RESUMO
Phospholipase A2 (PLA2) has found extensive use in industry. However, recombinant PLA2 production in different expression systems is a difficult task because of its toxicity to cell membranes. We report here the development of an effective method for production of highly active PLA2 from Streptomyces violaceoruber strain A-2688 in the yeast Saccharomyces cerevisiae. The method is based on the use of the PRP8 mini-intein (from Penicillium chrysogenum) inserted into the phospholipase sequence with the purpose of temporal inactivation of the enzyme and its subsequent delayed autoactivation. We demonstrate that the most effective site for intein insertion is Ser76 of the mature phospholipase. As a result of intein-containing precursor secretion from yeast cells and its subsequent autocatalytic splicing, highly active enzyme accumulated in the yeast culture fluid. The properties of the obtained recombinant phospholipase A2 protein were similar to those of the native Streptomyces violaceoruber PLA2 protein. A possible evolutionary role of delayed autoactivation of intein-containing proteins is also discussed.
Assuntos
Proteínas de Bactérias/metabolismo , Fosfolipases A2/metabolismo , Proteínas de Bactérias/genética , Inteínas/genética , Inteínas/fisiologia , Fosfolipases/genética , Fosfolipases/metabolismo , Fosfolipases A2/genética , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Streptomyces/genética , Streptomyces/metabolismoRESUMO
The effects of hybrid lectins--full-sized pea Pisum sativum lectin (PSL) with the carbohydrate-binding region of white melilot Melilotus albus lectin or wild licorice Astragalus glycyphyllos lectin substituted for the corresponding PSL region (PSL/MAL and PSL/AGL, correspondingly)--on the legume-rhizobium symbiosis were studied. The treatment of the Rhizobium leguminosarum bv. viciae in the alfalfa (Medicago sativa) rhizosphere with PSL induced formation of uninfected pseudonodules on its roots, whereas the treatment of the bacteria from Astragalus cicer nodules with PSL/AGL rendered these bacteria able to form infective nodules on alfalfa roots. This ability is associated with expanded and unusual carbohydrate-binding properties (combined specificity for Gal and Glc) of this hybrid protein as compared with the natural legume lectins.
Assuntos
Astrágalo/microbiologia , Galega/microbiologia , Medicago sativa/microbiologia , Lectinas de Plantas/farmacologia , Rhizobium/fisiologia , Astrágalo/fisiologia , Galega/fisiologia , Medicago sativa/fisiologia , Lectinas de Plantas/genética , Raízes de Plantas/microbiologia , Proteínas Recombinantes de Fusão/genética , Proteínas Recombinantes de Fusão/farmacologia , Nódulos Radiculares de Plantas/microbiologia , SimbioseRESUMO
Competitiveness and genetic variation of the Rhizobium galegae strains from the collection of the All-Russia Institute of Agricultural Microbiology, Russian Academy of Agricultural Sciences, causing nodulation of oriental goat's rue under conditions of Bashkortostan soils (lacking this rhizobial species) were studied. It was demonstrated that of all the tested strains, the strains CIAM 0702 and CIAM 0704, each carrying two megaplasmids of 1500 and 2000 MDa, were the most competitive. RAPD (random amplified polymorphic DNA) analysis showed that R. galegae strains were able to intensively exchange the genetic material in the host plant rhizosphere. We did not succeed in detecting the local root nodule bacteria that were either initially able to infect oriental goat's rue or had adapted to infecting this species due to various genetic rearrangements.
Assuntos
Galega/microbiologia , Plasmídeos/genética , Rhizobium/genética , Nódulos Radiculares de Plantas/microbiologia , Bashkiria , Galega/genética , Plasmídeos/metabolismo , Rhizobium/crescimento & desenvolvimento , Nódulos Radiculares de Plantas/genética , Especificidade da EspécieRESUMO
The population genetic diversity and phylogeny of the bacteria entering the symbiosis with sainfoin that grows on the Chesnokovskaya Mountain, Ufa region, Republic of Bashkortostan, have been studied. RAPD analysis of DNA polymorphism of the microbial strains grown from the nodules of 20 plants using several random primers detected a high degree of genetic homogeneity in their population as compared with the populations of rhizobia of other leguminous plants growing at the same site. Sequencing of 16S rRNA genes of the three most different samples have demonstrated that these genes were identical and display 99.9% homology with the sequence of Phyllobacterium trifolii 16S rRNA gene.
Assuntos
Alphaproteobacteria/classificação , Alphaproteobacteria/isolamento & purificação , Fabaceae/microbiologia , Alphaproteobacteria/genética , Técnicas de Tipagem Bacteriana , Genes Bacterianos/genética , Genes de RNAr/genética , Filogenia , RNA Ribossômico 16S/genética , Técnica de Amplificação ao Acaso de DNA PolimórficoRESUMO
The carbohydrate-binding sequences (CBS) in the lectin genes of Trijilium repens, T. pratense, and T. tri-chocephalum were sequenced. The gene regions encoding lectin CBS of T. pratense and T. repens displayed a considerable similarity; however, the CBS of these species differed essentially. Moreover, T. repens formed a compact cluster with Melilotus albus and M. officinalis in the phylogenetic trees constructed according to the nucleotide sequences and the corresponding CBS of legume lectins. T. trichocephalum does not fall into the group of the tribe Trifolieae members according to both the amino acid sequence of lectin carbohydrate-binding region and the nucleotide sequence of lectin gene.
Assuntos
Genes de Plantas , Filogenia , Lectinas de Plantas/genética , Trifolium/genética , Sequência de Aminoácidos/genética , Estrutura Terciária de Proteína/genéticaRESUMO
Method of site-directed mutagenesis that gave an opportunity to the site-independent manipulation with enough extended exposed mutagenesis fragments of gene cloning in plasmid vector was developed. With the help of this method hybrid constructions representing peas Pisum sativum (PSL) lectine with carbohydrate-binding fragment changing on that lectines from another legume plants were created. This method is suitable for obtaining the expression constructions in plasmid vectors, when is necessary to put a coding part under the control of promoter in a certain reading frame.
Assuntos
Genes de Plantas , Mutagênese Sítio-Dirigida , Pisum sativum/genética , Lectinas de Plantas/genética , Reação em Cadeia da Polimerase , Lectinas de Plantas/biossíntese , Plasmídeos/genética , Estrutura Terciária de Proteína/genética , Proteínas Recombinantes/biossíntese , Proteínas Recombinantes/genéticaAssuntos
Carboidratos/química , Lectinas/química , Lectinas/metabolismo , Proteínas de Plantas/química , Proteínas de Plantas/metabolismo , Engenharia de Proteínas/métodos , Lectinas/genética , Proteínas de Plantas/genética , Ligação Proteica , Isoformas de Proteínas/química , Isoformas de Proteínas/genética , Isoformas de Proteínas/metabolismo , Proteínas Recombinantes/química , Proteínas Recombinantes/genética , Proteínas Recombinantes/metabolismoRESUMO
A significant heterogeneity between bacteria Rhizobium galegae bv. officinalis and R. galegae bv. orientalis forming the nitrogen-fixing symbiosis with Galega officinalis and G. orientalis, respectively, and not forming any single cross-inoculation group, was found by means of RAPD and RFEL methods. The high level of sequence similiraty between lectins of these plants indicates at their close relationship. However the sequences of lectin sugar binding peptides (SBP) of G. orientalis (TYCNPGWDPRDR) and G. officinalis (TFYNEEWDLVIKDEH) were highly diverged. Amino acids of SBP which are involved in linkage of Ca2+ and Mn2+ ions responsible for stabilization of spatial structure of carbohydrate-binding "pocket", keep their position in peptide. It suggests that lectins participate in Rhizobium-legume symbiosis and that carbohydrate-binding site plays a key role in this process.