Analysis of gut microbial regulation of host gene expression along the length of the gut and regulation of gut microbial ecology through MyD88

BackgroundThe gut microbiota has profound effects on host physiology but local host-microbial interactions in the gut are only poorly characterised and are likely to vary from the sparsely colonised duodenum to the densely colonised colon. Microorganisms are recognised by pattern recognition receptors such as Toll-like receptors, which signal through the adaptor molecule MyD88.MethodsTo identify host responses induced by gut microbiota along the length of the gut and whether these required MyD88, transcriptional profiles of duodenum, jejunum, ileum and colon were compared from germ-free and conventionally raised wild-type and Myd88-/- mice. The gut microbial ecology was assessed by 454-based pyrosequencing and viruses were analysed by PCR.ResultsThe gut microbiota modulated the expression of a large set of genes in the small intestine and fewer genes in the colon but surprisingly few microbiota-regulated genes required MyD88 signalling. However, MyD88 was essential for microbiota-induced colonic expression of the antimicrobial genes Reg3β and Reg3γ in the epithelium, and Myd88 deficiency was associated with both a shift in bacterial diversity and a greater proportion of segmented filamentous bacteria in the small intestine. In addition, conventionally raised Myd88-/- mice had increased expression of antiviral genes in the colon, which correlated with norovirus infection in the colonic epithelium.ConclusionThis study provides a detailed description of tissue-specific host transcriptional responses to the normal gut microbiota along the length of the gut and demonstrates that the absence of MyD88 alters gut microbial ecology

Heteromeric Transposase Elements Target Insertions Into Specific Genomic Loci And Methods To Study Transposition

Transposons are mobile genetic elements found in all domains of life that are capable of moving between positions in a genome. The bacterial transposon Tn7 and related elements accumulate in specific insertion sites of the chromosome called genomic islands. The ability to recognize a specific insertion site and control the frequency of insertion involves five transposon encoded genes (tnsABCDE). The Tn7 core machinery is comprised of the atypical heteromeric transposase, TnsAB, and a regulator, TnsC. By using the core machinery and one of two targeting proteins, TnsD or TnsE, Tn7 is capable of inserting into either a chromosomal locus (attTn7) or horizontally transferred DNA. Tn7 is one of at least three families of transposons containing a heteromeric transposase that are found in diverse bacteria across a range of environments. In this work, I analyze ten elements from each of these three heteromeric transposase families and discuss how the conserved proteins within each family relate to TnsD and how those proteins may be responsible for targeting a specific conserved site on the chromosome. I then shift my focus to the other protein involved in target site selection, TnsE, which has been suggested to be species-specific in its interactions with the [beta]-clamp. I find that cells expressing [beta]-clamps from foreign hosts are viable in the absence of the dnaN allele and that TnsE-mediated transposition in strains containing foreign clamps only occurs in the presence of both proteins from the same host. In order to study how TnsE interacts with other host proteins, such as SeqA, a greater set of experimental tools is required. I develop an expression vector to express SeqA and a set of six mutants and suggest an in vitro assay to analyze multiple SeqA- TnsE interactions within one experiment. I then work to develop a high-throughput method for mapping TnsE-mediated transposition events. I use a dam- strain and draw a few conclusions based on where insertions map in seqA- and wild type strains. More importantly, I suggest improvements for the method moving forward that will enable future generations of scientists to map millions of insertion events within a single experiment

A Small-Group Activity Introducing the Use and Interpretation of BLAST

As biological sequence data are generated at an ever increasing rate, the role of bioinformatics in biological research also grows. Students must be trained to complete and interpret bioinformatic searches to enable them to effectively utilize the trove of sequence data available. A key bioinformatic tool for sequence comparison and genome database searching is BLAST (Basic Local Alignment Search Tool). BLAST identifies sequences in a database that are similar to the entered query sequence, and ranks them based on the length and quality of the alignment. Our goal was to introduce sophomore and junior level undergraduate students to the basic functions and uses of BLAST with a small group activity lasting a single class period. The activity provides students an opportunity to perform a BLAST search, interpret the data output, and use the data to make inferences about bacterial cell envelope structure. The activity consists of two parts. Part 1 is a handout to be completed prior to class, complete with video tutorial, that reviews cell envelope structure, introduces key terms, and allows students to familiarize themselves with the mechanics of a BLAST search. Part 2 consists of a hands-on, web-based small group activity to be completed during the class period. Evaluation of the activity through student performance assessments suggests that students who complete the activity can better interpret the BLAST output parameters % query coverage and % max identity. While the topic of the activity is bacterial cell wall structure, it could be adapted to address other biological concepts.<br /

<i>Dehalococcoides mccartyi</i> Strain JNA in Pure Culture Extensively Dechlorinates Aroclor 1260 According to Polychlorinated Biphenyl (PCB) Dechlorination Process N

We isolated <i>Dehalococcoides mccartyi</i> strain JNA from the JN mixed culture which was enriched and maintained using the highly chlorinated commercial PCB mixture Aroclor 1260 for organohalide respiration. For isolation we grew the culture in minimal liquid medium with 2,2′,3,3′,6,6′-hexachlorobiphenyl (236–236-CB)­(20 μM) as respiratory electron acceptor. We repeatedly carried out serial dilutions to extinction and recovered dechlorination activity from transfers of 10^–7 and 10^–8 dilutions. Fluorescence microscopy, DGGE and RFLP analysis of PCR amplified16S rRNA genes, and multilocus sequence typing of three housekeeping genes confirmed culture purity. No growth occurred on complex media. JNA dechlorinated most hexa- and heptachlorobiphenyls in Aroclor 1260 (50 μg/mL) leading to losses of 51% and 20%, respectively. Dechlorination was predominantly from flanked <i>meta</i> positions of <u>3</u>4-, 2<u>3</u>4-, 2<u>3</u>5-, 2<u>3</u>6-, 24<u>5</u>-, 2<u>3</u>45-, 2<u>3</u>46-, and 23<u>5</u>6-chlorophenyl rings, as indicated by the underscores. The major products were 24–24-CB, 24–26-CB, 24–25-CB, and 25–26-CB. We identified 85 distinct PCB dechlorination reactions and 56 different PCB dechlorination pathways catalyzed by JNA. Dechlorination pathways were confirmed by mass balance of substrates and products. This dechlorination pattern matches PCB Dechlorination Process N. JNA is the first pure culture demonstrated to carry out this extensive and environmentally relevant PCB dechlorination pattern

Isolation of Uncultured Bacteria from Antarctica Using Long Incubation Periods and Low Nutritional Media

Uncultured microorganisms comprise most of the microbial diversity existing on our planet. Despite advances in environmental sequencing and single-cell genomics, in-depth studies about bacterial metabolism and screening of novel bioproducts can only be assessed by culturing microbes in the laboratory. Here we report uncultured, or recalcitrant, microorganisms from an Antarctic soil sample, using relatively simple methods: oligotrophic media, extended incubation periods, observation under stereo microscopy, and selection of slow-growing bacteria. We managed to isolate several rare microorganisms belonging to infrequently isolated or recently described genera, for example Lapillicoccus, Flavitalea, Quadrisphaera, Motilibacter, and Polymorphobacter. Additionally, we obtained isolates presenting 16S rRNA sequence similarity ranging from 92.08 to 94.46% with any other known cultured species, including two distinct isolates from the class Thermoleophilia, that although common in Antarctic soils (as identified by metagenomics), was never reported to be isolated from such samples. Our data indicates that simple methods are still useful for cultivating recalcitrant microorganisms, even when dealing with samples from extreme environments

<i>Dehalococcoides mccartyi</i> Strain JNA Dechlorinates Multiple Chlorinated Phenols Including Pentachlorophenol and Harbors at Least 19 Reductive Dehalogenase Homologous Genes

Pentachlorophenol and other chlorinated phenols are highly toxic ubiquitous environmental pollutants. Using gas chromatographic analysis we determined that <i>Dehalococcoides mccartyi</i> strain JNA in pure culture dechlorinated pentachlorophenol to 3,5-dichlorophenol (DCP) via removal of the <i>ortho</i> and <i>para</i> chlorines in all of the three possible pathways. In addition, JNA dechlorinated 2,3,4,6-tetrachlorophenol via 2,4,6-trichlorophenol (TCP) and 2,4,5-TCP to 2,4-DCP and 3,4-DCP, respectively, and dechlorinated 2,3,6-TCP to 3-chlorophenol (CP) via 2,5-DCP. JNA converted 2,3,4-TCP to 3,4-DCP and 2,4-DCP by <i>ortho</i> and <i>meta</i> dechlorination, respectively. 2,3-DCP was dechlorinated to 3-CP, and, because cultures using it could be transferred with a low inoculum (0.5 to 1.5% vol/vol), it may act as an electron acceptor to support growth. Using PCR amplification with targeted and degenerate primers followed by cloning and sequencing, we determined that JNA harbors at least 19 reductive dehalogenase homologous (<i>rdh</i>) genes including orthologs of <i>pcb</i>A4 and <i>pcb</i>A5, <i>pce</i>A, and <i>mbr</i>A, but not <i>tce</i>A or <i>vcr</i>A. Many of these genes are shared with <i>D. mccartyi</i> strains CBDB1, DCMB5, GT, and CG5. Strain JNA has previously been shown to extensively dechlorinate the commercial polychlorinated biphenyl (PCB) mixture Aroclor 1260. Collectively the data suggest that strain JNA may be well adapted to survive in sites contaminated with chlorinated aromatics and may be useful for <i>in situ</i> bioremediation