Lead and cadmium in the Weser Estuary and the German Bight: Correlations between bacteria populations, heavy metals and organic carbon

Organic carbon, lead and cadmium contents of 20 sediments were determined and compared with the colony counts of anaerobic heterotrophic, anaerobic nitrogen fixing, chitinoclastic and cellulolytic bacteria. Organic carbon content, which is dependent on the sediment type, was positively correlated with lead and cadmium as well as with colony counts of all 4 physiological groups of bacteria. Even the sediments with the highest concentrations of 251.7 ppm Pb and 3.1 ppm Cd showed no reduction in their colony counts. From 2 different sediment sampIes with lead contents of 140 ppm and 21 ppm lead tolerance of the aerobic heterotrophic bacteria was investigated. However, no significant difference in lead tolerance of the 2 heterotrophic populations was found. Water from 6 stations was analysed for dissolved and particulate organic carbon, lead and cadmium. Dissolved lead concentrations were in the range of 0.2-0.5 µg/l and the particulate lead contents were between 0.05 and 4.3 µg/l. The concentrations of total lead for the stations off-shore were only one order of magnitude from the concentrations of the near-shore stations. The same phenomenon was observed for dissolved cadmium (0.02 - 0.25 µg/l) and particulate cadmium (0.003 - 0.15 µg/I) concentrations. Correlations between dissolved (1.6 - 10.8 mg/I) and particulate organic carbon (0.25 - 1.53 mg/I) with dissolved and particulate lead or cadmium were not found.

Seawater carbonate chemistry and processes during experiments with cyanobacterium Nodularia spumigena, 2009

The surface ocean absorbs large quantities of the CO2 emitted to the atmosphere from human activities. As this CO2 dissolves in seawater, it reacts to form carbonic acid. While this phenomenon, called ocean acidification, has been found to adversely affect many calcifying organisms, some photosynthetic organisms appear to benefit from increasing [CO2]. Among these is the cyanobacterium Trichodesmium, a predominant diazotroph (nitrogen-fixing) in large parts of the oligotrophic oceans, which responded with increased carbon and nitrogen fixation at elevated pCO2. With the mechanism underlying this CO2 stimulation still unknown, the question arises whether this is a common response of diazotrophic cyanobacteria. In this study we therefore investigate the physiological response of Nodularia spumigena, a heterocystous bloom-forming diazotroph of the Baltic Sea, to CO2-induced changes in seawater carbonate chemistry. N. spumigena reacted to seawater acidification/carbonation with reduced cell division rates and nitrogen fixation rates, accompanied by significant changes in carbon and phosphorus quota and elemental composition of the formed biomass. Possible explanations for the contrasting physiological responses of Nodularia compared to Trichodesmium may be found in the different ecological strategies of non-heterocystous (Trichodesmium) and heterocystous (Nodularia) cyanobacteria.

Seasonal evolution of soil respiration in a mixed forestry plantation of walnuts (Juglans × intermedia Carr.) and alders (Alnus cordata (Loisel.) Duby) in Domaine de Restinclières (France)

The ecological intensification of crops is proposed as a solution to the growing demand of agricultural and forest resources, in opposition to intensive monocultures. The introduction of mixed cultures as mixtures between nitrogen fixing species and non nitrogen fixing species intended to increase crop yield as a result of an improvement of the available nitrogen and phosphorus in soil. Relationship between crops have received little attention despite the wide range of advantages that confers species diversity to these systems, such as increased productivity, resilience to disruption and ecological sustainability. Forests and forestry plantations can develop an important role in storing carbon in their tissues, especially in wood which become into durable product. A simplifying parameter to analyze the amount allocated carbon by plantation is the TBCA (total belowground carbon allocation), whereby, for short periods and mature plantations, is admitted as the subtraction between soil carbon efflux and litterfall. Soil respiration depends on a wide range of factors, such as soil temperature and soil water content, soil fertility, presence and type of vegetation, among others. The studied orchard is a mixed forestry plantation of hybrid walnuts(Juglans × intermedia Carr.) for wood and alders (Alnus cordata (Loisel.) Duby.), a nitrogen fixing specie through the actinomycete Frankia alni ((Woronin, 1866) Von Tubeuf 1895). The study area is sited at Restinclières, a green area near Montpellier (South of France). In the present work, soil respiration varied greatly throughout the year, mainly influenced by soil temperature. Soil water content did not significantly influence the response of soil respiration as it was constant during the measurement period and under no water stress conditions. Distance between nearest walnut and measurement was also a highly influential factor in soil respiration. Generally there was a decreasing trend in soil respiration when the distance to the nearest tree increased. It was also analyzed the response of soil respiration according to alder presence and fertilizer management (50 kg N·ha-1·año-1 from 1999 to 2010). None of these treatments significantly influenced soil respiration, although previous studies noticed an inhibition in rates of soil respiration under fertilized conditions and high rates of available nitrogen. However, treatments without fertilization and without alder presence obtained higher respiration rates in those cases with significant differences. The lack of significant differences between treatments may be due to the high coefficient of variation experienced by soil respiration measurements. Finally an asynchronous fluctuation was observed between soil respiration and litterfall during senescence period. This is possibly due to the slowdown in the emission of exudates by roots during senescence period, which are largely related to microbial activity.

Native bradyrhizobial symbionts of Lupinus mariae-josephae, a unique endemism thriving in alkaline soils in Eastern Spain

Lupinus mariae-josephae (Lmj) es una especie de lupino endémica de una pequeña y específica área de Comunidad Valenciana (Este de España), donde prospera en suelos alcalinoscalcáreos, un hábitat singular para los altramuces, que crecen preferentemente en suelos ácidos o neutros. Esto hace de Lmj una especie de lupino única. Cuando se inició este trabajo, la extensión conocida de este endemismo abarcaba unos 700 kilómetros cuadrados, confinados en la provincia de Valencia. En esta área, Lmj prospera en pequeñas poblaciones aisladas que contienen un número reducido de plantas por lo que se la consideró una especie en peligro de extinción. Todos los esfuerzos, utilizando estrategias clásicas dirigidas a ampliar el área de crecimiento de Lmj y garantizar su conservación, han tenido un éxito limitado. El trabajo que se presenta está dirigido a mejorar el conocimiento de la ecología de Lmj, en particular la interacción simbiótica que establece con bacterias del suelo denominadas rizobios y se centra en la caracterización fenotípica, filogenética y genómica de esos rizobios. También se investiga la posible contribución de la simbiosis en mejorar la conservación de Lmj. Para este fin, se han estudiado diferentes aspectos que se describen a continuación. El primero objetivo se centró en aislar y estudiar de la diversidad genética de las bacterias endosimbióticas de Lmj. . Se realizó un análisis filogenético de genes esenciales que mostró que las cepas de Lmj pertenecen al género Bradyrhizobium y que presentan una gran diversidad con características fenotípicas y simbióticas diferentes de cepas de Bradyrhizobium que nodulan otras especies de lupinos nativos de España (cepas ISLU). Las cepas estudiadas se dividieron en dos grupos (Clado I y Clado II). El Clado I, incluye a las cepas Lmj, definiendo un nuevo linaje, filogenéticamente relacionado con otras especies de Bradyrhizobium, como B. jicamae y B. elkanii. El Clado II contiene cepas ISLU relacionadas con cepas de B. canariense y B. japonicum que establecen simbiosis con lupinos de suelos ácidos. Otro análisis filogenético basado en genes simbióticos, distribuyó las cepas de Lmj en sólo dos grupos diferentes. La singularidad y gran diversidad de estas cepas en una pequeña área geográfica, hacen de este, un atractivo sistema para el estudio de la evolución y adaptación de las bacterias simbióticas a su respectiva planta huésped. Adicionalmente, se estudio la presencia de bacterias capaces de nodular Lmj en suelos básicos de Chiapas, México. Sorprendentemente, estos suelos contienen bacterias capaces establecer interacciones simbióticas eficientes con Lmj en ensayos de invernadero. A continuación se investigó la taxonomía de los endosimbiontes de Lmj analizando la secuencia de cuatro genes esenciales (16S rRNA, recA, glnII y atpD) y el promedio de identidad de nucleótidos de genomas completos de algunas cepas representativas de la diversidad (ANIm). Se identificaron nuevas especies de Bradyrhizobium dentro del Clado I y se definió una de ellas: 'Bradyrhizobium valentinum' sp. nov (cepa tipo LmjM3T = CECT 8364T, LMG 2761T). También se abordó cómo conservar Lmj en su hábitat natural mediante inoculación con alguna de las cepas aisladas. Se demostró la ausencia de bacterias capaces de nodular Lmj en suelos rojos alcalinos o ‘‘terra rossa’’ de la Península Ibérica y Baleares. Dos cepas, altamente eficientes en cuanto a la fijación de nitrógeno, LmjC y LmjM3T, fueron seleccionadas para ser empleadas como inoculantes. Dos experimentos de campo llevados a cabo en años consecutivos en áreas con características edafoclimáticas similares a las que presentan las poblaciones de Lmj, lograron la reproducción exitosa de la planta. Se concluyó que un ciclo reproductivo exitoso de Lmj es absolutamente dependiente de la inoculación con sus simbiontes naturales y que la simbiosis debe ser considerada un factor esencial en estrategias de conservación de leguminosas en peligro. La obtención de varias secuencias genómicas de cepas aisladas de Lmj y de otras cepas de Bradyrhizobium reveló una alta similitud entre los genomas de las cepas del Clado I, y permitió la identificación de cinco posibles nuevas especies. Además, se estudiaron tres agrupaciones de genes relacionados con la simbiosis (nod, nif y fix) definiendo un nuevo linaje para las cepas de Lmj, diferente del symbiovar “genistearum” de B. canariense y B. japonicum. La baja diversidad encontrada en el análisis filogenético de los genes simbióticos contrasta con la gran diversidad asociada a genes esenciales. La presencia de plásmidos en cepas del género Bradyrhizobium ha sido descrita en muy pocas ocasiones, sin embargo el análisis de la secuencia genómica de la cepa ISLU101, aislada de Lupinus angustifolius, reveló la presencia de un origen de replicación extracromosómico homólogo al operón repABC, presente en el plásmido de Bradyrhizobium sp BTAi1. Gracias a esta secuencia se identificaron genes homólogos en 19 de 72 cepas ISLU. Filogenéticamente, las secuencias de repABC se agruparon en un grupo monofilético con las de pBTAi1 y separadas de los rizobios de crecimiento rápido. Finalmente, se identificaron sistemas de secreción de proteínas de tipo III (T3SS) en nueve genomas de cepas de Lmj. Los T3SS pueden inyectar proteínas efectoras al interior de células vegetales. Su presencia en rizobios se ha relacionado con la gama de hospedador que pueden nodular y puede tener un efecto beneficioso, neutro o perjudicial en la simbiosis. Los T3SS de las cepas de Lmj codifican para una proteína efectora similar a NopE, un efector dependiente de T3SS descrito en B. diazoefficiens USDA 110T. La proteína NopE de la cepa LmjC se ha caracterizado bioquímicamente. ABSTRACT Lupinus mariae-josephae (Lmj) is a lupine species endemic of a unique small area in Valencia region (Eastern Spain) where the lupine plants thrive in alkaline-limed soils, which preferentially grow in acid or neutral soils. This is the type of soils native lupines of Spain. When this work was initiated, the extension of the endemic area of Lmj was of about 700 squared kilometers confined to the Valencia province. In this area, Lmj thrives in small, isolated patches containing a reduced number of plants, and points to an endemism that can easily became endangered or extinct. Consequently, the Valencia Community authorities gave a ‘‘microreserve” status for conservation of the species. All efforts, using classical strategies directed to extend the area of Lmj growth and ensure its conservation have been so far unsuccessful. The work presented here is directed to improve our knowledge of Lmj ecology and it is centered in the characterization of the rhizobial symbiosis by phenotypic, phylogenetic and genomic analysis as well as in investigate the potential contribution of the symbiosis to improve its conservation. To this end, five different topics have been studied, and results are briefly described here. Extensive details can be followed en the attached, published articles. The first topic deals with the indigenous rhizobial symbionts of the Lmj endemism, and its genetic diversity was investigated. The Lmj root symbionts belong to the Bradyrhizobium genus, and phylogenetic analysis based on core genes identified a large diversity of Bradyrhizobium strains with phenotypic and symbiotic characteristics different from rhizobia nodulating other Lupinus spp. native of Spain. The strains were split in two clades. Clade II contained strains close to classical B. canariense and B. japonicum lineages that establish symbioses with lupines in acid soils of the Mediterranean area. Clade I included Lmj strains that define a new lineage, close to other Bradyrhizobium species as B. jicamae and B. elkanii. The phylogenetic analysis based on symbiotic genes identified only two distinct clusters. The singularity and large diversity of these strains in such a small geographical area makes this an attractive system for studying the evolution and adaptation of the rhizobial symbiont to the plant host. Additionally, the presence of bacteria able to nodulate Lmj in basic soils from Chiapas, Mexico was investigated. Surprisingly, these soils contain bacteria able to effectively nodulate and fix nitrogen with Lmj plants in greenhouse assays. In the second topic, the taxonomic status of the endosymbiotic bacteria of Lmj from Valencia endemism and Chiapas was investigated. Results from phylogenetic analysis of core genes and Average Nucleotide Identity (ANIm) using draft genomic sequences identified new Bradyrhizobium species within strains of Clade I of Lmj endosymbiotic bacteria. Only one of these potentially new species has been defined, meanwhile the others are under process of characterization. The name ‘Bradyrhizobium valentinum’ sp. nov. was proposed for the defined species (type strain LmjM3T= CECT 8364T, LMG 2761T). The third topic was directed to conservation of endangered Lmj in its natural habitat. The relevant conclusion of this experimentation is that the symbiosis should be considered as a relevant factor in the conservation strategies for endangered legumes. First, we showed absence of bacteria able to nodulate Lmj in all the inspected ‘‘terra rossa’’ or alkaline red soils of the Iberian Peninsula and Balearic Islands. Then, two efficient nitrogen fixing strains with Lmj plants, LmjC and LmjM3T, were selected as inoculum for seed coating. Two planting experiments were carried out in consecutive years under natural conditions in areas with edapho-climatic characteristics identical to those sustaining natural Lmj populations, and successful reproduction of the plant was achieved. The relevant conclusion from these assays was that the successful reproductive cycle was absolutely dependent on seedling inoculation with effective bradyrhizobia The forth topic deep into the analysis of the genomic of Lmj representative strains. To this end, draft genomic sequences of selected Lmj strains and type strains of Bradyrhizobium spp. were assembled. The comparison analysis of the draft genomic sequences of Lmj strains and related Bradyrhizobium species grouped in Clade I, revealed a high genomic homology among them, and allowed the definition of five potentially new species of Lmj nodulating bacteria. Also, based on the available draft genomic sequences, only three clusters of nod, fix and nif genes from Lmj strains were identified and showed to define a new symbiotic lineage, distant from that of B. canariense and B. japonicum bv. genistearum. The low diversity exhibited by the phylogenetic analysis of symbiotic genes contrast with the large diversity of strains as regards the housekeeping genes analyzed. Besides, the genomic analysis of a Lupinus angustifolius strain ISLU101, revealed the presence of an extrachromosomal replication origin homologous to repABC cluster from plasmid present in Bradyrhizobium spp BTAi1. This repABC cluster gene sequence allowed the identification of extrachromosomic replication origin in 19 out of 72 Bradyrhizobium strains from Lupinus spp., a highly significant result since the absence of plasmids in the Bradyrhizobium genus was traditionally assumed. The repABC gene sequences of these strains grouped them in a unique monophyletic group, related to B. sp. BTAi1 plasmid, but differentiated from the repABC gene cluster of plasmids in fast growing rhizobium strains. The last topic was focused on characterization of type III secreted effectors present in Lmj endosymbiotic bacteria. Type III secretion systems (T3SS) are specialized protein export machineries which can deliver effector proteins into plant cells. The presence of T3SS in rhizobia has frequently been related to the symbiotic nodulation host-range and may have a beneficial or detrimental effect on the symbiosis with legumes. In this context, the presence of T3SS in genomes of nine Lmj strains was investigated, and it was shown the presence of clusters encoding NopE type III-secreted protein similar to the NopE1 and NopE2 of B. diazoefficiens USDA 110T. The putative NopE protein of LmjC strain is at present being characterized regarding its structure and function.

PoolSeq analysis of the selection of the Rhizobium genotypes by the legume host plant

Rhizobium leguminosarum establishes highly specific nitrogen-fixing symbioses. We have applied a Pool-Seq approach to study plant host selection of genotypes. Our results confirm, at the genomic level, previous observations regarding plant selection of specific genotypes

Metagenomic Anlaysis of microsymbiont selection by the legume plant host

Rhizobium leguminosarum bv.viciae is able to establish nitrogen-fixing symbioses with legumes of the genera Pisum, Lens, Lathyrus and Vicia. Classic studies using trap plants (Laguerre et al., Young et al.) provided evidence that different plant hosts are able to select different rhizobial genotypes among those available in a given soil. However, these studies were necessarily limited by the paucity of relevant biodiversity markers. We have now reappraised this problem with the help of genomic tools. A well-characterized agricultural soil (INRA Bretennieres) was used as source of rhizobia. Plants of Pisum sativum, Lens culinaris, Vicia sativa and V. faba were used as traps. Isolates from 100 nodules were pooled, and DNA from each pool was sequenced (BGI-Hong Kong; Illumina Hiseq 2000, 500 bp PE libraries, 100 bp reads, 12 Mreads). Reads were quality filtered (FastQC, Trimmomatic), mapped against reference R. leguminosarum genomes (Bowtie2, Samtools), and visualized (IGV). An important fraction of the filtered reads were not recruited by reference genomes, suggesting that plant isolates contain genes that are not present in the reference genomes. For this study, we focused on three conserved genomic regions: 16S-23S rDNA, atpD and nodDABC, and a Single Nucleotide Polymorphism (SNP) analysis was carried out with meta / multigenomes from each plant. Although the level of polymorphism varied (lowest in the rRNA region), polymorphic sites could be identified that define the specific soil population vs. reference genomes. More importantly, a plant-specific SNP distribution was observed. This could be confirmed with many other regions extracted from the reference genomes (data not shown). Our results confirm at the genomic level previous observations regarding plant selection of specific genotypes. We expect that further, ongoing comparative studies on differential meta / multigenomic sequences will identify specific gene components of the plant-selected genotypes

enod40 induces dedifferentiation and division of root cortical cells in legumes

Under nitrogen-limiting conditions Rhizobium meliloti can establish symbiosis with Medicago plants to form nitrogen-fixing root nodules. Nodule organogenesis starts with the dedifferentiation and division of root cortical cells. In these cells the early nodulin gene enod40, which encodes an unusually small peptide (12 or 13 amino acids), is induced from the beginning of this process. Herein we show that enod40 expression evokes root nodule initiation. (i) Nitrogen-deprived transgenic Medicago truncatula plants overexpressing enod40 exhibit extensive cortical cell division in their roots in the absence of Rhizobium. (ii) Bombardment of Medicago roots with an enod40-expressing DNA cassette induces dedifferentiation and division of cortical cells and the expression of another early nodulin gene, Msenod12A. Moreover, transient expression of either the enod40 region spanning the oligopeptide sequence or only the downstream region without this sequence induces these responses. Our results suggest that the cell-specific growth response elicited by enod40 is involved in the initiation of root nodule organogenesis.

Rhizobium gone native: Unexpected plasmid stability of indigenous Rhizobium leguminosarum

Lateral transfer of bacterial plasmids is thought to play an important role in microbial evolution and population dynamics. However, this assumption is based primarily on investigations of medically or agriculturally important bacterial species. To explore the role of lateral transfer in the evolution of bacterial systems not under intensive, human-mediated selection, we examined the association of genotypes at plasmid-encoded and chromosomal loci of native Rhizobium, the nitrogen-fixing symbiont of legumes. To this end, Rhizobium leguminosarum strains nodulating sympatric species of native Trifolium were characterized genetically at plasmid-encoded symbiotic (sym) regions (nodulation AB and nodulation CIJT loci) and a repeated chromosomal locus not involved in the symbiosis with legumes. Restriction fragment length polymorphism analysis was used to distinguish genetic groups at plasmid and chromosomal loci. The correlation between major sym and chromosomal genotypes and the distribution of genotypes across host plant species and sampling location were determined using χ2 analysis. In contrast to findings of previous studies, a strict association existed between major sym plasmid and chromosomal genetic groups, suggesting a lack of successful sym plasmid transfer between major Rhizobium chromosomal types. These data indicate that previous observations of sym plasmid transfer in agricultural settings may seriously overestimate the rates of successful conjugation in systems not impacted by human activities. In addition, a nonrandom distribution of Rhizobium genotypes across host plant species and sampling site demonstrates the importance of both factors in shaping Rhizobium population dynamics.

Genetic analysis of calcium spiking responses in nodulation mutants of Medicago truncatula

The symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti results in the formation of nitrogen-fixing nodules on the roots of the host plant. The early stages of nodule formation are induced by bacteria via lipochitooligosaccharide signals known as Nod factors (NFs). These NFs are structurally specific for bacterium–host pairs and are sufficient to cause a range of early responses involved in the host developmental program. Early events in the signal transduction of NFs are not well defined. We have previously reported that Medicago sativa root hairs exposed to NF display sharp oscillations of cytoplasmic calcium ion concentration (calcium spiking). To assess the possible role of calcium spiking in the nodulation response, we analyzed M. truncatula mutants in five complementation groups. Each of the plant mutants is completely Nod− and is blocked at early stages of the symbiosis. We defined two genes, DMI1 and DMI2, required in common for early steps of infection and nodulation and for calcium spiking. Another mutant, altered in the DMI3 gene, has a similar mutant phenotype to dmi1 and dmi2 mutants but displays normal calcium spiking. The calcium behavior thus implies that the DMI3 gene acts either downstream of calcium spiking or downstream of a common branch point for the calcium response and the later nodulation responses. Two additional mutants, altered in the NSP and HCL genes, which show root hair branching in response to NF, are normal for calcium spiking. This system provides an opportunity to use genetics to study ligand-stimulated calcium spiking as a signal transduction event.

Regulation of Soybean Nodulation Independent of Ethylene Signaling1

Leguminous plants regulate the number of Bradyrhizobium- or Rhizobium-infected sites that develop into nitrogen-fixing root nodules. Ethylene has been implicated in the regulation of nodule formation in some species, but this role has remained in question for soybean (Glycine max). The present study used soybean mutants with decreased responsiveness to ethylene, soybean mutants with defective regulation of nodule number, and Ag+ inhibition of ethylene perception to examine the role of ethylene in the regulation of nodule number. Nodule numbers on ethylene-insensitive mutants and plants treated with Ag+ were similar to those on wild-type plants and untreated plants, respectively. Hypernodulating mutants displayed wild-type ethylene sensitivity. Suppression of nodule numbers by high nitrate was also similar between ethylene-insensitive plants, wild-type plants, and plants treated with Ag+. Ethylene insensitivity of the roots of etr1-1 mutants was confirmed using assays for sensitivity to 1-aminocyclopropane-1-carboxylic acid and for ethylene-stimulated root-hair formation. Additional phenotypes of etr1-1 roots were also characterized. Ethylene-dependent pathways regulate the number of nodules that form on species such as pea and Medicago truncatula, but our data indicate that ethylene is less significant in regulating the number of nodules that form on soybean.

Expressed Sequence Tags from a Root-Hair-Enriched Medicago truncatula cDNA Library1

The root hair is a specialized cell type involved in water and nutrient uptake in plants. In legumes the root hair is also the primary site of recognition and infection by symbiotic nitrogen-fixing Rhizobium bacteria. We have studied the root hairs of Medicago truncatula, which is emerging as an increasingly important model legume for studies of symbiotic nodulation. However, only 27 genes from M. truncatula were represented in GenBank/EMBL as of October, 1997. We report here the construction of a root-hair-enriched cDNA library and single-pass sequencing of randomly selected clones. Expressed sequence tags (899 total, 603 of which have homology to known genes) were generated and made available on the Internet. We believe that the database and the associated DNA materials will provide a useful resource to the community of scientists studying the biology of roots, root tips, root hairs, and nodulation.