"Invisible in the Most Tragic of Ways": Exploring Internalized Transphobia and Coping Through Photovoice.

Transgender and gender diverse (TGD) individuals experience minority stress that includes internalizing the negative attitudes that exist in society about those whose identities rest outside of the gender binary. The purpose of this study was to gain a better understanding of the experience of internalized stigma and associated coping methods among six TGD adults utilizing photovoice. Thematic and iconographic analysis of 35 works produced by the participants revealed themes involving metaphors for internalized transphobia, internalized social messages, the psychological debt or emotional cost of internalized transphobia, and a range of coping strategies. Specifically, invisibility was a common metaphor used to suggest that a TGD person's experience may be associated with actively hiding, masking their identity, or failing to be seen as their true self. Participants expressed concern with being accepted by others and noted anxiety about outward expressions of their gender and personal characteristics. The results also revealed that participants experienced loneliness, fear of rejection, and low self-esteem related to internalized transphobia and their TGD identities. Participants reported receiving messages that TGD individuals are often viewed negatively and generally lack support from society and close relations. Coping strategies utilized by participants included withdrawing from others, hiding their TGD identity to avoid rejection or violence, self-exploration, accepting self and others, and connecting to a community of TGD persons and/or allies. Focus group feedback revealed benefits to participants, including an enhanced sense of community and self-esteem, as well as recommendations for future group structure. Implications and future research directions are discussed.

Species- and site-specific genome editing in complex bacterial communities.

Understanding microbial gene functions relies on the application of experimental genetics in cultured microorganisms. However, the vast majority of bacteria and archaea remain uncultured, precluding the application of traditional genetic methods to these organisms and their interactions. Here, we characterize and validate a generalizable strategy for editing the genomes of specific organisms in microbial communities. We apply environmental transformation sequencing (ET-seq), in which nontargeted transposon insertions are mapped and quantified following delivery to a microbial community, to identify genetically tractable constituents. Next, DNA-editing all-in-one RNA-guided CRISPR-Cas transposase (DART) systems for targeted DNA insertion into organisms identified as tractable by ET-seq are used to enable organism- and locus-specific genetic manipulation in a community context. Using a combination of ET-seq and DART in soil and infant gut microbiota, we conduct species- and site-specific edits in several bacteria, measure gene fitness in a nonmodel bacterium and enrich targeted species. These tools enable editing of microbial communities for understanding and control.

Deciphering Microbial Metal Toxicity Responses via Random Bar Code Transposon Site Sequencing and Activity-Based Metabolomics.

To uncover metal toxicity targets and defense mechanisms of the facultative anaerobe Pantoea sp. strain MT58 (MT58), we used a multiomic strategy combining two global techniques, random bar code transposon site sequencing (RB-TnSeq) and activity-based metabolomics. MT58 is a metal-tolerant Oak Ridge Reservation (ORR) environmental isolate that was enriched in the presence of metals at concentrations measured in contaminated groundwater at an ORR nuclear waste site. The effects of three chemically different metals found at elevated concentrations in the ORR contaminated environment were investigated: the cation Al3+, the oxyanion CrO42-, and the oxycation UO22+. Both global techniques were applied using all three metals under both aerobic and anaerobic conditions to elucidate metal interactions mediated through the activity of metabolites and key genes/proteins. These revealed that Al3+ binds intracellular arginine, CrO42- enters the cell through sulfate transporters and oxidizes intracellular reduced thiols, and membrane-bound lipopolysaccharides protect the cell from UO22+ toxicity. In addition, the Tol outer membrane system contributed to the protection of cellular integrity from the toxic effects of all three metals. Likewise, we found evidence of regulation of lipid content in membranes under metal stress. Individually, RB-TnSeq and metabolomics are powerful tools to explore the impact various stresses have on biological systems. Here, we show that together they can be used synergistically to identify the molecular actors and mechanisms of these pertubations to an organism, furthering our understanding of how living systems interact with their environment. IMPORTANCE Studying microbial interactions with their environment can lead to a deeper understanding of biological molecular mechanisms. In this study, two global techniques, RB-TnSeq and activity metabolomics, were successfully used to probe the interactions between a metal-resistant microorganism, Pantoea sp. strain MT58, and metals contaminating a site where the organism can be located. A number of novel metal-microbe interactions were uncovered, including Al3+ toxicity targeting arginine synthesis, which could lead to a deeper understanding of the impact Al3+ contamination has on microbial communities as well as its impact on higher-level organisms, including plants for whom Al3+ contamination is an issue. Using multiomic approaches like the one described here is a way to further our understanding of microbial interactions and their impacts on the environment overall.

Selective carbon sources influence the end products of microbial nitrate respiration.

Respiratory and catabolic genes are differentially distributed across microbial genomes. Thus, specific carbon sources may favor different respiratory processes. We profiled the influence of 94 carbon sources on the end products of nitrate respiration in microbial enrichment cultures from diverse terrestrial environments. We found that some carbon sources consistently favor dissimilatory nitrate reduction to ammonium (DNRA/nitrate ammonification) while other carbon sources favor nitrite accumulation or denitrification. For an enrichment culture from aquatic sediment, we sequenced the genomes of the most abundant strains, matched these genomes to 16S rDNA exact sequence variants (ESVs), and used 16S rDNA amplicon sequencing to track the differential enrichment of functionally distinct ESVs on different carbon sources. We found that changes in the abundances of strains with different genetic potentials for nitrite accumulation, DNRA or denitrification were correlated with the nitrite or ammonium concentrations in the enrichment cultures recovered on different carbon sources. Specifically, we found that either L-sorbose or D-cellobiose enriched for a Klebsiella nitrite accumulator, other sugars enriched for an Escherichia nitrate ammonifier, and citrate or formate enriched for a Pseudomonas denitrifier and a Sulfurospirillum nitrate ammonifier. Our results add important nuance to the current paradigm that higher concentrations of carbon will always favor DNRA over denitrification or nitrite accumulation, and we propose that, in some cases, carbon composition can be as important as carbon concentration in determining nitrate respiratory end products. Furthermore, our approach can be extended to other environments and metabolisms to characterize how selective parameters influence microbial community composition, gene content, and function.

Dual-barcoded shotgun expression library sequencing for high-throughput characterization of functional traits in bacteria.

A major challenge in genomics is the knowledge gap between sequence and its encoded function. Gain-of-function methods based on gene overexpression are attractive avenues for phenotype-based functional screens, but are not easily applied in high-throughput across many experimental conditions. Here, we present Dual Barcoded Shotgun Expression Library Sequencing (Dub-seq), a method that uses random DNA barcodes to greatly increase experimental throughput. As a demonstration of this approach, we construct a Dub-seq library with Escherichia coli genomic DNA, performed 155 genome-wide fitness assays in 52 experimental conditions, and identified overexpression phenotypes for 813 genes. We show that Dub-seq data is reproducible, accurately recapitulates known biology, and identifies hundreds of novel gain-of-function phenotypes for E. coli genes, a subset of which we verified with assays of individual strains. Dub-seq provides complementary information to loss-of-function approaches and will facilitate rapid and systematic functional characterization of microbial genomes.