FEMS Microbiology Ecology

Published by Oxford University Press (OUP)
Online ISSN: 1574-6941
Publications
DGGE separation patterns (A) and its schematic representation (B) of 16S rDNA fragments obtained after enzymatic ampli¢cation of genomic DNA extracted from di¡erent ¢ltered fractions of seawater samples near Calvi (Corsica/France). (A) DGGE pro¢les of di¡erent ¢ltered seawater samples. Lanes 1^3 collected in winter (February), lanes 4^6 in summer (July) 1997 ; lanes 1 and 4 show total bacterial population (P total ) of the corresponding sample event; lanes 2 and 5 represent the bacterial population which was retained on a 0.2 Wm ¢lter (P s0X2 Wm ); lanes 3 and 6 show the fraction of bacteria which passed a 0.2 Wm ¢lter and was retained on a 0.1 Wm ¢lter (P 0X1À0X2 Wm ). (B) Schematic representation of original DGGE pro¢les of winter and summer samples. The labeled DGGE bands in lane 3 (CFW (Calvi, 0.2 Wm ¢lterable, winter sample)-1, 2, 3, 5, 7, 10) and lane 6 (CFS (Calvi, 0.2 Wm ¢lterable, summer sample)-1, 3, 4, 5, 6, 7, 9, 10) were bands for which sequence information was obtained. Arrows (lanes 4 and 6) indicate those bands in corresponding sample fractions which occurred in both, winter and summer samples.
Article
Although the existence of 0.2 µm filterable bacteria has been known since the early 80's, they are not taken into consideration when modeling microbial food webs, due to an overall lack of information concerning this specific size class. According to physiological studies on starvation forms and investigations on small bacterial cells in marine ecosystems, a 0.2 µm filtrate may consist of different phenotypes: starvation forms of typical marine bacteria, ultramicrobacteria or bacterial cells, even larger than 0.2 µm, but flexible enough to pass the nominal filter pore-size. In this pilot study we examined three filtered seawater fractions from the Western Mediterranean Sea (Bay of Calvi, Corsica/France) - the total bacterial population, the bacterial fraction above 0.2 µm and the 0.2 µm filtrate - to investigate the bacterial community structure of each of those fractions by the molecular approach of denaturing gradient gel electrophoresis (DGGE) of 16S rDNA fragments. The analysis of the resulting DGGE profiles revealed different patterns of dominant bands for the 0.2 µm filterable and the total bacterial populations within the samples. Additionally the 0.2 µm filterable bacterial compartment exhibited obvious differences in band patterns for winter and summer samples, which were not observed for the total bacterial fraction. According to the current knowledge concerning the status of 0.2 µm filterable bacteria, DGGE patterns indicate that most of the fragments representing 0.2 µm filterable bacteria were rather starvation forms of marine bacteria than ultramicrobacteria. The sequencing of excised and cloned DNA bands of the DGGE profiles characterized the phylogenetic affiliation of the corresponding 0.2 µm filterable bacteria, clustering mainly with known, typical marine isolates of both alpha-subclass and gamma-subclass of the Proteobacteria and the Cytophaga-Flavobacterium-Bacteroides branch.
 
Site descriptions and depositional ages of the soil chronosequences 
Distribution and phylogenetic affiliation of the most abundant OTUs 
Phylogeny of representative Chloroflexi clones from the dune soils of southeast Georgia. Classification of the subdivisions is according to Costello & Schmidt (2006). Other notations are as described for Fig. 2. 
Diversity indices for the chronolibraries 
Article
The origins of the biological complexity and the factors that regulate the development of community composition, diversity and richness in soil remain largely unknown. To gain a better understanding of how bacterial communities change during soil ecosystem development, their composition and diversity in soils that developed over c. 77 000 years of intermittent aeolian deposition were studied. 16S rRNA gene clone libraries and fatty acid methyl ester (FAME) analyses were used to assess the diversity and composition of the communities. The bacterial community composition changed with soil age, and the overall diversity, richness and evenness of the communities increased as the soil habitat matured. When analysed using a multivariate Bray-Curtis ordination technique, the distribution of ribotypes showed an orderly pattern of bacterial community development that was clearly associated with soil and ecosystem development. Similarly, changes in the composition of the FAMEs across the chronosequence were associated with biomarkers for fungi, actinomycetes and Gram-positive bacteria. The development of the soil ecosystem promoted the development of distinctive microbial communities that were reminiscent of successional processes often evoked to describe change during the development of plant communities in terrestrial ecosystems.
 
Article
The fermentability of rice bran (RB), alone or in combination with one of two probiotics, by canine faecal microbiota was evaluated in stirred, pH-controlled, anaerobic batch cultures. RB enhanced the levels of bacteria detected by probes Bif164 (bifidobacteria) and Lab158 (lactic acid bacteria); however, addition of the probiotics did not have a significant effect on the predominant microbial counts compared with RB alone. RB sustained levels of Bifidobacterium longum 05 throughout the fermentation; in contrast, Lactobacillus acidophilus 14 150B levels decreased significantly after 5-h fermentation. RB fermentation induced changes in the short-chain fatty acid (SCFA) profile. However, RB combined with probiotics did not alter the SCFA levels compared with RB alone. Denaturing gradient gel electrophoresis analysis of samples obtained at 24 h showed a treatment effect with RB, which was not observed in the RB plus probiotic systems. Overall, the negative controls displayed lower species richness than the treatment systems and their banding profiles were distinct. This study illustrates the ability of a common ingredient found in pet food to modulate the canine faecal microbiota and highlights that RB may be an economical alternative to prebiotics for use in dog food.
 
Relative abundances of bacterial phyla and classes. Relative abundances (%) of bacteria were determined in feces from human adults with type 2 diabetes (green triangles, N = 10) and non-diabetic controls (blue dots, N = 10) by pyrosequencing analysis of the V4 region of the 16S rRNA gene. Mean values are denoted by red crosses and numbers. Values out of scale are shown in brackets. doi:10.1371/journal.pone.0009085.g002 
Correlation between OGTT or BMI and bacterial estimates by pyrosequencing. Correlation between OGTT plasma glucose and (A) ratios of Bacteroidetes to Firmicutes, (B) relative abundance of Clostridia, (C) relative abundance of Betaproteobacteria. (D) Correlation between body mass indices and ratios of Bacteroidetes to Firmicutes. The Spearman Rank probability (P) and correlation (R) are shown in the graphs. Bacterial abundances were determined by pyrosequencing of the V4 region of the 16S rRNA gene in fecal bacterial DNA from human adults with type 2 diabetes (N = 10) and non-diabetic controls (N = 10). doi:10.1371/journal.pone.0009085.g003 
PCA plots of bacterial phyla and classes. PCA plots showing the grouping of human adults with type 2 diabetes (m, D1-D10) and non-diabetic controls ( N , C1-C10) according to the abundances of bacterial phyla (A) and classes (B) in the fecal bacterial DNA as determined by pyrosequencing of the V4 region of the 16S rRNA gene. doi:10.1371/journal.pone.0009085.g004 
Box-and-whisker plots of bacterial groups quantified by qPCR. Bacterial groups quantified by SYBR Green qPCR and expressed as Log 10 bacteria per g stool in human adults with type 2 diabetes (black and white boxes; N = 18) and non-diabetic controls (grey and white boxes; N = 18). The median counts are presented by numbers. Boxes show the upper (75%) and the lower (25%) percentiles of the data. Whiskers indicate the highest and the smallest values. doi:10.1371/journal.pone.0009085.g005 
Article
The effect of probiotic bacteria Lactobacillus acidophilus NCFM and Bifidobacterium lactis Bi-07 on the composition of the Lactobacillus group, Bifidobacterium and the total bacterial population in feces from young children with atopic dermatitis was investigated. The study included 50 children randomized to intake of one of the probiotic strain or placebo. Microbial composition was characterized by denaturing gradient gel electrophoresis, quantitative PCR and, in a subset of subjects, by pyrosequencing of the 16S rRNA gene. The core population of the Lactobacillus group was identified as Lactobacillus gasseri, Lactobacillus fermentum, Lactobacillus oris, Leuconostoc mesenteroides, while the bifidobacterial community included Bifidobacterium adolescentis, Bifidobacterium bifidum, Bifidobacterium longum and Bifidobacterium catenulatum. The fecal numbers of L. acidophilus and B. lactis increased significantly after intervention, indicating survival of the ingested bacteria. The levels of Bifidobacterium correlated positively (P=0.03), while the levels of the Lactobacillus group negatively (P=0.01) with improvement of atopic eczema evaluated by the Severity Scoring of Atopic Dermatitis index. This correlation was observed across the whole study cohort and not attributed to the probiotic intake. The main conclusion of the study is that administration of L. acidophilus NCFM and B. lactis Bi-07 does not affect the composition and diversity of the main bacterial populations in feces.
 
Article
Micromanipulated filamentous bacteria from bulking and foaming activated sludge morphologically identified as Eikelboom type 0803 were shown to be affiliated to the genus Caldilinea within the phylum Chloroflexi. Specific FISH probes were designed for their in situ detection and quantification in seven Danish wastewater treatment plants with biological nutrient removal. The survey applied all species-specific probes for Chloroflexi of relevance in activated sludge treatment plants as well as the phylum-specific probes. Type 0803 filaments constituted around 20% of the total Chloroflexi population. In four of the treatment plants, type 0803 and type 0092 co-occurred and were the dominating fraction of the Chloroflexi population. In the other plants, most Chloroflexi could not be identified beyond the phylum level, suggesting a yet far larger diversity. On average, for all plants, the total Chloroflexi population constituted 12% of the entire microbial population and seems to play an important structural role in the sludge floc formation. Ecophysiological characterization of type 0803 showed their potential role in macromolecule conversion as evident by high levels of exoenzyme expression. Acetate was not consumed. Glucose was consumed with oxygen, nitrite and nitrite as electron acceptors, suggesting that type 0803 may be a denitrifier. Their surfaces were hydrophobic, explaining their occasional occurrence in foaming incidents.
 
Article
Evidence is presented demonstrating the ability of Ralstonia eutropha A5 to degrade 1,1-dichloro-2,2-bis(4-chlorophenyl)ethane (DDD) aerobically. Strain A5 was able to effect significant transformation of [(14)C]DDD: the hexane extractable radioactivity decreased to approximately 50% of the controls while more than 25% of the total radioactivity became associated with the acidified culture supernatant. There was also an increase in the amount of radioactivity associated with the cell pellet when compared to the biotic control. A meta-fission pathway for the degradation of DDD is proposed based on the recovery of seven chlorinated metabolites identified by gas chromatography-mass spectrometry analysis.
 
Article
Thermodynamic calculations were coupled with time-series measurements of chemical species (parent and daughter chlorinated solvents, H(2), sulfite, sulfate and methane) to predict the anaerobic transformation of cis-1,2-dichloroethene (cis-1,2-DCE) and 1,2-dichloroethane (1,2-DCA) in constructed wetland soil microcosms inoculated with a dehalorespiring culture. For cis-1,2-DCE, dechlorination occurred simultaneously with sulfite and sulfate reduction but competitive exclusion of methanogenesis was observed due to the rapid H(2) drawdown by the dehalorespiring bacteria. Rates of cis-1,2-DCE dechlorination decreased proportionally to the free energy yield of the competing electron acceptor and proportionally to the rate of H(2) drawdown, suggesting that H(2) competition between dehalorespirers and other populations was occurring, affecting the dechlorination rate. For 1,2-DCA, dechlorination occurred simultaneously with methanogenesis and sulfate reduction but occurred only after sulfite was completely depleted. Rates of 1,2-DCA dechlorination were unaffected by the presence of competing electron-accepting processes. The absence of a low H(2) threshold suggests that 1,2-DCA dechlorination is a cometabolic transformation, occurring at a higher H(2) threshold, despite the high free energy yields available for dehalorespiration of 1,2-DCA. We demonstrate the utility of kinetic and thermodynamic calculations to understand the complex, H(2)-utilizing reactions occurring in the wetland bed and their effect on rates of dechlorination of priority pollutants.
 
Article
The bacterial diversity of an anaerobic 1,2-dichloropropane (DCP) dechlorinating bioreactor consortium derived from river sediment has been investigated by a combined molecular approach. By using rDNA clone libraries, denaturing gradient gel electrophoresis and quantitative real-time PCR, both Dehalococcoides ethenogenes- and Dehalobacter restrictus-like 16S rDNA sequences were found within the community. Both species are known for reductive dechlorination of tetrachloroethene. Furthermore, numerous yet-uncultured members of the Green non-sulfur bacteria occurred within the consortium. The community analyses over a period of 14 months revealed a clear population shift. D. restrictus 16S rDNA was enriched significantly and became the most abundant rDNA sequence type, suggesting that Dehalobacter spp. play a key role within the reductive dechlorination of DCP in this consortium. We propose the use of this species as an indicator to monitor the transformation process within the bioreactor.
 
Article
The dynamics and composition of microbial communities in the aqueous phase of a model wetland supplied with cis- and trans-1,2-dichloroethenes (DCE)-contaminated groundwater was characterized. PCR-denaturing gradient gel electrophoresis analysis of water samples obtained from different parts of the wetland revealed that changes of the bacterial community structure coincided with a succession of the hydrochemical conditions in the wetland, from oxic towards anoxic conditions. During this transition phase, the appearance of vinyl chloride and ethene correlated with the presence of putative dechlorinating bacteria (Dehalococcoides spp., Geobacter spp. and Dehalobacter spp.). Additionally, a shift of the DCE isotopic composition indicated the progressive prevalence of reductive dechlorination in the wetland. Although the DCE degradation processes varied over time, biodegradation activity was maintained in the wetland system. 16S rRNA gene libraries revealed that Proteobacteria accounted for >50% of 16S rRNA genes clone libraries, whereas approximately 17% of the sequences from the wetland were related to sulphate reducers. Based on a multiple-method approach, this study illustrates the linkage between microbial community dynamics and composition, changes of hydrochemical conditions and processes of DCE degradation in a wetland system.
 
Article
Gene sequence alignments of the reductive dehalogenases PceA (Dehalospirillum multivorans) and CprA (Desulfitobacterium dehalogenans) were used to develop specific PCR primers binding to conserved regions of these sequences. These primers enabled us to amplify and subsequently sequence cprA-like gene fragments from the chlororespiring species Dehalobacter restrictus, Desulfitobacterium sp. strain PCE1, and D. hafniense. No specific amplicons were obtained from the chlororespiring species D. frappieri, D. chlororespirans, and Desulfomonile tiedjei. Furthermore, we were able to amplify and sequence cprA/pceA-like gene fragments from both trichlorobenzene (TCB)- and 1,2-dichloropropane (DCP)-dechlorinating microbial consortia using the novel primers. Subsequent sequence analysis of the fragments obtained from the microbial consortia revealed a group of four clusters (I-IV). Of these, clusters I and II showed the highest similarities to the cprA-like gene of Dehalobacter restrictus (79.0 and 96.2%, respectively). Cluster III comprised cprA-like sequences found in both the TCB- and the DCP-dechlorinating consortia, whereas sequences of cluster IV were most similar to the pceA gene of Dehalospirillum multivorans (97.8%). Our detection of genes encoding reductive dehalogenases, the key enzymes of chlororespiration, supports the hypothesis that reductive dechlorination of TCB and DCP occurs via a respiratory pathway.
 
Article
Many organisms have been found to readily oxidize the prevalent contaminant 1,2-dichloroethane (1,2-DCA) to CO2 under aerobic conditions. Some organisms have also been isolated that can reduce 1,2-DCA to ethene via dihaloelimination under anaerobic, fermentative conditions. However, none have been described that can metabolize 1,2-DCA under anoxic, nitrate-reducing conditions. In microcosms prepared from aquifer material and groundwater samples from a contaminated site in eastern Louisiana, USA, 1,2-DCA was observed to degrade with nitrate as the terminal electron acceptor. Nitrate-dependent enrichment cultures were developed from these microcosms that sustained rapid 1,2-DCA degradation rates of up to 500 microM day(-1). This degradation was tightly coupled to complete reduction of nitrate via nitrite to nitrogen gas. A novel 1,2-DCA-degrading organism belonging to the Betaproteobacteria (affiliated with the genus Thauera) was isolated from this enrichment culture. However, degradation rates were much slower in cultures of the isolate than observed in the parent mixed culture. Complete mineralization of 1,2-DCA to CO2 was linked to cell growth and to nitrate reduction in both enrichment and isolated cultures. Monochloroacetate, a putative metabolite of 1,2-DCA degradation, could also be mineralized by these cultures.
 
Article
A suite of experiments were conducted to ascertain whether dehalogenation of a model dioxin compound could be stimulated in marine sediments by supplementation with halogenated analogues to enrich for dehalogenating bacteria and if growth by members of the Chloroflexi-like group was associated with dioxin removal. Five halogenated compounds (tetrachlorobenzene, tetrachloroanisole, tetrachlorophenol, tetrachlorobenzoic acid and trichloroacetophenone) were added with 1,2,3,4-tetrachlorodibenzo-p-dioxin (TeCDD) to estuarine sediments from four sites in San Diego Bay and the coast of southern New Jersey to test for dioxin dehalogenation. Most of the halogenated additives were found to stimulate dechlorination of the model dioxin. Molecular analysis of the bacterial population using 16S rRNA and reductive dehalogenase genes indicated that distinct microbial populations were enriched with each halogenated co-amendment. Additionally, Chloroflexi-like ribosomal genes associated with dehalogenation were detected. For example, quantitative real-time PCR analysis of 16S rRNA and reductive dehalogenase gene copy number in the microcosms showed a positive correlation with 1,2,3,4-TeCDD reductive dechlorination in coastal sediments amended with different halogenated additives. These results suggest that specific Chloroflexi-like microorganisms related to Dehalococcoides are involved in 1,2,3,4-TeCDD reductive dechlorination.
 
Article
Five obligate anaerobes that were most closely related to Clostridium bifermentans, Clostridium celerecrescens, Clostridium saccharolyticum, Clostridium butyricum and Desulfovibrio desulfuricans by their 16S rRNA genes sequences were isolated from enrichment cultures using hexahydro-1,3,5-trinitro-1,3,5-triazine (RDX) as a nitrogen source. The above isolates transformed RDX at rates of 24.0, 5.4, 6.2, 2.5, 5.5 mumol h(-1) g (dry weight) of cells(-1), respectively, to nitrite, formaldehyde, methanol, and nitrous oxide. The present results indicate that clostridia are major strains responsible for RDX removal, and all isolates seemed to mainly transform RDX via its initial reduction to MNX and subsequent denitration. Since clostridia are commonly present in soil, we suggest that they may contribute to the removal of RDX in the subsurface (anoxic) soil.
 
Article
Octahydro-1,3,5,7-tetranitro-1,3,5,7-tetrazocine (HMX) is a toxic explosive known to be resistant to biodegradation. In this study, we found that sediment collected from two unexploded ordnance (UXO) disposal sites (UXO-3, UXO-5) and one nearby reference site (midref) in Hawaii contained anaerobic bacteria capable of removing HMX. Two groups of HMX-removing bacteria were found in UXO-5: group I contained aerotolerant anaerobes and microaerophiles, and group II contained facultative anaerobes. In UXO-3 and midref sediments, HMX-metabolizing bacteria were strictly anaerobic (group III and group IV). Using 16S rRNA sequencing, group I was assigned to a novel phylogenetic cluster of Clostridiales, and groups II and III were related to Paenibacillus and Tepidibacter of Firmicutes, respectively. Group IV bacteria were identified as Desulfovibrio of Deltaproteobacteria. Using [UL-(14)C]-HMX, group IV isolates were found to mineralize HMX (26.8% in 308 d) as determined by liberated (14)CO(2), but negligible mineralization was observed in groups I-III. Resting cells of isolates metabolized HMX to N(2)O and HCHO via the intermediary formation of 1-nitroso-octahydro-3,5,7-trinitro-1,3,5,7-tetrazocine together with methylenedinitramine. These experimental findings suggest that HMX biotransformation occurred either via initial denitration followed by ring cleavage or via reduction of one or more of the N-NO(2) group(s) to the corresponding N-NO bond(s) prior to ring cleavage.
 
Article
The distributions of bacterial form IA and form IC ribulose-1,5-bisphosphate carboxylase/oxygenase (RuBisCO) were investigated using Lowes Cove intertidal mudflat and Damariscotta Lake littoral sediments by PCR amplification of 492-495 bp fragments of the large subunit RuBisCO gene, cbbL. Genomic extracts for amplification were obtained from lake surface (upper 2 mm), mudflat surface (upper 2 mm), subsurface (5-7 cm), and soft-shell clam (Mya arenaria) burrow-wall sediments, as well as from a sulfide-oxidizing mat. Phylogenetic analyses of cbbL clone libraries revealed that Lowes Cove sediments were dominated by form IA cbbL-containing sequences most closely related to cbbL genes of sulfur-oxidizing bacteria or sulfide-oxidizing mats. In contrast, Damariscotta Lake cbbL clones contained primarily form IC cbbL sequences, which typify aerobic CO- and hydrogen-oxidizing facultative chemolithotrophs. Statistical analyses supported clear differentiation of intertidal and lake chemolithotroph communities, and provided evidence for some differentiation among intertidal communities. amova and libshuff analyses of Lowes Cove libraries suggested that M. arenaria burrow-wall sediments did not harbour distinct communities compared with surface and subsurface sediments, but that surface and subsurface libraries displayed moderate differences. The results collectively support a conceptual model in which the relative distribution of form IA- and IC-containing bacterial chemolithotrophs depends on sulfide availability, which could reflect the role of sulfate reduction in sediment organic matter metabolism, or the presence of geothermal sulfide sources.
 
Article
The Calvin cycle is known to be the major pathway for CO2 fixation, but our current understanding of its occurrence and importance in paddy soils is poor. In this study, the diversity of three ribulose-1,5-bisphosphate carboxylase/oxygenase large-subunit genes (cbbLG, cbbLR, cbbM) was investigated by clone library, T-RFLP, qPCR, and enzyme assay in five paddy soils in China. The cbbLG sequences revealed a relatively low level of diversity and were mostly related to the sequences of species from Thiobacillus. In contrast, highly diverse cbbLR and cbbM sequences were dispersed on the phylogenetic trees, and most of them were distantly related to known sequences, even forming separate clusters. Abundances of three cbbL genes ranged from 10(6) to 10(9) copies g(-1) soil, and cbbLR outnumbered cbbM and cbbLG in all soil samples, indicating that cbbLR may play a more important role than other two cbbL genes. Soil properties significantly influenced cbbL diversity in five paddy soils, of which clay content, C/N ratio, CEC, pH, and SOC correlated well with variations in microbial composition and abundance. In summary, this study provided a comparison of three cbbL genes, advancing our understanding of their role in carbon sequestration and nutrient turnover in the paddy soil.
 
Article
Archaeal communities in many acidic forest soil systems are dominated by a distinct crenarchaeal lineage Group 1.1c. In addition, they are found consistently in other acidic soils including grassland pasture, moorland and alpine soils. To determine whether soil pH is a major factor in determining their presence and abundance, Group 1.1c community size and composition were investigated across a pH gradient from 4.5 to 7.5 that has been maintained for > 40 years. The abundances of Group 1.1c Crenarchaeota, total Crenarchaeota and total bacteria were assessed by quantitative PCR (qPCR) targeting 16S rRNA genes and the diversity of Group 1.1c crenarchaeal community was investigated by denaturing gradient gel electrophoresis (DGGE) and phylogenetic analysis. The abundance of Group 1.1c Crenarchaeota declined as the pH increased, whereas total Crenarchaeota and Bacteria showed no clear trend. Community diversity of Group 1.1c Crenarchaeota was also influenced with different DGGE bands dominating at different pH. Group 1.1c Crenarchaeota were also quantified in 13 other soils representing a range of habitats, soil types and pH. These results exhibited the same trend as that shown across the pH gradient with Group 1.1c Crenarchaeota representing a greater proportion of total Crenarchaeota in the most acidic soils.
 
Article
The aim of this study was to investigate the diversity and variability of bacterial communities associated with the marine sponge Halichondria panicea with respect to tissue compartmentalization as well as seasonal and small-scale geographic variation. Diversity of microorganisms in sponges was investigated recently, but work on the variability and succession of associated bacterial communities is rare. Despite some information on Pacific and Mediterranean sponges, it is still uncertain whether bacteria and sponges are specifically associated. In this study, H. panicea specimens were sampled throughout the year at different stations around the island of Helgoland (North Sea) and investigated using molecular tools. The bacterial community associated with H. panicea was diverse, consisting of one denaturing gradient gel electrophoresis (DGGE) band occurring in most 'tissue' samples and additional variable bands. Variability was observed between different sponge fractions (i.e. the aquiferous system and the 'tissue'), sampling locations, and sampling dates. A PCR-DGGE specific for the Roseobacter group of marine Alphaproteobacteria displayed low diversity and a marked similarity between all samples. Phylogenetic analysis also pointed to specific Alphaproteobacteria of the Roseobacter group, which was predominant in most sponge 'tissue' samples. We conclude that H. panicea harbour a specific Roseobacter population with varying bacterial co-populations occurring seasonally or on a small-scale geographically, sometimes even dominating the bacterial community.
 
Rarefaction curves at different similarity cut-off values. The distance matrix was calculated using the maximum composite likelihood method as implemented in the mega 4.0 software (Tamura et al., 2007). The curves were calculated using the dotur software (Schloss & Handelsman, 2005).
A phylogenetic tree including all obtained sequences from Zerka Ma'in (isolates as well as environmental samples). The tree was rooted with Escherichia coli K-12. The topology of the tree was tested with the bootstrap method using 1000 replicates.
A phylogenetic tree including all obtained Synechococcus sp. sequences from Zerka Ma'in together with relevant sequences from the database. The tree was rooted with Escherichia coli k12. The various sequences are color coded according to their origin as described in the global map legend. The topology of the tree was tested with the bootstrap method using 1000 replicates.
A phylogenetic tree including all obtained Spirulina-like sequences from Zerka Ma'in, together with relevant sequences from the database. The tree was rooted with Escherichia coli K-12. The various sequences are color coded according to their origin as described in the global map legend. The topology of the tree was tested with the bootstrap method using 1000 replicates.
Article
The thermal springs of Zerka Ma'in, with waters emerging at temperatures up to 63 degrees C, have been of interest to biologists already from the beginning of the 19th century. These waters, springing out from below ground and flowing into the hypersaline Dead Sea, form an isolated environment from a biogeographic point of view. We have investigated the molecular diversity of the cyanobacteria in the springs. The diversity discovered was large, defining operational taxonomic units (OTUs) by a cutoff of 97% similarity; 10 major OTUs were found, including an as yet unidentified cluster of cyanobacteria. The various patterns of similarities of our sequences to others obtained from different thermal environments worldwide led us to rethink the common theories in biogeography. Based on the data obtained, we suggest that there is no constant geographical separation of microorganisms; however, local speciation does occur at a rate dictated mainly by local community dynamics and the rate of entrance of new organisms into the ecosystem.
 
Article
Bacterial populations and pathways involved in acetate and propionate consumption were studied in anoxic brackish sediment from the Grosser Jasmunder Bodden, German Baltic Sea. Uptake of acetate and propionate from the porewater was studied using stable carbon isotope-labeled compounds. Labeled acetate was not produced as an intermediate during propionate uptake experiments, and propionate consumption was not affected by the addition of acetate. In parallel, incorporation of labeled acetate and propionate into phospholipid-derived fatty acids (PLFA) was studied to indicate bacterial populations involved in the consumption of these substrates. The (13)C-acetate label was mainly recovered in even-numbered PLFA (16:1omega7c, 16:0 and 18:1omega7c). In contrast, primarily odd-numbered PLFA (a15:0, 15:0, 17:1omega6 and 17:0) and the even-numbered i16:0 were labeled after incubation with (13)C-propionate. Although single PLFA labeled with propionate are commonly found in sulfate reducers, the complete PLFA-labeling pattern does not resemble any of the know strains. However, the acetate-labeling pattern is similar to Desulfotomaculum acetoxidans and Desulfofrigus spp., two acetate-consuming, sulfate reducers. In conclusion, our data suggest that acetate and propionate were predominantly consumed by different, specialized groups of sulfate-reducing bacteria.
 
Article
Vibrio cholerae, the causative agent of cholera, is a natural inhabitant of the aquatic ecosystem. Chironomid (nonbiting midges) egg masses were recently found to harbour V. cholerae non-O1 and non-O139, providing a natural reservoir for the cholera bacterium. Chironomid populations and the presence of V. cholerae in chironomid egg masses were monitored. All V. cholerae isolates were able to degrade chironomid egg masses. The following virulence associated genes were detected in the bacterial isolates: hapA (100%), toxR (100%), hlyA (72%) and ompU (28%). The chironomid populations and the V. cholerae in their egg masses followed the phenological succession and interaction of host-pathogen population dynamics. A peak in the chironomid population was followed by a peak in the V. cholerae population. If such a connection is further substantiated for the pathogenic serogroups of V. cholerae in endemic areas of the disease, it may lead to a better understanding of the role of chironomids as a host for the cholera bacterium.
 
Article
Abstract Alterations in soil microfungal community structure across a transect between a semi-natural upland grassland and an agriculturally improved enclosure were assessed using an indirect measurement of active fungal biomass (ergosterol), together with a nucleic acid approach, terminal restriction fragment length polymorphism (TRFLP), which was compared to a commonly used but less sensitive community fingerprinting technique, denaturing gradient gel electrophoresis (DGGE). These techniques indicated that there was no reduction in numbers of fungal ribotypes across the floristic transect, despite decreased floristic diversity and a reduction of more than two-fold in ergosterol concentration. Although there were no differences in ribotype number, there was a decrease in diversity and an increase in dominance in only one of the transitional areas. The highest degree of variability within fungal communities was also found in this transitional area, with 84% of ribotypes only being detected in one of three replicates. Comparison of the two fungal community fingerprinting approaches indicated that TRFLP (26-33 ribotypes) was more sensitive for monitoring alterations in fungal community structure than DGGE (13-18 ribotypes). Using a measurement of the relative percentage of each ribotype within communities, a decrease in abundance of prominent ribotypes of the natural grassland soil fungal community was indicated together with an emergence of previously undetected ribotypes towards the improved area. This may have important implications for ecosystem stability or productivity, particularly if agricultural inputs to managed grasslands are suspended.
 
Article
In order to develop effective bioremediation strategies for radionuclide contaminants, the composition and metabolic potential of microbial communities need to be better understood, especially in highly contaminated subsurface sediments for which little cultivation-independent information is available. In this study, we characterized metabolically active and total microbial communities associated with uranium-contaminated subsurface sediments along geochemical gradients. DNA and RNA were extracted and amplified from four sediment-depth intervals representing moderately acidic (pH 3.7) to near-neutral (pH 6.7) conditions. Phylotypes related to Proteobacteria (Alpha-, Beta-, Delta- and Gammaproteobacteria), Bacteroidetes, Actinobacteria, Firmicutes and Planctomycetes were detected in DNA- and RNA-derived clone libraries. Diversity and numerical dominance of phylotypes were observed to correspond to changes in sediment geochemistry and rates of microbial activity, suggesting that geochemical conditions have selected for well-adapted taxa. Sequences closely related to nitrate-reducing bacteria represented 28% and 43% of clones from the total and metabolically active fractions of the microbial community, respectively. This study provides the first detailed analysis of total and metabolically active microbial communities in radionuclide-contaminated subsurface sediments. Our microbial community analysis, in conjunction with rates of microbial activity, points to several groups of nitrate-reducers that appear to be well adapted to environmental conditions common to radionuclide-contaminated sites.
 
Article
Autotrophic ammonia-oxidizing bacteria were considered to be responsible for the majority of ammonia oxidation in soil until the recent discovery of the autotrophic ammonia-oxidizing archaea. To assess the relative contributions of bacterial and archaeal ammonia oxidizers to soil ammonia oxidation, their growth was analysed during active nitrification in soil microcosms incubated for 30 days at 30 degrees C, and the effect of an inhibitor of ammonia oxidation (acetylene) on their growth and soil nitrification kinetics was determined. Denaturing gradient gel electrophoresis (DGGE) analysis of bacterial ammonia oxidizer 16S rRNA genes did not detect any change in their community composition during incubation, and quantitative PCR (qPCR) analysis of bacterial amoA genes indicated a small decrease in abundance in control and acetylene-containing microcosms. DGGE fingerprints of archaeal amoA and 16S rRNA genes demonstrated changes in the relative abundance of specific crenarchaeal phylotypes during active nitrification. Growth was also indicated by increases in crenarchaeal amoA gene copy number, determined by qPCR. In microcosms containing acetylene, nitrification and growth of the crenarchaeal phylotypes were suppressed, suggesting that these crenarchaea are ammonia oxidizers. Growth of only archaeal but not bacterial ammonia oxidizers occurred in microcosms with active nitrification, indicating that ammonia oxidation was mostly due to archaea in the conditions of the present study.
 
Article
Uncultured bacteria affiliated with the CL500-11 cluster (phylum Chloroflexi) were first reported from the oxygenated hypolimnion of Crater Lake (USA) as a predominant bacterioplankton, although this dominance has not been reported in other environments. In this study, we showed that CL500-11 is also dominant in the oxygenated hypolimnion of Lake Biwa (Japan) and followed its spatiotemporal succession using fluorescent in situ hybridization. CL500-11 cells were almost absent [< 1% of 4',6-diamidino-2-phenylindole (DAPI)-stained cells] at the beginning of the stratification period, dominated (> 10% of DAPI-stained cells; maximum = 16.5%) in the hypolimnion during the stratification period, and decreased to below the detection limit with the collapse of the thermocline. This pattern was observed over two annual cycles. A longitudinal assessment also showed that CL500-11 was the dominant bacterium in the hypolimnion over the whole lake, but was generally undetectable in the stratified epilimnion. These data suggest that CL500-11 is acclimated to the oxygenated hypolimnion and is a potentially important component of the pelagic biogeochemical cycling of the lake. A comparative analysis of 16S rRNA gene sequences revealed that almost all CL500-11 sequences previously deposited in the database were detected from hypolimnion or holomictic water in deep oxic freshwater lakes, suggesting that the bacteria may form one of the common lineages residing in an aerobic hypolimnetic niche.
 
Article
Crop plants genetically modified for the expression of Bacillus thuringiensis (Bt) insecticidal toxins have broad appeal for reducing insect damage in agricultural systems, yet questions remain about the impact of Bt plants on symbiotic soil organisms. Here, arbuscular mycorrhizal fungal (AMF) colonization of transgenic maize isoline Bt 11 (expressing Cry1Ab) and its non-Bt parental line (Providence) was evaluated under different fertilizer level and spore density scenarios. In a three-way factorial design, Bt 11 and non-Bt maize were inoculated with 0, 40, or 80 spores of Glomus mosseae and treated weekly with 'No' (0 g L(-1) ), 'Low' (0.23 g L(-1) ), or 'High' (1.87 g L(-1) ) levels of a complete fertilizer and grown for 60 days in a greenhouse. While no difference in AMF colonization was detected between the Bt 11 and Providence maize cultivars in the lower spore/higher fertilizer treatments, microcosm experiments demonstrated a significant reduction in AMF colonization in Bt 11 maize roots in the 80 spore treatments when fertilizer was limited. These results confirm previous work indicating an altered relationship between this Bt 11 maize isoline and AMF and demonstrate that the magnitude of this response is strongly dependent on both nutrient supply and AMF spore inoculation level.
 
Article
Ammonia oxidation, the first step in nitrification, is performed by autotrophic bacteria and thaumarchaea, whose relative contributions vary in different soils. Distinctive environmental niches for the two groups have not been identified, but evidence from previous studies suggests that activity of thaumarchaea, unlike that of bacterial ammonia oxidizers, is unaffected by addition of inorganic N fertilizer and that they preferentially utilize ammonia generated from the mineralization of organic N. This hypothesis was tested by determining the influence of both inorganic and organic N sources on nitrification rate and ammonia oxidizer growth and community structure in microcosms containing acidic, forest soil in which ammonia oxidation was dominated by thaumarchaea. Nitrification rate was unaffected by the incubation of soil with inorganic ammonium but was significantly stimulated by the addition of organic N. Oxidation of ammonia generated from native soil organic matter or added organic N, but not added inorganic N, was accompanied by increases in abundance of the thaumarchaeal amoA gene, a functional gene for ammonia oxidation, but changes in community structure were not observed. Bacterial amoA genes could not be detected. Ammonia oxidation was completely inhibited by 0.01% acetylene in all treatments, indicating ammonia monooxygenase-dependent activity. The findings have implications for current models of soil nitrification and for nitrification control strategies to minimize fertilizer loss and nitrous oxide production.
 
Article
Abstract The prokaryotic community inhabiting the deep subsurface sediments in the Forearc Basin of the Nankai Trough southeast of Japan (ODP Site 1176) was analyzed by 16S rDNA sequencing. Sediment samples from 1.15, 51.05, 98.50 and 193.96 m below sea floor (mbsf) harbored highly diverse bacterial communities. The most frequently retrieved clones included members of the Green non-sulfur bacteria whose closest relatives come from deep subsurface environments, a new epsilon-proteobacterial phylotype, and representatives of a cluster of closely related bacterial sequences from hydrocarbon- and methane-rich sediments around the world. Archaeal clones were limited to members of the genus Thermococcus, and were only obtained from the two deepest samples.
 
Article
One alternative to mineral fertilization is to use organic fertilizers. Our aim was to compare the impacts of 7-year applications of composted pig manure and ammonium nitrate on the structure and activity of the denitrifying community. Mineralization and organization of N, denitrification rates and N2O/N2 ratio were also investigated. Fourteen months after the last application, the potential denitrifying activity (+319%), N mineralization (+110%) and organization (+112%) were higher under pig compost than under ammonium nitrate fertilization. On the other hand, the N2O/(N2O+N2) ratio was lower (P<0.05, n=5) under organic fertilization. These effects of organic fertilization were in accordance with its higher total carbon content and microbial biomass. Fingerprints and clone library analyses showed that the structure of the denitrifying community was affected by the fertilization regime. Our results reveal that organic or mineral fertilizer applications could affect both structure and activity of the denitrifying community, with a possible influence on in situ N2O fluxes. These effects of the fertilization regime persisted for at least 14 months after the last application.
 
Article
Abstract The methanogenic archaeal communities in the sediment of two basins of an artificially partitioned acidic bog lake were studied. In the northeast basin, which was separated from a peat bog, a high methane production rate was measured only in the upper layers of the sediment. In contrast, methane production was detected at various depths of the sediment in the southwest basin, which continuously receiving humic acids from the bog. Ten bands were observed in the denaturing gradient gel electrophoresis (DGGE) fingerprint of the 16S rDNA archaeal amplicons from the NE basin top (0-5 cm) sediment layer, which reflected the presence of at least 10 ribotypes. Seventy clones of the 16S rDNA amplicons were obtained from the NE basin top sediment layer, and were grouped into 10 operational taxonomy units (OTUs) according to their positions on the DGGE gel. Seven of these OTUs could be matched with the bands of the community fingerprint. Phylogenetic analyses indicated that the sequences clustered into three groups: five of the OTUs were related to Methanosaeta, four OTUs to Methanomicrobiales and one OTU to Methanobacterium. Among the OTUs, sequences with high similarities (>96%) were retrieved. The sequence data suggested that the diversity of the methanogenic archaeal community was limited. Bands corresponding to those three phylogenetic groups were found in the DGGE fingerprints of both NE and SW basins, which reflected the presence of the same dominant methanogenic archaeal groups in both basins. However, differences in the distribution of the ribotypes that had high 16S rRNA sequence identities were observed in the fingerprints between the two basins at various depths. The microdiversity decreased along the sediment depth in the NE basin, and vice versa in the SW basin. The greater variety of the archaeal ribotypes, apparently, correlated with the higher methanogenesis rate.
 
Article
The spatial distribution of the tremendous bacterial diversity in soil partially depends on the broad range of scales of soil physical structures and the size of bacteria. The aim of this article is to collect information on spatial distribution of bacteria, the genetic structure of bacterial populations and communities, and on spatial constraints that operate in soil. This has been addressed by studying the spatial pattern of micro-habitats for various bacterial types and the spatial spread of clones in soil environment. The clones were considered as the units of genetic population structure. Experimental findings from a number of studies provide evidence that in soils a clone and a micro-colony are not necessarily identical. For some bacterial types, members of the same clone have been found far apart. Besides, micro-colonies of a few cells have also been reported. Short-range cell movements seem to be common in soil, in agreement with the observation of high small-scale diversity (millimetre scale). The mechanisms for the spread of clones are complex and probably operate at different spatial scales, even for soil bacteria with no specific vectors. The hypothesis underlying the study of the spatial dimension of diversity is that it can reveal mechanisms of diversity maintenance and contribute to their evaluation, complementing available knowledge of genetic processes.
 
Aroclor 1242 total removal rates (%) in various sampling zones as a function of proximity to ryegrass roots grown in the soil treated with 8.0 mg kg−1 of Aroclor 1242. Bars are the SE of means of three replicates. RC, root compartment.
Different PLFAs concentrations at various zone layers in planted treatments. The number of 0–5 and >5 mm are the distances from the root surface, which denote RC 1, 2, 3, 4, 5 mm near-rhizosphere and >5 mm for far-rhizosphere, respectively.
Article
Polychlorinated biphenyls in a commercial mixture (Aroclor 1242) were added to soil at 8.0 mg kg(-1) with and without ryegrass (Lolium multiflorum L.) planted in a specially designed rhizobox. At the end of 90 days, the presence of plants significantly increased Aroclor 1242 degradation compared with soils without ryegrass. Phospholipid fatty acids (PLFAs) profiles were affected by the distance from the rhizosphere, indicating a distance-dependent selective enrichment of competent species that may be responsible for efficient Aroclor 1242 degradation. The highest concentration of total PLFAs also occurred at 3 mm from the root zone in planted soils. The numbers of bacteria (cy17:0, 16:0), gram-positive bacteria (a15:0, i16:1, a17:0) and actinomycete (18:2 omega 6,9c) were significantly higher in planted soils than in unplanted soils. Furthermore, individual PLFAs [i16:0, 16:0 N alcohol, 18:0(10Me), i16:1, a15:0, i14:1, 14:0 2OH, 18:1 omega 9c, a17:0, 14:0 3OH, i14:0, a16:0, 16:1 omega 5c] were strongly correlated with the Aroclor 1242 degradation rates (%) (P<0.05) in planted treatments, whereas individual PLFAs of i16:1, 12:0 3OH, 15:0, a15:0 had significant correlations with the Aroclor 1242 degradation rates (%) (P<0.05) in unplanted soils. In particular, individual PLFAs i16:1 had strong correlations with Aroclor 1242 degradation in treatments both with and without ryegrass.
 
Article
Microbial populations in 16 groundwater samples from six Fennoscandian Shield sites in Finland and Sweden were investigated. The average total cell number was 3.7x10(5) cells ml(-1), and there was no change in the mean of the total cell numbers to a depth of 1390 m. Culture media were designed based on the chemical composition of each groundwater sample and used successfully to culture anaerobic microorganisms from all samples between 65 and 1350 m depth. Between 0.0084 and 14.8% of total cells were cultured from groundwater samples. Sulfate-reducing bacteria, iron-reducing bacteria and heterotrophic acetogenic bacteria were cultured from groundwater sampled at 65-686 m depth in geographically distant sites. Different microbial populations were cultured from deeper, older and more saline groundwater from 863 to 1350 m depth. Principal component analysis of groundwater chemistry data showed that sulfate- and iron-reducing bacteria were not detected in the most saline groundwater. Iron-reducing bacteria and acetogens were cultured from deep groundwater that contained 0.35-3.5 mM sulfate, while methanogens and acetogens were cultured from deep sulfate-depleted groundwater. In one borehole from which autotrophic methanogens were cultured, dissolved inorganic carbon was enriched in (13)C compared to other Fennoscandian Shield groundwater samples, suggesting that autotrophs were active. It can be concluded that a diverse microbial community is present from the surface to over 1300 m depth in the Fennoscandian Shield.
 
Article
In sustainable, low-input cropping systems the natural roles of microorganisms in maintaining soil fertility and biocontrol of plant pathogens may be more important than in conventional agriculture where their significance has been marginalised by high inputs of agrochemicals. Better understanding of the interactions between arbuscular mycorrhizal fungi and other microorganisms is necessary for the development of sustainable management of soil fertility and crop production. Many studies of the influence of mycorrhizal colonisation on associated bacterial communities have been conducted, however, the mechanisms of interaction are still poorly understood. Novel approaches including PCR-based methods, stable isotope profiling, and molecular markers have begun to shed light on the activity, identity and spatiotemporal location of bacteria in the mycorrhizosphere. This paper reviews current knowledge concerning the interactions between arbuscular mycorrhizal fungi and other microorganisms, particularly bacteria, and discusses the implications these interactions may have in sustainable agriculture.
 
Article
Cellulose hydrolysis often limits the kinetics and efficiency of anaerobic degradation in industrial digesters. In animal digestive systems, specialized microorganisms enable cellulose biodegradation at significantly higher rates. This study aims to assess the potential of ruminal microbial communities to settle and to express their cellulolytic properties in anaerobic digesters. Cellulose-degrading batch incubations were co-inoculated with municipal solid waste digester sludge and ruminal content. (13) C-labeled cellulose degradation was described over time with GC-C-IRMS. Results were linked to the identification of the microorganisms assimilating (13) C and to the monitoring of their relative dynamics. Cellulose degradation in co-inoculated incubations was efficient but not significantly improved. Transient disturbances in degradation pathways occurred, as revealed by propionate accumulation. ARISA dynamics and pyrosequencing revealed that expected classes of Bacteria and Archaea were active and degraded cellulose. However, despite favorable co-inoculation conditions, molecular tools also revealed that no ruminal species settled in the bioreactors. Other specific parameters were probably needed for this to happen. This study shows that exploiting the rumen's cellulolytic properties in anaerobic digesters is not straightforward. Co-inoculation can only be successful if ruminal microorganisms manage to thrive in the anaerobic digester and out-compete native microorganisms, which requires specific nutritional and environmental parameters, and a meticulous reproduction of the selection pressure encountered in the rumen. This article is protected by copyright. All rights reserved.
 
Article
More and more investigations indicate that genetic modification has no significant or persistent effects on microbial community composition in the rice rhizosphere. Very few studies however, have focused on its impact on functional microorganisms. This study completed a (13) C-CO2 pulse-chase labeling experiment comparing the potential effects of cry1Ab gene transformation on (13) C tissue distribution and rhizosphere methanogenic archaeal community composition with its parental rice variety (Ck) and a distant parental rice variety (Dp). Results showed that (13) C partitioning in aboveground biomass (mainly in stems) and roots of Dp was significantly lower than that of Ck. However, there were no significant differences in (13) C partitioning between the Bt transgenic rice line (Bt) and Ck. RNA-stable isotope probing combined with clone library analyses inferred that the group Methanosaetaceae was the predominant methanogenic Archaea in all three rice rhizospheres. The active methanogenic archaeal community in the Bt rhizosphere was dominated by Methanosarcinaceae, Methanosaetaceae and Methanomicrobiaceae, while there were only two main methanogenic clusters (Methanosaetaceae and Methanomicrobiaceae) in the Ck and Dp rhizospheres. These results indicate that the insertion of cry1Ab gene into the rice genome has the potential to result in the modification of methanogenic community composition in its rhizosphere. This article is protected by copyright. All rights reserved.
 
Article
Plasmid-mediated horizontal gene transfer influences bacterial community structure and evolution. However, an understanding of the forces which dictate the fate of plasmids in bacterial populations remains elusive. This is in part due to the enormous diversity of plasmids, in terms of size, structure, transmission, evolutionary history and accessory phenotypes, coupled with the lack of a standard theoretical framework within which to investigate them. This review discusses how ecological factors, such as spatial structure and temporal fluctuations, shape both the population dynamics and the physical features of plasmids. Novel data indicate that larger plasmids are more likely to be harboured by hosts in complex environments. Plasmid size may therefore be determined by environmentally mediated fitness trade-offs. As the correlation between replicon size and complexity of environment is similar for plasmids and chromosomes, plasmids could be used as tractable tools to investigate the influence of ecological factors on chromosomes. Parallels are drawn between plasmids and bacterial facultative symbionts, including the evolution of some members of both groups to a more obligate relationship with their host. The similarity between the influences of ecological factors on plasmids and bacterial symbionts suggests that it may be appropriate to study plasmids within a classical ecological framework.
 
Article
The following study evaluated the diversity and biogeography of 83 new atrazine-degrading bacteria and the composition of their atrazine degradation genes. These strains were isolated from 13 agricultural soils and grouped according to rep-PCR genomic fingerprinting into 11 major clusters, which showed biogeographic patterns. Three clusters (54 strains) belonged to the genus Arthrobacter, seven clusters (28 strains) were similar to the genus Nocardioides and only one strain was a gram-negative from the genus Ancylobacter. PCR assays for the detection of the genes atzA, B, C, D, E, F and trzN conducted with each of the 83 strains revealed that 82 strains (all gram positive) possessed trzN, 74 of them possessed the combination of trzN, atzB and atzC, while only the gram-negative strain had atzA. A similar PCR assay for the two analogous genes, atzA and trzN, responsible for the first step of atrazine degradation, was performed with DNA extracted directly from the enrichment cultures and microcosms spiked with atrazine. In these assays, the gene trzN was detected in each culture, while atzA was detected in only six out of 13 soils. These results raise an interesting hypothesis on the evolutionary ecology of the two atrazine chlorohydrolase genes (i.e. atzA and trzN) and about the biogeography of atrazine-degrading bacteria.
 
Article
Microorganisms capable of dissimilatory Fe(III) reduction in the temperature range of 52-90 degrees C were enriched from outer and inner parts of chimney-like structures, tubes of the polychaetous annelid Alvinella sp., and hydrothermal fluids collected at 13 degrees N hydrothermal vent sites on the East Pacific Rise at a depth of 2650 m. Numbers of culturable Fe(III)-reducing thermophiles estimated by the serial dilution technique varied from 10 to 10(7) cells per cm(3) of sample. Phylogenetic analysis of bacterial and archaeal PCR-amplified 16S rDNA genes obtained from Fe(III)-reducing enrichments and separated by denaturing gradient gel electrophoresis revealed sequences related to Deferibacter, Thermotogales (Bacteria) and Thermococcus (Archaea) for which the capacity for Fe(III) reduction had been reported. This was confirmed by isolating a hyperthermophilic iron reducer that belongs to the genus Thermococcus. Other bacterial thermophiles found in the enrichments were related to so far uncultured members of the Clostridiaceae, and epsilon-subdivision of the Proteobacteria.
 
Article
Exclusion of combined nitrogen (NaNO3) from the growth medium caused certain changes in metabolic processes leading to cessation in growth of the non-heterocystous, non nitrogen-fixing marine cyanobacterium Oscillatoria willei BDU 130511. But antioxidative enzymes, namely superoxide dismutase and peroxidase, helped the organism to survive the nitrogen stress. Prominent effects observed during nitrogen starvation/limitation were: (i) reduction of major and accessory photosynthetic pigments, (ii) impairment of photosynthesis due to loss of one major Rubisco isoenzyme, (iii) reduced synthesis of lipids and fatty acids, (iv) modifications of protein synthesis leading to the repression of three polypeptides and synthesis of two new polypeptides, (v) enhanced glutamine synthetase and reduced nitrate reductase activities, (vi) enhanced production of hydrogen peroxide and (vii) induced appearance of four new peroxidase isoenzymes. The observed metabolic changes were reversible, and the arrested growth under prolonged nitrogen deficiency could be fully restored upon subculturing in freshly prepared ASN III medium containing nitrogen (NaNO3). The present study demonstrates the capability of a non-nitrogen-fixer to withstand nitrogen stress making it an ecologically successful organism in the marine environment. The above pleiotropic effects of nitrogen deficiency also demonstrate that nitrogen plays a crucial role in growth and metabolism of marine cyanobacteria.
 
Article
Abstract The influences of the arbuscular mycorrhizal fungus Glomus intraradices on the culturable aerobic-heterotrophic bacterial communities in the rhizosphere and hyphosphere of cucumber plants (Cucumis satvius) were investigated. Mycorrhizal and non-mycorrhizal plants were grown in compartmentalised growth units, each with a root compartment and two lateral root-free compartments. Samples representing rhizosphere, root-free soil, root-free sand and washed sand extract were collected 52 days after sowing from treatments both with and without mycorrhiza. No significant difference in total bacterial number was observed between the mycorrhizal and non-mycorrhizal treatment. Fourteen hundred bacterial colonies were isolated and identified by fatty acid methyl ester analysis using the Sherlock system (MIDI Inc.), 87 species within 48 genera were identified with a similarity index >0.30. Pseudomonas, Arthrobacter and Burkholderia were the genera most frequently encountered. Large differences in bacterial community structure were observed between rhizosphere soil, root-free soil/sand and washed sand extract, whereas major differences between mycorrhizal and non-mycorrhizal treatments were observed for a few bacterial species only. Isolates identified as Paenibacillus spp. were more frequently found in the mycorrhizal treatment and especially in the washed sand extract with mycelium of G. intraradices, indicating that bacteria within this genus may live in close association with mycelium of these fungi.
 
Article
The dissolution of sulfide minerals such as pyrite (FeS2), arsenopyrite (FeAsS), chalcopyrite (CuFeS2), sphalerite (ZnS), and marcasite (FeS2) yields hot, sulfuric acid-rich solutions that contain high concentrations of toxic metals. In locations where access of oxidants to sulfide mineral surfaces is increased by mining, the resulting acid mine drainage (AMD) may contaminate surrounding ecosystems. Communities of autotrophic and heterotrophic archaea and bacteria catalyze iron and sulfur oxidation, thus may ultimately determine the rate of release of metals and sulfur to the environment. AMD communities contain fewer prokaryotic lineages than many other environments. However, it is notable that at least two archaeal and eight bacterial divisions have representatives able to thrive under the extreme conditions typical of AMD. AMD communities are characterized by a very limited number of distinct species, probably due to the small number of metabolically beneficial reactions available. The metabolisms that underpin these communities include organoheterotrophy and autotrophic iron and sulfur oxidation. Other metabolic activity is based on anaerobic sulfur oxidation and ferric iron reduction. Evidence for physiological synergy in iron, sulfur, and carbon flow in these communities is reviewed. The microbial and geochemical simplicity of these systems makes them ideal targets for quantitative, genomic-based analyses of microbial ecology and evolution and community function.
 
Photographs of the in situ collector deployed for 5 days at Genesis vent site. (a) Deposition of the collector near a Riftia pachyptila colony; the syntactic foam float (FL) is attached to the collector; white tubes of Alvinella pompejana (AL) are visible around the vent orifice (VE); bar, 10 cm (copyright Ifremer/PHARE 2002). (b) Recovery of the in situ collector after 5 days of incubation; the dense white biofilm formed on the syntactic foam float was scrapped once onboard.
Dot blot analysis of the probe specificities. Hybridization responses of radioactively labeled probes to nucleic acids from 27 target and nontarget DNAs (left) of environmental clones (light grey background) and strains (dark grey background). The blots were hybridized with the following probes: BACT338 (middle) and EPSI682 (right). The layout of nucleic acid samples on the blot is the following: 1A, VC1.2 clone 01; 1B, VC1.2 clone 10; 1C, VC1.2 clone 42; 2A, VC1.2 clone 26; 2B, VC1.2 clone 32; 2C, VC1.2 clone D; 3A, VC1.2 clone 21; 3B, VC1.2 clone 57; 3C, VC1.2 clone 31; 4A, VC1.2 clone 69; 4B, VC2.2 clone 01; 4C, VC2.2 clone 02; 5A, VC2.2 clone 03; 5B, Alvinella epibiont APB13B; 5C, Campylobacter jejuni; 6A, Campylobacter coli; 6B, Geospirillum barnesii; 6C, Arcobacter nitrofigilis; 7A, Thiomicrospira denitrificans; 7B, Wolinella succinogenes; 7C, Helicobacter pylori; 8A, Escherichia coli; 8B, Zobellia galactanivorans; 8C, Bacillus sp. strain SG9).
Whole cell hybridizations of different mixtures of fixed cells hybridized simultaneously with fluorescein-labeled (green fluorescence) and a rhodamine- or Cy3-labeled probe (red fluorescence). Epifluorescence photomicrographs (double exposures) are shown at a magnification of × 1140 (scale bar, 10 μm). Cells that hybridized with both types of probes appeared yellowish or orange on the double-exposed film. (a) Cells of Campylobacter jejuni and Nautilia lithotrophica simultaneously hybridized with probes EPSI682 (green fluorescence) and EUB338 (red fluorescence). (b) Cells of Arcobacter skirrowii and Desulfurobacterium thermolithotrophum simultaneously hybridized with probes EPSI682 (red fluorescence) and EUB338 (green fluorescence). (c) Cells of A. skirrowii and C. jejuni simultaneously hybridized with probes EPSI682 (green fluorescence) and ARC94 (red fluorescence). (d) Cells of A. skirrowii and C. jejuni simultaneously hybridized with probe EUB338 (green fluorescence) and ARC94 (red fluorescence).
Whole-cell hybridizations of cells from the mat collected on the 13°N EPR with probes EPSI682 (green fluorescence) and EUB338 (red fluorescence) (a–b), with probes EPSI682 (green fluorescence) and probe ARC94 (red fluorescence) (c) and with probes ARC94 (red fluorescence) and EUB338 (green fluorescence) (d and e). Epifluorescence photomicrographs (double exposures) are shown at a magnification of × 1,140 (scale bar, 10 μm).
Phylogenetic affiliations of bacterial type sequences detected in the hydrothermal mat sample
Article
Rapid growth of microbial sulphur mats have repeatedly been observed during oceanographic cruises to various deep-sea hydrothermal vent sites. The microorganisms involved in the mat formation have not been phylogenetically characterized, although the production of morphologically similar sulphur filaments by a Arcobacter strain coastal marine has been documented. An in situ collector deployed for 5 days at the 13 degrees N deep-sea hydrothermal vent site on the East Pacific Rise (EPR) was rapidly colonized by a filamentous microbial mat. Microscopic and chemical analyses revealed that the mat consisted of a network of microorganisms embedded in a mucous sulphur-rich matrix. Molecular surveys based on 16S rRNA gene and aclB genes placed all the environmental clone sequences within the Epsilonproteobacteria. Although few 16S rRNA gene sequences were affiliated with that of cultured organisms, the majority was related to uncultured representatives of the Arcobacter group (< or = 95% sequence similarity). A probe designed to target all of the identified lineages hybridized with more than 95% of the mat community. Simultaneous hybridizations with the latter probe and a probe specific to Arcobacter spp. confirmed the numerical dominance of Arcobacter-like bacteria. This study provides the first example of the prevalence and ecological significance of free-living Arcobacter at deep-sea hydrothermal vents.
 
Article
Stable carbon isotope analysis of chironomid larvae gave rise to the hypothesis that methane-oxidizing bacteria can provide an important food source for higher trophic levels in lakes. To investigate the importance of the methane cycle for the larval stable carbon signatures, isotope analysis and microbiological and biogeochemical investigations were combined. The study was based on comparison of a dimictic lake (Holzsee) and a polymictic, shallow lake (Großer Binnensee), both located in northern Germany. Both lakes are inhabited by Chironomus plumosus larvae, which exhibited a stronger 13C-depletion in Holzsee than in Großer Binnensee, indicating a greater contribution of methane–carbon in the former. Indeed, the processes involved in the microbial methane cycle were found to be more active in Holzsee, showing higher potential methane production and methane oxidation rates. Consistently, cell numbers of methane-oxidizing bacteria were with 0.5 − 1.7 × 106 cells about one order of magnitude higher in Holzsee than in Großer Binnensee. Molecular analysis of the microbial community structure revealed no differences in the methanotrophic community between the two lakes, with a clear dominance of type I methanotrophs. The methanogenic population seemed to be adapted to the prevailing substrate in the respective lake (H2/CO2 in Holzsee and acetate in Großer Binnensee), even though differences were minor. In conclusion, the stronger larval 13C-depletion in Holzsee was not reflected in differences in the microbial community structure, but in the activity and size of the methanogenic and methanotrophic populations in the lake sediment.
 
Distribution and quantity of PLFAs in rhizosphere soil of Bt and Ck at different stages of rice development at: (a) seedling; (b) booting; (c) heading; and (d) maturing stages. ‘*’ Indicate significant differences. Scale bars denote the SEs of the means.
PCA of the composition of 18 PLFAs in the rhizosphere soil of Bt and Ck at different stages of rice development. 7, Seedling; 8, booting; 9, heading; and 10, maturing stages. Scale bars denote the SEs of the means.
Percent 13C incorporated into individual PLFAs in rhizosphere soil of Bt and Ck at different stages of rice development. (a) Seeding; (b) booting; (c) heading; and (d) maturing stages. Scale bars denote the SEs of the means.
Article
Photosynthetic assimilation of CO2 is a primary source of carbon in soil and root exudates and can influence the community dynamics of rhizosphere organisms. Thus, if carbon partitioning is affected in transgenic crops, rhizosphere microbial communities may also be affected. In this study, the temporal effects of gene transformation on carbon partitioning in rice and rhizosphere microbial communities were investigated under greenhouse conditions using the 13C pulse-chase labeling method and phospholipid fatty acid (PLFA) analysis. The 13C contents in leaves of transgenic (Bt) and nontransgenic (Ck) rice were significantly different at the seedling, booting and heading stages. There were no detectable differences in 13C distribution in rice roots and rhizosphere microorganisms at any point during rice development. Although a significantly lower amount of Gram-positive bacterial PLFAs and a higher amount of Gram-negative bacterial PLFAs were observed in Bt rice rhizosphere as compared with Ck at all plant development stages, there were no significant differences in the amount of individual 13C-PLFA between Bt and Ck rhizospheres at any growing stage. These findings indicate that the insertion of cry1Ab and marker genes into rice had no persistent or adverse effect on the photosynthate distribution in rice or the microbial community composition in its rhizosphere.
 
Article
Reconstituted municipal solid waste (MSW) with varying contents of putrescible and cellulosic waste was incubated anaerobically under mesophilic conditions. Standard physicochemical parameters were monitored, together with stable isotopic signatures of produced CH(4) and CO(2). delta(13)C values for CH(4) indicated a change of methanogenic metabolism with time. CH(4) was predominantly produced from H(2)/CO(2) at the beginning of the incubations. This period was associated with important shifts in archaeal communities monitored by automated ribosomal intergenic spacer analysis (ARISA) and FISH of oligonucleotidic probes targeting specifically 16S rRNA gene of various methanogenic groups. The onset of the active methane generation phase was characterized by an increase of CH(4)delta(13)C, indicating a progressive shift toward an aceticlastic metabolism. When the methane production levelled off, a decrease in the isotopic signature was observed toward values characteristics of hydrogenotrophic metabolism. ARISA profiles were, however, found to be stable from the beginning of the active methane generation phase until the end of the experiment. FISH observation indicated that members of the family Methanosarcinaceae were predominant in the archaeal community during this period, suggesting that these methanogens might exhibit a high metabolic versatility during methanization of waste.
 
Article
Anaerobic microbial activity has a major influence on the subsurface environment. We investigated the denitrification and methanogenesis in anoxic groundwater at a depth of 140 m in two boreholes drilled in a sedimentary geological setting, where the redox potential fluctuated. The average maximum potential denitrification rates, measured under anaerobic conditions in the two boreholes using an (15) N tracer, were 0.060 and 0.085 nmol (30) N2 mL(-1) h(-1) . The nirS sequences obtained from in situ samples were similar to those of isolates belonging to the α-, β-, and γ-Proteobacteria, and the Firmicutes (72%-100% similarity at the amino acid level). Based on the nirS gene, the same operational taxonomic unit dominated incubated samples from each borehole. Methanogenesis candidates were detected by 16S rRNA gene analysis, but no sequence was detected using primers for the functional methanogenesis gene mcrA. Although the stable isotope signatures suggested that some of the dissolved methane was of biogenic origin, no potential for methane production was evident during the incubations. The groundwater at 140 m depth did not contain oxygen, had an Eh ranging from -144 to 6.8 mV, and was found to be a potential field for denitrification. This article is protected by copyright. All rights reserved.
 
Article
A combination of culture-independent and culturing methods was used to determine the impacts of hydrocarbon contamination on the diversity of bacterial communities in coastal soil from Ross Island, Antarctica. While numbers of culturable aerobic heterotrophic microbes were 1-2 orders of magnitude higher in the hydrocarbon-contaminated soil than control soil, the populations were less diverse. Members of the divisions Fibrobacter/Acidobacterium, Cytophaga/Flavobacterium/Bacteroides, Deinococcus/Thermus, and Low G+C gram positive occurred almost exclusively in control soils whereas the contaminated soils were dominated by Proteobacteria; specifically, members of the genera Pseudomonas, Sphingomonas and Variovorax, some of which degrade hydrocarbons. Members of the Actinobacteria were found in both soils.
 
Correspondence analysis of method usage in various scientific fields. In this symmetrical scaling of CA scores, the first two axes explained 47.3% and 35.8% of the total inertia of Table 1, respectively. The gray areas were drawn to facilitate the interpretation. Complete row names (scientific fields; full circles) and column names (methods; white triangles) are given in Table 1. Methods (triangles) located close to each other correspond to methods often occurring together in studies. The distance between a scientific field point and a method point approximates the probability of method usage in the field.
Ordination diagrams in two dimensions. (a) In a PCA biplot representation, samples are represented by dots and species by arrows. The arrows point in the direction of maximal variation in the species abundances, and their lengths are proportional to their maximal rate of change. Long arrows correspond to species contributing more to the data set variation. Right-angle projection of a sample dot on a species arrow gives approximate species abundance in the sample. (b) In a CA joint plot representation focusing on species distance, both samples and species are depicted as dots. Species dots correspond to the center of gravity (inertia) of the samples where they mostly occur. Distances between sample and species points give an indication of the probability of species composition in samples (see Table 2 for more details about diagram interpretation).
Partitioning biological variation into the effects of two factors. The large rectangle represents the total variation in the biological data table, which is partitioned among two sets of explanatory variables (a, b). Fraction 4 shows the unexplained part of the biological variation. Fractions 1 and 3 are obtained by partial constrained ordination or partial regression, and can be tested for significance. For instance, fraction 1 corresponds to the amount of biological variation that can be exclusively explained by (a) effects when (b) effects are taken into consideration (i.e., when b is considered as a covariable). Fraction 2 [i.e., variation indifferently attributed to (a) and (b) or a covariation of (a) and (b)] is obtained by subtracting fractions 1 and 3 from the total explained variance, and cannot be tested for statistical significance.
Relationships between numerical methods. Exploratory tools such as PCA, CA, PCoA, NMDS, or cluster analysis can be applied to a sample-by-species table to extract the main patterns of variation, to identify groups or clusters of samples, or specific species interactions. Sample scores on the main axes of variation can be related to variation in environmental variables using indirect gradient analyses. When a constrained analysis is desired (i.e. direct gradient analysis), RDA, db-RDA, CCA, or linear discriminant analysis can be used as extensions of the unconstrained methods. Mantel tests are appropriate to test the significance of the correlation between two distance matrices (e.g. one based on species data and the other on environmental variables). Raw data may be transformed, normalised or standardised as appropriate before analysis.
Combination of ordination and cluster analysis. On a same distance matrix, NMDS or PCOA can be applied to represent the major axes of variation among objects in a two-dimensional space. The superimposition of the results of cluster analysis (primary connections) onto the ordination diagram can help identify the structure in the data set as discontinuities (clusters) into a continuous space (ordination). Adapted from Legendre & Legendre (1998).
Article
Environmental microbiology is undergoing a dramatic revolution due to the increasing accumulation of biological information and contextual environmental parameters. This will not only enable a better identification of diversity patterns, but will also shed more light on the associated environmental conditions, spatial locations, and seasonal fluctuations, which could explain such patterns. Complex ecological questions may now be addressed using multivariate statistical analyses, which represent a vast potential of techniques that are still underexploited. Here, well-established exploratory and hypothesis-driven approaches are reviewed, so as to foster their addition to the microbial ecologist toolbox. Because such tools aim at reducing data set complexity, at identifying major patterns and putative causal factors, they will certainly find many applications in microbial ecology.
 
Article
The activity of the human armpit microbiota triggers the formation of body odor. We used differential 16S rRNA gene (rDNA)- and rRNA-based terminal-restriction fragment length polymorphism fingerprinting in combination with cloning and sequencing to identify active members of the human armpit microbiota. DNA and RNA were isolated from skin scrub samples taken from both armpits of 10 preconditioned, healthy males. The fingerprint profiles indicated pronounced similarities between the armpit microbiota in the right and the left axillae of an individual test person, but larger differences between the axilla microbiota of different individuals. Using 16S rDNA and rRNA sequence data, the majority of peaks in the armpit profiles were assigned to bacteria affiliated with well-known genera of skin bacteria. The relative abundances of all groups were similar among the rDNA and rRNA samples, suggesting that all groups of armpit bacteria were active. Surprisingly, the relative abundance of sequences affiliated with Peptoniphilus sp. was by far and with statistical significance the highest in the rRNA samples of the right armpits. Thus, bacteria affiliated with Peptoniphilus sp. might have been particularly active in the right axillae of the test persons, possibly owing to the handedness of the test persons, which might cause different environmental conditions in the right axillae.
 
Top-cited authors
Kornelia Smalla
  • Julius Kühn-Institut
J.G. Kuenen
  • Delft University of Technology
Angela Sessitsch
  • AIT Austrian Institute of Technology
Hendrikus (Riks) J Laanbroek
  • Koninklijke Nederlandse Akademie van Wetenschappen
Søren J Sørensen
  • University of Copenhagen