ABSTRACT
Nickel acts as cofactor for a number of enzymes of many bacteria species. Its homeostasis is ensured by proteins working as ion efflux or accumulation systems. These mechanisms are also generally adopted to counteract life-threatening high extra-cellular Ni2+ concentrations. Little is known regarding nickel tolerance in the genus Sphingobium. We studied the response of the novel Sphingobium sp. ba1 strain, able to adapt to high Ni2+ concentrations. Differential gene expression in cells cultured in 10 mM Ni2+, investigated by RNA-seq analysis, identified 118 differentially expressed genes. Among the 90 up-regulated genes, a cluster including genes coding for nickel and other metal ion efflux systems (similar to either cnrCBA, nccCBA or cznABC) and for a NreB-like permease was found. Comparative analyses among thirty genomes of Sphingobium species show that this cluster is conserved only in two cases, while in the other genomes it is partially present or even absent. The differential expression of genes encoding proteins which could also work as Ni2+-accumulators (HupE/UreJ-like protein, NreA and components of TonB-associated transport and copper-homeostasis systems) was also detected. The identification of Sphingobium sp. ba1 strain adaptive mechanisms to nickel ions, can foster its possible use for biodegradation of poly-aromatic compounds in metal-rich environments.
Subject(s)
Nickel/adverse effects , Sphingomonadaceae/drug effects , Sphingomonadaceae/genetics , Biodegradation, Environmental , Gene Expression Profiling , Gene Expression Regulation, Bacterial/drug effects , Genomics/methods , High-Throughput Nucleotide Sequencing , Sequence Analysis, RNA , Sphingomonadaceae/growth & development , Sphingomonadaceae/metabolism , TranscriptomeABSTRACT
Food allergies are recognized as an increasing health concern. Proteins commonly identified as food allergens tend to have one of about 30 different biochemical activities. This leads to the assumption that food allergens must have specific structural features which causes their allergenicity. But these structural features are not completely understood. Uncovering the structural basis of allergenicity would allow improved diagnosis and therapy of allergies and would provide insights for safer food production. The availability of recombinant food allergens can accelerate their structural analysis and benefit specific studies in allergology. Plant chitinases are an example of food allergenic proteins for which structural analysis of allergenicity has only partially been reported. The recombinant maize chitinase, rChiA, was purified from Pichia pastoris extracellular medium by differential precipitation and cation exchange chromatography. Enzyme activity was evaluated by halo-assays and microcalorimetric procedures. rChiA modeling was performed by a two-step procedure, using the Swiss-Model server and Modeller software. Allergenicity of rChiA was verified by immunoblot assays with sera from allergic subjects. rChiA is active in the hydrolysis of glycol chitin and tetra-N-acetylchitotetraose and maintains its activity at high temperatures (70°C) and low pH (pH 3). The molecule is also reactive with IgE from sera of maize-allergic subjects. rChiA is a valuable molecule for further studies on structure-allergenicity relationships and as a tool for diagnosing allergies.
Subject(s)
Antigens, Plant/immunology , Chitinases/immunology , Food Hypersensitivity , Allergens , Chitinases/chemistry , Chitinases/isolation & purification , Humans , Immunoglobulin E , Pichia , Plant Proteins/immunology , Recombinant Proteins/chemistry , Structure-Activity Relationship , Zea maysABSTRACT
PLANT-PIs is a database developed to facilitate retrieval of information on plant protease inhibitors (PIs) and related genes. For each PI, links to sequence databases are reported together with a summary of the functional properties of the molecule (and its mutants) as deduced from literature. PLANT-PIs contains information for 351 plant PIs, plus several isoinhibitors. The database is accessible at http://bighost.area.ba.cnr.it/PLANT-PIs.
Subject(s)
Databases, Protein , Genes, Plant , Plants/enzymology , Protease Inhibitors/chemistry , Amino Acid Sequence , Binding Sites , DNA Mutational Analysis , DNA, Plant/analysis , Gene Expression , Information Storage and Retrieval , Internet , Plant Proteins/chemistry , Plant Proteins/genetics , Plants/genetics , Structure-Activity RelationshipABSTRACT
The mustard trypsin inhibitor MTI-2 is a potential tool in the study of interactions between pest insects and plants. It can be applied to study the adaptations of digestive proteases in pest insects. Phage display allows a rapid and exhaustive system for the selection of heterologous protein variants with novel specificities. Here we describe a bacteriophage expression system which permits functional expression of MTI-2 variants. Active and inactive mutants of MTI-2 are constructed and displayed on phage. These are used to demonstrate that an active variant can be selected from a background of 10,000 inactive mutants in four rounds of selection and amplification.
Subject(s)
Mustard Plant/genetics , Plant Proteins/genetics , Plants, Medicinal , Trypsin Inhibitors/genetics , Animals , Base Sequence , DNA Primers/genetics , Escherichia coli/genetics , Gene Expression , Genetic Variation , Peptide Library , Pichia/genetics , Plant Proteins/metabolism , Protein Engineering , Trypsin Inhibitors/metabolismABSTRACT
The mustard trypsin inhibitor MTI2 was expressed as secretory protein in the yeast Pichia pastoris. In order to evaluate the influence of the C-terminal amino acids of the precursor form on the inhibitor activity, the C-terminal precursor and the mature protein were both expressed. A third His-tagged construct was also designed to compare alternative purification procedures. Proteins were efficiently expressed at levels of 40-160 mg/l in shake flasks. Equilibrium dissociation constants demonstrated that the mature protein was a stronger inhibitor of bovine beta-trypsin compared to the precursor and His-tagged forms (0.01 nM vs. 0.58 nM and 0.71 nM, respectively). The recombinant proteins were active inhibitors of Spodoptera exigua gut proteases.
Subject(s)
Mustard Plant/metabolism , Plant Proteins/pharmacology , Plants, Medicinal , Trypsin Inhibitors/chemistry , Trypsin/metabolism , Amino Acid Sequence , Animals , Base Sequence , Cattle , Cloning, Molecular , Fermentation , Kinetics , Molecular Sequence Data , Mustard Plant/genetics , Pichia/growth & development , Plant Proteins/chemistry , Plant Proteins/genetics , Recombinant Proteins/chemistry , Recombinant Proteins/isolation & purification , Recombinant Proteins/pharmacology , Trypsin Inhibitors/genetics , Trypsin Inhibitors/pharmacologyABSTRACT
The PLMItRNA database contains information and multialignments of tRNA genes and molecules detected in higher plant mitochondria. It has been developed from a previous compilation of higher plant mitochondrial tRNA genes [Sagliano,A., Volpicella,M., Gallerani,R. and Ceci,L.R. (1998) Nucleic Acids Res., 26, 154-155] and implemented with data and sequences of tRNA molecules retrieved from the literature. The current version of the database reports information on 171 genes and 16 tRNA molecules from 24 plants. PLMItRNA is accessible via WWW at http://bio-www.ba.cnr.it:8000/srs/
Subject(s)
Databases, Factual , Genes, Plant , Mitochondria/genetics , Plants/genetics , RNA, Transfer/genetics , Base Sequence , DNA, Mitochondrial/genetics , DNA, Plant/genetics , Information Storage and Retrieval , Internet , RNA, Plant/chemistry , RNA, Plant/genetics , RNA, Transfer/chemistry , Sequence AlignmentABSTRACT
A new version of the compilation of higher plant mitochondrial tRNA genes (http://www.ebi.ac.uk/service ) has been obtained by means of the FastA program for similarity searching in nucleotide sequence Databases. This approach improves the previous collection, which was based on literature data analysis. The current compilation contains 158 sequences with an increase of 43 units. In this paper, some interesting features of the new entries are briefly presented.
