Application of gas chromatography-mass spectrometry (GC-MS) can significantly improve trace analyses of compounds in complex matrices from natural environments compared to gas chromatography only. A GC-MS/MS technique for determination of poly-beta-hydroxybutyrate (PHB), a bacterial storage compound, has been developed and used for analysis of two soils stored for up to 319 d, fresh samples of sewage sludge, as well as a pure culture of Bacillus megaterium. Specific derivatization of beta-hydroxybutyrate (3-OH C4:0) PHB monomer units by N-tert-butyl-dimethylsilyl-N-methyltrifluoracetamide (MTBSTFA) improved chromatographic and mass spectrometric properties of the analyte. The diagnostic fragmentation scheme of the derivates tert-butyldimethylsilyl ester and ether of beta-hydroxybutyric acid (MTBSTFA-HB) essential for the PHB identification was shown. The ion trap MS was used, therefore the scan gave the best sensitivity and with MS/MS the noise decreased, so the S/N was better and also with second fragmentation the amount of ions increased compared to SIM. The detection limit for MTBSTFA-HB by GC-MS/MS was about 10(-13) g microL(-1) of injected volume, while by GC (FID) and GC-MS (scan) it was around 10(-10) g microL(-1) of injected volume. Sensitivity of GC-MS/MS measurements of PHB in arable soil and activated sludge samples was down to 10 pg of PHB g(-1) dry matter. Comparison of MTBSTFA-HB detection in natural soil sample by GC (FID), GC-MS (scan) and by GC-MS/MS demonstrated potentials and limitations of the individual measurement techniques.

• MeSH
• Bacillus megaterium chemie   růst a vývoj   MeSH
• glykolipidy analýza   MeSH
• hydroxybutyráty analýza   MeSH
• látky znečišťující půdu analýza   MeSH
• látky znečišťující životní prostředí analýza   MeSH
• odpadní vody analýza   MeSH
• plynová chromatografie s hmotnostně spektrometrickou detekcí metody   MeSH
• polyestery analýza   MeSH
• senzitivita a specificita MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• glykolipidy MeSH
• hydroxybutyráty MeSH
• látky znečišťující půdu MeSH
• látky znečišťující životní prostředí MeSH
• odpadní vody MeSH
• poly-beta-hydroxybutyrate MeSH Prohlížeč
• polyestery MeSH

Elevated concentration of NaCl in liquid medium caused a concentration-dependent growth delay (adaptation lag) and decrease in the maximal growth rate of Bacillus megaterium. The adaptation to salt stress was accompanied by transformation of some otherwise stable (long-lived; LLP) cell proteins into quickly degraded (short-lived; SLP) ones. Exposure to the strongly growth-reducing 1 M NaCl increased the size of the SLP 'pool' of intracellular proteins from about 5 to about 15% of total protein. The major intracellular proteolytic capacity of B. megaterium is represented by intracellular serine proteinases (ISP). Paradoxically, their specific activity was lowered or masked during the adaptation phase marked by increased catabolism of short-lived and/or destabilized proteins by the stress. This documents that intracellular proteolytic activity cannot be a key regulator of protein catabolism during adaptation to stress.

• MeSH
• Bacillus megaterium růst a vývoj   metabolismus   ultrastruktura   MeSH
• bakteriální proteiny metabolismus   MeSH
• chlorid sodný farmakologie   MeSH
• elektronová mikroskopie MeSH
• fyziologická adaptace * MeSH
• kinetika MeSH
• kultivační média MeSH
• osmotický tlak MeSH
• serinové endopeptidasy metabolismus   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chlorid sodný MeSH
• kultivační média MeSH
• serinové endopeptidasy MeSH

The total proteolytic activity of ISP1 determined after its partial purification by size exclusion HPLC increased 3.0, 7.3 and 27.3 times in sporulating, growing and netropsin-treated cells, respectively, as compared with the corresponding original activity in crude cytoplasmic preparations at the same CaCl2 concentration of 3 mM. A similar rise in proteolytic activity occurred on increasing the Ca2+ concentration in the crude cytoplasm from 3 to 30 mM. This activation in the cytoplasm of netropsin-treated and sporulating cells was not reversed by subsequent lowering of CaCl2 concentration back to 3 mM by dialysis. Moreover, a similar activation appeared even after the same dialysis of cytoplasm that had not been exposed to 30 mM CaCl2. The activation was probably due to the processing of ISP1, as established by SDS-PAGE and immunoblotting, but an involvement of additional regulation factor(s), e.g. an inhibitor or other molecules, is possible.

• MeSH
• aktivace enzymů MeSH
• antibakteriální látky farmakologie   MeSH
• Bacillus megaterium enzymologie   růst a vývoj   fyziologie   MeSH
• chlorid vápenatý farmakologie   MeSH
• fenylmethylsulfonylfluorid farmakologie   MeSH
• molekulová hmotnost MeSH
• netropsin farmakologie   MeSH
• serinové endopeptidasy chemie   izolace a purifikace   metabolismus   MeSH
• spory bakteriální MeSH
• vápník metabolismus   farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH
• Názvy látek
• antibakteriální látky MeSH
• chlorid vápenatý MeSH
• fenylmethylsulfonylfluorid MeSH
• netropsin MeSH
• serinové endopeptidasy MeSH
• vápník MeSH

Cells of Bacillus megaterium 27 were challenged by a 30-min heat shock at 45 degrees C during various sporulation stages and then shifted back to a temperature permissive for sporulation (27 degrees C), at which they developed spores. Heat shock applied at 120 min after the end of the exponential phase induced synthesis of heat shock proteins (HSPs) in the sporangia and delayed the inactivation of spores at 85 degrees C. Several HSPs, mainly HSP 70, could be detected in the cytoplasm of these spores. An analogous HSP, the main HSP induced by increased temperature during growth, belongs to the GroEL group according to its N-terminal sequence. The identity of this protein was confirmed by Western blot (immunoblot) analysis with polyclonal antibodies against B. subtilis GroEL. Sporangia treated by heat shock immediately or 240 min after exponential phase also synthesized HSPs, but none of them could be detected in the spores in an appreciable amount. These spores showed only a slightly increased heat resistance.

• MeSH
• autoradiografie MeSH
• Bacillus megaterium růst a vývoj   metabolismus   fyziologie   MeSH
• bakteriální proteiny biosyntéza   izolace a purifikace   MeSH
• časové faktory MeSH
• chaperon hsp60 MeSH
• cytoplazma metabolismus   MeSH
• elektroforéza v polyakrylamidovém gelu MeSH
• kinetika MeSH
• molekulární sekvence - údaje MeSH
• molekulová hmotnost MeSH
• proteiny tepelného šoku biosyntéza   izolace a purifikace   MeSH
• radioizotopy síry MeSH
• sekvence aminokyselin MeSH
• sírany metabolismus   MeSH
• spory bakteriální fyziologie   MeSH
• vysoká teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• chaperon hsp60 MeSH
• proteiny tepelného šoku MeSH
• radioizotopy síry MeSH
• sírany MeSH
• sodium sulfate MeSH Prohlížeč

The anticancer drug netropsin increases the synthesis of an exocellular metalloproteinase during exponential growth as well as in the stationary phase of a sporulating strain of Bacillus megaterium. Its effect is due to a stimulation of the synthesis of the mRNA coding for the proteinase, determined as a residual synthesis of the enzyme in the presence of actinomycin D. The half-life of the proteinase mRNA (5-6 min at 35 degrees C) is not affected by netropsin. Netropsin relieves partially the repression of the proteinase mRNA caused by amino acids, whereas the repression brought about by an increased temperature is almost unaffected by the drug.

• MeSH
• aminokyseliny farmakologie   MeSH
• Bacillus megaterium účinky léků   enzymologie   genetika   růst a vývoj   fyziologie   MeSH
• genetická transkripce účinky léků   MeSH
• guanidiny farmakologie   MeSH
• kultivační média MeSH
• messenger RNA biosyntéza   MeSH
• metaloendopeptidasy biosyntéza   genetika   MeSH
• netropsin farmakologie   MeSH
• poločas MeSH
• proteosyntéza účinky léků   MeSH
• regulace genové exprese účinky léků   MeSH
• spory bakteriální MeSH
• teplota MeSH
• tetracyklin farmakologie   MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• guanidiny MeSH
• kultivační média MeSH
• messenger RNA MeSH
• metaloendopeptidasy MeSH
• netropsin MeSH
• tetracyklin MeSH

Batch cultures of Bacillus megaterium grown in phosphate-limited media were compared with control cultures grown in phosphate-sufficient media. The effects of phosphate limitation on growth were determined by viable cells counts. Intracellular levels of protein, RNA, poly-3-hydroxybutyrate, carbohydrate and oxygen uptake were significantly affected by phosphate limitation. Electron micrographs of sectioned cells revealed differences in the structure; in particular the thick, rigid cell wall was absent from cells grown in phosphate-limited media, and such cells were larger, pleomorphic, and after 2 d were insensitive to lysozyme.

• MeSH
• Bacillus megaterium analýza   růst a vývoj   fyziologie   ultrastruktura   MeSH
• bakteriální proteiny analýza   MeSH
• bakteriální RNA analýza   MeSH
• buněčná stěna ultrastruktura   MeSH
• časové faktory MeSH
• elektronová mikroskopie MeSH
• fosfáty metabolismus   farmakologie   MeSH
• kultivační média MeSH
• muramidasa farmakologie   MeSH
• spory bakteriální fyziologie   MeSH
• spotřeba kyslíku MeSH
• Publikační typ
• časopisecké články MeSH
• srovnávací studie MeSH
• Názvy látek
• bakteriální proteiny MeSH
• bakteriální RNA MeSH
• fosfáty MeSH
• kultivační média MeSH
• muramidasa MeSH

Germinated spores of Bacillus megaterium were mutagenized with ethyl methanesulphonate and spread on test agar with caseinate. Colonies with altered proteolytic zones or morphology were isolated and tested in liquid media. The mutants can be divided into four groups: A) those producing more proteinase in both growth and sporulation media, B) those producing the same amount of the enzyme in growth medium but higher amount in sporulation medium, C) those producing less proteinase in the growth medium and more in the sporulation one, D) those producing less or no enzyme. Clones of the first three groups were phenotypically asporogenic. All mutants producing more enzyme during growth retained their sensitivity to repression by amino acids. Isolation of mutants of types B) and C) supports the idea of differences in the control of proteinase synthesis during growth and during sporulation.

• MeSH
• Bacillus megaterium enzymologie   genetika   růst a vývoj   MeSH
• endopeptidasy biosyntéza   MeSH
• kultivační média MeSH
• mutace * MeSH
• neprilysin MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• endopeptidasy MeSH
• kultivační média MeSH
• neprilysin MeSH

Protein turnover was followed in populations of Bacillus megaterium growing in temperature range of 17-48 degrees C in different media. Higher temperature stimulated the protein turnover (expressed as the amount of protein degraded during 3.3 h) in all the media tested up to the optimal growth temperature (40-42 degrees C). Protein turnover in a medium containing amino acids continued to be stimulated by temperature even above this point; no further significant increase of turnover was found in the other media.

• MeSH
• aminokyseliny farmakologie   MeSH
• Bacillus megaterium růst a vývoj   metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• kinetika MeSH
• kultivační média MeSH
• teplota MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• bakteriální proteiny MeSH
• kultivační média MeSH

Synthesis of exocellular metalloprotease and cellular and exocellular proteins in the sporogenic strain Bacillus megaterium J-27 and asporogenic strain KM 1 was investigated. Both organisms excrete the enzyme into the medium during growth and during the stationary phase. In the asporogenic strain the excretion decreases at the end of the exponential phase. In the sporogenic strain it continues during the transition to the stationary phase at the original rate and proteolytic activity in the medium increases two to three times during 2 h after the end of the exponential phase. Both organisms synthesize relatively more exocellular proteins during the exponential phase than during the stationary phase. The proportion of exocellular protein synthesized during the exponential phase does not exceed 3 % of total proteins, during the stationary phase this proportion usually decreases to less than 1 %.

• MeSH
• Bacillus megaterium růst a vývoj   metabolismus   MeSH
• bakteriální proteiny biosyntéza   MeSH
• druhová specificita MeSH
• endopeptidasy biosyntéza   MeSH
• metaloendopeptidasy MeSH
• spory bakteriální MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• bakteriální proteiny MeSH
• endopeptidasy MeSH
• metaloendopeptidasy MeSH

The rate of protein turnover in asporogenic Bacillus megaterium decreases continuously during incubation in a sporulation medium. The capability of equilibration of external amino acids with amino acids in the metabolic pool of non-growing cells was retained for at least 5 h. Leucine, while repressing the synthesis of the exocellular protease, does not significantly influence the course of protein degradation in vivo. Transfer of non-growing cells after 4 h to a fresh sporulation medium does not influence the rate of protein degradation. The gradual decrease of the rate of protein turnover in non-growing cells of the asporogenic variant is thus not an artifact caused by a decreased uptake of amino acids by cells or by conditions under which the protein turnover is determined.

• MeSH
• aminokyseliny metabolismus   MeSH
• Bacillus megaterium růst a vývoj   metabolismus   MeSH
• bakteriální proteiny metabolismus   MeSH
• kinetika MeSH
• kultivační média MeSH
• leucin metabolismus   MeSH
• spory bakteriální MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• aminokyseliny MeSH
• bakteriální proteiny MeSH
• kultivační média MeSH
• leucin MeSH