Commensal bacterium Clostridium paraputrificum J4 produces several extracellular chitinolytic enzymes including a 62 kDa chitinase Chit62J4 active toward 4-nitrophenyl N,N'-diacetyl-β-d-chitobioside (pNGG). We characterized the crude enzyme from bacterial culture fluid, recombinant enzyme rChit62J4, and its catalytic domain rChit62J4cat. This major chitinase, securing nutrition of the bacterium in the human intestinal tract when supplied with chitin, has a pH optimum of 5.5 and processes pNGG with Km = 0.24 mM and kcat = 30.0 s-1. Sequence comparison of the amino acid sequence of Chit62J4, determined during bacterial genome sequencing, characterizes the enzyme as a family 18 glycosyl hydrolase with a four-domain structure. The catalytic domain has the typical TIM barrel structure and the accessory domains-2x Fn3/Big3 and a carbohydrate binding module-that likely supports enzyme activity on chitin fibers. The catalytic domain is highly homologous to a single-domain chitinase of Bacillus cereus NCTU2. However, the catalytic profiles significantly differ between the two enzymes despite almost identical catalytic sites. The shift of pI and pH optimum of the commensal enzyme toward acidic values compared to the soil bacterium is the likely environmental adaptation that provides C. paraputrificum J4 a competitive advantage over other commensal bacteria.
- MeSH
- bakteriální proteiny genetika metabolismus MeSH
- chitin metabolismus MeSH
- chitinasy chemie genetika metabolismus MeSH
- Clostridium růst a vývoj izolace a purifikace metabolismus MeSH
- katalytická doména MeSH
- koncentrace vodíkových iontů MeSH
- lidé MeSH
- rekombinantní proteiny genetika metabolismus MeSH
- střevní mikroflóra MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
UNLABELLED: The need for suitable selective cultivation media for the isolation of Bifidobacterium spp. continues to be a real concern in the field of intestinal microbiology. Isolation of bifidobacteria from human and animal faecal samples using selective agar plating may be problematic especially in samples with increased clostridial counts than bifidobacterial counts. Due to the absence of anticlostridial agents in existing selective media, clostridia can displace bifidobacteria resulting in incorrect estimation of their counts. Therefore, we supplemented the existing selective medium 'modified Wilkins Chalgren agar with mupirocin' (MWM) with 90 mg l(-1) of 8-hydroxyquinoline (8HQ), which was recently proved to act selectively against clostridia. The newly composed 'modified Wilkins-Chalgren agar with 8HQ' (MWMQ) was tested on pure bifidobacterial and clostridial strains, their mixtures, and using faecal samples of mammalian origin; its selectivity was evaluated by genus-specific identification of isolates. The results demonstrated that the presence of 8HQ in this agar eliminated the growth of nonbifidobacterial strains on MWMQ compared to that on MWM, whereas the recovery of bifidobacterial counts was at satisfactory levels. In conclusion, MWMQ could be recommended for bifidobacterial isolation from human and animal faeces especially when bifidobacteria are not numerically dominant and there are chances of clostridial contamination. SIGNIFICANCE AND IMPACT OF THE STUDY: Routine isolation of bifidobacteria from mammalian faeces does not use a reliable selective agar with an anticlostridial agent. Overgrowth of clostridia may result in incorrect estimation of bifidobacterial counts. Thus, in order to improve the selectivity of existing media for bifidobacterial isolation, we chose the modified Wilkins-Chalgren agar with mupirocin and supplemented it with 8-hydroxyquinoline (8HQ), a molecule that shows anticlostridial activity without affecting the growth of bifidobacteria. This newly composed medium showed enhanced selectivity and specificity compared to the original medium and therefore, can be recommended for the isolation of bifidobacteria from mammal faeces.
- MeSH
- agar farmakologie MeSH
- bakteriální nálož účinky léků MeSH
- Bifidobacterium růst a vývoj izolace a purifikace MeSH
- Clostridium účinky léků růst a vývoj MeSH
- feces mikrobiologie MeSH
- kultivační média farmakologie MeSH
- lidé MeSH
- mupirocin farmakologie MeSH
- oxychinolin farmakologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
As the number of biogas plants has grown rapidly in the last decade, the amount of potentially contaminated wastes with pathogenic Clostridium spp. has increased as well. This study reports the results from examining 203 biogas plant wastes (BGWs). The following Clostridium spp. with different frequencies could be isolated via a new enrichment medium (Krüne medium) and detected by matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS): Clostridium perfringens (58 %) then Clostridium bifermentans (27 %), Clostridium tertium (23 %) and Clostridium butyricum (19 %), Clostridium cadaveris (15 %), Clostridium parapurificum (6 %), Clostridium glycolicum (5 %), Clostridium baratii (4 %), Clostridium sporogenes (2 %), Clostridium sordellii (1 %) and Clostridium subterminale (0.5 %). The mean most probable number (MPN) count of sulfite reducing bacteria was between 10(3) and 10(4)/mL, and the higher the MPN, the more pathogenic Clostridium spp. were present. Also, real-time PCR was used to be compared with culture method for C. perfringens, C. bifermentans, C. butyricum, C. sporogenes/Clostridium botulinum and C. sordellii. Although real-time PCR was more sensitive than the culture method, both systems improve the recovery rate but in different ways and are useful to determine pathogenic clostridia in biogas plants. In conclusion, BGWs could present a biohazard risk of clostridia for humans and animals.
Changes in membrane lipid composition of Clostridium pasteurianum NRRL B-598 were studied during butanol fermentation by lipidomic analysis, performed by high resolution electrospray ionization tandem mass spectrometry. The highest content of plasmalogen phospholipids correlated with the highest butanol productivity, which indicated a probable role of these compounds in the complex responses of cells toward butanol stress. A difference in the ratio of saturated to unsaturated fatty acids was found between the effect of butanol produced by the cells and butanol added to the medium. A decrease in the proportion of saturated fatty acids during conventional butanol production was observed while a rise in the content of these acids appeared when butanol was added to the culture. The largest change in total plasmalogen content was observed one hour after butanol addition i.e. at the 7th hour of cultivation. When butanol is produced by bacterial cells, then the cells are not subjected to severe stress and responded to it by relatively slowly changing the content of fatty acids and plasmalogens, while after a pulse addition of external butanol (to a final non-lethal concentration of 0.5 % v/v) the cells reacted relatively quickly (within a time span of tens of minutes) by increasing the total plasmalogen content.
- MeSH
- biomasa MeSH
- Clostridium účinky léků růst a vývoj metabolismus MeSH
- hmotnostní spektrometrie s elektrosprejovou ionizací MeSH
- mastné kyseliny analýza MeSH
- membránové lipidy chemie MeSH
- n-butanol metabolismus farmakologie MeSH
- nenasycené mastné kyseliny analýza MeSH
- plasmalogeny analýza MeSH
- techniky vsádkové kultivace MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Highly undesirable microbial contaminants of processed cheese are endospore-forming bacteria of the genera Bacillus and Clostridium. Survival of Bacillus subtilis, B. cereus, Clostridium butyricum and C. sporogenes was examined in model processed cheese samples supplemented with monoacylglycerols. In processed cheese samples, monoacylglycerols of undecanoic, undecenoic, lauric and adamantane-1-carboxylic acid at concentration of 0.15% w/w prevented the growth and multiplication of both Bacillus species throughout the storage period. The two species of Clostridium were less affected by monoacylglycerols in processed cheese samples and only partial inhibition was observed. The effect of milk fat content on microbial survival in processed cheese was also evaluated. The growth of Bacillus sp. was affected by the fat level of processed cheese while population levels of Clostridium sp. did not differ in processed cheese samples with 30, 40 and 50% fat in dry matter.
- MeSH
- antiinfekční látky farmakologie MeSH
- Bacillus účinky léků MeSH
- Clostridium účinky léků růst a vývoj MeSH
- monoglyceridy farmakologie MeSH
- potravinářská mikrobiologie * MeSH
- potravinářské přísady farmakologie MeSH
- sýr mikrobiologie MeSH
- tuky metabolismus farmakologie MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
We endeavored to develop a method for viability determination of solventogenic clostridia and to apply it for monitoring acetone-butanol-ethanol (ABE) fermentation. Six fluorescent probes (propidium iodide [PI], ethidium bromide, fluorescein diacetate, carboxyfluorescein diacetate [cFDA], rhodamine 123, bis-(1,3-dibutylbarbituric acid)trimethine oxonol [BOX]) were tested in order to distinguish two subpopulations of live and dead clostridial cells in suspension. Three of them were found to be appropriate (PI, BOX and cFDA) for this purpose. Developed fluorescent staining methods were applied to batch fermentation processes of Clostridium pasteurianum and C. beijerinckii carried out in a laboratory bioreactor under anaerobic conditions. Whereas PI was found to be applicable to both strains, BOX was convenient only for viability determination of C. pasteurianum. Although cFDA can distinguish two cell subpopulations in suspension, it was found to be unsuitable for viability determination under tested conditions, since it reflected more variable esterase activity during sporulation cell cycle than viability. Flow cytometry in combination with convenient fluorescent probe has been proved to be a valuable tool for viability determination. We assume this rapid and simple method can help to obtain more complex and precise information about ABE fermentation.
- MeSH
- aceton metabolismus MeSH
- barvení a značení MeSH
- butanoly metabolismus MeSH
- Clostridium chemie růst a vývoj metabolismus MeSH
- ethanol metabolismus MeSH
- fermentace MeSH
- fluorescenční barviva chemie metabolismus MeSH
- mikrobiální viabilita MeSH
- průtoková cytometrie metody MeSH
- rozpouštědla metabolismus MeSH
- Publikační typ
- časopisecké články MeSH
- hodnotící studie MeSH
- práce podpořená grantem MeSH
The object of this study are chitinolytic enzymes produced by bacterium Clostridium paraputrificum J4 isolated from the gastrointestinal tract of a healthy human. In particular, we focus on the development of purification protocols, determination of properties of the enzymes and their activity profiles. The process of bacteria cultivation and isolation of chitinolytic complex of enzymes showing specific activities of endo-, exo-chitinase and N-acetyl-β-glucosaminidase was optimized. A range of various purification procedures were used such as ultrafiltration, precipitation, chromatographic separations (ion-exchange, size exclusion, chromatofocusing) in altered combinations. The optimal purification protocol comprises two or three steps. Individual samples were analyzed by SDS/PAGE electrophoresis and after renaturation their activity could be detected using zymograms. Mass spectroscopy peptide fragment analysis and MALDI analysis of the purest samples indicate presence of endochitinase B (molecular mass about 85 kDa) and of 60-kDa endo- and exochitinases.
- MeSH
- anaerobióza MeSH
- bakteriální proteiny izolace a purifikace metabolismus sekrece MeSH
- chitin metabolismus MeSH
- chitinasy izolace a purifikace metabolismus sekrece MeSH
- Clostridium enzymologie růst a vývoj MeSH
- gastrointestinální trakt mikrobiologie MeSH
- glykosidhydrolasy izolace a purifikace metabolismus sekrece MeSH
- hexosaminidasy izolace a purifikace metabolismus sekrece MeSH
- kultivační techniky metody MeSH
- lidé MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
Membrane ultrafiltration (UF) was used in sample preparation of the culture fluids of the human intestinal bacterium Clostridium paraputrificum strain J4 containing seven extracellular chitinolytic isoenzymes (38-90 kDa). The subsequent filtration of the bacteria-free supernatants was carried out through Millipore membranes with cut-off 100 and 30 kDa for separation of undigested components of the culture medium and bacterial metabolites with molecular weight higher and lower than that of the target enzymes. The chitinolytic enzymes, which were the minor components in the culture fluids, were concentrated at UF as well. The aim of the research consisted in evaluation of the effect of component composition of bacteria-free supernatants and the chemical nature of membrane active layer on partial fractionation of the chitinolytic enzymes, their recovery in retentates and purification degree. On the basis of the obtained experimental results, the sample preparation procedure of the culture fluids of C. paraputrificum J4 was established to be used further in chromatographic separations of the chitinolytic enzymes.
- MeSH
- bakteriální proteiny izolace a purifikace metabolismus MeSH
- chitinasy izolace a purifikace metabolismus MeSH
- Clostridium enzymologie růst a vývoj MeSH
- elektroforéza v polyakrylamidovém gelu MeSH
- kultivační média MeSH
- lidé MeSH
- membrány umělé MeSH
- průmyslová mikrobiologie MeSH
- střeva mikrobiologie MeSH
- ultrafiltrace metody MeSH
- Check Tag
- lidé MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
V posledních desetiletích je snahou Evropského společenství snížit závislost svých členských států na ropných zdrojích, což vede k zájmu O procesy, které využívají obnovitelné zdroje energií. Jedním z nich je proces výroby 1-butanolu, který je realizován pomocí různých solventogenních druhů klostridií. Butanol se fermentačním způsobem produkuje při aceton-butanol-ethanolové fermentaci, kdy v první fázi procesu dochází k tvorbě organických kyselin a v druhé fázi procesu, často spojené se sporulací produkčního kmene, se tvoří rozpouštědla. Dvoufázový charakter fermentace výrazně komplikuje regulaci procesu a toxicita produktů brání dosažení jejich vysoké koncentrace. Aby byla zvýšena efektivita tohoto procesu, je prováděn intenzivní výzkum, zaměřený hlavně na zlepšení produkčních vlastností kmenů, zvýšení jejich odolnosti k 1-butanolu, využití levných surovin a zvýšení celkové produktivity procesu. Pokud se podaří výrobní cenu biobutanolu snížit, mohl by díky svým výhodným vlastnostem sloužit jako kosolvent do motorového paliva, do kterého se dnes již přidává ethanol a oddálit tak nevyhnutelné vyčerpání fosilních paliv.
An effort of European Community to reduce its dependence on fossil fuels provokes increased interest on processes using renewable energy resources. One of them is the fermentation process of 1-butanol production using various types of solventogenic clostridia. Biobutanol is produced by aceton-butanol-ethanol fermentation process consisting of two phases - acidogenic and solventogenic, which is associated with sporulation. Biphasic nature of fermentation significantly complicates the process control and product toxicity prevents the achievement of high concentration of solvents. To increase the efficiency of this process, an inventive research focused on improving the production characteristics of strains, increasing their resistance to 1-butanol, the use of cheap raw materials and amelioration of overall productivity of the process is carried out. If the production cost of biobutanol would be reduced, it could be advantageously used as an additive to petrol which could postpone the depletion of fossil fuels.
Both bifidobacteria and clostridia are part of the natural gut microflora and while clostridia may be responsible for severe intestinal infections, bifidobacteria are probiotic microorganisms belonging to the most important prospective bacteria in the bowel. The antimicrobial activity of biochanin A was tested in vitro against six Bifidobacterium spp., and eight Clostridium spp. using the broth microdilution method. Biochanin A showed an inhibition against all clostridia in the range of minimum inhibitory concentrations (MIC) from 64 microg/mL (for Cl. clostridioforme, strains DSM 933 and I3) to 1,024 microg/mL (for Cl. perfringens, DSM 11778). No bifidobacteria were suppressed at four-fold higher concentration (MICs > 4,096) than MIC of Cl. perfringens. These results indicate selective growth inhibition of biochanin A and its potential use in antimicrobial prevention and/or protection.
- MeSH
- antibakteriální látky farmakologie MeSH
- Bifidobacterium klasifikace účinky léků růst a vývoj MeSH
- Clostridium klasifikace účinky léků růst a vývoj MeSH
- druhová specificita MeSH
- genistein farmakologie MeSH
- lidé MeSH
- mikrobiální testy citlivosti MeSH
- střeva mikrobiologie MeSH
- zvířata MeSH
- Check Tag
- lidé MeSH
- zvířata MeSH
- Publikační typ
- časopisecké články MeSH
- práce podpořená grantem MeSH
