Browsing by Subject "Denaturierende Gradienten-Gelelektrophorese"

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    Ecological studies of the Lactobacillus biota in the human digestive tract and adaptation of intestinal lactobacilli to the sourdough ecosystem
    (2005) Dal Bello, Fabio; Hertel, Christian
    Among the bacteria inhabiting the human gut, lactobacilli have received considerable attention, due to their putative health promoting effects (Reid, 1999; Vaughan et al., 1999). Cultivation of lactobacilli is considered to be reliable and numerous studies using plating on selective media have been performed to investigate these bacteria in intestinal ecosystems (Tannock, 1995; Reuter, 2001). Recently, the application of PCR-DGGE in combination with primers specific for lactic acid bacteria (LAB) detected species which are not considered to be intestinal inhabitants but food-associated, such as Lactobacillus curvatus, Lactobacillus sakei, Leuconostoc mesenteroides and Pediococcus pentosaceus (Walter et al., 2001; Heilig et al., 2002). Remarkably, these species could not be recovered by traditional bacteriological culture on Rogosa SL agar (Walter et al., 2001). In Chapter III, different cultivation media, as well as new incubation conditions were applied to overcome these difficulties. Human faecal samples were plated on selective and non-selective media and incubated under standard condition (37°C, anaerobiosis) for faecal LAB as well as alternative condition (30°C, 2% O2). PCR-DGGE analyses of resuspended bacterial biomass (RBB) obtained from agar plates revealed that the species composition of the recovered LAB was affected stronger by the incubation condition than by the used medium. It was observed that food-associated LAB such as L. sakei and Lc. mesenteroides, hitherto not described as intestinal inhabitants, are more easily selected when the alternative incubation condition is used. Identification of randomly picked colonies grown under the alternative condition on Rogosa SL agar showed that L. sakei is one of the predominant food-associated LAB species in faecal samples, reaching counts of up to 106 CFU per gram faeces. Comparison of the results of bacteriological culture with those obtained by PCR-DGGE analysis of the RBB showed that investigation of RBB is a fast and reliable method to gain insight into the species composition of culturable LAB in faeces. Examination of the faecal Lactobacillus populations over longer periods has revealed marked variation in the complexity and stability of these populations among human subjects (Vanhoutte et al., 2004, Walter et al., 2001). Ecological studies indicate that most Lactobacillus species found in the human gastrointestinal tract (GIT) are likely to be transient (allochthonous), originating from either the oral cavity or food (reviewed in Bibiloni et al., 2004). In order to investigate if oral lactobacilli constitute a part of the faecal Lactobacillus biota, the Lactobacillus biota of saliva and faeces of three human subjects were investigated and compared at two time-points in a three months interval (Chapter IV). The species composition of the Lactobacillus biota of human saliva and faeces was found to be subject-specific and fluctuated to some degree, but the species Lactobacillus gasseri, Lactobacillus paracasei, Lactobacillus rhamnosus and Lactobacillus vaginalis were detected at both time-points in saliva and faecal samples of individual subjects. RAPD-PCR analysis indicated that several strains of these species were present both in the oral cavity and in the faecal samples of the same subject. Oral isolates of the species L. gasseri and L. vaginalis showing identical RAPD types were found to persist over time, suggesting that these species are autochthonous to the oral cavity. The results of Chapter IV, together with recently published data (reviewed in Bibiloni et al., 2004), give strong evidence that some lactobacilli found in human faeces are allochthonous to the intestine and originate from the oral cavity. Lactobacilli have been detected in diverse environments and have been the subject of considerable research due to their commercial use in the food industry (reviewed in Hammes and Hertel, 2003). Several Lactobacillus species are commonly detected in both fermented food and the human GIT, but the genetic background for this ecological versatility is poorly understood. Lactobacillus reuteri is a dominant member of the microbiota of type II sourdough fermentations (Meroth et al., 2003) and is considered one of the truly autochthonous Lactobacillus species in humans (Reuter, 2001). The in vivo expression technology (IVET) developed by Walter et al. (2003) was used to identify genes (so-called ivi genes) of the sourdough isolate L. reuteri LTH5531 that show elevated levels of expression during growth of this organism in a type II sourdough fermentation (Chapter V) and during passage through the GIT of mice (Chapter VI). Thirty-eight induced fusions were found to be highly expressed during the sourdough fermentation (Chapter V), and 29 genes could be identified on the basis of the available sequence information. Four genes encoded stress-related functions (e.g. acid and general stress response) reflecting the harsh conditions prevailing during sourdough fermentation. Further eight genes were involved in acquisition and synthesis of amino acids and nucleotides, indicating their limited availability in sourdough. The remaining genes were either part of functionally unrelated pathways or encoded hypothetical proteins. The identification of a putative proteinase and a component of the arginine deiminase pathway are of technological interest, as they are potentially involved in the formation of aroma precursors. Remarkably, IVET with the genomic library that was successfully used in the sourdough study (Chapter V) did not detect ivi promoters when LTH5531 inhabited the GIT of mice (Chapter VI). With IVET, active promoters are selected by expression of an "essential growth factor" (in our system the erythromycin resistance mediated by ErmGT) that allows the organism to colonize and/ or grow in the ecosystem (Rainey, 1999, Walter et al., 2003). Expression of ivi promoters in particular ecosystems must therefore be permanent and strong in order to allow comparable growth rates of ivi clones and clones bearing constitutive promoters, especially in the GIT, where inactive bacteria are washed out. The findings of Chapter V and VI indicate that L. reuteri LTH5531 does not possess strongly expressed "GIT inducible" genes, while possessing 38 ones specifically induced in sourdough. Ivi genes are more likely to contribute to the ecological performance of an organism in a specific environment than genes expressed equally in a broad range of habitats (Rainey, 1999, Gal et al, 2003, Walter et al., 2005). Therefore, traits encoded by ivi genes are likely to be adaptive and the extent of their expression would be shaped by natural selection to improve ecological fitness. The presence of thirty-eight "sourdough specific" ivi fusions in L. reuteri LTH5531 probably reflects the long term adaptation of LTH5531 to the sourdough environment, just as ivi genes detected in strain 100-23 reflect adaptation of this GIT isolate to the rodent GIT (Walter et al., 2003). Indeed, LTH5531 was isolated from an experimental sourdough that had been inoculated with an industrial starter. This industrial starter has been propagated over several years, giving the organisms present sufficient time to adapt. In accordance with this, by using RAPD-PCR, Meroth et al. (2003) showed that strain LTH5531 was present in a commercial type II sourdough starter collected 10 years prior isolation of LTH5531, thus indicating that this strain has adapted to the sourdough environment for at least 10 years. The results of Chapter V clearly demonstrated that knowledge of gene expression and metabolic activities of bacteria during food fermentations can be obtained by applying IVET. The results collected provide an important molecular basis on which improved starter strains can be developed for industrial exploitation. Moreover, the results of Chapter VI show the importance of working with highly adapted, autochthonous strains in studies of microbial ecology in order to reveal the adaptive interactions responsible for the ecological success of these bacteria in their natural environment or during food fermentations.
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    Function and composition of the soil microbial community in calcareous grassland exposed to elevated atmospheric carbon dioxide
    (2003) Ebersberger, Diana; Kandeler, Ellen
    Terrestrial ecosystems generally respond to rising atmospheric carbon dioxide (CO2) concentrations with increased net primary productivity and increased water use efficiency. This may change the amount and quality of organic substances entering the soil and fuelling microbial metabolism. Soil microorganisms and their activity might also be affected by increased soil moisture at elevated CO2. This thesis was designed to analyse the response of the soil microbial community in a species-rich calcareous grassland in the Swiss Jura Mountains, which had been exposed to ambient and elevated CO2 concentrations (365 and 600 ppm) for six growing seasons. In the first study, laboratory incubation experiments were conducted to explore the relationship between litter quality under elevated carbon dioxide and enzymes involved in carbon cycling. Naturally senescent, mixed litter from the long-term field experiment was incubated with soil material for 10, 30 and 60 days. Soil samples were then obtained close to the litter layer using a microtome cutting device. Litter and soil samples were analysed for invertase and xylanase activity. The lower litter quality produced under elevated CO2, i.e. wider C/N ratio, yielded lower invertase and xylanase activities of litter. Litter addition stimulated activities in adjacent soil. Invertase activities of adjacent soil were not affected by litter quality, while soil xylanase activity was higher in soil compartments adjacent to litter from elevated CO2 plots. The reduced enzyme activities of litter produced under elevated CO2 can slow decomposition, at least during the initial stages. Since the effects of litter quality on enzyme activities in adjacent soil were small, we conclude that CO2-induced belowground C-inputs (e.g. increased root mass) and altered moisture conditions are more important controls of enzyme activities than altered litter quality. In the second study, functional diversity of the soil microbial community was assessed by analysing N-mineralisation and activities of enzymes of the C-, N-, P- and S-cycle of soil samples taken in spring and summer 1999, in the 6th season of CO2 exposure. In spring, N-mineralisation increased significantly by 30% at elevated CO2, while there was no significant difference between treatments in summer. The response of soil enzymes to CO2 enrichment was also more pronounced in spring, when alkaline phosphatase and urease activities were increased most strongly, by 32% and 21%, respectively. In summer, activity differences between CO2 treatments were greatest in the case of urease and protease (+21% and +17% at elevated CO2). The significant stimulation of N-mineralisation and enzyme activities at elevated CO2 was probably caused by higher soil moisture and/or increased root biomass. In the third study, soil microbial community structure of soil samples taken in spring and summer 1999 was analysed by means of PLFA profiles and 16S rDNA fingerprints obtained by PCR-DGGE. PLFA profiles were not affected by elevated CO2. Ordination analysis of DNA fingerprints revealed a significant relation between CO2 enrichment and variation in DNA fingerprints. This variation must be attributed to low intensity bands because dominant bands did not differ between treatments. Diversity of the bacterial community (number of bands in DNA fingerprints and Shannon indices) was not affected. The observed minute, but significant changes in the structure of the soil bacterial community might be caused by changes in the quality of rhizodeposits at elevated CO2. These could either result from altered rhizodeposition of individual plants or from altered species composition of the calcareous grassland.The 4th part of the thesis compiles data on soil microorganisms, soil fauna, soil structure and nitrogen cycle of calcareous grassland after CO2 exposure for six growing seasons. Microbial biomass, soil basal respiration and the metabolic quotient were not altered significantly. PLFA analysis revealed no significant shift in the ratio of fungi to bacteria. Protozoans, bacterivorous and fungivorous nematodes, acarians, collembolans, and root-feeding nematodes were not affected by elevated CO2. Total nematode numbers averaged slightly lower (-16%) and nematode mass was significantly reduced (by 43%) due to fewer large-diameter nematodes classified as omnivorous and predacious. CO2 exposure resulted in a shift towards smaller aggregate sizes; this was caused by higher soil moisture. Reduced aggregate sizes result in reduced pore neck diameters. This can confine the locomotion of large-diameter nematodes and possibly accounts for their decrease. The CO2 enrichment also affected the nitrogen cycle. N stocks in living plants and surface litter increased, but N in soil organic matter and microorganisms remained unaltered. N mineralisation increased considerably, but microbial N did not differ between treatments, indicating that net N immobilization rates were unaltered.