Antonis Chatzinotas

Analysis of bacterial community structure in bulk soil by in situ hybridization

In situ hybridization with rRNA-targeted, fluorescent (Cy3-labeled) oligonucleotide probes was used to analyze bacterial community structure in ethanol- or paraformaldehyde-fixed bulk soil after homogenization of soil samples in 0.1% pyrophosphate by mild ultrasonic treatment. In ethanol-fixed samples 37 ± 7%, and in paraformaldehyde 41 ± 8% of the 4′, 6-diamidino-2-phenylindole(DAPI)-stained cells were detected with the bacterial probe Eub338. The yield could not be increased by enzymatic and/or chemical pretreatments known to enhance the permeability of bacterial cells for probes. However, during storage in ethanol for 7 months, the detectability of bacteria increased in both ethanol- and paraformaldehyde-fixed samples to up to 47 ± 8% due to an increase in the detection yield of members of the α-subdivision of Proteobacteria from 2 ± 1% to 10 ± 3%. Approximately half of the bacteria detected by probe Eub338 could be affiliated to major phylogenetic groups such as the α-, β-, γ-, and δ-subdivisions of Proteobacteria, gram-positive bacteria with a high G+C DNA content, bacteria of the Cytophaga-Flavobacterium cluster of the CFB phylum, and the planctomycetes. The analysis revealed that bacteria of the α- and δ-subdivision of Proteobacteria and the planctomycetes were predominant. Here, members of the α-subdivision of Proteobacteria accounted for approximately 10 ± 3% of DAPI-stained cells, which corresponded to 44 ± 16 × 108 cells (g soil, dry wt.)–1, while members of the δ-subdivision of Proteobacteria made up 4 ± 2% of DAPI-stained cells [17 ± 9 × 108 cells (g soil, dry wt.)–1]. A large population of bacteria in bulk soil was represented by the planctomycetes, which accounted for 7 ± 3% of DAPI-stained cells [32 ± 12 × 108 cells (g soil, dry wt.)–1]. The detection of planctomycetes in soil confirms previous reports on the occurrence of planctomycetes in soil and indicates a yet unknown ecological significance of this group, which to date has never been isolated from terrestrial environments.

Impacts of heavy metal contamination and phytoremediation on a microbial community during a twelve-month microcosm experiment

The effects of heavy metals and phytoextraction practices on a soil microbial community were studied during 12 months using a hyperaccumulating plant (Thlaspi caerulescens) grown in an artificially contaminated soil. The 16S ribosomal RNA genes of the Bacteria and the beta-Proteobacteria and the amoA gene (encoding the alpha-subunit of ammonia monooxygenase) were PCR-amplified and analysed by denaturing gradient gel electrophoresis (DGGE). Principal component analysis (PCA) of the DGGE data revealed that: (i) the heavy metals had the most drastic effects on the bacterial groups targeted, (ii) the plant induced changes which could be observed in the amoA and in the Bacteria 16S rRNA gene patterns, (iii) the changes observed during 12 months in the DGGE-patterns of the planted contaminated soil did not indicate recovery of the initial bacterial community present in the non-contaminated soil. The potential function of the microbial community was assessed recording community level physiological profiles (CLPP) and analysing them by PCA. The lower capability of the bacterial community to degrade the substrates provided in the BIOLOG plates, in particular the amino acids, amides and amines, as well as a delay in the average well colour development (AWCD) differentiated the bacterial community of the contaminated samples from that of the non-contaminated ones. However, the plant had a positive effect on substrate utilization as shown by the greater number of substrates used in all planted samples compared to unplanted ones. Finally, the measurement of the potential ammonia oxidation indicated that ammonia oxidising bacteria were completely inhibited in the contaminated soil. The stimulation of ammonia oxidation by the plant observed in the non-contaminated samples was surpassed by the inhibitory effect of the heavy metals in the contaminated soil. This study emphasises the combined use of culture-independent techniques with conventional methods to investigate the ecology of bacteria in their natural habitats.

Interannual variation in land-use intensity enhances grassland multidiversity

Significance Land-use intensification is a major threat to biodiversity. So far, however, studies on biodiversity impacts of land-use intensity (LUI) have been limited to a single or few groups of organisms and have not considered temporal variation in LUI. Therefore, we examined total ecosystem biodiversity in grasslands varying in LUI with a newly developed index called multidiversity, which integrates the species richness of 49 different organism groups ranging from bacteria to birds. Multidiversity declined strongly with increasing LUI, but changing LUI across years increased multidiversity, particularly of rarer species. We conclude that encouraging farmers to change the intensity of their land use over time could be an important strategy to maintain high biodiversity in grasslands. Although temporal heterogeneity is a well-accepted driver of biodiversity, effects of interannual variation in land-use intensity (LUI) have not been addressed yet. Additionally, responses to land use can differ greatly among different organisms; therefore, overall effects of land-use on total local biodiversity are hardly known. To test for effects of LUI (quantified as the combined intensity of fertilization, grazing, and mowing) and interannual variation in LUI (SD in LUI across time), we introduce a unique measure of whole-ecosystem biodiversity, multidiversity. This synthesizes individual diversity measures across up to 49 taxonomic groups of plants, animals, fungi, and bacteria from 150 grasslands. Multidiversity declined with increasing LUI among grasslands, particularly for rarer species and aboveground organisms, whereas common species and belowground groups were less sensitive. However, a high level of interannual variation in LUI increased overall multidiversity at low LUI and was even more beneficial for rarer species because it slowed the rate at which the multidiversity of rare species declined with increasing LUI. In more intensively managed grasslands, the diversity of rarer species was, on average, 18% of the maximum diversity across all grasslands when LUI was static over time but increased to 31% of the maximum when LUI changed maximally over time. In addition to decreasing overall LUI, we suggest varying LUI across years as a complementary strategy to promote biodiversity conservation.

Effects of long-term differential fertilization on eukaryotic microbial communities in an arable soil: a multiple barcoding approach.

To understand the fine‐scale effects of changes in nutrient availability on eukaryotic soil microorganisms communities, a multiple barcoding approach was used to analyse soil samples from four different treatments in a long‐term fertilization experiment. We performed PCR amplification on soil DNA with primer pairs specifically targeting the 18S rRNA genes of all eukaryotes and three protist groups (Cercozoa, Chrysophyceae‐Synurophyceae and Kinetoplastida) as well as the ITS gene of fungi and the 23S plastid rRNA gene of photoautotrophic microorganisms. Amplicons were pyrosequenced, and a total of 88 706 quality filtered reads were clustered into 1232 operational taxonomic units (OTU) across the six data sets. Comparisons of the taxonomic coverage achieved based on overlapping assignment of OTUs revealed that half of the eukaryotic taxa identified were missed by the universal eukaryotic barcoding marker. There were only little differences in OTU richness observed between organic‐ (farmyard manure), mineral‐ and nonfertilized soils. However, the community compositions appeared to be strongly structured by organic fertilization in all data sets other than that generated using the universal eukaryotic 18S rRNA gene primers, whereas mineral fertilization had only a minor effect. In addition, a co‐occurrence based network analysis revealed complex potential interaction patterns between OTUs from different trophic levels, for example between fungivorous flagellates and fungi. Our results demonstrate that changes in pH, moisture and organic nutrients availability caused shifts in the composition of eukaryotic microbial communities at multiple trophic levels.

Analysis of broad-scale differences in microbial community composition of two pristine forest soils

Broad-scale differences in soil microbial community composition were analyzed in two contrasting soils using DNA reassociation and % G + C profiles for analysis on the community-level, and filter- and whole cell hybridization techniques for a coarse-level characterization of larger phylogenetic groups of bacteria. Reassociation analysis of DNA from bacterial fractions extracted from the organic soil Seim and the mineral soil Hau revealed similar complexity of the communities with 5700 and 4900 different bacterial genomes (g soil [dry wt])-1, respectively. Thermal denaturation studies showed wide % G + C distributions in DNA from bacteria of both soils. Differences in the median % G + C with 55 to 61% for the bacterial community in soil Seim and 61 to 66% for that in soil Hau indicated a higher proportion of bacteria with a high DNA G + C content in soil Hau. In situ hybridization with fluorescent (Cy3-labeled) probes targeting larger phylogenetic groups showed minor differences between both soils, and between direct detection of bacteria in dispersed soil slurries and in bacterial fractions extracted from soils through about 90% of the total bacteria were lost during extraction. In dispersed slurries of both soils, only probes ALF1b, SRB385, and PLA46 hybridized to cells accounting for more than 1% of the DAPI-stained cells, while numbers obtained after hybridization with probes ARCH915, BET42a, GAM42a, HGC69a, and CF319a were below the detection limit set at < 1%. These results were confirmed by in situ hybridization with horseradish peroxidase (HRP)-labeled probes and subsequent Cy3-tyramide signal amplification. In contrast, dot blot hybridization with probe HGC69a indicated significant amounts of Gram-positive bacteria with a high DNA G + C content in both soils. These could subsequently be visualized in non-dispersed soil slurries by in situ hybridization with HRP-labeled probe HGC69a and Cy3-tyramide signal amplification. Filamentous Gram-positive bacteria with a high DNA G + C content, likely actinomycetes, which are present in soil Hau in significant numbers are obviously destroyed by procedures used for soil dispersion.

Differential enumeration and in situ localization of microorganisms in the hindgut of the lower termite Mastotermes darwiniensis by hybridization with rRNA-targeted probes

Abstract We examined the abundance and spatial distribution of major phylogenetic groups of the domain Bacteria in hindguts of the Australian lower termite Mastotermes darwiniensis by using in situ hybridization with group-specific, fluorescently labeled, rRNA-targeted oligonucleotide probes. Between 32.0 ± 7.2% and 52.3 ± 8.2% of the DAPI-stained cells in different hindgut fractions were detected with probe EUB338, specific for members of the domain Bacteria. About 85% of the prokaryotic cells were associated with the flagellates of the thin-walled anterior region (P3a) and the thick wall of the posterior region (P3b/P4) of the hindgut, as shown by DAPI staining. At most, half of the EUB338-detected cells hybridized with one of the other probes that targeted a smaller assemblage within the bacterial domain. In most fractions, cells were found in varying numbers with probe ALF1b, which targeted members of the α-Proteobacteria, whereas substantial amounts of sulfate-reducing bacteria, gram-positive bacteria with a high DNA G+C content and members of the Cytophaga-Flavobacterium cluster of the Cytophaga-Flavobacterium-Bacteroides (CFB) phylum could be detected only in the wall fraction of P3b/P4. This clearly indicates that the hindgut microhabitats differ in the composition of their microbial community. In situ hybridization of cryosections through the hindgut showed only low numbers of bacteria attached to the P3a wall. In contrast, the wall of P3b was densely colonized by rod- and coccus-shaped bacteria, which could be assigned to the Cytophaga-Flavobacterium cluster of the CFB phylum and to the group of gram-positive bacteria with a high DNA G+C content, respectively. Oxygen concentration profiles determined with microelectrodes revealed steep oxygen gradients both in P3a and P3b. Oxygen was consumed within 100 μm below the gut surface, and anoxic conditions prevailed in the central portions of both gut regions, indicating that oxygen consumption in the hindgut does not depend on the presence of a biofilm on the hindgut wall.

Enrichment and characterization of a sulfate-reducing toluene-degrading microbial consortium by combining in situ microcosms and stable isotope probing techniques

A toluene-degrading microbial consortium was enriched directly in a BTEX-contaminated aquifer under sulfate-reducing conditions using in situ microcosms consisting of toluene-loaded activated carbon pellets. Degradation of toluene and concomitant sulfide production by the consortium was subsequently demonstrated in laboratory microcosms. The consortium was physiologically and phylogenetically characterized by isotope tracer experiments using nonlabeled toluene, [(13)C]-alpha-toluene or [(13)C(7)]-toluene as growth substrates. Cells incubated with [(13)C]-alpha-toluene or [(13)C(7)]-toluene incorporated 8-15 at.%(13)C and 51-57 at.%(13)C into total lipid fatty acids, respectively, indicating a lower specific incorporation of (13)C from [(13)C(7)]-toluene. In order to identify the toluene-assimilating bacteria, the incorporation of carbon from both [(13)C]-alpha-toluene and [(13)C(7)]-toluene into rRNA was analyzed by stable isotope probing. Time and buoyant density-resolved 16S rRNA gene-based terminal restriction fragment length polymorphism profiles, combined with cloning and sequencing, revealed that an uncultured bacterium (99% sequence similarity) related to the genus Desulfocapsa was the main toluene-degrading organism in the consortium. The ratio of the respective terminal restriction fragments changed over time, indicating trophic interactions within this consortium.

Evidence for Geographic Isolation and Signs of Endemism within a Protistan Morphospecies

ABSTRACT The possible existence of endemism among microorganisms resulting from and preserved by geographic isolation is one of the most controversial topics in microbial ecology. We isolated 31 strains of “Spumella-like” flagellates from remote sampling sites from all continents, including Antarctica. These and another 23 isolates from a former study were characterized morphologically and by small-subunit rRNA gene sequence analysis and tested for the maximum temperature tolerance. Only a minority of the Spumella morpho- and phylotypes from the geographically isolated Antarctic continent follow the worldwide trend of a linear correlation between ambient (air) temperature during strain isolation and heat tolerance of the isolates. A high percentage of the Antarctic isolates, but none of the isolates from locations on all other continents, were obligate psychrophilic, although some of the latter were isolated at low ambient temperatures. The drastic deviation of Antarctic representatives of Spumella from the global trend of temperature adaptation of this morphospecies provides strong evidence for geographic transport restriction of a microorganism; i.e., Antarctic protistan communities are less influenced by transport of protists to and from the Antarctic continent than by local adaptation, a subtle form of endemism.

Where less may be more: how the rare biosphere pulls ecosystems strings

Rare species are increasingly recognized as crucial, yet vulnerable components of Earth’s ecosystems. This is also true for microbial communities, which are typically composed of a high number of relatively rare species. Recent studies have demonstrated that rare species can have an over-proportional role in biogeochemical cycles and may be a hidden driver of microbiome function. In this review, we provide an ecological overview of the rare microbial biosphere, including causes of rarity and the impacts of rare species on ecosystem functioning. We discuss how rare species can have a preponderant role for local biodiversity and species turnover with rarity potentially bound to phylogenetically conserved features. Rare microbes may therefore be overlooked keystone species regulating the functioning of host-associated, terrestrial and aquatic environments. We conclude this review with recommendations to guide scientists interested in investigating this rapidly emerging research area.

Use of isotopic and molecular techniques to link toluene degradation in denitrifying aquifer microcosms to specific microbial populations.

Abstract. Microcosms were inoculated with sediments from both a petroleum-hydrocarbon (PHC)-contaminated aquifer and from a nearby pristine aquifer and incubated under anoxic denitrifying conditions with [methyl-13C]toluene. These microcosms served as a laboratory model system to evaluate the combination of isotope (13C-labeling of polar-lipid-derived fatty acids) and molecular techniques (16S rRNA-targeting gene probes) to identify the toluene-metabolizing population. After total depletion of toluene, the following bacterial phospholipid fatty acids (PLFA) were 13C-enriched: 16:1ω7c, 16:1ω7t, 16:0, cy17:0, and 18:1ω7c. Pure culture experiments demonstrated that these compounds were also found in PLFA profiles of PHC-degrading Azoarcus spp. (β-Proteobacteria) and related species. The origin of the CO2 evolved in the microcosms was determined by measurements of stable carbon isotope ratios. Toluene represented 11% of the total pool of mineralized substrates in the contaminated sediment and 54% in the pristine sediment. The microbial community in the microcosm incubations was characterized by using DAPI staining and whole-cell hybridization with specific fluorescently labeled 16S rRNA-targeted oligonucleotide probes. Results revealed that 6% of the DAPI-stained cells in the contaminated sediment and 32% in the pristine sediment were PHC-degrading Azoarcus spp. In biotic control microcosms (incubated under denitrifying conditions, no toluene added), Azoarcus spp. cells remained at less than 1% of the DAPI-stained cells. The results show that isotope analysis in combination with whole-cell hybridization is a promising approach to identify and to quantify denitrifying toluene degraders within microbial communities.