Congruent population genetic structures and divergence histories in anther‐smut fungi and their host plants Silene italica and the Silene nutans species complex

Study of the congruence of population genetic structure between hosts and pathogens gives important insights into their shared phylogeographical and coevolutionary histories. We studied the population genetic structure of castrating anther‐smut fungi (genus Microbotryum) and of their host plants, the Silene nutans species complex, and the morphologically and genetically closely related Silene italica, which can be found in sympatry. Phylogeographical population genetic structure related to persistence in separate glacial refugia has been recently revealed in the S. nutans plant species complex across Western Europe, identifying several distinct lineages. We genotyped 171 associated plant–pathogen pairs of anther‐smut fungi and their host plant individuals using microsatellite markers and plant chloroplastic single nucleotide polymorphisms. We found clear differentiation between fungal populations parasitizing S. nutans and S. italica plants. The population genetic structure of fungal strains parasitizing the S. nutans plant species complex mirrored the host plant genetic structure, suggesting that the pathogen was isolated in glacial refugia together with its host and/or that it has specialized on the plant genetic lineages. Using random forest approximate Bayesian computation (ABC‐RF), we found that the divergence history of the fungal lineages on S. nutans was congruent with that previously inferred for the host plant and probably occurred with ancient but no recent gene flow. Genome sequences confirmed the genetic structure and the absence of recent gene flow between fungal genetic lineages. Our analyses of individual host–pathogen pairs contribute to a better understanding of co‐evolutionary histories between hosts and pathogens in natural ecosystems, in which such studies remain scarce.     

Dictyosphaerium-like morphotype in terrestrial algae: what is Xerochlorella (Trebouxiophyceae,Chlorophyta)?1

Several strains of terrestrial algae isolated from biological soil crusts in Germany and Ukraine were identified by morphological methods as the widely distributed species Dictyosphaerium minutum (=Dictyosphaerium chlorelloides). Investigation of the phylogeny showed their position unexpectedly outside of Chlorellaceae (Trebouxiophyceae) and distantly from Chlorella chlorelloides, to which this taxon was attributed after revision of the genus Chlorella based on an integrative approach. SSU rRNA phylogeny determined the position of our strains inside a clade recently described as a new genus of the cryptic alga Xerochlorella olmiae isolated from desert biological soil crusts in the United States. Investigation of the morphology of the authentic strain of X. olmiae showed Dictyosphaerium-like morphology, as well as some other characters, common for our strains and morphospecies D. minutum. The latter alga was described as terrestrial and subsequently united with the earlier described aquatic representative D. chlorelloides because of their similar morphology. The revision of Chlorella mentioned above provided only one aquatic strain (D. chlorelloides), which determined its position in the genus. But terrestrial strains of the morphospecies were not investigated phylogenetically. Our study showed that the terrestrial D. minutum is not related to the morphologically similar D. chlorelloides (=Chlorella chlorelloides, Chlorellaceae), and instead represented a separate lineage in the Trebouxiophyceae, recently described as genus Xerochlorella. Therefore, revision of Xerochlorella is proposed, including nomenclatural combinations, epitypifications, and emendations of two species: X. minuta and X. dichotoma. New characters of the genus based on investigation of morphology and ultrastructure were determined.     

eIF3j facilitates loading of release factors into the ribosome

eIF3j is one of the eukaryotic translation factors originally reported as the labile subunit of the eukaryotic translation initiation factor eIF3. The yeast homolog of this protein, Hcr1, has been implicated in stringent AUG recognition as well as in controlling translation termination and stop codon readthrough. Using a reconstituted mammalian in vitro translation system, we showed that the human protein eIF3j is also important for translation termination. We showed that eIF3j stimulates peptidyl-tRNA hydrolysis induced by a complex of eukaryotic release factors, eRF1-eRF3. Moreover, in combination with the initiation factor eIF3, which also stimulates peptide release, eIF3j activity in translation termination increases. We found that eIF3j interacts with the pre-termination ribosomal complex, and eRF3 destabilises this interaction. In the solution, these proteins bind to each other and to other participants of translation termination, eRF1 and PABP, in the presence of GTP. Using a toe-printing assay, we determined the stage at which eIF3j functions – binding of release factors to the A-site of the ribosome before GTP hydrolysis. Based on these data, we assumed that human eIF3j is involved in the regulation of translation termination by loading release factors into the ribosome.     

LKB1 tumor suppressor protein regulates actin filament assembly through Rho and its exchange factor Dbl independently of kinase activity

Background  

Germline mutations in LKB1 result in Peutz-Jeghers Syndrome characterized by intestinal hamartomas and increased incidence of epithelial cancers. LKB1 encodes a serine/threonine kinase that plays an important role in regulating energy metabolism through the AMPK/mTOR signaling pathway. In addition, LKB1 is homologous to PAR-4, a polarity protein first described in C. elegans, while activation of LKB1 in mammalian epithelial cells induces the polarized assembly of actin filaments.     

Activation of sodium transport in rat erythrocytes by inhibition of protein phosphatases 1 and 2A

Four structurally different protein phosphatases (PPs) inhibitors - fluoride, calyculin A, okadaic acid and cantharidin--were tested for their ability to modulate unidirectional Na(+) influx in rat red blood cells. Erythrocytes were incubated at 37 degrees C in isotonic and hypertonic media containing 1 mM ouabain and (22)Na in the absence or presence of PP inhibitors. Exposure of the cells to 20 mM fluoride or 50 nM calyculin A for 1 h under isosmotic conditions caused a significant stimulation of Na(+) influx, whereas addition of 200 microM cantharidin or 100 nM okadaic acid had no effect. After 2 h of treatment, however, all these PPs blockers significantly enhanced Na(+) transport in rat erythrocytes. Selective inhibitors of PP-1 and PP-2A types, calyculin A, cantharidin and okadaic acid, produced similar ( approximately 1.2-1.4-fold) stimulatory effects on Na(+) influx in the cells. Activation of Na(+) influx was unchanged with increasing calyculin A concentration from 50 to 200 nM. No additive stimulation of Na(+) influx was observed when the cells were treated with combination of 20 mM fluoride and 50 nM calyculin A. Na(+) influx induced by PPs blockers was inhibited by 1 mM amiloride and 200 muM bumetanide approximately in the equal extent, indicating the involvement of Na(+)/H(+) exchange and Na-K-2Cl cotransport in sodium transport through rat erythrocytes membrane. Activation of Na(+) transport in the cells induced by calyculin A and fluoride was associated with increase of intracellular Na(+) content. Shrinkage of the rat erythrocytes resulted in 2-fold activation of Na(+) influx. All tested PPs inhibitors additionally activated the Na(+) influx by 70-100% above basal shrinkage-induced level. Amiloride and bumetanide have diminished both the shrinkage-induced and PPs-inhibitors-induced Na(+) influxes. Thus, our observations clearly indicate that activities of Na(+)/H(+) exchanger and Na-K-2Cl cotransporter in rat erythrocytes are regulated by protein phosphatases and stimulated when protein dephosphorylation is inhibited.     

Characterization of Exiguobacterium isolates from the Siberian permafrost. Description of Exiguobacterium sibiricum sp. nov.

Three Gram-positive bacterial strains, 7-3, 255-15 and 190-11, previously isolated from Siberian permafrost, were characterized and taxonomically classified. These microorganisms are rod-shaped, facultative aerobic, motile with peritrichous flagella and their growth ranges are from -2.5 to 40 degrees C. The chemotaxonomic markers indicated that the three strains belong to the genus Exiguobacterium. Their peptidoglycan type was A3alpha L-Lys-Gly. The predominant menaquinone detected in all three strains was MK7. The polar lipids present were phosphatidyl-glycerol, diphosphatidyl-glycerol and phosphatidyl-ethanolamine. The major fatty acids were iso-C13:0, anteiso-C13:0, iso-C15:0, C16:0 and iso-C17:0. Phylogenetic analysis based on 16S rRNA and six diverse genes, gyrB (gyrase subunit B), rpoB (DNA-directed RNA polymerase beta subunit), recA (homologous recombination), csp (cold shock protein), hsp70 (ClassI-heat shock protein-chaperonin) and citC (isocitrate dehydrogenase), indicated that the strains were closely related to Exiguobacterium undae (DSM 14481(T)) and Exiguobacterium antarcticum (DSM 14480(T)). On the basis of the phenotypic characteristics, phylogenetic data and DNA-DNA reassociation data, strain 190-11 was classified as E. undae, while the other two isolates, 7-3 and 255-15, comprise a novel species, for which the name Exiguobacterium sibiricum sp. nov. is proposed.     

Aqueous two-phase systems extraction of alpha-toxin from Clostridium perfringens type A

Two sequential half-fraction designs were applied to studying the alpha-toxin partition produced by Clostridium perfringens type A in aqueous two phase systems (ATPS), as a function of four factors: PEG molar mass and concentration, phosphate concentration and pH. The highest purification factor, yield and partition coefficient results were obtained with PEG 8000 (15%, w/w), phosphate at 20% (w/w) and pH 8.0. This system allows, in a single step, an alpha-toxin purification of 4.6-fold with final activity yield of 230% and partition coefficient of 113.9 in the PEG rich phase.     

PRINS-labeled knobs are not associated with increased chromosomal stickiness in the maize st1 mutant

In maize, the st1 mutant has been observed to result in chromosomes that stick together during both mitotic and meiotic anaphase. These sticky chromosomes result in abnormal chromosome separation at anaphase. Although the mechanism producing the st1 mutant phenotype is unknown, delayed replication of knob heterochromatin has been implicated in similar phenomena that result in sticky chromosomes. Primed in situ labeling (PRINS) was used to locate the 180-bp knob DNA sequences on mitotic metaphase chromosomes of several maize lines. The chromosomal regions labeled by PRINS corresponded to the reported C bands found in these lines. Additionally, PRINS was used to identify knob-bearing regions in anaphase spreads of a line carrying the st1 mutant and a nonmutant line having a similar number of chromosome knobs. The increase in abnormal anaphase figures in the st1 mutant was not accompanied by an increase in association of knob DNA with abnormal anaphases. Thus, the increase in chromosomal stickiness appears to be due to an increase in stickiness of knob and nonknob chromosomal regions. The mechanism responsible for the st1 mutant, therefore, is hypothesized to be different from that implicated in the other previously described sticky chromosomes situations.     

Antiamoebin I in methanol solution: rapid exchange between right-handed and left-handed 3(10)-helical conformations

Antiamoebin I (Aam-I) is a membrane-active peptaibol antibiotic isolated from fungal species belonging to the genera Cephalosporium, Emericellopsis, Gliocladium, and Stilbella. Antiamoebin I has the amino acid sequence: Ac-Phe(1)-Aib-Aib-Aib-Iva-Gly-Leu-Aib(8)-Aib-Hyp-Gln-Iva-Hyp-Aib-Pro-Phl(16). By using the uniformly (13)C,(15)N-labeled sample of Aam-I, the set of conformationally dependent J couplings and (3h)J(NC) couplings through H-bonds were measured. Analysis of these data along with the data on magnetic nonequivalence of the (13)C(beta) nuclei (Deltadelta((13)C(beta))) in Aib and Iva residues allowed us to draw the univocal conclusion that the N-terminal part (Phe(1)-Gly(6)) of Aam-I in MeOH solution is in fast exchange between the right-handed and left-handed 3(10)-helical conformations, with an approximately equal population of both states. An additional conformational exchange process was found at the Aib(8) residue. The (15)N-NMR-relaxation and CD-spectroscopy measurements confirmed these findings. Molecular modeling and Monte Carlo simulations revealed that both exchange processes are correlated and coupled with significant hinge-bending motions around the Aib(8) residue. Our results explain relatively low activity of Aam-I with respect to other 15-amino acid residue peptaibols (for example, zervamicin) in functional and biological tests. The high dynamic 'propensity' possibly prevents both initial binding of the antiamoebin to the membrane and subsequent formation of stable ionic channels according to the barrel-stave mechanism.