Undergraduate Urban Metagenomics Research Module†

The undergraduate urban metagenomics research module is an adaptable and tractable project that can be incorporated into exisiting microbiology, ecology, bioinformatics, introductory biology or other courses.  The module takes advantage of recent advances in metagenomics and next generation sequencing, giving students an opportunity to apply these current technologies to novel questions regarding microbial community diversity and dynamics

Microbiomes for All

Microbiome research projects are often interdisciplinary, involving fields such as microbiology, genetics, ecology, evolution, bioinformatics, and statistics. These research projects can be an excellent fit for undergraduate courses ranging from introductory biology labs to upper-level capstone courses. Microbiome research projects can attract the interest of students majoring in health and medical sciences, environmental sciences, and agriculture, and there are meaningful ties to real-world issues relating to human health, climate change, and environmental sustainability and resilience in pristine, fragile ecosystems to bustling urban centers. In this review, we will discuss the potential of microbiome research integrated into classes using a number of different modalities. Our experience scaling-up and implementing microbiome projects at a range of institutions across the US has provided us with insight and strategies for what works well and how to diminish common hurdles that are encountered when implementing undergraduate microbiome research projects. We will discuss how course-based microbiome research can be leveraged to help faculty make advances in their own research and professional development and the resources that are available to support faculty interested in integrating microbiome research into their courses

T-DNA-Genome junctions form early after infection and are influenced by the chromatin state of the host genome

Agrobacterium tumefaciens mediated T-DNA integration is a common tool for plant genome manipulation. However, there is controversy regarding whether T-DNA integration is biased towards genes or randomly distributed throughout the genome. In order to address this question, we performed high-throughput mapping of T-DNA-genome junctions obtained in the absence of selection at several time points after infection. T-DNA-genome junctions were detected as early as 6 hours post-infection. T-DNA distribution was apparently uniform throughout the chromosomes, yet local biases toward AT-rich motifs and T-DNA border sequence micro-homology were detected. Analysis of the epigenetic landscape of previously isolated sites of T-DNA integration in Kanamycin-selected transgenic plants showed an association with extremely low methylation and nucleosome occupancy. Conversely, non-selected junctions from this study showed no correlation with methylation and had chromatin marks, such as high nucleosome occupancy and high H3K27me3, that correspond to three-dimensional-interacting heterochromatin islands embedded within euchromatin. Such structures may play a role in capturing and silencing invading T-DNA

Green Infrastructure Design Influences Communities of Urban Soil Bacteria

The importance of natural ecosystem processes is often overlooked in urban areas. Green Infrastructure (GI) features have been constructed in urban areas as elements to capture and treat excess urban runoff while providing a range of ancillary benefits, e.g., ecosystem processes mediated by microorganisms that improve air and water quality, in addition to the associations with plant and tree rhizospheres. The objective of this study was to characterize the bacterial community and diversity in engineered soils (Technosols) of five types of GI in New York City; vegetated swales, right of way bioswales (ROWB; including street-side infiltration systems and enhanced tree pits), and an urban forest. The design of ROWB GI features directly connects with the road to manage street runoff, which can increase the Technosol saturation and exposure to urban contaminants washed from the street and carried into the GI feature. This GI design specifically accommodates dramatic pulses of water that influence the bacterial community composition and diversity through the selective pressure of contaminants or by disturbance. The ROWB had the highest biodiversity, but no significant correlation with levels of soil organic matter and microbially-mediated biogeochemical functions. Another important biogeochemical parameter for soil bacterial communities is pH, which influenced the bacterial community composition, consistent with studies in non-urban soils. Bacterial community composition in GI features showed signs of anthropogenic disturbance, including exposure to animal feces and chemical contaminants, such as petroleum products. Results suggest the overall design and management of GI features with a channeled connection with street runoff, such as ROWB, have a comprehensive effect on soil parameters (particularly organic matter) and the bacterial community. One key consideration for future assessments of GI microbial community would be to determine the source of organic matter and elucidate the relationship between vegetation, Technosol, and bacteria in the designed GI features

Green Infrastructure Design Influences Communities of Urban Soil Bacteria

The importance of natural ecosystem processes is often overlooked in urban areas. Green Infrastructure (GI) features have been constructed in urban areas as elements to capture and treat excess urban runoff while providing a range of ancillary benefits, e.g., ecosystem processes mediated by microorganisms that improve air and water quality, in addition to the associations with plant and tree rhizospheres. The objective of this study was to characterize the bacterial community and diversity in engineered soils (Technosols) of five types of GI in New York City; vegetated swales, right of way bioswales (ROWB; including street-side infiltration systems and enhanced tree pits), and an urban forest. The design of ROWB GI features directly connects with the road to manage street runoff, which can increase the Technosol saturation and exposure to urban contaminants washed from the street and carried into the GI feature. This GI design specifically accommodates dramatic pulses of water that influence the bacterial community composition and diversity through the selective pressure of contaminants or by disturbance. The ROWB had the highest biodiversity, but no significant correlation with levels of soil organic matter and microbially-mediated biogeochemical functions. Another important biogeochemical parameter for soil bacterial communities is pH, which influenced the bacterial community composition, consistent with studies in non-urban soils. Bacterial community composition in GI features showed signs of anthropogenic disturbance, including exposure to animal feces and chemical contaminants, such as petroleum products. Results suggest the overall design and management of GI features with a channeled connection with street runoff, such as ROWB, have a comprehensive effect on soil parameters (particularly organic matter) and the bacterial community. One key consideration for future assessments of GI microbial community would be to determine the source of organic matter and elucidate the relationship between vegetation, Technosol, and bacteria in the designed GI features

Geospatial Resolution of Human and Bacterial Diversity with City-Scale Metagenomics

The panoply of microorganisms and other species present in our environment influence human health and disease, especially in cities, but have not been profiled with metagenomics at a city-wide scale. We sequenced DNA from surfaces across the entire New York City (NYC) subway system, the Gowanus Canal, and public parks. Nearly half of the DNA (48%) does not match any known organism; identified organisms spanned 1,688 bacterial, viral, archaeal, and eukaryotic taxa, which were enriched for harmless genera associated with skin (e.g., Acinetobacter). Predicted ancestry of human DNA left on subway surfaces can recapitulate U.S. Census demographic data, and bacterial signatures can reveal a station’s history, such as marine-associated bacteria in a hurricane-flooded station. Some evidence of pathogens was found (Bacillus anthracis), but a lack of reported cases in NYC suggests that the pathogens represent a normal, urban microbiome. This baseline metagenomic map of NYC could help long-term disease surveillance, bioterrorism threat mitigation, and health management in the built environment of citie

Sequence motifs associated with T-DNA–genome junctions sites.

<p>The motifs CACCAC (P-value = 1e-147, HOMER, E-value = 1.7e-234, MEME) and A-rich (P-value < 1e-21, HOMER, E-value = 2.3e-483, MEME) were associated with T-DNA–genome junction sites.</p

T-DNA-genome junctions form early after infection and are influenced by the chromatin state of the host genome - Table 1

<p>T-DNA-genome junctions form early after infection and are influenced by the chromatin state of the host genome</p> - Table

Experimental design–<i>Arabidopsis</i> roots were infected with <i>A</i>. <i>tumefaciens</i>.

<p>DNA was extracted at 0, 6 and 24 hours post infection. Extracted DNA was digested with 3 restriction enzymes: <i>Eco</i>RI (RI), <i>Hind</i>III (H3) and <i>Xba</i>I (Xb). An adapter was ligated to the overhang end of the digested DNA. T-DNA-genomic junctions were amplified using three different primers from within the T-DNA and one primer from the adapter (primers—black arrows, LB–left border, RB–right border). Amplicons were sequenced using high throughput sequencing. Adapter to adapter products were reduced as detailed in O’Malley et al. 2007 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006875#pgen.1006875.ref027" target="_blank">27</a>].</p

Association of genomic features with T-DNA-genome junctions under selective and non-selective conditions.

<p>A–The genomic distribution of unselected and selected T-DNA–genome junctions across chromosome 4. The numbers of T-DNA–genome junctions (circles, and smoothed blue line) do not show a correlation with the distribution of transposons (TE, red line) and promoters (green line). T-DNA integrations under selective conditions (orange line) correlates with genes/promoters [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006875#pgen.1006875.ref013" target="_blank">13</a>]. B- The portion of each genomic feature: TE (red), genes (purple), promoters (green) and the remaining regions (other, grey). The portion of genomic features is represented across all the genome (genome wide) and according to the number of T-DNA–genome junctions: without selection (Unselected) and with selection (Selected- data from Alonso et al., 2003 [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006875#pgen.1006875.ref013" target="_blank">13</a>]). Selected events [<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1006875#pgen.1006875.ref013" target="_blank">13</a>] show an enrichment in promoters (χ² test, compared to unselected events, p = 2.88E-24) and a decrease in TE regions (χ2 test, compared to unselected events, p = 0.005).</p