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Bunk B, Schulenburg H, Yang W, Leippe M, Hoeppner MP, Zimmermann J, Sproer C, Aidley J, Obeng N, Pees B, Kaleta C, Dierking K, Kissoyan KA, Petersen C, Waschina S
[
ISME J,
2019]
The microbiota is generally assumed to have a substantial influence on the biology of multicellular organisms. The exact functional contributions of the microbes are often unclear and cannot be inferred easily from 16S rRNA genotyping, which is commonly used for taxonomic characterization of bacterial associates. In order to bridge this knowledge gap, we here analyzed the metabolic competences of the native microbiota of the model nematode Caenorhabditis elegans. We integrated whole-genome sequences of 77 bacterial microbiota members with metabolic modeling and experimental characterization of bacterial physiology. We found that, as a community, the microbiota can synthesize all essential nutrients for C. elegans. Both metabolic models and experimental analyses revealed that nutrient context can influence how bacteria interact within the microbiota. We identified key bacterial traits that are likely to influence the microbe's ability to colonize C. elegans (i.e., the ability of bacteria for pyruvate fermentation to acetoin) and affect nematode fitness (i.e., bacterial competence for hydroxyproline degradation). Considering that the microbiota is usually neglected in C. elegans research, the resource presented here will help our understanding of this nematode's biology in a more natural context. Our integrative approach moreover provides a novel, general framework to characterize microbiota-mediated functions.
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Kuenzel S, Sieber M, Schulenburg H, Petersen C, Traulsen A, Zimmermann J, Moitinho-Silva L, Kaleta C, Bluhm L, Piecyk A, Johnke J
[
mBio,
2024]
The microbiome expresses a variety of functions that influence host biology. The range of functions depends on the microbiome's composition, which can change during the host's lifetime due to neutral assembly processes, host-mediated selection, and environmental conditions. To date, the exact dynamics of microbiome assembly, the underlying determinants, and the effects on host-associated functions remain poorly understood. Here, we used the nematode Caenorhabditis elegans and a defined community of fully sequenced, naturally associated bacteria to study microbiome dynamics and functions across a major part of the worm's lifetime of hosts under controlled experimental conditions. Bacterial community composition initially shows strongly declining levels of stochasticity, which increases during later time points, suggesting selective effects in younger animals as opposed to more random processes in older animals. The adult microbiome is enriched in genera Ochrobactrum and Enterobacter compared to the direct substrate and a host-free control environment. Using pathway analysis, metabolic, and ecological modeling, we further find that the lifetime assembly dynamics increase competitive strategies and gut-associated functions in the host-associated microbiome, indicating that the colonizing bacteria benefit the worm. Overall, our study introduces a framework for studying microbiome assembly dynamics based on stochastic, ecological, and metabolic models, yielding new insights into the processes that determine host-associated microbiome composition and function.Importance: The microbiome plays a crucial role in host biology. Its functions depend on the microbiome composition that can change during a host's lifetime. To date, the dynamics of microbiome assembly and the resulting functions still need to be better understood. This study introduces a new approach to characterize the functional consequences of microbiome assembly by modeling both the relevance of stochastic processes and metabolic characteristics of microbial community changes. The approach was applied to experimental time-series data obtained for the microbiome of the nematode Caenorhabditis elegans across the major part of its lifetime. Stochastic processes played a minor role, whereas beneficial bacteria as well as gut-associated functions enriched in hosts. This indicates that the host might actively shape the composition of its microbiome. Overall, this study provides a framework for studying microbiome assembly dynamics and yields new insights into C. elegans microbiome functions.
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[
J Biol Chem,
2002]
The glioma-amplified sequence (GAS) 41 protein has been proposed to be a transcription factor. To investigate its functional role in vivo, we attempted to knock out the GAS41 gene by targeted disruption in the chicken pre-lymphoid cell line DT40. Heterozygous GAS41+/- cell lines generated by the first round of homologous recombination express approximately half the normal level of GAS41 mRNA. However, a homozygous GAS41-/- cell line with both GAS41 alleles disrupted was not obtained following the second round of transfection, indicating that the GAS41 gene is essential for cell viability. Indeed, homozygous GAS41-/- cell lines with two disrupted GAS41 alleles can be generated following substitution of the endogenous gene by stable integration of GAS41 cDNA controlled by a tetracycline-regulated CMV promoter. Inactivation of this promoter by tetracycline withdrawal results in rapid depletion of GAS41, causing a significant decrease in RNA synthesis and subsequently cell death. Thus, our results indicate that GAS41 is required for RNA transcription.
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[
Biochemistry,
1987]
The major intestinal esterase from the nematode Caenorhabditis elegans has been purified to essential homogeneity. Starting from whole worms, the overall purification is 9000-fold with a 10% recovery of activity. The esterase is a single polypeptide chain of Mr 60,000 and is stoichiometrically inhibited by organophosphates. Substrate preferences and inhibition patterns classify the enzyme as a carboxylesterase (EC 3.1.1.1), but the physiological function is unknown. The sequence of 13 amino acid residues at the esterase N- terminus has been determined. This partial sequence shows a surprisingly high degree of similarity to the N-terminal sequence of two carboxylesterases recently isolated from Drosophila mojavensis [Pen, J., van Beeumen, J., & Beintema, J. J. (1986) Biochem. J. 238, 691-699].
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Berynskyy M, Morimoto RI, Bukau B, Stengel F, Kirstein J, Szlachcic A, Arnsburg K, Stank A, Scior A, Nillegoda NB, Gao X, Guilbride DL, Aebersold R, Wade RC, Mayer MP
[
Nature,
2015]
Protein aggregates are the hallmark of stressed and ageing cells, and characterize several pathophysiological states. Healthy metazoan cells effectively eliminate intracellular protein aggregates, indicating that efficient disaggregation and/or degradation mechanisms exist. However, metazoans lack the key heat-shock protein disaggregase HSP100 of non-metazoan HSP70-dependent protein disaggregation systems, and the human HSP70 system alone, even with the crucial HSP110 nucleotide exchange factor, has poor disaggregation activity in vitro. This unresolved conundrum is central to protein quality control biology. Here we show that synergic cooperation between complexed J-protein co-chaperones of classes A and B unleashes highly efficient protein disaggregation activity in human and nematode HSP70 systems. Metazoan mixed-class J-protein complexes are transient, involve complementary charged regions conserved in the J-domains and carboxy-terminal domains of each J-protein class, and are flexible with respect to subunit composition. Complex formation allows J-proteins to initiate transient higher order chaperone structures involving HSP70 and interacting nucleotide exchange factors. A network of cooperative class A and B J-protein interactions therefore provides the metazoan HSP70 machinery with powerful, flexible, and finely regulatable disaggregase activity and a further level of regulation crucial for cellular protein quality control.
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[
Mol Immunol,
1999]
Invertebrate cells lack the
p53 recombination checkpoint but contain mobile DNA sequences that transpose by a mechanism in part shared with excision of the V(D)J recombination signal sequences (RSS). In this work, inversion, deletion, and duplication of sequences associated with an invertebrate C. elegans Tc6 element is described. The structure of this C. elegans sequence and other dispersed Tc6 elements suggests that covalently closed 'hairpin' structures are not unique to excision of the V(D)J RSS by the RAG proteins, but rather can be generated by transposases at transposon termini leading to characteristic inversion and duplication events. Comparative analysis of recombination events at invertebrate sequences resembling the vertebrate V(D)J RSS may be useful in understanding V(D)J recombination-mediated recombination events in malignant vertebrate cells or genetic diseases such as ataxia telangectasia, in which the
p53 recombination checkpoint is defective.
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[
Phytother Res,
2008]
A bioassay-guided fractionation of Juniperus procera berries yielded antiparasitic, nematicidal and antifouling constituents, including a wide range of known abietane, pimarane and labdane diterpenes. Among these, abieta-7,13-diene (1) demonstrated in vitro antimalarial activity against Plasmodium falciparum D6 and W2 strains (IC(50) = 1.9 and 2.0 microg/mL, respectively), while totarol (6), ferruginol (7) and 7beta-hydroxyabieta-8,13-diene-11,12-dione (8) inhibited Leishmania donovani promastigotes with IC(50) values of 3.5-4.6 microg/mL. In addition, totarol demonstrated nematicidal and antifouling activities against Caenorhabditis elegans and Artemia salina at a concentration of 80 microg/mL and 1 microg/mL, respectively. The resinous exudate of J. virginiana afforded known antibacterial E-communic acid (4) and 4-epi-abietic acid (5), while the volatile oil from its trunk wood revealed large quantities of cedrol (9). Using GC/MS, the two known abietanes totarol (6) and ferruginol (7) were identified from the berries of J. procera, J. excelsa and J. phoenicea.
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[
Aging Cell,
2017]
Protein aggregation is enhanced upon exposure to various stress conditions and aging, which suggests that the quality control machinery regulating protein homeostasis could exhibit varied capacities in different stages of organismal lifespan. Recently, an efficient metazoan disaggregase activity was identified invitro, which requires the Hsp70 chaperone and Hsp110 nucleotide exchange factor, together with single or cooperating J-protein co-chaperones of classes A and B. Here, we describe how the orthologous Hsp70s and J-protein of Caenorhabditis elegans work together to resolve protein aggregates both invivo and invitro to benefit organismal health. Using an RNAi knockdown approach, we show that class A and B J-proteins cooperate to form an interactive flexible network that relocalizes to protein aggregates upon heat shock and preferentially recruits constitutive Hsc70 to disaggregate heat-induced protein aggregates and polyQ aggregates that form in an age-dependent manner. Cooperation between class A and B J-proteins is also required for organismal health and promotes thermotolerance, maintenance of fecundity, and extended viability after heat stress. This disaggregase function of J-proteins and Hsc70 therefore constitutes a powerful regulatory network that is key to Hsc70-based protein quality control mechanisms in metazoa with a central role in the clearance of aggregates, stress recovery, and organismal fitness in aging.
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[
Nat Genet,
2002]
Mice that are homozygous with respect to a mutation (ax(J)) in the ataxia (ax) gene develop severe tremors by 2-3 weeks of age followed by hindlimb paralysis and death by 6-10 weeks of age. Here we show that ax encodes ubiquitin-specific protease 14 (Usp14). Ubiquitin proteases are a large family of cysteine proteases that specifically cleave ubiquitin conjugates. Although Usp14 can cleave a ubiquitin-tagged protein in vitro, it is unable to process polyubiquitin, which is believed to be associated with the protein aggregates seen in Parkinson disease, spinocerebellar ataxia type 1 (SCA1; ref. 4) and gracile axonal dystrophy (GAD). The physiological substrate of Usp14 may therefore contain a mono-ubiquitin side chain, the removal of which would regulate processes such as protein localization and protein activity. Expression of Usp14 is significantly altered in ax(J)/ax(J) mice as a result of the insertion of an intracisternal-A particle (IAP) into intron 5 of Usp14. In contrast to other neurodegenerative disorders such as Parkinson disease and SCA1 in humans and GAD in mice, neither ubiquitin-positive protein aggregates nor neuronal cell loss is detectable in the central nervous system (CNS) of ax(J) mice. Instead, ax(J) mice have defects in synaptic transmission in both the central and peripheral nervous systems. These results suggest that ubiquitin proteases are important in regulating synaptic activity in mammals.
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[
Development,
1991]
In wild-type Caenorhabditis elegans hermaphrodites, two bilaterally symmetric sex myoblasts (SMs) migrate anteriorly to flank the precise center of the gonad, where they divide to generate the muscles required for egg laying (J. E. Sulston and H. R. Horvitz (1977) Devl Biol. 56, 110-156). Although this migration is largely independent of the gonad, a signal from the gonad attracts the SMs to their precise final positions (J. H. Thomas, M. J. Stern and H. R. Horvitz (1990) Cell 62, 1041-1052). Here we show that mutations in either of two genes,
egl-15 and
egl-17, cause the premature termination of the migrations of the SMs. This incomplete migration is caused by the repulsion of the SMs by the same cells in the somatic gonad that are the source of the attractive signal in wild-type animals.