The recently published genome sequence of Bacillus anthracis Ames has facilitated the prediction of proteins associated with the virulence of this bacterium. The aim of this study was to define reference maps for the extracellular and cytoplasmic proteomes of the avirulent B. anthracis strain UM23C1-2 that are useful for physiological studies and the development of improved vaccines. Using 2-DE and subsequent MALDI-TOF-TOF MS, 64 proteins were identified in the extracellular proteome, only 29 of which were predicted to be exported into the culture medium. The latter included chitinases, proteases, nucleotidases, sulfatases, phosphatases and proteins of unknown function. Of the remaining proteins in the culture medium, 18 were predicted to be associated with the cell wall or anchored on the trans side of the cytoplasmic membrane while 17 other proteins lacked identifiable export signals and were predicted to be cytoplasmic proteins. Among the S-layer proteins, Sap and Eag account for 10% of the total extracellular proteome. Many of the proteins are predicted to contribute to the virulence and antigenic signature of B. anthracis. We have also studied the composition of the cytoplasmic proteome, identifying 300 distinct proteins. The most abundant cytoplasmic proteins are primarily those involved in glycolysis, amino acid metabolism, protein translation, protein folding and stress adaptation. The presence of a variety of proteases, peptidases, peptide binding proteins, as well as enzymes required for the metabolism of amino acids, suggests that B. anthracis is adapted to life in a protein-rich environment rather than the soil. We therefore speculate that proteases and peptidases could be useful targets for the development of improved vaccines. In addition, both of these B. anthracis compartment-specific proteomes can be used as reference maps to monitor changes in the production of secreted and cytosolic proteins that occur, for example, during growth in macrophages.
Protective antigen (PA) is a component of the Bacillus anthracis lethal and edema toxins and the basis of the current anthrax vaccine. In its heptameric form, PA targets host cells and internalizes the enzymatically active components of the toxins, namely lethal and edema factors. PA and other toxin components are secreted from B. anthracis using the Sec-dependent secretion pathway. This requires them to be translocated across the cytoplasmic membrane in an unfolded state and then to be folded into their native configurations on the trans side of the membrane, prior to their release from the environment of the cell wall. In this study we show that recombinant PA (rPA) requires the extracellular chaperone PrsA for efficient folding when produced in the heterologous host, B. subtilis; increasing the concentration of PrsA leads to an increase in rPA production. To determine the likelihood of PrsA being required for PA production in its native host, we have analyzed the B. anthracis genome sequence for the presence of genes encoding homologues of B. subtilis PrsA. We identified three putative B. anthracis PrsA proteins (PrsAA, PrsAB, and PrsAC) that are able to complement the activity of B. subtilis PrsA with respect to cell viability and rPA secretion, as well as that of AmyQ, a protein previously shown to be PrsA-dependent.
Mouse lemurs (Microcebus) are a radiation of morphologically cryptic primates distributed throughout Madagascar for which the number of recognized species has exploded in the past two decades. This taxonomic explosion has prompted understandable concern that there has been substantial oversplitting in the mouse lemur clade. Here, we take an integrative approach to investigate species diversity in two pairs of sister lineages that occur in a region in northeastern Madagascar with high levels of microendemism and predicted habitat loss. We analyzed RADseq data with multispecies coalescent (MSC) species delimitation methods for three named species and an undescribed lineage previously identified to have divergent mtDNA. Marked differences in effective population sizes, levels of gene flow, patterns of isolation-by-distance, and species delimitation results were found among them. Whereas all tests support the recognition of the presently undescribed lineage as a separate species, the species-level distinction of two previously described species, M. mittermeieri and M. lehilahytsara is not supported – a result that is particularly striking when using the genealogical discordance index (gdi). Non-sister lineages occur sympatrically in two of the localities sampled for this study, despite an estimated divergence time of less than 1 Ma. This suggests rapid evolution of reproductive isolation in the focal lineages, and in the mouse lemur clade generally. The divergence time estimates reported here are based on the MSC and calibrated with pedigree-based mutation rates and are considerably more recent than previously published fossil-calibrated concatenated likelihood estimates, however. We discuss the possible explanations for this discrepancy, noting that there are theoretical justifications for preferring the MSC estimates in this case.
Madagascar is a biodiversity hotspot that is facing rapid rates of deforestation, habitat destruction, and poverty. Urgent action is required to document the status of biodiversity to facilitate efficacious conservation plans. With the recent advent of portable and affordable genetic technologies, it is now possible to take genomic approaches out of the lab and into the field. Mobile genetics labs can produce scientifically reproducible data under field conditions, dramatically minimizing the time between sample collection and data analysis. Here, we show “proof of concept” by deploying miniPCR bio’s miniaturized thermal cycler alongside Oxford Nanopore’s MinION DNA sequencer in Madagascar. Specifically, we deployed this technology at Anjajavy, northwestern Madagascar for rapid biodiversity assessment. We successfully extracted mouse lemur DNA, amplified and sequenced a phylogenetically informative mitochondrial gene (cytochrome-b; cytb), and thereby confirmed the presence of Danfoss’ mouse lemur (M. danfossi) within the Anjajavy Reserve. We show that a mobile genetics lab can provide expeditious results, and allow scientists to conduct genetic analyses, potentially allowing for rapid interventions under emergency conditions in situ. Additionally, mobile labs offer powerful training opportunities for in-country scientists for whom training opportunities were previously confined to ex-situ locations. By bringing genomic technologies to Madagascar and other economically challenged and biodiverse regions of the world, the next generation of scientists and conservationists can more fully implement their leadership roles. Local laboratory and training facilities are changing the polarity of research programs in Madagascar and empowering national researchers to take charge of environmental stewardship.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.