Showing papers in "Applied and Environmental Microbiology in 1999"

Direct Analysis of Genes Encoding 16S rRNA from Complex Communities Reveals Many Novel Molecular Species within the Human Gut

Abstract: The human intestinal tract harbors a complex microbial ecosystem which plays a key role in nutrition and health. Although this microbiota has been studied in great detail by culture techniques, microscopic counts on human feces suggest that 60 to 80% of the observable bacteria cannot be cultivated. Using comparative analysis of cloned 16S rRNA gene (rDNA) sequences, we have investigated the bacterial diversity (both cultivated and noncultivated bacteria) within an adult-male fecal sample. The 284 clones obtained from 10-cycle PCR were classified into 82 molecular species (at least 98% similarity). Three phylogenetic groups contained 95% of the clones: the Bacteroides group, the Clostridium coccoides group, and the Clostridium leptum subgroup. The remaining clones were distributed among a variety of phylogenetic clusters. Only 24% of the molecular species recovered corresponded to described organisms (those whose sequences were available in public databases), and all of these were established members of the dominant human fecal flora (e.g., Bacteroides thetaiotaomicron, Fusobacterium prausnitzii, and Eubacterium rectale). However, the majority of generated rDNA sequences (76%) did not correspond to known organisms and clearly derived from hitherto unknown species within this human gut microflora.

Bactericidal activity of photocatalytic TiO(2) reaction: toward an understanding of its killing mechanism.

Abstract: When titanium dioxide (TiO(2)) is irradiated with near-UV light, this semiconductor exhibits strong bactericidal activity. In this paper, we present the first evidence that the lipid peroxidation reaction is the underlying mechanism of death of Escherichia coli K-12 cells that are irradiated in the presence of the TiO(2) photocatalyst. Using production of malondialdehyde (MDA) as an index to assess cell membrane damage by lipid peroxidation, we observed that there was an exponential increase in the production of MDA, whose concentration reached 1.1 to 2.4 nmol. mg (dry weight) of cells(-1) after 30 min of illumination, and that the kinetics of this process paralleled cell death. Under these conditions, concomitant losses of 77 to 93% of the cell respiratory activity were also detected, as measured by both oxygen uptake and reduction of 2,3,5-triphenyltetrazolium chloride from succinate as the electron donor. The occurrence of lipid peroxidation and the simultaneous losses of both membrane-dependent respiratory activity and cell viability depended strictly on the presence of both light and TiO(2). We concluded that TiO(2) photocatalysis promoted peroxidation of the polyunsaturated phospholipid component of the lipid membrane initially and induced major disorder in the E. coli cell membrane. Subsequently, essential functions that rely on intact cell membrane architecture, such as respiratory activity, were lost, and cell death was inevitable.

Key Physiology of Anaerobic Ammonium Oxidation

Abstract: The physiology of anaerobic ammonium oxidizing (anammox) aggregates grown in a sequencing batch reactor was investigated quantitatively. The physiological pH and temperature ranges were 6.7 to 8.3 and 20 to 43°C, respectively. The affinity constants for the substrates ammonium and nitrite were each less than 0.1 mg of nitrogen per liter. The anammox process was completely inhibited by nitrite concentrations higher than 0.1 g of nitrogen per liter. Addition of trace amounts of either of the anammox intermediates (1.4 mg of nitrogen per liter of hydrazine or 0.7 mg of nitrogen per liter of hydroxylamine) restored activity completely.

Evaluation and Optimization of DNA Extraction and Purification Procedures for Soil and Sediment Samples

Abstract: We compared and statistically evaluated the effectiveness of nine DNA extraction procedures by using frozen and dried samples of two silt loam soils and a silt loam wetland sediment with different organic matter contents. The effects of different chemical extractants (sodium dodecyl sulfate [SDS], chloroform, phenol, Chelex 100, and guanadinium isothiocyanate), different physical disruption methods (bead mill homogenization and freeze-thaw lysis), and lysozyme digestion were evaluated based on the yield and molecular size of the recovered DNA. Pairwise comparisons of the nine extraction procedures revealed that bead mill homogenization with SDS combined with either chloroform or phenol optimized both the amount of DNA extracted and the molecular size of the DNA (maximum size, 16 to 20 kb). Neither lysozyme digestion before SDS treatment nor guanidine isothiocyanate treatment nor addition of Chelex 100 resin improved the DNA yields. Bead mill homogenization in a lysis mixture containing chloroform, SDS, NaCl, and phosphate-Tris buffer (pH 8) was found to be the best physical lysis technique when DNA yield and cell lysis efficiency were used as criteria. The bead mill homogenization conditions were also optimized for speed and duration with two different homogenizers. Recovery of high-molecular-weight DNA was greatest when we used lower speeds and shorter times (30 to 120 s). We evaluated four different DNA purification methods (silica-based DNA binding, agarose gel electrophoresis, ammonium acetate precipitation, and Sephadex G-200 gel filtration) for DNA recovery and removal of PCR inhibitors from crude extracts. Sephadex G-200 spin column purification was found to be the best method for removing PCR-inhibiting substances while minimizing DNA loss during purification. Our results indicate that for these types of samples, optimum DNA recovery requires brief, low-speed bead mill homogenization in the presence of a phosphate-buffered SDS-chloroform mixture, followed by Sephadex G-200 column purification.

Screening of Probiotic Activities of Forty-Seven Strains of Lactobacillus spp. by In Vitro Techniques and Evaluation of the Colonization Ability of Five Selected Strains in Humans

Abstract: It is well known that the presence of lactobacilli is important for the maintenance of the intestinal microbial ecosystem (39). They have been shown to possess inhibitory activity toward the growth of pathogenic bacteria such as Listeria monocytogenes (3, 25, 36, 42), Escherichia coli, Salmonella spp. (8, 16, 27), and others (4, 13, 37). This inhibition could be due to the production of inhibitory compounds such as organic acids, hydrogen peroxide, bacteriocins (30), or reuterin (4) or to competitive adhesion to the epithelium. In order to survive in and colonize the gastrointestinal tract, probiotic bacteria should express high tolerance to acid and bile and have the ability to adhere to intestinal surfaces (31, 34). Survival in and temporary colonization of the human gastrointestinal tract have been demonstrated for some lactic acid bacteria (1, 23, 29). However, in vivo testing is expensive and time consuming and requires approval by ethical committees. Therefore, reliable in vitro methods for selection of promising strains are required. Enterocyte-like Caco-2 cells (38) have been successfully used for in vitro studies on the mechanism of cellular adhesion of nonpathogenic lactobacilli (10, 24, 40, 43) and bifidobacteria (5, 15). Moreover, this cell line has been used to examine the mechanism of cellular adhesion and invasion of pathogenic bacteria such as L. monocytogenes (21), Salmonella typhimurium (20), and E. coli (32). Recently, Caco-2 cells have been used to examine the antimicrobial activity of lactobacilli (6, 13, 27) and bifidobacteria (5) against pathogenic bacteria. Antimicrobial properties of lactobacilli have been determined by using three methods: inhibitory activity toward the growth of test bacteria in vitro (7, 13, 14), inhibitory activity toward cell association, and invasion of pathogens using cultured human intestinal cells (6, 7, 12–14, 27), as well as protection of conventional or germfree mice against bacterial infection (7, 13, 14, 27). These showed how antimicrobial activities observed by in vitro methods could be confirmed in vivo as well. In the present study, the Caco-2 cell line was used to study the adhesive properties of 47 potentially probiotic cultures in vitro. The cultures were also examined for antimicrobial properties toward pathogenic bacteria along with tolerance to low pH and bile salts. Among these cultures, five promising strains were examined by in vivo studies. The abilities of the selected strains to survive passage through the gastrointestinal tract and maintain colonization was tested in fecal samples using API 50CHL and internal transcribed spacer PCR (ITS-PCR) for primary selection of strains and restriction enzyme analysis (REA) combined with pulsed-field gel electrophoresis (PFGE) for confirmation of isolates recovered from fecal samples during and after administration. It was the main objective of this study to compare the in vitro evaluation of certain properties of various Lactobacillus spp. that are important for their survival in the gastrointestinal tract with their actual ability to survive in vivo.

Automated approach for ribosomal intergenic spacer analysis of microbial diversity and its application to freshwater bacterial communities.

Abstract: An automated method of ribosomal intergenic spacer analysis (ARISA) was developed for the rapid estimation of microbial diversity and community composition in freshwater environments. Following isolation of total community DNA, PCR amplification of the 16S-23S intergenic spacer region in the rRNA operon was performed with a fluorescence-labeled forward primer. ARISA-PCR fragments ranging in size from 400 to 1,200 bp were next discriminated and measured by using an automated electrophoresis system. Database information on the 16S-23S intergenic spacer was also examined, to understand the potential biases in diversity estimates provided by ARISA. In the analysis of three natural freshwater bacterial communities, ARISA was rapid and sensitive and provided highly reproducible community-specific profiles at all levels of replication tested. The ARISA profiles of the freshwater communities were quantitatively compared in terms of both their relative diversity and similarity level. The three communities had distinctly different profiles but were similar in their total number of fragments (range, 34 to 41). In addition, the pattern of major amplification products in representative profiles was not significantly altered when the PCR cycle number was reduced from 30 to 15, but the number of minor products (near the limit of detection) was sensitive to changes in cycling parameters. Overall, the results suggest that ARISA is a rapid and effective community analysis technique that can be used in conjunction with more accurate but labor-intensive methods (e.g., 16S rRNA gene cloning and sequencing) when fine-scale spatial and temporal resolution is needed.

Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization.

Abstract: Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea, and of these, about half could be identified at the subdomain level with a set of group-specific probes. Beta subclass proteobacteria constituted a dominant fraction in freshwater systems, accounting for 16% (range, 3 to 32%) of the cells, although they were essentially absent in the marine samples examined. Members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in the marine systems, accounting for 18% (range, 2 to 72%) of the 4′,6-diamidino-2-phenylindole (DAPI) counts, and they were also important in freshwater systems (7%, range 0 to 18%). Furthermore, members of the alpha and gamma subclasses of Proteobacteria as well as members of the Planctomycetales were detected in both freshwater and marine water in abundances <7%.

Solubilization of phosphates and micronutrients by the plant-growth-promoting and biocontrol fungus trichoderma harzianum rifai 1295-22

Abstract: We investigated the capability of the plant-growth-promoting and biocontrol fungus Trichoderma harzianum Rifai 1295-22 (T-22) to solubilize in vitro some insoluble or sparingly soluble minerals via three possible mechanisms: acidification of the medium, production of chelating metabolites, and redox activity. T-22 was able to solubilize MnO2, metallic zinc, and rock phosphate (mostly calcium phosphate) in a liquid sucrose-yeast extract medium, as determined by inductively coupled plasma emission spectroscopy. Acidification was not the major mechanism of solubilization since the pH of cultures never fell below 5.0 and in cultures containing MnO2 the pH rose from 6.8 to 7.4. Organic acids were not detected by high-performance thin-layer chromatography in the culture filtrates. Fe2O3, MnO2, Zn, and rock phosphate were also solubilized by cell-free culture filtrates. The chelating activity of T-22 culture filtrates was determined by a method based on measurement of the equilibrium concentration of the chrome azurol S complex in the presence of other chelating substances. A size exclusion chromatographic separation of the components of the culture filtrates indicated the presence of a complexed form of Fe but no chelation of Mn. In liquid culture, T. harzianum T-22 also produced diffusible metabolites capable of reducing Fe(III) and Cu(II), as determined by the formation of Fe(II)-Na2-bathophenanthrolinedisulfonic acid and Cu(I)-Na2-2, 9-dimethyl-4,7-diphenyl-1,10-phenanthrolinedisulfonic acid complexes. This is the first report of the ability of a Trichoderma strain to solubilize insoluble or sparingly soluble minerals. This activity may explain, at least partially, the ability of T-22 to increase plant growth. Solubilization of metal oxides by Trichoderma involves both chelation and reduction. Both of these mechanisms also play a role in biocontrol of plant pathogens, and they may be part of a multiple-component action exerted by T-22 to achieve effective biocontrol under a variety of environmental conditions.

Mechanisms of action of carvacrol on the food-borne pathogen Bacillus cereus.

Abstract: Carvacrol, a naturally occurring compound mainly present in the essential oil fraction of oregano and thyme, was studied for its effect on bioenergetic parameters of vegetative cells of the food-borne pathogen Bacillus cereus. Incubation for 30 min in the presence of 1 to 3 mM carvacrol reduced the viable cell numbers exponentially. Carvacrol (2 mM) significantly depleted the intracellular ATP pool to values close to 0 within 7 min. No proportional increase of the extracellular ATP pool was observed. Depletion of the internal ATP pool was associated with a change of the membrane potential (Deltapsi). At concentrations of 0.01 mM carvacrol and above, a significant reduction of Deltapsi was observed, leading to full dissipation of Deltapsi at concentrations of 0.15 mM and higher. Finally, an increase of the permeability of the cytoplasmic membrane for protons and potassium ions was observed (at 0.25 and 1 mM carvacrol, respectively). From this study, it could be concluded that carvacrol interacts with the membranes of B. cereus by changing its permeability for cations like H(+) and K(+). The dissipation of ion gradients leads to impairment of essential processes in the cell and finally to cell death.

Induction of defense responses in cucumber plants (Cucumis sativus L. ) By the biocontrol agent trichoderma harzianum

Abstract: The potential of the biocontrol agent Trichoderma harzianum T-203 to trigger plant defense responses was investigated by inoculating roots of cucumber seedlings with Trichoderma in an aseptic, hydroponic system. Trichoderma-treated plants were more developed than nontreated plants throughout the experiment. Electron microscopy of ultrathin sections from Trichoderma-treated roots revealed penetration of Trichoderma into the roots, restricted mainly to the epidermis and outer cortex. Strengthening of the epidermal and cortical cell walls was observed, as was the deposition of newly formed barriers. These typical host reactions were found beyond the sites of potential fungal penetration. Wall appositions contained large amounts of callose and infiltrations of cellulose. The wall-bound chitin in Trichoderma hyphae was preserved, even when the hyphae had undergone substantial disorganization. Biochemical analyses revealed that inoculation with Trichoderma initiated increased peroxidase and chitinase activities within 48 and 72 h, respectively. These results were observed for both the roots and the leaves of treated seedlings, providing evidence that T. harzianum may induce systemic resistance mechanisms in cucumber plants.

Bacterial leaching of metal sulfides proceeds by two indirect mechanisms via thiosulfate or via polysulfides and sulfur

Abstract: The acid-insoluble metal sulfides FeS2, MoS2, and WS2 are chemically attacked by iron(III) hexahydrate ions, generating thiosulfate, which is oxidized to sulfuric acid. Other metal sulfides are attacked by iron(III) ions and by protons, resulting in the formation of elemental sulfur via intermediary polysulfides. Sulfur is biooxidized to sulfuric acid. This explains leaching of metal sulfides by Thiobacillus thiooxidans.

Combination of fluorescent in situ hybridization and microautoradiography-a new tool for structure-function analyses in microbial ecology

Abstract: A new microscopic method for simultaneously determining in situ the identities, activities, and specific substrate uptake profiles of individual bacterial cells within complex microbial communities was developed by combining fluorescent in situ hybridization (FISH) performed with rRNA-targeted oligonucleotide probes and microautoradiography. This method was evaluated by using defined artificial mixtures of Escherichia coli and Herpetosiphon aurantiacus under aerobic incubation conditions with added [H-3]glucose. Subsequently, we were able to demonstrate the potential of this method by visualizing the uptake of organic and inorganic radiolabeled substrates ([C-14]acetate, [C-14]butyrate, [C-14]bicarbonate, and P-33(i)) in probe-defined populations from complex activated sludge microbial communities by using aerobic incubation conditions and anaerobic incubation conditions (with and without nitrate). For both defined cell mixtures and activated sludge, the method proved to be useful for simultaneous identification and analysis of the uptake of labeled substrates under the different experimental conditions used. Optimal results were obtained when fluorescently labeled oligonucleotides were applied prior to the microautoradiographic developing procedure. For single-cell resolution of FISH and microautoradiographic signals within activated sludge flocs, cryosectioned sample material was examined with a confocal laser scanning microscope. The combination of in situ rRNA hybridization techniques, cryosectioning, microautoradiography, and confocal laser scanning microscopy provides a unique opportunity for obtaining cultivation-independent insights into the structure and function of bacterial communities.

Phylogenetic Analysis of Cryptosporidium Parasites Based on the Small-Subunit rRNA Gene Locus

Abstract: Biological data support the hypothesis that there are multiple species in the genus Cryptosporidium, but a recent analysis of the available genetic data suggested that there is insufficient evidence for species differentiation. In order to resolve the controversy in the taxonomy of this parasite genus, we characterized the small-subunit rRNA genes of Cryptosporidium parvum, Cryptosporidium baileyi, Cryptosporidium muris, and Cryptosporidium serpentis and performed a phylogenetic analysis of the genus Cryptosporidium. Our study revealed that the genus Cryptosporidium contains the phylogenetically distinct species C. parvum, C. muris, C. baileyi, and C. serpentis, which is consistent with the biological characteristics and host specificity data. The Cryptosporidium species formed two clades, with C. parvum and C. baileyi belonging to one clade and C. muris and C. serpentis belonging to the other clade. Within C. parvum, human genotype isolates and guinea pig isolates (known as Cryptosporidium wrairi) each differed from bovine genotype isolates by the nucleotide sequence in four regions. A C. muris isolate from cattle was also different from parasites isolated from a rock hyrax and a Bactrian camel. Minor differences were also detected between C. serpentis isolates from snakes and lizards. Based on the genetic information, a species- and strain-specific PCR-restriction fragment length polymorphism diagnostic tool was developed.

Phylogenetic Analysis of Particle-Attached and Free-Living Bacterial Communities in the Columbia River, Its Estuary, and the Adjacent Coastal Ocean

Abstract: The Columbia River estuary is a dynamic system in which estuarine turbidity maxima trap and extend the residence time of particles and particle-attached bacteria over those of the water and free-living bacteria. Particle-attached bacteria dominate bacterial activity in the estuary and are an important part of the estuarine food web. PCR-amplified 16S rRNA genes from particle-attached and free-living bacteria in the Columbia River, its estuary, and the adjacent coastal ocean were cloned, and 239 partial sequences were determined. A wide diversity was observed at the species level within at least six different bacterial phyla, including most subphyla of the class Proteobacteria. In the estuary, most particle-attached bacterial clones (75%) were related to members of the genus Cytophaga or of the a, g ,o rd subclass of the class Proteobacteria. These same clones, however, were rare in or absent from either the particle-attached or the free-living bacterial communities of the river and the coastal ocean. In contrast, about half (48%) of the free-living estuarine bacterial clones were similar to clones from the river or the coastal ocean. These free-living bacteria were related to groups of cosmopolitan freshwater bacteria (b-proteobacteria, gram-positive bacteria, and Verrucomicrobium spp.) and groups of marine organisms (gram-positive bacteria and a-proteobacteria [SAR11 and Rhodobacter spp.]). These results suggest that rapidly growing particle-attached bacteria develop into a uniquely adapted estuarine community and that free-living estuarine bacteria are similar to members of the river and the coastal ocean microbial communities. The high degree of diversity in the estuary is the result of the mixing of bacterial communities from the river, estuary, and coastal ocean.

Piriformospora indica, a Cultivable Plant-Growth-Promoting Root Endophyte

Abstract: Piriformospora indica (Hymenomycetes, Basidiomycota) is a newly described cultivable endophyte that colonizes roots. Inoculation with the fungus and application of fungal culture filtrate promotes plant growth and biomass production. Due to its ease of culture, this fungus provides a model organism for the study of beneficial plant-microbe interactions and a new tool for improving plant production systems.

Enumeration of Marine Viruses in Culture and Natural Samples by Flow Cytometry

Abstract: Flow cytometry (FCM) was successfully used to enumerate viruses in seawater after staining with the nucleic acid-specific dye SYBR Green-I. The technique was first optimized by using the Phaeocystis lytic virus PpV-01. Then it was used to analyze natural samples from different oceanic locations. Virus samples were fixed with 0.5% glutaraldehyde and deep frozen for delayed analysis. The samples were then diluted in Tris-EDTA buffer and analyzed in the presence of SYBR Green-I. A duplicate sample was heated at 80°C in the presence of detergent before analysis. Virus counts obtained by FCM were highly correlated to, although slightly higher than, those obtained by epifluorescence microscopy or by transmission electron microscopy (r 5 0.937, n 5 14, and r 5 0.96, n 5 8, respectively). Analysis of a depth profile from the Mediterranean Sea revealed that the abundance of viruses displayed the same vertical trend as that of planktonic cells. FCM permits us to distinguish between at least two and sometimes three virus populations in natural samples. Because of its speed and accuracy, FCM should prove very useful for studies of virus infection in cultures and should allow us to better understand the structure and dynamics of virus populations in natural waters.

Microbial Population Changes during Bioremediation of an Experimental Oil Spill

Abstract: Three crude oil bioremediation techniques were applied in a randomized block field experiment simulating a coastal oil spill. Four treatments (no oil control, oil alone, oil plus nutrients, and oil plus nutrients plus an indigenous inoculum) were applied. In situ microbial community structures were monitored by phospholipid fatty acid (PLFA) analysis and 16S rDNA PCR-denaturing gradient gel electrophoresis (DGGE) to (i) identify the bacterial community members responsible for the decontamination of the site and (ii) define an end point for the removal of the hydrocarbon substrate. The results of PLFA analysis demonstrated a community shift in all plots from primarily eukaryotic biomass to gram-negative bacterial biomass with time. PLFA profiles from the oiled plots suggested increased gram-negative biomass and adaptation to metabolic stress compared to unoiled controls. DGGE analysis of untreated control plots revealed a simple, dynamic dominant population structure throughout the experiment. This banding pattern disappeared in all oiled plots, indicating that the structure and diversity of the dominant bacterial community changed substantially. No consistent differences were detected between nutrient-amended and indigenous inoculum-treated plots, but both differed from the oil-only plots. Prominent bands were excised for sequence analysis and indicated that oil treatment encouraged the growth of gram-negative microorganisms within the α-proteobacteria and Flexibacter-Cytophaga-Bacteroides phylum. α-Proteobacteria were never detected in unoiled controls. PLFA analysis indicated that by week 14 the microbial community structures of the oiled plots were becoming similar to those of the unoiled controls from the same time point, but DGGE analysis suggested that major differences in the bacterial communities remained.

Genetic diversity within cryptosporidium parvum and related cryptosporidium species

Abstract: To assess the genetic diversity in Cryptosporidium parvum, we have sequenced the small subunit (SSU) rRNA gene of seven Cryptosporidium spp., various isolates of C. parvum from eight hosts, and a Cryptosporidium isolate from a desert monitor. Phylogenetic analysis of the SSU rRNA sequences confirmed the multispecies nature of the genus Cryptosporidium, with at least four distinct species (C. parvum, C. baileyi, C. muris, and C. serpentis). Other species previously defined by biologic characteristics, including C. wrairi, C. meleagridis, and C. felis, and the desert monitor isolate, clustered together or within C. parvum. Extensive genetic diversities were present among C. parvum isolates from humans, calves, pigs, dogs, mice, ferrets, marsupials, and a monkey. In general, specific genotypes were associated with specific host species. A PCR-restriction fragment length polymorphism technique previously developed by us could differentiate most Cryptosporidium spp. and C. parvum genotypes, but sequence analysis of the PCR product was needed to differentiate C. wrairi and C. meleagridis from some of the C. parvum genotypes. These results indicate a need for revision in the taxonomy and assessment of the zoonotic potential of some animal C. parvum isolates.

Analysis of fungal diversity in the wheat rhizosphere by sequencing of cloned PCR-amplified genes encoding 18S rRNA and temperature gradient gel electrophoresis.

Abstract: Like bacteria, fungi play an important role in the soil ecosystem. As only a small fraction of the fungi present in soil can be cultured, conventional microbiological techniques yield only limited information on the composition and dynamics of fungal communities in soil. DNA-based methods do not depend on the culturability of microorganisms, and therefore they offer an attractive alternative for the study of complex fungal community structures. For this purpose, we designed various PCR primers that allow the specific amplification of fungal 18S-ribosomal-DNA (rDNA) sequences, even in the presence of nonfungal 18S rDNA. DNA was extracted from the wheat rhizosphere, and 18S rDNA gene banks were constructed in Escherichia coli by cloning PCR products generated with primer pairs EF4-EF3 (1.4 kb) and EF4-fung5 (0.5 kb). Fragments of 0.5 kb from the cloned inserts were sequenced and compared to known rDNA sequences. Sequences from all major fungal taxa were amplified by using both primer pairs. As predicted by computer analysis, primer pair EF4-EF3 appeared slightly biased to amplify Basidiomycota and Zygomycota, whereas EF4-fung5 amplified mainly Ascomycota. The 61 clones that were sequenced matched the sequences of 24 different species in the Ribosomal Database Project (RDP) database. Similarity values ranged from 0.676 to 1. Temperature gradient gel electrophoresis (TGGE) analysis of the fungal community in the wheat rhizosphere of a microcosm experiment was carried out after amplification of total DNA with both primer pairs. This resulted in reproducible, distinctive fingerprints, confirming the difference in amplification specificity. Clear banding patterns were obtained with soil and rhizosphere samples by using both primer sets in combination. By comparing the electrophoretic mobility of community fingerprint bands to that of the bands obtained with separate clones, some could be tentatively identified. While 18S-rDNA sequences do not always provide the taxonomic resolution to identify fungal species and strains, they do provide information on the diversity and dynamics of groups of related species in environmental samples with sufficient resolution to produce discrete bands which can be separated by TGGE. This combination of 18S-rDNA PCR amplification and TGGE community analysis should allow study of the diversity, composition, and dynamics of the fungal community in bulk soil and in the rhizosphere.

Persistence of Colonization of Human Colonic Mucosa by a Probiotic Strain, Lactobacillus rhamnosus GG, after Oral Consumption

Abstract: Lactobacillus rhamnosus GG is one of the most thoroughly studied probiotic strains. Its advantages in the treatment of gastrointestinal disorders are well documented. The aim of the present study was to demonstrate with colonic biopsies the attachment of strain GG to human intestinal mucosae and the persistence of the attachment after discontinuation of GG administration. A whey drink fermented with strain GG was fed to human volunteers for 12 days. Fecal samples were collected before, during, and after consumption. L. rhamnosus GG-like colonies were detected in both fecal and colonic biopsy samples. Strain GG was identified by its characteristic colony morphology, a lactose fermentation test, and PCR. This study showed that strain GG was able to attach in vivo to colonic mucosae and, although the attachment was temporary, to remain for more than a week after discontinuation of GG administration. The results demonstrate that the study of fecal samples alone is not sufficient in evaluating colonization by a probiotic strain.

In Situ Analysis of Nitrifying Biofilms as Determined by In Situ Hybridization and the Use of Microelectrodes

Abstract: We investigated the in situ spatial organization of ammonia-oxidizing and nitrite-oxidizing bacteria in domestic wastewater biofilms and autotrophic nitrifying biofilms by using microsensors and fluorescent in situ hybridization (FISH) performed with 16S rRNA-targeted oligonucleotide probes. The combination of these techniques made it possible to relate in situ microbial activity directly to the occurrence of nitrifying bacterial populations. In situ hybridization revealed that bacteria belonging to the genus Nitrosomonas were the numerically dominant ammonia-oxidizing bacteria in both types of biofilms. Bacteria belonging to the genus Nitrobacter were not detected; instead, Nitrospira-like bacteria were the main nitrite-oxidizing bacteria in both types of biofilms. Nitrospira-like cells formed irregularly shaped aggregates consisting of small microcolonies, which clustered around the clusters of ammonia oxidizers. Whereas most of the ammonia-oxidizing bacteria were present throughout the biofilms, the nitrite-oxidizing bacteria were restricted to the active nitrite-oxidizing zones, which were in the inner parts of the biofilms. Microelectrode measurements showed that the active ammonia-oxidizing zone was located in the outer part of a biofilm, whereas the active nitrite-oxidizing zone was located just below the ammonia-oxidizing zone and overlapped the location of nitrite-oxidizing bacteria, as determined by FISH.

Quantification of bias related to the extraction of DNA directly from soils.

Abstract: In recent years, several protocols based on the extraction of nucleic acids directly from the soil matrix after lysis treatment have been developed for the detection of microorganisms in soil. Extraction efficiency has often been evaluated based on the recovery of a specific gene sequence from an organism inoculated into the soil. The aim of the present investigation was to improve the extraction, purification, and quantification of DNA derived from as large a portion of the soil microbial community as possible, with special emphasis placed on obtaining DNA from gram-positive bacteria, which form structures that are difficult to disrupt. Furthermore, we wanted to identify and minimize the biases related to each step in the procedure. Six soils, covering a range of pHs, clay contents, and organic matter contents, were studied. Lysis was carried out by soil grinding, sonication, thermal shocks, and chemical treatments. DNA was extracted from the indigenous microflora as well as from inoculated bacterial cells, spores, and hyphae, and the quality and quantity of the DNA were determined by gel electrophoresis and dot blot hybridization. Lysis efficiency was also estimated by microscopy and viable cell counts. Grinding increased the extracellular DNA yield compared with the yield obtained without any lysis treatment, but none of the subsequent treatments clearly increased the DNA yield. Phage λ DNA was inoculated into the soils to mimic the fate of extracellular DNA. No more than 6% of this DNA could be recovered from the different soils. The clay content strongly influenced the recovery of DNA. The adsorption of DNA to clay particles decreased when the soil was pretreated with RNA in order to saturate the adsorption sites. We also investigated different purification techniques and optimized the PCR methods in order to develop a protocol based on hybridization of the PCR products and quantification by phosphorimaging.

How stable is stable? Function versus community composition.

Abstract: The microbial community dynamics of a functionally stable, well-mixed, methanogenic reactor fed with glucose were analyzed over a 605-day period. The reactor maintained constant pH and chemical oxygen demand removal during this period. Thirty-six rrn clones from each of seven sampling events were analyzed by amplified ribosomal DNA restriction analysis (ARDRA) for the Bacteria and Archaea domains and by sequence analysis of dominant members of the community. Operational taxonomic units (OTUs), distinguished as unique ARDRA patterns, showed reproducible distribution for three sample replicates. The highest diversity was observed in the Bacteria domain. The 16S ribosomal DNA Bacteria clone library contained 75 OTUs, with the dominant OTU accounting for 13% of the total clones, but just 21 Archaea OTUs were found, and the most prominent OTU represented 50% of the clones from the respective library. Succession in methanogenic populations was observed, and two periods were distinguished: in the first, Methanobacterium formicicum was dominant, and in the second, Methanosarcina mazei and a Methanobacterium bryantii-related organism were dominant. Higher variability in Bacteria populations was detected, and the temporal OTU distribution suggested a chaotic pattern. Although dominant OTUs were constantly replaced from one sampling point to the next, phylogenetic analysis indicated that inferred physiologic changes in the community were not as dramatic as were genetic changes. Seven of eight dominant OTUs during the first period clustered with the spirochete group, although a cyclic pattern of substitution occurred among members within this order. A more flexible community structure characterized the second period, since a sequential replacement of a Eubacterium-related organism by an unrelated deep-branched organism and finally by a Propionibacterium-like species was observed. Metabolic differences among the dominant fermenters detected suggest that changes in carbon and electron flow occurred during the stable performance and indicate that an extremely dynamic community can maintain a stable ecosystem function.

Significance of size and nucleic acid content heterogeneity as measured by flow cytometry in natural planktonic bacteria.

Abstract: Total bacterial abundances estimated with different epifluorescence microscopy methods (4′,6-diamidino-2-phenylindole [DAPI], SYBR Green, and Live/Dead) and with flow cytometry (Syto13) showed good correspondence throughout two microcosm experiments with coastal Mediterranean water. In the Syto13-stained samples we could differentiate bacteria with apparent high DNA (HDNA) content and bacteria with apparent low DNA (LDNA) content. HDNA bacteria, “live” bacteria (determined as such with the Molecular Probes Live/Dead BacLight bacterial viability kit), and nucleoid-containing bacteria (NuCC) comprised similar fractions of the total bacterial community. Similarly, LDNA bacteria and “dead” bacteria (determined with the kit) comprised a similar fraction of the total bacterial community in one of the experiments. The rates of change of each type of bacteria during the microcosm experiments were also positively correlated between methods. In various experiments where predator pressure on bacteria had been reduced, we detected growth of the HDNA bacteria without concomitant growth of the LDNA bacteria, such that the percentage contribution of HDNA bacteria to total bacterial numbers (%HDNA) increased. This indicates that the HDNA bacteria are the dynamic members of the bacterial assemblage. Given how quickly and easily the numbers of HDNA and LDNA bacteria can be obtained, and given the similarity to the numbers of “live” cells and NuCC, the %HDNA is suggested as a reference value for the percentage of actively growing bacteria in marine planktonic environments.

Environmental Factors Modulating Antibiotic and Siderophore Biosynthesis by Pseudomonas fluorescens Biocontrol Strains

Abstract: Understanding the environmental factors that regulate the biosynthesis of antimicrobial compounds by disease-suppressive strains of Pseudomonas fluorescens is an essential step toward improving the level and reliability of their biocontrol activity. We used liquid culture assays to identify several minerals and carbon sources which had a differential influence on the production of the antibiotics 2,4-diacetylphloroglucinol (PHL), pyoluteorin (PLT), and pyrrolnitrin and the siderophores salicylic acid and pyochelin by the model strain CHA0, which was isolated from a natural disease-suppressive soil in Switzerland. Production of PHL was stimulated by Zn2+, NH4Mo2+, and glucose; the precursor compound mono-acetylphloroglucinol was stimulated by the same factors as PHL. Production of PLT was stimulated by Zn2+, Co2+, and glycerol but was repressed by glucose. Pyrrolnitrin production was increased by fructose, mannitol, and a mixture of Zn2+ and NH4Mo2+. Pyochelin production was increased by Co2+, fructose, mannitol, and glucose. Interestingly, production of its precursor salicylic acid was increased by different factors, i.e., NH4Mo2+, glycerol, and glucose. The mixture of Zn2+ and NH4Mo2+ with fructose, mannitol, or glycerol further enhanced the production of PHL and PLT compared with either the minerals or the carbon sources used alone, but it did not improve siderophore production. Extending fermentation time from 2 to 5 days increased the accumulation of PLT, pyrrolnitrin, and pyochelin but not of PHL. When findings with CHA0 were extended to an ecologically and genetically diverse collection of 41 P. fluorescens biocontrol strains, the effect of certain factors was strain dependent, while others had a general effect. Stimulation of PHL by Zn2+ and glucose was strain dependent, whereas PLT production by all strains that can produce this compound was stimulated by Zn2+ and transiently repressed by glucose. Inorganic phosphate reduced PHL production by CHA0 and seven other strains tested but to various degrees. Production of PLT but not pyrrolnitrin by CHA0 was also reduced by 100 mM phosphate. The use of 1/10-strength nutrient broth-yeast extract, compared with standard nutrient broth-yeast extract, amended with glucose and/or glycerol resulted in dramatically increased accumulations of PHL (but not PLT), pyochelin, and salicylic acid, indicating that the ratio of carbon source to nutrient concentration played a key role in the metabolic flow. The results of this study (i) provide insight into the biosynthetic regulation of antimicrobial compounds, (ii) limit the number of factors for intensive study in situ, and (iii) indicate factors that can be manipulated to improve bacterial inoculants.

Molecular Characterization of Functional and Phylogenetic Genes from Natural Populations of Methanotrophs in Lake Sediments

Abstract: The 16S rRNA and pmoA genes from natural populations of methane-oxidizing bacteria (methanotrophs) were PCR amplified from total community DNA extracted from Lake Washington sediments obtained from the area where peak methane oxidation occurred. Clone libraries were constructed for each of the genes, and approximately 200 clones from each library were analyzed by using restriction fragment length polymorphism (RFLP) and the tetrameric restriction enzymes MspI, HaeIII, and HhaI. The PCR products were grouped based on their RFLP patterns, and representatives of each group were sequenced and analyzed. Studies of the 16S rRNA data obtained indicated that the existing primers did not reveal the total methanotrophic diversity present when these data were compared with pure-culture data obtained from the same environment. New primers specific for methanotrophs belonging to the genera Methylomonas, Methylosinus, and Methylocystis were developed and used to construct more complete clone libraries. Furthermore, a new primer was designed for one of the genes of the particulate methane monooxygenase in methanotrophs, pmoA. Phylogenetic analyses of both the 16S rRNA and pmoA gene sequences indicated that the new primers should detect these genes over the known diversity in methanotrophs. In addition to these findings, 16S rRNA data obtained in this study were combined with previously described phylogenetic data in order to identify operational taxonomic units that can be used to identify methanotrophs at the genus level.

Microscale distribution of populations and activities of Nitrosospira and Nitrospira spp. along a macroscale gradient in a nitrifying bioreactor: quantification by in situ hybridization and the use of microsensors.

Abstract: The change of activity and abundance of Nitrosospira and Nitrospira spp. along a bulk water gradient in a nitrifying fluidized bed reactor was analyzed by a combination of microsensor measurements and fluorescence in situ hybridization. Nitrifying bacteria were immobilized in bacterial aggregates that remained in fixed positions within the reactor column due to the flow regimen. Nitrification occurred in a narrow zone of 100 to 150 mm on the surface of these aggregates, the same layer that contained an extremely dense community of nitrifying bacteria. The central part of the aggregates was inactive, and significantly fewer nitrifiers were found there. Under conditions prevailing in the reactor, i.e., when ammonium was limiting, ammonium was completely oxidized to nitrate within the active layer of the aggregates, the rates decreasing with increasing reactor height. To analyze the nitrification potential, profiles were also recorded in aggregates subjected to a shortterm incubation under elevated substrate concentrations. This led to a shift in activity from ammonium to nitrite oxidation along the reactor and correlated well with the distribution of the nitrifying population. Along the whole reactor, the numbers of ammonia-oxidizing bacteria decreased, while the numbers of nitriteoxidizing bacteria increased. Finally, volumetric reaction rates were calculated from microprofiles and related to cell numbers of nitrifying bacteria in the active shell. Therefore, it was possible for the first time to estimate the cell-specific activity of Nitrosospira spp. and hitherto-uncultured Nitrospira-like bacteria in situ.

Ubiquity and diversity of dissimilatory (per)chlorate-reducing bacteria.

Abstract: Environmental contamination with compounds containing oxyanions of chlorine, such as perchlorate or chlorate [(per)chlorate] or chlorine dioxide, has been a constantly growing problem over the last 100 years. Although the fact that microbes reduce these compounds has been recognized for more than 50 years, only six organisms which can obtain energy for growth by this metabolic process have been described. As part of a study to investigate the diversity and ubiquity of microorganisms involved in the microbial reduction of (per)chlorate, we enumerated the (per)chlorate-reducing bacteria (ClRB) in very diverse environments, including pristine and hydrocarbon-contaminated soils, aquatic sediments, paper mill waste sludges, and farm animal waste lagoons. In all of the environments tested, the acetate-oxidizing ClRB represented a significant population, whose size ranged from 2.31 × 10 3 to 2.4 × 10 6 cells per g of sample. In addition, we isolated 13 ClRB from these environments. All of these organisms could grow anaerobically by coupling complete oxidation of acetate to reduction of (per)chlorate. Chloride was the sole end product of this reductive metabolism. All of the isolates could also use oxygen as a sole electron acceptor, and most, but not all, could use nitrate. The alternative electron donors included simple volatile fatty acids, such as propionate, butyrate, or valerate, as well as simple organic acids, such as lactate or pyruvate. Oxidized-minus-reduced difference spectra of washed whole-cell suspensions of the isolates had absorbance maxima close to 425, 525, and 550 nm, which are characteristic of type c cytochromes. In addition, washed cell suspensions of all of the ClRB isolates could dismutate chlorite, an intermediate in the reductive metabolism of (per)chlorate, into chloride and molecular oxygen. Chlorite dismutation was a result of the activity of a single enzyme which in pure form had a specific activity of approximately 1,928 μmol of chlorite per mg of protein per min. Analyses of the 16S ribosomal DNA sequences of the organisms indicated that they all belonged to the alpha, beta, or gamma subclass of the Proteobacteria . Several were closely related to members of previously described genera that are not recognized for the ability to reduce (per)chlorate, such as the genera Pseudomonas and Azospirllum . However, many were not closely related to any previously described organism and represented new genera within the Proteobacteria . The results of this study significantly increase the limited number of microbial isolates that are known to be capable of dissimilatory (per)chlorate reduction and demonstrate the hitherto unrecognized phylogenetic diversity and ubiquity of the microorganisms that exhibit this type of metabolism.

Ethanol Synthesis by Genetic Engineering in Cyanobacteria

Abstract: Cyanobacteria are autotrophic prokaryotes which carry out oxygenic photosynthesis and accumulate glycogen as the major form of stored carbon. In this research, we introduced new genes into a cyanobacterium in order to create a novel pathway for fixed carbon utilization which results in the synthesis of ethanol. The coding sequences of pyruvate decarboxylase (pdc) and alcohol dehydrogenase II (adh) from the bacterium Zymomonas mobilis were cloned into the shuttle vector pCB4 and then used to transform the cyanobacterium Synechococcus sp. strain PCC 7942. Under control of the promoter from the rbcLS operon encoding the cyanobacterial ribulose-1,5-bisphosphate carboxylase/oxygenase, the pdc and adh genes were expressed at high levels, as demonstrated by Western blotting and enzyme activity analyses. The transformed cyanobacterium synthesized ethanol, which diffused from the cells into the culture medium. As cyanobacteria have simple growth requirements and use light, CO2, and inorganic elements efficiently, production of ethanol by cyanobacteria is a potential system for bioconversion of solar energy and CO2 into a valuable resource. Cyanobacteria, also known as blue-green algae, are autotrophic prokaryotes which exhibit diversity in metabolism, structure, morphology, and habitat. However, all of these organisms perform oxygenic photosynthesis, and this photosynthesis is similar to that performed by higher plants (29, 32). As the cyanobacteria have simple growth requirements, grow to high densities, and use light, carbon dioxide, and other inorganic nutrients efficiently, they could be attractive hosts for production of valuable organic products. In fact, many cyanobacteria can be used directly as food and fodder since they are nonpathogenic and have high nutrient value (27). Some cyanobacteria also synthesize secondary metabolites which have been reported to have significant therapeutic effects (4). In addition, mass cultivation for commercial production of some cyanobacteria can be performed efficiently. Synechococcus sp. strain PCC 7942 (previously referred to as Anacystis nidulans R2), a unicellular cyanobacterium that lives in freshwater, is one of the few cyanobacterial strains which have been relatively well-characterized in terms of physiology, biochemistry, and genetics. This organism is able to take up foreign DNA and can be transformed either by using shuttle vectors capable of replicating in both Escherichia coli and the cyanobacterium or by integrating foreign DNA into the chromosome through homologous recombination at targeted sites (14, 36). In recent years, workers have achieved limited success in expressing foreign genes in this cyanobacterium, as well as other transformable strains. For example, the human carbonic anhydrase gene caII used to investigate CO2-concentrating mechanisms (26), E. coli and human superoxide dismutase genes used to investigate oxidative stress (15, 34), E. coli pet genes used to increase salt stress resistance (25), and Bacillus thuringiensis larvicidal genes used to develop bioinsecticidal hosts (33, 35) have all been expressed in Synechococcus sp. at sufficiently high levels to generate discernible phenotypes. In this paper, we describe our attempts to transform Synechococcus sp. strain PCC 7942 with bacterial genes in order to create a novel pathway for ethanol production in cyanobacteria.

Biochemical Characterization of Fungal Phytases (myo-Inositol Hexakisphosphate Phosphohydrolases): Catalytic Properties

Abstract: The phosphatases are a diverse class of enzymes. According to one classification, alkaline phosphatases, purple acid phosphatases, high-molecular-weight acid phosphatases, low-molecular-weight acid phosphatases, and protein phosphatases can be distinguished (13). These classes differ in their pH optima, metal ion requirements, substrate specificities, and possibly even reaction mechanisms. The phytases (myo-inositol hexakisphosphate phosphohydrolases; EC 3.1.3.8 and 3.1.3.26) are a subfamily of the high-molecular-weight histidine acid phosphatases. The phytase reaction mechanism is a two-step mechanism which includes a covalent phosphohistidine adduct as an obligatory reaction intermediate (6). Phytases are found naturally in plants and microorganisms, particularly fungi (for a review see reference 15). They catalyze phosphate monoester hydrolysis of phytic acid (myo-inositol hexakisphosphate), which results in the stepwise formation of myo-inositol pentakis-, tetrakis-, tris-, bis-, and monophosphates, as well as the liberation of inorganic phosphate. Phytic acid is the major storage form of phosphorus in plant seeds and, thus, in seed-based animal feed (for reviews see references 1 and 8). Monogastric animals, such as pigs and poultry, are not able to utilize phytic acid phosphorus, since they have only low levels of phytase activity in their digestive tracts and since phytic acid cannot be resorbed. Therefore, pig and poultry feed commonly is supplemented with either inorganic phosphate or a phytase of fungal origin. Despite considerable economic interest, only limited data on the catalytic properties of fungal phytases are available. In order to get an impression of the natural diversity of phytases, the enzymatic properties of six fungal phytases (phytases from Aspergillus niger, two strains of Aspergillus terreus, Aspergillus fumigatus, Emericella nidulans, and Myceliophthora thermophila) and of Escherichia coli phytase were characterized in more detail by addressing the following questions. (i) What are the specific activities and pH optima of wild-type phytases? (ii) What are the kinetics of phytic acid degradation, and what are the end products? (iii) Does the substrate specificity profile correlate with the results of in vitro experiments performed to determine phosphate liberation from feed samples? And (iv) what is the potential influence of modulators of enzymatic activity?