Project description:Here, we present chimera assembly by plasmid recovery and restriction enzyme site insertion (CAPRRESI). CAPRRESI benefits from many strengths of the original plasmid recovery method and introduces restriction enzyme digestion to ease DNA ligation reactions (required for chimera assembly). For this protocol, users clone wildtype genes into the same plasmid (pUC18 or pUC19). After the in silico selection of amino acid sequence regions where chimeras should be assembled, users obtain all the synonym DNA sequences that encode them. Ad hoc Perl scripts enable users to determine all synonym DNA sequences. After this step, another Perl script searches for restriction enzyme sites on all synonym DNA sequences. This in silico analysis is also performed using the ampicillin resistance gene (ampR) found on pUC18/19 plasmids. Users design oligonucleotides inside synonym regions to disrupt wildtype and ampR genes by PCR. After obtaining and purifying complementary DNA fragments, restriction enzyme digestion is accomplished. Chimera assembly is achieved by ligating appropriate complementary DNA fragments. pUC18/19 vectors are selected for CAPRRESI because they offer technical advantages, such as small size (2,686 base pairs), high copy number, advantageous sequencing reaction features, and commercial availability. The usage of restriction enzymes for chimera assembly eliminates the need for DNA polymerases yielding blunt-ended products. CAPRRESI is a fast and low-cost method for fusing protein-coding genes.
Project description:We report here the isolation and recombinational cloning of a large plasmid, pZL12, from endophytic Streptomyces sp. 9R-2. pZL12 comprises 90,435 bp, encoding 112 genes, 30 of which are organized in a large operon resembling bacteriophage genes. A replication locus (repA) and a conjugal transfer locus (traA-traC) were identified in pZL12. Surprisingly, the supernatant of a 9R-2 liquid culture containing partially purified phage particles infected 9R-2 cured of pZL12 (9R-2X) to form plaques, and a phage particle (phiZL12) was observed by transmission electron microscopy. Major structural proteins (capsid, portal, and tail) of phiZL12 virions were encoded by pZL12 genes. Like bacteriophage P1, linear phiZL12 DNA contained ends from a largely random pZL12 sequence. There was also a hot end sequence in linear phiZL12. phiZL12 virions efficiently infected only one host, 9R-2X, but failed to infect and form plaques in 18 other Streptomyces strains. Some 9R-2X spores rescued from lysis by infection of phiZL12 virions contained a circular pZL12 plasmid, completing a cycle comprising autonomous plasmid pZL12 and lytic phage phiZL12. These results confirm pZL12 as the first example of a plasmid-phage in Streptomyces.
Project description:Coevolution with bacteriophages is a major selective force shaping bacterial populations and communities. A variety of both environmental and genetic factors has been shown to influence the mode and tempo of bacteria-phage coevolution. Here, we test the effects that carriage of a large conjugative plasmid, pQBR103, had on antagonistic coevolution between the bacterium Pseudomonas fluorescens and its phage, SBW25ϕ2. Plasmid carriage limited bacteria-phage coevolution; bacteria evolved lower phage-resistance and phages evolved lower infectivity in plasmid-carrying compared with plasmid-free populations. These differences were not explained by effects of plasmid carriage on the costs of phage resistance mutations. Surprisingly, in the presence of phages, plasmid carriage resulted in the evolution of high frequencies of mucoid bacterial colonies. Mucoidy can provide weak partial resistance against SBW25ϕ2, which may have limited selection for qualitative resistance mutations in our experiments. Taken together, our results suggest that plasmids can have evolutionary consequences for bacteria that go beyond the direct phenotypic effects of their accessory gene cargo.
Project description:Bacterial viruses (phages) are potent agents of lateral gene transfer and thus are important drivers of evolution. A group of mobile genetic elements, referred to as phage satellites, exploits phages to disseminate their own genetic material. Here, we isolated a novel member of the family Inoviridae, Shewanella phage Dolos, along with an autonomously replicating plasmid, pDolos. Dolos causes a chronic infection in its host Shewanella oneidensis by phage production with only minor effects on the host cell proliferation. When present, plasmid pDolos hijacks Dolos functions to be predominantly packaged into phage virions and released into the environment and, thus, acts as a phage satellite. pDolos can disseminate further genetic material encoding, e.g., resistances or fluorophores to host cells sensitive to Dolos infection. Given the rather simple requirements of a plasmid for takeover of an inovirus and the wide distribution of phages of this group, we speculate that similar phage-satellite systems are common among bacteria.IMPORTANCEPhage satellites are mobile genetic elements, which hijack phages to be transferred to other host cells. The vast majority of these phage satellites integrate within the host's chromosome, and they all carry remaining phage genes. Here, we identified a novel phage satellite, pDolos, which uses an inovirus for dissemination. pDolos (i) remains as an autonomously replicating plasmid within its host, (ii) does not carry recognizable phage genes, and (iii) is smaller than any other phage satellites identified so far. Thus, pDolos is the first member of a new class of phage satellites, which resemble natural versions of phagemids.
Project description:Leaf color change of variegated leaves from chimera species is regulated by fine-tuned molecular mechanisms. Hosta "Gold Standard" is a typical chimera Hosta species with golden-green variegated leaves, which is an ideal material to investigate the molecular mechanisms of leaf variegation. In this study, the margin and center regions of young and mature leaves from Hosta "Gold Standard", as well as the leaves from plants after excess nitrogen fertilization were studied using physiological and comparative proteomic approaches. We identified 31 differentially expressed proteins in various regions and development stages of variegated leaves. Some of them may be related to the leaf color regulation in Hosta "Gold Standard". For example, cytosolic glutamine synthetase (GS1), heat shock protein 70 (Hsp70), and chloroplastic elongation factor G (cpEF-G) were involved in pigment-related nitrogen synthesis as well as protein synthesis and processing. By integrating the proteomics data with physiological results, we revealed the metabolic patterns of nitrogen metabolism, photosynthesis, energy supply, as well as chloroplast protein synthesis, import and processing in various leaf regions at different development stages. Additionally, chloroplast-localized proteoforms involved in nitrogen metabolism, photosynthesis and protein processing implied that post-translational modifications were crucial for leaf color regulation. These results provide new clues toward understanding the mechanisms of leaf color regulation in variegated leaves.
Project description:PRD1, a lipid-containing double-stranded DNA bacteriophage, uses the mating pair formation (Mpf) complex encoded by conjugative IncP plasmids as a receptor. Functions responsible for conjugative transfer of IncP plasmids are encoded by two distinct regions, Tra1 and Tra2. Ten Tra2 region gene products (TrbB to TrbL) and one from the Tra1 region (TraF) form the Mpf complex. We carried out a mutational analysis of the PRD1 receptor complex proteins by isolating spontaneous PRD1-resistant mutants. The mutations were distributed among the trb genes in the Tra2 region and accumulated predominantly in three genes, trbC, trbE, and trbL. Three of 307 phage-resistant mutants were weakly transfer proficient. Mutations causing a phage adsorption-deficient, transfer-positive phenotype were analyzed by sequencing.
Project description:Plasmids and temperate phages are key contributors to bacterial evolution. They are usually regarded as very distinct. However, some elements, termed phage-plasmids, are known to be both plasmids and phages, e.g. P1, N15 or SSU5. The number, distribution, relatedness and characteristics of these phage-plasmids are poorly known. Here, we screened for these elements among ca. 2500 phages and 12000 plasmids and identified 780 phage-plasmids across very diverse bacterial phyla. We grouped 92% of them by similarity of gene repertoires to eight defined groups and 18 other broader communities of elements. The existence of these large groups suggests that phage-plasmids are ancient. Their gene repertoires are large, the average element is larger than an average phage or plasmid, and they include slightly more homologs to phages than to plasmids. We analyzed the pangenomes and the genetic organization of each group of phage-plasmids and found the key phage genes to be conserved and co-localized within distinct groups, whereas genes with homologs in plasmids are much more variable and include most accessory genes. Phage-plasmids are a sizeable fraction of the sequenced plasmids (∼7%) and phages (∼5%), and could have key roles in bridging the genetic divide between phages and other mobile genetic elements.
Project description:Tumor-derived cachectic factors such as proinflammatory cytokines and neuromodulators not only affect skeletal muscle but also affect other organs, including the heart, in the form of cardiac muscle atrophy, fibrosis, and eventual cardiac dysfunction, resulting in poor quality of life and reduced survival. This article reviews the holistic approaches of existing diagnostic, pathophysiological, and multimodal therapeutic interventions targeting the molecular mechanisms that are responsible for cancer-induced cardiac cachexia. The major drivers of cardiac muscle wasting in cancer patients are autophagy activation by the cytokine-NFkB, TGF β-SMAD3, and angiotensin II-SOCE-STIM-Ca2+ pathways. A lack of diagnostic markers and standard treatment protocols hinder the early diagnosis of cardiac dysfunction and the initiation of preventive measures. However, some novel therapeutic strategies, including the use of Withaferin A, have shown promising results in experimental models, but Withaferin A's effectiveness in human remains to be verified. The combined efforts of cardiologists and oncologists would help to identify cost effective and feasible solutions to restore cardiac function and to increase the survival potential of cancer patients.