The genome of Desulfotalea psychrophila, a sulfate-reducing bacterium from permanently cold Arctic sediments.

Desulfotalea psychrophila is a marine sulfate-reducing delta-proteobacterium that is able to grow at in situ temperatures below 0 degrees C. As abundant members of the microbial community in permanently cold marine sediments, D. psychrophila-like bacteria contribute to the global cycles of carbon and sulfur. Here, we describe the genome sequence of D. psychrophila strain LSv54, which consists of a 3 523 383 bp circular chromosome with 3118 predicted genes and two plasmids of 121 586 bp and 14 663 bp. Analysis of the genome gave insight into the metabolic properties of the organism, e.g. the presence of TRAP-T systems as a major route for the uptake of C(4)-dicarboxylates, the unexpected presence of genes from the TCA cycle, a TAT secretion system, the lack of a beta-oxidation complex and typical Desulfovibrio cytochromes, such as c(553), c(3) and ncc. D. psychrophila encodes more than 30 two-component regulatory systems, including a new Ntr subcluster of hybrid kinases, nine putative cold shock proteins and nine potentially cold shock-inducible proteins. A comparison of D. psychrophila's genome features with those of the only other published genome from a sulfate reducer, the hyperthermophilic archaeon Archaeoglobus fulgidus, revealed many striking differences, but only a few shared features.

Identification of planctomycetes with order-, genus-, and strain-specific 16S rRNA-targeted probes.

A specific 16S rRNA-targeted oligonucleotide probe (PIR1223) for the genus Pirellula and a species-specific probe (RB454) for Pirellula sp. strain SH1 have been designed and optimized. Together with the already existing order-specific probe PLA886, the two newly designed probes were used to detect and identify planctomycetes, pirellulae, and close relatives of Pirellula sp. strain SH1 in different habitats. With the help of these probes for detection and identification, bacteria of the genus Pirellula were detected and cultivated from tissue of the Mediterranean sponge Aplysina aerophoba and from the water column of the Kiel Fjord. An unexpected result was the close phylogenetic relationship of the isolate from the sponge and the brackish water habitat Kiel Fjord as revealed by DNA/DNA hybridization.

Complete genome sequence of the marine planctomycete Pirellula sp. strain 1.

Pirellula sp. strain 1 ("Rhodopirellula baltica") is a marine representative of the globally distributed and environmentally important bacterial order Planctomycetales. Here we report the complete genome sequence of a member of this independent phylum. With 7.145 megabases, Pirellula sp. strain 1 has the largest circular bacterial genome sequenced so far. The presence of all genes required for heterolactic acid fermentation, key genes for the interconversion of C1 compounds, and 110 sulfatases were unexpected for this aerobic heterotrophic isolate. Although Pirellula sp. strain 1 has a proteinaceous cell wall, remnants of genes for peptidoglycan synthesis were found. Genes for lipid A biosynthesis and homologues to the flagellar L- and P-ring protein indicate a former Gram-negative type of cell wall. Phylogenetic analysis of all relevant markers clearly affiliates the Planctomycetales to the domain Bacteria as a distinct phylum, but a deepest branching is not supported by our analyses.

Isolation of novel pelagic bacteria from the German bight and their seasonal contributions to surface picoplankton.

We tested new strategies for the isolation of abundant bacteria from coastal North Sea surface waters, which included reducing by several orders of magnitude the concentrations of inorganic N and P compounds in a synthetic seawater medium. Agar plates were resampled over 37 days, and slowly growing colonies were allowed to develop by repeatedly removing all newly formed colonies. A fivefold increase of colonies was observed on plates with reduced nutrient levels, and the phylogenetic composition of the culture collection changed over time, towards members of the Roseobacter lineage and other alpha-proteobacteria. Novel gamma-proteobacteria from a previously uncultured but cosmopolitan lineage (NOR5) formed colonies only after 12 days of plate incubation. A time series of German Bight surface waters (January to December 1998) was screened by fluorescence in situ hybridization (FISH) with isolate-specific and general probes. During spring and early summer, a prominent fraction of FISH-detectable bacteria (mean, 51%) were affiliated with the Cytophaga-Flavobacterium group (CF) of the Bacteroidetes. One Cytophaga sp. lineage with cultured representatives formed almost 20% of the CF group. Members of the Roseobacter cluster constituted approximately 50% of alpha-proteobacteria, but none of the Roseobacter-related isolates formed populations of >1% in the environment. Thus, the readily culturable members of this clade are probably not representative of Roseobacter species that are common in the water column. In contrast, members of NOR5 were found at high abundances (>10(5) cells ml(-1)) in the summer plankton. Some abundant pelagic bacteria are apparently able to form colonies on solid media, but appropriate isolation techniques for different species need to be developed.

Predator-specific enrichment of actinobacteria from a cosmopolitan freshwater clade in mixed continuous culture.

We investigated whether individual populations of freshwater bacteria in mixed experimental communities may exhibit specific responses to the presence of different bacterivorous protists. In two successive experiments, a two-stage continuous cultivation system was inoculated with nonaxenic batch cultures of the cryptophyte Cryptomonas sp. Algal exudates provided the sole source of organic carbon for growth of the accompanying microflora. The dynamics of several 16S rRNA-defined bacterial populations were followed in the experimental communities. Although the composition and stability of the two microbial communities differed, numerous members of the first assemblage could again be detected during the second experiment. The introduction of a size-selectively feeding mixotrophic nanoflagellate (Ochromonas sp.) always resulted in an immediate bloom of a single phylotype population of members of the class Actinobacteria (Ac1). These bacteria were phylogenetically affiliated with an uncultured lineage of gram-positive bacteria that have been found in freshwater habitats only. The Ac1 cells were close to the average size of freshwater bacterioplankton and significantly smaller than any of the other experimental community members. In contrast, no increase of the Ac1 population was observed in vessels exposed to the bacterivorous ciliate Cyclidium glaucoma. However, when the Ochromonas sp. was added after the establishment of C. glaucoma, the proportion of population Ac1 within the microbial community rapidly increased. Populations of a beta proteobacterial phylotype related to an Aquabacterium sp. decreased relative to the total bacterial communities following the addition of either predator, albeit to different extents. The community structure of pelagic microbial assemblages can therefore be influenced by the taxonomic composition of the predator community.

Dethiosulfovibrio russensis sp. nov., Dethosulfovibrio marinus sp. nov. and Dethosulfovibrio acidaminovorans sp. nov., novel anaerobic, thiosulfate- and sulfur-reducing bacteria isolated from 'Thiodendron' sulfur mats in different saline environments.

Four strains of strictly anaerobic, sulfur- and thiosulfate-reducing bacteria, SR12T, SR13, SR15T and WS100T, were isolated from 'Thiodendron' sulfur mats obtained from different saline environments. All isolates were motile, Gram-negative, non-spore-forming curved rods with pointed or rounded ends. The sizes of cells varied from 0.9 x 3-5 microm for strains SR12T, SR13 and SR15T to 0.9 x 4.8 microm for strain WS100T. All strains could form long spiral filamentous cells up to 70-110 microm during the early stage of growth. All strains were motile by a tumbling movement and possessed lateral flagella arranged at the concave side of cells. Incomplete cross-septa were distinctive features of all strains. Growth occurred at temperatures of 10-40 degrees C with an optimum at 28 degrees C. The pH limits for growth were 5.5 to 8.0, with optimal growth at pH 6.5-7.0. All isolates were obligately anaerobic and slightly halophilic and grew in media containing 0.5-5% NaCl with an optimum at 2% NaCl. All strains were chemoorganoheterotrophic, having a fermentative type of metabolism and utilized proteins, peptides, amino acids and some organic acids, but not sugars, fatty acids or alcohols. Some organic substrates (isoleucine, valine, alanine, glutamate) were utilized only by strain SR12T in the presence of sulfur or thiosulfate. Fermentation of citrate yielded mainly acetate, CO2 and H2. Sulfur and thiosulfate were reduced to hydrogen sulfide during the fermentation of organic substances, which increased cell yields and growth rates. Sulfate, sulfite, fumarate, nitrate, Fe2O3, MnO2, DMSO and elemental selenium were not used as electron acceptors by these strains. The G+C contents of the DNA were 51 mol% for strains SR12T, SR13 and SR15T and 52 mol% for strain WS100T. Based on morphological, physiological and phylogenetic similarities, all four isolates could be assigned to three new species of the genus Dethiosulfovibrio, named Dethiosulfovibrio russensis (type strain DSM 12538T), Dethiosulfovibrio marinus (type strain DSM 12537T) and Dethiosulfovibrio acidaminovorans (type strain DSM 12590T).

Comparative 16S rRNA analysis of lake bacterioplankton reveals globally distributed phylogenetic clusters including an abundant group of actinobacteria.

In a search for cosmopolitan phylogenetic clusters of freshwater bacteria, we recovered a total of 190 full and partial 16S ribosomal DNA (rDNA) sequences from three different lakes (Lake Gossenköllesee, Austria; Lake Fuchskuhle, Germany; and Lake Baikal, Russia). The phylogenetic comparison with the currently available rDNA data set showed that our sequences fall into 16 clusters, which otherwise include bacterial rDNA sequences of primarily freshwater and soil, but not marine, origin. Six of the clusters were affiliated with the alpha, four were affiliated with the beta, and one was affiliated with the gamma subclass of the Proteobacteria; four were affiliated with the Cytophaga-Flavobacterium-Bacteroides group; and one was affiliated with the class Actinobacteria (formerly known as the high-G+C gram-positive bacteria). The latter cluster (hgcI) is monophyletic and so far includes only sequences directly retrieved from aquatic environments. Fluorescence in situ hybridization (FISH) with probes specific for the hgcI cluster showed abundances of up to 1.7 x 10(5) cells ml(-1) in Lake Gossenköllesee, with strong seasonal fluctuations, and high abundances in the two other lakes investigated. Cell size measurements revealed that Actinobacteria in Lake Gossenköllesee can account for up to 63% of the bacterioplankton biomass. A combination of phylogenetic analysis and FISH was used to reveal 16 globally distributed sequence clusters and to confirm the broad distribution, abundance, and high biomass of members of the class Actinobacteria in freshwater ecosystems.

Unlabeled helper oligonucleotides increase the in situ accessibility to 16S rRNA of fluorescently labeled oligonucleotide probes.

Target site inaccessibility represents a significant problem for fluorescence in situ hybridization (FISH) of 16S rRNA with oligonucleotide probes. Here, unlabeled oligonucleotides (helpers) that bind adjacent to the probe target site were evaluated for their potential to increase weak probe hybridization signals in Escherichia coli DSM 30083(T). The use of helpers enhanced the fluorescence signal of all six probes examined at least fourfold. In one case, the signal of probe Eco474 was increased 25-fold with the use of a single helper probe, H440-2. In another case, four unlabeled helpers raised the FISH signal of a formerly weak probe, Eco585, to the level of the brightest monolabeled oligonucleotide probes available for E. coli. The temperature of dissociation and the mismatch discrimination of probes were not significantly influenced by the addition of helpers. Therefore, using helpers should not cause labeling of additional nontarget organisms at a defined stringency of hybridization. However, the helper action is based on sequence-specific binding, and there is thus a potential for narrowing the target group which must be considered when designing helpers. We conclude that helpers can open inaccessible rRNA regions for FISH with oligonucleotide probes and will thereby further improve the applicability of this technique for in situ identification of microorganisms.

16S rRNA-targeted oligonucleotide probes for the in situ detection of members of the phylum Cytophaga-Flavobacterium-Bacteroides.

Bacteria of the Cytophaga-Flavobacterium-Bacteroides phylum (CFB-phylum) are numerically important members of many microbial communities. A suite of five 16S rRNA-targeted oligonucleotide probes for members of this group is described which was designed to dominantly target bacteria of the CFB-phylum that are found in particular habitats. For this we initially performed a literature survey-for the sources and sites of isolation of hitherto described members of the CFB-phylum. Probe CFB286 is mostly complementary to the 16S rRNA of species originally isolated from freshwater habitats, however, detects some marine and soil isolates and is the only probe which includes some food isolates. Probe CFB563 detects marine as well as animal-associated isolates. Probe CFB719, which also detects some environmental isolates, and probe CFB972 are mostly targeting animal-associated isolates. All probes are complementary to a variety of human-associated species within the CFB-phylum which, in the data set investigated (October 1998), made up 46% of all 16S rRNA sequences from the CFB-phylum. We conclude that it is difficult to find habitat-specific probes for members of the CFB-phylum and that the design of probes for monophyletic groups should remain the standard approach. Applicability of the probes for fluorescence in situ hybridization and specificity for single cell detection were evaluated for the four mentioned probes whereas the fifth, probe CFB1082, which almost exclusively targets human-associated species, was not further characterized. The new probes are of limited determinative value and should be used together with the already established probes for the CFB-phylum. It is the hybridization pattern observed for a given cell or culture with the enlarged probe set that is suggestive for its affiliation with a defined group within the CFB-phylum.

Culturability and In situ abundance of pelagic bacteria from the North Sea.

The culturability of abundant members of the domain Bacteria in North Sea bacterioplankton was investigated by a combination of various cultivation strategies and cultivation-independent 16S rRNA-based techniques. We retrieved 16S rRNA gene (rDNA) clones from environmental DNAs and determined the in situ abundance of different groups and genera by fluorescence in situ hybridization (FISH). A culture collection of 145 strains was established by plating on oligotrophic medium. Isolates were screened by FISH, amplified ribosomal DNA restriction analysis (ARDRA), and sequencing of representative 16S rDNAs. The majority of isolates were members of the genera Pseudoalteromonas, Alteromonas, and Vibrio. Despite being readily culturable, they constituted only a minor fraction of the bacterioplankton community. They were not detected in the 16S rDNA library, and FISH indicated rare (<1% of total cell counts) occurrence as large, rRNA-rich, particle-associated bacteria. Conversely, abundant members of the Cytophaga-Flavobacteria and gamma proteobacterial SAR86 clusters, identified by FISH as 17 to 30% and up to 10% of total cells in the North Sea bacterioplankton, respectively, were cultured rarely or not at all. Whereas SAR86-affiliated clones dominated the 16S rDNA library (44 of 53 clones), no clone affiliated to the Cytophaga-Flavobacterum cluster was retrieved. The only readily culturable abundant group of marine bacteria was related to the genus Roseobacter. The group made up 10% of the total cells in the summer, and the corresponding sequences were also present in our clone library. Rarefaction analysis of the ARDRA patterns of all of the isolates suggested that the total culturable diversity by our method was high and still not covered by the numbers of isolated strains but was almost saturated for the gamma proteobacteria. This predicts a limit to the isolation of unculturable marine bacteria, particularly the gamma-proteobacterial SAR86 cluster, as long as no new techniques for isolation are available and thus contrasts with more optimistic accounts of the culturability of marine bacterioplankton.

Bacterioplankton compositions of lakes and oceans: a first comparison based on fluorescence in situ hybridization.

Fluorescence in situ hybridization (FISH) with rRNA-targeted oligonucleotide probes was used to investigate the phylogenetic composition of bacterioplankton communities in several freshwater and marine samples. An average of about 50% of the cells were detected by probes for the domains Bacteria and Archaea, and of these, about half could be identified at the subdomain level with a set of group-specific probes. Beta subclass proteobacteria constituted a dominant fraction in freshwater systems, accounting for 16% (range, 3 to 32%) of the cells, although they were essentially absent in the marine samples examined. Members of the Cytophaga-Flavobacterium cluster were the most abundant group detected in the marine systems, accounting for 18% (range, 2 to 72%) of the 4',6-diamidino-2-phenylindole (DAPI) counts, and they were also important in freshwater systems (7%, range 0 to 18%). Furthermore, members of the alpha and gamma subclasses of Proteobacteria as well as members of the Planctomycetales were detected in both freshwater and marine water in abundances <7%.

Phylogeny and diversity of Achromatium oxaliferum.

Achromatium oxaliferum was first described in 1893 by Schewiakoff as an unusually large bacterium living in freshwater sediments. Up to now no pure culture is available. Physical enrichments of achromatia collected from the acidic Lake Fuchskuhle, which houses a peculiar, smaller variety, and the neutral Lake Stechlin were investigated by the cultivation-independent rRNA approach. PCR in combination with cloning and sequencing was used for the retrieval of 24 partial and 4 nearly full-length 16S rRNA sequences that formed two distinct phylogenetic clusters. Fluorescence-in-situ-hybridization (FISH) with four 16S rRNA-targeted oligonucleotide probes unambiguously assigned the different sequences to either regular, large A. oxaliferum cells or to the smaller Lake Fuchskuhle population, tentatively named "A. minus". The two Achromatium sp. 16S rRNA sequence clusters form a stable deep branch in the gamma subclass of the class Proteobacteria. The closest cultivated relatives are Chromatium vinosum, Rhabdochromatium marinum and Ectothiorhodospira halophila with 16S rRNA similarities of 86.2 to 90.5%. Profound differences in the population structure of achromatia were revealed in the two lakes by FISH. In one sample from Lake Stechlin three genotypes could be visualized, and 49% of the cells were assigned to A. oxaliferum clone AST01, 28% to Achromatium sp. genotype AFK192/AFK433 and 23% to Achromatium sp. genotype AFK192/AST433. In contrast, a morphologically and phylogenetically homogeneous population of "A. minus". was present in Lake Fuchskuhle.

Phylogeny and identification in situ of Nevskia ramosa.

An enrichment of the neuston bacterium Nevskia ramosa was investigated by the cultivation-independent rRNA approach. N. ramosa was first described by Famintzin in 1892 as a rod-shaped, slightly bent bacterium forming typical flat rosettes on the surface of shallow freshwater habitats by unilateral slime formation. PCR in combination with cloning and sequencing was used for retrieving 21 partial and 5 nearly full-length 16S rRNA sequences forming three tight clusters. In situ hybridization with rRNA-targeted oligonucleotide probes allowed us to assign the three sequence clusters to three distinct bacterial populations abundant in the enrichment. The two probes that unambiguously identified the N. ramosa morphotype were derived from a 16S rRNA sequence that had similarities of 87.9 to 88.9% to the rRNA sequences of the most closely related group in the database, Xanthomonas sp. and relatives. N. ramosa currently is the only representative of an independent, deep branch of the gamma subclass of the class Proteobacteria. The two other populations abundant in the enrichment were affiliated with the alpha subclass of the class Proteobacteria. They were most closely related to Blastobacter sp. (97.2% similarity) and Mycoplana bullata (97.6% similarity) and might represent new species in the respective genera.