Jennifer Pett-Ridge

A Bacterium That Can Grow by Using Arsenic Instead of Phosphorus

Evidence is offered for arsenate replacing phosphate as a molecular building block in a Mono Lake, California, bacterium. Life is mostly composed of the elements carbon, hydrogen, nitrogen, oxygen, sulfur, and phosphorus. Although these six elements make up nucleic acids, proteins, and lipids and thus the bulk of living matter, it is theoretically possible that some other elements in the periodic table could serve the same functions. Here, we describe a bacterium, strain GFAJ-1 of the Halomonadaceae, isolated from Mono Lake, California, that is able to substitute arsenic for phosphorus to sustain its growth. Our data show evidence for arsenate in macromolecules that normally contain phosphate, most notably nucleic acids and proteins. Exchange of one of the major bio-elements may have profound evolutionary and geochemical importance.

Linking microbial phylogeny to metabolic activity at the single-cell level by using enhanced element labeling-catalyzed reporter deposition fluorescence in situ hybridization (EL-FISH) and NanoSIMS.

ABSTRACT To examine phylogenetic identity and metabolic activity of individual cells in complex microbial communities, we developed a method which combines rRNA-based in situ hybridization with stable isotope imaging based on nanometer-scale secondary-ion mass spectrometry (NanoSIMS). Fluorine or bromine atoms were introduced into cells via 16S rRNA-targeted probes, which enabled phylogenetic identification of individual cells by NanoSIMS imaging. To overcome the natural fluorine and bromine backgrounds, we modified the current catalyzed reporter deposition fluorescence in situ hybridization (FISH) technique by using halogen-containing fluorescently labeled tyramides as substrates for the enzymatic tyramide deposition. Thereby, we obtained an enhanced element labeling of microbial cells by FISH (EL-FISH). The relative cellular abundance of fluorine or bromine after EL-FISH exceeded natural background concentrations by up to 180-fold and allowed us to distinguish target from non-target cells in NanoSIMS fluorine or bromine images. The method was optimized on single cells of axenic Escherichia coli and Vibrio cholerae cultures. EL-FISH/NanoSIMS was then applied to study interrelationships in a dual-species consortium consisting of a filamentous cyanobacterium and a heterotrophic alphaproteobacterium. We also evaluated the method on complex microbial aggregates obtained from human oral biofilms. In both samples, we found evidence for metabolic interactions by visualizing the fate of substrates labeled with 13C-carbon and 15N-nitrogen, while individual cells were identified simultaneously by halogen labeling via EL-FISH. Our novel approach will facilitate further studies of the ecophysiology of known and uncultured microorganisms in complex environments and communities.

Integrating microbial ecology into ecosystem models: challenges and priorities

Microbial communities can potentially mediate feedbacks between global change and ecosystem function, owing to their sensitivity to environmental change and their control over critical biogeochemical processes. Numerous ecosystem models have been developed to predict global change effects, but most do not consider microbial mechanisms in detail. In this idea paper, we examine the extent to which incorporation of microbial ecology into ecosystem models improves predictions of carbon (C) dynamics under warming, changes in precipitation regime, and anthropogenic nitrogen (N) enrichment. We focus on three cases in which this approach might be especially valuable: temporal dynamics in microbial responses to environmental change, variation in ecological function within microbial communities, and N effects on microbial activity. Four microbially-based models have addressed these scenarios. In each case, predictions of the microbial-based models differ—sometimes substantially—from comparable conventional models. However, validation and parameterization of model performance is challenging. We recommend that the development of microbial-based models must occur in conjunction with the development of theoretical frameworks that predict the temporal responses of microbial communities, the phylogenetic distribution of microbial functions, and the response of microbes to N enrichment.

Fixation and fate of C and N in the cyanobacterium Trichodesmium using nanometer-scale secondary ion mass spectrometry

The marine cyanobacterium Trichodesmium is ubiquitous in tropical and subtropical seas and is an important contributor to global N and C cycling. We sought to characterize metabolic uptake patterns in individual Trichodesmium IMS-101 cells by quantitatively imaging 13C and 15N uptake with high-resolution secondary ion mass spectrometry (NanoSIMS). Trichodesmium fix both CO2 and N2 concurrently during the day and are, thus, faced with a balancing act: the O2 evolved during photosynthesis inhibits nitrogenase, the key enzyme in N2 fixation. After performing correlated transmission electron microscopy (TEM) and NanoSIMS analysis on trichome thin-sections, we observed transient inclusion of 15N and 13C into discrete subcellular bodies identified as cyanophycin granules. We speculate that Trichodesmium uses these dynamic storage bodies to uncouple CO2 and N2 fixation from overall growth dynamics. We also directly quantified both CO2 and N2 fixation at the single cell level using NanoSIMS imaging of whole cells in multiple trichomes. Our results indicate maximal CO2 fixation rates in the morning, compared with maximal N2 fixation rates in the afternoon, bolstering the argument that segregation of CO2 and N2 fixation in Trichodesmium is regulated in part by temporal factors. Spatial separation of N2 and CO2 fixation may also have a role in metabolic segregation in Trichodesmium. Our approach in combining stable isotope labeling with NanoSIMS and TEM imaging can be extended to other physiologically relevant elements and processes in other important microbial systems.

Carbon and nitrogen fixation and metabolite exchange in and between individual cells of Anabaena oscillarioides

Filamentous nitrogen fixing cyanobacteria are key players in global nutrient cycling, but the relationship between CO2- and N2-fixation and intercellular exchange of these elements remains poorly understood in many genera. Using high-resolution nanometer-scale secondary ion mass spectrometry (NanoSIMS) in conjunction with enriched H13CO3− and 15N2 incubations of Anabaena oscillarioides, we imaged the cellular distributions of C, N and P and 13C and 15N enrichments at multiple time points during a diurnal cycle as proxies for C and N assimilation. The temporal and spatial distributions of the newly fixed C and N were highly heterogeneous at both the intra- and inter-cellular scale, and indicative of regions performing active assimilation and biosynthesis. Subcellular components such as the neck region of heterocycts, cell division septae and putative cyanophycin granules were clearly identifiable by their elemental composition. Newly fixed nitrogen was rapidly exported from heterocysts and was evenly allocated among vegetative cells, with the exception of the most remote vegetative cells between heterocysts, which were N limited based on lower 15N enrichment. Preexisting functional heterocysts had the lowest levels of 13C and 15N enrichment, while heterocysts that were inferred to have differentiated during the experiment had higher levels of enrichment. This innovative approach, combining stable isotope labeling and NanoSIMS elemental and isotopic imaging, allows characterization of cellular development (division, heterocyst differentiation), changes in individual cell composition and cellular roles in metabolite exchange.

An arbuscular mycorrhizal fungus significantly modifies the soil bacterial community and nitrogen cycling during litter decomposition

Arbuscular mycorrhizal fungi (AMF) perform an important ecosystem service by improving plant nutrient capture from soil, yet little is known about how AMF influence soil microbial communities during nutrient uptake. We tested whether an AMF modifies the soil microbial community and nitrogen cycling during litter decomposition. A two-chamber microcosm system was employed to create a root-free soil environment to control AMF access to (13) C- and (15) N-labelled root litter. Using a 16S rRNA gene microarray, we documented that approximately 10% of the bacterial community responded to the AMF, Glomus hoi. Taxa from the Firmicutes responded positively to AMF, while taxa from the Actinobacteria and Comamonadaceae responded negatively to AMF. Phylogenetic analyses indicate that AMF may influence bacterial community assembly processes. Using nanometre-scale secondary ion mass spectrometry (NanoSIMS) we visualized the location of AMF-transported (13) C and (15) N in plant roots. Bulk isotope ratio mass spectrometry revealed that the AMF exported 4.9% of the litter (15) N to the host plant (Plantago lanceolata L.), and litter-derived (15) N was preferentially exported relative to litter-derived (13) C. Our results suggest that the AMF primarily took up N in the inorganic form, and N export is one mechanism by which AMF could modify the soil microbial community and decomposition processes.

PLANT AND MICROBIAL CONTROLS ON NITROGEN RETENTION AND LOSS IN A HUMID TROPICAL FOREST

Humid tropical forests are generally characterized by the lack of nitrogen (N) limitation to net primary productivity, yet paradoxically have high potential for N loss. We conducted an intensive field experiment with 15 NH4 and 15 NO3 additions to highly weathered tropical forest soils in Puerto Rico to determine the relative importance of N retention and loss mechanisms. Over one-half of all the NH4 + produced was rapidly converted to NO3 - via the process of gross nitrification. During the first 24 hours, plant roots took up 28% of the inorganic N produced, dominantly as NH4 + , and were a greater sink for N than soil microbial biomass. Soil microbes were not a significant sink for added 15 NH4 + or 15 NO3 - during the first 24 hours, and only for 15 NH4 + after 7 days. Patterns of microbial community composition, as determined by terminal restriction fragment length polymorphism analysis (TRFLP), were weakly but significantly correlated with nitrification and denitrification to N2 O. Rates of dissimilatory NO3 - reduction to NH4 + (DNRA) were high in this forest, accounting for up to 25% of gross NH4 + production and 35% of gross nitrification. DNRA was a major sink for NO3 - , which may have contributed to the lower rates of N2 O and leaching losses. Despite considerable N conservation via DNRA and plant NH4 + uptake, the fate of ∼45% of the NO3 - produced and 4% of the NH4 + produced were not measured in our fluxes, suggesting that other important pathways for N retention and loss (e.g., denitrification to N2 ) are important in this system. The high proportion of mineralized N that was rapidly nitrified and the fates of that NO3 - highlight the key role of gross nitrification as a proximate control on N retention and loss in humid tropical forest soils. Furthermore, our results demonstrate the importance of the coupling between DNRA and plant uptake of NH4 + as a potential N-conserving mechanism within tropical forests.

Subcellular metal imaging identifies dynamic sites of Cu accumulation in Chlamydomonas

We identified a Cu accumulating structure with a dynamic role in intracellular Cu homeostasis. During Zn limitation, Chlamydomonas reinhardtii hyperaccumulated Cu, dependent on the nutritional Cu sensor CRR1, but was functionally Cu-deficient. Visualization of intracellular Cu revealed major Cu accumulation sites coincident with electron-dense structures that stained positive for low pH and polyphosphate, suggesting that they are lysosome-related organelles. NanoSIMS showed colocalization of Ca and Cu, and X-ray absorption spectroscopy (XAS) was consistent with Cu+ accumulation in an ordered structure. Zn resupply restored Cu homeostasis concomitant with reduced abundance of these structures. Cu isotope labeling demonstrated that sequestered Cu+ became bio-available for the synthesis of plastocyanin, and transcriptome profiling indicated that mobilized Cu became visible to CRR1. Cu trafficking to intracellular accumulation sites may be a strategy for preventing protein mis-metallation during Zn deficiency and enabling efficient cuproprotein (re)-metallation upon Zn resupply.

Successional Trajectories of Rhizosphere Bacterial Communities over Consecutive Seasons

ABSTRACT It is well known that rhizosphere microbiomes differ from those of surrounding soil, and yet we know little about how these root-associated microbial communities change through the growing season and between seasons. We analyzed the response of soil bacteria to roots of the common annual grass Avena fatua over two growing seasons using high-throughput sequencing of 16S rRNA genes. Over the two periods of growth, the rhizosphere bacterial communities followed consistent successional patterns as plants grew, although the starting communities were distinct. Succession in the rhizosphere was characterized by a significant decrease in both taxonomic and phylogenetic diversity relative to background soil communities, driven by reductions in both richness and evenness of the bacterial communities. Plant roots selectively stimulated the relative abundance of Alphaproteobacteria, Betaproteobacteria, and Bacteroidetes but reduced the abundance of Acidobacteria, Actinobacteria, and Firmicutes. Taxa that increased in relative abundance in the rhizosphere soil displayed phylogenetic clustering, suggesting some conservation and an evolutionary basis for the response of complex soil bacterial communities to the presence of plant roots. The reproducibility of rhizosphere succession and the apparent phylogenetic conservation of rhizosphere competence traits suggest adaptation of the indigenous bacterial community to this common grass over the many decades of its presence. IMPORTANCE We document the successional patterns of rhizosphere bacterial communities associated with a “wild” annual grass, Avena fatua, which is commonly a dominant plant in Mediterranean-type annual grasslands around the world; the plant was grown in its grassland soil. Most studies documenting rhizosphere microbiomes address “domesticated” plants growing in soils to which they are introduced. Rhizosphere bacterial communities exhibited a pattern of temporal succession that was consistent and repeatable over two growing seasons. There are few studies assessing the reproducibility over multiple seasons. Through the growing season, the rhizosphere community became progressively less diverse, likely reflecting root homogenization of soil microniches. Phylogenetic clustering of the rhizosphere dynamic taxa suggests evolutionary adaptation to Avena roots. The reproducibility of rhizosphere succession and the apparent phylogenetic conservation of rhizosphere competence traits suggest adaptation of the indigenous bacterial community to this common grass over the many decades of its presence. We document the successional patterns of rhizosphere bacterial communities associated with a “wild” annual grass, Avena fatua, which is commonly a dominant plant in Mediterranean-type annual grasslands around the world; the plant was grown in its grassland soil. Most studies documenting rhizosphere microbiomes address “domesticated” plants growing in soils to which they are introduced. Rhizosphere bacterial communities exhibited a pattern of temporal succession that was consistent and repeatable over two growing seasons. There are few studies assessing the reproducibility over multiple seasons. Through the growing season, the rhizosphere community became progressively less diverse, likely reflecting root homogenization of soil microniches. Phylogenetic clustering of the rhizosphere dynamic taxa suggests evolutionary adaptation to Avena roots. The reproducibility of rhizosphere succession and the apparent phylogenetic conservation of rhizosphere competence traits suggest adaptation of the indigenous bacterial community to this common grass over the many decades of its presence.

Identification of a novel cyanobacterial group as active diazotrophs in a coastal microbial mat using NanoSIMS analysis.

N2 fixation is a key process in photosynthetic microbial mats to support the nitrogen demands associated with primary production. Despite its importance, groups that actively fix N2 and contribute to the input of organic N in these ecosystems still remain largely unclear. To investigate the active diazotrophic community in microbial mats from the Elkhorn Slough estuary, Monterey Bay, CA, USA, we conducted an extensive combined approach, including biogeochemical, molecular and high-resolution secondary ion mass spectrometry (NanoSIMS) analyses. Detailed analysis of dinitrogenase reductase (nifH) transcript clone libraries from mat samples that fixed N2 at night indicated that cyanobacterial nifH transcripts were abundant and formed a novel monophyletic lineage. Independent NanoSIMS analysis of 15N2-incubated samples revealed significant incorporation of 15N into small, non-heterocystous cyanobacterial filaments. Mat-derived enrichment cultures yielded a unicyanobacterial culture with similar filaments (named Elkhorn Slough Filamentous Cyanobacterium-1 (ESFC-1)) that contained nifH gene sequences grouping with the novel cyanobacterial lineage identified in the transcript clone libraries, displaying up to 100% amino-acid sequence identity. The 16S rRNA gene sequence recovered from this enrichment allowed for the identification of related sequences from Elkhorn Slough mats and revealed great sequence diversity in this cluster. Furthermore, by combining 15N2 tracer experiments, fluorescence in situ hybridization and NanoSIMS, in situ N2 fixation activity by the novel ESFC-1 group was demonstrated, suggesting that this group may be the most active cyanobacterial diazotroph in the Elkhorn Slough mat. Pyrotag sequences affiliated with ESFC-1 were recovered from mat samples throughout 2009, demonstrating the prevalence of this group. This work illustrates that combining standard and single-cell analyses can link phylogeny and function to identify previously unknown key functional groups in complex ecosystems.