Jörg Overmann

Ecological Significance of Microdiversity: Identical 16S rRNA Gene Sequences Can Be Found in Bacteria with Highly Divergent Genomes and Ecophysiologies

ABSTRACT A combination of cultivation-based methods with a molecular biological approach was used to investigate whether planktonic bacteria with identical 16S rRNA gene sequences can represent distinct eco- and genotypes. A set of 11 strains of Brevundimonas alba were isolated from a bacterial freshwater community by conventional plating or by using a liquid most-probable-number (MPN) dilution series. These strains had identical 16S rRNA gene sequences and represented the dominant phylotype in the plateable fraction, as well as in the highest positive dilutions of the MPN series. However, internally transcribed spacer and enterobacterial repetitive intergenic consensus PCR fingerprinting analyses, as well as DNA-DNA hybridization analyses, revealed great genetic diversity among the 11 strains. Each strain utilized a specific combination of 59 carbon substrates, and the niche overlap indices were low, suggesting that each strain occupied a different ecological niche. In dialysis cultures incubated in situ, each strain had a different growth rate and cell yield. We thus demonstrated that the B. alba strains represent distinct populations with genetically determined adaptations and probably occupy different ecological niches. Our results have implications for assessment of the diversity and biogeography of bacteria and increase the perception of natural diversity beyond the level of 16S rRNA gene sequences.

Cyclic AMP and Acyl Homoserine Lactones Increase the Cultivation Efficiency of Heterotrophic Bacteria from the Central Baltic Sea

ABSTRACT The effect of signal molecules on the cultivation efficiency of bacteria from the Gotland Deep in the central Baltic Sea was investigated. Numbers of cultivated cells were determined by the most-probable-number (MPN) technique. Artificial brackish water supplemented with different carbon substrates at low concentrations (200 μM each) was employed as the growth medium. Compared to the results of previous studies, this approach yielded significantly higher cultivation efficiencies (up to 11% in fluid media). A further and pronounced increase in cultivation success was accomplished by the addition of cyclic AMP (cAMP), N-butyryl homoserine lactone, or N-oxohexanoyl-dl-homoserine lactone at a low concentration of 10 μM. The most effective inducer was cAMP, which led to cultivation efficiencies of up to 100% of total bacterial counts. From the highest positive dilutions of these latter MPN series, several strains were isolated in pure culture and one strain (G100) was used to study the physiological effect of cAMP. Dot blot hybridization revealed, however, that strain G100 represented only a small fraction of the total bacterial community. This points towards an inherent limitation of the MPN approach, which does not necessarily recover abundant species from highly diverse communities. Bacterial cells of strain G100 that were starved for 6 weeks attained a higher growth rate and a higher biomass yield when resuscitated in the presence of cAMP instead of AMP.

Physiology and phylogeny of green sulfur bacteria forming a monospecific phototrophic assemblage at a depth of 100 meters in the Black Sea.

ABSTRACT The biomass, phylogenetic composition, and photoautotrophic metabolism of green sulfur bacteria in the Black Sea was assessed in situ and in laboratory enrichments. In the center of the western basin, bacteriochlorophyll e (BChl e) was detected between depths of 90 and 120 m and reached maxima of 54 and 68 ng liter−1. High-pressure liquid chromatography analysis revealed a dominance of farnesyl esters and the presence of four unusual geranyl ester homologs of BChl e. Only traces of BChl e (8 ng liter−1) were found at the northwestern slope of the Black Sea basin, where the chemocline was positioned at a significantly greater depth of 140 m. Stable carbon isotope fractionation values of farnesol indicated an autotrophic growth mode of the green sulfur bacteria. For the first time, light intensities in the Black Sea chemocline were determined employing an integrating quantum meter, which yielded maximum values between 0.0022 and 0.00075 μmol quanta m−2 s−1 at the top of the green sulfur bacterial layer around solar noon in December. These values represent by far the lowest values reported for any habitat of photosynthetic organisms. Only one 16S rRNA gene sequence type was detected in the chemocline using PCR primers specific for green sulfur bacteria. This previously unknown phylotype groups with the marine cluster of the Chlorobiaceae and was successfully enriched in a mineral medium containing sulfide, dithionite, and freshly prepared yeast extract. Under precisely controlled laboratory conditions, the enriched green sulfur bacterium proved to be capable of exploiting light intensities as low as 0.015 μmol quanta m−2 s−1 for photosynthetic 14CO2 fixation. Calculated in situ doubling times of the green sulfur bacterium range between 3.1 and 26 years depending on the season, and anoxygenic photosynthesis contributes only 0.002 to 0.01% to total sulfide oxidation in the chemocline. The stable population of green sulfur bacteria in the Black Sea chemocline thus represents the most extremely low-light-adapted and slowest-growing type of phototroph known to date.

Microbial Communities in the Chemocline of a Hypersaline Deep-Sea Basin (Urania Basin, Mediterranean Sea)

ABSTRACT The Urania basin is a hypersaline sulfidic brine lake at the bottom of the eastern Mediterranean Sea. Since this basin is located at a depth of ∼3,500 m below the sea surface, it receives only a small amount of phytoplankton organic carbon. In the present study, the bacterial assemblages at the interface between the hypersaline brine and the overlaying seawater were investigated. The sulfide concentration increased from 0 to 10 mM within a vertical interval of 5 m across the interface. Within this chemocline, the total bacterial cell counts and the exoenzyme activities were elevated. Employing 11 cultivation methods, we isolated a total of 70 bacterial strains. The 16S ribosomal DNA sequences of 32 of the strains were identical to environmental sequences detected in the chemocline by culture-independent molecular methods. These strains were identified as flavobacteria, Alteromonas macleodii, and Halomonas aquamarina. All 70 strains could grow chemoorganoheterotrophically under oxic conditions. Sixty-six strains grew on peptone, casein hydrolysate, and yeast extract, whereas only 15 strains did not utilize polymeric carbohydrates. Twenty-one of the isolates could grow both chemoorganotrophically and chemolithotrophically. While the most probable numbers in most cases ranged between 0.006 and 4.3% of the total cell counts, an unsually high value of 54% was determined above the chemocline with media containing amino acids as the carbon and energy source. Our results indicate that culturable bacteria thriving at the oxic-anoxic interface of the Urania basin differ considerably from the chemolithoautotrophic bacteria typical of other chemocline habitats.

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.

Effect of Signal Compounds and Incubation Conditions on the Culturability of Freshwater Bacterioplankton

ABSTRACT The effect of signal compounds and of different incubation conditions on the culturability (i.e., the fraction of all cells capable of growth) of natural bacterioplankton from the eutrophic lake Zwischenahner Meer was investigated over a period of 20 months. Numbers of growing cells were determined by the most-probable-number technique in liquid media containing low concentrations (10 μM) of the signal compounds N-(oxohexanoyl)-dl-homoserine lactone, N-(butyryl)-dl-homoserine lactone, cyclic AMP (cAMP), or ATP. cAMP was the most effective signal compound, leading to significantly increased cultivation efficiencies of up to 10% of the total bacterial counts. Microautoradiography with [2,8-3H]cAMP, combined with fluorescence in situ hybridization, demonstrated that cAMP was taken up by 18% of all cells. The bacterial cAMP uptake systems had a very low Km value of ≤1 nM. Analysis of the cultured bacteria by 16S rRNA gene fingerprinting showed that different bacterial phylotypes were recovered in the presence and in the absence of cAMP. Consequently, the addition of cAMP caused a stimulation of otherwise nonculturable bacteria. Phylogenetically different bacteria were also recovered at different temperatures and oxygen partial pressures. Throughout the study period, mainly members of the β-subclass of the Proteobacteria were cultivated. In addition, some members of the Actinomycetales were enriched. Quantification by culture-independent fluorescence in situ hybridization demonstrated that β-Proteobacteria and Actinomycetales also dominated the natural bacterioplankton assemblage. Sequence comparison revealed that two members of the Actinomycetales which reached high numbers in the natural bacterioplankton assemblage could actually be enriched by our cultivation approach.

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.

Characterization of abundance and diversity of lactic acid bacteria in the hindgut of wood- and soil-feeding termites by molecular and culture-dependent techniques

Abstract Lactic acid bacteria have been identified as typical and numerically significant members of the gut microbiota of Reticulitermes flavipes and other wood-feeding lower termites. We found that also in the guts of the higher termites Nasutitermes arborum (wood-feeding), Thoracotermes macrothorax, and Anoplotermes pacificus (both soil-feeding), lactic acid bacteria represent the largest group of culturable carbohydrate-utilizing bacteria (3.6–5.2×104 bacteria per gut; 43%–54% of all colonies). All isolates were coccoid and phenotypically difficult to distinguish, but their enterobacterial repetitive intergenic consensus sequence (ERIC) fingerprint patterns showed a significant genetic diversity. Six different genotypes each were identified among the isolates from R. flavipes and T. macrothorax, and representative strains were selected for further characterization. By 16S rRNA gene sequence analysis, strain RfL6 from R. flavipes was classified as a close relative of Enterococcus faecalis, whereas strain RfLs4 from R. flavipes and strain TmLO5 from T. macrothorax were closely related to Lactococcus lactis. All strains consumed oxygen during growth on glucose and cellobiose; oxygen consumption of these and other isolates from both termite species was due to NADH and pyruvate oxidase activities, but did not result in H2O2 formation. In order to assess the significance of the isolates in the hindgut, denaturing gradient gel electrophoresis was used to compare the fingerprints of 16S rRNA genes in the bacterial community of R. flavipes with those of representative isolates. The major DNA band from the hindgut bacterial community was further separated by bis-benzimide-polyethylene glycol electrophoresis, and the two resulting bands were sequenced. Whereas one sequence belonged to a spirochete, the second sequence was closely related to the sequences of the Lactococcus strains RfLs4 and TmLO5. Apparently, those isolates represent strains of a new Lactococcus species which forms a significant fraction of the complex hindgut community of the lower termite R. flavipes and possibly also of other termites.

Phylogeny and molecular fingerprinting of green sulfur bacteria

Abstract The 16S rDNA sequences of nine strains of green sulfur bacteria (Chlorobiaceae) were determined and compared to the four known sequences of Chlorobiaceae and to sequences representative for all eubacterial phyla. The sequences of the Chlorobiaceae strains were consistent with the secondary structure model proposed earlier for Chlorobium vibrioforme strain 6030. Similarity values > 90.1% and Knuc values < 0.11 indicate a close phylogenetic relatedness among the green sulfur bacteria. As a group, these bacteria represent an isolated branch within the eubacterial radiation. In Chlorobiaceae, a similar morphology does not always reflect a close phylogenetic relatedness. While ternary fission is a morphological trait of phylogenetic significance, gas vesicle formation occurs also in distantly related species. Pigment composition is not an indicator of phylogenetic relatedness since very closely related species contain different bacteriochlorophylls and carotenoids. Two different molecular fingerprinting techniques for the rapid differentiation of Chlorobiaceae species were investigated. The 16S rDNA fragments of several species could not be separated by denaturing gradient gel electrophoresis. In contrast, all strains investigated during the present work gave distinct banding patterns when dispersed repetitive DNA sequences were used as targets in PCR. The latter technique is, therefore, well suited for the rapid screening of isolated pure cultures of green sulfur bacteria.

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.