Cysteine of sequence motif VI is essential for nucleophilic catalysis by yeast tRNA m5C methyltransferase

Sequence comparison of several RNA m(5)C methyltransferases identifies two conserved cysteine residues that belong to signature motifs IV and VI of RNA and DNA methyltransferases. While the cysteine of motif IV is used as the nucleophilic catalyst by DNA m(5)C methyltransferases, this role is fulfilled by the cysteine of motif VI in Escherichia coli 16S rRNA m(5)C967 methyltransferase, but whether this conclusion applies to other RNA m(5)C methyltransferases remains to be verified. Yeast tRNA m(5)C methyltransferase Trm4p is a multisite-specific S-adenosyl-L-methionine-dependent enzyme that catalyzes the methylation of cytosine at C5 in several positions of tRNA. Here, we confirm that Cys310 of motif VI in Trm4p is essential for nucleophilic catalysis, presumably by forming a covalent link with carbon 6 of cytosine. Indeed, the enzyme is able to form a stable covalent adduct with the 5-fluorocytosine-containing RNA substrate analog, whereas the C310A mutant protein is inactive and unable to form the covalent complex.     

Human T cell clones recognize two abundant Mycobacterium tuberculosis protein antigens expressed in Escherichia coli

Human T cells reactive to Mycobacterium tuberculosis were cloned from peripheral blood mononuclear cells (PBMC) of four tuberculosis patients by using whole irradiated bacilli as the in vitro stimulatory agent. Twenty-two T cell clones (CD4+) were tested for their reactivity to 12 different mycobacterial species and showed a distribution from limited to broad cross-reactivity. These T cell clones were also tested for their reactivity to three abundant M. tuberculosis proteins of 65, 19, and 14 kD, each expressed from recombinant DNA in Escherichia coli. The three proteins were expressed from DNA clones that were previously isolated from a lambda gt11 genomic DNA library of M. tuberculosis by using monoclonal antibodies directed against this pathogen. A T cell clone from one patient was stimulated by an E. coli lysate containing the 65 kD antigen, and a T cell clone from a second patient was stimulated by an E. coli lysate containing the 19 kD antigen in an in vitro proliferation assay. Both T cell clones showed very limited cross-reactivity when tested against other mycobacteria. We conclude that some patients with tuberculosis exhibit a T cell response to the 65 and 19 kD M. tuberculosis proteins defined by these isolated genes.     

A multispectral optical illumination system with precise spatiotemporal control for the manipulation of optogenetic reagents

Optogenetics is an excellent tool for noninvasive activation and silencing of neurons and muscles. Although they have been widely adopted, illumination techniques for optogenetic tools remain limited and relatively nonstandardized. We present a protocol for constructing an illumination system capable of dynamic multispectral optical targeting of micrometer-sized structures in both stationary and moving objects. The initial steps of the protocol describe how to modify an off-the-shelf video projector by insertion of optical filters and modification of projector optics. Subsequent steps involve altering the microscope's epifluorescence optical train as well as alignment and characterization of the system. When fully assembled, the illumination system is capable of dynamically projecting multispectral patterns with a resolution better than 10 μm at medium magnifications. Compared with other custom-assembled systems and commercially available products, this protocol allows a researcher to assemble the illumination system for a fraction of the cost and can be completed within a few days.     

Evidence of Sundaland’s subsidence requires revisiting its biogeography

It is widely accepted that sea level changes intermittently inundated the Sunda Shelf throughout the Pleistocene, separating Java, Sumatra and Borneo from the Malay Peninsula and from each other. On this basis, the dynamics of the biodiversity hotspot of Sundaland is consistently regarded as solely contingent on glacial sea level oscillations, with interglacial highstands creating intermittent dispersal barriers between disjunct landmasses. However, recent findings on the geomorphology of the currently submerged Sunda shelf suggest that it subsided during the Pleistocene and that, over the Late Pliocene and Quaternary, is was never submerged prior to Marine Isotope Stage 11 (MIS 11, 400 ka). This would have enabled the dispersal of terrestrial organisms regardless of sea level variations until 400 ka and hampered movements thereafter, at least during interglacial periods. Existing phylogeographic data for terrestrial organisms conform to this scenario: available divergence time estimates reveal an 8- to 9-fold increase in the rate of vicariance between landmasses of Sundaland after 400 ka, corresponding to the onset of episodic flooding of the Sunda shelf. These results highlight how reconsidering the paleogeographic setting of Sundaland challenges understanding the mechanisms generating Southeast Asian biodiversity.     

Optimization of a real-time PCR assay for the detection of the quarantine pathogen Melampsora medusae f. sp. deltoidae

Melampsora medusae (Mm), one of the causal agents of poplar rust, is classified as an A2 quarantine pest for European Plant Protection Organization (EPPO) and its presence in Europe is strictly controlled. Two formae speciales have been described within Mm, Melampsora medusae f. sp. deltoidae (Mmd), and Melampsora medusae f. sp. tremuloidae (Mmt) on the basis of their pathogenicity on Populus species from the section Aigeiros (e.g. Populus deltoides) or Populus (e.g. Populus tremuloides), respectively. In this study, a real-time polymerase chain reaction (PCR) assay was developed allowing the detection of Mmd, the forma specialis that is economically harmful. A set of primers and hydrolysis probe were designed based on sequence polymorphisms in the large ribosomal RNA subunit (28S). The real-time PCR assay was optimized and performance criteria of the detection method, i.e. sensitivity, specificity, repeatability, reproducibility, and robustness, were assessed. The real-time PCR method was highly specific and sensitive and allowed the detection of one single urediniospore of Mmd in a mixture of 2 mg of urediniospores of other Melampsora species. This test offers improved specificity over currently existing conventional PCR tests and can be used for specific surveys in European nurseries and phytosanitary controls, in order to avoid introduction and spread of this pathogen in Europe.     

Phosphorylation regulates mycobacterial proteasome

Mycobacterium tuberculosis possesses a proteasome system that is required for the microbe to resist elimination by the host immune system. Despite the importance of the proteasome in the pathogenesis of tuberculosis, the molecular mechanisms by which proteasome activity is controlled remain largely unknown. Here, we demonstrate that the α-subunit (PrcA) of the M. tuberculosis proteasome is phosphorylated by the PknB kinase at three threonine residues (T84, T202, and T178) in a sequential manner. Furthermore, the proteasome with phosphorylated PrcA enhances the degradation of Ino1, a known proteasomal substrate, suggesting that PknB regulates the proteolytic activity of the proteasome. Previous studies showed that depletion of the proteasome and the proteasome-associated proteins decreases resistance to reactive nitrogen intermediates (RNIs) but increases resistance to hydrogen peroxide (H₂O₂). Here we show that PknA phosphorylation of unprocessed proteasome β-subunit (pre-PrcB) and α-subunit reduces the assembly of the proteasome complex and thereby enhances the mycobacterial resistance to H₂O₂ and that H₂O₂ stress diminishes the formation of the proteasome complex in a PknA-dependent manner. These findings indicate that phosphorylation of the M. tuberculosis proteasome not only modulates proteolytic activity of the proteasome, but also affects the proteasome complex formation contributing to the survival of M. tuberculosis under oxidative stress conditions.     

Real-time multimodal optical control of neurons and muscles in freely behaving Caenorhabditis elegans

The ability to optically excite or silence specific cells using optogenetics has become a powerful tool to interrogate the nervous system. Optogenetic experiments in small organisms have mostly been performed using whole-field illumination and genetic targeting, but these strategies do not always provide adequate cellular specificity. Targeted illumination can be a valuable alternative but it has only been shown in motionless animals without the ability to observe behavior output. We present a real-time, multimodal illumination technology that allows both tracking and recording the behavior of freely moving C. elegans while stimulating specific cells that express channelrhodopsin-2 or MAC. We used this system to optically manipulate nodes in the C. elegans touch circuit and study the roles of sensory and command neurons and the ultimate behavioral output. This technology enhances our ability to control, alter, observe and investigate how neurons, muscles and circuits ultimately produce behavior in animals using optogenetics.