Proteome study on bacterium Paracoccus denitrificans and construction of 2-DE protein database
| Authors | |
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| Year of publication | 2004 |
| Type | Article in Proceedings |
| Conference | VIII. Pracovní setkání biochemiků a molekulárních biologů. Sborník příspěvků. |
| MU Faculty or unit | |
| Citation | |
| Field | Biochemistry |
| Keywords | Paracoccus denitrificans; Proteome; Database |
| Description | Paracoccus denitrificans is a non-fermentative, facultatively autotrophic soil bacterium often studied in the field of bioenergetics, particularly due to resemblance of its aerobic respiratory chain to that of mitochondria. Also an aspect of a great nutritional adaptability was discovered, related to the ability of exploiting various electron donors and electron acceptors for maintenance and growth. To discuss mechanisms underlying regulation of gene expression by growth conditions, a high-throughput proteome mapping is one of the most effective tools to-date. For 2-DE gel analysis of whole-cell extract and its membrane fraction, newly optimized methods using isoelectric focusation with immobilized pH gradient in the first dimension and denaturing SDS-PAGE in the second dimension were used. Using this approach, more than 800 protein spots of total cell extract and membrane fraction were detected in the range pI 3-10 and Mr 15-100 kDa. In order to follow the dynamics of protein expression under different growth conditions, we compared complex protein composition of whole cells of P. denitrificans cultivated (i) aerobically, (ii) anaerobically with nitrate (as electron acceptor), (iii) anaerobically with nitrite, (iv) anaerobically with nitrous oxide. We also observed effect of azide on protein expression, which is known to induce some denitrification enzymes under aerobic conditions. For this reason, we compared protein composition of whole cells as well as its membrane fraction grown (i) aerobically, (ii) aerobically with addition of 0.4 mM sodium azide, (iii) anaerobically with nitrate, (iv) anaerobically with nitrite (whole cells only), (v) anaerobically with nitrite and addition of 0.4 mM sodium azide (whole cells only). By combined statistical and quantitative evaluation of set 30 large format 2-DE gels, we followed alterations in the quantity of protein spots. We detected significant differences related to the growth on various terminal electron acceptors and sodium azide; the results will be presented and discussed. Anotace anglicky (rozsah pro RIV: max. 1000 znaků)Údaje pro RIV Obor49 protein spots have been submitted for analysis by peptide mass fingerprinting using MALDI-TOF MS. However, the genome of P. denitrificans have not been completely sequenced yet and, currently, information about only 98+126 proteins is available from Swiss?Prot/TrEMBL database. Due to this fact, we were able to identify only 8 proteins up to-date. Among them, nitrite reductase and succinate dehydrogenase are key enzymes in P. denitrificans metabolism. We also compared expression data of nitrite reductase obtained by proteome analysis with measurement of nitrite reductase enzyme activity. These data were in a good agreement. The quantitative data and protein maps are kept in a database in PDQUEST format. This database is going to become web-accessible. |
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