Project description:Drosophila larvae were infected with Erwinia bacteria (Ecc15) by introducing concentrated bacterial pellet into the fly medium. The tracheae were dissected 24h later. Three genotypes of larvae were compared: wt (CantonS), RelE20 and PGRP-LA, each present in unchallenged and infected conditions. 3 independent repeats were performed.
Project description:In order to gain insight into gene regulation in the Drosophila gut following oral infection, we performed the genome-wide DNA Polymerase II binding data for Polymerase II in the fly gut.
Project description:Six bacterial genomes, Geobacter metallireducens GS-15, Chromohalobacter salexigens, Vibrio breoganii 1C-10, Bacillus cereus ATCC 10987, Campylobacter jejuni subsp. jejuni 81-176 and Campylobacter jejuni NCTC 11168, all of which had previously been sequenced using other platforms were re-sequenced using single-molecule, real-time (SMRT) sequencing specifically to analyze their methylomes. In every case a number of new N6-methyladenine (m6A) and N4-methylcytosine (m4C) methylation patterns were discovered and the DNA methyltransferases (MTases) responsible for those methylation patterns were assigned. In 15 cases it was possible to match MTase genes with MTase recognition sequences without further sub-cloning. Two Type I restriction systems required sub-cloning to differentiate their recognition sequences, while four MTases genes that were not expressed in the native organism were sub-cloned to test for viability and recognition sequences. No attempt was made to detect 5-methylcytosine (m5C) recognition motifs from the SMRT sequencing data because this modification produces weaker signals using current methods. However, all predicted m6A and m4C MTases were detected unambiguously. This study shows that the addition of SMRT sequencing to traditional sequencing approaches gives a wealth of useful functional information about a genome showing not only which MTase genes are active, but also revealing their recognition sequences. Examination of the methylomes of six different strains of bacteria using kinetic data from single-molecule, real-time (SMRT) sequencing on the PacBio RS.
Project description:The host genotype has been proposed to contribute to individually composed bacterial communities in the gut. To provide deeper insight into interactions between gut bacteria and their host, we associated germ-free C3H and C57BL/10 mice with intestinal bacteria from a C57BL/10 donor mouse. Analysis of microbiota similarity between the experimental animals with denaturing gradient gel electrophoresis (DGGE) 13 weeks after association revealed the development of a mouse strain specific microbiota. Gene expression in the colonic mucosa was analyzed with a microarray approach taking advantage of a modified Affymetrix mouse genome chip. We detected 202 genes whose expression differed significantly by a factor of < 2. Application of bioinformatics tools demonstrated that functional terms including signaling/secretion, lipid degradation/catabolism, guanine nucleotide/guanylate binding and immune response were significantly enriched in differentially expressed genes. We had a closer look at the 56 genes with expression differences of < 4 and observed a higher expression in C57BL/10 mice of the genes coding for toll-like receptor 1 (4-fold) and angiogenin 4 (33-fold) which are involved in the recognition and response to gut bacteria. In contrast, a 70-fold higher expression of the phospholipase A2, group IIA-encoding gene (Pla2g2a) was detected in C3H mice. In addition, a number of interferon-inducible genes were higher expressed in C3H than in C57BL/10 mice including Gbp1 (18-fold), Mal (7-fold), Oasl2 (7-fold), Ifi202b (7-fold), Rtp4 (6-fold), Ly6g6c (5-fold), Ifi27l2a (5-fold), Usp18 (5-fold), Ifit1 (5-fold), Ifi44 (4-fold), and Ly6g (4-fold) indicating that these cytokines play an essential role in microbiota regulation. However, genes coding for interferons, their receptors or factors involved in interferon signaling pathways were not differentially expressed between the two mouse strains. Taken together, our study confirms that the host genotype is involved in the establishment of host-specific bacterial communities in the gut. Based on expression differences after colonization with the same bacterial inoculum, we propose that Pla2g2a and interferon-dependent genes may contribute to this phenomenon. Total RNA was extracted from the colonic mucosa and hybridization was performed using 12.5M-BM- M-bM-^@M-^SM-BM- 20M-BM- M-BM-5g of cDNA on a customized Affymetrix nugomm 1a520177 chip.