La face cachée de la dune : communautés fongiques du sol: dynamique, succession et interactions avec la végétation d’un écosystème dunaire côtier aux Îles de la Madeleine, Qc

Les écosystèmes dunaires remplissent plusieurs fonctions écologiques essentielles comme celle de protéger le littoral grâce à leur capacité d’amortissement face aux vents et vagues des tempêtes. Les dunes jouent aussi un rôle dans la filtration de l’eau, la recharge de la nappe phréatique, le maintien de la biodiversité, en plus de présenter un attrait culturel, récréatif et touristique. Les milieux dunaires sont très dynamiques et incluent plusieurs stades de succession végétale, passant de la plage de sable nu à la dune bordière stabilisée par l’ammophile à ligule courte, laquelle permet aussi l’établissement d’autres herbacées, d’arbustes et, éventuellement, d’arbres. Or, la survie de ces végétaux est intimement liée aux microorganismes du sol. Les champignons du sol interagissent intimement avec les racines des plantes, modifient la structure des sols, et contribuent à la décomposition de la matière organique et à la disponibilité des nutriments. Ils sont donc des acteurs clés de l’écologie des sols et contribuent à la stabilisation des dunes. Malgré cela, la diversité et la structure des communautés fongiques, ainsi que les mécanismes influençant leur dynamique écologique, demeurent relativement méconnus. Le travail présenté dans cette thèse explore la diversité des communautés fongiques à travers le gradient de succession et de conditions édaphiques d’un écosystème dunaire côtier afin d’améliorer la compréhension de la dynamique des sols en milieux dunaires. Une vaste collecte de données sur le terrain a été réalisée sur une plaine de dunes reliques se trouvant aux Îles de la Madeleine, Qc. J’ai échantillonné plus de 80 sites répartis sur l’ensemble de ce système dunaire et caractérisé les champignons du sol grâce au séquençage à haut débit. Dans un premier temps, j’ai dressé un portait d’ensemble des communautés fongiques du sol à travers les différentes zones des dunes. En plus d’une description taxonomique, les modes de vie fongiques ont été prédits afin de mieux comprendre comment les variations au niveau des communautés de champignons du sol peuvent se traduire en changements fonctionnels. J’ai observé un niveau de diversité fongique élevé (plus de 3400 unités taxonomiques opérationnelles au total) et des communautés taxonomiquement et fonctionnellement distinctes à travers un gradient de succession et de conditions édaphiques. Ces résultats ont aussi indiqué que toutes les zones des dunes, incluant la zone pionière, supportent des communautés fongiques diversifiées. Ensuite, le lien entre les communautés végétales et fongiques a été étudié à travers l’ensemble de la séquence dunaire. Ces résultats ont montré une augmentation claire de la richesse spécifique végétale, ainsi qu’une augmentation de la diversité des stratégies d’acquisition de nutriments (traits souterrains lié à la nutrition des plantes, soit mycorhizien à arbuscule, ectomycorhizien, mycorhizien éricoide, fixateur d’azote ou non spécialisé). J’ai aussi pu établir une forte corrélation entre les champignons du sol et la végétation, qui semblent tous deux réagir de façon similaire aux conditions physicochimiques du sol. Le pH du sol influençait fortement les communautés végétales et fongiques. Le lien observé entre les communautés végétales et fongiques met l’emphase sur l’importance des interactions biotiques positives au fil de la succession dans les environnements pauvres en nutriments. Finalement, j’ai comparé les communautés de champignons ectomycorhiziens associées aux principales espèces arborescentes dans les forêts dunaires. J’ai observé une richesse importante, avec un total de 200 unités taxonomiques opérationnelles ectomycorhiziennes, appartenant principalement aux Agaricomycètes. Une analyse de réseaux n’a pas permis de détecter de modules (c'est-à-dire des sous-groupes d’espèces en interaction), ce qui indique un faible niveau de spécificité des associations ectomycorhiziennes. De plus, je n’ai pas observé de différences en termes de richesse ou de structure des communautés entre les quatre espèces hôtes. En conclusion, j’ai pu observer à travers la succession dunaire des communautés diversifiées et des structures distinctes selon la zone de la dune, tant chez les champignons que chez les plantes. La succession semble toutefois moins marquée au niveau des communautés fongiques, par rapport aux patrons observés chez les plantes. Ces résultats ont alimenté une réflexion sur le potentiel et les perspectives, mais aussi sur les limitations des approches reposant sur le séquençage à haut-débit en écologie microbienne.

The Sinorhizobium meliloti EmrR Regulator Is Required for Efficient Colonization of Medicago sativa Root Nodules

The nitrogen-fixing bacterium Sinorhizobium meliloti must adapt to diverse conditions encountered during its symbiosis with leguminous plants. We characterized a new symbiotically relevant gene, emrR (SMc03169), whose product belongs to the TetR family of repressors and is divergently transcribed from emrAB genes encoding a putative major facilitator superfamily-type efflux pump. An emrR deletion mutant produced more succinoglycan, displayed increased cell-wall permeability, and exhibited higher tolerance to heat shock. It also showed lower tolerance to acidic conditions, a reduced production of siderophores, and lower motility and biofilm formation. The simultaneous deletion of emrA and emrR genes restored the mentioned traits to the wild-type phenotype, except for survival under heat shock, which was lower than that displayed by the wild-type strain. Furthermore, the ΔemrR mutant as well as the double ΔemrAR mutant was impaired in symbiosis with Medicago sativa; it formed fewer nodules and competed poorly with the wild-type strain for nodule colonization. Expression profiling of the ΔemrR mutant showed decreased expression of genes involved in Nod-factor and rhizobactin biosynthesis and in stress responses. Expression of genes directing the biosynthesis of succinoglycan and other polysaccharides were increased. EmrR may therefore be involved in a regulatory network targeting membrane and cell wall modifications in preparation for colonization of root hairs during symbiosis.

Heat shock response in bacteria with large genomes: lessons from rhizobia

Rhizobia are important soil bacteria due to their ability to establish nitrogen-fixing symbioses with legume plants. In this dual lifestyle, as free-living bacteria or as plant symbiont, rhizobia are often exposed to different environmental stresses. The present chapter overviews the current knowledge on the heat shock response of rhizobia, highlighting how these large genome bacteria respond to heat from a transcriptional point of view. Response to heat shock in rhizobia involves genome wide changes in the transcriptome that may affect more than 30% of the genome and involve all replicons. In addition to the expected upregulation of genes already known to be involved in stress response (dnaK, groEL, ibpA, clpB), the reports on the heat shock response in rhizobia also showed particular aspects of stress response in these resourceful bacteria. The transcriptional response to heat in rhizobia includes the overexpression of a large number of genes involved in transcription and carbohydrate transport and metabolism. Additional studies are needed in order to better understand the transcriptional regulation of stress response in bacteria with large genomes.

Agronomic efficiency of Bradyrhizobium in peanut under different environments in Brazilian Northeast.

Several legumes have natural ability to associate with nitrogen - fixing bacteria known as rhizobia. The efficiency of this association depends on the plant and bacterial genotype and the edaphoclimatic conditions. Peanut is a tropical legume able to associate with a wide range of rhizobia and the selection of efficient bacteria is important to increase the nitrogen fixation in this crop. In order to investigate the agronomic efficiency of two Bradyrhizobiumstrains, two peanut genotypes were used in field trails carried out in three environments located at Brazilian Northeast. The genotypes (BR1 and L7 Bege) were submitted to rhizobial inoculation (SEMIA 6144 or ESA 123, both Bradyrhizobium strains, and chemical nitrogen fertilization in randomized block design experiments. The following traits were analyzed: flowering (F), main axis height (MAH), number of nodules/plant (NN), number of pods/plant (NP) and weight of pods (WP). Differential responses were found in all to treatments to NN, NP and WP, in the three environments studied. Overall, ESA 123 showed good agronomic performance inducing higher pod production. The results supportthe evaluation of the Bradyrhizobium in further experiments aiming at its recommendation to commercial inoculants in Brazilian Northeast region.

Ocorrência de bactérias diazotroficas e fungos micorrizicos arbusculares na cultura da mandioca.

Este trabalho teve como objetivo avaliar a ocorrencia, isolar e identificar fungos micorrízicos carbunculares associados a cultura da mandioca (Manihot esculenta). Amostras de solo rizosférico e de varias partes da planta (raízes, tubérculos, manivas e folhas) de locais nos Estados do Rio de Janeiro, São Paulo e Paraná, foram inoculadas nos meios LGI-P, NFb-malato e NFb-GOC, avaliando-se o numero mais provável de células e a atividade de redução de acetileno. Bactérias diazotróficas foram isoladas de todas as partes da planta, com exceção das folhas, sendo identificadas como Klebsiella sp., Azospirillum lipoferum e uma bactéria denominada "E", provavelmente pertencente ao gênero Burkholderia. A bactéria E acumulou de 7,63 mg a 14,84 mg de N/g de C em meio semi-solido, isento de N, e conseguiu manter a capacidade de fixação biológica de N, mesmo apos uma dezena de repicagens consecutivas. A colonização micorrízica variou de 31% a 69%, e a densidade de esporos de 10 a 384 esporos/100 mL de solo, predominando as espécies Entrophospora colombiana e Acaulospora scrobiculata no Rio de Janeiro, A. scrobiculata e Scutellospora heterogama no Paraná e em Piracicaba (São Paulo) e A. appendicula e S. pellucida em Campinas (São Paulo). This study was performed to evaluate the occurrence and to isolate and identify diazotrophic bacteria and arbuscular mycorrhizal fungi associated with the cassava (Manihot esculenta) crop. Samples from rhizosperical soil, roots, tubers, stems and leaves from several localities of the States of Rio de Janeiro, Sao Paulo and Parana, in Brazil, were inoculated in three media specific for diazotrophic associative bacteria, LG1-P, NFb-malate and NFb-GOC, evaluating the most probable number of cells and the acetylene-reducing activity. Diazotrophic bacteria were detected in all plant parts except for the leaves, and were identified as Klebsiella sp., Azospirillum lipoferum and a bacterium called "E", probably belonging to the Burkholderia genus. Bacterium E was able to accumulate, in the N-free semi-solid media, from 7.63 to 14.84 mg of N/g of C and to maintain N fixation capacity after ten consecutive transferences. Mycorrhizal root colonization varied from 31% to 69% and spore density from 10 to 384 spores/100 mL of soil, predominanting the species Entrophospora colombiana and Acaulospora scrobiculata in Rio de Janeiro, A. scrobiculata and Scutellospora heterogama in Parana and in Piracicaba (Sao Paulo), and A. appendicula and S. pellucida in Campinas (Sao Paulo).

Soil N2O emissions under N2-fixing legumes and N-fertilised canola: A reappraisal of emissions factor calculations

Introducing nitrogen (N)-fixing legumes into cereal-based crop rotations reduces synthetic fertiliser-N use and may mitigate soil emissions of nitrous oxide (N2O). Current IPCC calculations assume 100% of legume biomass N as the anthropogenic N input and use 1% of this as an emission factor (EF)—the percentage of input N emitted as N2O. However, legumes also utilise soil inorganic N, so legume-fixed N is typically less than 100% of legume biomass N. In two field experiments, we measured soil N2O emissions from a black Vertosol in sub-tropical Australia for 12 months after sowing of chickpea (Cicer arietinum L.), canola (Brassica napus L.), faba bean (Vicia faba L.), and field pea (Pisum sativum L.). Cumulative N2O emissions from N-fertilised canola (624 g N2O-N ha−1) greatly exceeded those from chickpea (127 g N2O-N ha−1) in Experiment 1. Similarly, N2O emitted from canola (385 g N2O-N ha−1) in Experiment 2 was significantly greater than chickpea (166 g N2O-N ha−1), faba bean (166 g N2O-N ha−1) or field pea (135 g N2O-N ha−1). Highest losses from canola were recorded during the growing season, whereas 75% of the annual N2O losses from the legumes occurred post-harvest. Legume N2-fixation provided 37–43% (chickpea), 54% (field pea) and 64% (faba bean) of total plant biomass N. Using only fixed-N inputs, we calculated EFs for chickpea (0.13–0.31%), field pea (0.18%) and faba bean (0.04%) that were significantly less than N-fertilised canola (0.48–0.78%) (P < 0.05), suggesting legume-fixed N is a less emissive form of N input to the soil than fertiliser N. Inputs of legume-fixed N should be more accurately quantified to properly gauge the potential for legumes to mitigate soil N2O emissions. EF’s from legume crops need to be revised and should include a factor for the proportion of the legume’s N derived from the atmosphere.

The effect of pasture utilization rate on stocks of soil organic carbon and total nitrogen in a semi-arid tropical grassland

The influence of grazing management on total soil organic carbon (SOC) and soil total nitrogen (TN) in tropical grasslands is an issue of considerable ecological and economic interest. Here we have used linear mixed models to investigate the effect of grazing management on stocks of SOC and TN in the top 0.5 m of the soil profile. The study site was a long-term pasture utilization experiment, 26 years after the experiment was established for sheep grazing on native Mitchell grass (Astrebla spp.) pasture in northern Australia. The pasture utilization rates were between 0% (exclosure) and 80%, assessed visually. We found that a significant amount of TN had been lost from the top 0.1 m of the soil profile as a result of grazing, with 80% pasture utilization resulting in a loss of 84 kg ha−1 over the 26-year period. There was no significant effect of pasture utilization rate on TN when greater soil depths were considered. There was no significant effect of pasture utilization rate on stocks of SOC and soil particulate organic carbon (POC), or the C:N ratio at any depth; however, visual trends in the data suggested some agreement with the literature, whereby increased grazing pressure appeared to: (i) decrease SOC and POC stocks; and, (ii) increase the C:N ratio. Overall, the statistical power of the study was limited, and future research would benefit from a more comprehensive sampling scheme. Previous studies at the site have found that a pasture utilization rate of 30% is sustainable for grazing production on Mitchell grass; however, given our results, we conclude that N inputs (possibly through management of native N2-fixing pasture legumes) should be made for long-term maintenance of soil health, and pasture productivity, within this ecosystem.

Ocean acidification impacts on nitrogen fixation in the coastal western Mediterranean Sea

The effects of ocean acidification on nitrogen (N2) fixation rates and on the community composition of N2-fixing microbes (diazotrophs) were examined in coastal waters of the North-Western Mediterranean Sea. Nine experimental mesocosm enclosures of ∼50 m3 each were deployed for 20 days during June-July 2012 in the Bay of Calvi, Corsica, France. Three control mesocosms were maintained under ambient conditions of carbonate chemistry. The remainder were manipulated with CO2 saturated seawater to attain target amendments of pCO2 of 550, 650, 750, 850, 1000 and 1250 μatm. Rates of N2 fixation were elevated up to 10 times relative to control rates (2.00 ± 1.21 nmol L-1d-1) when pCO2 concentrations were >1000 μatm and pHT (total scale) < 7.74. Diazotrophic phylotypes commonly found in oligotrophic marine waters, including the Mediterranean, were not present at the onset of the experiment and therefore, the diazotroph community composition was characterised by amplifying partial nifH genes from the mesocosms. The diazotroph community was comprised primarily of cluster III nifH sequences (which include possible anaerobes), and proteobacterial (α and γ) sequences, in addition to small numbers of filamentous (or pseudo-filamentous) cyanobacterial phylotypes. The implication from this study is that there is some potential for elevated N2 fixation rates in the coastal western Mediterranean before the end of this century as a result of increasing ocean acidification. Observations made of variability in the diazotroph community composition could not be correlated with changes in carbon chemistry, which highlights the complexity of the relationship between ocean acidification and these keystone organisms.

Introducing Acacia mangium trees in Eucalyptus grandis plantations: consequences for soil organic matter stocks and nitrogen mineralization

Background and aims Eucalyptus plantations cover 20 million hectares on highly weathered soils. Large amounts of nitrogen (N) exported during harvesting lead to concerns about their sustainability. Our goal was to assess the potential of introducing A. mangium trees in highly productive Eucalyptus plantations to enhance soil organic matter stocks and N availability. Methods A randomized block design was set up in a Brazilian Ferralsol soil to assess the effects of mono-specific Eucalyptus grandis (100E) and Acacia mangium (100A) stands and mixed plantations (50A:50E)on soil organic matter stocks and net N mineralization. Results A 6-year rotation of mono-specific A. mangium plantations led to carbon (C) and N stocks in the forest floor that were 44% lower and 86% higher than in pure E. grandis stands, respectively. Carbon and N stocks were not significantly different between the three treatments in the 0-15 cm soil layer. Field incubations conducted every 4 weeks for the two last years of the rotation estimated net soil N mineralization in 100A and 100E at 124 and 64 kg ha(-1) yr(-1), respectively. Nitrogen inputs to soil with litterfall were of the same order as net N mineralization. Conclusions Acacia mangium trees largely increased the turnover rate of N in the topsoil. Introducing A. mangium trees might improve mineral N availability in soils where commercial Eucalyptus plantations have been managed for a long time.

Database of diazotrophs in global ocean: abundances, biomass and nitrogen fixation rates

[EN] Marine N2 fixing microorganisms, termed diazotrophs, are a key functional group in marine pelagic ecosystems. The biological fixation of dinitrogen (N2) to bioavailable nitrogen provides an important new source of nitrogen for pelagic marine ecosystems 5 and influences primary productivity and organic matter export to the deep ocean. As one of a series of efforts to collect biomass and rates specific to different phytoplankton functional groups, we have constructed a database on diazotrophic organisms in the global pelagic upper ocean by compiling about 12 000 direct field measurements of cyanobacterial diazotroph abundances (based on microscopic cell counts or qPCR 10 assays targeting the nifH genes) and N2 fixation rates. Biomass conversion factors are estimated based on cell sizes to convert  abundance data to diazotrophic biomass. The database is limited spatially, lacking large regions of the ocean especially in the Indian Ocean. The data are approximately log-normal distributed, and large variances exist in most sub-databases with non-zero values differing 5 to 8 orders of magnitude. 15 Lower mean N2 fixation rate was found in the North Atlantic Ocean than the Pacific Ocean. Reporting the geometric mean and the range of one geometric standard error below and above the geometric mean, the pelagic N2 fixation rate in the global ocean is estimated to be 62 (53–73) TgNyr−1 and the pelagic diazotrophic biomass in the global ocean is estimated to be 4.7 (2.3–9.6) TgC from cell counts and to 89 (40–20 200) TgC from nifH-based abundances. Uncertainties related to biomass conversion factors can change the estimate of geometric mean pelagic diazotrophic biomass in the global ocean by about ±70 %. This evolving database can be used to study spatial and temporal distributions and variations of marine N2 fixation, to validate geochemical estimates and to parameterize and validate biogeochemical models. The database is 25 stored in PANGAEA (http://doi.pangaea.de/10.1594/PANGAEA.774851).

Combined effects of CO2 and light on large and small isolates of the unicellular N2-fixing cyanobacterium Crocosphaera watsonii from the western tropical Atlantic Ocean

We examined the combined effects of light and pCO2 on growth, CO2-fixation and N2-fixation rates by strains of the unicellular marine N2-fixing cyanobacterium Crocosphaera watsonii with small (WH0401) and large (WH0402) cells that were isolated from the western tropical Atlantic Ocean. In low-pCO2-acclimated cultures (190 ppm) of WH0401, growth, CO2-fixation and N2-fixation rates were significantly lower than those in cultures acclimated to higher (present-day 385 ppm, or future 750 ppm) pCO2 treatments. Growth rates were not significantly different, however, in low-pCO2-acclimated cultures of WH0402 in comparison with higher pCO2 treatments. Unlike previous reports for C. watsonii (strain WH8501), N2-fixation rates did not increase further in cultures of WH0401 or WH0402 when acclimated to 750 ppm relative to those maintained at present-day pCO2. Both light and pCO2 had a significant negative effect on gross : net N2-fixation rates in WH0402 and trends were similar in WH0401, implying that retention of fixed N was enhanced under elevated light and pCO2. These data, along with previously reported results, suggest that C. watsonii may have wide-ranging, strain-specific responses to changing light and pCO2, emphasizing the need for examining the effects of global change on a range of isolates within this biogeochemically important genus. In general, however, our data suggest that cellular N retention and CO2-fixation rates of C. watsonii may be positively affected by elevated light and pCO2 within the next 100 years, potentially increasing trophic transfer efficiency of C and N and thereby facilitating uptake of atmospheric carbon by the marine biota.

Seawater carbonate chemistry and combined mechanistic effects of CO2 and light on the N2-fixing cyanobacterium Trichodesmium IMS101, 2010

The marine diazotrophic cyanobacterium Trichodesmium responds to elevated atmospheric CO2 partial pressure (pCO2) with higher N2 fixation and growth rates. To unveil the underlying mechanisms, we examined the combined influence of pCO2(150 and 900 µatm) and light (50 and 200 µmol photons m-2 s-1) on TrichodesmiumIMS101. We expand on a complementary study that demonstrated that while elevated pCO2 enhanced N2 fixation and growth, oxygen evolution and carbon fixation increased mainly as a response to high light. Here, we investigated changes in the photosynthetic fluorescence parameters of photosystem II, in ratios of the photosynthetic units (photosystem I:photosystem II), and in the pool sizes of key proteins involved in the fixation of carbon and nitrogen as well as their subsequent assimilation. We show that the combined elevation in pCO2 and light controlled the operation of the CO2-concentrating mechanism and enhanced protein activity without increasing their pool size. Moreover, elevated pCO2 and high light decreased the amounts of several key proteins (NifH, PsbA, and PsaC), while amounts of AtpB and RbcL did not significantly change. Reduced investment in protein biosynthesis, without notably changing photosynthetic fluxes, could free up energy that can be reallocated to increase N2 fixation and growth at elevated pCO2 and light. We suggest that changes in the redox state of the photosynthetic electron transportchain and posttranslational regulation of key proteins mediate the high flexibility in resources and energy allocation in Trichodesmium. This strategy should enableTrichodesmium to flourish in future surface oceans characterized by elevated pCO2, higher temperatures, and high light.

Nitrogen fuelling of the pelagic food web of the Tropical Atlantic

We estimated the relative contribution of atmospheric Nitrogen (N) input (wet and dry deposition and N fixation) to the epipelagic food web by measuring N isotopes of different functional groups of epipelagic zooplankton along 23°W (17°N-4°S) and 18°N (20-24°W) in the Eastern Tropical Atlantic. Results were related to water column observations of nutrient distribution and vertical diffusive flux as well as colony abundance of Trichodesmium obtained with an Underwater Vision Profiler (UVP5). The thickness and depth of the nitracline and phosphocline proved to be significant predictors of zooplankton stable N isotope values. Atmospheric N input was highest (61% of total N) in the strongly stratified and oligotrophic region between 3 and 7°N, which featured very high depth-integrated Trichodesmium abundance (up to 9.4×104 colonies m-2), strong thermohaline stratification and low zooplankton delta15N (~2 per mil). Relative atmospheric N input was lowest south of the equatorial upwelling between 3 and 5°S (27%). Values in the Guinea Dome region and north of Cape Verde ranged between 45 and 50%, respectively. The microstructure-derived estimate of the vertical diffusive N flux in the equatorial region was about one order of magnitude higher than in any other area (approximately 8 mmol m-2 d 1). At the same time, this region received considerable atmospheric N input (35% of total). In general, zooplankton delta15N and Trichodesmium abundance were closely correlated, indicating that N fixation is the major source of atmospheric N input. Although Trichodesmium is not the only N fixing organism, its abundance can be used with high confidence to estimate the relative atmospheric N input in the tropical Atlantic (r2 = 0.95). Estimates of absolute N fixation rates are two- to tenfold higher than incubation-derived rates reported for the same regions. Our approach integrates over large spatial and temporal scales and also quantifies fixed N released as dissolved inorganic and organic N. In a global analysis, it may thus help to close the gap in oceanic N budgets.

Nitrogen fixation by native Bradyrhizobia in symbiosis with Lupinus mariae-josephae requires a T3SS encoding a NopE-like effector

Several bradyrhizobial isolates from L. mariae-josephae root nodules [1] contain a type III secretion system (T3SS) within a cluster of about 30 genes. Among those genes, ttsI codes for the transcriptional activator of the system. Mutation of ttsI resulted in the formation of white, non-fixing nodules with the natural legume host, L. mariae-josephae. The T3SS cluster also contains a gene coding for a NopE-like protein. NopE proteins have been demonstrated to be effectors in the Bradyrhizobium-soybean symbiosis [2] and belong to a small group of poorly characterized proteins from plant-associated bacteria that contain one or two autocleavage motifs known as DUF1521 (Schirrmeister et al. 2011). The amino acid sequence of a NopE-like protein in the L. mariae-josephae strain LmjC contains just one autocatalytic motif. This is unlike NopE1 and NopE2 proteins secreted by the T3SS of B. japonicum, that contain two motifs [3]. The autocleavage of LmjC NopE protein was analyzed after expression in E. coli and purification. Two protein fragments of the predicted sizes appeared in the presence of Ca2+, Cu2+, Cd2+, Zn2+ and Mn2+ cations. In contrast, autocleavage did not take place in the presence of Ni2+, Co2+ or Mg2+. Site-directed mutagenesis of the DUF1521 motif in LmjC NopE abolished self-cleavage in vitro. Symbiotic competence of a NopE- mutant with the L. mariae-josephae host was not affected. Possible roles of NopE are discussed.

Nitrogen isotope ratios of Cretaceous Atlantic sedimentary sequences

At two locations in the Atlantic Ocean (DSDP Sites 367 and 530) early to middle Cretaceous organic-carbon-rich beds (black shales) were found to have significantly lower delta15N values (lower 15N/14N ratios) than adjacent organic-carbon-poor beds (white limestones or green claystones). While these lithologies are of marine origin, the black strata in particular have delta15N values that are significantly lower than those previously found in the marine sediment record and most contemporary marine nitrogen pools. In contrast, black, organic-carbon-rich beds at a third site (DSDP Site 603) contain predominantly terrestrial organic matter and have C- and N-isotopic compositions similar to organic matter of modern terrestrial origin. The recurring 15N depletion in the marine-derived Cretaceous sequences prove that the nitrogen they contain is the end result of an episodic and atypical biogeochemistry. Existing isotopic and other data indicate that the low 15N relative abundance is the consequence of pelagic rather than post-depositional processes. Reduced ocean circulation, increased denitrification, and, hence, reduced euphoric zone nitrate availability may have led to Cretaceous phytoplankton assemblages that were periodically dominated by N2-fixing blue-green algae, a possible source of this sediment 15N-depletion. Lack of parallel isotopic shifts in Cretaceous terrestrially-derived nitrogen (Site 603) argues that the above change in nitrogen cycling during this period did not extend beyond the marine environment.