Christophe Mougel

Relationships between Staphylococcus aureus Genetic Background, Virulence Factors, agr Groups (Alleles), and Human Disease

ABSTRACT The expression of most Staphylococcus aureus virulence factors is controlled by the agr locus, which encodes a two-component signaling pathway whose activating ligand is an agr-encoded autoinducing peptide (AIP). A polymorphism in the amino acid sequence of the AIP and of its corresponding receptor divides S. aureus strains into four major groups. Within a given group, each strain produces a peptide that can activate the agr response in the other member strains, whereas the AIPs belonging to different groups are usually mutually inhibitory. We investigated a possible relationship between agr groups and human S. aureus disease by studying 198 S. aureus strains isolated from 14 asymptomatic carriers, 66 patients with suppurative infection, and 114 patients with acute toxemia. The agr group and the distribution of 24 toxin genes were analyzed by PCR, and the genetic background was determined by means of amplified fragment length polymorphism (AFLP) analysis. The isolates were relatively evenly distributed among the four agrgroups, with 61 strains belonging to agr group I, 49 belonging to group II, 43 belonging to group III, and 45 belonging to group IV. Principal coordinate analysis performed on the AFLP distance matrix divided the 198 strains into three main phylogenetic groups, AF1 corresponding to strains of agr group IV, AF2 corresponding to strains of agr groups I and II, and AF3 corresponding to strains of agr group III. This indicated that the agr type was linked to the genetic background. A relationship between genetic background, agr group, and disease type was observed for several toxin-mediated diseases: for instance, agr group IV strains were associated with generalized exfoliative syndromes, and phylogenetic group AF1 strains with bullous impetigo. Among the suppurative infections, endocarditis strains mainly belonged to phylogenetic group AF2 and agr groups I and II. While these results do not show a direct role of the agr type in the type of human disease caused by S. aureus, the agr group may reflect an ancient evolutionary division of S. aureus in terms of this species’ fundamental biology.

Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability

ABSTRACT Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334–5339, 2000) and fungi.

egc, A Highly Prevalent Operon of Enterotoxin Gene, Forms a Putative Nursery of Superantigens in Staphylococcus aureus

The recently described staphylococcal enterotoxins (SE) G and I were originally identified in two separate strains of Staphylococcus aureus. We have previously shown that the corresponding genes seg and sei are present in S. aureus in tandem orientation, on a 3.2-kb DNA fragment (Jarraud, J. et al. 1999. J. Clin. Microbiol. 37:2446–2449). Sequence analysis of seg-sei intergenic DNA and flanking regions revealed three enterotoxin-like open reading frames related to seg and sei, designated sek, sel, and sem, and two pseudogenes, ψ ent1 and ψ ent2. RT-PCR analysis showed that all these genes, including seg and sei, belong to an operon, designated the enterotoxin gene cluster (egc). Recombinant SEG, SEI, SEK, SEL, and SEM showed superantigen activity, each with a specific Vβ pattern. Distribution studies of genes encoding superantigens in clinical S. aureus isolates showed that most strains harbored such genes and in particular the enterotoxin gene cluster, whatever the disease they caused. Phylogenetic analysis of enterotoxin genes indicated that they all potentially derived from this cluster, identifying egc as a putative nursery of enterotoxin genes.

Plant-microbe-soil interactions in the rhizosphere: an evolutionary perspective

Soils are the product of the activities of plants, which supply organic matter and play a pivotal role in weathering rocks and minerals. Many plant species have a distinct ecological amplitude that shows restriction to specific soil types. In the numerous interactions between plants and soil, microorganisms also play a key role. Here we review the existing literature on interactions between plants, microorganisms and soils, and include considerations of evolutionary time scales, where possible. Some of these interactions involve intricate systems of communication, which in the case of symbioses such as the arbuscular mycorrhizal symbiosis are several hundreds of millions years old; others involve the release of exudates from roots, and other products of rhizodeposition that are used as substrates for soil microorganisms. The possible reasons for the survival value of this loss of carbon over tens or hundreds of millions of years of evolution of higher plants are discussed, taking a cost-benefit approach. Co-evolution of plants and rhizosphere microorganisms is discussed, in the light of known ecological interactions between various partners in terrestrial ecosystems. Finally, the role of higher plants, especially deep-rooted plants and associated microorganisms in the weathering of rocks and minerals, ultimately contributing to pedogenesis, is addressed. We show that rhizosphere processes in the long run are central to biogeochemical cycles, soil formation and Earth history. Major anticipated discoveries will enhance our basic understanding and allow applications of new knowledge to deal with nutrient deficiencies, pests and diseases, and the challenges of increasing global food production and agroecosystem productivity in an environmentally responsible manner.

Metaproteomics: A New Approach for Studying Functional Microbial Ecology

In the postgenomic era, there is a clear recognition of the limitations of nucleic acid-based methods for getting information on functions expressed by microbial communities in situ. In this context, the large-scale study of proteins expressed by indigenous microbial communities (metaproteome) should provide information to gain insights into the functioning of the microbial component in ecosystems. Characterization of the metaproteome is expected to provide data linking genetic and functional diversity of microbial communities. Studies on the metaproteome together with those on the metagenome and the metatranscriptome will contribute to progress in our knowledge of microbial communities and their contribution in ecosystem functioning. Effectiveness of the metaproteomic approach will be improved as increasing metagenomic information is made available thanks to the environmental sequencing projects currently running. More specifically, analysis of metaproteome in contrasted environmental situations should allow (1) tracking new functional genes and metabolic pathways and (2) identifying proteins preferentially associated with specific stresses. These proteins considered as functional bioindicators should contribute, in the future, to help policy makers in defining strategies for sustainable management of our environment.

A mathematical method for determining genome divergence and species delineation using AFLP.

The delineation of bacterial species is presently achieved using direct DNA-DNA relatedness studies of whole genomes. It would be helpful to obtain the same genomically based delineation by indirect methods, provided that descriptions of individual genome composition of bacterial genomes are obtained and included in species descriptions. The amplified fragment length polymorphism (AFLP) technique could provide the necessary data if the nucleotides involved in restriction and amplification are fundamental to the description of genomic divergences. Firstly, in order to verify that AFLP analysis permits a realistic exploration of bacterial genome composition, the strong correspondence between predicted and experimental AFLP data was demonstrated using Agrobacterium strain C58 as a model system. Secondly, a method is proposed for determining current genome mispairing and evolutionary genome divergences between pairs of bacteria, based on arbitrary sampling of genomes by using AFLP. The measure of current genome mispairing was validated by comparison with DNA-DNA relatedness data, which itself correlates with base mispairing. The evolutionary genome divergence is the estimated rate of nucleotide substitution that has occurred since the strains diverged from a common ancestor. Current genome mispairing and evolutionary genome divergence were used to compare members of Agrobacterium, used as a model of closely related genomic species. A strong and highly significant correlation was found between calculated genome mispairing and DNA-DNA relatedness values within genomic species. The canonical 70% DNA-DNA hybridization value used to delineate genomic species was found to correspond to a range of current genome mispairing of 13-13.6%. These limits correspond to 0.097 and 0.104 nucleotide substitutions per site, respectively. In addition, experimental data showed that the large Ti and cryptic plasmids of Agrobacterium had little effect on the estimation of genome divergence. Evolutionary genome divergence was used for phylogenetic inferences. Data showed that members of the same genomic species clustered consistently, as supported by bootstrap resampling. On the basis of these results, it is proposed that the genomic delineation of bacterial species could be based, in future, on phylogenetic groups supported by bootstraps and genome descriptions of individual strains, obtained by AFLP analysis, recorded in accessible databases; this approach might eventually replace DNA-DNA hybridization studies.

Validation and Application of a PCR Primer Set to Quantify Fungal Communities in the Soil Environment by Real-Time Quantitative PCR

Fungi constitute an important group in soil biological diversity and functioning. However, characterization and knowledge of fungal communities is hampered because few primer sets are available to quantify fungal abundance by real-time quantitative PCR (real-time Q-PCR). The aim in this study was to quantify fungal abundance in soils by incorporating, into a real-time Q-PCR using the SYBRGreen® method, a primer set already used to study the genetic structure of soil fungal communities. To satisfy the real-time Q-PCR requirements to enhance the accuracy and reproducibility of the detection technique, this study focused on the 18S rRNA gene conserved regions. These regions are little affected by length polymorphism and may provide sufficiently small targets, a crucial criterion for enhancing accuracy and reproducibility of the detection technique. An in silico analysis of 33 primer sets targeting the 18S rRNA gene was performed to select the primer set with the best potential for real-time Q-PCR: short amplicon length; good fungal specificity and coverage. The best consensus between specificity, coverage and amplicon length among the 33 sets tested was the primer set FR1 / FF390. This in silico analysis of the specificity of FR1 / FF390 also provided additional information to the previously published analysis on this primer set. The specificity of the primer set FR1 / FF390 for Fungi was validated in vitro by cloning - sequencing the amplicons obtained from a real time Q-PCR assay performed on five independent soil samples. This assay was also used to evaluate the sensitivity and reproducibility of the method. Finally, fungal abundance in samples from 24 soils with contrasting physico-chemical and environmental characteristics was examined and ranked to determine the importance of soil texture, organic carbon content, C∶N ratio and land use in determining fungal abundance in soils.

Molecular biomass and MetaTaxogenomic assessment of soil microbial communities as influenced by soil DNA extraction procedure

Three soil DNA extraction procedures (homemade protocols and commercial kit) varying in their practicability were applied to contrasting soils to evaluate their efficiency in recovering: (i) soil DNA and (ii) bacterial diversity estimated by 16S rDNA pyrosequencing. Significant differences in DNA yield were systematically observed between tested procedures. For certain soils, 10 times more DNA was recovered with one protocol than with the others. About 15 000 sequences of 16S rDNA were obtained for each sample which were clustered to draw rarefaction curves. These curves, as well as the PCA ordination of community composition based on OTU clustering, did not reveal any significant difference between procedures. Nevertheless, significant differences between procedures were highlighted by the taxonomic identification of sequences obtained at the phylum to genus levels. Depending on the soil, differences in the number of genera detected ranged from 1% to 26% between the most and least efficient procedures, mainly due to a poorer capacity to recover populations belonging to Actinobacteria, Firmicutes or Crenarchaeota. This study enabled us to rank the relative efficiencies of protocols for their recovery of soil molecular microbial biomass and bacterial diversity and to help choosing an appropriate soil DNA extraction procedure adapted to novel sequencing technologies.

Soil microbial diversity: Methodological strategy, spatial overview and functional interest

Since the development of industrialization, urbanization and agriculture, soils have been subjected to numerous variations in environmental conditions, which have resulted in modifications of the taxonomic diversity and functioning of the indigenous microbial communities. As a consequence, the functional significance of these losses/modifications of biodiversity, in terms of the capacity of ecosystems to maintain the functions and services on which humanity depends, is now of pivotal importance. In this context, one of the main challenges in soil microbial ecology is to better understand and predict the processes that drive soil microbial diversity and the link between diversity and ecosystem process. This review describes past, present and ongoing conceptual and methodological strategies employed to better assess and understand the distribution and evolution of soil microbial diversity with the aim of increasing our capacity to translate such diversity into soil biological functioning and, more widely, into ecosystem services.

Bacterial effects on arbuscular mycorrhizal fungi and mycorrhiza development as influenced by the bacteria, fungi, and host plant

Bacterial strains from mycorrhizal roots (three belonging to Comamonadaceae and one to Oxalobacteraceae) and from non-mycorrhizal roots (two belonging to Comamonadaceae) of Medicago truncatula and two reference strains (Collimonas fungivorans Ter331 and Pseudomonas fluorescens C7R12) were tested for their effect on the in vitro saprophytic growth of Glomus mosseae BEG12 and on its colonization of M. truncatula roots. Only the Oxalobacteraceae strain, isolated from barrel medic mycorrhizal roots, and the reference strain P. fluorescens C7R12 promoted both the saprophytic growth and root colonization of G. mosseae BEG12, indicating that they acted as mycorrhiza helper bacteria. Greatest effects were achieved by P. fluorescens C7R12 and its influence on the saprophytic growth of G. mosseae was compared to that on Gigaspora rosea BEG9 to determine if the bacterial stimulation was fungal specific. This fungal specificity, together with plant specificity, was finally evaluated by comparing bacterial effects on arbuscular mycorrhizal symbiosis when each of the fungal species was inoculated to two different plant species (M. truncatula and Lycopersicon esculentum). The results obtained showed that promotion of saprophytic growth by P. fluorescens C7R12 was expressed in vitro towards G. mosseae but not towards G. rosea. Bacterial promotion of mycorhization was also expressed towards G. mosseae, but not G. rosea, in roots of M. truncatula and L. esculentum. Taken together, results indicated that enhancement of arbuscular mycorrhiza development was only induced by a limited number of bacteria, promotion by the most efficient bacterial strain being fungal and not plant specific.