Integrative and conjugative elements (ICEs) are self-mobile genetic elements found in the genomes of some bacteria. These elements may confer a fitness advantage upon their host bacteria through the cargo genes that they carry. Salmonella pathogenicity island 7 (SPI-7), found within some pathogenic strains of Salmonella enterica, possesses features indicative of an ICE and carries genes implicated in virulence. We aimed to identify and fully analyze ICEs related to SPI-7 within the genus Salmonella and other Enterobacteriaceae. We report the sequence of two novel SPI-7-like elements, found within strains of Salmonella bongori, which share 97% nucleotide identity over conserved regions with SPI-7 and with each other. Although SPI-7 within Salmonella enterica serovar Typhi appears to be fixed within the chromosome, we present evidence that these novel elements are capable of excision and self-mobility. Phylogenetic analyses show that these Salmonella mobile elements share an ancestor which existed approximately 3.6 to 15.8 million years ago. Additionally, we identified more distantly related ICEs, with distinct cargo regions, within other strains of Salmonella as well as within Citrobacter, Erwinia, Escherichia, Photorhabdus, and Yersinia species. In total, we report on a collection of 17 SPI-7 related ICEs within enterobacterial species, of which six are novel. Using comparative and mutational studies, we have defined a core of 27 genes essential for conjugation. We present a growing family of SPI-7-related ICEs whose mobility, abundance, and cargo variability indicate that these elements may have had a large impact on the evolution of the Enterobacteriaceae. Bacterial genomes are often represented as a core of conserved genes interspersed with accessory regions which are variably present throughout members of a species. The ability of an organism to sample variation through accessing a pan-genome can be strongly associated with a change in lifestyle or adaptation to a new niche (29). In some cases, acquired accessory functions become fixed and go on to define a particular lineage (30). A classical example of this is the acquisition of the Salmonella pathogenicity islands (SPIs), considered to have been fundamental to the evolution of the genus Salmonella (26). The largest of these islands identified to date is SPI-7, found within the genomes of Salmonella enterica subsp. enterica serovar Typhi (S. Typhi) (52), Salmonella enterica subsp. enterica serovar Paratyphi C (S. Paratyphi C) (43), and some strains of Salmonella enterica subsp. enterica serovar Dublin (S. Dublin) (55). SPI-7 is 120 kb in length and encodes important virulence functions, including the major virulence antigen (Vi) and type IVB pili (24, 52). Additionally, integrated within SPI-7 from strains of S. Typhi is a prophage harboring the type 3 secretion system effector protein-encoding gene sopE (47).SPI-7 possesses many similarities to integrative and conjugative elements (ICEs), a class of self-transmissible elements (7) also known as conjugativ...
In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1,000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons, the extent of synapse diversity across the brain is largely unknown. This is mainly due to the limitations of current techniques. Here, we report an efficient method for the purification of synaptic protein complexes, fusing a high‐affinity tag to endogenous PSD95 in specific cell types. We also developed a strategy, which enables the visualisation of endogenous PSD95 with fluorescent‐protein tag in Cre‐recombinase‐expressing cells. We demonstrate the feasibility of proteomic analysis of synaptic protein complexes and visualisation of these in specific cell types. We find that the composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. The results suggest that there might be differential interactions in the PSD95 complexes in different brain regions. We have detected differentially interacting proteins by comparing data sets from the whole hippocampus and the CA3 subfield of the hippocampus. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific brain regions and subsets of neuronal cells, but also provide an opportunity to better understand brain region‐ and cell‐type‐specific alterations associated with various psychiatric/neurological diseases. These newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will facilitate research on the molecular complexity of synapses.
In recent years, the remarkable molecular complexity of synapses has been revealed, with over 1000 proteins identified in the synapse proteome. Although it is known that different receptors and other synaptic proteins are present in different types of neurons and synapses, the extent of synapse diversity across the brain is largely unknown, mainly owing to technical limitations. Combining mouse genetics and proteomics we have previously reported highly efficient methods for purification of synaptic protein complexes under native conditions. In that approach, tandem affinity purification (TAP) tags were fused to the carboxyl terminus of PSD95 using gene targeting in mice. Here we report an approach that restricts tagging of endogenous PSD95 to cells expressing Cre recombinase. In addition, we developed a labelling strategy enabling visualization of endogenous PSD95 tagged by fluorescent proteins in Cre-expressing cells. We demonstrate the feasibility of proteomic characterisation of synapse proteomes and visualization of synapse proteins in specific cell types. We find that composition of PSD95 complexes purified from specific cell types differs from those extracted from tissues with diverse cellular composition. Therefore, these novel conditional PSD95 tagging lines will not only serve as powerful tools for precisely dissecting synapse diversity in specific subsets of regions/neuronal cells, but also provide an opportunity to better understand brain region-specific alterations associated with various psychiatric/neurological diseases. The newly developed conditional gene tagging methods can be applied to many different synaptic proteins and will thus facilitate research on the molecular complexity of synapses.
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