Impediments to DNA replication are known to induce gross chromosomal rearrangements (GCRs) and copy-number variations (CNVs). GCRs and CNVs underlie human genomic disorders and are a feature of ...cancer. During cancer development, environmental factors and oncogene-driven proliferation promote replication stress. Resulting GCRs and CNVs are proposed to contribute to cancer development and therapy resistance. When stress arrests replication, the replisome remains associated with the fork DNA (stalled fork) and is protected by the inter-S-phase checkpoint. Stalled forks efficiently resume when the stress is relieved. However, if the polymerases dissociate from the fork (fork collapse) or the fork structure breaks (broken fork), replication restart can proceed either by homologous recombination or microhomology-primed re-initiation. Here we ascertain the consequences of replication with a fork restarted by homologous recombination in fission yeast. We identify a new mechanism of chromosomal rearrangement through the observation that recombination-restarted forks have a considerably high propensity to execute a U-turn at small inverted repeats (up to 1 in 40 replication events). We propose that the error-prone nature of restarted forks contributes to the generation of GCRs and gene amplification in cancer, and to non-recurrent CNVs in genomic disorders.
Bacteria belonging to the newly classified candidate phylum "Atribacteria" (formerly referred to as "OP9" and "JS1") are common in anoxic methane-rich sediments. However, the metabolic functions and ...biogeochemical role of these microorganisms in the subsurface remains unrealized due to the lack of pure culture representatives. In this study of deep sediment from Antarctica's Adélie Basin, collected during Expedition 318 of the Integrated Ocean Drilling Program (IODP), Atribacteria-related sequences of the 16S rRNA gene were abundant (up to 51% of the sequences) and steadily increased in relative abundance with depth throughout the methane-rich zones. To better understand the metabolic potential of Atribacteria within this environment, and to compare with phylogenetically distinct Atribacteria from non-deep-sea environments, individual cells were sorted for single cell genomics from sediment collected from 97.41 m below the seafloor from IODP Hole U1357C. As observed for non-marine Atribacteria, a partial single cell genome suggests a heterotrophic metabolism, with Atribacteria potentially producing fermentation products such as acetate, ethanol, and CO2. These products may in turn support methanogens within the sediment microbial community and explain the frequent occurrence of Atribacteria in anoxic methane-rich sediments. This first report of a single cell genome from deep sediment broadens the known diversity within the Atribacteria phylum and highlights the potential role of Atribacteria in carbon cycling in deep sediment.
The exploration of Earth's terrestrial subsurface biosphere has led to the discovery of several new archaeal lineages of evolutionary significance. Similarly, the deep subseafloor crustal biosphere ...also harbors many unique, uncultured archaeal taxa, including those belonging to Candidatus Hydrothermarchaeota, formerly known as Marine Benthic Group-E. Recently, Hydrothermarchaeota was identified as an abundant lineage of Juan de Fuca Ridge flank crustal fluids, suggesting its adaptation to this extreme environment. Through the investigation of single-cell and metagenome-assembled genomes, we provide insight into the lineage's evolutionary history and metabolic potential. Phylogenomic analysis reveals the Hydrothermarchaeota to be an early-branching archaeal phylum, branching between the superphylum DPANN, Euryarchaeota, and Asgard lineages. Hydrothermarchaeota genomes suggest a potential for dissimilative and assimilative carbon monoxide oxidation (carboxydotrophy), as well as sulfate and nitrate reduction. There is also a prevalence of chemotaxis and motility genes, indicating adaptive strategies for this nutrient-limited fluid-rock environment. These findings provide the first genomic interpretations of the Hydrothermarchaeota phylum and highlight the anoxic, hot, deep marine crustal biosphere as an important habitat for understanding the evolution of early life.
Despite accounting for the majority of sedimentary methane, the physiology and relative abundance of subsurface methanogens remain poorly understood. We combined intact polar lipid and metagenome ...techniques to better constrain the presence and functions of methanogens within the highly reducing, organic-rich sediments of Antarctica's Adélie Basin. The assembly of metagenomic sequence data identified phylogenic and functional marker genes of methanogens and generated the first Methanosaeta sp. genome from a deep subsurface sedimentary environment. Based on structural and isotopic measurements, glycerol dialkyl glycerol tetraethers with diglycosyl phosphatidylglycerol head groups were classified as biomarkers for active methanogens. The stable carbon isotope (δ
C) values of these biomarkers and the Methanosaeta partial genome suggest that these organisms are acetoclastic methanogens and represent a relatively small (0.2%) but active population. Metagenomic and lipid analyses suggest that Thaumarchaeota and heterotrophic bacteria co-exist with Methanosaeta and together contribute to increasing concentrations and δ
C values of dissolved inorganic carbon with depth. This study presents the first functional insights of deep subsurface Methanosaeta organisms and highlights their role in methane production and overall carbon cycling within sedimentary environments.
A rapid Listeria detection method was developed utilizing A511 bacteriophage amplification combined with surface-enhanced Raman spectroscopy (SERS) and lateral flow immunochromatography (LFI). ...Anti-A511 antibodies were covalently linked to SERS nanoparticles and printed onto nitrocellulose membranes. Antibody-conjugated SERS nanoparticles were used as quantifiable reporters. In the presence of A511, phage-SERS nanoparticle complexes were arrested and concentrated as a visible test line, which was interrogated quantitatively by Raman spectroscopy. An increase in SERS intensity correlated to an increase in captured phage-reporter complexes. SERS limit of detection was 6 × 10(6) pfu·mL(-1), offering detection below that obtainable by the naked eye (LOD 6 × 10(7) pfu·mL(-1)). Phage amplification experiments were carried out at a multiplicity of infection (MOI) of 0.1 with 4 different starting phage concentrations monitored over time using SERS-LFI and validated by spot titer assay. Detection of L. monocytogenes concentrations of 1 × 10(7) colony forming units (cfu)·mL(-1), 5 × 10(6) cfu·mL(-1), 5 × 10(5) cfu·mL(-1) and 5 × 10(4) cfu·mL(-1) was achieved in 2, 2, 6, and 8 h, respectively. Similar experiments were conducted at a constant starting phage concentration (5 × 10(5) pfu·mL(-1)) with MOIs of 1, 2.5, and 5 and were detected in 2, 4, and 5 h, respectively.
Our current knowledge of host-virus interactions in biofilms is limited to computational predictions based on laboratory experiments with a small number of cultured bacteria. However, natural ...biofilms are diverse and chiefly composed of uncultured bacteria and archaea with no viral infection patterns and lifestyle predictions described to date. Herein, we predict the first DNA sequence-based host-virus interactions in a natural biofilm. Using single-cell genomics and metagenomics applied to a hot spring mat of the Cone Pool in Mono County, California, we provide insights into virus-host range, lifestyle and distribution across different mat layers. Thirty-four out of 130 single cells contained at least one viral contig (26%), which, together with the metagenome-assembled genomes, resulted in detection of 59 viruses linked to 34 host species. Analysis of single-cell amplification kinetics revealed a lack of active viral replication on the single-cell level. These findings were further supported by mapping metagenomic reads from different mat layers to the obtained host-virus pairs, which indicated a low copy number of viral genomes compared to their hosts. Lastly, the metagenomic data revealed high layer specificity of viruses, suggesting limited diffusion to other mat layers. Taken together, these observations indicate that in low mobility environments with high microbial abundance, lysogeny is the predominant viral lifestyle, in line with the previously proposed "Piggyback-the-Winner" theory.
In this article, we describe curricular materials developed to engage undergraduate students in evolutionary thinking around antibiotic resistance using the MEGA-plate experiment (Microbial Evolution ...and Growth Arena). This elegant and visual experiment, developed by the Kishony Lab, shows the development of antibiotic resistance on the timescale of hours and days. It not only provides important biological insights but also captures students’ attention, making it a very useful tool for education. While a short video describing the method and major results has already been widely used in the classroom setting, our case study connects details of the methods and results of the MEGA-plate experiment and antibiotic resistance to core biological concepts. The interrupted case study consists of four major parts: 1) an opening hook activity to capture students’ attention and introduce the antibiotic crisis, 2) a jigsaw activity to research different classes of antibiotic targets and the resistance mechanisms that can arise, 3) a discussion of antibiotic resistance in real-time using the MEGA-plate experiment video, and 4) three different options for students to dive deeper into the experimental data from the MEGA-plate research article. These components are modular and can be used in many different combinations to reach different audiences or connect to other topics related to microbiology, evolution, or genetics.
Serial multi-omic analysis of proteome, phosphoproteome, and acetylome provides insights into changes in protein expression, cell signaling, cross-talk and epigenetic pathways involved in disease ...pathology and treatment. However, ubiquitylome and HLA peptidome data collection used to understand protein degradation and antigen presentation have not together been serialized, and instead require separate samples for parallel processing using distinct protocols. Here we present MONTE, a highly sensitive multi-omic native tissue enrichment workflow, that enables serial, deep-scale analysis of HLA-I and HLA-II immunopeptidome, ubiquitylome, proteome, phosphoproteome, and acetylome from the same tissue sample. We demonstrate that the depth of coverage and quantitative precision of each 'ome is not compromised by serialization, and the addition of HLA immunopeptidomics enables the identification of peptides derived from cancer/testis antigens and patient specific neoantigens. We evaluate the technical feasibility of the MONTE workflow using a small cohort of patient lung adenocarcinoma tumors.
The Universal Design for Learning (UDL) framework has been shown to support instructors in the design of inclusive instructional learning environments. However, few studies have examined the effects ...of UDL professional development programmes for community college faculty and staff. The present study examined the effects of a four-day Universal Design for Learning (UDL) training on community college faculty and staff. Three paired samples t tests were run on the pretest and posttest scores of participants. The results indicated that, after the training, faculty and staff had a statistically significantly greater knowledge of UDL and were better able to provide examples of how to apply it to their work on campus. The results provide support for training community college faculty and staff to apply the UDL framework to their instruction.
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