D 2004

Proteome study on bacterium Paracoccus denitrificans and construction of 2-DE protein database

BOUCHAL, Pavel, Petra PŘECECHTĚLOVÁ, Zbyněk ZDRÁHAL and Igor KUČERA

Basic information

Original name

Proteome study on bacterium Paracoccus denitrificans and construction of 2-DE protein database

Name in Czech

Proteomová analýza bakterie Paracoccus denitrificans a konstrukce 2-DE databáze bílkovin

Authors

BOUCHAL, Pavel (203 Czech Republic, guarantor), Petra PŘECECHTĚLOVÁ (203 Czech Republic), Zbyněk ZDRÁHAL (203 Czech Republic) and Igor KUČERA (203 Czech Republic)

Edition

Brno, VIII. Pracovní setkání biochemiků a molekulárních biologů. Sborník příspěvků. p. 5-5, 2004

Publisher

Masarykova univerzita v Brně

Other information

Language

English

Type of outcome

Stať ve sborníku

Field of Study

10600 1.6 Biological sciences

Country of publisher

Czech Republic

Confidentiality degree

není předmětem státního či obchodního tajemství

RIV identification code

RIV/00216224:14310/04:00019741

Organization unit

Faculty of Science

ISBN

80-210-3321-5

Keywords in English

Paracoccus denitrificans; Proteome; Database
Změněno: 25/5/2006 12:04, doc. Mgr. Pavel Bouchal, Ph.D.

Abstract

V originále

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.

In Czech

Celobuněčné lyzáty a membránové frakce bakterie Paracoccus denitrificans byly analyzovány pomocí dvourozměrné elektroforézy s imobilizovanými pH gradienty s cílem charakterizovat rozdíly v expresi proteinů během růstu za aerobních a několika typech anaerobních podmínek (s dusičnanem, dusitanem a oxidem dusným) a dále během růstu v přítomnosti respiračního inhibitoru azidu. Komparativní obrazová analýza 2-D gelů odhalila několik skupin z celkového počtu 800 proteinových skvrn, které byly charakteristicky indukovány anebo potlačeny v odpovědi na daný terminální akceptor elektronů. Také respirační inhibitor azid vykazoval významné ovlivnění proteinových profilů. Omezená informace o genomu studované bakterie neumožnila identifikovat více než 8 fyziologicky významných proteinů. V současné době se připravuje veřejné zpřístupění 2-DE databáze P.denitrificans.

Links

GA203/01/1589, research and development project
Name: Mechanizmy regulace respiračních systémů denitrifikačních bakterií faktory prostředí
Investor: Czech Science Foundation, Mechanisms involved in regulation of respiratory systems in denitrification bacteria by environmental factors
MSM 143100005, plan (intention)
Name: Strukturně-funkční vztahy biomolekul a jejich role v metabolismu
Investor: Ministry of Education, Youth and Sports of the CR, Biomolecular Structure-function Relationships and their role in the Metabolism
MSM 143100008, plan (intention)
Name: Genomy a jejich funkce
Investor: Ministry of Education, Youth and Sports of the CR, Genomes and their functions