Trampling, defoliation and physiological integration affect growth, morphological and mechanical properties of a root-suckering clonal tree

Background and Aims

Grazing is a complex process involving the simultaneous occurrence of both trampling and defoliation. Clonal plants are a common feature of heavily grazed ecosystems where large herbivores inflict the simultaneous pressures of trampling and defoliation on the vegetation. We test the hypothesis that physiological integration (resource sharing between interconnected ramets) may help plants to deal with the interactive effects of trampling and defoliation.

Methods

In a field study, small and large ramets of the root-suckering clonal tree Populus simonii were subjected to two levels of trampling and defoliation, while connected or disconnected to other ramets. Plant responses were quantified via survival, growth, morphological and stem mechanical traits.

Key Results

Disconnection and trampling increased mortality, especially in small ramets. Trampling increased stem length, basal diameter, fibrous root mass, stem stiffness and resistance to deflection in connected ramets, but decreased them in disconnected ones. Trampling decreased vertical height more in disconnected than in connected ramets, and reduced stem mass in disconnected ramets but not in connected ramets. Defoliation reduced basal diameter, leaf mass, stem mass and leaf area ratio, but did not interact with trampling or disconnection.

Conclusions

Although clonal integration did not influence defoliation response, it did alleviate the effects of trampling. We suggest that by facilitating resource transport between ramets, clonal integration compensates for trampling-induced damage to fine roots.     

Mutation of the arginine finger in the active site of Escherichia coli DbpA abolishes ATPase and helicase activity and confers a dominant slow growth phenotype

Escherichia coli DEAD-box protein A (DbpA) is an ATP-dependent RNA helicase with specificity for 23S ribosomal RNA. Although DbpA has been extensively characterized biochemically, its biological function remains unknown. Previous work has shown that a DbpA deletion strain is viable with little or no effect on growth rate. In attempt to elucidate a phenotype for DbpA, point mutations were made at eleven conserved residues in the ATPase active site, which have exhibited dominant-negative phenotypes in other DExD/H proteins. Biochemical analysis of these DbpA mutants shows the expected decrease in RNA-dependent ATPase activity and helix unwinding activity. Only the least biochemically active mutation, R331A, produces small colony phenotype and a reduced growth rate. This dominant slow growth mutant will be valuable to determine the cellular function of DbpA.     

Bacteriophages ofBacillus subtilis: Comparison of different isolation techniques and possible use for classification ofBacillus subtilis strains

When different techniques are used for the isolation of bacteriophages ofBacillus subtilis a number of different phages may be obtained. Furthermore defective phages are found in old cultures of all strains ofB. subtilis tested so far. The possible use of the phages and the defective phages for classifyingB. subtilis strains into a number of groups according to their susceptibility to different phages and according to the presence of certain defective phages in the cells is discussed.     

Piperazine-based CCR5 antagonists as HIV-1 inhibitors. I: 2(S)-methyl piperazine as a key pharmacophore element

Optimization of the piperidino-piperazines 1 and 2 provided early leads 3 and 4, which showed good activity in the CCR5-RANTES binding assay and in antiviral assays. A systematic study around these structures showed that the 2(S)-methyl piperazine is essential for CCR5 affinity, which is further enhanced by forming the 2,6-dimethyl benzamide of the piperidine.     

From microbial gene essentiality to novel antimicrobial drug targets

Background

Bacterial respiratory tract infections, mainly caused by Streptococcus pneumoniae, Haemophilus influenzae and Moraxella catarrhalis are among the leading causes of global mortality and morbidity. Increased resistance of these pathogens to existing antibiotics necessitates the search for novel targets to develop potent antimicrobials.

Result

Here, we report a proof of concept study for the reliable identification of potential drug targets in these human respiratory pathogens by combining high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics. Approximately 20% of all genes in these three species were essential for growth and viability, including 128 essential and conserved genes, part of 47 metabolic pathways. By comparing these essential genes to the human genome, and a database of genes from commensal human gut microbiota, we identified and excluded potential drug targets in respiratory tract pathogens that will have off-target effects in the host, or disrupt the natural host microbiota. We propose 249 potential drug targets, 67 of which are targets for 75 FDA-approved antimicrobials and 35 other researched small molecule inhibitors. Two out of four selected novel targets were experimentally validated, proofing the concept.

Conclusion

Here we have pioneered an attempt in systematically combining the power of high-density transposon mutagenesis, high-throughput sequencing, and integrative genomics to discover potential drug targets at genome-scale. By circumventing the time-consuming and expensive laboratory screens traditionally used to select potential drug targets, our approach provides an attractive alternative that could accelerate the much needed discovery of novel antimicrobials.

Electronic supplementary material

The online version of this article (doi:10.1186/1471-2164-15-958) contains supplementary material, which is available to authorized users.     

Genetic and environmental influences on migraine: a twin study across six countries.

Migraine is a common neurovascular brain disorder that is manifested in recurrent episodes of disabling headache. The aim of the present study was to compare the prevalence and heritability of migraine across six of the countries that participate in GenomEUtwin project including a total number of 29,717 twin pairs. Migraine was assessed by questionnaires that differed between most countries. It was most prevalent in Danish and Dutch females (32% and 34%, respectively), whereas the lowest prevalence was found in the younger and older Finnish cohorts (13% and 10%, respectively). The estimated genetic variance (heritability) was significant and the same between sexes in all countries. Heritability ranged from 34% to 57%, with lowest estimates in Australia, and highest estimates in the older cohort of Finland, the Netherlands, and Denmark. There was some indication that part of the genetic variance was non-additive, but this was significant in Sweden only. In addition to genetic factors, environmental effects that are non-shared between members of a twin pair contributed to the liability of migraine. After migraine definitions are homogenized among the participating countries, the GenomEUtwin project will provide a powerful resource to identify the genes involved in migraine.     

Characterization of the yeast BMH1 gene encoding a putative protein homologous to mammalian protein kinase II activators and protein kinase C inhibitors.

We describe the identification and characterization of the BMH1 gene from the yeast Saccharomyces cerevisiae. The gene encodes a putative protein of 292 amino acids which is more than 50% identical with the bovine brain 14-3-3 protein and proteins isolated from sheep brain which are strong inhibitors of protein kinase C. Disruption mutants and strains with the BMH1 gene on multicopy plasmids have impaired growth on minimal medium with glucose as carbon source, i.e. a 30-50% increase in generation time. These observations suggest a regulatory function of the bmh1 protein. In contrast to strains with an intact or a disrupted BMH1 gene, strains with the BMH1 gene on multicopy plasmids hardly grew on media with acetate or glycerol as carbon source.     

Identification of enantioselective extractants for chiral separation of amines and aminoalcohols

The major obstacle for the introduction of fractional reactive extraction as a chiral separation method in the chemical and pharmaceutical industries is the lack of versatile enantioselective extractants. Therefore, a rational approach is developed to transfer the extensive knowledge of chiral selectors reported in the literature on chiral recognition and other chiral separation techniques to extraction. Based on a similarity in separation mechanisms, it was expected that chiral selectors originating from a technique in which chiral recognition takes place in the liquid phase are most likely to function as enantioselective extractant. Using this approach, a selection of promising extractants was made from the literature and experimentally evaluated for the enantioseparation of aminoalcohols and amines. As a result, four enantioselective extractant systems, namely, dibutyl-L-tartrate with boric acid, N-(2-hydroxydodecyl)-L-hydroxyproline Cu(II) complex, N-dodecyl-L-hydroxyproline Cu(II) complex, and azophenolic crown ether, have been identified. The azophenolic crown ether system performed the best and demonstrated an enantioselectivity between 1.3-5.0 for five out of six test compounds. Identification of the enantioselective extractant systems was highly facilitated by the developed rational transfer approach that, although partially qualitative, appeared capable of reducing more than 50 encountered candidates to only three promising systems for further experimental evaluation. Therefore, it is expected that this approach can be successfully applied to identify enantioselective extractants for other classes of enantiomers as well.     

Carbonic Anhydrase Is Essential for Streptococcus pneumoniae Growth in Environmental Ambient Air

The respiratory tract pathogen Streptococcus pneumoniae needs to adapt to the different levels of carbon dioxide (CO₂) it encounters during transmission, colonization, and infection. Since CO₂ is important for various cellular processes, factors that allow optimal CO₂ sequestering are likely to be important for pneumococcal growth and survival. In this study, we showed that the putative pneumococcal carbonic anhydrase (PCA) is essential for in vitro growth of S. pneumoniae under the CO₂-poor conditions found in environmental ambient air. Enzymatic analysis showed that PCA catalyzes the reversible hydration of CO₂ to bicarbonate (HCO₃⁻), an essential step to prevent the cellular release of CO₂. The addition of unsaturated fatty acids (UFAs) reversed the CO₂-dependent in vitro growth inhibition of S. pneumoniae strains lacking the pca gene (Δpca), indicating that PCA-mediated CO₂ fixation is at least associated with HCO₃⁻-dependent de novo biosynthesis of UFAs. Besides being necessary for growth in environmental ambient conditions, PCA-mediated CO₂ fixation pathways appear to be required for intracellular survival in host cells. This effect was especially pronounced during invasion of human brain microvascular endothelial cells (HBMEC) and uptake by murine J774 macrophage cells but not during interaction of S. pneumoniae with Detroit 562 pharyngeal epithelial cells. Finally, the highly conserved pca gene was found to be invariably present in both CO₂-independent and naturally circulating CO₂-dependent strains, suggesting a conserved essential role for PCA and PCA-mediated CO₂ fixation pathways for pneumococcal growth and survival.The Gram-positive bacterium Streptococcus pneumoniae, or pneumococcus, is a human respiratory tract pathogen that contributes significantly to global mortality and morbidity. In addition, it is an important asymptomatic colonizer of the human nasopharynx, with carriage rates around 10% in adults and over 40% in children (6). Pneumococcal colonization and infection are closely linked, but knowledge of the factors that contribute to transmission, carriage, disease, and transition from carriage to disease is still limited. Research on components that physically contribute to host-pathogen interactions, such as capsular polysaccharides, adhesins, and toxins, has provided valuable insights into the process of pneumococcal pathogenesis (20). In contrast, the influence of environmental factors on pneumococcal growth and survival remains fairly unexplored.S. pneumoniae needs to adapt to various aerobic and anaerobic conditions, reflecting the different niches it occupies during transmission, colonization, and invasive disease. During niche transition, oxygen (O₂) levels change considerably. Levels of O₂ are 21% in ambient air, decrease to 10 to 15% in the alveoli of the lungs, and are about 5% in resting cells. In O₂-rich conditions, S. pneumoniae expresses pyruvate oxidase (SpxB), which generates acetyl-phosphate as a source of ATP and hydrogen peroxide (H₂O₂) for interspecies competition at the mucosal surfaces of the nasopharynx (41). The presence of O₂ is also a prerequisite for the pneumococcal X state (4, 14), which is a physiological condition that allows for genetic transformation and an adequate response to environmental stress (32). Recently, it was shown that the fatty acid (FA) content of the pneumococcal cell membrane (31) and the expression of 69 genes (8) change in response to the availability of O₂. Finally, changes in O₂ levels can also affect production of the polysaccharide capsule (48), which is the major pneumococcal virulence determinant.Similar to those of O₂, the levels of carbon dioxide (CO₂) vary considerably among the different pneumococcal niches inside and outside the host. Ambient levels of CO₂ in the environment are 0.038%, while CO₂ levels inside the human body, in particular in the lower respiratory tract, can reach 5% or more. The importance of this gaseous compound for S. pneumoniae is illustrated by the observation that the depletion of CO₂ from ambient air completely inhibits pneumococcal growth (21). Moreover, about 8% of all clinical isolates require a CO₂-enriched environment for growth in laboratory conditions (3). This intrinsic CO₂ dependence of S. pneumoniae and many other (micro)organisms is most likely related to an anabolic need for CO₂ or bicarbonate (HCO_3⁻) during biosynthesis of nucleic acids, amino acids, and FAs (1). Pathogens can often sequester CO₂ directly from host tissues, but in the absence of sufficient levels of extracellular CO₂, endogenous CO₂ needs to be enzymatically fixated. Carbonic anhydrases (CAs; EC 4.2.1.1) are enzymes that catalyze the reversible reaction CO₂ + H₂O ↔ HCO₃⁻ + H⁺. Because HCO₃⁻ cannot passively diffuse across biological membranes, its formation significantly delays the release of intracellular CO₂. At least five different classes of CAs have been described, and most eukaryotic, prokaryotic, and archaeal species express at least one CA class (39, 40).Genome analysis (39) has revealed that S. pneumoniae has one putative CA, a β-class CA that is highly conserved in all available pneumococcal genome sequences. Pneumococcal CA (PCA) is highly homologous to CAs in other streptococcal species, such as Streptococcus pyogenes. The closest nonstreptococcal PCA homologs are found in Mycobacterium species, while PCA homologs in other respiratory tract pathogens such as Neisseria meningitidis and Haemophilus influenzae are more divergent (40). The aim of this study was to investigate the functional characteristics of the pca gene and the encoded PCA enzyme in S. pneumoniae and to establish the relevance of PCA for pneumococcal growth and survival under CO₂-poor conditions in vitro. Further, we examined the importance of PCA during host-pathogen interaction.