability to assemble DNA sequences de novo through efficient and powerful DNA fabrication methods is one of the foundational technologies of synthetic biology. Gene synthesis, in particular, has been considered the main driver for the emergence of this new scientific discipline. Here we describe RapGene, a rapid gene assembly technique which was successfully tested for the synthesis and cloning of both prokaryotic and eukaryotic genes through a ligation independent approach. The method developed in this study is a complete bacterial gene synthesis platform for the quick, accurate and cost effective fabrication and cloning of gene-length sequences that employ the widely used host Escherichia coli.

Neurofibromin controls macropinocytosis and phagocytosis in Dictyostelium.

Cells use phagocytosis and macropinocytosis to internalise bulk material, which in phagotrophic organisms supplies the nutrients necessary for growth. Wildtype Dictyostelium amoebae feed on bacteria, but for decades laboratory work has relied on axenic mutants that can also grow on liquid media. We used forward genetics to identify the causative gene underlying this phenotype. This gene encodes the RasGAP Neurofibromin (NF1). Loss of NF1 enables axenic growth by increasing fluid uptake. Mutants form outsized macropinosomes which are promoted by greater Ras and PI3K activity at sites of endocytosis. Relatedly, NF1 mutants can ingest larger-than-normal particles using phagocytosis. An NF1 reporter is recruited to nascent macropinosomes, suggesting that NF1 limits their size by locally inhibiting Ras signalling. Our results link NF1 with macropinocytosis and phagocytosis for the first time, and we propose that NF1 evolved in early phagotrophs to spatially modulate Ras activity, thereby constraining and shaping their feeding structures.

Coupled cryoconite ecosystem structure-function relationships are revealed by comparing bacterial communities in alpine and Arctic glaciers.

Cryoconite holes are known as foci of microbial diversity and activity on polar glacier surfaces, but are virtually unexplored microbial habitats in alpine regions. In addition, whether cryoconite community structure reflects ecosystem functionality is poorly understood. Terminal restriction fragment length polymorphism and Fourier transform infrared metabolite fingerprinting of cryoconite from glaciers in Austria, Greenland and Svalbard demonstrated cryoconite bacterial communities are closely correlated with cognate metabolite fingerprints. The influence of bacterial-associated fatty acids and polysaccharides was inferred, underlining the importance of bacterial community structure in the properties of cryoconite. Thus, combined application of T-RFLP and FT-IR metabolite fingerprinting promises high throughput, and hence, rapid assessment of community structure-function relationships. Pyrosequencing revealed Proteobacteria were particularly abundant, with Cyanobacteria likely acting as ecosystem engineers in both alpine and Arctic cryoconite communities. However, despite these generalities, significant differences in bacterial community structures, compositions and metabolomes are found between alpine and Arctic cryoconite habitats, reflecting the impact of local and regional conditions on the challenges of thriving in glacial ecosystems.

Draft Genome Sequence of Rhodococcus rhodnii Strain LMG5362, a Symbiont of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), the Principle Vector of Trypanosoma cruzi.

We report the 4,385,577-bp high-quality draft assembly of the bacterial symbiont Rhodococcus rhodnii strain LMG5362, isolated from the gut of Rhodnius prolixus (Hemiptera, Reduviidae, Triatominae), the principle vector of the protozoan Trypanosoma cruzi, the etiological agent of Chagas disease. This sequence might provide useful information for subsequent studies of the symbiotic relationship between Rd. prolixus and Rc. rhodnii, while also providing a starting point for the development of biotechnological applications for the control of Rd. prolixus.

The dynamic bacterial communities of a melting High Arctic glacier snowpack.

Snow environments can occupy over a third of land surface area, but little is known about the dynamics of snowpack bacteria. The effect of snow melt on bacterial community structure and diversity of surface environments of a Svalbard glacier was examined using analyses of 16S rRNA genes via T-RFLP, qPCR and 454 pyrosequencing. Distinct community structures were found in different habitat types, with changes over 1 week apparent, in particular for the dominant bacterial class present, Betaproteobacteria. The differences observed were consistent with influences from depositional mode (snowfall vs aeolian dusts), contrasting snow with dust-rich snow layers and near-surface ice. Contrary to that, slush as the decompositional product of snow harboured distinct lineages of bacteria, further implying post-depositional changes in community structure. Taxa affiliated to the betaproteobacterial genus Polaromonas were particularly dynamic, and evidence for the presence of betaproteobacterial ammonia-oxidizing bacteria was uncovered, inviting the prospect that the dynamic bacterial communities associated with snowpacks may be active in supraglacial nitrogen cycling and capable of rapid responses to changes induced by snowmelt. Furthermore the potential of supraglacial snowpack ecosystems to respond to transient yet spatially extensive melting episodes such as that observed across most of Greenland's ice sheet in 2012 merits further investigation.