Karine Alain

Cultivating the uncultured: limits, advances and future challenges

Since the invention of the Petri dish, there have been continuous efforts to improve efficiency in microbial cultivation. These efforts were devoted to the attainment for diverse growth conditions, simulation of in situ conditions and achievement of high-throughput rates. As a result, prokaryotes catalysing novel redox reactions as well as representatives of abundant, but not-yet cultured taxa, were isolated. Significant insights into microbial physiology have been made by studying the small number of prokaryotes already cultured. However, despite these numerous breakthroughs, microbial cultivation is still a low-throughput process. The main hindrance to cultivation is likely due to the prevailing lack of knowledge on targeted species. In this review, we focus on the limiting factors surrounding cultivation. We discuss several cultivation obstacles, including the loss of microbial cell–cell communication following species isolation. Future research directions, including the refinement of culture media, strategies based on cell–cell communication and high-throughput innovations, are reviewed. We further propose that a combination of these approaches is urgently required to promote cultivation of uncultured species, thereby dawning a new era in the field.

Caminibacter hydrogeniphilus gen. nov., sp. nov., a novel thermophilic, hydrogen-oxidizing bacterium isolated from an East Pacific Rise hydrothermal vent

A novel thermophilic, anaerobic, hydrogen-oxidizing bacterium, designated strain AM1116T, was isolated from an East Pacific Rise hydrothermal vent sample. The cells were rod-shaped (1.01-5 x 0.5 microm), motile with polar flagella. They grew at temperatures between 50 and 70 degrees C (optimum 60 degrees C; doubling time approximately 1.5 h), at between pH 5.0 and 7.5 (optimum around pH 5.5-6.0) and in between 10 and 40 g NaCl l(-1) (optimum 20-25 g l(-1)). Cells grew chemolithoautotrophically in a H2/CO2 atmosphere (80:20; 200 kPa). Poor heterotrophic growth was observed on complex organic substrates. Elemental sulphur and nitrate served as electron acceptors, respectively yielding hydrogen sulphide and ammonia (doubling times were equal with the two electron acceptors). In contrast, when cystine was used as electron acceptor, growth was poor. The G+C content of the genomic DNA was 29 +/- 1 mol %. Phylogenetic analyses of the 16S rRNA gene located the strain within the epsilon-Proteobacteria, in the bacterial domain. On the basis of 16S rDNA sequence comparisons, physiological and biochemical characteristics, it is proposed that the isolate should be described as the type species of a new genus, Caminibacter gen. nov., as Caminibacter hydrogeniphilus sp. nov. The type strain is strain AM1116T (= DSM 14510T = CIP 107140T).

Phylogenetic characterization of the bacterial assemblage associated with mucous secretions of the hydrothermal vent polychaete Paralvinella palmiformis.

As part of an ongoing examination of microbial diversity associated with hydrothermal vent polychaetes of the family Alvinellidae, we undertook a culture-independent molecular analysis of the bacterial assemblage associated with mucous secretions of the Northeastern Pacific vent polychaete Paralvinella palmiformis. Using a molecular 16S rDNA-based phylogenetic approach, clone libraries were constructed from two samples collected from active sulfide edifices in two hydrothermal vent fields. In both cases, clone libraries were largely dominated by epsilon-Proteobacteria. Phylotypes belonging to the Cytophaga-Flavobacteria and to the Verrucomicrobia were also largely represented within the libraries. The remaining sequences were related to the taxonomic groups Fusobacteria, Green non-sulfur bacteria, Firmicutes, gamma- and delta-Proteobacteria. To our knowledge, this is the first report of the presence of Verrucomicrobia, Fusobacteria and green non-sulfur bacteria on hydrothermal edifices. The potential functions of the detected bacteria are discussed in terms of productivity, recycling of organic matter and detoxification within the P. palmiformis microhabitat.

Marinitoga piezophila sp. nov., a rod-shaped, thermo-piezophilic bacterium isolated under high hydrostatic pressure from a deep-sea hydrothermal vent

A thermophilic, anaerobic, piezophilic, chemo-organotrophic sulfur-reducing bacterium, designated as KA3T, was isolated from a deep-sea hydrothermal chimney sample collected at a depth of 2630 m on the East-Pacific Rise (13 degrees N). When grown under elevated hydrostatic pressure, the cells are rod-shaped with a sheath-like outer structure, motile, have a mean length of 1-1.5 microm and stain Gram-negative. They appear singly or in short chains. When grown at lower, or atmospheric, pressures, the cells elongate and become twisted. Growth is enhanced by hydrostatic pressure; the optimal pressure for growth is 40 MPa (26 MPa pressure at sampling site). The temperature range for growth is 45-70 degrees C, the optimum being around 65 degrees C (doubling time is approximately 20 min at 40 MPa). Growth is observed from pH 5 to pH 8, the optimum being at pH 6. The salinity range for growth is 10-50 g NaCl l(-1), the optimum being at 30 g l(-1). The isolate is able to grow on a broad spectrum of carbohydrates or complex proteinaceous substrates, and growth is stimulated by L-cystine and elemental sulfur. The G+C content of the genomic DNA is 29 +/- 1 mol%. According to phylogenetic analysis of the 16S rDNA gene, the strain is placed within the order Thermotogales, in the bacterial domain. On the basis of 16S rDNA sequence comparisons and morphological, physiological and genotypic characteristics, it is proposed that the isolate be described as a novel species of the genus Marinitoga, with Marinitoga piezophila sp. nov. as the type species. The type strain is KA3T (= DSM 14283T = JCM 11233T).

Microorganisms persist at record depths in the subseafloor of the Canterbury Basin

The subsurface realm is colonized by microbial communities to depths of >1000 meters below the seafloor (m.b.sf.), but little is known about overall diversity and microbial distribution patterns at the most profound depths. Here we show that not only Bacteria and Archaea but also Eukarya occur at record depths in the subseafloor of the Canterbury Basin. Shifts in microbial community composition along a core of nearly 2 km reflect vertical taxa zonation influenced by sediment depth. Representatives of some microbial taxa were also cultivated using methods mimicking in situ conditions. These results suggest that diverse microorganisms persist down to 1922 m.b.sf. in the seafloor of the Canterbury Basin and extend the previously known depth limits of microbial evidence (i) from 159 to 1740 m.b.sf. for Eukarya and (ii) from 518 to 1922 m.b.sf. for Bacteria.

Caminicella sporogenes gen. nov., sp. nov., a novel thermophilic spore-forming bacterium isolated from an East-Pacific Rise hydrothermal vent

A novel thermophilic, anaerobic, strictly chemoorganoheterotrophic bacterium, designated as AM1114T, was isolated from a deep-sea hydrothermal vent sample from the East-Pacific Rise (EPR 13 degrees N). The cells were long (3-10 microm) rods, motile with peritrichous flagella, and exhibited a gram-negative cell wall ultrastructure. In the late stationary phase of growth, cells formed an ovoid, refractile, terminal endospore. They grew at 45-65 degrees C inclusive (optimum 55-60 degrees C; doubling time approx. 45 min), at pH 4.5-8.0 inclusive (optimum pH 7.5-8.0) and at sea salt concentrations of 20-60 g l(-1) inclusive (optimum 25-30 g l(-1)). Strain AM1114T was an obligately heterotrophic bacterium able to ferment a mixture of 20 amino acids, complex proteinaceous substrates (such as yeast extract, brain-heart infusion or peptone), and carbohydrates such as glucose, galactose or maltose. The main fermentation products on glucose/yeast extract/peptone/sulfur medium were hydrogen, carbon dioxide, butyrate, ethanol, acetate, formate and L-alanine. The G+C content of the genomic DNA (determined by thermal denaturation) was 24.2+/-1 mol%. Phylogenetic analyses of the 16S rRNA gene located the strain within cluster XI of the lineage encompassing the genus Clostridium and related genera (sensu Collins et al., 1994), in the bacterial domain. On the basis of 16S rDNA sequence comparisons and physiological and biochemical characteristics, it is proposed that the isolate should be described as a novel genus, namely Caminicella gen. nov., of which Caminicella sporogenes sp. nov. is the type species. The type strain is AM1114T (= DSM 14501T = CIP 107141T).

Microbial diversity in Tunisian geothermal springs as detected by molecular and culture-based approaches

Prokaryotic diversities of 12 geothermal hot springs located in Northern, Central and Southern Tunisia were investigated by culture-based and molecular approaches. Enrichment cultures for both aerobic and anaerobic microorganisms were successfully obtained at temperatures ranging from 50 to 75°C. Fourteen strains including four novel species were cultivated and assigned to the phyla Firmicutes (9), Thermotogae (2), Betaproteobacteria (1), Synergistetes (1) and Bacteroidetes (1). Archaeal or universal oligonucleotide primer sets were used to generate 16S rRNA gene libraries. Representative groups included Proteobacteria, Firmicutes, Deinococcus-Thermus, Thermotogae, Synergistetes, Bacteroidetes, Aquificae, Chloroflexi, candidate division OP9 in addition to other yet unclassified strains. The archaeal library showed a low diversity of clone sequences belonging to the phyla Euryarchaeota and Crenarchaeota. Furthermore, we confirmed the occurrence of sulfate reducers and methanogens by amplification and sequencing of dissimilatory sulfite reductase (dsrAB) and methyl coenzyme M reductase α-subunit (mcrA) genes. Altogether, we discuss the diverse prokaryotic communities arising from the 12 geothermal hot springs studied and relate these findings to the physico-chemical features of the hot springs.

Thermodesulfatator atlanticus sp. nov., a thermophilic, chemolithoautotrophic, sulfate- reducing bacterium isolated from a Mid-Atlantic Ridge hydrothermal vent

A novel, strictly anaerobic, thermophilic, sulfate-reducing bacterium, designated strain AT1325(T), was isolated from a deep-sea hydrothermal vent at the Rainbow site on the Mid-Atlantic Ridge. This strain was subjected to a polyphasic taxonomic analysis. Cells were Gram-negative motile rods (approximately 2.4 x 0.6 microm) with a single polar flagellum. Strain AT1325(T) grew at 55-75 degrees C (optimum, 65-70 degrees C), at pH 5.5-8.0 (optimum, 6.5-7.5) and in the presence of 1.5-4.5 % (w/v) NaCl (optimum, 2.5 %). Cells grew chemolithoautotrophically with H2 as an energy source and SO4(2-) as an electron acceptor. Alternatively, the novel isolate was able to use methylamine, peptone or yeast extract as carbon sources. The dominant fatty acids (>5 % of the total) were C(16 : 0), C(18 : 1)omega7c, C(18 : 0) and C(19 : 0) cyclo omega8c. The G+C content of the genomic DNA of strain AT1325(T) was 45.6 mol%. Phylogenetic analyses based on 16S rRNA gene sequences placed strain AT1325(T) within the family Thermodesulfobacteriaceae, in the bacterial domain. Comparative 16S rRNA gene sequence analysis indicated that strain AT1325(T) belonged to the genus Thermodesulfatator, sharing 97.8 % similarity with the type strain of Thermodesulfatator indicus, the unique representative species of this genus. On the basis of the data presented, it is suggested that strain AT1325(T) represents a novel species of the genus Thermodesulfatator, for which the name Thermodesulfatator atlanticus sp. nov. is proposed. The type strain is AT1325(T) (=DSM 21156(T)=JCM 15391(T)).

Anaerobic utilization of toluene by marine alpha- and gammaproteobacteria reducing nitrate

Aromatic hydrocarbons are among the main constituents of crude oil and represent a major fraction of biogenic hydrocarbons. Anthropogenic influences as well as biological production lead to exposure and accumulation of these toxic chemicals in the water column and sediment of marine environments. The ability to degrade these compounds in situ has been demonstrated for oxygen- and sulphate-respiring marine micro-organisms. However, if and to what extent nitrate-reducing bacteria contribute to the degradation of hydrocarbons in the marine environment and if these organisms are similar to their well-studied freshwater counterparts has not been investigated thoroughly. Here we determine the potential of marine prokaryotes from different sediments of the Atlantic Ocean and Mediterranean Sea to couple nitrate reduction to the oxidation of aromatic hydrocarbons. Nitrate-dependent oxidation of toluene as an electron donor in anoxic enrichment cultures was elucidated by analyses of nitrate, nitrite and dinitrogen gas, accompanied by cell proliferation. The metabolically active members of the enriched communities were identified by RT-PCR of their 16S rRNA genes and subsequently quantified by fluorescence in situ hybridization. In all cases, toluene-grown communities were dominated by members of the Gammaproteobacteria, followed in some enrichments by metabolically active alphaproteobacteria as well as members of the Bacteroidetes. From these enrichments, two novel denitrifying toluene-degrading strains belonging to the Gammaproteobacteria were isolated. Two additional toluene-degrading denitrifying strains were isolated from sediments from the Black Sea and the North Sea. These isolates belonged to the Alphaproteobacteria and Gammaproteobacteria. Serial dilutions series with marine sediments indicated that up to 2.2×10(4) cells cm(-3) were able to degrade hydrocarbons with nitrate as the electron acceptor. These results demonstrated the hitherto unrecognized capacity of alpha- and gammaproteobacteria in marine sediments to oxidize toluene using nitrate.

DNA extractions from deep subseafloor sediments: novel cryogenic-mill-based procedure and comparison to existing protocols.

Extracting DNA from deep subsurface sediments is challenging given the complexity of sediments types, low biomasses, resting structures (spores, cysts) frequently encountered in deep sediments, and the potential presence of enzymatic inhibitors. Promising results for cell lysis efficiency were recently obtained by use of a cryogenic mill (Lipp et al., 2008). These findings encouraged us to devise a DNA extraction protocol using this tool. Thirteen procedures involving a combination of grinding in liquid nitrogen (for various durations and beating rates) with different chemical solutions (phenol, chloroform, SDS, sarkosyl, proteinase, GTC), or with use of DNA recovery kits (MagExtractor®) were compared. Effective DNA extraction was evaluated in terms of cell lysis efficiency, DNA extraction efficiency, DNA yield and determination of prokaryotic diversity. Results were compared to those obtained by standard protocols: the FastDNA®SPIN kit for soil and the Zhou protocol. For most sediment types grinding in a cryogenic mill at a low beating rate in combination with direct phenol-chloroform extraction resulted in much higher DNA yields than those obtained using classical procedures. In general (except for clay-rich sediments), this procedure provided high-quality crude extracts for direct downstream nested-PCR, from cell numbers as low as 1.1×10(6) cells/cm(3). This procedure is simple, rapid, low-cost, and could be used with minor modifications for large-scale DNA extractions for a variety of experimental goals.