Quantitative morphological analysis of Deinococcus radiodurans elucidates complex dose-dependent nucleoid condensation during recovery from ionizing radiation.

The extremophile Deinococcus radiodurans maintains a highly organized and condensed nucleoid as its default state, possibly contributing to its high tolerance to ionizing radiation (IR). Previous studies of the D. radiodurans nucleoid were limited by reliance on manual image annotation and qualitative metrics. Here, we introduce a high-throughput approach to quantify the geometric properties of cells and nucleoids using confocal microscopy, digital reconstructions of cells, and computational modeling. We utilize this novel approach to investigate the dynamic process of nucleoid condensation in response to IR stress. Our quantitative analysis reveals that at the population level, exposure to IR induced nucleoid compaction and decreased the size of D. radiodurans cells. Morphological analysis and clustering identified six distinct sub-populations across all tested experimental conditions. Results indicate that exposure to IR induced fractional redistributions of cells across sub-populations to exhibit morphologies associated with greater nucleoid condensation and decreased the abundance of sub-populations associated with cell division. Nucleoid-associated proteins (NAPs) may link nucleoid compaction and stress tolerance, but their roles in regulating compaction in D. radiodurans are unknown. Imaging of genomic mutants of known and suspected NAPs that contribute to nucleoid condensation found that deletion of nucleic acid-binding proteins, not previously described as NAPs, can remodel the nucleoid by driving condensation or decondensation in the absence of stress and that IR increased the abundance of these morphological states. Thus, our integrated analysis introduces a new methodology for studying environmental influences on bacterial nucleoids and provides an opportunity to further investigate potential regulators of nucleoid condensation.IMPORTANCEDeinococcus radiodurans, an extremophile known for its stress tolerance, constitutively maintains a highly condensed nucleoid. Qualitative studies have described nucleoid behavior under a variety of conditions. However, a lack of quantitative data regarding nucleoid organization and dynamics has limited our understanding of the regulatory mechanisms controlling nucleoid organization in D. radiodurans. Here, we introduce a quantitative approach that enables high-throughput quantitative measurements of subcellular spatial characteristics in bacterial cells. Applying this to wild-type or single-protein-deficient populations of D. radiodurans subjected to ionizing radiation, we identified significant stress-responsive changes in cell shape, nucleoid organization, and morphology. These findings highlight this methodology's adaptability and capacity for quantitatively analyzing the cellular response to stressors for screening cellular proteins involved in bacterial nucleoid organization.

Exposure of Shewanella oneidensis MR-1 to Sublethal Doses of Ionizing Radiation Triggers Short-Term SOS Activation and Longer-Term Prophage Activation.

Currently, there is a lack of bacterial biomarkers indicative of exposure to ionizing radiation (IR). IR biomarkers have applications for medical treatment planning, population exposure surveillance, and IR sensitivity studies. In this study, we compared the utility of signals originating from prophages and the SOS regulon as biomarkers of IR exposure in the radiosensitive bacterium Shewanella oneidensis. Using RNA sequencing, we demonstrated that 60 min after exposure to acute doses of IR (40, 1, 0.5, and 0.25 Gy), the transcriptional activation of the SOS regulon and the lytic cycle of the T-even lysogenic prophage So Lambda are comparable. Using quantitative PCR (qPCR), we showed that 300 min after exposure to doses as low as 0.25 Gy, the fold change of transcriptional activation of the So Lambda lytic cycle surpassed that of the SOS regulon. We observed an increase in cell size (a phenotype of SOS activation) and plaque production (a phenotype of prophage maturation) 300 min after doses as low as 1 Gy. While the transcriptional responses of the SOS and So Lambda regulons have been examined in S. oneidensis after lethal IR exposures, the potential of these (and other transcriptome-wide) responses as biomarkers of sublethal levels of IR (<10 Gy) and the longer-term activity of these two regulons have not been investigated. A major finding is that after exposure to sublethal doses of IR, the most upregulated transcripts are associated with a prophage regulon and not with a DNA damage response. Our findings suggest that prophage lytic cycle genes are a promising source of biomarkers of sublethal DNA damage. IMPORTANCE The bacterial minimum threshold of sensitivity to ionizing radiation (IR) is poorly understood, which hinders our understanding of how living systems recover from the doses of IR experienced in medical, industrial, and off-world environments. Using a transcriptome-wide approach, we studied how in the highly radiosensitive bacterium S. oneidensis, genes (including the SOS regulon and the So Lambda prophage) are activated after exposure to low doses of IR. We found that 300 min after exposure to doses as low as 0.25 Gy, genes within the So Lambda regulon remained upregulated. As this is the first transcriptome-wide study of how bacteria respond to acute sublethal doses of IR, these findings serve as a benchmark for future bacterial IR sensitivity studies. This is the first work to highlight the utility of prophages as biomarkers of exposure to very low (i.e., sublethal) doses of IR and to examine the longer-term impacts of sublethal IR exposure on bacteria.

Familial deletion of the HOXA gene cluster associated with Hand-Foot-Genital syndrome and phenotypic variability.

Hand-Foot-Genital syndrome is a rare autosomal dominant condition characterized by distal limb anomalies and urogenital malformations. This disorder is associated with loss-of-function mutations in the HOXA13 gene. HOXA13 plays an important role in the development of distal limbs and lower genitourinary tract of the fetus. We report a novel familial 589 kb deletion in the 7p15.2 region identified in a male toddler and his mother. The proband had severe penoscrotal hypospadias, mild skeletal anomalies of the hands and feet, cardiac, renal, and gastrointestinal anomalies. His mother had a bicornuate uterus, cervical incompetence, and minor anomalies of her hands and feet. This family was found to have the smallest reported deletion of 7p15.2 to date, and presented with features typical of Hand-Foot-Genital syndrome in the mother, but much more severe phenotype in her son. This deletion included the entire HOXA cluster in addition to the SKAP2 and EVX1 genes. An RT-PCR analysis was performed to determine the expression of the HOXA genes in the proband and to explore a parent-of-origin effect. Our expression studies did not support the hypothesis of an imprinted status of the HOXA2, HOXA3, HOXA5, and HOXA11 genes in peripheral blood. To our knowledge, this is the first familial 7p15.2 deletion. This family raises possibility for sexual dimorphism as a mechanism for phenotypic variability in patients with the HOXA gene cluster deletions. © 2016 Wiley Periodicals, Inc.

TolRad, a model for predicting radiation tolerance using Pfam annotations, identifies novel radiosensitive bacterial species from reference genomes and MAGs.

The trait of ionizing radiation (IR) tolerance is variable between bacterium, with species succumbing to acute doses as low as 60 Gy and extremophiles able to survive doses exceeding 10,000 Gy. While survival screens have identified multiple highly radioresistant bacteria, such systemic searches have not been conducted for IR-sensitive bacteria. The taxonomy-level diversity of IR sensitivity is poorly understood, as are genetic elements that influence IR sensitivity. Using the protein domain (Pfam) frequencies from 61 bacterial species with experimentally determined D10 values (the dose at which only 10% of the population survives), we trained TolRad, a random forest binary classifier, to distinguish between radiosensitive (D10 < 200 Gy) and radiation-tolerant (D10 > 200 Gy) bacteria. On untrained species, TolRad had an accuracy of 0.900. We applied TolRad to 152 UniProt-hosted bacterial proteomes associated with the human microbiome, including 37 strains from the ATCC Human Microbiome Collection, and classified 34 species as radiosensitive. Whereas IR-sensitive species (D10 < 200 Gy) in the training data set had been confined to the phylum Proteobacterium, this initial TolRad screen identified radiosensitive bacteria in two additional phyla. We experimentally validated the predicted radiosensitivity of a Bacteroidota species from the human microbiome. To demonstrate that TolRad can be applied to metagenome-assembled genomes (MAGs), we tested the accuracy of TolRad on Egg-NOG assembled proteomes (0.965) and partial proteomes. Finally, three collections of MAGs were screened using TolRad, identifying further phyla with radiosensitive species and suggesting that environmental conditions influence the abundance of radiosensitive bacteria. IMPORTANCE: Bacterial species have vast genetic diversity, allowing for life in extreme environments and the conduction of complex chemistry. The ability to harness the full potential of bacterial diversity is hampered by the lack of high-throughput experimental or bioinformatic methods for characterizing bacterial traits. Here, we present a computational model that uses de novo-generated genome annotations to classify a bacterium as tolerant of ionizing radiation (IR) or as radiosensitive. This model allows for rapid screening of bacterial communities for low-tolerance species that are of interest for both mechanistic studies into bacterial sensitivity to IR and biomarkers of IR exposure.

Leveraging anthropological expertise to respond to the COVID-19 global mental health syndemic.

This commentary asks anthropologists to work within communities to actively address the global mental health impact of COVID-19 and contribute to the pandemic response. Multiple social and physical losses, worsened by numerous factors, have produced syndemic traumatic stress and suffering across populations, highlighting persistent inequalities further amplified by the effects of COVID-19. Specifically, anthropologists can work to contribute to the development of mental health programs; confront the racialization of COVID-19 alongside marginalized communities; support real-time policy making with community responses; and innovate transparent collaborative research methods through open science. This pandemic can serve as an opportunity to prioritize research endeavors, public service, and teaching to better align with societal needs while providing new opportunities for synergy and collaborations between anthropologists in and outside the academy. Anthropologists collaborating directly with mental health clinicians and the public can contribute to knowledge specifically through direct program development and implementation of interventions designed to improve mental well-being. Innovating to find impactful solutions in response to the unprecedented mental health challenges exacerbated by the COVID-19 pandemic has the potential to promote more equitable recovery around the world.

A small RNA regulates pprM, a modulator of pleiotropic proteins promoting DNA repair, in Deinococcus radiodurans under ionizing radiation.

Networks of transcriptional and post-transcriptional regulators are critical for bacterial survival and adaptation to environmental stressors. While transcriptional regulators provide rapid activation and/or repression of a wide-network of genes, post-transcriptional regulators, such as small RNAs (sRNAs), are also important to fine-tune gene expression. However, the mechanisms of sRNAs remain poorly understood, especially in less-studied bacteria. Deinococcus radiodurans is a gram-positive bacterium resistant to extreme levels of ionizing radiation (IR). Although multiple unique regulatory systems (e.g., the Radiation and Desiccation Response (RDR)) have been identified in this organism, the role of post-transcriptional regulators has not been characterized within the IR response. In this study, we have characterized an sRNA, PprS (formerly Dsr2), as a post-transcriptional coordinator of IR recovery in D. radiodurans. PprS showed differential expression specifically under IR and knockdown of PprS resulted in reduced survival and growth under IR, suggesting its importance in regulating post-radiation recovery. We determined a number of potential RNA targets involved in several pathways including translation and DNA repair. Specifically, we confirmed that PprS binds within the coding region to stabilize the pprM (DR_0907) transcript, a RDR modulator. Overall, these results are the first to present an additional layer of sRNA-based control in DNA repair pathways associated with bacterial radioresistance.