Genome Sequence of the Atypical Symbiotic Frankia R43 Strain, a Nitrogen-Fixing and Hydrogen-Producing Actinobacterium.

Frankia strain R43 is a nitrogen-fixing and hydrogen-producing symbiotic actinobacterium that was isolated from nodules of Casuarina cunninghamiana but infects only Elaeagnaceae. This communication reports the genome of the strain R43 and provides insights into the microbe genomics and physiological potentials.

Aspects of nitrogen-fixing Actinobacteria, in particular free-living and symbiotic Frankia.

Studies of nitrogen-fixing properties among the Gram-positive Actinobacteria revealed that some species of Arthrobacter, Agromyces, Corynebacterium, Mycobacterium, Micromonospora, Propionibacteria and Streptomyces have nitrogen-fixing capacity. This is also valid for Frankia that fix nitrogen both in free-living and in symbiotic conditions. Frankia symbiosis results from interaction between the Frankia bacteria and dicotyledonous plants, that is, actinorhiza. These plants, which are important in forestry and agroforestry, form, together with the legumes (Fabales), a single nitrogen-fixing clade. It has been shown that a receptor-like kinase gene, SymRK, is necessary for nodulation in actinorhizal plants as well as in legumes and arbuscular mycorrhizal fungi. Recently, the involvement of isoflavonoids as signal molecules during nodulation of an actinorhizal plant was shown. The genome sizes of three Frankia species, Frankia EANpec, ACN14a and CcI3, are different, revealing a relationship between genome size and geographical distribution. Recent genomic sequencing data of Frankia represent genomes from cluster I to IV, indicating that the genome of DgI is one of the smallest genomes in Frankia. In addition, nonsymbiotic Frankiales such as Acidothermus cellulolyticus, Blastococcus saxoobsidens, Geodermatophilus obscurus and Modestobacter marinus have a variety of genome sizes ranging from 2.4 to 5.57 Mb.

The antimalarial natural product symplostatin 4 is a nanomolar inhibitor of the food vacuole falcipains.

The marine natural product symplostatin 4 (Sym4) has been recognized as a potent antimalarial agent. However, its mode of action and, in particular, direct targets have to date remained elusive. We report a chemical synthesis of Sym4 and show that Sym4-treatment of P. falciparum-infected red blood cells (RBCs) results in the generation of a swollen food vacuole phenotype and a reduction of parasitemia at nanomolar concentrations. We furthermore demonstrate that Sym4 is a nanomolar inhibitor of the P. falciparum falcipains in infected RBCs, suggesting inhibition of the hemoglobin degradation pathway as Sym4's mode of action. Finally, we reveal a critical influence of the unusual methyl-methoxypyrrolinone (mmp) group of Sym4 for potent inhibition, indicating that Sym4 derivatives with such a mmp moiety might represent viable lead structures for the development of antimalarial falcipain inhibitors.

Subclassification and biochemical analysis of plant papain-like cysteine proteases displays subfamily-specific characteristics.

Papain-like cysteine proteases (PLCPs) are a large class of proteolytic enzymes associated with development, immunity, and senescence. Although many properties have been described for individual proteases, the distribution of these characteristics has not been studied collectively. Here, we analyzed 723 plant PLCPs and classify them into nine subfamilies that are present throughout the plant kingdom. Analysis of these subfamilies revealed previously unreported distinct subfamily-specific functional and structural characteristics. For example, the NPIR and KDEL localization signals are distinctive for subfamilies, and the carboxyl-terminal granulin domain occurs in two PLCP subfamilies, in which some individual members probably evolved by deletion of the granulin domains. We also discovered a conserved double cysteine in the catalytic site of SAG12-like proteases and two subfamily-specific disulfides in RD19A-like proteases. Protease activity profiling of representatives of the PLCP subfamilies using novel fluorescent probes revealed striking polymorphic labeling profiles and remarkably distinct pH dependency. Competition assays with peptide-epoxide scanning libraries revealed common and unique inhibitory fingerprints. Finally, we expand the detection of PLCPs by identifying common and organ-specific protease activities and identify previously undetected proteases upon labeling with cell-penetrating probes in vivo. This study provides the plant protease research community with tools for further functional annotation of plant PLCPs.

Mining the Active Proteome of Arabidopsis thaliana.

Assigning functions to the >30,000 proteins encoded by the Arabidopsis genome is a challenging task of the Arabidopsis Functional Genomics Network. Although genome-wide technologies like proteomics and transcriptomics have generated a wealth of information that significantly accelerated gene annotation, protein activities are poorly predicted by transcript or protein levels as protein activities are post-translationally regulated. To directly display protein activities in Arabidopsis proteomes, we developed and applied activity-based protein profiling (ABPP). ABPP is based on the use of small molecule probes that react with the catalytic residues of distinct protein classes in an activity-dependent manner. Labeled proteins are separated and detected from proteins gels and purified and identified by mass spectrometry. Using probes of six different chemotypes we have displayed activities of 76 Arabidopsis proteins. These proteins represent over 10 different protein classes that contain over 250 Arabidopsis proteins, including cysteine, serine, and metalloproteases, lipases, acyltransferases, and the proteasome. We have developed methods for identification of in vivo labeled proteins using click chemistry and for in vivo imaging with fluorescent probes. In vivo labeling has revealed additional protein activities and unexpected subcellular activities of the proteasome. Labeling of extracts displayed several differential activities, e.g., of the proteasome during immune response and methylesterases during infection. These studies illustrate the power of ABPP to display the functional proteome and testify to a successful interdisciplinary collaboration involving chemical biology, organic chemistry, and proteomics.

Proteasome activity profiling: a simple, robust and versatile method revealing subunit-selective inhibitors and cytoplasmic, defense-induced proteasome activities.

The proteasome plays essential roles in nearly all biological processes in plant defense and development, yet simple methods for displaying proteasome activities in extracts and living tissues are not available to plant science. Here, we introduce an easy and robust method to simultaneously display the activities of all three catalytic proteasome subunits in plant extracts or living plant tissues. The method is based on a membrane-permeable, small-molecule fluorescent probe that irreversibly reacts with the catalytic site of the proteasome catalytic subunits in an activity-dependent manner. Activities can be quantified from fluorescent protein gels and used to study proteasome activities in vitro and in vivo. We demonstrate that proteasome catalytic subunits can be selectively inhibited by aldehyde-based inhibitors, including the notorious caspase-3 inhibitor DEVD. Furthermore, we show that the proteasome activity, but not its abundance, is significantly increased in Arabidopsis upon treatment with benzothiadiazole (BTH). This upregulation of proteasome activity depends on NPR1, and occurs mostly in the cytoplasm. The simplicity, robustness and versatility of this method will make this method widely applicable in plant science.

Chelation by histidine inhibits the vacuolar sequestration of nickel in roots of the hyperaccumulator Thlaspi caerulescens.

* The mechanisms of enhanced root to shoot metal transport in heavy metal hyperaccumulators are incompletely understood. Here, we compared the distribution of nickel (Ni) over root segments and tissues in the hyperaccumulator Thlaspi caerulescens and the nonhyperaccumulator Thlaspi arvense, and investigated the role of free histidine in Ni xylem loading and Ni transport across the tonoplast. * Nickel accumulation in mature cortical root cells was apparent in T. arvense and in a high-Ni-accumulating T. caerulescens accession, but not in a low-accumulating T. caerulescens accession. * Compared with T. arvense, the concentration of free histidine in T. caerulescens was 10-fold enhanced in roots, but was only slightly higher in leaves, regardless of Ni exposure. Nickel uptake in MgATP-energized root- and shoot-derived tonoplast vesicles was almost completely blocked in T. caerulescens when Ni was supplied as a 1 : 1 Ni-histidine complex, but was uninhibited in T. arvense. Exogenous histidine supply enhanced Ni xylem loading in T. caerulescens but not in T. arvense. * The high rate of root to shoot translocation of Ni in T. caerulescens compared with T. arvense seems to depend on the combination of two distinct characters, that is, a greatly enhanced root histidine concentration and a strongly decreased ability to accumulate histidine-bound Ni in root cell vacuoles.