| 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 we optimized new methods using immobilized pH gradient. Before the whole-cell proteome analysis, cells were disrupted using french-press. Released proteins were precipitated by ice-cold acetone and the pellet was solubilized using 7 M urea, 2 M thiourea, 4% (w/v) CHAPS, 2 mM tributylphosphine, 15 mM TRIS base, 0.8 % (v/v) Bio-Lyte 3/10 and 0.8 % (v/v) Bio?Lyte 8/10. For analysis of membrane fraction, cells were disintegrated by osmotic and enzymatic lysis, membrane proteins were than extracted using sample solution containing 7 M urea, 2 M thiourea, 1 % ASB 14, 1 % TRITON X-100, 2 mM tributylphosphine, 15 mM TRIS base, 1 % (v/v) Pharmalyte 3/10 and 0.5 % (v/v) Pharmalyte 8/10. Mixture of broad range (3-10) and basic (8-10) ampholytes improved resolution of some basic proteins, especially in the membrane fraction. After in-gel rehydration, proteins were separated by isoelectric focusation using nonlinear immobilized pH gradient (pH 3-10). In the second dimension, 12 % homogenous SDS-PAGE gels were used. Whereas the analysis of whole-cell extract using Laemmli buffer system provided good results, it was necessary to use taurine buffer system for the successful analysis of membrane fraction. Image analysis was performed and spot database created by PDQUEST software. We were able to detect 306 protein spots in the standard whole-cell gel and 515 protein spots in the standard membrane gel. Among them, 36 protein spots have been already analyzed by peptide mass fingerprinting, eventually also by MALDI-post source decay or liquid chromatography-tandem mass spectrometry. However, the genome of P. denitrificans have not been completely sequenced yet and, currently, information about only 97+127 proteins is available from Swiss?Prot/TrEMBL database. Due to this fact, we were able to identify only 6 proteins up to-date. Among them, nitrite reductase is a well-known enzyme involved in denitrification. 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) anearobically 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). Student t-test that is implemented in PDQUEST 6.2 was used for determination of significant differences at the levels of p < 0.05; the results are shown. For example, we found that azide induces 5 proteins of the whole-cell extract and 10 membrane proteins. 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.
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