Cell size, density, and nutrient dependency of unicellular algal gravitational sinking velocities.

Eukaryotic phytoplankton, also known as algae, form the basis of marine food webs and drive marine carbon sequestration. Algae must regulate their motility and gravitational sinking to balance access to light at the surface and nutrients in deeper layers. However, the regulation of gravitational sinking remains largely unknown, especially in motile species. Here, we quantify gravitational sinking velocities according to Stokes' law in diverse clades of unicellular marine microalgae to reveal the cell size, density, and nutrient dependency of sinking velocities. We identify a motile algal species, Tetraselmis sp., that sinks faster when starved due to a photosynthesis-driven accumulation of carbohydrates and a loss of intracellular water, both of which increase cell density. Moreover, the regulation of cell sinking velocities is connected to proliferation and can respond to multiple nutrients. Overall, our work elucidates how cell size and density respond to environmental conditions to drive the vertical migration of motile algae.

Physiological basis for atmospheric methane oxidation and methanotrophic growth on air.

Atmospheric methane oxidizing bacteria (atmMOB) constitute the sole biological sink for atmospheric methane. Still, the physiological basis allowing atmMOB to grow on air is not well understood. Here we assess the ability and strategies of seven methanotrophic species to grow with air as sole energy, carbon, and nitrogen source. Four species, including three outside the canonical atmMOB group USCα, enduringly oxidized atmospheric methane, carbon monoxide, and hydrogen during 12 months of growth on air. These four species exhibited distinct substrate preferences implying the existence of multiple metabolic strategies to grow on air. The estimated energy yields of the atmMOB were substantially lower than previously assumed necessary for cellular maintenance in atmMOB and other aerobic microorganisms. Moreover, the atmMOB also covered their nitrogen requirements from air. During growth on air, the atmMOB decreased investments in biosynthesis while increasing investments in trace gas oxidation. Furthermore, we confirm that a high apparent specific affinity for methane is a key characteristic of atmMOB. Our work shows that atmMOB grow on the trace concentrations of methane, carbon monoxide, and hydrogen present in air and outlines the metabolic strategies that enable atmMOB to mitigate greenhouse gases.

Low-level resource partitioning supports coexistence among functionally redundant bacteria during successional dynamics.

Members of microbial communities can substantially overlap in substrate use. However, what enables functionally redundant microorganisms to coassemble or even stably coexist remains poorly understood. Here, we show that during unstable successional dynamics on complex, natural organic matter, functionally redundant bacteria can coexist by partitioning low-concentration substrates even though they compete for one simple, dominant substrate. We allowed ocean microbial communities to self-assemble on leachates of the brown seaweed Fucus vesiculosus and then analyzed the competition among 10 taxonomically diverse isolates representing two distinct stages of the succession. All, but two isolates, exhibited an average of 90% ± 6% pairwise overlap in resource use, and functional redundancy of isolates from the same assembly stage was higher than that from between assembly stages, leading us to construct a simpler four-isolate community with two isolates from each of the early and late stages. We found that, although the short-term dynamics of the four-isolate communities in F. vesiculosus leachate was dependent on initial isolate ratios, in the long term, the four isolates stably coexist in F. vesiculosus leachate, albeit with some strains at low abundance. We therefore explored the potential for nonredundant substrate use by genomic content analysis and RNA expression patterns. This analysis revealed that the four isolates mainly differed in peripheral metabolic pathways, such as the ability to degrade pyrimidine, leucine, and tyrosine, as well as aromatic substrates. These results highlight the importance of fine-scale differences in metabolic strategies for supporting the frequently observed coexistence of large numbers of rare organisms in natural microbiomes.

Single-cell mass distributions reveal simple rules for achieving steady-state growth.

IMPORTANCE: Microbiologists have watched clear liquid turn cloudy for over 100 years. While the cloudiness of a culture is proportional to its total biomass, growth rates from optical density measurements are challenging to interpret when cells change size. Many bacteria adjust their size at different steady-state growth rates, but also when shifting between starvation and growth. Optical density cannot disentangle how mass is distributed among cells. Here, we use single-cell mass measurements to demonstrate that a population of cells in batch culture achieves a stable mass distribution for only a short period of time. Achieving steady-state growth in rich medium requires low initial biomass concentrations and enough time for individual cell mass accumulation and cell number increase via cell division to balance out. Steady-state growth is important for reliable cell mass distributions and experimental reproducibility. We discuss how mass variation outside of steady-state can impact physiology, ecology, and evolution experiments.

Epilithic Bacterial Assemblages on Subtidal Rocky Reefs: Variation Among Alternative Habitats at Ambient and Enhanced Nutrient Levels.

Temperate rocky reefs often support mosaics of alternative habitats such as macroalgal forests, algal turfs and sea urchin barrens. Although the composition of epilithic microbial biofilms (EMBs) is recognized as a major determinant of macroalgal recruitment, their role in regulating the stability of alternative habitats on temperate rocky reefs remains unexplored. On shallow rocky reefs of the Island of Capraia (NW Mediterranean), we compared EMB structure among canopy stands formed by the fucoid Ericaria brachycarpa, algal turfs, and urchin barrens under ambient versus experimentally enhanced nutrient levels. The three habitats shared a core microbial community consisting of 21.6 and 25.3% of total ASVs under ambient and enhanced nutrient conditions, respectively. Although Gammaproteobacteria, Alphaproteobacteria and Flavobacteriia were the most abundant classes across habitats, multivariate analyses at the ASV level showed marked differences in EMB composition among habitats. Enhancing nutrient level had no significant effect on EMBs, although it increased their similarity between macroalgal canopy and turf habitats. At both ambient and enriched nutrient levels, ASVs mostly belonging to Proteobacteria and Bacteroidetes were more abundant in EMBs from macroalgal canopies than barrens. In contrast, ASVs belonging to the phylum of Proteobacteria and, in particular, to the families of Rhodobacteraceae and Flavobacteriaceae at ambient nutrient levels and of Rhodobacteraceae and Bacteriovoracaceae at enhanced nutrient levels were more abundant in turf than canopy habitats. Our results show that primary surfaces from alternative habitats that form mosaics on shallow rocky reefs in oligotrophic areas host distinct microbial communities that are, to some extent, resistant to moderate nutrient enhancement. Understanding the role of EMBs in generating reinforcing feedback under different nutrient loading regimes appears crucial to advance our understanding of the mechanisms underpinning the stability of habitats alternative to macroalgal forests as well as their role in regulating reverse shifts.

Differential carbon utilization enables co-existence of recently speciated Campylobacteraceae in the cow rumen epithelial microbiome.

The activities of different microbes in the cow rumen have been shown to modulate the host's ability to utilize plant biomass, while the host-rumen interface has received little attention. As datasets collected worldwide have pointed to Campylobacteraceae as particularly abundant members of the rumen epithelial microbiome, we targeted this group in a subset of seven cows with meta- and isolate genome analysis. We show that the dominant Campylobacteraceae lineage has recently speciated into two populations that were structured by genome-wide selective sweeps followed by population-specific gene import and recombination. These processes led to differences in gene expression and enzyme domain composition that correspond to the ability to utilize acetate, the main carbon source for the host, at the cost of inhibition by propionate. This trade-off in competitive ability further manifests itself in differential dynamics of the two populations in vivo. By exploring population-level adaptations that otherwise remain cryptic in culture-independent analyses, our results highlight how recent evolutionary dynamics can shape key functional roles in the rumen microbiome.

Phage-host coevolution in natural populations.

Coevolution between bacteriophages (phages) and their bacterial hosts occurs through changes in resistance and counter-resistance mechanisms. To assess phage-host evolution in wild populations, we isolated 195 Vibrio crassostreae strains and 243 vibriophages during a 5-month time series from an oyster farm and combined these isolates with existing V. crassostreae and phage isolates. Cross-infection studies of 81,926 host-phage pairs delineated a modular network where phages are best at infecting co-occurring hosts, indicating local adaptation. Successful propagation of phage is restricted by the ability to adsorb to closely related bacteria and further constrained by strain-specific defence systems. These defences are highly diverse and predominantly located on mobile genetic elements, and multiple defences are active within a single genome. We further show that epigenetic and genomic modifications enable phage to adapt to bacterial defences and alter host range. Our findings reveal that the evolution of bacterial defences and phage counter-defences is underpinned by frequent genetic exchanges with, and between, mobile genetic elements.

Resolving the structure of phage-bacteria interactions in the context of natural diversity.

Microbial communities are shaped by viral predators. Yet, resolving which viruses (phages) and bacteria are interacting is a major challenge in the context of natural levels of microbial diversity. Thus, fundamental features of how phage-bacteria interactions are structured and evolve in the wild remain poorly resolved. Here we use large-scale isolation of environmental marine Vibrio bacteria and their phages to obtain estimates of strain-level phage predator loads, and use all-by-all host range assays to discover how phage and host genomic diversity shape interactions. We show that lytic interactions in environmental interaction networks (as observed in agar overlay) are sparse-with phage predator loads being low for most bacterial strains, and phages being host-strain-specific. Paradoxically, we also find that although overlap in killing is generally rare between tailed phages, recombination is common. Together, these results suggest that recombination during cryptic co-infections is an important mode of phage evolution in microbial communities. In the development of phages for bioengineering and therapeutics it is important to consider that nucleic acids of introduced phages may spread into local phage populations through recombination, and that the likelihood of transfer is not predictable based on lytic host range.

Rapid evolutionary turnover of mobile genetic elements drives bacterial resistance to phages.

Although it is generally accepted that phages drive bacterial evolution, how these dynamics play out in the wild remains poorly understood. We found that susceptibility to viral killing in marine Vibrio is mediated by large and highly diverse mobile genetic elements. These phage defense elements display exceedingly fast evolutionary turnover, resulting in differential phage susceptibility among clonal bacterial strains while phage receptors remain invariant. Protection is cumulative, and a single bacterial genome can harbor 6 to 12 defense elements, accounting for more than 90% of the flexible genome among close relatives. The rapid turnover of these elements decouples phage resistance from other genomic features. Thus, resistance to phages in the wild follows evolutionary trajectories alternative to those predicted from laboratory-based evolutionary experiments.

Successional dynamics and alternative stable states in a saline activated sludge microbial community over 9 years.

BACKGROUND: Microbial communities in both natural and applied settings reliably carry out myriads of functions, yet how stable these taxonomically diverse assemblages can be and what causes them to transition between states remains poorly understood. We studied monthly activated sludge (AS) samples collected over 9 years from a full-scale wastewater treatment plant to answer how complex AS communities evolve in the long term and how the community functions change when there is a disturbance in operational parameters. RESULTS: Here, we show that a microbial community in activated sludge (AS) system fluctuated around a stable average for 3 years but was then abruptly pushed into an alternative stable state by a simple transient disturbance (bleaching). While the taxonomic composition rapidly turned into a new state following the disturbance, the metabolic profile of the community and system performance remained remarkably stable. A total of 920 metagenome-assembled genomes (MAGs), representing approximately 70% of the community in the studied AS ecosystem, were recovered from the 97 monthly AS metagenomes. Comparative genomic analysis revealed an increased ability to aggregate in the cohorts of MAGs with correlated dynamics that are dominant after the bleaching event. Fine-scale analysis of dynamics also revealed cohorts that dominated during different periods and showed successional dynamics on seasonal and longer time scales due to temperature fluctuation and gradual changes in mean residence time in the reactor, respectively. CONCLUSIONS: Our work highlights that communities can assume different stable states under highly similar environmental conditions and that a specific disturbance threshold may lead to a rapid shift in community composition. Video Abstract.

Prokaryotic viruses impact functional microorganisms in nutrient removal and carbon cycle in wastewater treatment plants.

As one of the largest biotechnological applications, activated sludge (AS) systems in wastewater treatment plants (WWTPs) harbor enormous viruses, with 10-1,000-fold higher concentrations than in natural environments. However, the compositional variation and host-connections of AS viruses remain poorly explored. Here, we report a catalogue of ~50,000 prokaryotic viruses from six WWTPs, increasing the number of described viral species of AS by 23-fold, and showing the very high viral diversity which is largely unknown (98.4-99.6% of total viral contigs). Most viral genera are represented in more than one AS system with 53 identified across all. Viral infection widely spans 8 archaeal and 58 bacterial phyla, linking viruses with aerobic/anaerobic heterotrophs, and other functional microorganisms controlling nitrogen/phosphorous removal. Notably, Mycobacterium, notorious for causing AS foaming, is associated with 402 viral genera. Our findings expand the current AS virus catalogue and provide reference for the phage treatment to control undesired microorganisms in WWTPs.

Degradation of host translational machinery drives tRNA acquisition in viruses.

Viruses are traditionally thought to be under selective pressure to maintain compact genomes and thus depend on host cell translational machinery for reproduction. However, some viruses encode abundant tRNA and other translation-related genes, potentially optimizing for codon usage differences between phage and host. Here, we systematically interrogate selective advantages that carrying 18 tRNAs may convey to a T4-like Vibriophage. Host DNA and RNA degrade upon infection, including host tRNAs, which are replaced by those of the phage. These tRNAs are expressed at levels slightly better adapted to phage codon usage, especially that of late genes. The phage is unlikely to randomly acquire as diverse an array of tRNAs as observed (p = 0.0017). Together, our results support that the main driver behind phage tRNA acquisition is pressure to sustain translation as host machinery degrades, a process resulting in a dynamically adapted codon usage strategy during the course of infection.

Microbiome assembly for sulfonamide subsistence and the transfer of genetic determinants.

Antibiotic subsistence in bacteria represents an alternative resistance machinery, while paradoxically, it is also a cure for environmental resistance. Antibiotic-subsisting bacteria can detoxify antibiotic-polluted environments and prevent the development of antibiotic resistance in environments. However, progress toward efficient in situ engineering of antibiotic-subsisting bacteria is hindered by the lack of mechanistic and predictive understanding of the assembly of the functioning microbiome. By top-down manipulation of wastewater microbiomes using sulfadiazine as the single limiting source, we monitored the ecological selection process that forces the wastewater microbiome to perform efficient sulfadiazine subsistence. We found that the community-level assembly selects for the same three families rising to prominence across different initial pools of microbiomes. We further analyzed the assembly patterns using a linear model. Detailed inspections of the sulfonamide metabolic gene clusters in individual genomes of isolates and assembled metagenomes reveal limited transfer potential beyond the boundaries of the Micrococcaceae lineage. Our results open up new possibilities for engineering specialist bacteria for environmental applications.

Conjugative plasmids interact with insertion sequences to shape the horizontal transfer of antimicrobial resistance genes.

It is well established that plasmids play an important role in the dissemination of antimicrobial resistance (AMR) genes; however, little is known about the role of the underlying interactions between different plasmid categories and other mobile genetic elements (MGEs) in shaping the promiscuous spread of AMR genes. Here, we developed a tool designed for plasmid classification, AMR gene annotation, and plasmid visualization and found that most plasmid-borne AMR genes, including those localized on class 1 integrons, are enriched in conjugative plasmids. Notably, we report the discovery and characterization of a massive insertion sequence (IS)-associated AMR gene transfer network (245 combinations covering 59 AMR gene subtypes and 53 ISs) linking conjugative plasmids and phylogenetically distant pathogens, suggesting a general evolutionary mechanism for the horizontal transfer of AMR genes mediated by the interaction between conjugative plasmids and ISs. Moreover, our experimental results confirmed the importance of the observed interactions in aiding the horizontal transfer and expanding the genetic range of AMR genes within complex microbial communities.

Coastal Bacterial Community Response to Glacier Melting in the Western Antarctic Peninsula.

Current warming in the Western Antarctic Peninsula (WAP) has multiple effects on the marine ecosystem, modifying the trophic web and the nutrient regime. In this study, the effect of decreased surface salinity on the marine microbial community as a consequence of freshening from nearby glaciers was investigated in Chile Bay, Greenwich Island, WAP. In the summer of 2016, samples were collected from glacier ice and transects along the bay for 16S rRNA gene sequencing, while in situ dilution experiments were conducted and analyzed using 16S rRNA gene sequencing and metatranscriptomic analysis. The results reveal that certain common seawater genera, such as Polaribacter, Pseudoalteromonas and HTCC2207, responded positively to decreased salinity in both the bay transect and experiments. The relative abundance of these bacteria slightly decreased, but their functional activity was maintained and increased the over time in the dilution experiments. However, while ice bacteria, such as Flavobacterium and Polaromonas, tolerated the increased salinity after mixing with seawater, their gene expression decreased considerably. We suggest that these bacterial taxa could be defined as sentinels of freshening events in the Antarctic coastal system. Furthermore, these results suggest that a significant portion of the microbial community is resilient and can adapt to disturbances, such as freshening due to the warming effect of climate change in Antarctica.

Redox-informed models of global biogeochemical cycles.

Microbial activity mediates the fluxes of greenhouse gases. However, in the global models of the marine and terrestrial biospheres used for climate change projections, typically only photosynthetic microbial activity is resolved mechanistically. To move forward, we argue that global biogeochemical models need a theoretically grounded framework with which to constrain parameterizations of diverse microbial metabolisms. Here, we explain how the key redox chemistry underlying metabolisms provides a path towards this goal. Using this first-principles approach, the presence or absence of metabolic functional types emerges dynamically from ecological interactions, expanding model applicability to unobserved environments."Nothing is less real than realism. It is only by selection, by elimination, by emphasis, that we get at the real meaning of things." -Georgia O'Keefe.

Verrucomicrobia use hundreds of enzymes to digest the algal polysaccharide fucoidan.

Brown algae are important players in the global carbon cycle by fixing carbon dioxide into 1 Gt of biomass annually, yet the fate of fucoidan-their major cell wall polysaccharide-remains poorly understood. Microbial degradation of fucoidans is slower than that of other polysaccharides, suggesting that fucoidans are more recalcitrant and may sequester carbon in the ocean. This may be due to the complex, branched and highly sulfated structure of fucoidans, which also varies among species of brown algae. Here, we show that 'Lentimonas' sp. CC4, belonging to the Verrucomicrobia, acquired a remarkably complex machinery for the degradation of six different fucoidans. The strain accumulated 284 putative fucoidanases, including glycoside hydrolases, sulfatases and carbohydrate esterases, which are primarily located on a 0.89-megabase pair plasmid. Proteomics reveals that these enzymes assemble into substrate-specific pathways requiring about 100 enzymes per fucoidan from different species of brown algae. These enzymes depolymerize fucoidan into fucose, which is metabolized in a proteome-costly bacterial microcompartment that spatially constrains the metabolism of the toxic intermediate lactaldehyde. Marine metagenomes and microbial genomes show that Verrucomicrobia including 'Lentimonas' are abundant and highly specialized degraders of fucoidans and other complex polysaccharides. Overall, the complexity of the pathways underscores why fucoidans are probably recalcitrant and more slowly degraded, since only highly specialized organisms can effectively degrade them in the ocean.