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.

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.

Quantification of Agrobacterium tumefaciens C58 attachment to Arabidopsis thaliana roots.

Agrobacterium tumefaciens is the causal agent of crown gall disease and is a vector for DNA transfer in transgenic plants. The transformation process by A. tumefaciens has been widely studied, but the attachment stage has not been well characterized. Most measurements of attachment have used microscopy and colony counting, both of which are labor and time intensive. To reduce the time and effort required to analyze bacteria attaching to plant tissues, we developed a quantitative real-time PCR (qPCR) assay to quantify attached A. tumefaciens using the chvE gene as marker for the presence of the bacteria. The qPCR detection threshold of A. tumefaciens from pure culture was 104 cell equivalents/ml. The A. tumefaciens minimum threshold concentration from root-bound populations was determined to be 105 cell equivalents/ml inoculum to detect attachment above background. The qPCR assay can be used for measuring A. tumefaciens attachment in applications such as testing the effects of mutations on bacterial adhesion molecules or biofilm formation, comparing attachment across various plant species and ecotypes, and detecting mutations in putative attachment receptors expressed in plant roots.

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.

Development and Validation of the Microbiology for Health Sciences Concept Inventory.

Identifying misconceptions in student learning is a valuable practice for evaluating student learning gains and directing educational interventions. By accurately identifying students' knowledge and misconceptions about microbiology concepts, instructors can design effective classroom practices centered on student understanding. Following the development of ASM's Curriculum Guidelines in 2012, we developed a concept inventory, the Microbiology for Health Sciences Concept Inventory (MHSCI), that measures learning gains and identifies student misconceptions in health sciences microbiology classrooms. The 23-question MHSCI was delivered to a wide variety of students at multiple institution types. Psychometric analysis identified that the MHSCI instrument is both discriminatory and reliable in measuring student learning gains. The MHSCI results correlated with course outcomes, showing the value of using the instrument alongside course level assessments to measure student learning. The MHSCI is a reliable and efficient way to measure student learning in microbiology and can be used both as a faculty development tool and an effective student assessment tool.

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 cities.

Auranofin and related heterometallic gold(I)-thiolates as potent inhibitors of methicillin-resistant Staphylococcus aureus bacterial strains.

A series of new heterometallic gold(I) thiolates containing ferrocenyl-phoshines were synthesized. Their antimicrobial properties were studied and compared to that of FDA-approved drug, auranofin (Ridaura), prescribed for the treatment of rheumatoid arthritis. MIC in the order of one digit micromolar were found for most of the compounds against Gram-positive bacteria Staphylococcus aureus and CA MRSA strains US300 and US400. Remarkably, auranofin inhibited S. aureus, US300 and US400 in the order of 150-300 nM. This is the first time that the potent inhibitory effect of auranofin on MRSA strains has been described. The effects of a selected heterometallic compound and auranofin were also studied in a non-tumorigenic human embryonic kidney cell line (HEK-293).

Luminescent di- and polynuclear organometallic gold(I)-metal (Au2, {Au2Ag}n and {Au2Cu}n) compounds containing bidentate phosphanes as active antimicrobial agents.

The reaction of new dinuclear gold(I) organometallic complexes containing mesityl ligands and bridging bidentate phosphanes [Au(2)(mes)(2)(µ-LL)] (LL=dppe: 1,2-bis(diphenylphosphano)ethane 1a, and water-soluble dppy: 1,2-bis(di-3-pyridylphosphano)ethane 1b) with Ag(+) and Cu(+) lead to the formation of a family of heterometallic clusters with mesityl bridging ligands of the general formula [Au(2)M(µ-mes)(2) (µ-LL)][A] (M=Ag, A=ClO(4)(-), LL=dppe 2a, dppy 2b; M=Ag, A=SO(3)CF(3)(-), LL=dppe 3a, dppy 3b; M=Cu, A=PF(6)(-), LL=dppe 4a, dppy 4b). The new compounds were characterized by different spectroscopic techniques and mass spectrometry The crystal structures of [Au(2)(mes)(2)(µ-dppy)] (1b) and [Au(2)Ag(µ-mes)(2)(µ-dppe)][SO(3)CF(3)] (3a) were determined by a single-crystal X-ray diffraction study. 3a in solid state is not a cyclic trinuclear Au(2)Ag derivative but it gives an open polymeric structure instead, with the {Au(2)(µ-dppe)} fragments "linked" by {Ag(µ-mes)(2)} units. The very short distances of 2.7559(6) Š(Au-Ag) and 2.9229(8) Š(Au-Au) are indicative of gold-silver (metallophilic) and aurophilic interactions. A systematic study of their luminescence properties revealed that all compounds are brightly luminescent in solid state, at room temperature (RT) and at 77 K, or in frozen DMSO solutions with lifetimes in the microsecond range and probably due to the self-aggregation of [Au(2)M(µ-mes)(2)(µ-LL)](+) units (M=Ag or Cu; LL=dppe or dppy) into an extended chain structure, through Au-Au and/or Au-M metallophilic interactions, as that observed for 3a. In solid state the heterometallic Au(2)M complexes with dppe (2a-4a) show a shift of emission maxima (from ca. 430 to the range of 520-540 nm) as compared to the parent dinuclear organometallic product 1a while the complexes with dppy (2b-4b) display a more moderate shift (505 for 1b to a max of 563 nm for 4b). More importantly, compound [Au(2)Ag(µ-mes)(2)(µ-dppy)]ClO(4) (2b) resulted luminescent in diluted DMSO solution at room temperature. Previously reported compound [Au(2)Cl(2)(µ-LL)] (LL dppy 5b) was also studied for comparative purposes. The antimicrobial activity of 1-5 and Ag[A] (A=ClO(4)(-), SO(3)CF(3)(-)) against gram-positive and gram-negative bacteria and yeast was evaluated. Most tested compounds displayed moderate to high antibacterial activity while heteronuclear Au(2)M derivatives with dppe (2a-4a) were the more active (minimum inhibitory concentration 10 to 1 µg mL(-1)). Compounds containing silver were ten times more active to gram-negative bacteria than the parent dinuclear compound 1a or silver salts. Au(2)Ag compounds with dppy (2b, 3b) were also potent against fungi.

Protein trafficking in polarized cells.

Epithelial cells line the lumens of organs and thus constitute the interface between the body's interior and exterior surfaces. This position endows these cells with the important task of regulating what enters and what is exported from the body. In order to accomplish this function, epithelia must have structurally and functionally distinct membrane surfaces: the apical surface exposed to the lumen, and the basolateral surface in contact with the laterally adjacent epithelial cells, and the connective tissue and capillary network below the epithelia. The specific lipid and protein contents of the apical and basolateral membrane surfaces are determined by a number of sorting and retention mechanisms. Many of these sorting and retention mechanisms are shared with other polarized cell types including neurons and certain cells of the immune system. This chapter focuses on recent advances in understanding how these various mechanisms facilitate the generation, maintenance, and dynamic regulation of protein and lipid trafficking within epithelial cells.

The COOH-terminal tail of the GAT-2 GABA transporter contains a novel motif that plays a role in basolateral targeting.

The ability of polarized epithelia to perform vectorial transport depends on the asymmetrical distribution of transmembrane proteins among their plasma membrane domains. The establishment and maintenance of these polar distributions relies on molecular signals embedded in the proteins themselves and the interpretation of these signals by cellular sorting machinery. Using Madin-Darby canine kidney (MDCK) cells as an in vitro model of polarized epithelia, our laboratory has previously shown that the COOH-terminal cytoplasmic 22 amino acids of the GAT-2 isoform of the gamma-amino butyric acid (GABA) transporter are necessary for its basolateral distribution. We demonstrate that the COOH-terminal tail of the transporter can function as an autonomous basolateral distribution signal, independently of the rest of the transporter. We find that the three-amino acid PDZ domain-interacting motif at the COOH-terminus of GAT-2 is not necessary for its basolateral distribution. Instead, the more proximal seven amino acids are necessary both for targeting and for steady-state distribution. Because this sequence resembles no other known basolateral sorting information, we conclude that these seven amino acids contain a novel basolateral targeting and distribution motif.

Transport protein trafficking in polarized cells.

In order to carry out their physiological functions, ion transport proteins must be targeted to the appropriate domains of cell membranes. Regulation of ion transport activity frequently involves the tightly controlled delivery of intracellular populations of transport proteins to the plasma membrane or the endocytic retrieval of transport proteins from the cell surface. Transport proteins carry signals embedded within their structures that specify their subcellular distributions and endow them with the capacity to participate in regulated membrane trafficking processes. Recently, a great deal has been learned about the biochemical nature of these signals, as well as about the cellular machinery that interprets them and acts upon their messages.