The Mycobacterium tuberculosis Secreted Protein Rv0203 Transfers Heme to Membrane Proteins MmpL3 and MmpL11

Mycobacterium tuberculosis is the causative agent of tuberculosis, which is becoming an increasingly global public health problem due to the rise of drug-resistant strains. While residing in the human host, M. tuberculosis needs to acquire iron for its survival. M. tuberculosis has two iron uptake mechanisms, one that utilizes non-heme iron and another that taps into the vast host heme-iron pool. To date, proteins known to be involved in mycobacterial heme uptake are Rv0203, MmpL3, and MmpL11. Whereas Rv0203 transports heme across the bacterial periplasm or scavenges heme from host heme proteins, MmpL3 and MmpL11 are thought to transport heme across the membrane. In this work, we characterize the heme-binding properties of the predicted extracellular soluble E1 domains of both MmpL3 and MmpL11 utilizing absorption, electron paramagnetic resonance, and magnetic circular dichroism spectroscopic methods. Furthermore, we demonstrate that Rv0203 transfers heme to both MmpL3-E1 and MmpL11-E1 domains at a rate faster than passive heme dissociation from Rv0203. This work elucidates a key step in the mycobacterial uptake of heme, and it may be useful in the development of anti-tuberculosis drugs targeting this pathway.     

Structures of the Substrate-free and Product-bound Forms of HmuO,a Heme Oxygenase from Corynebacterium diphtheriae: X-RAY CRYSTALLOGRAPHY AND MOLECULAR DYNAMICS INVESTIGATION*?

Heme oxygenase catalyzes the degradation of heme to biliverdin, iron, and carbon monoxide. Here, we present crystal structures of the substrate-free, Fe³⁺-biliverdin-bound, and biliverdin-bound forms of HmuO, a heme oxygenase from Corynebacterium diphtheriae, refined to 1.80, 1.90, and 1.85 Å resolution, respectively. In the substrate-free structure, the proximal and distal helices, which tightly bracket the substrate heme in the substrate-bound heme complex, move apart, and the proximal helix is partially unwound. These features are supported by the molecular dynamic simulations. The structure implies that the heme binding fixes the enzyme active site structure, including the water hydrogen bond network critical for heme degradation. The biliverdin groups assume the helical conformation and are located in the heme pocket in the crystal structures of the Fe³⁺-biliverdin-bound and the biliverdin-bound HmuO, prepared by in situ heme oxygenase reaction from the heme complex crystals. The proximal His serves as the Fe³⁺-biliverdin axial ligand in the former complex and forms a hydrogen bond through a bridging water molecule with the biliverdin pyrrole nitrogen atoms in the latter complex. In both structures, salt bridges between one of the biliverdin propionate groups and the Arg and Lys residues further stabilize biliverdin at the HmuO heme pocket. Additionally, the crystal structure of a mixture of two intermediates between the Fe³⁺-biliverdin and biliverdin complexes has been determined at 1.70 Å resolution, implying a possible route for iron exit.     

The contribution of small individuals to density-body size relationships: examination of energetic equivalence in reef fishes

A key relationship in ecology is that between density and body size, with the emphasis placed on energetic rules constraining the abundance of larger organisms below that of smaller organisms. Most studies have focused upon the density-body size relationship at the species level. However, energy is gathered at an individual level. We therefore examined this relationship in a coral reef fish assemblage, focusing on individuals. Using a comprehensive data set, with over 14,000 observations we found that the relationship between local density and adult body size differs from the linear relationship predicted by the energetic equivalence rule. However, excluding the smallest size classes, the relationship between body size and individual abundance for intermediate to larger fish did not depart from the predicted –0.75. Unlike plants and intermediate to large reef fishes, the smallest fishes appear to have constraints that may reflect different patterns of resource acquisition.     

Clausal order and the acquisition of Dutch deverbal compounds

The purpose of this study was to explore how Dutch-speaking children acquire deverbal compounds, particularly in ordering verbs and nouns. English-speaking children form compounds like bottle breaker around 5–6 years of age and make noun-verb reversal errors at younger ages. These errors have been attributed to clausal ordering. Dutch allows more variations in clausal ordering, so Dutch-speaking children might acquire deverbal compounds differently from English-speaking children. In Study 1, we examined the input to a Dutch-speaking child between 4;8 and 5;2 and her compound acquisition. She heard a variety of clausal orderings, mostly with verbs before objects, and her deverbal compounds were already well acquired. In Study 2, we tested 24 Dutch-speaking preschool children’s production and comprehension of novel compounds. They produced many of the same forms as have been reported for English-speaking children, making reversal errors at around the same age. In Study 3, we compared a subset of the Dutch-speaking children with age-matched English monolingual children. We found a slight advantage for the Dutch-speaking children on production but no difference on comprehension. We argue that children’s ordering errors with OV-er compounds are not due to clausal word order but to ordering of other complex word forms.     

On phylogenetic tests of irreversible evolution

"Dollo's law" states that, following loss, a complex trait cannot reevolve in an identical manner. Although the law has previously fallen into disrepute, it has only recently been challenged with statistical phylogenetic methods. We employ simulation studies of an irreversible binary character to show that rejections of Dollo's law based on likelihood-ratio tests of transition rate constraints or on reconstructions of ancestral states are frequently incorrect. We identify two major causes of errors: incorrect assignment of root state frequencies, and neglect of the effect of the character state on rates of speciation and extinction. Our findings do not necessarily overturn the conclusions of phylogenetic studies claiming reversals, but we demonstrate devastating flaws in the methods that are the foundation of all such studies. Furthermore, we show that false rejections of Dollo's law can be reduced by the use of appropriate existing models and model selection procedures. More powerful tests of irreversibility require data beyond phylogenies and character states of extant taxa, and we highlight empirical work that incorporates additional information.     

Effect of tall-grass invasion on the flowering-related functional pattern of submediterranean hay-meadows

Several studies demonstrated that abandonment changes the functional composition of grasslands; nevertheless, little is known about the effects of grassland abandonment on the flowering-related functional pattern. We hypothesized that invasion by tall grasses affects this pattern. We counted the number of flowering shoots per species at five times during the growing season, in 80 plots placed in mown and in abandoned grasslands (central Apennines), and assessed the differences in the trait composition of flowering species between the two treatments. The selected traits were linked to resource acquisition and stress tolerance strategies. Our results indicated that abiotic environmental control is prevalent in determining the phenological pattern in both conditions and in accordance with the phenological “mid-domain hypothesis”. We demonstrated that when the dominant species is a tall grass with competitive behaviour, the magnitude of this phenomenon is amplified due to the abiotic changes yielded by the tall grass invasion. Indeed, in the central and late phases of the growing season (when invasive tall grasses are growing and blooming), abandoned grasslands were marked by a set of traits devoted to stress tolerance or underlying a long reproductive cycle or linked to competition for light.     

Chronic lymphocytic leukemia (CLL) cells genetically modified to express B7-1, ICAM-1, and LFA-3 confer APC capacity to T cells from CLL patients

In chronic lymphocytic leukemia (CLL), malignant B cells and nonmalignant T cells exhibit dysfunction. We previously demonstrated that infection of CLL cells with modified vaccinia Ankara (MVA) expressing the costimulatory molecules B7-1, ICAM-1, and LFA-3 (designated TRICOM) increased expression of these costimulatory molecules on the surface of CLL cells and thus augmented their antigen-presenting capability. Here, we evaluate the effect of MVA-TRICOM-modified CLL cells on T cells. Following incubation with irradiated MVA-TRICOM-modified CLL cells, allogeneic and autologous CD4⁺ and CD8⁺ T cells expressed significantly higher levels of B7-1, ICAM-1, and LFA-3. We show that this increase was the result of physical acquisition from the antigen-presenting cells (APCs), and that purified T cells that acquired costimulatory molecules from MVA-TRICOM-modified CLL cells were able to stimulate the proliferation of untreated T cells. These results demonstrate for the first time that T cells from CLL patients can acquire multiple costimulatory molecules from autologous CLL cells and can then act as APCs themselves. Given the immunodeficiencies characteristic of CLL, enhancing the antigen-presenting function of CLL cells and T cells simultaneously could be a distinct advantage in the effort to elicit antitumor immune responses. Electronic supplementary material  The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

Spontaneous alternation in marine crabs: Invasive versus native species

Organisms ranging from paramecia to humans tend to explore places that have been least recently explored, which is referred to as spontaneous alternation. Although organisms rely on different sources of information in alternating between places, the emergent behavioral pattern is likely advantageous during exploration and foraging. Under this rationale, continuous spontaneous alternation performance of the invasive green crab, Carcinus maenas was assessed and compared with the native blue crab, Callinectes sapidus in a plus-maze submerged in seawater. For the first time spontaneous alternation behavior was demonstrated in Crustacea (i.e., C. maenas) and significant interspecific differences in alternation performance were observed between the invasive versus the native species. Carcinus maneas exhibited a pronouncedly higher spontaneous alternation performance than C. sapidus. Carcinus maneas on average alternated at levels higher than chance, which was not the case for C. sapidus. These observations point to an additional behavioral mechanism that might result in the competitive success of green crabs over blue crabs in areas where they co-occur. Most of the subjects exhibited asymptotic alternation performance from the onset; there was no improvement in their performance over the course of the experimental session. This finding implies the innate nature of this behavioral policy.