Radu Popa

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.

Pyrite Framboids as Biomarkers for Iron-Sulfur Systems

The role of microorganisms in the formation of pyrite framboids has been suggested, but remains unresolved. We identified subsurface habitats from a sulfidic aquifer present in carbonate rock in Romania, where abundant pyrite framboids were found associated with microbial mats. Three types of subsurface microbial communities were studied: submerged mats overlaying clay-rich sediments, floating mats developed at the water-gas (sulfide/carbon dioxide) interface, and detrital FeS-rich sediments associated with floating microbial mats. All three habitats are anoxic, low temperature, low salinity, saturated with bicarbonate, and contain hydrogen sulfide, thiosulphate, and elemental sulfur. Sulfur stable isotope fractionation suggests that pyrite framboids from the floating microbial mats have a biogenic origin. Pyrite framboids characteristics, such as nucleation, growth, and spheroidicity, depend on conditions within the microbial mats and the inheritance of shape during the replacement of spheroidal greigite. We hypothesize that biogenic pyrite is formed in these microbial communities via a sulfide/ferrous iron pyritization mechanism. Some of the signatures of this type of biogenic pyritization are a similarity in size distribution between FeS spheroids and FeS2 framboids, the presence of microorganisms using sulfide and ferrous iron as a source of energy, reversed iron sulfide bilayers, a narrow size distribution of the pyrite framboids, a small numbers of large microcrystallites within the framboids, a S-fractionation up to 11.14 between H2S and FeS and up to 16.24 between H2S and FeS2. These signatures are relevant for the study of the biologically controlled pyritization and thus they are valuable tools for geomicrobiology, paleo-microbiology and exobiology.

Low-Temperature Growth of Shewanella oneidensis MR-1

ABSTRACT Shewanella oneidensis MR-1 is a mesophilic bacterium with a maximum growth temperature of ≈35°C but the ability to grow over a wide range of temperatures, including temperatures near zero. At room temperature (≈22°C) MR-1 grows with a doubling time of about 40 min, but when moved from 22°C to 3°C, MR-1 cells display a very long lag phase of more than 100 h followed by very slow growth, with a doubling time of ≈67 h. In comparison to cells grown at 22°C, the cold-grown cells formed long, motile filaments, showed many spheroplast-like structures, produced an array of proteins not seen at higher temperature, and synthesized a different pattern of cellular lipids. Frequent pilus-like structures were observed during the transition from 3 to 22°C.

Organization and Elemental Analysis of P-, S-, and Fe-rich Inclusions in a Population of Freshwater Magnetococci

We characterized a population of bilophotrichously flagellated freshwater magnetotactic cocci (MC), referred to as ARB-1, morphologically, chemically, and phylogenetically. Cells examined using light microscopy, fluorescence microscopy, environmental scanning electron microscopy (ESEM), and transmission electron microscopy (TEM) contained three types of intracellular inclusions placed in a specific arrangement within the cell. Elemental compositions of the inclusions were determined using energy dispersive X-ray spectroscopy (EDS) from both ESEM and TEM. The spherical to ovoid cells contained two large phosphorus-rich inclusions that occupied most of the cell volume and appeared to be enclosed in a membrane or coating. Several smaller sulfur-rich inclusions were located at the end of the cell opposite the flagellar bundles. The magnetosomes, arranged either as a cluster, a chain, or a combination of both, were located proximal to the two flagellar bundles. Magnetite was identified as the mineral phase of the magnetosomes using selected area electron diffraction (SAED) and by measuring lattice fringe spacings of the crystals. The magnetite crystals were hexagonal prisms that averaged 82 nm in length and thus fit into the single-magnetic-domain size range. Phylogenetic analysis of the 16S rRNA gene sequences suggests that it is a mixed population of MC that form a monophyletic clade distinct from but similar to other uncultured MC.

Microbiological characterization of a sulfide‐rich groundwater ecosystem

Movile Cave, recently discovered in southern Romania, contains sulfide‐rich thermal waters in submerged passages, as well as isolated air pockets. The water surfaces within the air pockets are covered by substantial microbial biofilms, while the air bells contain an abundant and diverse community of terrestrial and aquatic animal species. Based on the results of dehydrogenase activity, fecal streptococci counts, and stable carbon isotope ratios, we propose that the cave community is biologically isolated and receives little, if any, organic carbon inputs from the surface environment. Several sulfide‐oxidizing chemoautotrophic bacteria were isolated from the cave waters. One putative Thiosphaera sp. strain, LV‐43, was further characterized. The presence and high level activity of RuBisCO was clearly demonstrated in this strain.

Boron-Containing Compounds as Preventive and Chemotherapeutic Agents for Cancer

In the last few years boron (B) compounds became increasingly frequent in the chemotherapy of some forms of cancer with high malignancy and of inoperable cancers. As more B-based therapy chemicals are developed it is necessary to review the correlation between B and the incidence of different forms of cancer, the biochemical and molecular mechanisms influenced by B and to explore the relevance of B in the chemoprevention of cancer. This minireview analyzes dietary and therapeutic principles based on the chemistry of B compounds. We summarize studies correlating B-rich diets or B-rich environments with regional risks of specific forms of cancers, and studies about the utilization of natural and synthetic B-containing compounds as anticancer agents. We review mechanisms where B-containing compounds interfere with the physiology and reproduction of cancer cells. Types of cancers most frequently impacted by B-containing compounds include prostate, breast, cervical and lung cancer. Mechanisms involving B activity on cancer cells are based on the inhibition of a variety of enzymatic activities, including serine proteases, NAD-dehydrogenases, mRNA splicing and cell division, but also receptor binding mimicry, and the induction of apoptosis. Boron-enriched diets resulted in significant decrease in the risk for prostate and cervical cancer, and decrease in lung cancer in smoking women. Boron-based compounds show promising effects for the chemotherapy of specific forms of cancer, but due to specific benefits should also be included in cancer chemopreventive strategies.

Heterogeneous Occupancy and Density Estimates of the Pathogenic Fungus Batrachochytrium dendrobatidis in Waters of North America

Biodiversity losses are occurring worldwide due to a combination of stressors. For example, by one estimate, 40% of amphibian species are vulnerable to extinction, and disease is one threat to amphibian populations. The emerging infectious disease chytridiomycosis, caused by the aquatic fungus Batrachochytrium dendrobatidis (Bd), is a contributor to amphibian declines worldwide. Bd research has focused on the dynamics of the pathogen in its amphibian hosts, with little emphasis on investigating the dynamics of free-living Bd. Therefore, we investigated patterns of Bd occupancy and density in amphibian habitats using occupancy models, powerful tools for estimating site occupancy and detection probability. Occupancy models have been used to investigate diseases where the focus was on pathogen occurrence in the host. We applied occupancy models to investigate free-living Bd in North American surface waters to determine Bd seasonality, relationships between Bd site occupancy and habitat attributes, and probability of detection from water samples as a function of the number of samples, sample volume, and water quality. We also report on the temporal patterns of Bd density from a 4-year case study of a Bd-positive wetland. We provide evidence that Bd occurs in the environment year-round. Bd exhibited temporal and spatial heterogeneity in density, but did not exhibit seasonality in occupancy. Bd was detected in all months, typically at less than 100 zoospores L−1. The highest density observed was ∼3 million zoospores L−1. We detected Bd in 47% of sites sampled, but estimated that Bd occupied 61% of sites, highlighting the importance of accounting for imperfect detection. When Bd was present, there was a 95% chance of detecting it with four samples of 600 ml of water or five samples of 60 mL. Our findings provide important baseline information to advance the study of Bd disease ecology, and advance our understanding of amphibian exposure to free-living Bd in aquatic habitats over time.

Olivine-Respiring Bacteria Isolated from the Rock-Ice Interface in a Lava-Tube Cave, a Mars Analog Environment

The boundary between ice and basalt on Earth is an analogue for some near-surface environments of Mars. We investigated neutrophilic iron-oxidizing microorganisms from the basalt-ice interface in a lava tube from the Oregon Cascades with perennial ice. One of the isolates (Pseudomonas sp. HerB) can use ferrous iron Fe(II) from the igneous mineral olivine as an electron donor and O(2) as an electron acceptor. The optimum growth temperature is ∼12-14°C, but growth also occurs at 5°C. Bicarbonate is a facultative source of carbon. Growth of Pseudomonas sp. HerB as a chemolithotrophic iron oxidizer with olivine as the source of energy is favored in low O(2) conditions (e.g., 1.6% O(2)). Most likely, microbial oxidation of olivine near pH 7 requires low O(2) to offset the abiotic oxidation of iron. The metabolic capabilities of this bacterium would allow it to live in near-surface, icy, volcanic environments of Mars in the present or recent geological past and make this type of physiology a prime candidate in the search for life on Mars.

Discrimination among iron sulfide species formed in microbial cultures

A quantitative method for the study of iron sulfides precipitated in liquid cultures of bacteria is described. This method can be used to quantify and discriminate among amorphous iron sulfide (FeS(amorph)), iron monosulfide minerals such as mackinawite or greigite (FeS(min)), and iron disulfide minerals such as pyrite or marcasite (FeS(2min)) formed in liquid cultures. Degradation of iron sulfides is performed using a modified Cr(2+) reduction method with reflux distillation. The basic steps of the method are: first, separation of FeS(amorph); second, elimination of interfering species of S such as colloidal sulfur (S(c) degrees ), thiosulphate (S(2)O(3)(2-)) and polysulfides (S(x)(2-)); third, separation of FeS(min); and fourth, separation of FeS(2min). The final product is H(2)S which is determined after trapping. The efficiency of recovery is 96-99% for FeS(amorph), 76-88% for FeS(min), and >97% for FeS(2min). This method has a high reproducibility if the experimental conditions are rigorously applied and only glass conduits are used. A well ventilated fume hood must be used because of the toxicity and volatility of several reagents and products. The advantage relative to previously described methods are better resolution for iron sulfide species and use of the same bottles for both incubation of cultures and acid degradation. The method can also be used for Fe/S stoichiometry with sub-sampling and Fe analysis.