Stefan Spring

A phylogeny-driven genomic encyclopaedia of Bacteria and Archaea.

Sequencing of bacterial and archaeal genomes has revolutionized our understanding of the many roles played by microorganisms. There are now nearly 1,000 completed bacterial and archaeal genomes available, most of which were chosen for sequencing on the basis of their physiology. As a result, the perspective provided by the currently available genomes is limited by a highly biased phylogenetic distribution. To explore the value added by choosing microbial genomes for sequencing on the basis of their evolutionary relationships, we have sequenced and analysed the genomes of 56 culturable species of Bacteria and Archaea selected to maximize phylogenetic coverage. Analysis of these genomes demonstrated pronounced benefits (compared to an equivalent set of genomes randomly selected from the existing database) in diverse areas including the reconstruction of phylogenetic history, the discovery of new protein families and biological properties, and the prediction of functions for known genes from other organisms. Our results strongly support the need for systematic ‘phylogenomic’ efforts to compile a phylogeny-driven ‘Genomic Encyclopedia of Bacteria and Archaea’ in order to derive maximum knowledge from existing microbial genome data as well as from genome sequences to come.

Halobacillus gen. nov., with Descriptions of Halobacillus litoralis sp. nov. and Halobacillus trueperi sp. nov., and Transfer of Sporosarcina halophila to Halobacillus halophilus comb. nov.

Two moderately halophilic, gram-positive, heterotrophic bacterial strains were isolated from hypersaline sediments of the Great Salt Lake in Utah. These two strains, designated SL-4T (T = type strain) and SL-5T, were motile, spore-forming, strictly aerobic rods which contained peptidoglycan of the Orn-D-Asp type in their vegetative cell walls. The guanine-plus-cytosine contents of the DNAs of strains SL-4T and SL-5T were 42 and 43 mol%, respectively. A detailed investigation of the phenotypic and phylogenetic characteristics of these organisms revealed that each isolate represents a new species that is closely related to Sporosarcina halophila, a moderately halophilic, spore-forming coccus. Phylogenetic data indicate that there is only a distant relationship between Sporosarcina halophila and Sporosarcina ureae, the type species of the genus Sporosarcina. The sequences of the 16S rRNA genes of strain SL-4T and Salinicoccus roseus DSM 5351 were determined. We propose that a new genus, Halobacillus, should be created; this genus includes Halobacillus halophilus (formerly Sporosarcina halophila) as the type species, as well as Halobacillus litoralis DSM 10405T (= SL-4T) and Halobacillus trueperi DSM 10404T (= SL-5T).

The Genus Magnetospirillum gen. nov. Description of Magnetospirillum gryphiswaldense sp. nov. and Transfer of Aquaspirillum magnetotacticum to Magnetospirillum magnetotacticum comb. nov.

Summary A magnetotactic spirillum (strain MSR-1) was isolated from the mud of the eutrophic river Ryck near Greifswald. Its morphological, ultrastructural and biochemical characteristics were compared with those of Aquaspirillum magnetotacticum. A cell suspension of strain MSR-1 was used to amplify in vitro 16S rRNA genes (rDNA). The amplified 16S rDNA was cloned and sequenced. For comparison 16S rDNA was also directly amplified from a stock culture of Aquaspirillum magnetotacticum DSM 3856T. It was cloned, sequenced and compared with the 16S rDNA sequences of strain MSR-1 and other bacteria. An oligonucleotide probe complementary to a 16S rRNA region unique to the two magnetic spirilla was synthesized. Specific hybridization of the fluorescent oligonucleotide to whole cells of both strains demonstrated that the 16S rDNA clones originated from strain MSR-1 and A. magnetotacticum. Aquaspirillum magnetotacticum and strain MSR-1 are related and belong to the alpha subclass of Proteobacteria, whereas Aquaspirillum serpens, the type species of the genus Aquaspirillum, is a member of the beta subclass of Proteobacteria. A new genus Magnetospirillum sp. gen. with the type species Magnetospirillum gryphiswaldense sp. nov., and the transfer of A. magnetotacticum to Magnetospirillum magnetotacticum comb. nov. are proposed. The type strain of M. gryphiswaldense is DSM 6361.

Phylogenetic Diversity and Identification of Nonculturable Magnetotactic Bacteria

Summary Magnetotactic bacteria show a diverse morphology and are widely distributed in nature. Currently, only a few of them can be grown in axenic culture. To identify and characterize also nonculturable magnetotactic bacteria we examined enriched populations from freshwater sediment with a combination of 16S rRNA sequence retrieval and fluorescent whole cell hybridization. Comparative sequence analyses revealed an unexpectedly high diversity within the retrieved rRNA molecules exceeding the number of observed morphotypes. Fluorescent oligonucleotides complementary to unique signature regions of distinct rRNA sequences assigned three frequently occurring sequences to three populations of magnetotactic cocci. The analysed cocci group with the alpha-subclass of the proteobacterial phylum and form there a separate lineage of descent. Hardly discriminated on a morphological basis the three genotypes also demonstrated partially different tactic behaviour. The combination of 16S rRNA sequence retrieval and fluorescent whole cell hybridization has opened a new dimension to the structural analysis of natural communities. It is for the first time that nonculturable bacteria from multispecies enrichments could be identified on the single cell level.

Molecular Characterization of a Dechlorinating Community Resulting from In Situ Biostimulation in a Trichloroethene-Contaminated Deep, Fractured Basalt Aquifer and Comparison to a Derivative Laboratory Culture

ABSTRACT Sodium lactate additions to a trichloroethene (TCE) residual source area in deep, fractured basalt at a U.S. Department of Energy site have resulted in the enrichment of the indigenous microbial community, the complete dechlorination of nearly all aqueous-phase TCE to ethene, and the continued depletion of the residual source since 1999. The bacterial and archaeal consortia in groundwater obtained from the residual source were assessed by using PCR-amplified 16S rRNA genes. A clone library of bacterial amplicons was predominated by those from members of the class Clostridia (57 of 93 clones), of which a phylotype most similar to that of the homoacetogen Acetobacterium sp. strain HAAP-1 was most abundant (32 of 93 clones). The remaining Bacteria consisted of phylotypes affiliated with Sphingobacteria, Bacteroides, Spirochaetes, Mollicutes, and Proteobacteria and candidate divisions OP11 and OP3. The two proteobacterial phylotypes were most similar to those of the known dechlorinators Trichlorobacter thiogenes and Sulfurospirillum multivorans. Although not represented by the bacterial clones generated with broad-specificity bacterial primers, a Dehalococcoides-like phylotype was identified with genus-specific primers. Only four distinct phylotypes were detected in the groundwater archaeal library, including predominantly a clone affiliated with the strictly acetoclastic methanogen Methanosaeta concilii (24 of 43 clones). A mixed culture that completely dechlorinates TCE to ethene was enriched from this groundwater, and both communities were characterized by terminal restriction fragment length polymorphism (T-RFLP). According to T-RFLP, the laboratory enrichment community was less diverse overall than the groundwater community, with 22 unique phylotypes as opposed to 43 and a higher percentage of Clostridia, including the Acetobacterium population. Bioreactor archaeal structure was very similar to that of the groundwater community, suggesting that methane is generated primarily via the acetoclastic pathway, using acetate generated by lactate fermentation and acetogenesis in both systems.

Characterization of a marine gammaproteobacterium capable of aerobic anoxygenic photosynthesis

Members of the gammaproteobacterial clade NOR5/OM60 regularly form an abundant part, up to 11%, of the bacterioplankton community in coastal systems during the summer months. Here, we report the nearly complete genome sequence of one cultured representative, Congregibacter litoralis strain KT71, isolated from North Sea surface water. Unexpectedly, a complete photosynthesis superoperon, including genes for accessory pigments, was discovered. It has a high sequence similarity to BAC clones from Monterey Bay [Beja O, Suzuki MT, Heidelberg JF, Nelson WC, Preston CM, et al. (2002) Nature 415:630–633], which also share a nearly identical gene arrangement. Although cultures of KT71 show no obvious pigmentation, bacteriochlorophyll a and spirilloxanthin-like carotenoids could be detected by HPLC analysis in cell extracts. The presence of two potential BLUF (blue light using flavin adenine dinucleotide sensors), one of which was found adjacent to the photosynthesis operon in the genome, indicates a light- and redox-dependent regulation of gene expression. Like other aerobic anoxygenic phototrophs (AAnPs), KT71 is able to grow neither anaerobically nor photoautotrophically. Cultivation experiments and genomic evidence show that KT71 needs organic substrates like carboxylic acids, oligopeptides, or fatty acids for growth. The strain grows optimally under microaerobic conditions and actively places itself in a zone of ≈10% oxygen saturation. The genome analysis of C. litoralis strain KT71 identifies the gammaproteobacterial marine AAnPs, postulated based on BAC sequences, as members of the NOR5/OM60 clade. KT71 enables future experiments investigating the importance of this group of gammaproteobacterial AAnPs in coastal environments.

Phylogenetic affiliation and ultrastructure of uncultured magnetic bacteria with unusually large magnetosomes

Abstract Natural enrichments of magnetic bacteria from the Itaipu lagoon near Rio de Janeiro were dominated by coccoid-to-ovoid morphotypes that produced unusually large magnetosomes. To determine the phylogenetic position of these unusual microorganisms, 16S rRNA genes were retrieved from bacteria magnetically separated from sediment of the Itaipu lagoon by in vitro amplification and cloning of PCR products into a plasmid vector. Partial sequencing of the obtained clones revealed two clusters of closely related sequences affiliated to a distinct lineage consisting exclusively of magnetic bacteria within the α-subclass of Proteobacteria. For a detailed phylogenetic analysis, several almost complete sequences of the 16S rRNA genes were determined. One representative clone of each cluster provided a PCR template for the in vitro transcription of group-specific polynucleotide probes complementary to a variable region of the 16S rRNA molecule. At least three different morphotypes of magnetic bacteria were reliably identified by post-embedding hybridization of ultra-thin sections. Electron microscopic analyses of hybridized cells enabled for the first time a detailed description of the morphological variety and ultrastructure of phylogenetically identified, uncultured magnetic bacteria. Two distinct coccoid bacteria were identified by the transcript probe complementary to the 16S rRNA sequence mabrj12, whereas the probe complementary to the sequence mabrj58 allowed the identification of an ovoid morphotype that displayed magnetosomes with the largest volumes observed to date.

Ferribacterium limneticum, gen. nov., sp. nov., an Fe(III)-reducing microorganism isolated from mining-impacted freshwater lake sediments

Abstract A dissimilatory Fe(III)-reducing bacterium was isolated from mining-impacted lake sediments and designated strain CdA-1. The strain was isolated from a 4-month enrichment culture with acetate and Fe(III)-oxyhydroxide. Strain CdA-1 is a motile, obligately anaerobic rod, capable of coupling the oxidation of acetate and other organic acids to the reduction of ferric iron. Fe(III) reduction was not observed using methanol, ethanol, isopropanol, propionate, succinate, fumarate, H2, citrate, glucose, or phenol as potential electron donors. With acetate as an electron donor, strain CdA-1 also grew by reducing nitrate or fumarate. Growth was not observed with acetate as electron donor and O2, sulfoxyanions, nitrite, trimethylamine N-oxide, Mn(IV), As(V), or Se(VI) as potential terminal electron acceptors. Comparative 16 S rRNA gene sequence analyses show strain CdA-1 to be most closely related (93.6% sequence similarity) to Rhodocyclus tenuis. However, R. tenuis did not grow heterotrophically by Fe(III) reduction, nor did strain CdA-1 grow photrophically. We propose that strain CdA-1 represents a new genus and species, Ferribacterium limneticum. Strain CdA-1 represents the first dissimilatory Fe(III) reducer in the β subclass of Proteobacteria, as well as the first Fe(III) reducer isolated from mine wastes.

Genotypic Diversity of Acidovorax Strains Isolated from Activated Sludge and Description of Acidovorax defluvii sp. nov.

Fluorescence in situ hybridization of activated sludge samples from a municipal wastewater treatment plant using oligonucleotide probes specific for Acidovorax demonstrated that these bacteria are highly abundant in this environment. For the targeted cultivation of representatives belonging to this genus, isolates grown on agar plates after serial dilution were screened by whole-cell hybridization with specific probes. The obtained strains clustered in two phylogenetic groups as determined by 16S rRNA gene sequence analyses. The isolates of one cluster were phylogenetically and genotypically closely related to A. delafieldii. In contrast, the strains of the other cluster were genotypically and phenotypically distinct from the hitherto known Acidovorax species. Therefore, a new species, Acidovorax defluvii sp. nov., was proposed for these strains. The main characteristics of the newly defined species are as follows: Gram-negative, motile or non-motile rods with rounded ends, often with large polyhydroxybutyrate granules. In broth cultures flocs are formed. Test for cytochrome oxidase is positive with all strains. The majority of strains is catalase positive and reduces nitrate. All strains are metabolically inactive against most carbohydrates and organic acids. Fatty acid patterns are typical for the genus Acidovorax. The guanine-plus-cytosine content of DNAs varies between 62 and 64 mol%. The type strain of A. defluvii is BSB411T (DSM 12644). A new 16S rRNA-targeted oligonucleotide probe reacting by in situ hybridization with all known Acidovorax species, including A. defluvii sp. nov., was designed.

rRNA-Targeted Oligonucleotide Probes for the Identification of Genuine and Former Pseudomonads

Summary The specificity of previously published oligonucleotide probes war re-evaluated based on the expanded 16S and 23S rRNA databases. Moreover, new genus- and (sub) species-specific 16S rRNA-targeted oligonucleotide probes were designed in a computer-assisted strategy using the PROBE DESIGN tool of the software package ARB. Probes for Pseudomonas stutzeri and for the genera Acidovorax, Comamonas and Hydrogenophaga were evaluated. Optimal hybridization conditions for the probes have been determined. Using these conditions the probes were tested for binding to 16S rRNA of reference cultures. Furthermore, fluorescently labeled derivatives of these probes were tested for their applicability for whole cell hybridization. These rRNA-targeted probes are the basis for high-resolution, rapid, automated microbial identification.