Molecular cloning, characterization, and homologous expression of an endochitinase gene from Bacillus thuringiensis serovar morrisoni

The endochitinase gene (chi3023) of Bacillus thuringiensis (Bt) serovar morrisoni strain 3023 was amplified via polymerase chain reaction (PCR) and cloned in Escherichia coli. The ORF of chi3023 (GenBank Accession Number: DQ993175) consists of 2031 nucleotides encoding a 676-residue protein with a calculated molecular mass of 74.5 kDa and a pI value of 6.0. The amino acid sequence of Chi3023 was compared with previously sequenced Bt chitinases and the phylogenetic relationships among them were determined. The deduced N-terminal 34 amino acids of the premature Chi3023 exhibited a typical signal peptide. The E. coli-Bt shuttle vector pHT315 was used for homologous expression of chi3023. Introduction of recombinant pHT315BTC, carrying chi3023 into Bt serovar morrisoni 3023, resulted in a 23-fold increase in endochitinase activity (0.185 U/mg versus 4.256 U/mg)

IMMUNE RESPONSES AGAINST CHIMERIC DNA AND PROTEIN VACCINES COMPOSED OF PLPEN-OMPH AND PLPEC-OMPH FROM PASTEURELLA MULTOCIDA A:3 IN MICE

Pasteurella multocida is a pathogenic bacterium causing many diseases that are of significant economic importance to livestock industries. Outer membrane protein H (ompH) gene and two fragments of Pasteurella lipoprotein E (plpE) gene, namely plpEN and plpEC, were cloned from P. multocida A:3. Three DNA vaccine formulations, namely pCMV-ompH, pCMV-plpEN-ompH and pCMV-plpEC-ompH and two protein-based prototype vaccines, alum adjuvanted PlpEN-OmpH and PlpEC-OmpH, were generated. Antibody levels were induced in mice vaccinated with chimeric DNA or protein vaccines. A significant (p<0.05) increase in serum IFN-gamma titer was obtained by vaccination with 100 mu g of pCMV-ompH, pCMV-plpEC-ompH and PlpEC-OmpH. DNA vaccines did not provide protection upon intraperitoneal challenge with 10 LD50 of live P. multocida A: 3. However, 40% protection was conferred by 100 mu g of PlpEC-OmpH which was not statistically significant. These results showed that plpEN-ompH and plpEC-ompH chimeric DNA vaccines and alum adjuvanted PlpEN-OmpH or PlpEC-OmpH protein vaccines were immunogenic but not protective against P. multocida A: 3 in mice. Prime-boost strategies, i.e. priming with DNA vaccines and boost with protein formulations or different adjuvants can be utilized to obtain significant protection

Immune responses against chimeric DNA and protein vaccines composed of plpEN-OmpH and PlpEC-OmpH from Pasteurella multocida A:3 in mice

Pasteurella multocida is a pathogenic bacterium causing many diseases that are of significant economic importance to livestock industries. Outer membrane protein H (ompH) gene and two fragments of Pasteurella lipoprotein E (plpE) gene, namely plpEN and plpEC, were cloned from P. multocida A:3. Three DNA vaccine formulations, namely pCMV-ompH, pCMV-plpEN-ompH and pCMV-plpEC-ompH and two protein-based prototype vaccines, alum adjuvanted PlpEN-OmpH and PlpEC-OmpH, were generated. Antibody levels were induced in mice vaccinated with chimeric DNA or protein vaccines. A significant (p < 0.05) increase in serum IFN-g titer was obtained by vaccination with 100 μg of pCMV-ompH, pCMV-plpEC-ompH and PlpEC-OmpH. DNA vaccines did not provide protection upon intraperitoneal challenge with 10 LD50 of live P. multocida A:3. However, 40% protection was conferred by 100 μg of PlpEC-OmpH which was not statistically significant. These results showed that plpEN-ompH and plpEC-ompH chimeric DNA vaccines and alum adjuvanted PlpEN-OmpH or PlpEC-OmpH protein vaccines were immunogenic but not protective against P. multocida A:3 in mice. Prime-boost strategies, i.e. priming with DNA vaccines and boost with protein formulations or different adjuvants can be utilized to obtain significant protection

Expression of chitinase A (chiA) gene from a local isolate of Serratia marcescens in Coleoptera-specific Bacillus thuringiensis

Aims: The present study focused on cloning and expression of chiA gene from a highly chitinolytic local isolate of Serratia marcescens in an anti-Coleopteran Bacillus thuringiensis and comparison of the characteristics of the native and recombinant ChiAs

Dynamic proteomic analysis of Phanerochaete chrysosporium under copper stress

The model white rot fungus Phanerochaete chrysosporium is frequently preferred for heavy metal accumulation studies due to its high resistance to heavy metals, including copper (Cu). Here, the response of P. chrysosporium under Cu stress at different time points was investigated for the first time by a detailed proteomic analysis using 2DE MALDI-TOF/MS and nanoLC-MS/MS techniques. A total of 123 Cu-responsive protein spots were determined using 2DE approach, and 104 of them were corresponded to 73 distinct open reading frames (ORFs). Of identified ones, 88 spots were over-, and 16 spots were underrepresented. The majority of these proteins showed to the strongest response at 8th h of Cu exposure. Using nanoLC-MS/MS analysis, a total of 167 differentially produced proteins were identified from Cu-exposed cultures after enrichment of the membrane proteins followed by SILAC. Seventy four, 66, and 69 overrepresented, and 56, 71, and 64 underrepresented proteins were identified at 2 h, 4 h, and 8 h of Cu exposure, respectively. The bioinformatic analysis of these proteins revealed that intracellular trafficking proteins such as Ran GTPase and a p24 family protein, and certain proteins involved in posttranslational modification, protein turnover and folding were Cu-responsive. Three important transcription factors (TFs), NAC, BTF3, and homeobox TFs, 40S and 60S ribosomal proteins, chaperones such as Hsp26/Hsp42 and mortalin, as well as 20S proteasome, 14-3-3 proteins and Hsp90 involve in Cu-stress response of P. chrysosporium. Moreover, certain elements of translation machinery, the proteins related with aspartate, methionine, and pyruvate metabolisms, transketolase, and trehalase related with carbohydrate metabolism, citrate synthase, fumarase, V-ATPase, and F0F1-type ATPase playing role in energy production and conversion, transport proteins such as multidrug resistance and p24 family proteins as well as actin-related proteins involved in cytoskeleton remodeling were determined to be Cu-responsive. The present proteome analysis revealed that P. chrysosporium mainly regulates translational and posttranslational processes, certain transport processes, many metabolic pathways and cytoskeleton to overcome the Cu-induced oxidative stress

Homologous expression of aspartokinase (ask) gene in Streptomyces clavuligerus and its hom-deleted mutant: Effects on cephamycin C production

In this study, the effect of homologous multiple copies of the ask gene, which encodes aspartokinase catalyzing the first step of the aspartate pathway, on cephamycin C biosynthesis in S. clavuligerus NRRL 3585 and its hom mutant was investigated. The intracellular pool levels of aspartate pathway amino acids accorded well with the Ask activity levels in TB3585 and AK39. When compared with the control strain carrying vector alone without any gene insert, amplification of the ask gene in the wild strain resulted in a maximum of 3.1- and 3.3-fold increase in specific, 1.7- and 1.9-fold increase in volumetric cephamycin C production when grown in trypticase soy broth (TSB) and a modified chemically defined medium (mCDM), respectively. However, expression of multicopy ask gene in a hom-deleted background significantly decreased cephamycin C yields when the cells were grown in either TSB or mCDM, most probably due to physiological disturbance resulting from enzyme overexpression and high copy number plasmid burden in an auxotrophic host, respectively

Genetik manipulasyonlarla Bacillus Thuringiensis alttür Tenebrionis'in larvisidal aktivitesinin ve spektrumunun artırılması

TÜBİTAK TBAG Proje01.07.200