Agnieszka Klonowska

Biochemical and Molecular Characterization of a Laccase from Marasmius quercophilus

ABSTRACT The basidiomycete Marasmius quercophilus is commonly found during autumn on the decaying litter of the evergreen oak (Quercus ilex L.), a plant characteristic of Mediterranean forest. This white-rot fungus colonizes the leaf surface with rhizomorphs, causing a total bleaching of the leaf. In synthetic liquid media, this white-rot fungus has strong laccase activity. From a three-step chromatographic procedure, we purified a major isoform to homogeneity. The gene encodes a monomeric glycoprotein of approximately 63 kDa, with a 3.6 isoelectric point, that contains 12% carbohydrate. Spectroscopic analysis of the purified enzyme (UV/visible and electron paramagnetic resonance, atomic absorption) confirmed that it belongs to the “blue copper oxidase” family. With syringaldazine as the substrate, the enzymes pH optimum was 4.5, the optimal temperature was 75°C, and the Km was 7.1 μM. The structural gene, lac1, was cloned and sequenced. This gene encodes a 517-amino-acid protein 99% identical to a laccase produced by PM1, an unidentified basidiomycete previously isolated from wastewater from a paper factory in Spain. This similarity may be explained by the ecological distribution of the evergreen oak in Mediterranean forest.

Genetic diversity of Mimosa pudica rhizobial symbionts in soils of French Guiana: investigating the origin and diversity of Burkholderia phymatum and other beta‐rhizobia

The genetic diversity of 221 Mimosa pudica bacterial symbionts trapped from eight soils from diverse environments in French Guiana was assessed by 16S rRNA PCR-RFLP, REP-PCR fingerprints, as well as by phylogenies of their 16S rRNA and recA housekeeping genes, and by their nifH, nodA and nodC symbiotic genes. Interestingly, we found a large diversity of beta-rhizobia, with Burkholderia phymatum and Burkholderia tuberum being the most frequent and diverse symbiotic species. Other species were also found, such as Burkholderia mimosarum, an unnamed Burkholderia species and, for the first time in South America, Cupriavidus taiwanensis. The sampling site had a strong influence on the diversity of the symbionts sampled, and the specific distributions of symbiotic populations between the soils were related to soil composition in some cases. Some alpha-rhizobial strains taxonomically close to Rhizobium endophyticum were also trapped in one soil, and these carried two copies of the nodA gene, a feature not previously reported. Phylogenies of nodA, nodC and nifH genes showed a monophyly of symbiotic genes for beta-rhizobia isolated from Mimosa spp., indicative of a long history of interaction between beta-rhizobia and Mimosa species. Based on their symbiotic gene phylogenies and legume hosts, B. tuberum was shown to contain two large biovars: one specific to the mimosoid genus Mimosa and one to South African papilionoid legumes.

Biodiversity of Mimosa pudica rhizobial symbionts (Cupriavidus taiwanensis, Rhizobium mesoamericanum) in New Caledonia and their adaptation to heavy metal‐rich soils

Rhizobia are soil bacteria able to develop a nitrogen-fixing symbiosis with legumes. They are taxonomically spread among the alpha and beta subclasses of the Proteobacteria. Mimosa pudica, a tropical invasive weed, has been found to have an affinity for beta-rhizobia, including species within the Burkholderia and Cupriavidus genera. In this study, we describe the diversity of M. pudica symbionts in the island of New Caledonia, which is characterized by soils with high heavy metal content, especially of Ni. By using a plant-trapping approach on four soils, we isolated 96 strains, the great majority of which belonged to the species Cupriavidus taiwanensis (16S rRNA and recA gene phylogenies). A few Rhizobium strains in the newly described species Rhizobium mesoamericanum were also isolated. The housekeeping and nod gene phylogenies supported the hypothesis of the arrival of the C. taiwanensis and R. mesoamericanum strains together with their host at the time of the introduction of M. pudica in New Caledonia (NC) for its use as a fodder. The C. taiwanensis strains exhibited various tolerances to Ni, Zn and Cr, suggesting their adaptation to the specific environments in NC. Specific metal tolerance marker genes were found in the genomes of these symbionts, and their origin was investigated by phylogenetic analyses.

Local and systemic N signaling are involved in Medicago truncatula preference for the most efficient Sinorhizobium symbiotic partners

• Responses of the Medicago truncatula-Sinorhizobium interaction to variation in N₂-fixation of the bacterial partner were investigated. • Split-root systems were used to discriminate between local responses, at the site of interaction with bacteria, and systemic responses related to the whole plant N status. • The lack of N acquisition by a half-root system nodulated with a nonfixing rhizobium triggers a compensatory response enabling the other half-root system nodulated with N₂-fixing partners to compensate the local N limitation. This response is mediated by a stimulation of nodule development (number and size) and involves a systemic signaling mechanism related to the plant N demand. In roots co-infected with poorly and highly efficient strains, partner choice for nodule formation was not modulated by the plant N status. However, the plant N demand induced preferential expansion of nodules formed with the most efficient partners when the symbiotic organs were functional. The response of nodule expansion was associated with the stimulation of symbiotic plant cell multiplication and of bacteroid differentiation. • A general model where local and systemic N signaling mechanisms modulate interactions between Medicago truncatula and its Sinorhizobium partners is proposed.

Complete Genome sequence of Burkholderia phymatum STM815 T , a broad host range and efficient nitrogen-fixing symbiont of Mimosa species

Burkholderia phymatum is a soil bacterium able to develop a nitrogen-fixing symbiosis with species of the legume genus Mimosa, and is frequently found associated specifically with Mimosa pudica. The type strain of the species, STM 815T, was isolated from a root nodule in French Guiana in 2000. The strain is an aerobic, motile, non-spore forming, Gram-negative rod, and is a highly competitive strain for nodulation compared to other Mimosa symbionts, as it also nodulates a broad range of other legume genera and species. The 8,676,562 bp genome is composed of two chromosomes (3,479,187 and 2,697,374 bp), a megaplasmid (1,904,893 bp) and a plasmid hosting the symbiotic functions (595,108 bp).

The geographical patterns of symbiont diversity in the invasive legume Mimosa pudica can be explained by the competitiveness of its symbionts and by the host genotype

Variations in the patterns of diversity of symbionts have been described worldwide on Mimosa pudica, a pan-tropical invasive species that interacts with both α and β-rhizobia. In this study, we investigated if symbiont competitiveness can explain these variations and the apparent prevalence of β- over α-rhizobia. We developed an indirect method to measure the proportion of nodulation against a GFP reference strain and tested its reproducibility and efficiency. We estimated the competitiveness of 54 strains belonging to four species of β-rhizobia and four of α-rhizobia, and the influence of the host genotype on their competitiveness. Our results were compared with biogeographical patterns of symbionts and host varieties. We found: (i) a strong strain effect on competitiveness largely explained by the rhizobial species, with Burkholderia phymatum being the most competitive species, followed by B. tuberum, whereas all other species shared similar and reduced levels of competitiveness; (ii) plant genotype can increase the competitiveness of Cupriavidus taiwanensis. The latter data support the likelihood of the strong adaptation of C. taiwanensis with the M. pudica var. unijuga and help explain its prevalence as a symbiont of this variety over Burkholderia species in some environments, most notably in Taiwan.

Genetic and Genomic Diversity Studies of Acacia Symbionts in Senegal Reveal New Species of Mesorhizobium with a Putative Geographical Pattern

Acacia senegal (L) Willd. and Acacia seyal Del. are highly nitrogen-fixing and moderately salt tolerant species. In this study we focused on the genetic and genomic diversity of Acacia mesorhizobia symbionts from diverse origins in Senegal and investigated possible correlations between the genetic diversity of the strains, their soil of origin, and their tolerance to salinity. We first performed a multi-locus sequence analysis on five markers gene fragments on a collection of 47 mesorhizobia strains of A. senegal and A. seyal from 8 localities. Most of the strains (60%) clustered with the M. plurifarium type strain ORS 1032T, while the others form four new clades (MSP1 to MSP4). We sequenced and assembled seven draft genomes: four in the M. plurifarium clade (ORS3356, ORS3365, STM8773 and ORS1032T), one in MSP1 (STM8789), MSP2 (ORS3359) and MSP3 (ORS3324). The average nucleotide identities between these genomes together with the MLSA analysis reveal three new species of Mesorhizobium. A great variability of salt tolerance was found among the strains with a lack of correlation between the genetic diversity of mesorhizobia, their salt tolerance and the soils samples characteristics. A putative geographical pattern of A. senegal symbionts between the dryland north part and the center of Senegal was found, reflecting adaptations to specific local conditions such as the water regime. However, the presence of salt does not seem to be an important structuring factor of Mesorhizobium species.

Draft Genome Sequence of Rhizobium mesoamericanum STM3625, a Nitrogen-Fixing Symbiont of Mimosa pudica Isolated in French Guiana (South America).

ABSTRACT Rhizobium mesoamericanum STM3625 is a Mimosa pudica symbiont isolated in French Guiana. This strain serves as a model bacterium for comparison of adaptation to mutualism (symbiotic traits, bacterial genetic programs for plant infection) between alpha and beta rhizobial symbionts of Mimosa pudica.

Transcriptomic profiling of Burkholderia phymatum STM815, Cupriavidus taiwanensis LMG19424 and Rhizobium mesoamericanum STM3625 in response to Mimosa pudica root exudates illuminates the molecular basis of their nodulation competitiveness and symbiotic evolutionary history

BackgroundRhizobial symbionts belong to the classes Alphaproteobacteria and Betaproteobacteria (called “alpha” and “beta”-rhizobia). Most knowledge on the genetic basis of symbiosis is based on model strains belonging to alpha-rhizobia. Mimosa pudica is a legume that offers an excellent opportunity to study the adaptation toward symbiotic nitrogen fixation in beta-rhizobia compared to alpha-rhizobia. In a previous study (Melkonian et al., Environ Microbiol 16:2099–111, 2014) we described the symbiotic competitiveness of M. pudica symbionts belonging to Burkholderia, Cupriavidus and Rhizobium species.ResultsIn this article we present a comparative analysis of the transcriptomes (by RNAseq) of B. phymatum STM815 (BP), C. taiwanensis LMG19424 (CT) and R. mesoamericanum STM3625 (RM) in conditions mimicking the early steps of symbiosis (i.e. perception of root exudates). BP exhibited the strongest transcriptome shift both quantitatively and qualitatively, which mirrors its high competitiveness in the early steps of symbiosis and its ancient evolutionary history as a symbiont, while CT had a minimal response which correlates with its status as a younger symbiont (probably via acquisition of symbiotic genes from a Burkholderia ancestor) and RM had a typical response of Alphaproteobacterial rhizospheric bacteria. Interestingly, the upregulation of nodulation genes was the only common response among the three strains; the exception was an up-regulated gene encoding a putative fatty acid hydroxylase, which appears to be a novel symbiotic gene specific to Mimosa symbionts.ConclusionThe transcriptional response to root exudates was correlated to each strain nodulation competitiveness, with Burkholderia phymatum appearing as the best specialised symbiont of Mimosa pudica.

Ancient Heavy Metal Contamination in Soils as a Driver of Tolerant Anthyllis vulneraria Rhizobial Communities

ABSTRACT Anthyllis vulneraria is a legume associated with nitrogen-fixing rhizobia that together offer an adapted biological material for mine-soil phytostabilization by limiting metal pollution. To find rhizobia associated with Anthyllis at a given site, we evaluated the genetic and phenotypic properties of a collection of 137 rhizobia recovered from soils presenting contrasting metal levels. Zn-Pb mine soils largely contained metal-tolerant rhizobia belonging to Mesorhizobium metallidurans or to another sister metal-tolerant species. All of the metal-tolerant isolates harbored the cadA marker gene (encoding a metal-efflux PIB-type ATPase transporter). In contrast, metal-sensitive strains were taxonomically distinct from metal-tolerant populations and consisted of new Mesorhizobium genospecies. Based on the symbiotic nodA marker, the populations comprise two symbiovar assemblages (potentially related to Anthyllis or Lotus host preferences) according to soil geographic locations but independently of metal content. Multivariate analysis showed that soil Pb and Cd concentrations differentially impacted the rhizobial communities and that a rhizobial community found in one geographically distant site was highly divergent from the others. In conclusion, heavy metal levels in soils drive the taxonomic composition of Anthyllis-associated rhizobial populations according to their metal-tolerance phenotype but not their symbiotic nodA diversity. In addition to heavy metals, local soil physicochemical and topoclimatic conditions also impact the rhizobial beta diversity. Mesorhizobium communities were locally adapted and site specific, and their use is recommended for the success of phytostabilization strategies based on Mesorhizobium-legume vegetation. IMPORTANCE Phytostabilization of toxic mine spoils limits heavy metal dispersion and environmental pollution by establishing a sustainable plant cover. This eco-friendly method is facilitated by the use of selected and adapted cover crop legumes living in symbiosis with rhizobia that can stimulate plant growth naturally through biological nitrogen fixation. We studied microsymbiont partners of a metal-tolerant legume, Anthyllis vulneraria, which is tolerant to very highly metal-polluted soils in mining and nonmining sites. Site-specific rhizobial communities were linked to taxonomic composition and metal tolerance capacity. The rhizobial species Mesorhizobium metallidurans was dominant in all Zn-Pb mines but one. It was not detected in unpolluted sites where other distinct Mesorhizobium species occur. Given the different soil conditions at the respective mining sites, including their heavy-metal contamination, revegetation strategies based on rhizobia adapting to local conditions are more likely to succeed over the long term compared to strategies based on introducing less-well-adapted strains.