Blackburn, Dana et al. (2021) International Worm Meeting "Genetic determinants of host-microbiome interactions in Caenorhabditis elegans"

Samuel, Buck, Assie, Adrien, Deutschbauer, Adam, Vidal Vilchis, Daniela, Blackburn, Dana

[International Worm Meeting, 2021]

Microbes shape many aspects of host physiology, development and predisposition to disease. Yet the complexity of host-associated communities (microbiomes) makes studying host-microbe interactions challenging. As such, we employed the Caenorhabditis elegans system to investigate molecular mediators of host-microbial interactions with a simplified 12-member microbiome (CeMbio). Previous studies showed the microbiome exerts significant influence on the physiology and development of C. elegans, but lack of molecular tools has limited the ability to identify the microbial factors responsible. To address this challenge, we developed resources to enable genetic manipulation of CeMbio strains. Thus far, we identified several genetic determinants of microbe-microbe interactions, host-specific association and impact on physiology in the dominant microbiome member Ochrobactrum. For genetic manipulation of the CeMbio strains, we utilized broad host range vectors to make an effective panel of fluorescent reporter strains. We used these strains to test pairwise interactions with other microbiome members both in vitro and within the C. elegans gut. Ochrobactrum growth was inhibited by three strains while synergistic with one strain in rich media. In the C. elegans gut, however, microbial interactions were often dramatically different. Notably, Myroides suppresses Ochrobactrum growth in vitro, while Ochrobactrum benefits from the presence of Myroides to colonize the gut. This suggests that host factors may be driving the enrichment for and interactions between members of its microbiome. To examine the molecular determinants of host association and competition, we developed tools for random transposon mutagenesis in Ochrobactrum and several CeMbio strains. We screened a 96-clone mutant library of Ochrobactrum for fitness changes relative to the wild-type in ability to colonize C. elegans hosts with and without Myroides. None of the mutants altered Myroides inhibition of Ochrobactrum in vitro, but several (12) mutants exhibited host association and inter-microbial competition defects. These mutants highlight specific metabolic pathways that Ochrobactrum relies on to colonize and compete for nutrients in association with C. elegans. Our work has demonstrated the ability to use broad host range molecular tools to manipulate CeMbio strains allowing us to visualize and identify molecular mechanisms underlying microbiome assembly and impact upon the C. elegans host.

Assie, Adrien et al. (2021) International Worm Meeting "WormBiome: A pipeline to predict functional profiles of C. elegans associated microbial communities"

Samuel, Buck, Blackburn, Dana, Assie, Adrien, Zhang, Fan

[International Worm Meeting, 2021]

Teasing apart the dense network of molecular mechanisms that link digestive tract microbial community members to each other and their host is challenging in most systems due to complexity and tractability. Recently, Caenorhabditis elegans has emerged as a powerful model system to study host-microbe interactions. The simplicity of the C. elegans digestive tract, together with the nematode's genetic amenability, and the availability of relevant microbial collections make it ideal for the study of the fundamental mechanisms of host-microbiota interactions. To characterize those mechanisms, we focused on identifying genetic features important for bacteria colonizing the C. elegans gut. We developed "WormBiome" a pipeline that predicts the combined functional potential of defined C. elegans gut microbiome. The "Wormbiome" pipeline match predicted microbial abundance from 16S rRNA amplicon datasets to functional genomic annotations and output functional profiles for each microbial community present in the submitted dataset. Our functional annotations rely on curated metabolic (Metacyc) and functional annotations (KEGG) databases build on known C. elegans related bacteria. Then the pipeline predicts features significantly different between user-defined sample groups. We tested the pipeline using a defined and fully sequenced 12-member model microbiome (CeMbio) grown with and without N2 animals. With 10 replicates for each condition, we identified 1700 significantly different features, distributed across 180 KEGG and 63 Metacyc categories. The most abundant features belong to the lipid, amino acid, and cofactor metabolisms. Among genes predicted to be more abundant in worm-associated communities, we found the de novo synthesis of vitamin B12 and metabolic pathways for host-essential amino acids, such as proline, alanine, and arginine. We verified the pipeline's prediction by examining the impact of nutrient depletion on gut microbiome composition by selectively supplementing or removing amino acids individually or altogether. Our results show that a single change in amino acid can affect how bacteria interact with each other and promote the growth of certain community members and that complete removal of amino acids promotes colonization of metabolically flexible members of the microbiome like Ochrobactrum. This study establishes a robust framework for identifying microbial functions that govern affect host-microbe associations and beneficial interactions.

Coux O (2003) Biochem Soc Trans "An interaction map of proteasome subunits."

Coux O

[Biochem Soc Trans, 2003]

Despite the central role of the 26 S proteasome in eukaryotic cells, many facets of its structural organization and functioning are still poorly understood. To learn more about the interactions between its different subunits, as well as its possible functional partners in cells, we performed, with Marc Vidal's laboratory (Dana-Farber Cancer Institute, Boston, MA, U.S.A.), a systematic two-hybrid analysis using Caenorhaditis elegans 26 S proteasome subunits as baits (Davy, Bello, Thierry-Mieg, Vaglio, Hitti, Doucette-Stamm, Thierry-Mieg, Reboul, Boulton, Walhout et al. (2001) EMBO Rep. 2, 821-828). A pair-wise matrix of all subunit combinations allowed us to detect numerous possible intra-complex interactions, among which some had already been reported by others and eight were novel. Interestingly, four new interactions were detected between two ATPases of the 19 S regulatory complex and three alpha-subunits of the 20 S proteolytic core. Possibly, these interactions participate in the association of these two complexes to form the 26 S proteasome. Proteasome subunit sequences were also used to screen a cDNA library to identify new interactors of the complex. Among the interactors found, most (58) have no clear connection to the proteasome, and could be either substrates or potential cofactors of this complex. Few interactors (7) could be directly or indirectly linked to proteolysis. The others (12) interacted with more than one proteasome subunit, forming 'interaction clusters' of

Soderlund CA et al. (1990) Worm Breeder's Gazette "gm Sequence Analysis System Update"

Soderlund CA, Fields CA, Shanmugam P

[Worm Breeder's Gazette, 1990]

A number of improvements have been made in the gm automated DNA sequence analysis system: (1) The ratio of AT-containing to CG-containing dinucleotides has been added as a test for introns. This works better than AT frequency alone in C. elegans.(2) A branch site consensus sequence or an enhanced dinucleotide ratio can be required as an additional test on introns. (3) Predicted amino-acid sequence files are generated in a format appropriate for input to the Dana-Farber motif-identification program plsearch. (4) A graphic interface based on X-windows, version 11 is available as an option. (5) The complete analysis algorithm is significantly faster than the previous version. (6) A greedy model evaluation algorithm is available as an option. This algorithm generates the longest, non-overlapping models that cover a sequence and is much faster than the complete analysis algorithm. The program has been tested on Sun3, Sun4 and VAX machines running Unix (Ultrix on the VAX). Results for a series of tests run on a Sun 4/60 are shown in the table. [See Figure 1] gm can be run remotely on our machine, using the Internet. To do this, telnet to haywire.nmsu.edu, and logon as gm_guest with password gmuser. Read the README file for information on running gm. We are also distributing gm as C source code to nonprofit laboratories, either via anonymous ftp to haywire.nmsu.edu, or on tape. If you would like to receive gm on tape, send a 1/4' cartridge or 1/2' reel tape to: Chris Fields, Box 30001/3CRL, New Mexico State University, Las Cruces, New Mexico 88003-0001, USA; Telephone (505) 646-2848.

Magnenat L et al. (1994) Worm Breeder's Gazette "Telomerases in Nematodes"

Nilsen TW, Mueller F, Magnenat L, Tobler H

[Worm Breeder's Gazette, 1994]

Telomeres confer stability to the chromosome ends by protecting them from degradation and illegitimate recombination and might contribute to the spatial organization of the chromosomes in the nucleus. Furthermore, they affect the expression of telomeric proximal genes and might be involved in the mechanism of somatic cell aging (for review see Biessmann and Mason, 1992, Advances in Genetics 30, 185-249). The protecting telomeric extremities of the chromosomes are maintained by special enzymes, the telomerases, which add repeats of the telomeric sequence after each round of replication (for review see Blackburn, 1992, Ann. Rev. Biochem. 61, 113-129). Telomerases are ribonucleoproteins, acting as RNA dependent DNA polymerases, which contain their own RNA template as an integral part of the enzyme, unlike the conventional reverse transcriptases. The RNA component of telomerases from several Ciliates species has been identified and cloned, but all attempts to clone the gene(s) for the protein component(s) from any system have failed so far. We assume that new telomere formation during the process of chromatin diminution in A. Iumbricoides requires a strong telomerase activity to resynthetize several kilobases of telomeric sequences in somatic cells (for review see Tobler et al., 1992, TIG 8, 427-432). In vitro extracts from eliminating developmental stages might thus be well suited for the isolation of the telomerase protein (or other factors) and for the cloning of the corresponding genes. Therefore, extracts from 4-8 cell stages were established and their quality was assessed by the ability to transcribe 5S rRNA genes (pol III) and SL RNA genes (pol II). Faithful in vitro extracts were tested for telomerase activity; our preliminary results revealed a possible nonprocessive and RNAse sensitive telomerase activity, capable of adding specific nucleotide sequences to the oligonucleotide primer (TTAGGC)(4). Currently, these data are being confirmed and extended. In a parallel approach, we will try to identify the C. elegans telomerase gene genetically.

Reboul J et al. (2001) Nat Genet "Open-reading-frame sequence tags (OSTs) support the existence of at least 17,300 genes in C. elegans."

Doucette-Stamm L, Lamesch PE, Reboul J, Temple GF, Hartley JL, Brasch MA, Hill DE, Vaglio P, Thierry-Mieg N, Shin-i T, Lee H, Moore T, Vandenhaute J, Kohara Y, Vidal M, Jackson C, Thierry-Mieg J, Tzellas N, Thierry-Mieg D, Hitti J

[Nat Genet, 2001]

The genome sequences of Caenorhabditis elegans, Drosophila melanogaster and Arabidopsis thaliana have been predicted to contain 19,000, 13,600 and 25,500 genes, respectively. Before this information can be fully used for evolutionary and functional studies, several issues need to be addressed. First, the gene number estimates obtained in silico and not yet supported by any experimental data need to be verified. For example, it seems biologically paradoxical that C. elegans would have 50% more genes than Drosophilia. Second, intron/exon predictions need to be tested experimentally. Third, complete sets of open reading frames (ORFs), or "ORFeomes," need to be cloned into various expression vectors. To address these issues simultaneously, we have designed and applied to C. elegans the following strategy. Predicted ORFs are amplified by PCR from a highly representative cDNA library using ORF-specific primers, cloned by Gateway recombination cloning and then sequenced to generate ORF sequence tags (OSTs) as a way to verify identity and splicing. In a sample (n=1,222) of the nearly 10,000 genes predicted ab initio (that is, for which no expressed sequence tag (EST) is available so far), at least 70% were verified by OSTs. We also observed that 27% of these experimentally confirmed genes have a structure different from that predicted by GeneFinder. We now have experimental evidence that supports the existence of at least 17,300 genes in C. elegans. Hence we suggest that gene counts based primarily on ESTs may underestimate the number of genes in human and in other organisms.AD - Dana-Farber Cancer Institute and Department of Genetics, Harvard Medical School, Boston, Massachusetts, USA.FAU - Reboul, JAU - Reboul JFAU - Vaglio, PAU - Vaglio PFAU - Tzellas, NAU - Tzellas NFAU - Thierry-Mieg, NAU - Thierry-Mieg NFAU - Moore, TAU - Moore TFAU - Jackson, CAU - Jackson CFAU - Shin-i, TAU - Shin-i TFAU - Kohara, YAU - Kohara YFAU - Thierry-Mieg, DAU - Thierry-Mieg DFAU - Thierry-Mieg, JAU - Thierry-Mieg JFAU - Lee, HAU - Lee HFAU - Hitti, JAU - Hitti JFAU - Doucette-Stamm, LAU - Doucette-Stamm LFAU - Hartley, J LAU - Hartley JLFAU - Temple, G FAU - Temple GFFAU - Brasch, M AAU - Brasch MAFAU - Vandenhaute, JAU - Vandenhaute JFAU - Lamesch, P EAU - Lamesch PEFAU - Hill, D EAU - Hill DEFAU - Vidal, MAU - Vidal MLA - engID - R21 CA81658 A 01/CA/NCIID - RO1 HG01715-01/HG/NHGRIPT - Journal ArticleCY - United StatesTA - Nat GenetJID - 9216904SB - IM

Magnenat L et al. (1996) European Worm Meeting "TELOMERASE-MEDIATED CHROMOSOME HEALING IN NEMATODES"

Magnenat L, Tobler H, Jentsch S, Mueller F

[European Worm Meeting, 1996]

Telomeres are specialized terminal structures, that are necessary for the stability and complete replication of linear chromosomes. In most eukaryotic organisms, synthesis and maintenance of telomeric DNA sequences require a specialized telomere terminal transferase activity (telomerase). If the protecting extremities are lost during a chromosome fragmentation event, new telomere formation at the broken ends is essential to prevent genome rearrangements (reviewed in Telomeres, Blackburn & Greider, 1995). In nematodes, which have TTAGGC telomeric repeats similar to those of other eukaryotes, two interesting types of healing events have been reported: 1) An example of spontaneous chromosomal healing has been described for the Caenorhabditis elegans X-chromosome mutation me8, which disrupts meiotic crossing over and segregation. Molecular analysis of the me8 mutation revealed that it is a terminal chromosomal truncation healed by the de novo addition of telomeric repeats directly to the site of breakage. (C. Wicky et al., 1996. PNAS: in press ). 2) During the somatic differentiation of Ascaris suum, the developmentally-programmed phenomenon of chromatin diminution consists of chromosomal cleavage at specific breakage regions (CBRs), chromosome healing by new telomere formation and degradation of the eliminated chromatin. (F. Mueller et al., 1991. Cell 67: 815-822). Chromosome healing in both nematodes share the same molecular characteristics. First, the telomere addition sites share no sequence homology or secondary structures with each other. Second, no pre-existing telomeric sequences are found within CBRs, and third, all junctions contain 1-4 ambiguous bases which can be used as telomerase initiation primers. However, spontaneous chromosomal healing in C. elegans takes place randomly in germ cells with low efficiency, whereas the developmentally-programmed healing in A. suum is highly efficient and occurs in all presomatic cells. Also, a random distribution of the telomere addition sites within the CBRs suggests that chromatin diminution is initiated with a double-strand DNA break which is followed by exonuclease trimming of the unprotected ends until the telomerase- mediated healing machinery encounters the appropriate conditions to add new telomeric sequences. Surprisingly, telomeric repeats are not only added to the ends of the truncated chromosomes, but also to the eliminated chromatin fragments. This suggest that in A. suum, the same telomerase activity that is responsible for telomere maintenance and spontaneous healing may have been adapted to catalyze the highly efficient programmed healing process during chromatin diminution. To further analyze telomerase-mediated chromosome healing, we developed an in vitro telomerase system for both nematode species. Since we observed a DNA polymerization activity producing repeats of 6 bases that was sensitive to heat inactivation, proteinase K and RNase A digestions in the Ascaris cell-free extracts, we concluded that it represents telomerase activity. Its presence during early developmental stages may indicate a role for telomerase in the mechanism of chromatin diminution. Currently, different C. elegans extracts are being tested for telomerase in vitro and preliminary results suggest the presence of a similar activity. Given the abundance of the genetic tools and the advanced state of the physical and genetic map of the C. elegans genome, as well as the biochemical potential of A. suum, nematodes may serve as excellent model systems for the analysis of the telomere and telomerase function during chromosome healing processes among metazoan organisms.