tyrB-2 and phhC genes of Pseudomonas putida encode aromatic amino acid aminotransferase isozymes: evidence at the protein level

Two Pseudomonas putida aminotransferases (ArAT I and ArAT II) that exhibit activity toward l-tryptophan were purified 104- and 395-fold using a six-stage purification procedure involving ammonium sulfate fractionation and chromatographic separation on phenyl-Sepharose, Sephadex G-100 superfine, DEAE-cellulose and Protein-Pack Q8 HR columns. Mass spectrometry analysis resulted in the identification of 27 and 20 % of the total ArAT I and ArAT II amino acid sequences. In addition, N-terminal sequence fragments of ArAT I and ArAT II were determined using the Edman degradation method. Based on the analyses performed, the studied proteins were identified as products of the tyrB-2 and phhC genes, and the presence of these genes in the investigated bacterial strain was confirmed using molecular biology methods. Extensive analysis of the substrate specificities of ArAT I and ArAT II revealed that both enzymes most efficiently catalyzed reactions involving aromatic amino acids and 2-oxoacids followed by dicarboxylic compounds. The best substrates for ArAT I and ArAT II were l-phenylalanine and phenylpyruvate. Based on these results, the studied proteins were classified as aromatic amino acid aminotransferase isozymes.     

The rise of bilaterians: a few closing comments

Freeman (2009, 2010) argue that Ediacara “faunas” should be analysed using “promorphologies” of extant animals, that rangeomorphs and erniettomorphs are colonial animals, and that Vernanimalcula is a bilaterian animal. Recent studies of Ediacara fossils have provided multiple lines of evidence that these fossils represent a sample of phylogenetically diverse marine organisms that included crown-group animals as well as stem-group animals and non-animals. Thus, it is inappropriate to analyse Ediacara fossils using “promorphologies” of extant animals. The interpretation of rangeomorphs and erniettomorphs as colonial animals is inconsistent with the functional morphologies of these Ediacara fossils. Although Vernanimalcula is a fossil, the purported germ layers of Vernanimalcula are of diagenetic origin and there is no morphological evidence in support of its bilaterian affinity.     

The Effect of Fatal Carbon Monoxide Poisoning on the Surface Charge of Blood Cells

The objective of this investigation was to evaluate postmortem changes of electric charge of human erythrocytes and thrombocytes after fatal carbon monoxide (CO) poisoning. The surface charge density values were determined on the basis of the electrophoretic mobility measurements of the cells carried out at various pH values of electrolyte solution. The surface charge of erythrocyte membranes after fatal CO poisoning as well as after sudden unexpected death increased compared to the control group in the whole range of experimental pH values. Also, a slight shift of the isoelectric point of erythrocyte membranes to high pH values was observed. The surface charge of thrombocyte membranes after fatal CO poisoning decreased at low pH compared to the control group. However, at high pH, the values increased compared to the control group. The isoelectric point of thrombocyte membranes after fatal CO poisoning was considerably shifted toward low pH values compared to the control group. The observed changes are probably connected with the destruction of blood cell structure.     

Phosphorylation of HopQ1, a Type III Effector from Pseudomonas syringae,Creates a Binding Site for Host 14-3-3 Proteins

HopQ1 (for Hrp outer protein Q), a type III effector secreted by Pseudomonas syringae pv phaseolicola, is widely conserved among diverse genera of plant bacteria. It promotes the development of halo blight in common bean (Phaseolus vulgaris). However, when this same effector is injected into Nicotiana benthamiana cells, it is recognized by the immune system and prevents infection. Although the ability to synthesize HopQ1 determines host specificity, the role it plays inside plant cells remains unexplored. Following transient expression in planta, HopQ1 was shown to copurify with host 14-3-3 proteins. The physical interaction between HopQ1 and 14-3-3a was confirmed in planta using the fluorescence resonance energy transfer-fluorescence lifetime imaging microscopy technique. Moreover, mass spectrometric analyses detected specific phosphorylation of the canonical 14-3-3 binding site (RSXpSXP, where pS denotes phosphoserine) located in the amino-terminal region of HopQ1. Amino acid substitution within this motif abrogated the association and led to altered subcellular localization of HopQ1. In addition, the mutated HopQ1 protein showed reduced stability in planta. These data suggest that the association between host 14-3-3 proteins and HopQ1 is important for modulating the properties of this bacterial effector.A multicomponent defense response is initiated when plant pattern recognition receptors perceive microbially derived structural components (Nürnberger and Brunner, 2002), which are referred to as pathogen-associated molecular patterns. Many bacterial pathogens have developed type III secretion system (TTSS) effectors that can suppress or modulate pathogen-associated molecular pattern-triggered immunity (Jones and Dangl, 2006). Effector-triggered immunity represents a second layer of defense, whereby plants have evolved mechanisms that rely upon Resistance (R) proteins to sense and respond to cognate TTSS effectors. Thus, the expression of a specific bacterial effector can either sustain disease in susceptible plants or render the pathogen avirulent in resistant plants that express the corresponding R protein. Several lines of evidence suggest an involvement of scaffold proteins from the 14-3-3 family in mediating these defense responses at various levels (Yang et al., 2009; Oh et al., 2010). Some R proteins have been shown to bind 14-3-3 proteins directly. RPW2.8, which confers resistance to fungal pathogens of Golovinomyces spp., associates specifically with the 14-3-3 isoform λ (designated GF14λ) from Arabidopsis (Arabidopsis thaliana; Yang et al., 2009). Moreover, both types of resistance were compromised in Arabidopsis lacking the λ isoform. Consistently, ectopic overexpression of GF14λ in transgenic Arabidopsis results in enhanced resistance to powdery mildew (Golovinomyces cichoracearum; Yang et al., 2009). Tobacco (Nicotiana tabacum) N protein, which mediates resistance to Tobacco mosaic virus, also binds 14-3-3 protein (Ueda et al., 2006). The viral p50 replicase helicase domain is the cognate ligand for N protein. Since this domain also interacts with 14-3-3s, it is possible that 14-3-3s might function in the formation of the receptor-ligand recognition complex (Ueda et al., 2006). In addition, the tomato (Solanum lycopersicum) 14-3-3 protein TF7 has been shown to exhibit positive regulation on the mitogen-activated protein kinase cascade, which is activated rapidly by pathogen recognition (Oh et al., 2010; Oh and Martin, 2011).There is increasing evidence that many intracellular pathways are regulated by the modulation of scaffold protein properties rather than the activities of integral components in the signaling cascades (Good et al., 2011). This strategy enables signal transduction to be turned on or off rapidly via the assembly or disassembly of complexes. This same mechanism also allows the intensity and kinetics of a response to be fine-tuned to the stimulus (Good et al., 2011). It was recently suggested that the manipulation of scaffolding may be one strategy employed by pathogens to interfere with the host defense response. The best-characterized example of scaffolding manipulation is the phytotoxin fusicoccin, which is secreted by the fungus Fusicoccum amygdali. Fusicoccin targets a 14-3-3 protein that regulates guard cell H⁺-ATPases, and its activity results in stomatal opening, facilitating pathogen entry (Oecking et al., 1994). Some bacterial virulence factors simply require scaffold proteins to reach their destination within host cells or to become enzymatically active, while others target the host scaffold proteins to suppress defenses. Yersinia species secrete the TTSS effector YopK (for Yersinia outer protein K), which binds to the Receptor for Activated C Kinase1 in mammals (Thorslund et al., 2011). It is hypothesized that this interaction blocks phagocytosis, allowing efficient extracellular proliferation of the bacteria. Yersinia spp. has also acquired the virulence factor YopM, which mimics eukaryotic scaffolds and forces bridging of host kinases (McDonald et al., 2003). Similarly, enterohemorrhagic Escherichia coli strains use EspG to form an artificial complex that effectively reprograms host signaling (Selyunin et al., 2011).HopQ1 (for Hrp outer protein Q [also known as HopQ1-1]; {"type":"entrez-protein","attrs":{"text":"AAZ37975.1","term_id":"71558765","term_text":"AAZ37975.1"}}AAZ37975.1) is a type III effector that has been acquired recently by Pseudomonas syringae strains (Rohmer et al., 2004), whereas its xenologs from Ralstonia solanacearum and Xanthomonas spp. appear to be ancient. HopQ1 contributes to host specificity, but its exact role in pathogenesis remains undefined. This study shows that HopQ1 must undergo a specific phosphorylation event in planta as a prerequisite for its binding to host 14-3-3 and that its properties depend upon the formation of the effector-host protein complex.     

The long engagement of the emperor penguin

In birds, courtship is generally short relative to the whole breeding cycle. Emperor penguins (Aptenodytes forsteri), however, are an exception as their courtship period is much longer (ca. 6 weeks) than the courtship of other penguin species. This strategy may appear surprising, as it is especially costly to fast and endure drastic climatic conditions for long periods at the colony (1.5 and up to 4 months for females and males, respectively). We examined here the reasons of this extended courtship period and found that emperor penguins returned earlier to the colony when primary oceanic production before breeding was high. This suggests that emperor penguins return to the colony as soon as primary oceanic production in summer allows them to replenish their body reserves. The extended period of time spent at the colony during courtship may therefore result from an evolutionary process that confers advantages to emperor penguins that arrive earlier at the colony by reducing predation risks and offering better chances of securing a partner.     

Insights into eukaryotic Rubisco assembly — Crystal structures of RbcX chaperones from Arabidopsis thaliana

Background

Chloroplasts were formed by uptake of cyanobacteria into eukaryotic cells ca. 1.6 billion years ago. During evolution most of the cyanobacterial genes were transferred from the chloroplast to the nuclear genome. The rbcX gene, encoding an assembly chaperone required for Rubisco biosynthesis in cyanobacteria, was duplicated. Here we demonstrate that homologous eukaryotic chaperones (AtRbcX1 and AtRbcX2) demonstrate different affinities for the C-terminus of Rubisco large subunit and determine their crystal structures.

Methods

Three-dimensional structures of AtRbcX1 and AtRbcX2 were resolved by the molecular replacement method. Equilibrium binding constants of the C-terminal RbcL peptide by AtRbcX proteins were determined by spectrofluorimetric titration. The binding mode of RbcX–RbcL was predicted using molecular dynamic simulation.

Results

We provide crystal structures of both chaperones from Arabidopsis thaliana providing the first structural insight into Rubisco assembly chaperones form higher plants. Despite the low sequence homology of eukaryotic and cyanobacterial Rubisco chaperones the eukaryotic counterparts exhibit surprisingly high similarity of the overall fold to previously determined prokaryotic structures. Modeling studies demonstrate that the overall mode of the binding of RbcL peptide is conserved among these proteins. As such, the evolution of RbcX chaperones is another example of maintaining conserved structural features despite significant drift in the primary amino acid sequence.

General significance

The presented results are the approach to elucidate the role of RbcX proteins in Rubisco assembly in higher plants.     

Formant Frequencies are Acoustic Cues to Caller Discrimination and are a Weak Indicator of the Body Size of Corncrake Males

Source–filter theory assumes that calls are generated by a vocal source and are subsequently filtered by the vocal tract. The air in the vocal tract vibrates preferentially at certain resonant frequencies, called formants. Formant frequencies can be a good indicator of the caller's characteristics, such as sex, age, body size or individual identity. Although source–filter theory was originally proposed for mammals, formants are also observed in birds, and some bird species have been shown to perceive formants. In this study, we evaluated the hypotheses that formant frequencies (1) are an indicator of body size and (2) can be used for individual discrimination by a nocturnal bird species, the corncrake (Crex crex). We analysed calls of 104 males from Poland and the Czech Republic. Linear regression models showed that the males with a longer head (including the bill length) had a significantly lower formant dispersion and lower fourth and fifth formant frequencies. However, we found no significant relationships between body weight and any filter‐related acoustic measurement. The formant frequencies had smaller within‐ than between‐individual coefficients of variation. This characteristic of the formant frequencies implies a high potential for individual coding. A discriminant function analysis correctly assigned 94.8% of the calls to the caller based on formants from second to fifth. Our results indicated that the formant frequencies are a weak indicator of the body size of the sender in the corncrake. However, even weak dependence between body size and acoustic properties of signal may be important in natural selection process. Alternatively, such a weak dependence may be observed, because receivers ignore the acoustical, formant‐based cues of body size. Simultaneously, the formants might potentially provide acoustic cues to individual discrimination and could be used to census and monitoring tasks.     

Ozone fumigation results in accelerated growth and persistent changes in the antioxidant system of Brassica oleracea L. var. capitata f. alba

The growth response and antioxidant capacity of Brassica oleracea var. capitata f. alba plants treated with 70 ppb of ozone was examined. Four week old cabbage seedlings were fumigated with O₃ for 3 days before being transplanted into the growing field. The effect of O₃ treatment was determined directly after fumigation and over the course of field cultivation. Plants subjected to O₃ treatment had an increased diameter of rosettes and number of leaves after 3 and 7 weeks in agriculture, respectively. In addition, the vast majority of fumigated plants reached marketable quality faster than control plants, indicating a positive role of episodes of increased O₃ concentrations during vegetation on growth and yielding.