Stress proteomes of the cytoplasmic membrane fraction of Bacillus subtilis trp (C2)-exposed to acid pH and ethanol were characterized. Although these stress factors impair the cell function in a specific manner, they share the ability to denature proteins. Therefore, specific and general stress proteins in the membranes were investigated. Both ethanol (3 %) and pH 5.0 increase the doubling time from 17 to 25 min. Isolated cytoplasmic membranes were subjected to an optimized 2D PAGE analysis which permitted the separation and analysis of ?450 distinct protein spots. Two alternative methods of protein detection were compared, i.e. silver staining and (35)S-L-methionine pulse labeling; the stress induced proteins were identified by MALDI-TOF MS. After ethanol stress, five proteins were increased, viz. YdaP, Ctc, YfhM, YjcH and YwaC. Acid stress proteins were AcoB, YkwC, SodA, YjcH and YwaC. Proteins YjcH and YwaC were increased after ethanol as well as acid pH treatment.
- MeSH
- 2D gelová elektroforéza metody MeSH
- Bacillus subtilis fyziologie genetika metabolismus MeSH
- bakteriální proteiny genetika metabolismus MeSH
- buněčná membrána metabolismus MeSH
- ethanol farmakologie MeSH
- financování organizované MeSH
- koncentrace vodíkových iontů MeSH
- methionin MeSH
- proteiny teplotního šoku genetika metabolismus MeSH
- proteom MeSH
- reakce na tepelný šok MeSH
- regulace genové exprese u bakterií MeSH
- spektrometrie hmotnostní - ionizace laserem za účasti matrice MeSH
The IRESite (http://www.iresite.org) presents carefully curated experimental evidence of many eukaryotic viral and cellular internal ribosome entry site (IRES) regions. At the time of submission, IRESite stored >600 records. The IRESite gradually evolved into a robust tool providing (i) biologically meaningful information regarding the IRESs and their experimental background (including annotation of IRES secondary structures and IRES trans-acting factors) as well as (ii) thorough concluding remarks to stored database entries and regularly updated evaluation of the reported IRES function. A substantial portion of the IRESite data results purely from in-house bioinformatic analyses of currently available sequences, in silico attempts to repeat published cloning experiments, DNA sequencing and restriction endonuclease verification of received plasmid DNA. We also present a newly implemented tool for displaying RNA secondary structures and for searching through the structures currently stored in the database. The supplementary material contains an updated list of reported IRESs.
- MeSH
- databáze genetické MeSH
- databáze nukleových kyselin MeSH
- genom virový MeSH
- iniciace translace peptidového řetězce genetika MeSH
- internet MeSH
- konformace nukleové kyseliny MeSH
- lidé MeSH
- plazmidy metabolismus MeSH
- ribozomy genetika MeSH
- RNA virová genetika MeSH
- sekvenční analýza DNA MeSH
- software MeSH
- ukládání a vyhledávání informací metody MeSH
- výpočetní biologie metody trendy MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- práce podpořená grantem MeSH
Processes occurring in the cytoplasmic membrane of the surfactin producer Bacillus subtilis were examined during a 3-d cultivation. The fatty acid composition was found to be almost stable within this interval, except for the early stationary phase when the nonbranched, mostly C(16:0) and C(18:0) (high melting fatty acids), prevailed transiently in the membrane. As for phospholipids, phosphatidylglycerol and phosphatidylethanolamine, representing 73 % of the total in the membranes of exponential cells were partly replaced by cardiolipin, which gradually rose from 5 to 28 % at the end of cultivation. In parallel, steady-state fluorescence anisotropy (r (s)) measurements with 1,6-diphenyl-1,3,5-hexatriene (DPH) indicated a remarkable increase of r (s) DPH during the long-term cultivation and implied a continuous rigidization of the membrane interior. By contrast, the almost constant values of r (s) 1-(4-trimethylammoniumphenyl)-6-phenyl-1,3,5-hexatriene 4-toluenesulfonate (TMA-DPH) reflected stable microviscosity of the membrane surface region. Thus, the significant increase of high melting fatty acids and cardiolipin in the cytoplasmic membrane together with the progressive rigidization of the membrane interior reflected the cell adaptation to adverse conditions.
Lateral heterogeneity in the cytoplasmic membrane of Bacillus subtilis was found by using density gradient centrifugation. Crude membranes (CM) present in the whole cell lysate were separated into three fractions of increasing density (F, CI, CII). Substantial difference exists in the amount of protein recovered from these fractions, the relative ratio being 15 : 35 : 50. The qualitative protein composition (by SDS-PAGE) of the fractions varies markedly as well. The lipid components extracted from the fractions are also distributed in different proportions, viz. 40 : 40 : 20. The spectrum of fatty acids (FA), detected in lipids of F fraction and analyzed by GC-MS exhibits the same profile as that found in CM; in contrast, fractions CI and CII undergo extensive FA reconstruction. Thermotropic behavior of fractions measured by the steady-state fluorescence anisotropy of 1,6-diphenyl-1,3,5-hexatriene indicates significant variations of microviscosity (r(s)) within the F, CI and CII fractions. The protein-to-lipid ratio plays evidently a key role in affecting the physical state of the cytoplasmic membrane. Microdomains of different density coexist in the membrane and exhibit heterogeneity in both chemical composition and "physical state"; the increased de novo synthesis of FA induced by the cold exclusively in fractions CI and CII indicates correlation with an altered physiological state of bacterial metabolism.
- MeSH
- Bacillus subtilis izolace a purifikace růst a vývoj MeSH
- chromatografie plynová metody využití MeSH
- cytoplazmatické struktury chemie mikrobiologie MeSH
- elektroforéza v polyakrylamidovém gelu metody využití MeSH
- finanční podpora výzkumu jako téma MeSH
- fluorescenční polarizace metody využití MeSH
- membránové lipidy chemie izolace a purifikace MeSH
- membránové mikrodomény chemie mikrobiologie MeSH
- membránové proteiny chemie izolace a purifikace MeSH
