Arbuscular mycorrhizal community structure on co-existing tropical legume trees in French Guiana

Aims We aimed to characterise the arbuscular mycorrhizal fungal (AMF) community structure and potential edaphic determinants in the dominating, but poorly described, root-colonizing Paris-type AMF community on co-occurring Amazonian leguminous trees. Methods Three highly productive leguminous trees (Dicorynia guianensis, Eperua falcata and Tachigali melinonii were targeted) in species-rich forests on contrasting soil types at the Nouragues Research Station in central French Guiana. Abundant AMF SSU rRNA amplicons (NS31-AM1 & AML1-AML2 primers) from roots identified via trnL profiling were subjected to denaturing gradient gel electrophoresis (DGGE), clone library sequencing and phylogenetic analysis. Results Classical approaches targeting abundant SSU amplicons highlighted a diverse root-colonizing symbiotic AMF community dominated by members of the Glomeraceae. DGGE profiling indicated that, of the edaphic factors investigated, soil nitrogen was most important in influencing the AMF community and this was more important than any host tree species effect. Conclusions Dominating Paris-type mycorrhizal leguminous trees in Amazonian soils host diverse and novel taxa within the Glomeraceae that appear under edaphic selection in the investigated tropical forests. Linking symbiotic diversity of identified AMF taxa to ecological processes is the next challenge ahead

A Solve-RD ClinVar-based reanalysis of 1522 index cases from ERN-ITHACA reveals common pitfalls and misinterpretations in exome sequencing

Purpose Within the Solve-RD project (https://solve-rd.eu/), the European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies aimed to investigate whether a reanalysis of exomes from unsolved cases based on ClinVar annotations could establish additional diagnoses. We present the results of the “ClinVar low-hanging fruit” reanalysis, reasons for the failure of previous analyses, and lessons learned. Methods Data from the first 3576 exomes (1522 probands and 2054 relatives) collected from European Reference Network for Intellectual disability, TeleHealth, Autism and Congenital Anomalies was reanalyzed by the Solve-RD consortium by evaluating for the presence of single-nucleotide variant, and small insertions and deletions already reported as (likely) pathogenic in ClinVar. Variants were filtered according to frequency, genotype, and mode of inheritance and reinterpreted. Results We identified causal variants in 59 cases (3.9%), 50 of them also raised by other approaches and 9 leading to new diagnoses, highlighting interpretation challenges: variants in genes not known to be involved in human disease at the time of the first analysis, misleading genotypes, or variants undetected by local pipelines (variants in off-target regions, low quality filters, low allelic balance, or high frequency). Conclusion The “ClinVar low-hanging fruit” analysis represents an effective, fast, and easy approach to recover causal variants from exome sequencing data, herewith contributing to the reduction of the diagnostic deadlock

Cre-lox-Based System for Multiple Gene Deletions and Selectable-Marker Removal in Lactobacillus plantarum

The classic strategy to achieve gene deletion variants is based on double-crossover integration of nonreplicating vectors into the genome. In addition, recombination systems such as Cre-lox have been used extensively, mainly for eukaryotic organisms. This study presents the construction of a Cre-lox-based system for multiple gene deletions in Lactobacillus plantarum that could be adapted for use on gram-positive bacteria. First, an effective mutagenesis vector (pNZ5319) was constructed that allows direct cloning of blunt-end PCR products representing homologous recombination target regions. Using this mutagenesis vector, double-crossover gene replacement mutants could be readily selected based on their antibiotic resistance phenotype. In the resulting mutants, the target gene is replaced by a lox66-P(32)-cat-lox71 cassette, where lox66 and lox71 are mutant variants of loxP and P(32)-cat is a chloramphenicol resistance cassette. The lox sites serve as recognition sites for the Cre enzyme, a protein that belongs to the integrase family of site-specific recombinases. Thus, transient Cre recombinase expression in double-crossover mutants leads to recombination of the lox66-P(32)-cat-lox71 cassette into a double-mutant loxP site, called lox72, which displays strongly reduced recognition by Cre. The effectiveness of the Cre-lox-based strategy for multiple gene deletions was demonstrated by construction of both single and double gene deletions at the melA and bsh1 loci on the chromosome of the gram-positive model organism Lactobacillus plantarum WCFS1. Furthermore, the efficiency of the Cre-lox-based system in multiple gene replacements was determined by successive mutagenesis of the genetically closely linked loci melA and lacS2 in L. plantarum WCFS1. The fact that 99.4% of the clones that were analyzed had undergone correct Cre-lox resolution emphasizes the suitability of the system described here for multiple gene replacement and deletion strategies in a single genetic background

Genome-wide prediction and validation of sigma70 promoters in Lactobacillus plantarum WCFS1

Contains fulltext : 108029.pdf (publisher's version ) (Open Access)BACKGROUND: In prokaryotes, sigma factors are essential for directing the transcription machinery towards promoters. Various sigma factors have been described that recognize, and bind to specific DNA sequence motifs in promoter sequences. The canonical sigma factor sigma(70) is commonly involved in transcription of the cell's housekeeping genes, which is mediated by the conserved sigma(70) promoter sequence motifs. In this study the sigma(70)-promoter sequences in Lactobacillus plantarum WCFS1 were predicted using a genome-wide analysis. The accuracy of the transcriptionally-active part of this promoter prediction was subsequently evaluated by correlating locations of predicted promoters with transcription start sites inferred from the 5'-ends of transcripts detected by high-resolution tiling array transcriptome datasets. RESULTS: To identify sigma(70)-related promoter sequences, we performed a genome-wide sequence motif scan of the L. plantarum WCFS1 genome focussing on the regions upstream of protein-encoding genes. We obtained several highly conserved motifs including those resembling the conserved sigma(70)-promoter consensus. Position weight matrices-based models of the recovered sigma(70)-promoter sequence motif were employed to identify 3874 motifs with significant similarity (p-value<10(-4)) to the model-motif in the L. plantarum genome. Genome-wide transcript information deduced from whole genome tiling-array transcriptome datasets, was used to infer transcription start sites (TSSs) from the 5'-end of transcripts. By this procedure, 1167 putative TSSs were identified that were used to corroborate the transcriptionally active fraction of these predicted promoters. In total, 568 predicted promoters were found in proximity (</= 40 nucleotides) of the putative TSSs, showing a highly significant co-occurrence of predicted promoter and TSS (p-value<10(-263)). CONCLUSIONS: High-resolution tiling arrays provide a suitable source to infer TSSs at a genome-wide level, and allow experimental verification of in silico predicted promoter sequence motifs

Breast milk urea as a nitrogen source for urease positive Bifidobacterium infantis

Human milk stimulates a health-promoting gut microbiome in infants. However, it is unclear how the microbiota salvages and processes its required nitrogen from breast milk. Human milk nitrogen sources such as urea could contribute to the composition of this early life microbiome. Urea is abundant in human milk, representing a large part of the non-protein nitrogen (NPN). We found that B. longum subsp. infantis (ATCC17930) can use urea as a main source of nitrogen for growth in synthetic medium and enzyme activity was induced by the presence of urea in the medium. We furthermore confirmed the expression of both urease protein subunits and accessory proteins of B. longum subsp. infantis through proteomics. To the same end, metagenome data were mined for urease-related genes. It was found that the breastfed infant's microbiome possessed more urease-related genes than formula fed infants (51.4:22.1; 2.3-fold increase). Bifidobacteria provided a total of 106 of urease subunit alpha alignments, found only in breastfed infants. These experiments show how an important gut commensal that colonizes the infant intestine can metabolize urea. The results presented herein further indicate how dietary nitrogen can determine bacterial metabolism in the neonate gut and shape the overall microbiome.</p

Spatial and Temporal Expression of Lactobacillus plantarum Genes in the Gastrointestinal Tracts of Mice

Lactobacillus plantarum is a common inhabitant of mammalian gastrointestinal tracts, and L. plantarum strain WCFS1 is a human isolate with a known genome sequence. L. plantarum WCFS1 survives intestinal passage in an active form, and its transit time and transcriptional activities were monitored in 15 BALB/c mice at 2, 4, 6, 8, and 24 h after being fed a single intragastric dose of this organism. Enumeration of viable cells isolated from fecal material revealed that the majority of the L. plantarum inoculum transited the mouse intestine within 4 h after ingestion. Three mice were sacrificed at each time point, and total RNA was isolated from the mouse intestinal compartments (stomach through colon). Quantification of L. plantarum 16S rRNA by quantitative real-time reverse-transcription-PCR revealed that L. plantarum was present at elevated levels in the stomach and small intestine for at least 4 h following ingestion and for over 8 h in the cecum and colon. We also examined the expression of 9 L. plantarum housekeeping genes and 15 L. plantarum in vivo-inducible (ivi) genes previously identified by recombination-based in vivo expression technology to be induced in the mouse gastrointestinal tract. The relative expression levels of the ivi genes increased up to 350-fold in the mouse intestine compared to levels observed for L. plantarum WCFS1 cells grown in a rich laboratory medium. Moreover, several genes displayed intestinal compartment-specific (small intestine versus colon) activities. These results confirm that L. plantarum displays specific and differential responses at various sites along the mammalian intestine