The Wolbachia cytoplasmic incompatibility enzyme CidB targets nuclear import and protamine-histone exchange factors
TL;DR: CidB targets nuclear-protein import and protamine-histone exchange and that CidA rescues embryos by restricting CidB access to its targets and this is proposed to be a rescue mechanism for embryos.
Abstract: Intracellular Wolbachia bacteria manipulate arthropod reproduction to promote their own inheritance. The most prevalent mechanism, cytoplasmic incompatibility (CI), traces to a Wolbachia deubiquitylase, CidB, and CidA. CidB has properties of a toxin, while CidA binds CidB and rescues embryonic viability. CidB is also toxic to yeast where we identified both host effects and high-copy suppressors of toxicity. The strongest suppressor was karyopherin-α, a nuclear-import receptor; this required nuclear localization-signal binding. A protein-interaction screen of Drosophila extracts using a substrate-trapping catalytic mutant, CidB*, also identified karyopherin-α; the P32 protamine-histone exchange factor bound as well. When CidB* bound CidA, these host protein interactions disappeared. These associations would place CidB at the zygotic male pronucleus where CI defects first manifest. Overexpression of karyopherin-α, P32, or CidA in female flies suppressed CI. We propose that CidB targets nuclear-protein import and protamine-histone exchange and that CidA rescues embryos by restricting CidB access to its targets.
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TL;DR: The tight association of the CI genes with prophages provides clues to the possible evolutionary origin of this phenomenon and the levels of selection at play.
95 citations
TL;DR: The Two-by-One model in wMel-infected D. melanogaster is explicitly validated, established a robust system for transgenic studies of CI in a model system, and represents the first case of completely engineering male and female animal reproduction to depend upon bacteriophage gene products.
Abstract: Wolbachia are maternally inherited bacteria that infect arthropod species worldwide and are deployed in vector control to curb arboviral spread using cytoplasmic incompatibility (CI). CI kills embryos when an infected male mates with an uninfected female, but the lethality is rescued if the female and her embryos are likewise infected. Two phage WO genes, cifAwMel and cifBwMel from the wMel Wolbachia deployed in vector control, transgenically recapitulate variably penetrant CI, and one of the same genes, cifAwMel, rescues wild type CI. The proposed Two-by-One genetic model predicts that CI and rescue can be recapitulated by transgenic expression alone and that dual cifAwMel and cifBwMel expression can recapitulate strong CI. Here, we use hatch rate and gene expression analyses in transgenic Drosophila melanogaster to demonstrate that CI and rescue can be synthetically recapitulated in full, and strong, transgenic CI comparable to wild type CI is achievable. These data explicitly validate the Two-by-One model in wMel-infected D. melanogaster, establish a robust system for transgenic studies of CI in a model system, and represent the first case of completely engineering male and female animal reproduction to depend upon bacteriophage gene products.
76 citations
TL;DR: This review serves as a gateway to experimental, conceptual, and quantitative themes of CI and outlines significant gaps in understanding CI’s mechanism that are ripe for investigation from diverse subdisciplines in the life sciences.
Abstract: Cytoplasmic incompatibility (CI) is the most common symbiont-induced reproductive manipulation. Specifically, symbiont-induced sperm modifications cause catastrophic mitotic defects in the fertilized embryo and ensuing lethality in crosses between symbiotic males and either aposymbiotic females or females harboring a different symbiont strain. However, if the female carries the same symbiont strain, then embryos develop properly, thereby imparting a relative fitness benefit to symbiont-transmitting mothers. Thus, CI drives maternally-transmitted bacteria to high frequencies in arthropods worldwide. In the past two decades, CI experienced a boom in interest due to its (i) deployment in worldwide efforts to curb mosquito-borne diseases, (ii) causation by bacteriophage genes, cifA and cifB, that modify sexual reproduction, and (iii) important impacts on arthropod speciation. This review serves as a gateway to experimental, conceptual, and quantitative themes of CI and outlines significant gaps in understanding CI's mechanism that are ripe for investigation from diverse subdisciplines in the life sciences.
72 citations
TL;DR: It is shown that the Wolbachia cin operon constitutes another toxin–antidote system in which CinB is a nuclease toxin and CinA binds tightly to CInB and can rescue embryo viability, providing important insights into the molecular basis of CI.
Abstract: Wolbachia are endosymbiotic bacteria that infect nearly half of all arthropod species. This pandemic is due in part to their ability to increase their transmission through the female germline, most commonly by a mechanism called cytoplasmic incompatibility (CI). The Wolbachia cid operon, encoding 2 proteins, CidA and CidB, the latter a deubiquitylating enzyme (DUB), recapitulates CI in transgenic Drosophila melanogaster However, some CI-inducing Wolbachia strains lack a DUB-encoding cid operon; it was therefore proposed that the related cin operon codes for an alternative CI system. Here we show that the Wolbachia cin operon encodes a nuclease, CinB, and a second protein, CinA, that tightly binds CinB. Recombinant CinB has nuclease activity against both single-stranded and double-stranded DNA but not RNA under the conditions tested. Expression of the cin operon in transgenic male flies induces male sterility and embryonic defects typical of CI. Importantly, transgenic CinA can rescue defects in egg-hatch rates when expressed in females. Expression of CinA also rescues CinB-induced growth defects in yeast. CinB has 2 PD-(D/E)xK nuclease domains, and both are required for nuclease activity and for toxicity in yeast and flies. Our data suggest a distinct mechanism for CI involving a nuclease toxin and highlight the central role of toxin-antidote operons in Wolbachia-induced cytoplasmic incompatibility.
72 citations
TL;DR: It is shown here that the remarkable diversity of CI in the C. pipiens complex is due to the presence, in all tested wPip genomes, of several copies of the cidA-cidB operon, which undergoes diversification through recombination events, consistent with the hypothesis of a toxin–antitoxin system.
Abstract: Culex pipiens mosquitoes are infected with Wolbachia (wPip) that cause an important diversity of cytoplasmic incompatibilities (CIs). Functional transgenic studies have implicated the cidA-cidB operon from wPip and its homolog in wMel in CI between infected Drosophila males and uninfected females. However, the genetic basis of the CI diversity induced by different Wolbachia strains was unknown. We show here that the remarkable diversity of CI in the C. pipiens complex is due to the presence, in all tested wPip genomes, of several copies of the cidA-cidB operon, which undergoes diversification through recombination events. In 183 isofemale lines of C. pipiens collected worldwide, specific variations of the cidA-cidB gene repertoires are found to match crossing types. The diversification of cidA-cidB is consistent with the hypothesis of a toxin–antitoxin system in which the gene cidB co-diversifies with the gene cidA, particularly in putative domains of reciprocal interactions.
69 citations
References
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TL;DR: A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed and will reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications.
Abstract: A set of yeast strains based on Saccharomyces cerevisiae S288C in which commonly used selectable marker genes are deleted by design based on the yeast genome sequence has been constructed and analysed. These strains minimize or eliminate the homology to the corresponding marker genes in commonly used vectors without significantly affecting adjacent gene expression. Because the homology between commonly used auxotrophic marker gene segments and genomic sequences has been largely or completely abolished, these strains will also reduce plasmid integration events which can interfere with a wide variety of molecular genetic applications. We also report the construction of new members of the pRS400 series of vectors, containing the kanMX, ADE2 and MET15 genes.
3,448 citations
"The Wolbachia cytoplasmic incompati..." refers background in this paper
...Suppressor screens were performed in the BY4741 yeast background....
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...BY4741 [pRS416GAL1-His6CidBwPip] yeast were streaked out on synthetic defined (SD) glucose medium lacking uracil (SD-ura)....
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...For example, there may be lower levels in W303-1A compared to BY4741 of a key ubiquitin-protein conjugate that is essen- tial for growth and targeted by the CidB DUB....
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...BY4741 was discontinued after Figure 1a because W303-1A exhibited stronger sensitivity to CI factors....
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...All other serial dilution and Western blotting data used W303-1A except Figure 3d which was BY4741....
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TL;DR: To release plasmid DNA for the transformation of Escherichia coli, cells are subjected to vortex mixing in the presence of acid-washed glass beads, Triton X-100, sodium dodecyl sulfate, phenol and chloroform.
2,483 citations
"The Wolbachia cytoplasmic incompati..." refers methods in this paper
...Yeast genomic DNA was purified by lysing cells by glass bead disruption, followed by phenol/chloroform extraction and ethanol precipitation (Hoffman and Winston, 1987)....
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...for ‘smash-n-grab’ plasmid recovery (Hoffman and Winston, 1987)....
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...Cultures were used for ‘smash-n-grab’ plasmid recovery (Hoffman and Winston, 1987)....
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...Nucleic acid sources and construct preparation Yeast genomic DNA was purified by lysing cells by glass bead disruption, followed by phenol/chloroform extraction and ethanol precipitation (Hoffman and Winston, 1987)....
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TL;DR: The basic biology of Wolbachia is reviewed, with emphasis on recent advances in the authors' understanding of these fascinating endosymbionts, which are found in arthropods and nematodes.
Abstract: Wolbachia are common intracellular bacteria that are found in arthropods and nematodes. These alphaproteobacteria endosymbionts are transmitted vertically through host eggs and alter host biology in diverse ways, including the induction of reproductive manipulations, such as feminization, parthenogenesis, male killing and sperm-egg incompatibility. They can also move horizontally across species boundaries, resulting in a widespread and global distribution in diverse invertebrate hosts. Here, we review the basic biology of Wolbachia, with emphasis on recent advances in our understanding of these fascinating endosymbionts.
2,333 citations
"The Wolbachia cytoplasmic incompati..." refers background in this paper
...Wolbachia are obligate intracellular bacteria infecting arthropods and filarial nematodes (Werren et al., 2008)....
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TL;DR: A growing number of cellular regulatory mechanisms are being linked to protein modification by the polypeptide ubiquitin, including key transitions in the cell cycle, class I antigen processing, signal transduction pathways, and receptor-mediated endocytosis.
Abstract: ▪ Abstract A growing number of cellular regulatory mechanisms are being linked to protein modification by the polypeptide ubiquitin. These include key transitions in the cell cycle, class I antigen processing, signal transduction pathways, and receptor-mediated endocytosis. In most, but not all, of these examples, ubiquitination of a protein leads to its degradation by the 26S proteasome. Following attachment of ubiquitin to a substrate and binding of the ubiquitinated protein to the proteasome, the bound substrate must be unfolded (and eventually deubiquitinated) and translocated through a narrow set of channels that leads to the proteasome interior, where the polypeptide is cleaved into short peptides. Protein ubiquitination and deubiquitination are both mediated by large enzyme families, and the proteasome itself comprises a family of related but functionally distinct particles. This diversity underlies both the high substrate specificity of the ubiquitin system and the variety of regulatory mechanisms...
1,741 citations
"The Wolbachia cytoplasmic incompati..." refers background in this paper
...Post-translational ubiquitin modifications alter protein stability, localization, and interactions (Hochstrasser, 1996; Ronau et al., 2016)....
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TL;DR: Northern analysis of strains containing plasmid inserts with various promoter mutations suggests that the stimulation in recombination is mediated by events initiating within the integrated plasmID sequences.
1,641 citations
"The Wolbachia cytoplasmic incompati..." refers background in this paper
..., 1998) and W303-1A (Thomas and Rothstein, 1989), we noticed greater sensitivity to its expression in W303-1A (Figure 1a)....
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...BY4741 was discontinued after Figure 1a because W303-1A exhibited stronger sensitivity to CI factors....
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...All other serial dilution and Western blotting data used W303-1A except Figure 3d which was BY4741....
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...Yeast strain backgrounds used were W303-1A and BY4741....
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...For example, there may be lower levels in W303-1A compared to BY4741 of a key ubiquitin-protein conjugate that is essen- tial for growth and targeted by the CidB DUB....
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