Search for genes essential for pneumococcal transformation: the RADA DNA repair protein plays a role in genomic recombination of donor DNA

We applied a novel negative selection strategy called genomic array footprinting (GAF) to identify genes required for genetic transformation of the gram-positive bacterium Streptococcus pneumoniae. Genome-wide mariner transposon mutant libraries in S. pneumoniae strain R6 were challenged by transformation with an antibiotic resistance cassette and growth in the presence of the corresponding antibiotic. The GAF screen identified the enrichment of mutants in two genes, i.e., hexA and hexB, and the counterselection of mutants in 21 different genes during the challenge. Eight of the counterselected genes were known to be essential for pneumococcal transformation. Four other genes, i.e., radA, comGF, parB, and spr2011, have previously been linked to the competence regulon, and one, spr2014, was located adjacent to the essential competence gene comFA. Directed mutants of seven of the eight remaining genes, i.e., spr0459-spr0460, spr0777, spr0838, spr1259-spr1260, and spr1357, resulted in reduced, albeit modest, transformation rates. No connection to pneumococcal transformation could be made for the eighth gene, which encodes the response regulator RR03. We further demonstrated that the gene encoding the putative DNA repair protein RadA is required for efficient transformation with chromosomal markers, whereas transformation with replicating plasmid DNA was not significantly affected. The radA mutant also displayed an increased sensitivity to treatment with the DNA-damaging agent methyl methanesulfonate. Hence, RadA is considered to have a role in recombination of donor DNA and in DNA damage repair in S. pneumoniae.     

Unstable dynamics and population limitation in mountain hares

The regular large-scale population fluctuations that characterize many species of northern vertebrates have fascinated ecologists since the time of Charles Elton. There is still, however, no clear consensus on what drives these fluctuations. Throughout their circumpolar distribution, mountain hares Lepus timidus show regular and at times dramatic changes in density. There are distinct differences in the nature, amplitude and periodicity of these fluctuations between regions and the reasons for these population fluctuations and the geographic differences remain largely unknown. In this review we synthesize knowledge on the factors that limit or regulate mountain hare populations across their range in an attempt to identify the drivers of unstable dynamics. Current knowledge of mountain hare population dynamics indicates that trophic interactions--either predator-prey or host-parasite--appear to be the major factor limiting populations and these interactions may contribute to the observed unstable dynamics. There is correlative and experimental evidence that some mountain hare populations in Fennoscandia are limited by predation and that predation may link hare and grouse cycles to microtine cycles. Predation is unlikely to be important in mountain hare populations in Scotland as most hares occur on sporting estates where predators are controlled, but this hypothesis remains to be experimentally tested. There is, however, emerging experimental evidence that some Scottish mountain hare populations are limited by parasites and that host-parasite interactions contribute to unstable dynamics. By contrast, there is little evidence from Fennoscandia that parasitism is of any importance to mountain hare population dynamics, although disease may cause periodic declines. Although severe weather and food limitation may interact to cause periodic high winter mortality there is little evidence that food availability limits mountain hare populations. There is a paucity of information concerning the factors limiting or regulating mountain hare populations in the Alps of Central Europe or in the tundra and taiga belts of Russia. Future research on mountain hare population dynamics should focus on the interactions between predation, parasitism and nutrition with stochastic factors such as climate and anthropogenic management including harvesting.     

Retinoic acid signaling is essential for pancreas development and promotes endocrine at the expense of exocrine cell differentiation in Xenopus

How and when the vertebrate endoderm is first subdivided into discrete progenitor cell populations that will give rise to the different major organs, including pancreas and liver, are only poorly understood. We have used Xenopus laevis as a model system to characterize these events, since it is particularly suited to study the early embryonic patterning in vertebrates. Our experimental results support the notion that retinoic acid (RA) functions as an essential endodermal patterning signal in Xenopus and that it acts as early as during gastrulation. As a result of RA treatment, the expression of Sonic Hedgehog (Shh), a known inhibitor of pancreas development in other vertebrate systems, is negatively regulated in the dorsal prepancreatic endoderm. Furthermore, RA is found to promote endocrine at the expense of exocrine differentiation in the dorsal pancreas, correlating with a specific inhibition of Notch signaling activities in this territory. Conversely, RA enhances exocrine marker gene expression in the ventral pancreas.     

Mutation Analysis of Violaxanthin De-epoxidase Identifies Substrate-binding Sites and Residues Involved in Catalysis

Plants are able to deal with variable environmental conditions; when exposed to strong illumination, they safely dissipate excess energy as heat and increase their capacity for scavenging reacting oxygen species. Both these protection mechanisms involve activation of the xanthophyll cycle, in which the carotenoid violaxanthin is converted to zeaxanthin by violaxanthin de-epoxidase, using ascorbate as the source of reducing power. In this work, following determination of the three-dimensional structure of the violaxanthin de-epoxidase catalytic domain, we identified the putative binding sites for violaxanthin and ascorbate by in silico docking. Amino acid residues lying in close contact with the two substrates were analyzed for their involvement in the catalytic mechanism. Experimental results supported the proposed substrate-binding sites and point to two residues, Asp-177 and Tyr-198, which are suggested to participate in the catalytic mechanism, based on complete loss of activity in mutant proteins. The role of other residues and the mechanistic similarity to aspartic proteases and epoxide hydrolases are discussed.     

An experiment of fish spillover from a marine reserve in Cuba

Several studies on adult fish movement from marine protected areas to zones open to fishing activity conclude spillover is present, but most of these investigations use indirect evidence and small-sized species of little commercial importance. This paper reports the effects of manipulating a density gradient on movements of large-sized and commercially-important fish across “Jardines de la Reina” Marine Reserve boundaries, using tagging methods and visual census. Tagging was carried out using dart tags and modified spearguns at an experimental and a control site. Density of fish was experimentally manipulated on the unprotected side of the boundary. Before experimental manipulation, fish density was similar in both experimental and control sites and on both sides of the boundaries. After manipulation, fish density in the unprotected side of experimental site declined dramatically and a strong gradient was established through the boundary. One month later, this forced gradient disappeared, returning to the situation at the beginning of the study. This last result is due to spillover effect: the mean distance traveled by fish increased 1.5 times (mean from below 200 m to more than 300 m), the mean emigration rate doubled and the immigration rate decreased, allowing density levels to recover after manipulation.     

Characterization of the satellite DNA Msat-160 from species of Terricola (Microtus) and Arvicola (Rodentia, Arvicolinae)

In the subfamily Arvicolinae (Cricetidae, Rodentia) the satellite DNA Msat-160 has been so far described in only some species from the genus Microtus and in one species from another genus, Chionomys nivalis. Here we cloned and characterized this satellite in two new arvicoline species, Microtus (Terricola) savii and Arvicola amphibius (terrestris). We have also demonstrated, by PCR and FISH, its existence in the genomes of several other species from both genera. These results suggest that Msat-160 already occurred in the common ancestor of the four genera/subgenera of Arvicolinae (Microtus, Chionomys, Arvicola, and Terricola). In Arvicola and Terricola, Msat-160 showed the basic monomer length of 160 bp, although a higher-order repeat (HORs) of 640 bp could have been probably replacing the original monomeric unit in A. a. terrestris. Msat-160 was localized by FISH mostly on the pericentromeric regions of the chromosomes, but the signal intensity and the number of carrier chromosomes varied extremely even between closely related species, resulting in a species-specific pattern of chromosomal distribution of this satellite. Such a variable pattern most likely is a consequence of a rapid amplification and contraction of particular repeats in the pericentromeric regions of chromosomes. In addition, we proposed that the rapid variation of pericentromeric repeats is strictly related to the prolific species radiation and diversification of karyotypes that characterize Arvicolinae lineage. Finally, we performed phylogenetic analysis in this group of related species based on Msat-160 that results to be in agreement with previously reported phylogenies, derived from other molecular markers.     

Axial and Radial Oxylipin Transport

Jasmonates are oxygenated lipids (oxylipins) that control defense gene expression in response to cell damage in plants. How mobile are these potent mediators within tissues? Exploiting a series of 13-lipoxygenase (13-lox) mutants in Arabidopsis (Arabidopsis thaliana) that displays impaired jasmonic acid (JA) synthesis in specific cell types and using JA-inducible reporters, we mapped the extent of the transport of endogenous jasmonates across the plant vegetative growth phase. In seedlings, we found that jasmonate (or JA precursors) could translocate axially from wounded shoots to unwounded roots in a LOX2-dependent manner. Grafting experiments with the wild type and JA-deficient mutants confirmed shoot-to-root oxylipin transport. Next, we used rosettes to investigate radial cell-to-cell transport of jasmonates. After finding that the LOX6 protein localized to xylem contact cells was not wound inducible, we used the lox234 triple mutant to genetically isolate LOX6 as the only JA precursor-producing LOX in the plant. When a leaf of this mutant was wounded, the JA reporter gene was expressed in distal leaves. Leaf sectioning showed that JA reporter expression extended from contact cells throughout the vascular bundle and into extravascular cells, revealing a radial movement of jasmonates. Our results add a crucial element to a growing picture of how the distal wound response is regulated in rosettes, showing that both axial (shoot-to-root) and radial (cell-to-cell) transport of oxylipins plays a major role in the wound response. The strategies developed herein provide unique tools with which to identify intercellular jasmonate transport routes.Both animals and plants produce potently active lipid-derived mediators in response to wounding. These oxylipins (oxygenated lipid derivatives) include leukotrienes and prostaglandins in animals (Funk, 2001) and jasmonates in plants (Wasternack and Hause, 2013). Although these regulators frequently show structural similarities (many are cyclopentenone and cyclopentanone lipids), they operate through different signaling pathways often involving large protein complexes. For example, prostaglandins signal in part through G protein-coupled receptor complexes (Furuyashiki and Narumiya, 2011; Kalinski, 2012), and plant jasmonate signaling operates through the Skp/Cullin/F-box CORONATINE INSENSITIVE1 complex (Browse, 2009). Many oxylipins produced in response to tissue damage in metazoans act as paracrine signals to elicit defense responses in distal undamaged cells (Funk, 2001). Similarly, it is possible that jasmonates, including the biologically active derivative jasmonoyl-Ile (JA-Ile; Fonseca et al., 2009), might be transported from cell to cell in plants. However, to date, the majority of studies on oxylipin transport in plants have used exogenous jasmonates, and it remains unclear to what extent these compounds are transported between cells and tissues when produced endogenously.Based on the fact that jasmonic acid (JA) or methyl jasmonate treatments can affect defense gene expression at a distance to the sites of their application, JA was proposed to operate as a paracrine signal capable of being transported from cell to cell in tomato (Solanum lycopersicum) leaves (Farmer et al., 1992). Similar conclusions were drawn for JA in wild tobacco (Nicotiana sylvestris; Zhang and Baldwin, 1997). Isotope-labeling experiments using exogenous jasmonates have indicated JA/JA-Ile transport away from the site of application to distal tissues and even distal organs (Zhang and Baldwin, 1997; Thorpe et al., 2007; Sato et al., 2011). Additionally, grafting experiments in tomato were consistent with long-distance transport of JA/JA precursors (Li et al., 2002; Schilmiller and Howe, 2005), although other studies did not find evidence for JA transport from wounded leaves to distal unwounded leaves (Strassner et al., 2002). Concerning Arabidopsis (Arabidopsis thaliana), Koo et al. (2009) concluded that JA-Ile accumulation detected in leaves distal to the wound site resulted mainly from de novo synthesis in undamaged leaves rather than from the transport of JA/JA-Ile from the wound site. Recently, a transporter (GLUCOSINOLATE TRANSPORTER1) capable of importing JA-Ile (but not JA) into Xenopus laevis oocytes has been described (Saito et al., 2015), further supporting the possibility that jasmonates move between cells.In addition to the transport of jasmonates, there is much evidence consistent with other wound signaling mechanisms that lead to JA synthesis and JA-mediated defense responses at various distances from wounds. That is, wound-activated signaling pathways can be classified as those working near the damage site (i.e. local responses) and those operating distal to it (Rhodes et al., 2006; Wu et al., 2007). Both these types of wound responses can be difficult to study, because several types of events (including the transport of jasmonates) may contribute to JA signaling. However, there has been some progress in understanding long-distance signaling leading to distal wound responses. These mechanisms include electrical and potentially, hydraulic signaling (for review, see Koo and Howe, 2009; Farmer et al., 2014). Membrane hyperpolarizations have been recorded in wounded plants (Zimmermann et al., 2009); however, their relationship with JA synthesis or JA responses has not yet been reported. In Arabidopsis, wounding of adult-phase rosettes stimulates the leaf-to-leaf propagation of signals that (1) can be detected with surface electrodes as cell membrane depolarizations; (2) are propagated from leaf to leaf in a mechanism that requires several clade 3 GLUTAMATE RECEPTOR-LIKE (GLR) genes, including GLR3.3 and GLR3.6; and (3) can induce JA and JA-Ile accumulation in distal unwounded sites (Mousavi et al., 2013). However, even when electrical signals were compromised in both the wounded and distal leaves of a glr3.3 glr3.6 double mutant, JA responses were affected only in the distal leaf; local responses in the damaged leaf itself were similar to the wild type (Mousavi et al., 2013). Therefore, certain clade 3 GLRs operate in rosette-stage plants to extend the range of the wound response, and even if these genes are mutated, wounded rosette leaves still produce jasmonates. In summary, both jasmonates made near wounds and jasmonates made far from wounds in response to distal signals might be subject to transport within the plant.This study focused on the mobility of endogenous jasmonates produced in response to wounding. Here, we ask: how mobile are endogenous jasmonates generated in aboveground tissues in response to wounding? Our analysis was conducted throughout the vegetative phase and included different tissues that ranged from embryonic leaves (cotyledons) and roots to expanded rosette leaves. We investigated whether a glr3.3 glr3.6 double mutant that reduces leaf-to-leaf signal propagation in the adult phase (Mousavi et al., 2013) could also reduce cotyledon-to-root wound signaling in seedlings. Results from these electrophysiology experiments then led us to investigate whether JA (or JA precursors) can translocate from wounded cotyledons to roots. To do this, we used two approaches. One was based on mutants in 13-LIPOXYGENASEs (13-LOXs) that are necessary for an early step in the synthesis of the JA precursor oxophytodienoic acid. All four 13-LOXs in Arabidopsis (LOX2, LOX3, LOX4, and LOX6) are known to contribute to JA synthesis in vivo (Chauvin et al., 2013). First, LOX2 is responsible for the synthesis of a large pool of JA in wounded leaves (Bell et al., 1995), and it also produces precursors for the synthesis of arabidopside defense-related metabolites (Glauser et al., 2009). Second, LOX3 and LOX4 act together to produce the JA required for full male fertility (Caldelari et al., 2011). Third, LOX6 produces jasmonates in roots that are first separated from aerial tissues and then wounded (Grebner et al., 2013). We tested the impact of mutations in the different 13-LOXs on root JA signaling after cotyledon wounding. This was followed by grafting experiments between the wild type and the JA-deficient mutant allene oxide synthase (aos; Park et al., 2002) to test whether JA (or JA precursors) could translocate axially from wounded shoots into undamaged roots.In addition to its role in wounded roots (Grebner et al., 2013), LOX6 has been implicated in long-distance wound signaling in the adult-phase rosette, where it is necessary for most of the rapid distal expression of the JA-responsive gene JASMONATE ZIM-DOMAIN10 (JAZ10) when another leaf is wounded (Chauvin et al., 2013). This and the fact that the LOX6 promoter is active principally in xylem contact cells (Chauvin et al., 2013) provided us with the opportunity to investigate oxylipin transport within leaves. We confirmed the cellular localization of the LOX6 polypeptide with a LOX6-GUS fusion protein. We then used a lox234 triple mutant expressing a JAZ10 reporter to test whether jasmonates could be exported from xylem contact cells. These experiments led to unique insights into the transport of jasmonates across different leaf cell layers.