Role of Toxin ζ and Starvation Responses in the Sensitivity to Antimicrobials

A fraction of otherwise antimicrobial-sensitive Bacillus subtilis cells, called persisters, are phenotypically tolerant of antimicrobial treatment. We report that, independently of B. subtilis'' growth phase, transient ζ toxin expression induces a dormant state and alters cellular responses so that cells are more sensitive to antimicrobials with different modes of action. This outcome is modulated by fine tuning (p)ppGpp and GTP levels: i) in the presence of low “dysregulated” (p)ppGpp levels (as in relA⁻ cells) hyper-tolerance to both toxin and antimicrobials was observed; ii) physiological or low (p)ppGpp levels (as in the wild-type, sasA⁻, sasB⁻ and relA⁻sasA⁻ context) show a normal toxin and antimicrobial tolerance; and iii) lower levels (in relA⁻sasB⁻) or absence of (p)ppGpp (in the relA⁻sasA⁻sasB⁻ context), in concert with elevated GTP levels, potentiate the efficacy of both toxin and antimicrobial action, rendering tolerance vulnerable to eradication.     

Complex N-glycans: the story of the “yellow brick road”

The synthesis of complex asparagine-linked glycans (N-glycans) involves a multi-step process that starts with a five mannose N-glycan structure: [Manα1-6(Manα1-3)Manα1-6][Manα1-3]-R where R?=?Manβ1-4GlcNAcβ1-4GlcNAcβ1-Asn-protein. N-acetylglucosaminyltransferase I (GlcNAc-TI) first catalyzes addition of GlcNAc in β1-2 linkage to the Manα1-3-R terminus of the five-mannose structure. Mannosidase II then removes two Man residues exposing the Manα1-6 terminus that serves as a substrate for GlcNAc-T II and addition of a second GlcNAcβ1-2 residue. The resulting structure is the complex N-glycan: GlcNAcβ1-2Manα1-6(GlcNAcβ1-2Manα1-3)-R. This structure is the precursor to a large assortment of branched complex N-glycans involving four more N-acetylglucosaminyltransferases. This short review describes the experiments (done in the early 1970s) that led to the discovery of GlcNAc-TI and II.     

Exploring the Gastrointestinal “Nemabiome”: Deep Amplicon Sequencing to Quantify the Species Composition of Parasitic Nematode Communities

Parasitic helminth infections have a considerable impact on global human health as well as animal welfare and production. Although co-infection with multiple parasite species within a host is common, there is a dearth of tools with which to study the composition of these complex parasite communities. Helminth species vary in their pathogenicity, epidemiology and drug sensitivity and the interactions that occur between co-infecting species and their hosts are poorly understood. We describe the first application of deep amplicon sequencing to study parasitic nematode communities as well as introduce the concept of the gastro-intestinal “nemabiome”. The approach is analogous to 16S rDNA deep sequencing used to explore microbial communities, but utilizes the nematode ITS-2 rDNA locus instead. Gastro-intestinal parasites of cattle were used to develop the concept, as this host has many well-defined gastro-intestinal nematode species that commonly occur as complex co-infections. Further, the availability of pure mono-parasite populations from experimentally infected cattle allowed us to prepare mock parasite communities to determine, and correct for, species representation biases in the sequence data. We demonstrate that, once these biases have been corrected, accurate relative quantitation of gastro-intestinal parasitic nematode communities in cattle fecal samples can be achieved. We have validated the accuracy of the method applied to field-samples by comparing the results of detailed morphological examination of L3 larvae populations with those of the sequencing assay. The results illustrate the insights that can be gained into the species composition of parasite communities, using grazing cattle in the mid-west USA as an example. However, both the technical approach and the concept of the ‘nemabiome’ have a wide range of potential applications in human and veterinary medicine. These include investigations of host-parasite and parasite-parasite interactions during co-infection, parasite epidemiology, parasite ecology and the response of parasite populations to both drug treatments and control programs.     

The Possible Role of Polyamines to the Recalcitrance of “Kalamata” Olive Leafy Cuttings to Root

The objective of this study was to investigate the role of endogenous polyamines (PAs) (the sum of free plus soluble conjugate plus insoluble bound) on rooting potential of leafy cuttings of an easy, that is,“Arbequina” and a difficult-to-root olive cultivar, that is, “Kalamata”. Subsamples of cuttings were taken for PAs analysis before planting in the mist system and during the early phases of rhizogenesis (EPR). “Arbequina” exhibited higher initial free and total PA content than “Kalamata”. Spermidine (Spd) was the predominant PA observed in both cultivars. A low content of free putrescine (Put) and Spd was found in both cultivars, whereas spermine (Spm) was occasionally detected. “Arbequina” as well as “Kalamata” exhibited the highest free Put and free Spd in summer and Put was the predominant PA among the free PAs. “Arbequina” exhibited the highest individual and total PAs in spring, followed by those in summer and autumn. In contrast, “Kalamata” had the maximum PAs in summer and the lowest in autumn. Changes in the endogenous content of individual and total PAs during the EPR were also observed. Treatment of “Kalamata” cuttings in autumn with both indole-3-butyric acid (IBA) and Put increased rooting compared to IBA alone. Among the PAs administered, Put was the most effective, whereas Spd and Spm failed to promote rooting. PAs, especially in their free form, seem to be involved in the rooting process of olive cuttings; Put application enhanced the rooting response of the difficult-to-root “Kalamata” olive cultivar.     

Response to Dangl: Complex harmonies of the host–pathogen interaction

In the accompanying Research Focus article that summarises our recent research article [1], Jeffery Dangl likens the interplay of the intracellular pathogen Salmonella enterica serovar Typhimurium with the host resistance protein Nramp1 to the response of a congregation to the teachings of a preacher. In principle, this is a fair interpretation of what we believe to be occurring on the molecular level between host and pathogen within the limited scope of our experiments. However, as with most biological systems, we believe that this analogy might be too simplistic. As Dangl points out, Nramp1 ‘might have a multitude of messages that it sends to different vacuolar inhabitants’. This cannot exclude the fact that the effects of Nramp1 on any single pathogen are pleiotropic and that we focused solely on one aspect of Nramp1 function in our studies, that is, divalent cation limitation. Therefore, we suggest that the interplay between innate host defences and pathogens is far more complex than simple call-and-response. Instead, we propose that it is more like a musical round, where one instrument makes the initial statement of the tune followed by the response of a second instrument, building over successive entrances to a complex orchestral whole. As a case in point, we wish to draw the reader's attention to the complex influences of both Nramp1 and S. enterica Typhimurium on the generation of toxic oxygen and nitrogen intermediates by the host during infection.     

Testing the Role of Climate Change in Species Decline: Is the Eastern Quoll a Victim of a Change in the Weather?

To conserve a declining species we first need to diagnose the causes of decline. This is one of the most challenging tasks faced by conservation practitioners. In this study, we used temporally explicit species distribution models (SDMs) to test whether shifting weather can explain the recent decline of a marsupial carnivore, the eastern quoll (Dasyurus viverrinus). We developed an SDM using weather variables matched to occurrence records of the eastern quoll over the last 60 years, and used the model to reconstruct variation through time in the distribution of climatically suitable range for the species. The weather model produced a meaningful prediction of the known distribution of the species. Abundance of quolls, indexed by transect counts, was positively related to the modelled area of suitable habitat between 1990 and 2004. In particular, a sharp decline in abundance from 2001 to 2003 coincided with a sustained period of unsuitable weather over much of the species’ distribution. Since 2004, abundance has not recovered despite a return to suitable weather conditions, and abundance and area of suitable habitat have been uncorrelated. We suggest that fluctuations in weather account for the species’ recent decline, but other unrelated factors have suppressed recovery.     

Responses of disparate phenotypically-plastic,melanin-based traits to common cues: limits to the benefits of adaptive plasticity?

The evolution of perfect adaptive phenotypic plasticity of a given trait may be influenced by, among other things, phenotypic costs associated with the expression of a given trait value, relative to alternative trait values. One potential cause of such phenotypic costs is the allocation of limited resources to multiple traits. When multiple traits rely on the same resource, trait values for one adaptively plastic trait might be unavoidably associated with maladaptive trait values for other traits. I address this problem in three traits of Pieris rapae L. (the small cabbage white butterfly) that all rely on the pigment melanin and are adaptively plastic, but have very different functions: wing pattern, immune defense, and pupal color. Cool, short-day rearing conditions simultaneously increased total wing melanization and decreased a melanin-based immune response in females, consistent with predictions. However, cool, short days also reduced the melanin-based immune response in males, despite little effect on male wing melanization. Furthermore, contrary to predictions, these patterns were not altered by differences in dietary resources. Finally, dark-colored rearing backgrounds during pupation substantially increased pupal melanization in both sexes, but was not associated with differences in wing melanization. These results offer only mixed support for the hypothesis of melanin-based trade offs as a source of phenotypic costs to adaptive plasticity in these traits. However, patterns of sexual dimorphism for these traits suggest trade offs might be at work at another level: relative to males, females have consistently more heavily melanized wings but less heavily melanized pupae and immune responses. The reduced immune response under cool, short-day conditions may also have implications for the evolutionary ecology of these butterflies.     

Prooxidant Action of Maltol: Role of Transition Metals in the Generation of Reactive Oxygen Species and Enhanced Formation of 8-hydroxy-2′-deoxyguanosine Formation in DNA

Maltol (3-hydroxy-2-methyl-4-pyrone) produced reactive oxygen species as a complex with transition metals. Maltol/iron complex inactivated aconitase the most sensitive enzyme to oxidative stress. The inactivation of aconitase was iron-dependent, and prevented by TEMPOL, a scavenger of reactive oxygen species, suggesting that the maltol/iron-mediated generation of superoxide anion is responsible for the inactivation of aconitase. Addition of maltol effectively enhanced the ascorbate/copper-mediated formation of 8-hydroxy-2′-deoxyguanosine in DNA. Oxidation of ascorbic acid by CuSO₄ was effectively stimulated by addition of maltol, and the enhanced oxidation rate was markedly inhibited by the addition of catalase and superoxide dismutase. These results suggest that maltol can stimulate the copper reduction coupled with the oxidation of ascorbate, resulting in the production of superoxide radical which in turn converts to hydrogen peroxide and hydroxyl radical. Cytotoxic effect of maltol can be explained by its prooxidant properties: maltol/transition metal complex generates reactive oxygen species causing the inactivation of aconitase and the production of hydroxyl radical causing the formation of DNA base adduct.     

A Review of the Copitarsia decolora (Guenée) (Lepidoptera: Noctuidae) Species Complex with the Description of a New Species from Chile and Argentina

Copitarsia gibberosa n. sp. is described from Chile and Argentina. Morphological characters are discussed to differentiate it from Copitarsia decolora (Guenée), Copitarsia incommoda (Walker), and Copitarsia corruda (Pogue & Simmons). Copitarsia corruda has its status revised based on CO1 and morphology. Copitarsia paraturbata Castillo & Angulo is a new synonym of C. incommoda based on morphology. Copitarsia uncilata Burgos & Leiva is a new synonym of C. decolora based on morphology. A review of recent literature revealed a misunderstanding of the complex of species related to C. decolora, and these papers are evaluated and species are identified. Host plant utilization is discussed between C. decolora and C. corruda. Adults and male and female genitalia are illustrated to differentiate between the species in the C. decolora species complex. Keys to male and females based on genitalic morphology are given. Distribution maps of collected specimens are provided.     

PTB Binds to the 3’ Untranslated Region of the Human Astrovirus Type 8: A Possible Role in Viral Replication

The 3′ untranslated region (3′UTR) of human astroviruses (HAstV) consists of two hairpin structures (helix I and II) joined by a linker harboring a conserved PTB/hnRNP1 binding site. The identification and characterization of cellular proteins that interact with the 3′UTR of HAstV-8 virus will help to uncover cellular requirements for viral functions. To this end, mobility shift assays and UV cross-linking were performed with uninfected and HAstV-8-infected cell extracts and HAstV-8 3′UTR probes. Two RNA-protein complexes (CI and CII) were recruited into the 3′UTR. Complex CII formation was compromised with cold homologous RNA, and seven proteins of 35, 40, 45, 50, 52, 57/60 and 75 kDa were cross-linked to the 3′UTR. Supermobility shift assays indicated that PTB/hnRNP1 is part of this complex, and 3′UTR-crosslinked PTB/hnRNP1 was immunoprecipitated from HAstV-8 infected cell-membrane extracts. Also, immunofluorescence analyses revealed that PTB/hnRNP1 is distributed in the nucleus and cytoplasm of uninfected cells, but it is mainly localized perinuclearly in the cytoplasm of HAstV-8 infected cells. Furthermore, the minimal 3′UTR sequences recognized by recombinant PTB are those conforming helix I, and an intact PTB/hnRNP1-binding site. Finally, small interfering RNA-mediated PTB/hnRNP1 silencing reduced synthesis viral genome and virus yield in CaCo2 cells, suggesting that PTB/hnRNP1 is required for HAstV replication. In conclusion, PTB/hnRNP1 binds to the 3′UTR HAstV-8 and is required or participates in viral replication.     

Crystallographic studies of the binding of ligands to the dicarboxylate site of Complex II,and the identity of the ligand in the “oxaloacetate-inhibited” state

Mitochondrial Complex II (succinate:ubiquinone oxidoreductase) is purified in a partially inactivated state, which can be activated by removal of tightly bound oxaloacetate (E.B. Kearney, et al., Biochem. Biophys. Res. Commun. 49 1115–1121). We crystallized Complex II in the presence of oxaloacetate or with the endogenous inhibitor bound. The structure showed a ligand essentially identical to the “malate-like intermediate” found in Shewanella Flavocytochrome c crystallized with fumarate (P. Taylor, et al., Nat. Struct. Biol. 6 1108–1112) Crystallization of Complex II in the presence of excess fumarate also gave the malate-like intermediate or a mixture of that and fumarate at the active site. In order to more conveniently monitor the occupation state of the dicarboxylate site, we are developing a library of UV/Vis spectral effects induced by binding different ligands to the site. Treatment with fumarate results in rapid development of the fumarate difference spectrum and then a very slow conversion into a species spectrally similar to the OAA-liganded complex. Complex II is known to be capable of oxidizing malate to the enol form of oxaloacetate (Y.O. Belikova, et al., Biochim. Biophys. Acta 936 1–9). The observations above suggest it may also be capable of interconverting fumarate and malate. It may be useful for understanding the mechanism and regulation of the enzyme to identify the malate-like intermediate and its pathway of formation from oxaloacetate or fumarate.     

The Uncertainty of “Fitness”: What Prevents Understanding of the Role of Genetic Exchange

The evolutionary development of highly organized species is attained through an increase in average survival of individuals, whereas the evolution of primitive species involves only an increase in fecundity (Zavadsky, 1968). However, in population genetics, survival (or ecological resistance) and fecundity are regarded as components of a single character, fitness. Employment of the notion of fitness, which lacks a strict definition, hinders understanding of the mechanism of progressive evolution as the process that enhances ecological resistance of organisms. The notion of fitness also hinders understanding the role of genetic exchange, since the primary advantage of genetic recombination and sexual reproduction apparently is producing of progeny with high ecological resistance rather than with high genetic diversity as such. Thus, the regular genetic exchange ensures restoration of the level of ecological resistance characteristic for the species, and on the macroevolutionary scale leads to the formation of new genomes and new species with high ecological resistance.     

Responses of cyanobacteria to herbivorous zooplankton across predator regimes: who mows the bloom?

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