Role of D-tryptophan oxidase in D-tryptophan utilization by Escherichia coli.

Mutants of Escherichia coli K-12 that require L-tryptophan (trp) are normally unable to utilize D-tryptophan to fulfill their requirement. However, secondary mutations (dadR) that confer this ability can be isolated. In such strains two distinct enzymes are found to be produced at high levels: D-amino acid oxidase (EC 1.4.3.3) and D-tryptophan oxidase. A convenient assay procedure for D-tryptophan oxidase is described. The two enzymes could be distinguished on the basis of their sensitivity to inhibition by L-phenylalanine and L-tyrosine. Strains that were trp dadR could not grow with D-tryptophan in the presence of L-phenylalanine, but further mutations, Fyo, could be isolated that allowed growth under these conditions. Some of them were characterized by further increases in the level of D-tryptophan oxidase activity and a sharp decrease in D-amino acid oxidase. These kinds of Fyo mutations lay in or near the dadR gene. The substrate specificity of the two enzymes toward a large number of compounds was examined. The transamination of aromatic keto acids was investigated. In the wild-type strain only a single enzyme, transaminase A (EC 2.6.1.5), was found, and it was irreversibly activated when subjected to elevated temperatures. The present state of our knowledge on D-amino acid utilization in E. coli is summarized.     

Proteomic analysis of necroptotic extracellular vesicles

Necroptosis is a regulated and inflammatory form of cell death. We, and others, have previously reported that necroptotic cells release extracellular vesicles (EVs). We have found that necroptotic EVs are loaded with proteins, including the phosphorylated form of the key necroptosis-executing factor, mixed lineage kinase domain-like kinase (MLKL). However, neither the exact protein composition, nor the impact, of necroptotic EVs have been delineated. To characterize their content, EVs from necroptotic and untreated U937 cells were isolated and analyzed by mass spectrometry-based proteomics. A total of 3337 proteins were identified, sharing a high degree of similarity with exosome proteome databases, and clearly distinguishing necroptotic and control EVs. A total of 352 proteins were significantly upregulated in the necroptotic EVs. Among these were MLKL and caspase-8, as validated by immunoblot. Components of the ESCRTIII machinery and inflammatory signaling were also upregulated in the necroptotic EVs, as well as currently unreported components of vesicle formation and transport, and necroptotic signaling pathways. Moreover, we found that necroptotic EVs can be phagocytosed by macrophages to modulate cytokine and chemokine secretion. Finally, we uncovered that necroptotic EVs contain tumor neoantigens, and are enriched with components of antigen processing and presentation. In summary, our study reveals a new layer of regulation during the early stage of necroptosis, mediated by the secretion of specific EVs that influences the microenvironment and may instigate innate and adaptive immune responses. This study sheds light on new potential players in necroptotic signaling and its related EVs, and uncovers the functional tasks accomplished by the cargo of these necroptotic EVs.Subject terms: Necroptosis, Cell death and immune response     

G-protein beta subunit of Cochliobolus heterostrophus involved in virulence, asexual and sexual reproductive ability, and morphogenesis

Previous work established that mutations in mitogen-activated protein (MAP) kinase (CHK1) and heterotrimeric G-protein alpha (Galpha) subunit (CGA1) genes affect the development of several stages of the life cycle of the maize pathogen Cochliobolus heterostrophus. The effects of mutating a third signal transduction pathway gene, CGB1, encoding the Gbeta subunit, are reported here. CGB1 is the sole Gbeta subunit-encoding gene in the genome of this organism. cgb1 mutants are nearly wild type in vegetative growth rate; however, Cgb1 is required for appressorium formation, female fertility, conidiation, regulation of hyphal pigmentation, and wild-type virulence on maize. Young hyphae of cgb1 mutants grow in a straight path, in contrast to those of the wild type, which grow in a wavy pattern. Some of the phenotypes conferred by mutations in CGA1 are found in cgb1 mutants, suggesting that Cgb1 functions in a heterotrimeric G protein; however, there are also differences. In contrast to the deletion of CGA1, the loss of CGB1 is not lethal for ascospores, evidence that there is a Gbeta subunit-independent signaling role for Cga1 in mating. Furthermore, not all of the phenotypes conferred by mutations in the MAP kinase CHK1 gene are found in cgb1 mutants, implying that the Gbeta heterodimer is not the only conduit for signals to the MAP kinase CHK1 module. The additional phenotypes of cgb1 mutants, including severe loss of virulence on maize and of the ability to produce conidia, are consistent with CGB1 being unique in the genome. Fluorescent DNA staining showed that there is often nuclear degradation in mature hyphae of cgb1 mutants, while comparable wild-type cells have intact nuclei. These data may be genetic evidence for a novel cell death-related function of the Gbeta subunit in filamentous fungi.     

Endoplasmic Reticulum Stress Induces a Caspase-dependent N-terminal Cleavage of RBX1 Protein in B Cells

Endoplasmic reticulum (ER) stress develops when the ER is overloaded with too many proteins to fold. This elicits a signaling pathway called the unfolded protein response. The unfolded protein response is physiologically required for the terminal development of B cells into antibody-secreting plasma cells. Ring Box Protein 1 (RBX1) is a 14-kDa protein necessary for ubiquitin ligation activity of the multimeric cullin ring ubiquitin ligases (CRLs). As RBX1 is shared by a large number of CRLs, alterations in its activity may lead to global changes in protein stability. We discovered that RBX1 is cleaved in the course of LPS-induced plasma cell differentiation and in multiple myeloma cell lines upon induction of pharmacological ER stress. The cleavage is executed by several caspase proteases that cleave RBX1 eight amino acids from the N terminus. To address the possible implication of RBX1 cleavage for CRL activity, we replaced the endogenous RBX1 homolog of the yeast Saccharomyces cerevisiae, Roc1, with the wild type or the N-terminal Δ8 mutant human RBX1. We show that yeast expressing the cleaved RBX1 are hypersensitive to ER stress and are impaired in CRL-mediated ubiquitination and degradation. We propose a model by which N-terminal cleavage of RBX1 impairs its activity and promotes susceptibility to ER stress induction.     

High Functional Diversity in Mycobacterium tuberculosis Driven by Genetic Drift and Human Demography

Mycobacterium tuberculosis infects one third of the human world population and kills someone every 15 seconds. For more than a century, scientists and clinicians have been distinguishing between the human- and animal-adapted members of the M. tuberculosis complex (MTBC). However, all human-adapted strains of MTBC have traditionally been considered to be essentially identical. We surveyed sequence diversity within a global collection of strains belonging to MTBC using seven megabase pairs of DNA sequence data. We show that the members of MTBC affecting humans are more genetically diverse than generally assumed, and that this diversity can be linked to human demographic and migratory events. We further demonstrate that these organisms are under extremely reduced purifying selection and that, as a result of increased genetic drift, much of this genetic diversity is likely to have functional consequences. Our findings suggest that the current increases in human population, urbanization, and global travel, combined with the population genetic characteristics of M. tuberculosis described here, could contribute to the emergence and spread of drug-resistant tuberculosis.     

Short-term effects of rhizosphere microorganisms on fe uptake from microbial siderophores by maize and oat

Effects of rhizosphere microorganisms on Fe uptake by oat (Avena sativa) and maize (Zea mays) were studied in short-term (10 h) nutrient solution experiments. Fe was supplied either as microbial siderophores (pseudobactin [PSB] or ferrioxamine B [FOB]) or as phytosiderophores obtained as root exudates from barley (epi-3-hydroxy-mugineic acid [HMA]) under varied population densities of rhizosphere microorganisms (axenic, uninoculated, or inoculated with different microorganism cultures). When maize was grown under axenic conditions and supplied with FeHMA, Fe uptake rates were 100 to 300 times higher compared to those in plants supplied with Fe siderophores. Fe from both sources was taken up without the involvement of an extracellular reduction process. The supply of FeHMA enhanced both uptake rate and translocation rate to the shoot (more than 60% of the total uptake). However, increased density of microorganisms resulted in a decrease in Fe uptake rate (up to 65%), presumably due to microbial degradation of the FeHMA. In contrast, when FeFOB or FePSB was used as the Fe source, increased population density of microorganisms enhanced Fe uptake. The enhancement of Fe uptake resulted from the uptake of FeFOB and FePSB by microorganisms adhering to the rhizoplane or living in the free space of cortical cells. The microbial apoplastic Fe pool was not available for root to shoot transport or, thus, for utilization by the plants. These results, in addition to the low uptake rate under axenic conditions, are in contrast to earlier hypotheses suggesting the existence of a specific uptake system for Fe siderophores in higher plants. The bacterial siderophores PSB and FOB were inefficient as Fe sources for plants even when supplied by stem injection. It was concluded that microorganisms are involved in degradation processes of microbial siderophores, as well as in competition for Fe with higher plants.