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Integration of additional copies of Trichoderma reesei gene encoding protein O-mannosyltransferase I results in a decrease of the enzyme activity and alteration of cell wall composition.
Get PDFIn fungi, transfer of the first mannosyl residue to proteins during their O-glycosylation is catalyzed by protein O-mannosyltransferases. Integration of additional copies of the pmt1 gene into Trichoderma reesei genome unexpectedly resulted in the silencing of pmt1 expression. Strains carrying the additional copies of pmt1 gene exhibited lower total activity of protein O-mannosyltransferases, lower O- and N-glycosylation of secreted proteins and showed defects in their cell wall composition. Moreover, the strains grew slowly on solid medium and were hypersensitive to an antifungal reagent, Calcofluor white. These results indicate that protein O-manosyltransferases are required for proper cell wall formation, and their decreased activity influences not only O- but also N-glycosylation
Independent Subtilases Expansions in Fungi Associated with Animals
Get PDFMany socially important fungi encode an elevated number of subtilisin-like serine proteases, which have been shown to be
involved in fungal mutualisms with grasses and in parasitism of insects, nematodes, plants, other fungi, and mammalian
skin. These proteins have endopeptidase activities and constitute a significant part of fungal secretomes. Here, we use
comparative genomics to investigate the relationship between the quality and quantity of serine proteases and the ability
15 of fungi to cause disease in invertebrate and vertebrate animals. Our screen of previously unexamined fungi allowed us to
annotate and identify nearly 1000 subtilisin-containing proteins and to describe six new categories of serine proteases.
Architectures of predicted proteases reveal novel combinations of subtilisin domains with other, co-occurring domains.
Phylogenetic analysis of the most common clade of fungal proteases, proteinase K, showed that gene family size changed
independently in fungi, pathogenic to invertebrates (Hypocreales) and vertebrates (Onygenales). Interestingly,
20 simultaneous expansions in the S8 and S53 families of subtilases in a single fungal species are rare.
Our analysis finds that closely related systemic human pathogens may not show the same gene family expansions, and that
related pathogens and nonpathogens may show the same type of gene family expansion. Therefore, the number of
proteases does not appear to relate to pathogenicity. Instead, we hypothesize that the number of fungal serine proteases in
a species is related to the use of the animal as a food source, whether it is dead or alive
The Doping Effect of Fluorinated Aromatic Solvent on the Rate of Ruthenium Catalysed Olefin Metathesis
Get PDFA study concerning the effect of using a fluorinated aromatic solvent as the medium for olefin metathesis reactions catalysed by ruthenium complexes bearing N-heterocyclic carbene ligands is presented. The use of
fluorinated aromatic hydrocarbons (FAH) as solvents for olefin metathesis reactions catalysed by standard commercially available ruthenium pre-catalysts allows substantially higher yields of the desired products to be obtained,especially in the case of demanding polyfunctional molecules, including natural and biologically active compounds. Interactions between the FAH and the second-generation ruthenium catalysts, which apparently improve the
efficiency of the olefin metathesis transformation, have been studied by X-ray structure analysis and computations,
as well as by carrying out a number of metathesis experiments. The optimisation of reaction conditions by
using an FAH can be regarded as a complementary approach for the design of new improved ruthenium catalysts.
Fluorinated aromatic solvents are an attractive alternative medium for promoting challenging olefin metathesis reactions
SnRK2 Protein Kinases-Key Regulators of Plant Response to Abiotic Stresses
Get PDFAbstract The SnRK2 family members are plant-specific serine/threonine kinases involved in plant response to abiotic stresses and abscisic acid (ABA)-dependent plant development. SnRK2s have been classed into three groups; group 1 comprises kinases not activated by ABA, group 2 comprises kinases not activated or activated very weakly by ABA, and group 3 comprises kinases strongly activated by ABA. So far, the ABA-dependent kinases belonging to group 3 have been studied most thoroughly. They are considered major regulators of plant response to ABA. The regulation of the plant response to ABA via SnRK2s pathways occurs by direct phosphorylation of various downstream targets, for example, SLAC1, KAT1, AtRbohF, and transcription factors required for the expression of numerous stress response genes. Members of group 2 share some cellular functions with group 3 kinases; however, their contribution to ABA-related responses is not clear. There are strong indications that they are positive regulators of plant responses to water deficit. Most probably they complement the ABA-dependent kinases in plant defense against environmental stress. So far, data concerning the physiological role of ABA-independent SnRK2s are very limited; it is to be expected they will be studied extensively in the nearest future
Binding and Spreading of ParB on DNA Determine Its Biological Function in Pseudomonas aeruginosa.
Get PDFParB protein of Pseudomonas aeruginosa belongs to a widely represented ParB family of chromosomally and
plasmid-encoded partitioning type IA proteins. Ten putative parS sites are dispersed in the P. aeruginosa
chromosome, with eight of them localizing in the oriC domain. After binding to parS, ParB spreads on the DNA,
causing transcriptional silencing of nearby genes (A. A. Bartosik et al., J. Bacteriol. 186:6983–6998, 2004). We
have studied ParB derivatives impaired in spreading either due to loss of DNA-binding ability or oligomerization.
We defined specific determinants outside of the helix-turn-helix motif responsible for DNA binding.
Analysis confirmed the localization of the main dimerization domain in the C terminus of ParB but also
mapped another self-interactive domain in the N-terminal domain. Reverse genetics were used to introduce five
parB alleles impaired in spreading into the P. aeruginosa chromosome. The single amino acid substitutions in
ParB causing a defect in oligomerization but not in DNA binding caused a chromosome segregation defect,
slowed the growth rate, and impaired motilities, similarly to the pleiotropic phenotype of parB-null mutants,
indicating that the ability to spread is vital for ParB function in the cell. The toxicity of ParB overproduction
in Pseudomonas spp. is not due to the spreading since several ParB derivatives defective in oligomerization were
still toxic for P. aeruginosa when provided in excess
DNA Array
Get PDFThe DNA array technology provides efficient access to genetic information permitting studies of a wide range of analyses, on small as well as on a global scale, thus expanding popularity throughout the field of molecular biology, biotechnology and medicine. Primarily developed to enhance genome sequencing projects, the technology is rapidly evolving and has been adapted to an incredible set of applications. There is a lot of enthusiasm about its power, since it gives possibilities to study the molecular basis of interactions on a scale that would be impossible using conventional analyses. This technology has been successfully applied to investigate simultaneously expression of a few hundred of genes as well as many thousands. Its application has extended to the detection of mutations or polymorphisms, as well as their mapping and sequencing. But what exactly are these extremely useful life science tools? How are they created? Or what is their role – current and future? We hope that readers will find answers in this chapter, where we briefly review the format of DNA array, the technology used to fabricate and read them, and their applications. The diverse range of concepts required to master the experimental and data analysis aspects of DNA array technology is discussed. Moreover, although DNA array is a powerful technique, there are restrictions and problems which must be considered when planning experiments and analysing their results, and they will also be highlighted
A reliable method for storage of tailed phages
Get PDFA universal and effective method for long-term storage of bacteriophages has not yet been described. We show that randomly selected tailed phages could be stored inside the infected cells at−80 °C without a major loss of phage and host viability. Our results suggest the suitability of this method as a standard for phage preservation
Ion Mobility Separation Coupled with MS Detects Two Structural States of Alzheimer's Disease Aβ1-40 Peptide Oligomers.
Get PDFMounting evidence points to the soluble oligomers of amyloid β (Aβ) peptide as important neurotoxic species in Alzheimer's disease, causing synaptic dysfunction and neuronal injury, and finally leading to neuronal death. The mechanism of the Aβ peptide self-assembly is still under debate. Here, Aβ1-40 peptide oligomers were studied using mass spectrometry combined with ion mobility spectrometry, which allowed separation of the signals of numerous oligomers and measurement of their collisional cross-section values (Ω). For several oligomers, at least two different species of different Ω values were detected, indicating the presence of at least two families of conformers: compact and extended. The obtained results are rationalized by a set of molecular models of Aβ1-40 oligomer structure that provided a very good correlation between the experimental and theoretical Ω values, both for the compact and the extended forms. Our results indicate that mass spectrometry detects oligomeric species that are on-pathway in the process of fibril formation or decay, but also alternative structures which may represent off-pathway evolution of oligomers
Zn(II) Complexes of Glutathione Disulfide: Structural Basis of Elevated Stabilities
Get PDFGlutathione disulfide (GSSG), a long disregarded redox partner of glutathione (GSH), is thought to participate in intracellular zinc homeostasis. We performed a concerted potentiometric and NMR spectroscopic study of protonation and Zn(II) binding properties of GSSG ((γECG)2) and a series of its nine analogs with C-terminal modifications,
tripeptide disulfides: (γECS)2, (γECE)2, (γECG-NH2)2, (γECG-OEt)2, and (γEcG)2; dipeptide disulfides, (γEC)2and (γEC-OEt)2; and mixed disulfides, γECG-γEC and ECG-γEC-OEt. The acid-base and Zn(II) complexation properties in this group of compounds are strictly correlated to average C-terminal electrostatic charges. In particular, it was demonstrated that GSSG assumes a bent (head-to-tail) conformation in solution at neutral pH, which is controlled by electrostatic attraction between the protonated γ-amino groups of the Glu residue and the deprotonated C-terminal
Gly carboxylates. This interaction modulates the ability of GSSG to coordinate Zn(II), both indirectly, by affecting the
basicities of the amino groups, and directly, through the participation of the Gly carboxylates in the outer coordination sphere of the Zn(II) ion. A specific coiled structure of the major [Zn-GSSG]2- complex is additionally stabilized by the formation of hydrogen bonds between glycinyl carboxylates and two Zn(II)-coordinated water molecules. The elevated stability of Zn(II)-GSSG complexes was demonstrated by competition with FluoZin-3, a fluorescent sensor with high Zn(II) affinity, commonly used in in vitro and in vivo studies. The potential biological functions and reactivity of GSSG complexes of Zn(II) ions are discussed
The Centromere Site of the Segregation Cassette of Broad-Host-Range Plasmid RA3 Is Located at the Border of the Maintenance and Conjugative Transfer Modules
Get PDFRA3 is a low-copy-number, broad-host-range (BHR) conjugative plasmid of the IncU incompatibility group
isolated originally from Aeromonas spp. A 4.9-kb fragment of RA3 is sufficient to stabilize an otherwise unstable
replicon in Escherichia coli. This fragment specifies the korA-incC-korB-orf11 operon coding for an active
partition system related to the central control operon of IncP-1 plasmids and found also in BHR environmental
plasmids recently classified as the PromA group. All four genes in the cassette are necessary for segregation.
IncC and KorB of RA3 belong to the ParA and ParB families of partitioning proteins, respectively. In contrast
with IncP-1 plasmids, neither KorB nor IncC are involved in transcriptional autoregulation. Instead, KorA
exerts transcriptional control of the operon by binding to a palindromic sequence that overlaps the putative
�35 promoter motif of the cassette. The Orf11 protein is not required for regulation, but its absence decreases
the stabilization potential of the segregation module. A region discontiguous from the cassette harbors a set
of unrelated repeat motifs distributed over �300 bp. Dissection of this region identified the centromere
sequence that is vital for partitioning. The �300-bp fragment also encompasses the origin of conjugative
transfer, oriT, and the promoter that drives transcription of the conjugative transfer operon. A similar set of
cis-acting motifs are evident in the PromA group of environmental plasmids, highlighting a common evolutionary
origin of segregation and conjugative transfer modules in these plasmids and members of the IncU
group