Biology subjects

Richards, C.

Publications and source records attributed to Richards, C..

24 records · Page 2Linked to original sources

bioRxiv2020

DksA-dependent regulation of RpoS contributes to Borrelia burgdorferi tick-borne transmission and mammalian infectivity

Throughout its enzootic cycle, the Lyme disease spirochete Borreliella (Borrelia) burgdorferi, senses and responds to changes in its environment by using a small repertoire of transcription factors which coordinate the expression of genes required for infection of Ixodes ticks and various mammalian hosts. Among these transcription factors, the DnaK suppressor protein (DksA) plays a pivotal role in regulating gene expression in B. burgdorferi during periods of nutrient limitation and is required for mammalian infectivity. In many pathogenic bacteria, the gene regulatory activity of DksA along with the alarmone guanosine penta- and tetra-phosphate ((p)ppGpp) coordinates the stringent response to various environmental stresses including nutrient limitation. In this study, we sought to characterize the role of DksA in regulating the transcriptional activity of RNA polymerase and in the regulation of RpoS-dependent gene expression required for B. burgdorferi infectivity. Using in vitro transcription assays, we observed recombinant DksA inhibits RpoD-dependent transcription by B. burgdorferi RNA polymerase independent of ppGpp Additionally, we determined the pH-inducible expression of RpoS-dependent genes relies on DksA, but is independent of (p)ppGpp produced by Relbbu. Subsequent transcriptomic and western blot assays indicated DksA regulates the expression of BBD18, a protein previously implicated in the post-transcriptional regulation of RpoS. Moreover, we observed DksA was required for infection of mice following intraperitoneal inoculation or for transmission of B. burgdorferi by Ixodes scapularis nymphs. Together, these data suggest DksA plays a central role in coordinating transcriptional responses of B. burgdorferi required for infectivity through its interactions with RNA polymerase and post-transcriptional control of RpoS. Author SummaryLyme disease, caused by the spirochetal bacteria Borrelia burgdorferi, is the most common vector-borne illness in North America. The ability of B. burgdorferi to establish infection is predicated by its ability to coordinate the expression of virulence factors in response to diverse environmental stimuli encountered within Ixodes ticks and mammalian hosts. Previous studies have shown an essential role for the alternative sigma factor RpoS in regulating the expression of genes required for the successful transmission of B. burgdorferi by Ixodes ticks and infection of mammalian hosts. The DnaK suppressor protein (DksA) is a global gene regulator in B. burgdorferi that also contributes to the expression of RpoS-dependent genes. In this study, we determined DksA exerts its gene regulatory function through direct interactions with the B. burgdorferi RNA polymerase using in vitro transcription assays and controls the expression of RpoS-dependent genes required for mammalian infection by post-transcriptionally regulating cellular levels of RpoS. Our results demonstrate the utility of in vitro transcription assays to determine how gene regulatory proteins like DksA control gene expression in B. burgdorferi, and reveal a novel role for DksA in the infectious cycle of B. burgdorferi.

microbiology↗

bioRxiv2020

Biological traits of seabirds predict extinction risk and vulnerability to anthropogenic threats

AimHere we aim to: 1) test whether globally-threatened vs non-threatened seabirds are separated in trait space; 2) quantify the redundancy and uniqueness of species trait combinations per IUCN Red List Category; and 3) identify traits that render species vulnerable to anthropogenic threats. LocationGlobal Time periodContemporary Major taxa studiedSeabirds MethodsWe compile and impute eight traits that relate to species vulnerabilities and ecosystem functioning across 341 seabird species. Using these traits, we build a mixed data PCA of species trait space. We further quantify trait redundancy with a unique trait combinations (UTCs) approach. Finally, we employ a similarity of percentages analysis (SIMPER) to identify which traits explain the greatest difference between threat groups. ResultsWe find seabirds segregate in trait space based on threat status, indicating anthropogenic impacts are selectively removing large, long-lived, pelagic surface feeders with narrow habitat breadths. We further find that globally threatened species have higher trait redundancy, while non-threatened species have relatively unique ecological strategies and limited redundancy. Finally, we find that species with narrow habitat breadths, fast reproductive speeds, and omnivorous diets are more likely to be threatened by habitat-modifying processes (e.g., pollution and natural system modifications); whereas pelagic specialists with slow reproductive speeds and omnivorous diets are vulnerable to threats that directly impact survival and fecundity (e.g., invasive species and biological resource use). Main conclusionsOur results suggest both globally threatened and non-threatened species contribute unique ecological strategies. Consequently, conserving both threat groups, but with contrasting approaches may avoid potential changes in ecosystem functioning and stability.

ecology↗

bioRxiv2020

Induction of AmpC-mediated-βlactam resistance requires a single lytic transglycosylase in Agrobacterium tumefaciens

The remarkable ability of Agrobacterium tumefaciens to transfer DNA to plant cells has allowed the generation of important transgenic crops. One challenge of A. tumefaciens-mediated transformation is eliminating the bacteria after plant transformation to prevent detrimental effects to plants and the release of engineered bacteria to the environment. Here we use a reverse genetics approach to identify genes involved in ampicillin resistance with the goal of utilizing these antibiotic-sensitive strains for plant transformations. We show that treating A. tumefaciens C58 with ampicillin led to increased {beta}-lactamase production, a response dependent on the broad-spectrum {beta}-lactamase AmpC and its transcription factor AmpR. Loss of the putative ampD orthologue, atu2113, led to constitutive production of AmpC-dependent {beta}-lactamase activity and ampicillin resistance. Finally, one cell wall remodeling enzyme, MltB3, was necessary for the AmpC-dependent {beta}-lactamase activity and its loss elicited ampicillin and carbenicillin sensitivity in the A. tumefaciens C58 and GV3101 strains. Furthermore, GV3101{Delta} mltB3 transforms plants with comparable efficiency to wildtype but can be cleared with sub-lethal concentrations of ampicillin. The functional characterization of the genes involved in the inducible ampicillin resistance pathway of A. tumefaciens constitutes a major step forward in efforts to reduce the intrinsic antibiotic resistance of this bacterium. IMPORTANCEAgrobacterium tumefaciens, a significant biotechnological tool for production of transgenic plant lines, is highly resistant to a wide variety of antibiotics, posing challenges for various applications. One challenge is the efficient elimination of A. tumefaciens from transformed plant tissue without using levels of antibiotics that are toxic to the plants. Here, we present the functional characterization of genes involved in {beta}-lactam resistance in A. tumefaciens. Knowledge about proteins that promote or inhibit {beta}-lactam resistance will enable the development of strains to improve the efficiency of Agrobacterium-mediated plant genetic transformations. Effective removal of Agrobacterium from transformed plant tissue has the potential to maximize crop yield and food production, improving the outlook for global food security.

microbiology↗

bioRxiv2020

Longitudinal whole-exome sequencing of cell-free DNA unravels the metastatic evolutionary dynamics of BRCA2 mutated breast cancer

Little is known about the metastatic evolutionary dynamics of BRCA2-mutated cancers. Here, we applied whole-exome sequencing (WES) of primary tumor (PT), local relapse (LR) and eight serial plasma cfDNA samples collected from disease progression to depict the 12 years evolutionary trajectory of a metastatic BRCA2-mutated breast cancer. While longitudinal WES-cfDNA recapitulated clonal and subclonal mutations and copy number profiles detected in LR, emergence of plasma-exclusive mutations in TSC2 and HDAC9 cancer-related genes and loss of HLA loci as an immune escape mechanism were also detected. Lastly, mutation signature 3, associated with homologous recombination deficiency and response to platinum-based therapy raised profoundly from 19% in PT to 60% in late stage disease. In conclusion, we show for the first time that longitudinal WES-cfDNA enables the evolutionary trajectory of advanced cancer to be uncovered and that increment of MS3 and loss of HLA are key players in this BRCA2-mutated breast metastasis.

cancer biology↗

bioRxiv2018

Reduced-representation bisulfite sequencing finds novel epigenome-wide response to oil pollution in the foundation plant Spartina alterniflora

Theory predicts that environmental challenges can shape the composition of populations, which is manifest at the molecular level. Previously, we demonstrated that oil pollution affected gene expression patterns and altered genetic variation in natural populations of the foundation salt marsh grass, Spartina alterniflora. Here, we used a reduced representation bisulfite sequencing approach, epigenotyping by sequencing (epiGBS), to examine relationships among DNA sequence, DNA methylation, gene expression, and exposure to oil pollution. We documented genetic and methylation differentiation between oil-exposed and unexposed populations, suggesting that the Deepwater Horizon oil spill may have selected on genetic variation, and either selected on epigenetic variation or induced particular epigenotypes and expression patterns in exposed compared to unexposed populations. In support of the potential for differential response to the Deepwater Horizon oil spill, we demonstrate genotypic differences in response to oil under controlled conditions. Overall, these findings demonstrate genetic variation, epigenetic variation and gene expression are correlated to exposure to oil pollution, which may all contribute to the response to environmental stress.

ecology↗

bioRxiv2017

The Antiviral and Cancer Genomic DNA Deaminase APOBEC3H Is Regulated by a RNA-Mediated Dimerization Mechanism

Human APOBEC3H and homologous single-stranded DNA cytosine deaminases are unique to mammals. These DNA editing enzymes function in innate immunity by restricting the replication of viruses and transposons. Misregulated APOBEC3H also contributes to cancer mutagenesis. Here we address the role of RNA in APOBEC3H regulation. APOBEC3H co-purifies with RNA as an inactive protein, and RNase A treatment yields enzyme preparations with stronger DNA deaminase activity. RNA binding-defective mutants are DNA hypermutators. Chromatography profiles and crystallographic data demonstrate a mechanism in which double-stranded RNA mediates enzyme dimerization. RNA binding is required for APOBEC3H cytoplasmic localization and for packaging into HIV-1 particles and antiviral activity. Related DNA deaminases including other APOBEC3 family members and the antibody gene diversification enzyme AID also bind RNA and are predicted to have a similar RNA binding motif suggesting mechanistic conservation and relevance to innate and adaptive immunity and to multiple diseases.\n\nHIGHLIGHTSRNA inhibits human APOBEC3H DNA cytosine deaminase activity\n\nRNA binding mutants are DNA hypermutators\n\nX-ray structure demonstrates an RNA duplex-mediated APOBEC3H dimerization mechanism\n\nRNA binding is required for packaging into HIV-1 particles and antiviral activity

microbiology↗