Frederic Gich

Previously unknown and phylogenetically diverse members of the green nonsulfur bacteria are indigenous to freshwater lakes

Abstract. The phylogenetic diversity of green nonsulfur bacteria in nine stratified freshwater lakes was investigated. A set of oligonucleotide primers was developed that permitted the selective amplification of 16S rRNA gene sequences of this group. Subsequently, amplification products were separated by denaturing gradient gel electrophoresis (DGGE) and sequenced, which yielded a total of 19 novel sequence types. Ten of the sequences were related to those of different cultivated members of the Chloroflexus assemblage, whereas nine fell into the T78 group of environmental clones. For the latter subgroup of the green nonsulfur bacteria, no molecular isolate from freshwater plankton has been reported so far. Several of the sequence types occurred in more than one lake, indicating that not only relatives of the Chloroflexus assemblage, but also bacteria of the clone T78 group represent indigenous bacteria of nonthermal stratified freshwater ecosystems. Our results indicate that the natural diversity in the phylum of the green nonsulfur bacteria has been significantly underestimated in the past.

Edaphobacter modestus gen. nov., sp. nov., and Edaphobacter aggregans sp. nov., acidobacteria isolated from alpine and forest soils.

The phylum Acidobacteria is currently represented mostly by environmental 16S rRNA gene sequences, and the phylum so far contains only four species with validly published names, Holophaga foetida, Geothrix fermentans, Acidobacterium capsulatum and Terriglobus roseus. In the present study, two novel strains of acidobacteria were isolated. High-throughput enrichments were set up with the MicroDrop technique using an alpine calcareous soil sample and a mixture of polymeric carbon compounds supplemented with signal compounds. This approach yielded a novel, previously unknown acidobacterium, strain Jbg-1T. The second strain, Wbg-1T, was recovered from a co-culture with a methanotrophic bacterium established from calcareous forest soil. Both strains represent members of subdivision 1 of the phylum Acidobacteria and are closely related to each other (98.0 % 16S rRNA gene sequence similarity). At a sequence similarity of 93.8-94.7 %, strains Jbg-1T and Wbg-1T are only distantly related to the closest described relative, Terriglobus roseus KBS 63T, and accordingly are described as members of the novel genus Edaphobacter gen. nov. Based on the DNA-DNA relatedness between strains Jbg-1T and Wbg-1T of 11.5-13.6 % and their chemotaxonomic and phenotypic characteristics, the two strains are assigned to two separate species, Edaphobacter modestus sp. nov. (the type species), with strain Jbg-1T (=ATCC BAA-1329T =DSM 18101T) as the type strain, and Edaphobacter aggregans sp. nov., with strain Wbg-1T (=ATCC BAA-1497T =DSM 19364T) as the type strain. The two novel species are adapted to low carbon concentrations and to neutral to slightly acidic conditions.

Specific Detection, Isolation, and Characterization of Selected, Previously Uncultured Members of the Freshwater Bacterioplankton Community

ABSTRACT High-throughput cultivation was combined with rapid and group-specific phylogenetic fingerprinting in order to recover representatives of three freshwater bacterioplankton communities. A total of 570 bacterial cultures were obtained by employing the most probable number and MicroDrop techniques. The majority of the cultured bacteria were closely related to previously uncultured bacteria and grouped with the α-Proteobacteria, β-Proteobacteria, Actinobacteria, Firmicutes, or Flavobacteria-Cytophaga lineage. Correspondingly, the natural bacterioplankton community was analyzed by high-resolution phylogenetic fingerprinting of these five bacterial lineages. 16S rRNA gene fragments were generated for each lineage and subsequently separated by denaturing gradient gel electrophoresis. By the combination of five group-specific PCR protocols, the total number of 16S rRNA gene fingerprints generated from the natural communities was increased sixfold compared to conventional (eubacterial) fingerprinting. Four of the environmental α-Proteobacteria 16S rRNA gene sequences obtained from the natural community were found to be identical to those of bacterial isolates. One of these phylotypes was detected in 14 different cultures and hence represented the most frequently cultured bacterium. Three of these 14 strains were characterized in detail. Their complete 16S rRNA gene sequences showed only 93% similarity to that of Sandaracinobacter sibiricus, the closest relative described so far. The novel phylotype of bacterium is a strict aerobe capable of using numerous organic carbon substrates and contains bacteriochlorophyll a bound to two different photosynthetic light-harvesting complexes. Dot blot hybridization revealed that the strains occur in lakes of different trophic status and constitute up to 2% of the microbial community.

Vertical distribution of ammonia-oxidizing crenarchaeota and methanogens in the epipelagic waters of Lake Kivu (Rwanda-Democratic Republic of the Congo)

ABSTRACT Four stratified basins in Lake Kivu (Rwanda-Democratic Republic of the Congo) were sampled in March 2007 to investigate the abundance, distribution, and potential biogeochemical role of planktonic archaea. We used fluorescence in situ hybridization with catalyzed-reported deposition microscopic counts (CARD-FISH), denaturing gradient gel electrophoresis (DGGE) fingerprinting, and quantitative PCR (qPCR) of signature genes for ammonia-oxidizing archaea (16S rRNA for marine Crenarchaeota group 1.1a [MCG1] and ammonia monooxygenase subunit A [amoA]). Abundance of archaea ranged from 1 to 4.5% of total DAPI (4′,6-diamidino-2-phenylindole) counts with maximal concentrations at the oxic-anoxic transition zone (∼50-m depth). Phylogenetic analysis of the archaeal planktonic community revealed a higher level of richness of crenarchaeal 16S rRNA gene sequences (21 of the 28 operational taxonomic units [OTUs] identified [75%]) over euryarchaeotal ones (7 OTUs). Sequences affiliated with the kingdom Euryarchaeota were mainly recovered from the anoxic water compartment and mostly grouped into methanogenic lineages (Methanosarcinales and Methanocellales). In turn, crenarchaeal phylotypes were recovered throughout the sampled epipelagic waters (0- to 100-m depth), with clear phylogenetic segregation along the transition from oxic to anoxic water masses. Thus, whereas in the anoxic hypolimnion crenarchaeotal OTUs were mainly assigned to the miscellaneous crenarchaeotic group, the OTUs from the oxic-anoxic transition and above belonged to Crenarchaeota groups 1.1a and 1.1b, two lineages containing most of the ammonia-oxidizing representatives known so far. The concomitant vertical distribution of both nitrite and nitrate maxima and the copy numbers of both MCG1 16S rRNA and amoA genes suggest the potential implication of Crenarchaeota in nitrification processes occurring in the epilimnetic waters of the lake.

Determination of the topography and biometry of chlorosomes by atomic force microscopy

Isolated chlorosomes of several species of filamentous anoxygenic phototrophic bacteria (FAPB) and green sulfur bacteria (GSB) were examined by atomic force microscopy (AFM) to characterize their topography and biometry. Chlorosomes of Chloroflexusaurantiacus, Chloronema sp., and Chlorobium (Chl.) tepidum exhibited a smooth surface, whereas those of Chl. phaeobacteroides and Chl. vibrioforme showed a rough one. The potential artifactual nature of the two types of surfaces, which may have arisen because of sample manipulation or AFM processing, was ruled out when AFM images and transmission electron micrographs were compared. The difference in surface texture might be associated with the specific lipid and polypeptide composition of the chlorosomal envelope. The study of three-dimensional AFM images also provides information about the size and shape of individual chlorosomes. Chlorosomal volumes ranged from ca. 35 000 nm3 to 247 000 nm3 for Chl. vibrioforme and Chl. phaeobacteroides, respectively. The mean height was about 25 nm for all the species studied, except Chl. vibrioforme, which showed a height of only 14 nm, suggesting that GSB have 1–2 layers of bacteriochlorophyll (BChl) rods and GFB have ∼4. Moreover, the average number of BChl molecules per chlorosome was estimated according to models of BChl rod organisation. These calculations yielded upper limits ranging from 34 000 BChl molecules in Chl. vibrioforme to 240 000 in Chl. phaeobacteroides, values that greatly surpass those conventionally accepted.

Diversity of Miscellaneous Crenarchaeotic Group archaea in freshwater karstic lakes and their segregation between planktonic and sediment habitats.

The Miscellaneous Crenarchaeotic Group (MCG) is an archaeal lineage whose members are widespread and abundant in marine sediments. MCG archaea have also been consistently found in stratified euxinic lakes. In this work, we have studied archaeal communities in three karstic lakes to reveal potential habitat segregation of MCG subgroups between planktonic and sediment compartments. In the studied lakes, archaeal assemblages were strikingly similar to those of the marine subsurface with predominance of uncultured Halobacteria in the plankton and Thermoplasmata and MCG in anoxic, organic-rich sediments. Multivariate analyses identified sulphide and dissolved organic carbon as predictor variables of archaeal community composition. Quantification of MCG using a newly designed qPCR primer pair that improves coverage for MCG subgroups prevalent in the studied lakes revealed conspicuous populations in both the plankton and the sediment. Subgroups MCG-5a and -5b appear as planktonic specialists thriving in euxinic bottom waters, while subgroup MCG-6 emerges as a generalist group able to cope with varying reducing conditions. Besides, comparison of DNA- and cDNA-based pyrotag libraries revealed that rare subgroups in DNA libraries, i.e. MCG-15, were prevalent in cDNA-based datasets, suggesting that euxinic, organic-rich sediments of karstic lakes provide optimal niches for the activity of some specialized MCG subgroups.

Active bacteria and archaea cells fixing bicarbonate in the dark along the water column of a stratified eutrophic lagoon

We studied the carbon dioxide fixation activity in a stratified hypereutrophic karstic lagoon using a combination of fingerprinting techniques targeting bacterial and archaeal 16S rRNA genes, functional gene cloning [the acetyl-CoA carboxylase (accC)], and isotopic labelling ((14)C-bicarbonate) coupled to single-cell analyses [microautoradiography combined with catalyzed reported deposition-FISH (MAR-CARD-FISH)]. The microbial planktonic community was dominated by bacteria with maximal abundances of archaea just below the oxic/anoxic transition zone (7% of total cells). In situ incubations with radiolabelled bicarbonate showed maximal photoassimilation activity in the oxic epilimnion, whereas dark CO(2) fixation was consistently observed throughout the water column, with a maximum at the oxic/anoxic interface (8.6 mg C m(-3) h(-1)). The contributions of light and dark carbon fixation activities in the whole water column were 69% and 31% of the total C incorporated, respectively. MAR-CARD-FISH incubations corroborated these results and revealed that the highest fraction of bacterial and archaeal cells actively uptaking bicarbonate in the light was found at the surface. The bacterial community was mainly composed of green sulfur bacteria (Chlorobi) and members of the Betaproteobacteria and the Bacteroidetes. The archaeal assemblage was composed of phylotypes of the Miscellaneous Crenarchaeotic Group and a few methanogens. Clone libraries of the accC gene showed an absolute dominance of bacterial carboxylases. Our results suggest that the dark carbon fixation activity measured was mainly related to CO(2) incorporation by heterotrophs rather than to the activity of true chemoautotrophs.

Shifts in microbial community structure and function in light‐ and dark‐grown biofilms driven by warming

Biofilms are dynamic players in biogeochemical cycling in running waters and are subjected to environmental stressors like those provoked by climate change. We investigated whether a 2°C increase in flowing water would affect prokaryotic community composition and heterotrophic metabolic activities of biofilms grown under light or dark conditions. Neither light nor temperature treatments were relevant for selecting a specific bacterial community at initial phases (7-day-old biofilms), but both variables affected the composition and function of mature biofilms (28-day-old). In dark-grown biofilms, changes in the prokaryotic community composition due to warming were mainly related to rotifer grazing, but no significant changes were observed in functional fingerprints. In light-grown biofilms, warming also affected protozoan densities, but its effect on prokaryotic density and composition was less evident. In contrast, heterotrophic metabolic activities in light-grown biofilms under warming showed a decrease in the functional diversity towards a specialized use of several carbohydrates. Results suggest that prokaryotes are functionally redundant in dark biofilms but functionally plastic in light biofilms. The more complex and self-serving light-grown biofilm determines a more buffered response to temperature than dark-grown biofilms. Despite the moderate increase in temperature of only 2°C, warming conditions drive significant changes in freshwater biofilms, which responded by finely tuning a complex network of interactions among microbial populations within the biofilm matrix.

Enrichment of previously uncultured bacteria from natural complex communities by adhesion to solid surfaces

The adhesion to inert solid surfaces was explored as a novel approach for the enrichment of previously uncultured bacteria from natural microbial communities. Enrichments on solid steel, glass and synthetic polymeric surfaces were established using samples from five freshwater lakes, a marine microbial mat and an alpine soil, and were subsequently analysed by molecular fingerprinting and sequencing of their 16S rRNA gene fragments. The majority of the enriched phylotypes grouped with the Alphaproteobacteria, Betaproteobacteria or Bacteroidetes and in several cases were related to typical biofilm-forming species and genera. Most enrichments were most closely related to previously uncultured phylotypes and none had previously been cultivated from the original environments even when applying improved high throughput liquid cultivation techniques. Of the 13 phylotypes enriched from freshwater samples, seven were previously unknown, three matched so-far uncultured environmental clones, and three were identical to previously cultivated bacteria. Of the 17 phylotypes recovered from soil, 12 were previously unknown with five of these phylotypes representing novel genera, whereas five phylotypes were identical to previously cultured soil bacteria. The feasibility of the biofilm-enrichment approach was exemplified by the successful isolation of a not-yet cultured Betaproteobacterium that constituted a discernible component of the alpine soil microbial community in situ and exhibited only 93% similarity to its closest cultured relative. Based on these results, cultivation on solid surfaces represents a promising approach to recover isolates that have so far escaped cultivation as suspended cultures in liquid media.

A comparative study of bchG from green photosynthetic bacteria

The gene bchG, coding for bacteriochlorophyll a synthase from a variety of green sulfur bacteria and the filamentous anoxygenic phototrophic bacteria, Chloroflexus aurantiacus, Chloronema sp., and Roseiflexus castenholzii HL08, was partially sequenced and compared. The deduced amino acid consensus sequences for green sulfur bacteria and green filamentous anoxygenic phototrophic bacteria were found to belong to the UbiA enzyme family of polyprenyltransferases with the most similar sequences being those of photosynthetic organisms. All deduced amino acid sequences showed a highly conserved region, which includes the motif DRXXD, characteristic of polyprenyltransferases, which was extended to DREVDAINEP for green sulfur bacteria. Neighbor-joining analysis of a protein similitude matrix displayed a relatively high distance between green sulfur bacteria and the other groups. Sequences from green sulfur bacteria were more closely related to those of purple bacteria than to those of filamentous anoxygenic phototrophic bacteria. In addition, internal grouping within green sulfur bacteria was congruent regarding taxonomic features including cell shape, presence of gas vacuoles and NaCl requirement. In addition to bchlG, another gene encoding for a second chlorophyll synthetase, previously tentatively identified as chlG, was also found in Chlorobium tepidum, showing the highest similarities with polyprenyltransferases from chlorophyll-a-containing organisms.