The influence of alfalfa-switchgrass intercropping on microbial community structure and function.

The use of nitrogen fertilizer on bioenergy crops such as switchgrass results in increased costs, nitrogen leaching and emissions of N2 O, a potent greenhouse gas. Intercropping with nitrogen-fixing alfalfa has been proposed as an environmentally sustainable alternative, but the effects of synthetic fertilizer versus intercropping on soil microbial community functionality remain uncharacterized. We analysed 24 metagenomes from the upper soil layer of agricultural fields from Prosser, WA over two growing seasons and representing three agricultural practices: unfertilized switchgrass (control), fertilized switchgrass and switchgrass intercropped with alfalfa. The synthetic fertilization and intercropping did not result in major shifts of microbial community taxonomic and functional composition compared with the control plots, but a few significant changes were noted. Most notably, mycorrhizal fungi, ammonia-oxidizing archaea and bacteria increased in abundance with intercropping and fertilization. However, only betaproteobacterial ammonia-oxidizing bacteria abundance in fertilized plots significantly correlated to N2 O emission and companion qPCR data. Collectively, a short period of intercropping elicits minor but significant changes in the soil microbial community toward nitrogen preservation and that intercropping may be a viable alternative to synthetic fertilization.

Metagenomic Characterization of Soil Microbial Communities in the Luquillo Experimental Forest (Puerto Rico) and Implications for Nitrogen Cycling.

The phylogenetic and functional diversities of microbial communities in tropical rainforests and how these differ from those of temperate communities remain poorly described but are directly related to the increased fluxes of greenhouse gases such as nitrous oxide (N2O) from the tropics. Toward closing these knowledge gaps, we analyzed replicated shotgun metagenomes representing distinct life zones and an elevation gradient from four locations in the Luquillo Experimental Forest (LEF), Puerto Rico. These soils had a distinct microbial community composition and lower species diversity compared to those of temperate grasslands or agricultural soils. In contrast to the overall distinct community composition, the relative abundances and nucleotide sequences of N2O reductases (nosZ) were highly similar between tropical forest and temperate soils. However, respiratory NO reductase (norB) was 2-fold more abundant in the tropical soils, which might be relatable to their greater N2O emissions. Nitrogen fixation (nifH) also showed higher relative abundance in rainforest than in temperate soils, i.e., 20% versus 0.1 to 0.3% of bacterial genomes in each soil type harbored the gene, respectively. Finally, unlike temperate soils, LEF soils showed little stratification with depth in the first 0 to 30 cm, with ∼45% of community composition differences explained solely by location. Collectively, these results advance our understanding of spatial diversity and metabolic repertoire of tropical rainforest soil communities and should facilitate future ecological studies of these ecosystems. IMPORTANCE Tropical rainforests are the largest terrestrial sinks of atmospheric CO2 and the largest natural source of N2O emissions, two greenhouse gases that are critical for the climate. The microbial communities of rainforest soils that directly or indirectly, through affecting plant growth, contribute to these fluxes remain poorly described by cultured-independent methods. To close this knowledge gap, the present study applied shotgun metagenomics to samples selected from three distinct life zones within the Puerto Rico rainforest. The results advance our understanding of microbial community diversity in rainforest soils and should facilitate future studies of natural or manipulated perturbations of these critical ecosystems.

Metagenomics as a Public Health Risk Assessment Tool in a Study of Natural Creek Sediments Influenced by Agricultural and Livestock Runoff: Potential and Limitations.

Little is known about the public health risks associated with natural creek sediments that are affected by runoff and fecal pollution from agricultural and livestock practices. For instance, the persistence of foodborne pathogens such as Shiga toxin-producing Escherichia coli (STEC) originating from these practices remains poorly quantified. Towards closing these knowledge gaps, the water-sediment interface of two creeks in the Salinas River Valley of California was sampled over a 9-month period using metagenomics and traditional culture-based tests for STEC. Our results revealed that these sediment communities are extremely diverse and have functional and taxonomic diversity comparable to that observed in soils. With our sequencing effort (∼4 Gbp per library), we were unable to detect any pathogenic E. coli in the metagenomes of 11 samples that had tested positive using culture-based methods, apparently due to relatively low abundance. Furthermore, there were no significant differences in the abundance of human- or cow-specific gut microbiome sequences in the downstream impacted sites compared to that in upstream more pristine (control) sites, indicating natural dilution of anthropogenic inputs. Notably, the high number of metagenomic reads carrying antibiotic resistance genes (ARGs) found in all samples was significantly higher than ARG reads in other available freshwater and soil metagenomes, suggesting that these communities may be natural reservoirs of ARGs. The work presented here should serve as a guide for sampling volumes, amount of sequencing to apply, and what bioinformatics analyses to perform when using metagenomics for public health risk studies of environmental samples such as sediments.IMPORTANCE Current agricultural and livestock practices contribute to fecal contamination in the environment and the spread of food- and waterborne disease and antibiotic resistance genes (ARGs). Traditionally, the level of pollution and risk to public health are assessed by culture-based tests for the intestinal bacterium Escherichia coli However, the accuracy of these traditional methods (e.g., low accuracy in quantification, and false-positive signal when PCR based) and their suitability for sediments remain unclear. We collected sediments for a time series metagenomics study from one of the most highly productive agricultural regions in the United States in order to assess how agricultural runoff affects the native microbial communities and if the presence of Shiga toxin-producing Escherichia coli (STEC) in sediment samples can be detected directly by sequencing. Our study provided important information on the potential for using metagenomics as a tool for assessment of public health risk in natural environments.

Predominance of deterministic microbial community dynamics in salterns exposed to different light intensities.

While the dynamics of microbial community assembly driven by environmental perturbations have been extensively studied, our understanding is far from complete, particularly for light-induced perturbations. Extremely halophilic communities thriving in coastal solar salterns are mainly influenced by two environmental factors-salt concentrations and high sunlight irradiation. By experimentally manipulating light intensity through the application of shading, we showed that light acts as a deterministic factor that ultimately drives the establishment of recurrent microbial communities under near-saturation salt concentrations. In particular, the stable and highly change-resistant communities that established under high-light intensities were dominated (>90% of metagenomic reads) by Haloquadratum spp. and Salinibacter spp. On the other hand, under 37-fold lower light intensity, different, less stable and change-resistant communities were established, mainly dominated by yet unclassified haloarchaea and relatively diverse photosynthetic microorganisms. These communities harboured, in general, much lower carotenoid pigment content than their high-irradiation counterparts. Both assemblage types appeared to be highly resilient, re-establishing when favourable conditions returned after perturbation (i.e. high-irradiation for the former communities and low-irradiation for the latter ones). Overall, our results revealed that stochastic processes were of limited significance to explain these patterns.

ROCker: accurate detection and quantification of target genes in short-read metagenomic data sets by modeling sliding-window bitscores.

Functional annotation of metagenomic and metatranscriptomic data sets relies on similarity searches based on e-value thresholds resulting in an unknown number of false positive and negative matches. To overcome these limitations, we introduce ROCker, aimed at identifying position-specific, most-discriminant thresholds in sliding windows along the sequence of a target protein, accounting for non-discriminative domains shared by unrelated proteins. ROCker employs the receiver operating characteristic (ROC) curve to minimize false discovery rate (FDR) and calculate the best thresholds based on how simulated shotgun metagenomic reads of known composition map onto well-curated reference protein sequences and thus, differs from HMM profiles and related methods. We showcase ROCker using ammonia monooxygenase (amoA) and nitrous oxide reductase (nosZ) genes, mediating oxidation of ammonia and the reduction of the potent greenhouse gas, N2O, to inert N2, respectively. ROCker typically showed 60-fold lower FDR when compared to the common practice of using fixed e-values. Previously uncounted 'atypical' nosZ genes were found to be two times more abundant, on average, than their typical counterparts in most soil metagenomes and the abundance of bacterial amoA was quantified against the highly-related particulate methane monooxygenase (pmoA). Therefore, ROCker can reliably detect and quantify target genes in short-read metagenomes.

Year-Round Shotgun Metagenomes Reveal Stable Microbial Communities in Agricultural Soils and Novel Ammonia Oxidizers Responding to Fertilization.

The dynamics of individual microbial populations and their gene functions in agricultural soils, especially after major activities such as nitrogen (N) fertilization, remain elusive but are important for a better understanding of nutrient cycling. Here, we analyzed 20 short-read metagenomes collected at four time points during 1 year from two depths (0 to 5 and 20 to 30 cm) in two Midwestern agricultural sites representing contrasting soil textures (sandy versus silty loam) with similar cropping histories. Although the microbial community taxonomic and functional compositions differed between the two locations and depths, they were more stable within a depth/site throughout the year than communities in natural aquatic ecosystems. For example, among the 69 population genomes assembled from the metagenomes, 75% showed a less than 2-fold change in abundance between any two sampling points. Interestingly, six deep-branching Thaumarchaeota and three complete ammonia oxidizer (comammox) Nitrospira populations increased up to 5-fold in abundance upon the addition of N fertilizer. These results indicated that indigenous archaeal ammonia oxidizers may respond faster (are more copiotrophic) to N fertilization than previously thought. None of 29 recovered putative denitrifier genomes encoded the complete denitrification pathway, suggesting that denitrification is carried out by a collection of different populations. Altogether, our study identified novel microbial populations and genes responding to seasonal and human-induced perturbations in agricultural soils that should facilitate future monitoring efforts and N-related studies.IMPORTANCE Even though the impact of agricultural management on the microbial community structure has been recognized, an understanding of the dynamics of individual microbial populations and what functions each population carries are limited. Yet, this information is important for a better understanding of nutrient cycling, with potentially important implications for preserving nitrogen in soils and sustainability. Here, we show that reconstructed metagenome-assembled genomes (MAGs) are relatively stable in their abundance and functional gene content year round, and seasonal nitrogen fertilization has selected for novel Thaumarchaeota and comammox Nitrospira nitrifiers that are potentially less oligotrophic than their marine counterparts previously studied.

The low diverse gastric microbiome of the jellyfish Cotylorhiza tuberculata is dominated by four novel taxa.

Cotylorhiza tuberculata is an important scyphozoan jellyfish producing population blooms in the Mediterranean probably due to pelagic ecosystem's decay. Its gastric cavity can serve as a simple model of microbial-animal digestive associations, yet poorly characterized. Using state-of-the-art metagenomic population binning and catalyzed reporter deposition fluorescence in situ hybridization (CARD-FISH), we show that only four novel clonal phylotypes were consistently associated with multiple jellyfish adults. Two affiliated close to Spiroplasma and Mycoplasma genera, one to chlamydial 'Candidatus Syngnamydia', and one to bacteroidetal Tenacibaculum, and were at least one order of magnitude more abundant than any other bacteria detected. Metabolic modelling predicted an aerobic heterotrophic lifestyle for the chlamydia, which were found intracellularly in Onychodromopsis-like ciliates. The Spiroplasma-like organism was predicted to be an anaerobic fermenter associated to some jellyfish cells, whereas the Tenacibaculum-like as free-living aerobic heterotroph, densely colonizing the mesogleal axis inside the gastric filaments. The association between the jellyfish and its reduced microbiome was close and temporally stable, and possibly related to food digestion and protection from pathogens. Based on the genomic and microscopic data, we propose three candidate taxa: 'Candidatus Syngnamydia medusae', 'Candidatus Medusoplasma mediterranei' and 'Candidatus Tenacibaculum medusae'.

Detection and Diversity of Fungal Nitric Oxide Reductase Genes (p450nor) in Agricultural Soils.

UNLABELLED: Members of the Fungi convert nitrate (NO3 (-)) and nitrite (NO2 (-)) to gaseous nitrous oxide (N2O) (denitrification), but the fungal contributions to N loss from soil remain uncertain. Cultivation-based methodologies that include antibiotics to selectively assess fungal activities have limitations, and complementary molecular approaches to assign denitrification potential to fungi are desirable. Microcosms established with soils from two representative U.S. Midwest agricultural regions produced N2O from added NO3 (-) or NO2 (-) in the presence of antibiotics to inhibit bacteria. Cultivation efforts yielded 214 fungal isolates belonging to at least 15 distinct morphological groups, 151 of which produced N2O from NO2 (-) Novel PCR primers targeting the p450nor gene, which encodes the nitric oxide (NO) reductase responsible for N2O production in fungi, yielded 26 novel p450nor amplicons from DNA of 37 isolates and 23 amplicons from environmental DNA obtained from two agricultural soils. The sequences shared 54 to 98% amino acid identity with reference P450nor sequences within the phylum Ascomycota and expand the known fungal P450nor sequence diversity. p450nor was detected in all fungal isolates that produced N2O from NO2 (-), whereas nirK (encoding the NO-forming NO2 (-) reductase) was amplified in only 13 to 74% of the N2O-forming isolates using two separate nirK primer sets. Collectively, our findings demonstrate the value of p450nor-targeted PCR to complement existing approaches to assess the fungal contributions to denitrification and N2O formation. IMPORTANCE: A comprehensive understanding of the microbiota controlling soil N loss and greenhouse gas (N2O) emissions is crucial for sustainable agricultural practices and addressing climate change concerns. We report the design and application of a novel PCR primer set targeting fungal p450nor, a biomarker for fungal N2O production, and demonstrate the utility of the new approach to assess fungal denitrification potential in fungal isolates and agricultural soils. These new PCR primers may find application in a variety of biomes to assess the fungal contributions to N loss and N2O emissions.

Globally occurring pelagiphage infections create ribosome-deprived cells.

Phages play an essential role in controlling bacterial populations. Those infecting Pelagibacterales (SAR11), the dominant bacteria in surface oceans, have been studied in silico and by cultivation attempts. However, little is known about the quantity of phage-infected cells in the environment. Using fluorescence in situ hybridization techniques, we here show pelagiphage-infected SAR11 cells across multiple global ecosystems and present evidence for tight community control of pelagiphages on the SAR11 hosts in a case study. Up to 19% of SAR11 cells were phage-infected during a phytoplankton bloom, coinciding with a ~90% reduction in SAR11 cell abundance within 5 days. Frequently, a fraction of the infected SAR11 cells were devoid of detectable ribosomes, which appear to be a yet undescribed possible stage during pelagiphage infection. We dubbed such cells zombies and propose, among other possible explanations, a mechanism in which ribosomal RNA is used as a resource for the synthesis of new phage genomes. On a global scale, we detected phage-infected SAR11 and zombie cells in the Atlantic, Pacific, and Southern Oceans. Our findings illuminate the important impact of pelagiphages on SAR11 populations and unveil the presence of ribosome-deprived zombie cells as part of the infection cycle.

Acidimicrobiia, the actinomycetota of coastal marine sediments: Abundance, taxonomy and genomic potential.

Microbial communities in marine sediments represent some of the densest and most diverse biological communities known, with up to a billion cells and thousands of species per milliliter. Among this taxonomic diversity, the class Acidimicrobiia, within the phylum Actinomycetota, stands out for its consistent presence, yet its limited taxonomic understanding obscures its ecological role. We used metagenome-assembled genomes from a 5-year Arctic fjord sampling campaign and compared them to publicly available Acidimicrobiia genomes using 16S rRNA gene and whole-genome phylogenies, alongside gene prediction and annotation to study their taxonomy and genomic potential. Overall, we provide a taxonomic overview of the class Acidimicrobiia and show its significant prevalence in Isfjorden and Helgoland coastal sediments, representing over 90% of Actinomycetota 16S rRNA gene sequences, and 3-7% of Bacteria. We propose Benthobacter isfjordensis gen. nov., sp. nov., Hadalibacter litoralis gen. nov., sp. nov., and two new species from Ilumatobacter, following SeqCode guidelines. In addition, we report the first in situ quantification of the family Ilumatobacteraceae, revealing its substantial presence (1-6%) in coastal sediments. This work highlights the need of refining the taxonomy of Acidimicrobiia to better understand their ecological contributions.

Possible Role of CHAD Proteins in Copper Resistance.

Conserved Histidine Alpha-helical Domain (CHAD) proteins attached to the surface of polyphosphate (PolyP) have been studied in some bacteria and one archaeon. However, the activity of CHAD proteins is unknown beyond their interaction with PolyP granules. By using bioinformatic analysis, we report that several species of the biomining acidophilic bacteria contain orthologs of CHAD proteins with high sequence identity. Furthermore, the gene coding for the CHAD protein is in the same genetic context of the enzyme polyphosphate kinase (PPK), which is in charge of PolyP synthesis. Particularly, the group of ppk and CHAD genes is highly conserved. Metallosphaera sedula and other acidophilic archaea used in biomining also contain CHAD proteins. These archaea show high levels of identity in genes coding for a cluster having the same organization. Amongst these genes are chad and ppx. In general, both biomining bacteria and archaea contain high PolyP levels and are highly resistant to heavy metals. Therefore, the presence of this conserved genetic organization suggests a high relevance for their metabolism. It has been formerly reported that a crystallized CHAD protein contains a copper-binding site. Based on this previous knowledge, in the present report, it was determined that all analyzed CHAD proteins are very conserved at their structural level. In addition, it was found that the lack of YgiF, an Escherichia coli CHAD-containing protein, decreases copper resistance in this bacterium. This phenotype was not only complemented by transforming E. coli with YgiF but also by expressing CHAD from Acidithiobacillus ferrooxidans in it. Interestingly, the strains in which the possible copper-binding sites were mutated were also more metal sensitive. Based on these results, we propose that CHAD proteins are involved in copper resistance in microorganisms. These findings are very interesting and may eventually improve biomining operations in the future.

Niche differentiation within bacterial key-taxa in stratified surface waters of the Southern Pacific Gyre.

One of the most hostile marine habitats on Earth is the surface of the South Pacific Gyre (SPG), characterized by high solar radiation, extreme nutrient depletion, and low productivity. During the SO-245 "UltraPac" cruise through the center of the ultra-oligotrophic SPG, the marine alphaproteobacterial group AEGEAN169 was detected by fluorescence in situ hybridization at relative abundances up to 6% of the total microbial community in the uppermost water layer, with two distinct populations (Candidatus Nemonibacter and Ca. Indicimonas). The high frequency of dividing cells combined with high transcript levels suggests that both clades may be highly metabolically active. Comparative metagenomic and metatranscriptomic analyses of AEGEAN169 revealed that they encoded subtle but distinct metabolic adaptions to this extreme environment in comparison to their competitors SAR11, SAR86, SAR116, and Prochlorococcus. Both AEGEAN169 clades had the highest percentage of transporters per predicted proteins (9.5% and 10.6%, respectively). In particular, the high expression of ABC transporters in combination with proteorhodopsins and the catabolic pathways detected suggest a potential scavenging lifestyle for both AEGEAN169 clades. Although both AEGEAN169 clades may share the genomic potential to utilize phosphonates as a phosphorus source, they differ in their metabolic pathways for carbon and nitrogen. Ca. Nemonibacter potentially use glycine-betaine, whereas Ca. Indicimonas may catabolize urea, creatine, and fucose. In conclusion, the different potential metabolic strategies of both clades suggest that both are well adapted to thrive resource-limited conditions and compete well with other dominant microbial clades in the uppermost layers of SPG surface waters.

Taxonomic and functional stability overrules seasonality in polar benthic microbiomes.

Coastal shelf sediments are hot spots of organic matter mineralization. They receive up to 50% of primary production, which, in higher latitudes, is strongly seasonal. Polar and temperate benthic bacterial communities, however, show a stable composition based on comparative 16S rRNA gene sequencing despite different microbial activity levels. Here, we aimed to resolve this contradiction by identifying seasonal changes at the functional level, in particular with respect to algal polysaccharide degradation genes, by combining metagenomics, metatranscriptomics, and glycan analysis in sandy surface sediments from Isfjorden, Svalbard. Gene expressions of diverse carbohydrate-active enzymes changed between winter and spring. For example, ß-1,3-glucosidases (e.g. GH30, GH17, GH16) degrading laminarin, an energy storage molecule of algae, were elevated in spring, while enzymes related to α-glucan degradation were expressed in both seasons with maxima in winter (e.g. GH63, GH13_18, and GH15). Also, the expression of GH23 involved in peptidoglycan degradation was prevalent, which is in line with recycling of bacterial biomass. Sugar extractions from bulk sediments were low in concentrations during winter but higher in spring samples, with glucose constituting the largest fraction of measured monosaccharides (84% ± 14%). In porewater, glycan concentrations were ~18-fold higher than in overlying seawater (1107 ± 484 vs. 62 ± 101 µg C l-1) and were depleted in glucose. Our data indicate that microbial communities in sandy sediments digest and transform labile parts of photosynthesis-derived particulate organic matter and likely release more stable, glucose-depleted residual glycans of unknown structures, quantities, and residence times into the ocean, thus modulating the glycan composition of marine coastal waters.

An ANI gap within bacterial species that advances the definitions of intra-species units.

IMPORTANCE: Bacterial strains and clonal complexes are two cornerstone concepts for microbiology that remain loosely defined, which confuses communication and research. Here we identify a natural gap in genome sequence comparisons among isolate genomes of all well-sequenced species that has gone unnoticed so far and could be used to more accurately and precisely define these and related concepts compared to current methods. These findings advance the molecular toolbox for accurately delineating and following the important units of diversity within prokaryotic species and thus should greatly facilitate future epidemiological and micro-diversity studies across clinical and environmental settings.

Comparing genomes recovered from time-series metagenomes using long- and short-read sequencing technologies.

BACKGROUND: Over the past years, sequencing technologies have expanded our ability to examine novel microbial metabolisms and diversity previously obscured by isolation approaches. Long-read sequencing promises to revolutionize the metagenomic field and recover less fragmented genomes from environmental samples. Nonetheless, how to best benefit from long-read sequencing and whether long-read sequencing can provide recovered genomes of similar characteristics as short-read approaches remains unclear. RESULTS: We recovered metagenome-assembled genomes (MAGs) from the free-living fraction at four-time points during a spring bloom in the North Sea. The taxonomic composition of all MAGs recovered was comparable between technologies. However, differences consisted of higher sequencing depth for contigs and higher genome population diversity in short-read compared to long-read metagenomes. When pairing population genomes recovered from both sequencing approaches that shared ≥ 99% average nucleotide identity, long-read MAGs were composed of fewer contigs, a higher N50, and a higher number of predicted genes when compared to short-read MAGs. Moreover, 88% of the total long-read MAGs carried a 16S rRNA gene compared to only 23% of MAGs recovered from short-read metagenomes. Relative abundances for population genomes recovered using both technologies were similar, although disagreements were observed for high and low GC content MAGs. CONCLUSIONS: Our results highlight that short-read technologies recovered more MAGs and a higher number of species than long-read due to an overall higher sequencing depth. Long-read samples produced higher quality MAGs and similar species composition compared to short-read sequencing. Differences in the GC content recovered by each sequencing technology resulted in divergences in the diversity recovered and relative abundance of MAGs within the GC content boundaries.

Diversity, Taxonomic Novelty, and Encoded Functions of Salar de Ascotán Microbiota, as Revealed by Metagenome-Assembled Genomes.

Salar de Ascotán is a high-altitude arsenic-rich salt flat exposed to high ultraviolet radiation in the Atacama Desert, Chile. It hosts unique endemic flora and fauna and is an essential habitat for migratory birds, making it an important site for conservation and protection. However, there is limited information on the resident microbiota's diversity, genomic features, metabolic potential, and molecular mechanisms that enable it to thrive in this extreme environment. We used long- and short-read metagenomics to investigate the microbial communities in Ascotán's water, sediment, and soil. Bacteria predominated, mainly Pseudomonadota, Acidobacteriota, and Bacteroidota, with a remarkable diversity of archaea in the soil. Following hybrid assembly, we recovered high-quality bacterial (101) and archaeal (6) metagenome-assembled genomes (MAGs), including representatives of two putative novel families of Patescibacteria and Pseudomonadota and two novel orders from the archaeal classes Halobacteriota and Thermoplasmata. We found different metabolic capabilities across distinct lineages and a widespread presence of genes related to stress response, DNA repair, and resistance to arsenic and other metals. These results highlight the remarkable diversity and taxonomic novelty of the Salar de Ascotán microbiota and its rich functional repertoire, making it able to resist different harsh conditions. The highly complete MAGs described here could serve future studies and bioprospection efforts focused on salt flat extremophiles, and contribute to enriching databases with microbial genome data from underrepresented regions of our planet.

In situ cell division and mortality rates of SAR11, SAR86, Bacteroidetes, and Aurantivirga during phytoplankton blooms reveal differences in population controls.

Net growth of microbial populations, that is, changes in abundances over time, can be studied using 16S rRNA fluorescence in situ hybridization (FISH). However, this approach does not differentiate between mortality and cell division rates. We used FISH-based image cytometry in combination with dilution culture experiments to study net growth, cell division, and mortality rates of four bacterial taxa over two distinct phytoplankton blooms: the oligotrophs SAR11 and SAR86, and the copiotrophic phylum Bacteroidetes, and its genus Aurantivirga. Cell volumes, ribosome content, and frequency of dividing cells (FDC) co-varied over time. Among the three, FDC was the most suitable predictor to calculate cell division rates for the selected taxa. The FDC-derived cell division rates for SAR86 of up to 0.8/day and Aurantivirga of up to 1.9/day differed, as expected for oligotrophs and copiotrophs. Surprisingly, SAR11 also reached high cell division rates of up to 1.9/day, even before the onset of phytoplankton blooms. For all four taxonomic groups, the abundance-derived net growth (-0.6 to 0.5/day) was about an order of magnitude lower than the cell division rates. Consequently, mortality rates were comparably high to cell division rates, indicating that about 90% of bacterial production is recycled without apparent time lag within 1 day. Our study shows that determining taxon-specific cell division rates complements omics-based tools and provides unprecedented clues on individual bacterial growth strategies including bottom-up and top-down controls. IMPORTANCE The growth of a microbial population is often calculated from their numerical abundance over time. However, this does not take cell division and mortality rates into account, which are important for deriving ecological processes like bottom-up and top-down control. In this study, we determined growth by numerical abundance and calibrated microscopy-based methods to determine the frequency of dividing cells and subsequently calculate taxon-specific cell division rates in situ. The cell division and mortality rates of two oligotrophic (SAR11 and SAR86) and two copiotrophic (Bacteroidetes and Aurantivirga) taxa during two spring phytoplankton blooms showed a tight coupling for all four taxa throughout the blooms without any temporal offset. Unexpectedly, SAR11 showed high cell division rates days before the bloom while cell abundances remained constant, which is indicative of strong top-down control. Microscopy remains the method of choice to understand ecological processes like top-down and bottom-up control on a cellular level.

Verrucomicrobiota are specialist consumers of sulfated methyl pentoses during diatom blooms.

Marine algae annually sequester petagrams of carbon dioxide into polysaccharides, which are a central metabolic fuel for marine carbon cycling. Diatom microalgae produce sulfated polysaccharides containing methyl pentoses that are challenging to degrade for bacteria compared to other monomers, implicating these sugars as a potential carbon sink. Free-living bacteria occurring in phytoplankton blooms that specialise on consuming microalgal sugars, containing fucose and rhamnose remain unknown. Here, genomic and proteomic data indicate that small, coccoid, free-living Verrucomicrobiota specialise in fucose and rhamnose consumption during spring algal blooms in the North Sea. Verrucomicrobiota cell abundance was coupled with the algae bloom onset and accounted for up to 8% of the bacterioplankton. Glycoside hydrolases, sulfatases, and bacterial microcompartments, critical proteins for the consumption of fucosylated and sulfated polysaccharides, were actively expressed during consecutive spring bloom events. These specialised pathways were assigned to novel and discrete candidate species of the Akkermansiaceae and Puniceicoccaceae families, which we here describe as Candidatus Mariakkermansia forsetii and Candidatus Fucivorax forsetii. Moreover, our results suggest specialised metabolic pathways could determine the fate of complex polysaccharides consumed during algae blooms. Thus the sequestration of phytoplankton organic matter via methyl pentose sugars likely depend on the activity of specialised Verrucomicrobiota populations.

ROCker Models for Reliable Detection and Typing of Short-Read Sequences Carrying ß-Lactamase Genes.

Identification of genes encoding ß-lactamases (BLs) from short-read sequences remains challenging due to the high frequency of shared amino acid functional domains and motifs in proteins encoded by BL genes and related non-BL gene sequences. Divergent BL homologs can be frequently missed during similarity searches, which has important practical consequences for monitoring antibiotic resistance. To address this limitation, we built ROCker models that targeted broad classes (e.g., class A, B, C, and D) and individual families (e.g., TEM) of BLs and challenged them with mock 150-bp- and 250-bp-read data sets of known composition. ROCker identifies most-discriminant bit score thresholds in sliding windows along the sequence of the target protein sequence and hence can account for nondiscriminative domains shared by unrelated proteins. BL ROCker models showed a 0% false-positive rate (FPR), a 0% to 4% false-negative rate (FNR), and an up-to-50-fold-higher F1 score [2 × precision × recall/(precision + recall)] compared to alternative methods, such as similarity searches using BLASTx with various e-value thresholds and BL hidden Markov models, or tools like DeepARG, ShortBRED, and AMRFinder. The ROCker models and the underlying protein sequence reference data sets and phylogenetic trees for read placement are freely available through http://enve-omics.ce.gatech.edu/data/rocker-bla. Application of these BL ROCker models to metagenomics, metatranscriptomics, and high-throughput PCR gene amplicon data should facilitate the reliable detection and quantification of BL variants encoded by environmental or clinical isolates and microbiomes and more accurate assessment of the associated public health risk, compared to the current practice. IMPORTANCE Resistance genes encoding ß-lactamases (BLs) confer resistance to the widely prescribed antibiotic class ß-lactams. Therefore, it is important to assess the prevalence of BL genes in clinical or environmental samples for monitoring the spreading of these genes into pathogens and estimating public health risk. However, detecting BLs in short-read sequence data is technically challenging. Our ROCker model-based bioinformatics approach showcases the reliable detection and typing of BLs in complex data sets and thus contributes toward solving an important problem in antibiotic resistance surveillance. The ROCker models developed substantially expand the toolbox for monitoring antibiotic resistance in clinical or environmental settings.

A genomic island provides Acidithiobacillus ferrooxidans ATCC 53993 additional copper resistance: a possible competitive advantage.

There is great interest in understanding how extremophilic biomining bacteria adapt to exceptionally high copper concentrations in their environment. Acidithiobacillus ferrooxidans ATCC 53993 genome possesses the same copper resistance determinants as strain ATCC 23270. However, the former strain contains in its genome a 160-kb genomic island (GI), which is absent in ATCC 23270. This GI contains, amongst other genes, several genes coding for an additional putative copper ATPase and a Cus system. A. ferrooxidans ATCC 53993 showed a much higher resistance to CuSO(4) (>100 mM) than that of strain ATCC 23270 (<25 mM). When a similar number of bacteria from each strain were mixed and allowed to grow in the absence of copper, their respective final numbers remained approximately equal. However, in the presence of copper, there was a clear overgrowth of strain ATCC 53993 compared to ATCC 23270. This behavior is most likely explained by the presence of the additional copper-resistance genes in the GI of strain ATCC 53993. As determined by qRT-PCR, it was demonstrated that these genes are upregulated when A. ferrooxidans ATCC 53993 is grown in the presence of copper and were shown to be functional when expressed in copper-sensitive Escherichia coli mutants. Thus, the reason for resistance to copper of two strains of the same acidophilic microorganism could be determined by slight differences in their genomes, which may not only lead to changes in their capacities to adapt to their environment, but may also help to select the more fit microorganisms for industrial biomining operations.