Mock microbial community meta-analysis using different trimming of amplicon read lengths.

Trimming of sequencing reads is a pre-processing step that aims to discard sequence segments such as primers, adapters and low quality nucleotides that will interfere with clustering and classification steps. We evaluated the impact of trimming length of paired-end 16S and 18S rRNA amplicon reads on the ability to reconstruct the taxonomic composition and relative abundances of communities with a known composition in both even and uneven proportions. We found that maximizing read retention maximizes recall but reduces precision by increasing false positives. The presence of expected taxa was accurately predicted across broad trim length ranges but recovering original relative proportions remains a difficult challenge. We show that parameters that maximize taxonomic recovery do not simultaneously maximize relative abundance accuracy. Trim length represents one of several experimental parameters that have non-uniform impact across microbial clades, making it a difficult parameter to optimize. This study offers insights, guidelines, and helps researchers assess the significance of their decisions when trimming raw reads in a microbiome analysis based on overlapping or non-overlapping paired-end amplicons.

Production and utilization of pseudocobalamin in marine Synechococcus cultures and communities.

Cobalamin influences marine microbial communities because an exogenous source is required by most eukaryotic phytoplankton, and demand can exceed supply. Pseudocobalamin is a cobalamin analogue produced and used by most cyanobacteria but is not directly available to eukaryotic phytoplankton. Some microbes can remodel pseudocobalamin into cobalamin, but a scarcity of pseudocobalamin measurements impedes our ability to evaluate its importance for marine cobalamin production. Here, we perform simultaneous measurements of pseudocobalamin and methionine synthase (MetH), the key protein that uses it as a co-factor, in Synechococcus cultures and communities. In Synechococcus sp. WH8102, pseudocobalamin quota decreases in low temperature (17°C) and low nitrogen to phosphorus ratio, while MetH did not. Pseudocobalamin and MetH quotas were influenced by culture methods and growth phase. Despite the variability present in cultures, we found a comparably consistent quota of 300 ± 100 pseudocobalamin molecules per cyanobacterial cell in the Northwest Atlantic Ocean, suggesting that cyanobacterial cell counts may be sufficient to estimate pseudocobalamin inventories in this region. This work offers insights into cellular pseudocobalamin metabolism, environmental and physiological conditions that may influence it, and provides environmental measurements to further our understanding of when and how pseudocobalamin can influence marine microbial communities.

Physical mixing in coastal waters controls and decouples nitrification via biomass dilution.

Nitrification is a central process of the aquatic nitrogen cycle that controls the supply of nitrate used in other key processes, such as phytoplankton growth and denitrification. Through time series observation and modeling of a seasonally stratified, eutrophic coastal basin, we demonstrate that physical dilution of nitrifying microorganisms by water column mixing can delay and decouple nitrification. The findings are based on a 4-y, weekly time series in the subsurface water of Bedford Basin, Nova Scotia, Canada, that included measurement of functional (amoA) and phylogenetic (16S rRNA) marker genes. In years with colder winters, more intense winter mixing resulted in strong dilution of resident nitrifiers in subsurface water, delaying nitrification for weeks to months despite availability of ammonium and oxygen. Delayed regrowth of nitrifiers also led to transient accumulation of nitrite (3 to 8 µmol · kgsw-1) due to decoupling of ammonia and nitrite oxidation. Nitrite accumulation was enhanced by ammonia-oxidizing bacteria (Nitrosomonadaceae) with fast enzyme kinetics, which temporarily outcompeted the ammonia-oxidizing archaea (Nitrosopumilus) that dominated under more stable conditions. The study reveals how physical mixing can drive seasonal and interannual variations in nitrification through control of microbial biomass and diversity. Variable, mixing-induced effects on functionally specialized microbial communities are likely relevant to biogeochemical transformation rates in other seasonally stratified water columns. The detailed study reveals a complex mechanism through which weather and climate variability impacts nitrogen speciation, with implications for coastal ecosystem productivity. It also emphasizes the value of high-frequency, multiparameter time series for identifying complex controls of biogeochemical processes in aquatic systems.

Cobalamin producers and prokaryotic consumers in the Northwest Atlantic.

Cobalamin availability can influence primary productivity and ecological interactions in marine microbial communities. The characterization of cobalamin sources and sinks is a first step in investigating cobalamin dynamics and its impact on productivity. Here, we identify potential cobalamin sources and sinks on the Scotian Shelf and Slope in the Northwest Atlantic Ocean. Functional and taxonomic annotation of bulk metagenomic reads, combined with analysis of genome bins, were used to identify potential cobalamin sources and sinks. Cobalamin synthesis potential was mainly attributed to Rhodobacteraceae, Thaumarchaeota, and cyanobacteria (Synechococcus and Prochlorococcus). Cobalamin remodelling potential was mainly attributed to Alteromonadales, Pseudomonadales, Rhizobiales, Oceanospirilalles, Rhodobacteraceae, and Verrucomicrobia, while potential cobalamin consumers include Flavobacteriaceae, Actinobacteria, Porticoccaceae, Methylophiliaceae, and Thermoplasmatota. These complementary approaches identified taxa with the potential to be involved in cobalamin cycling on the Scotian Shelf and revealed genomic information required for further characterization. The Cob operon of Rhodobacterales bacterium HTCC2255, a strain with known importance in cobalamin cycling, was similar to a major cobalamin producer bin, suggesting that a related strain may represent a critical cobalamin source in this region. These results enable future inquiries that will enhance our understanding of how cobalamin shapes microbial interdependencies and productivity in this region.

Geomicrobiology of the carbon, nitrogen and sulphur cycles in Powell Lake: a permanently stratified water column containing ancient seawater.

We present the first geomicrobiological characterization of the meromictic water column of Powell Lake (British Columbia, Canada), a former fjord, which has been stably stratified since the last glacial period. Its deepest layers (300-350 m) retain isolated, relict seawater from that period. Fine-scale vertical profiling of the water chemistry and microbial communities allowed subdivision of the water column into distinct geomicrobiological zones. These zones were further characterized by phylogenetic and functional marker genes from amplicon and shotgun metagenome sequencing. Binning of metagenomic reads allowed the linkage of function to specific taxonomic groups. Statistical analyses (analysis of similarities, Bray-Curtis similarity) confirmed that the microbial community structure followed closely the geochemical zonation. Yet, our characterization of the genetic potential relevant to carbon, nitrogen and sulphur cycling of each zone revealed unexpected features, including potential for facultative anaerobic methylotrophy, nitrogen fixation despite high ammonium concentrations and potential micro-aerobic nitrifiers within the chemocline. At the oxic-suboxic interface, facultative anaerobic potential was found in the widespread freshwater lineage acI (Actinobacteria), suggesting intriguing ecophysiological similarities to the marine SAR11. Evolutionary divergent lineages among diverse phyla were identified in the ancient seawater zone and may indicate novel adaptations to this unusual environment.

Doubling of marine dinitrogen-fixation rates based on direct measurements.

Biological dinitrogen fixation provides the largest input of nitrogen to the oceans, therefore exerting important control on the ocean's nitrogen inventory and primary productivity. Nitrogen-isotope data from ocean sediments suggest that the marine-nitrogen inventory has been balanced for the past 3,000 years (ref. 4). Producing a balanced marine-nitrogen budget based on direct measurements has proved difficult, however, with nitrogen loss exceeding the gain from dinitrogen fixation by approximately 200 Tg N yr−1 (refs 5, 6). Here we present data from the Atlantic Ocean and show that the most widely used method of measuring oceanic N2-fixation rates underestimates the contribution of N2-fixing microorganisms (diazotrophs) relative to a newly developed method. Using molecular techniques to quantify the abundance of specific clades of diazotrophs in parallel with rates of 15N2 incorporation into particulate organic matter, we suggest that the difference between N2-fixation rates measured with the established method and those measured with the new method can be related to the composition of the diazotrophic community. Our data show that in areas dominated by Trichodesmium, the established method underestimates N2-fixation rates by an average of 62%. We also find that the newly developed method yields N2-fixation rates more than six times higher than those from the established method when unicellular, symbiotic cyanobacteria and γ-proteobacteria dominate the diazotrophic community. On the basis of average areal rates measured over the Atlantic Ocean, we calculated basin-wide N2-fixation rates of 14 ± 1 Tg N yr−1 and 24 ±1 Tg N yr−1 for the established and new methods, respectively. If our findings can be extrapolated to other ocean basins, this suggests that the global marine N2-fixation rate derived from direct measurements may increase from 103 ± 8 Tg N yr−1 to 177 ± 8 Tg N yr−1, and that the contribution of N2 fixers other than Trichodesmium is much more significant than was previously thought.

Fragment recruitment on metabolic pathways: comparative metabolic profiling of metagenomes and metatranscriptomes.

MOTIVATION: The sheer scale of the metagenomic and metatranscriptomic datasets that are now available warrants the development of automated protocols for organizing, annotating and comparing the samples in terms of their metabolic profiles. We describe a user-friendly java program FROMP (Fragment Recruitment on Metabolic Pathways) for mapping and visualizing enzyme annotations onto the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathways or custom-made pathways and comparing the samples in terms of their Pathway Completeness Scores, their relative Activity Scores or enzyme enrichment odds ratios. This program along with our fully configurable PERL-based annotation organization pipeline Meta2Pro (METAbolic PROfiling of META-omic data) offers a quick and accurate standalone solution for metabolic profiling of environmental samples or cultures from different treatments. Apart from pictorial comparisons, FROMP can also generate score matrices for multiple meta-omics samples, which can be used directly by other statistical programs.

Handling temperature bursts reaching 464°C: different microbial strategies in the sisters peak hydrothermal chimney.

The active venting Sisters Peak (SP) chimney on the Mid-Atlantic Ridge holds the current temperature record for the hottest ever measured hydrothermal fluids (400°C, accompanied by sudden temperature bursts reaching 464°C). Given the unprecedented temperature regime, we investigated the biome of this chimney with a focus on special microbial adaptations for thermal tolerance. The SP metagenome reveals considerable differences in the taxonomic composition from those of other hydrothermal vent and subsurface samples; these could be better explained by temperature than by other available abiotic parameters. The most common species to which SP genes were assigned were thermophilic Aciduliprofundum sp. strain MAR08-339 (11.8%), Hippea maritima (3.8%), Caldisericum exile (1.5%), and Caminibacter mediatlanticus (1.4%) as well as to the mesophilic Niastella koreensis (2.8%). A statistical analysis of associations between taxonomic and functional gene assignments revealed specific overrepresented functional categories: for Aciduliprofundum, protein biosynthesis, nucleotide metabolism, and energy metabolism genes; for Hippea and Caminibacter, cell motility and/or DNA replication and repair system genes; and for Niastella, cell wall and membrane biogenesis genes. Cultured representatives of these organisms inhabit different thermal niches; i.e., Aciduliprofundum has an optimal growth temperature of 70°C, Hippea and Caminibacter have optimal growth temperatures around 55°C, and Niastella grows between 10 and 37°C. Therefore, we posit that the different enrichment profiles of functional categories reflect distinct microbial strategies to deal with the different impacts of the local sudden temperature bursts in disparate regions of the chimney.

Marine microbiology: How to evolve a nitrogen-fixing organelle.

The evolution of intracellular organelles by endosymbiosis is considered rare. Two recent studies suggest that endosymbioses between nitrogen-fixing bacteria and eukaryotic algae are approaching levels of integration comparable to cellular organelles, helping to solve the problem of oceanic nitrogen limitation.

Factors associated with childhood non-vaccination against COVID-19 in Canada: A national survey analysis.

Background: COVID-19 vaccination efforts are critical in mitigating the impact of the virus, but despite proven safety and efficacy, vaccination rates among children in Canada are lower than in adults, prompting a need to explore determinants of childhood COVID-19 non-vaccination to improve uptake. Method: This study analyzed data from the Canadian COVID-19 Immunization Coverage Survey 2022. Using multivariable logistic regression, it examined the association between COVID-19 non-vaccination among children aged 5-17 and factors such as parental sociodemographic characteristics, vaccine knowledge, attitudes, and beliefs (KAB), and vaccination history. Results: The analysis revealed that negative KAB towards vaccines, reflected in higher KAB composite scores, significantly increased the likelihood of non-vaccination. Additionally, factors such as lower household incomes, rural residence, employment in sectors not at risk for vaccine-preventable diseases, and younger parental age were associated with higher non-vaccination. The study also highlighted ethnic disparities in vaccination odds and found that children with incomplete routine vaccinations or inconsistent flu vaccination histories were more likely to be unvaccinated against COVID-19. Surprisingly, children of parents who consistently received flu vaccinations were more likely to be unvaccinated against COVID-19. Furthermore, parental education levels showed a complex relationship with children's COVID-19 vaccination status, indicating nuanced influences on vaccination decisions. Conclusion: The findings offer vital insights into the factors influencing COVID-19 vaccination uptake among children in Canada, suggesting avenues for targeted strategies to improve vaccine coverage.

Determinants of influenza non-vaccination among Canadian children: insights from a nationwide survey.

Background: To identify determinants influencing Canadian parents' decision not to vaccinate their children aged 6 months to 17 years against seasonal influenza. Methods: Data from the 2022 Childhood COVID-19 Immunization Coverage Survey, a national survey of approximately 10,500 Canadian parents/guardians and their children, was analyzed. The survey examined influenza vaccine coverage, parental perspectives on vaccines, reasons for hesitancy, and factors influencing immunization. Socio-demographic characteristics, including ethnicity, household income, working sector, educational attainment, and prevalence of chronic medical conditions among children were considered. Historical vaccine uptake and the impact of the COVID-19 pandemic on immunization decisions were also reviewed. Key determinants of non-vaccination in the 2021-2022 influenza season were analyzed using multivariable logistic regression, with a statistical significance level set at p-value <0·05. Results: 70% of children aged 6 months to 17 years did not receive the seasonal influenza vaccine. Key predictors for non-vaccination included: residing in rural settings (aOR 1·35, 95% CI 1·13-1·60), parental education attainment of less than high school (aOR 2·48, 95% CI 1·24-4·97), and the absence of chronic medical conditions in children (aOR 1.60, 95% CI 1.34-1.91)· Other strong predictors included lower household income; deterrence due to the COVID-19 pandemic; and parental hesitancy stemming from concerns about the vaccine's safety, effectiveness, and by beliefs that their child was not at risk of contracting the influenza or severe consequences from the infection. Conclusion: This research underscores pivotal determinants of parental decisions not to vaccinate their children against seasonal influenza and sheds light on the impact of the COVID-19 pandemic. The results highlight the importance of addressing safety concerns and providing clear information to alleviate hesitancy.

Targeted metagenomics reveals pangenomic diversity of the nitroplast (UCYN-A) and its algal host plastid.

UCYN-A (Cand. Atelocyanobacterium thalassa) has recently been recognized as a globally-distributed, early stage, nitrogen-fixing organelle (the 'nitroplast') of cyanobacterial origin present in select species of haptophyte algae (e.g., Braarudosphaera bigelowii). Although the nitroplast was recognized as the UCYN-A2 sublineage, it is yet to be confirmed in other sublineages of the algal/UCYN-A complex. We used water samples collected from Halifax Harbour (Bedford Basin, Nova Scotia, Canada) and the offshore Scotian Shelf to further our understanding of B. bigelowii and UCYN-A in the coastal Northwest Atlantic. Sequencing data revealed UCYN-A-associated haptophyte signatures and yielded near-complete metagenome-assembled genomes (MAGs) for UCYN-A1, UCYN-A4, and the plastid of the A4-associated haptophyte. Comparative genomics provided new insights into the pangenome of UCYN-A. The UCYN-A4 MAG is the first genome sequenced from this sublineage and shares ~85% identity with the UCYN-A2 nitroplast. Genes missing in the reduced genome of the nitroplast were also missing in the A4 MAG supporting its likely classification as a nitroplast as well. The UCYN-A1 MAG was found to be nearly 100% identical to the reference genome despite coming from different ocean basins. Time-series data paired with the recurrence of specific microbes in enrichment cultures gave insight into the microbes that frequently co-occur with the algal/UCYN-A complex (e.g., Pelagibacter ubique). Overall, our study expands knowledge of UCYN-A and its host across major ocean basins and investigates their co-occurring microbes in the coastal Northwest Atlantic (NWA), thereby facilitating future studies on the underpinnings of haptophyte-associated diazotrophy in the sea.

Nitrogen fixation in the widely distributed marine γ-proteobacterial diazotroph Candidatus Thalassolituus haligoni.

The high diversity and global distribution of heterotrophic bacterial diazotrophs (HBDs) in the ocean has recently become apparent. However, understanding the role these largely uncultured microorganisms play in marine N2 fixation poses a challenge due to their undefined growth requirements and the complex regulation of the nitrogenase enzyme. We isolated and characterized Candidatus Thalassolituus haligoni, a member of a widely distributed clade of HBD belonging to the Oceanospirillales. Analysis of its nifH gene via amplicon sequencing revealed the extensive distribution of Cand. T. haligoni across the Pacific, Atlantic, and Arctic Oceans. Pangenome analysis indicates that the isolate shares >99% identity with an uncultured metagenome-assembled genome called Arc-Gamma-03, recently recovered from the Arctic Ocean. Through combined genomic, proteomic, and physiological approaches, we confirmed that the isolate fixes N2 gas. However, the mechanisms governing nitrogenase regulation in Cand. T. haligoni remain unclear. We propose Cand. T. haligoni as a globally distributed, cultured HBD model species within this understudied clade of Oceanospirillales.

Microevolutionary patterns in ecotypes of the symbiotic cyanobacterium UCYN-A revealed from a Northwest Atlantic coastal time series.

UCYN-A is a globally important nitrogen-fixing symbiotic microbe often found in colder regions and coastal areas where nitrogen fixation has been overlooked. We present a 3-year coastal Northwest Atlantic time series of UCYN-A by integrating oceanographic data with weekly nifH and16S rRNA gene sequencing and quantitative PCR assays for UCYN-A ecotypes. High UCYN-A relative abundances dominated by A1 to A4 ecotypes reoccurred annually in the coastal Northwest Atlantic. Although UCYN-A was detected every summer/fall, the ability to observe separate ecotypes may be highly dependent on sampling time given intense interannual and weekly variability of ecotype-specific occurrences. Additionally, much of UCYN-A's rarer diversity was populated by short-lived neutral mutational variants, therefore providing insight into UCYN-A's microevolutionary patterns. For instance, rare ASVs exhibited community composition restructuring annually, while also sharing a common connection to a dominant ASV within each ecotype. Our study provides additional perspectives for interpreting UCYN-A intraspecific diversity and underscores the need for high-resolution datasets when deciphering spatiotemporal ecologies within UCYN-A.

Short-term response to iron resupply in an iron-limited open ocean diatom reveals rapid decay of iron-responsive transcripts.

In large areas of the ocean, iron concentrations are insufficient to promote phytoplankton growth. Numerous studies have been conducted to characterize the effect of iron on algae and how algae cope with fluctuating iron concentrations. Fertilization experiments in low-iron areas resulted primarily in diatom-dominated algal blooms, leading to laboratory studies on diatoms comparing low- and high-iron conditions. Here, we focus on the short-term temporal response following iron addition to an iron-starved open ocean diatom, Thalassiosira oceanica. We employed the NanoString platform and analyzed a high-resolution time series on 54 transcripts encoding proteins involved in photosynthesis, N-linked glycosylation, iron transport, as well as transcription factors. Nine transcripts were iron-responsive, with an immediate response to the addition of iron. The fastest response observed was the decrease in transcript levels of proteins involved in iron uptake, followed by an increase in transcript levels of iron-containing enzymes and a simultaneous decrease in the transcript levels of their iron-free replacement enzymes. The transcription inhibitor actinomycin D was used to understand the underlying mechanisms of the decrease of the iron-responsive transcripts and to determine their half-lives. Here, Mn-superoxide dismutase (Mn-SOD), plastocyanin (PETE), ferredoxin (PETF) and cellular repressor of EA1-stimulated genes (CREGx2) revealed longer than average half-lives. Four iron-responsive transcripts showed statistically significant differences in their decay rates between the iron-recovery samples and the actD treatment. These differences suggest regulatory mechanisms influencing gene transcription and mRNA stability. Overall, our study contributes towards a detailed understanding of diatom cell biology in the context of iron fertilization response and provides important observations to assess oceanic diatom responses following sudden changes in iron concentrations.

Marine nitrogen-fixers in the Canadian Arctic Gateway are dominated by biogeographically distinct noncyanobacterial communities.

We describe diazotrophs present during a 2015 GEOTRACES expedition through the Canadian Arctic Gateway (CAG) using nifH metabarcoding. In the less studied Labrador Sea, Bradyrhizobium sp. and Vitreoscilla sp. nifH variants were dominant, while in Baffin Bay, a Stutzerimonas stutzeri variant was dominant. In comparison, the Canadian Arctic Archipelago (CAA) was characterized by a broader set of dominant variants belonging to Desulfobulbaceae, Desulfuromonadales, Arcobacter sp., Vibrio spp., and Sulfuriferula sp. Although dominant diazotrophs fell within known nifH clusters I and III, only a few of these variants were frequently recovered in a 5-year weekly nifH times series in the coastal NW Atlantic presented herein, notably S. stutzeri and variants belonging to Desulfobacterales and Desulfuromonadales. In addition, the majority of dominant Arctic nifH variants shared low similarity (< 92% nucleotide identities) to sequences in a global noncyanobacterial diazotroph catalog recently compiled by others. We further detected UCYN-A throughout the CAG at low-levels using quantitative-PCR assays. Temperature, depth, salinity, oxygen, and nitrate were most strongly correlated to the Arctic diazotroph diversity observed, and we found a stark division between diazotroph communities of the Labrador Sea versus Baffin Bay and the CAA, hence establishing that a previously unknown biogeographic community division can occur for diazotrophs in the CAG.

Compact and automated eDNA sampler for in situ monitoring of marine environments.

Using environmental DNA (eDNA) to monitor biodiversity in aquatic environments is becoming an efficient and cost-effective alternative to other methods such as visual and acoustic identification. Until recently, eDNA sampling was accomplished primarily through manual sampling methods; however, with technological advances, automated samplers are being developed to make sampling easier and more accessible. This paper describes a new eDNA sampler capable of self-cleaning and multi-sample capture and preservation, all within a single unit capable of being deployed by a single person. The first in-field test of this sampler took place in the Bedford Basin, Nova Scotia, Canada alongside parallel samples taken using the typical Niskin bottle collection and post-collection filtration method. Both methods were able to capture the same aquatic microbial community and counts of representative DNA sequences were well correlated between methods with R[Formula: see text] values ranging from 0.71-0.93. The two collection methods returned the same top 10 families in near identical relative abundance, demonstrating that the sampler was able to capture the same community composition of common microbes as the Niskin. The presented eDNA sampler provides a robust alternative to manual sampling methods, is amenable to autonomous vehicle payload constraints, and will facilitate persistent monitoring of remote and inaccessible sites.