Project description:Enterotoxin-producing C. perfringens type A is a common cause of food poisonings. The cpe encoding the enterotoxin can be chromosomal (genotype IS1470) or plasmid-borne (genotypes IS1470-like-cpe or IS1151-cpe). The chromosomal cpe-carrying C. perfringens are a more common cause of food poisonings than plasmid-borne cpe-genotypes. The chromosomal cpe-carrying C. perfringens type A strains are generally more resistant to most food-processing conditions than plasmid-borne cpe-carrying strains. On the other hand, the plasmid-borne cpe-positive genotypes are more commonly found in human feces than chromosomal cpe-positive genotypes, and humans seem to be a reservoir for plasmid-borne cpe-carrying strains. Thus, it is possible that the epidemiology of C. perfringes type A food poisonings caused by plasmid-borne and chromosomal cpe-carrying strains is different. A DNA microarray was designed for analysis of genetic relatedness between the different cpe-positive and cpe-negative genotypes of C. perfringens strains isolated from human, animal, environmental and food samples. The DNA microarray contained two probes for all protein-coding sequences in the three genome-sequenced strains (C. perfringens type A strains 13, ATCC13124, and SM101). The chromosomal and plasmid-borne C. perfringens genotypes were grouped into two distinct clusters, one consisting of the chromosomal cpe-genotypes and the other consisting of plasmid-borne cpe-genotypes. Analysis of the variable gene pool complemented with the growth studies demonstrate different carbohydrate and amine metabolism in the chromosomal and plasmid-borne cpe-carrying strains, suggesting different epidemiology of the cpe-positive C. perfringens strain groups.
Project description:We performed physiological recording and single cell RNAseq on mouse prefrontal cortical pyramidal and fast-spiking interneurons in the live brain slice preparation. One of the goals was to identify potential gene targets whose function might be disrupted in schizophrenia.
Project description:Low copy number plasmids must encode maintenance mechanisms, such as partitioning systems, to ensure that the plasmid is sustained through host generations. Plasmid partition systems segregate sister plasmid copies and are subdivided into different types based on the NTPase they encode. The characterisation and distribution of partition system types is well understood in Enterobacteriaceae plasmids. However, how these systems maintain plasmids and are distributed across wider bacterial diversity is poorly understood. We used the Streptomyces coelicolor A3(2) plasmid SCP1, which encodes two type Ia partition systems, as a model to investigate this. Sequence analysis of the SCP1 partition systems revealed that both ParB proteins contain less conserved CTP-binding pockets, suggesting one or both proteins may not behave like canonical ParB proteins. However, using a combination of chromatin immunoprecipitation with deep sequencing (ChIP-seq) we demonstrate that both the SCP1 ParB proteins, ParB1 and ParB2, bound to distinct parS sites on SCP1, and accumulate, or spread, on DNA approximately 20 kb away from their initial parS loading site. Together, our findings further our understanding of Streptomyces plasmid maintenance by providing the first functional characterisation of two type Ia partition systems coexisting on a single plasmid and offer new insights into the diversity and distribution of plasmid partition systems.
Project description:Extra-chromosomal selfish DNA elements can evade the risk of being lost at every generation by behaving as chromosome appendages, thereby ensuring high fidelity segregation and stable persistence in host cell populations. The yeast 2-micron plasmid and episomes of the mammalian gammaherpes and papilloma viruses that tether to chromosomes and segregate by hitchhiking on them exemplify this strategy. We document for the first time the utilization of a SWI/SNF type chromatin remodeling complex as a conduit for chromosome association by a selfish element. One principal mechanism for chromosome tethering by the 2-micron plasmid is the bridging interaction of the plasmid partitioning proteins (Rep1 and Rep2) with the yeast RSC2 complex and the plasmid partitioning locus STB. We substantiate this model by multiple lines of evidence derived from genomics, cell biology and in vivo and in vitro interaction analyses. We describe a Rep-STB bypass system in which a plasmid non-covalently associated with the RSC complex mimics segregation by chromosome hitchhiking. Given the ubiquitous prevalence of SWI/SNF family chromatin remodeling complexes among eukaryotes, it is likely that the 2-micron plasmid paradigm or analogous ones will be encountered among other eukaryotic selfish elements.
Project description:The type VI secretion system (T6SS) is a highly sophisticated nanomachine widely used by bacteria to achieve competitive advantage and to potentiate horizontal gene transfer. Plasmid conjugation plays crucial roles in bacterial evolution by driving adaptation to environmental stimuli and pathogenicity. The lethal effect mediated by T6SS is detrimental to horizontal gene transfer by conjugation, while bacteria have evolved T6SS repression mechanisms regulated by plasmid to accomplish conjugative transfer. Two TetR family regulators encoded by large conjugative plasmid (LCP) in Acinetobacter baumannii have been proved similar in T6SS restriction, which seems redundant in function. Here, the global regulation roles and multiple DNA binding sites of two plasmid-sourced TetRs were identified. The two TetRs showed distinct preferences in similar roles of T6SS inhibition and binding with DNA probes. Crystal structures of TetRs were solved for illuminating the regulatory mechanism and possible reasons for difference in functions. In addition, plasmid-sourced TetRs also significantly downregulated biofilm formation and bacterial colonization, as well as influenced bacterial virulence in cultured cells and murine pneumonia infection models. Taken together, this work comprehensively elucidates the roles and regulatory mechanisms of TetRs and clarifies their similarity and difference in function, providing insights into plasmid encoded chromosome regulation pathways.
Project description:Natural plasmids are common in prokaryotes, but few have been documented in eukaryotes. The natural 2µ plasmid present in the yeast Saccharomyces cerevisiae is one of these best-characterized exceptions. This highly stable genetic element has coexisted with its host for millions of years, faithfully segregating at each cell division through a mechanism that remains unclear. Using proximity ligation methods (such as Hi-C, Micro-C) to map the contacts between 2µ plasmid and yeast chromosomes under dozens of different biological conditions, we found that the plasmid is tethered preferentially to regions with low transcriptional activity, often corresponding to long, inactive genes. These contacts do not depend on common chromosome-structuring factors, such as members of the structural maintenance of chromosome complexes (SMC) but depend on a nucleosome-encoded signal associated with RNA Pol II depletion. They appear stable throughout the cell cycle and can be established within minutes. This chromosome hitchhiking strategy may extend beyond the 2µ plasmid/S. cerevisiae pair, as suggested by the binding pattern of the natural eukaryotic plasmid Ddp5 along silent chromosome regions of the amoeba Dictyostelium discoideum.
Project description:<p>Gut environments harbour dense microbial ecosystems in which plasmids are widely distributed. Plasmids facilitate the exchange of genetic material among microorganisms while enabling the transfer of a diverse array of accessory functions. However, their precise impact on microbial community composition and function remains largely unexplored. Here we identify a prevalent bacterial toxin and a plasmid-encoded resistance mechanism that mediates the interaction between Lactobacilli and Enterococci. This plasmid is widespread across ecosystems, including the rumen and human gut microbiota. Biochemical characterization of the plasmid revealed a defence mechanism against reuterin, a toxin produced by various gut microbes, such as Limosilactobacillus reuteri. Using a targeted metabolomic approach, we find reuterin to be prevalent across rumen ecosystems with impacts on microbial community structure. Enterococcus strains carrying the protective plasmid were isolated and their interactions with L. reuteri, the toxin producer, were studied in vitro. Interestingly, we found that by conferring resistance against reuterin, the plasmid mediates metabolic exchange between the defending and the attacking microbial species, resulting in a beneficial relationship or mutualism. Hence, we reveal here an ecological role for a plasmid-coded defence system in mediating a beneficial interaction. </p>
Project description:By following chromatin occupancy of RNAPII and TOP1 using ChIP-seq throughout mitosis, we found that TOP1 is required for RNAPII translocation along genes. The stimulation of TOP1 activity through its interaction with RNAPII during elongation, allowed RNAPII clearance from genes in prometaphase and enabled proper chromosomal segregation. Interference with the TOP1-RNAPII interaction or acute depletion of TOP1 at the onset of mitosis impaired RNAPII spiking at promoters, which is necessary for the first pioneering round of transcription during mitotic exit, and triggered defects in the transcriptional program of the post-mitotic cells.
Project description:Ventral subiculum (vSUB) is integral to the regulation of stress and reward, however the intrinsic connectivity and synaptic properties of the inhibitory microcircuit are poorly understood. Neurexin-3 (Nrxn3) is highly expressed in hippocampal inhibitory neurons, but its function at inhibitory synapses has remained elusive. Using slice electrophysiology, imaging, and single-cell RNA sequencing, we identify multiple roles for Nrxn3 at GABAergic parvalbumin (PV) interneuron synapses made onto vSUB regular spiking (RS) and burst spiking (BS) principal neurons. Surprisingly, we found that intrinsic connectivity of vSUB and synaptic function of Nrxn3 in vSUB are sexually dimorphic. We reveal that vSUB PVs make preferential contact with RS neurons in males, but BS neurons in females. Furthermore, we determined that despite comparable Nrxn3 isoform expression in male and female PV neurons, Nrxn3 maintains synapse density at PV-RS synapses in males, but suppresses presynaptic release at the same synapses in females.