A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary

Argonaute proteins of the PIWI clade complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.

Evidence for a conserved queen-worker genetic toolkit across slave-making ants and their ant hosts

The ecological success of social Hymenoptera (ants, bees, wasps) depends on the division of labour between the queen and workers. Each caste is highly specialized in their respective function in morphology, behaviour and life history traits, such as lifespan and fecundity. Despite strong defences against alien intruders, insect societies are vulnerable to social parasites, such as workerless inquilines or slave-making (dulotic) ants. Here, we investigate whether gene expression varies in parallel ways between slave-making ants and their host ants across five independent origins of ant slavery in the Formicoxenus-group of the ant tribe Crematogastrini. As caste differences are often less pronounced in slave-making ants than non-parasitic ants, we also compare the transcriptomes of queens and workers in these species. We demonstrate a substantial overlap in expression differences between queens and workers across taxa, irrespective of lifestyle. Caste affects the transcriptomes much more profoundly than lifestyle, as indicated by 37 times more genes being linked to caste than to lifestyle and by multiple caste-associated gene modules with strong connectivity. However, several genes and one gene module are linked to the slave-making lifestyle across the independent origins, pointing to some evolutionary convergence. Finally, we do not find evidence for an interaction between caste and lifestyle, indicating that caste differences remain consistent even when species switch to a parasitic lifestyle. Our findings are a strong indication for the existence of a core set of genes whose expression is linked to the queen and worker caste in this ant taxon, supporting the genetic-toolkit hypothesis.

A genetic toolkit and gene switches to limit Mycoplasma growth for a synthetic vaccine chassis

Mycoplasmas have exceptionally streamlined genomes and are strongly adapted to their many hosts, which provide them with essential nutrients. Owing to their relative genomic simplicity, Mycoplasmas have been used for the development of chassis to deploy tailored vaccines. However, the dearth of robust and precise toolkits for genomic manipulation and tight regulation has hindered any substantial advance. Herein we describe the construction of a robust genetic toolkit for M. pneumoniae, and its successful deployment to engineer synthetic gene switches that control and limit Mycoplasma growth, for biosafety containment applications. We found these synthetic gene circuits to be stable and robust in the long-term, in the context of a minimal cell. With this work, we lay a foundation to develop viable and robust biosafety systems to exploit a synthetic Mycoplasma chassis for live attenuated vaccines or even for live vectors for biotherapeutics.

Social complexity, life-history and lineage influence the molecular basis of caste in a major transition in evolution

Major evolutionary transitions describe how biological complexity arises; e.g. in the evolution of complex multicellular bodies, and superorganismal insect societies. Such transitions involve the evolution of division of labour, e.g. as queen and worker castes in insect societies. A key mechanistic hypothesis for the evolution of division of labour is that a shared set of genes co-opted from a common solitary ancestral ground plan - a so-called genetic toolkit for sociality - regulate insect castes across different levels of social complexity. The vespid wasps represent an excellent system in which to test this. Here, using conventional and machine learning analyses of brain transcriptome data from nine species of vespid wasps, we find evidence of a shared genetic toolkit across species representing different levels of social complexity, with a large suite of genes classifying castes correctly across species. However, we also found evidence of additional fine-scale differences in predictive gene sets, functional enrichment and rates of gene evolution that were related to level of social complexity, but also life-history traits (e.g. mode of colony founding). Thus, there appear to be shifts in the gene networks regulating social behaviour and rates of gene evolution that are influenced by innovations in both social complexity and life-history. These results suggest that the concept of a shared genetic toolkit for sociality may be too simplistic to fully describe the process of the major transition to sociality, even within a single lineage. Diversity in lineage, social complexity and life-history traits must be taken into account in the quest to uncover the molecular bases of the major transition to sociality.

A genetic toolkit for studying transposon control in the Drosophila melanogaster ovary

Argonaute proteins of the PIWI class complexed with PIWI-interacting RNAs (piRNAs) protect the animal germline genome by silencing transposable elements. One of the leading experimental systems for studying piRNA biology is the Drosophila melanogaster ovary. In addition to classical mutagenesis, transgenic RNA interference (RNAi), which enables tissue-specific silencing of gene expression, plays a central role in piRNA research. Here, we establish a versatile toolkit focused on piRNA biology that combines germline transgenic RNAi, GFP marker lines for key proteins of the piRNA pathway, and reporter transgenes to establish genetic hierarchies. We compare constitutive, pan-germline RNAi with an equally potent transgenic RNAi system that is activated only after germ cell cyst formation. Stage-specific RNAi allows us to investigate the role of genes essential for germline cell survival, for example nuclear RNA export or the SUMOylation pathway, in piRNA-dependent and independent transposon silencing. Our work forms the basis for an expandable genetic toolkit provided by the Vienna Drosophila Resource Center.

Improved dynamic range of a rhamnose-inducible promoter for gene expression in Burkholderia

A diverse genetic toolkit is critical for understanding bacterial physiology and genotype-phenotype relationships. Inducible promoter systems are an integral part of this toolkit. In Burkholderia and related species, the L-rhamnose-inducible promoter is among the first choices due to its tight control and the lack of viable alternatives. To improve upon its maximum activity and dynamic range, we explored the effect of promoter system modifications in B. cenocepacia with a LacZ-based reporter. By combining the bacteriophage T7 gene 10 stem loop and engineered rhaI transcription factor-binding sites, we obtained a rhamnose-inducible system with a 6.5-fold and 3.0-fold increase in maximum activity and dynamic range, respectively, compared to the native promoter. We then added the modified promoter system to pSCrhaB2 and pSC201, common genetic tools used for plasmid-based and chromosome-based gene expression, respectively, in Burkholderia, creating pSCrhaB2plus and pSC201plus. We demonstrated the utility of pSCrhaB2plus for gene expression in B. thailandensis , B. multivorans and B. vietnamiensis and used pSC201plus to control highly expressed essential genes from the chromosome of B. cenocepacia . The utility of the modified system was demonstrated as we recovered viable mutants to control ftsZ , rpoBC , and rpsF , whereas the unmodified promoter was unable to control rpsF . The modified expression system allowed control of an essential gene depletion phenotype at lower levels of L-rhamnose, the inducer. pSCRhaB2plus and pSC201plus are expected to be valuable additions to the genetic toolkit for Burkholderia and related species. Importance Species of Burkholderia are dually recognized as being of attractive biotechnological potential but also opportunistic pathogens for immunocompromised individuals. Understanding the genotype-phenotype relationship is critical for synthetic biology approaches in Burkholderia to disentangle pathogenic from beneficial traits. A diverse genetic toolkit, including inducible promoters, is the foundation for these investigations. We thus sought to improve on the commonly used rhamnose-inducible promoter system. Our modifications resulted in both higher levels of heterologous protein expression and broader control over highly-expressed essential genes in B. cenocepacia . The significance of our work is in expanding the genetic toolkit to enable more comprehensive studies into Burkholderia and related bacteria.

Multifunktionale bakterielle Nanomagnete für Biotechnologie und Medizin

Bacterial magnetosomes represent magnetic core-shell nanoparticles biomineralized by magnetotactic bacteria like Magnetospirillum gryphiswaldense. The establishment of fermentation regimes for high-yield particle production, standardized isolation procedures as well as the development of a genetic toolkit for the generation of “tailored” particles might soon pave the way for the application of engineered magnetosomes in the biomedical and biotechnological field.

Exon Shuffling Played a Decisive Role in the Evolution of the Genetic Toolkit for the Multicellular Body Plan of Metazoa

Division of labor and establishment of the spatial pattern of different cell types of multicellular organisms require cell type-specific transcription factor modules that control cellular phenotypes and proteins that mediate the interactions of cells with other cells. Recent studies indicate that, although constituent protein domains of numerous components of the genetic toolkit of the multicellular body plan of Metazoa were present in the unicellular ancestor of animals, the repertoire of multidomain proteins that are indispensable for the arrangement of distinct body parts in a reproducible manner evolved only in Metazoa. We have shown that the majority of the multidomain proteins involved in cell–cell and cell–matrix interactions of Metazoa have been assembled by exon shuffling, but there is no evidence for a similar role of exon shuffling in the evolution of proteins of metazoan transcription factor modules. A possible explanation for this difference in the intracellular and intercellular toolkits is that evolution of the transcription factor modules preceded the burst of exon shuffling that led to the creation of the proteins controlling spatial patterning in Metazoa. This explanation is in harmony with the temporal-to-spatial transition hypothesis of multicellularity that proposes that cell differentiation may have predated spatial segregation of cell types in animal ancestors.