Dynamic nitrogen fixation in an aerobic endophyte of Populus.

Biological nitrogen fixation by microbial diazotrophs can contribute significantly to nitrogen availability in non-nodulating plant species. In this study of molecular mechanisms and gene expression relating to biological nitrogen fixation, the aerobic nitrogen-fixing endophyte Burkholderia vietnamiensis, strain WPB, isolated from Populus trichocarpa served as a model for endophyte-poplar interactions. Nitrogen-fixing activity was observed to be dynamic on nitrogen-free medium with a subset of colonies growing to form robust, raised globular like structures. Secondary ion mass spectrometry (NanoSIMS) confirmed that N-fixation was uneven within the population. A fluorescent transcriptional reporter (GFP) revealed that the nitrogenase subunit nifH is not uniformly expressed across genetically identical colonies of WPB and that only ~11% of the population was actively expressing the nifH gene. Higher nifH gene expression was observed in clustered cells through monitoring individual bacterial cells using single-molecule fluorescence in situ hybridization. Through 15N2 enrichment, we identified key nitrogenous metabolites and proteins synthesized by WPB and employed targeted metabolomics in active and inactive populations. We cocultivated WPB Pnif-GFP with poplar within a RhizoChip, a synthetic soil habitat, which enabled direct imaging of microbial nifH expression within root epidermal cells. We observed that nifH expression is localized to the root elongation zone where the strain forms a unique physical interaction with the root cells. This work employed comprehensive experimentation to identify novel mechanisms regulating both biological nitrogen fixation and beneficial plant-endophyte interactions.

Tracking nitrogen allocation to proteome biosynthesis in a marine microbial community.

Microbial growth in many environments is limited by nitrogen availability, yet there is limited understanding of how complex communities compete for and allocate this resource. Here we develop a broadly applicable approach to track biosynthetic incorporation of 15N-labelled nitrogen substrates into microbial community proteomes, enabling quantification of protein turnover and N allocation to specific cellular functions in individual taxa. Application to oligotrophic ocean surface water identifies taxa-specific substrate preferences and a distinct subset of protein functions undergoing active biosynthesis. The cyanobacterium Prochlorococcus is the most effective competitor for acquisition of ammonium and urea and shifts its proteomic allocation of N over the day/night cycle. Our approach reveals that infrastructure and protein-turnover functions comprise substantial biosynthetic demand for N in Prochlorococcus and a range of other microbial taxa. The direct interrogation of the proteomic underpinnings of N limitation with 15N-tracking proteomics illuminates how nutrient stress differentially influences metabolism in co-existing microbes.

Metabolic and biogeochemical consequences of viral infection in aquatic ecosystems.

Ecosystems are controlled by 'bottom-up' (resources) and 'top-down' (predation) forces. Viral infection is now recognized as a ubiquitous top-down control of microbial growth across ecosystems but, at the same time, cell death by viral predation influences, and is influenced by, resource availability. In this Review, we discuss recent advances in understanding the biogeochemical impact of viruses, focusing on how metabolic reprogramming of host cells during lytic viral infection alters the flow of energy and nutrients in aquatic ecosystems. Our synthesis revealed several emerging themes. First, viral infection transforms host metabolism, in part through virus-encoded metabolic genes; the functions performed by these genes appear to alleviate energetic and biosynthetic bottlenecks to viral production. Second, viral infection depends on the physiological state of the host cell and on environmental conditions, which are challenging to replicate in the laboratory. Last, metabolic reprogramming of infected cells and viral lysis alter nutrient cycling and carbon export in the oceans, although the net impacts remain uncertain. This Review highlights the need for understanding viral infection dynamics in realistic physiological and environmental contexts to better predict their biogeochemical consequences.

Closely related viruses of the marine picoeukaryotic alga Ostreococcus lucimarinus exhibit different ecological strategies.

In marine ecosystems, viruses are major disrupters of the direct flow of carbon and nutrients to higher trophic levels. Although the genetic diversity of several eukaryotic phytoplankton virus groups has been characterized, their infection dynamics are less understood, such that the physiological and ecological implications of their diversity remain unclear. We compared genomes and infection phenotypes of the two most closely related cultured phycodnaviruses infecting the widespread picoprasinophyte Ostreococcus lucimarinus under standard- (1.3 divisions per day) and limited-light (0.41 divisions per day) nutrient replete conditions. OlV7 infection caused early arrest of the host cell cycle, coinciding with a significantly higher proportion of infected cells than OlV1-amended treatments, regardless of host growth rate. OlV7 treatments showed a near-50-fold increase of progeny virions at the higher host growth rate, contrasting with OlV1's 16-fold increase. However, production of OlV7 virions was more sensitive than OlV1 production to reduced host growth rate, suggesting fitness trade-offs between infection efficiency and resilience to host physiology. Moreover, although organic matter released from OlV1- and OlV7-infected hosts had broadly similar chemical composition, some distinct molecular signatures were observed. Collectively, these results suggest that current views on viral relatedness through marker and core gene analyses underplay operational divergence and consequences for host ecology.

Environmental science. Rethinking the marine carbon cycle: factoring in the multifarious lifestyles of microbes.

The profound influence of marine plankton on the global carbon cycle has been recognized for decades, particularly for photosynthetic microbes that form the base of ocean food chains. However, a comprehensive model of the carbon cycle is challenged by unicellular eukaryotes (protists) having evolved complex behavioral strategies and organismal interactions that extend far beyond photosynthetic lifestyles. As is also true for multicellular eukaryotes, these strategies and their associated physiological changes are difficult to deduce from genome sequences or gene repertoires­a problem compounded by numerous unknown function proteins. Here, we explore protistan trophic modes in marine food webs and broader biogeochemical influences. We also evaluate approaches that could resolve their activities, link them to biotic and abiotic factors, and integrate them into an ecosystems biology framework.

Phylogenetic constraints on elemental stoichiometry and resource allocation in heterotrophic marine bacteria.

The objective of this study was to evaluate the contribution of evolutionary history to variation in the biomass stoichiometry and underlying biochemical allocation patterns of heterotrophic marine bacteria. We hypothesized that phylogeny significantly constrains biochemical allocation strategy and elemental composition among taxa of heterotrophic marine bacteria. Using a 'common-garden' experimental design, we detected significant interspecific variation in stoichiometry, macromolecule allocation and growth rate among 13 strains of marine Proteobacteria. However, this variation was not well explained by 16S rRNA phylogenetic relationships or differences in growth rate. Heterotrophic bacteria likely experience C-limitation when consuming resources in Redfield proportions, which consequently decouples growth rate from allocation to rRNA and biomass P content. Accordingly, overall bacterial C : nutrient ratios (C : P = 77, C : N = 4.9) were lower than Redfield proportions, whereas the average N : P ratio of 17 was consistent with the Redfield ratio. Our results suggest that strain-level diversity is an important driver of variation in the C : N : P ratios of heterotrophic bacterial biomass and that the potential importance of non-nucleic acid pools of P warrants further investigation. Continued work clarifying the range and controls on the stoichiometry of heterotrophic marine bacteria will help improve understanding and predictions of global ocean C, N and P dynamics.

Microdiversity of extracellular enzyme genes among sequenced prokaryotic genomes.

Understanding the relationship between prokaryotic traits and phylogeny is important for predicting and modeling ecological processes. Microbial extracellular enzymes have a pivotal role in nutrient cycling and the decomposition of organic matter, yet little is known about the phylogenetic distribution of genes encoding these enzymes. In this study, we analyzed 3058 annotated prokaryotic genomes to determine which taxa have the genetic potential to produce alkaline phosphatase, chitinase and ß-N-acetyl-glucosaminidase enzymes. We then evaluated the relationship between the genetic potential for enzyme production and 16S rRNA phylogeny using the consenTRAIT algorithm, which calculated the phylogenetic depth and corresponding 16S rRNA sequence identity of clades of potential enzyme producers. Nearly half (49.2%) of the genomes analyzed were found to be capable of extracellular enzyme production, and these were non-randomly distributed across most prokaryotic phyla. On average, clades of potential enzyme-producing organisms had a maximum phylogenetic depth of 0.008004-0.009780, though individual clades varied broadly in both size and depth. These values correspond to a minimum 16S rRNA sequence identity of 98.04-98.40%. The distribution pattern we found is an indication of microdiversity, the occurrence of ecologically or physiologically distinct populations within phylogenetically related groups. Additionally, we found positive correlations among the genes encoding different extracellular enzymes. Our results suggest that the capacity to produce extracellular enzymes varies at relatively fine-scale phylogenetic resolution. This variation is consistent with other traits that require a small number of genes and provides insight into the relationship between taxonomy and traits that may be useful for predicting ecological function.