Laure Franqueville

Soil Bacterial Community Shifts after Chitin Enrichment: An Integrative Metagenomic Approach

Chitin is the second most produced biopolymer on Earth after cellulose. Chitin degrading enzymes are promising but untapped sources for developing novel industrial biocatalysts. Hidden amongst uncultivated micro-organisms, new bacterial enzymes can be discovered and exploited by metagenomic approaches through extensive cloning and screening. Enrichment is also a well-known strategy, as it allows selection of organisms adapted to feed on a specific compound. In this study, we investigated how the soil bacterial community responded to chitin enrichment in a microcosm experiment. An integrative metagenomic approach coupling phylochips and high throughput shotgun pyrosequencing was established in order to assess the taxonomical and functional changes in the soil bacterial community. Results indicate that chitin enrichment leads to an increase of Actinobacteria, γ-proteobacteria and β-proteobacteria suggesting specific selection of chitin degrading bacteria belonging to these classes. Part of enriched bacterial genera were not yet reported to be involved in chitin degradation, like the members from the Micrococcineae sub-order (Actinobacteria). An increase of the observed bacterial diversity was noticed, with detection of specific genera only in chitin treated conditions. The relative proportion of metagenomic sequences related to chitin degradation was significantly increased, even if it represents only a tiny fraction of the sequence diversity found in a soil metagenome.

Characterization of new bacterial catabolic genes and mobile genetic elements by high throughput genetic screening of a soil metagenomic library

A mix of oligonucleotide probes was used to hybridize soil metagenomic DNA from a fosmid clone library spotted on high density membranes. The pooled radio-labeled probes were designed to target genes encoding glycoside hydrolases GH18, dehalogenases, bacterial laccases and mobile genetic elements (integrases from integrons and insertion sequences). Positive hybridizing spots were affiliated to the corresponding clones in the library and the metagenomic inserts were sequenced. After assembly and annotation, new coding DNA sequences related to genes of interest were identified with low protein similarity against the closest hits in databases. This work highlights the sensitivity of DNA/DNA hybridization techniques as an effective and complementary way to recover novel genes from large metagenomic clone libraries. This study also supports that some of the identified catabolic genes might be associated with horizontal transfer events.

Linking environmental prokaryotic viruses and their host through CRISPRs

The ecological pressure that viruses place on microbial communities is not only based on predation, but also on gene transfer. In order to determine the potential impact of viruses and transduction, we need a better understanding of the dynamics of interactions between viruses and their hosts in the environment. Data on environmental viruses are scarce, and methods for tracking their interactions with prokaryotes are needed. Clustered regularly interspaced short palindromic repeats (CRISPRs), which contain viral sequences in bacterial genomes, might help document the history of virus-host interactions in the environment. In this study, a bioinformatics network linking viruses and their hosts using CRISPR sequences obtained from metagenomic data was developed and applied to metagenomes from Arctic glacial ice and soil. The application of our network approach showed that putative interactions were more commonly detected in the ice samples than the soil which would be consistent with the ice viral-bacterial interactions being more dynamic than those in soil. Further analysis of the viral sequences in the CRISPRs indicated that Ralstonia phages might be agents of transduction in the Arctic glacial ice.

nDEP-driven cell patterning and bottom-up construction of cell aggregates using a new bioelectronic chip

Creating cell aggregates of controlled size and shape and patterning cells on substrates using a bottom-up approach constitutes important challenges for tissue-engineering applications and studies of cell-cell interactions. In this paper, we report nDEP (negative dielectrophoresis) driven assembly of cells as compact aggregates or onto defined areas using a new bioelectronic chip. This chip is composed of a quadripolar electrode array obtained using coplanar electrodes partially covered with a thin, micropatterned PDMS membrane. This thin PDMS layer was coated with poly-L-lysine and played the role of adhesive substrate for cell patterning. For the formation of detachable cell aggregates, the PDMS was not pretreated and cells were simply immobilized into assemblies maintained by cell-cell adhesion after the electric field removal. Cell viability after exposition to DEP buffer was also assessed, as well as cell spreading activity following DEP-driven assembly.

Microbial soil community analyses for forensic science: Application to a blind test

Soil complexity, heterogeneity and transferability make it valuable in forensic investigations to help obtain clues as to the origin of an unknown sample, or to compare samples from a suspect or object with samples collected at a crime scene. In a few countries, soil analysis is used in matters from site verification to estimates of time after death. However, up to date the application or use of soil information in criminal investigations has been limited. In particular, comparing bacterial communities in soil samples could be a useful tool for forensic science. To evaluate the relevance of this approach, a blind test was performed to determine the origin of two questioned samples (one from the mock crime scene and the other from a 50:50 mixture of the crime scene and the alibi site) compared to three control samples (soil samples from the crime scene, from a context site 25m away from the crime scene and from the alibi site which was the suspects home). Two biological methods were used, Ribosomal Intergenic Spacer Analysis (RISA), and 16S rRNA gene sequencing with Illumina Miseq, to evaluate the discriminating power of soil bacterial communities. Both techniques discriminated well between soils from a single source, but a combination of both techniques was necessary to show that the origin was a mixture of soils. This study illustrates the potential of applying microbial ecology methodologies in soil as an evaluative forensic tool.