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Marine pelagic redoxclines are characterized by pronounced activities of chemolithoautotrophic microorganisms. As evidenced by the high dark CO2 fixation rates measured around the oxic–anoxic interface but also in the upper sulfidic zone, the accordant organisms participate in important biogeochemical transformations. Although Epsilonproteobacteria have been identified as an important chemoautotrophic group in these environments, detailed species-level information on the identity of actively involved prokaryotes is lacking. In the present study, active chemolithoautotrophic prokaryotic assemblages were identified in the sulfidic zone of a pelagic Black Sea redoxcline by applying rRNA-based stable isotope probing in combination with 16S rRNA gene single-strand conformation polymorphism analysis and 16S rRNA gene cloning. The results showed that a single epsilonproteobacterium, affiliated with the genus Sulfurimonas, and two different members of the gammaproteobacterial sulfur oxidizer (GSO) cluster were responsible for dark CO2 fixation activities in the upper sulfidic layer of the Black Sea redoxcline. Phylogenetically, these organisms were closely related to microorganisms, distributed worldwide, that are thought to be key players in denitrification and sulfide oxidation. Together, these findings emphasize the importance of chemolithoautotrophic members of the Sulfurimonas and GSO groups in the carbon, nitrogen, and sulfur cycles of oxic–anoxic pelagic transition zones. [ABSTRACT FROM AUTHOR]

Identification and functional analysis of key members of bacterial communities in marine and estuarine environments are major challenges for obtaining a mechanistic understanding of biogeochemical processes. In the Baltic Sea basins, as in many other marine environments with anoxic bodies of water, the oxic-anoxic interface is considered a layer of high bacterial turnover of sulfur, nitrogen, and carbon compounds that has a great impact on matter balances in the whole ecosystem. We focused on autotrophic denitrification by oxidation of reduced sulfur compounds as a biogeochemically important process mediating concomitant turnover of sulfur, nitrogen, and carbon. We used a newly developed approach consisting of molecular analyses in stimulation experiments and in situ abundance. The molecular approach was based on single-strand conformational polymorphism (SSCP) analysis of the bacterial community RNA, which allowed identification of potential denitrifiers based on the sequences of enhanced SSCP bands and monitoring of the overall bacterial community during the experiments. Sequences of the SSCP bands of interest were used to design highly specific primers that enabled (i) generation of almost complete 16S rRNA gene sequences using experimental and environmental DNA as templates and (ii) quantification of the bacteria of interest by real-time PCR. By using this approach we identified the bacteria responsible for autotrophic denitrification as a single taxon, an epsilonproteobacterium related to the autotrophic denitrifier Thiomicrospira denitrificans. This finding was confirmed by material balances in the experiments that were consistent with those obtained with continuous cultures of T. denitrificans. The presence and activity of a bacterium that is phylogenetically and physiologically closely related to T. denitrificans could be relevant for the carbon budget of the central Baltic Sea because T. denitrificans exhibits only one-half the efficiency for carbon dioxide fixation per mol of sulfide oxidized and mol of nitrate reduced of Thiobacillus denitrificans hypothesized previously for this function. [ABSTRACT FROM AUTHOR]

Anaerobic or microaerophilic chemolithoautotrophic bacteria have been considered to be responsible for CO2 dark fixation in different pelagic redoxclines worldwide, but their involvement in redox processes is still not fully resolved. We investigated the impact of 17 different electron donor/acceptor combinations in water of pelagic redoxclines from the central Baltic Sea on the stimulation of bacterial CO2 dark fixation as well as on the development of chemolithoautotrophic populations. In situ, the highest CO2 dark fixation rates, ranging from 0.7 to 1.4 μmol liter-1 day-1, were measured directly below the redoxcline. In enrichment experiments, chemolithoautotrophic CO2 dark fixation was maximally stimulated by the addition of thiosulfate, reaching values of up to 9.7 μmol liter-1 CO2 day-1. Chemolithoautotrophic nitrate reduction proved to be an important process, with rates of up to 33.5 μmol liter-1 NO3- day-1. Reduction of Fe(III) or Mn(IV) was not detected; nevertheless, the presence of these potential electron acceptors influenced the development of stimulated microbial assemblages. Potential chemolithoautotrophic bacteria in the enrichment experiments were displayed on 16S ribosomal complementary DNA single-strand-conformation polymorphism fingerprints and identified by sequencing of excised bands. Sequences were closely related to chemolithoautotrophic Thiomicrospira psychrophila and Maorithyas hadalis gill symbiont (both Gammaproteobacteria) and to an uncultured nitrate-reducing Helicobacteraceae bacterium (Epsilonproteobacteria). Our data indicate that this Helicobacteraceae bacterium could be of general importance or even a key organism for autotrophic nitrate reduction in pelagic redoxclines. [ABSTRACT FROM AUTHOR]

We have developed a highly sensitive approach to assess the abundance of uncultured bacteria in water samples from the central Baltic Sea by using a noncultured member of the “Epsilonproteobacteria” related to Thiomicrospira denitrificans as an example. Environmental seawater samples and samples enriched for the target taxon provided a unique opportunity to test the approach over a broad range of abundances. The approach is based on a combination of taxon- and domain-specific real-time PCR measurements determining the relative T. denitrificans-like 16S rRNA gene and 16S rRNA abundances, as well as the determination of total cell counts and environmental RNA content. It allowed quantification of T. denitrificans-like 16S rRNA molecules or 16S rRNA genes as well as calculation of the number of ribosomes per T. denitrificans-like cell. Every real-time measurement and its specific primer system were calibrated using environmental nucleic acids obtained from the original habitat for external standardization. These standards, as well as the respective samples to be measured, were prepared from the same DNA or RNA extract. Enrichment samples could be analyzed directly, whereas environmental templates had to be preamplified with general bacterial primers before quantification. Preamplification increased the sensitivity of the assay by more than 4 orders of magnitude. Quantification of enrichments with or without a preamplification step yielded comparable results. T. denitrificans-like 16S rRNA molecules ranged from 7.1 × 10³ to 4.4 × 109 copies ml-1 or 0.002 to 49.7% relative abundance. T. denitrificans-like 16S rRNA genes ranged from 9.0 × 10¹ to 2.2 × 106 copies ml-1 or 0.01 to 49.7% relative abundance. Detection limits of this real-time-PCR approach were 20 16S rRNA molecules or 0.2 16S rRNA gene ml-1. The number of ribosomes per T. denitrificans-like cell was estimated to range from 20 to 200 in seawater and reached up to 2,000 in the enrichments. The results indicate that our real-time PCR approach can be used to determine cellular and relative abundances of uncultured marine bacterial taxa and to provide information about their levels of activity in their natural environment. [ABSTRACT FROM AUTHOR]

Throughout the Baltic Sea redoxcline, virus production and the frequency of lytically-infected prokaryotic cells were estimated from parallel incubations of undiluted seawater and seawater that contained prokaryotes with substantially reduced numbers of viruses (virus dilution approach), effectively preventing viral reinfection during the incubation period. Undiluted seawater incubations resulted in much higher estimates of virus production (6–35×104 mL-1 h-1) and the frequency of infected cells (5–84%) than the virus dilution approach (virus production: 1–3×104 mL-1 h-1; frequency of infected cells: 1–11%). Viral production and the frequency of infected cells from both approaches, however, cannot be directly compared, as data obtained from undiluted incubations were biased by viral reinfection and other uncontrollable processes during the incubation period. High in situ viral abundance (1–2×107 mL-1) together with low virus production rates based on the virus dilution approach resulted in some of the longest viral turnover times (24–84 d) ever reported for the epipelagial. Throughout a wide range of environmental conditions, viral turnover time and burst size were negatively correlated. Given that viral decay estimated in ultra-filtered water was below the detection limit and the burst size was low (1–17), we conclude that prokaryotic viruses in the Baltic Sea redoxcline are investing most of their resources into stress defense (strong capsids) rather than proliferation (high burst size). In summary, the Baltic Sea redoxcline constitutes an environment where low virus production is found in combination with low viral decay, resulting in high viral abundance. [ABSTRACT FROM AUTHOR]

The abundance, vertical distribution, and diversity of aerobic anoxygenic phototrophic bacteria (AAP) were studied at four basins of the Baltic Sea. AAP were enumerated by infrared epifluorescence microscopy, and their diversity was analyzed by using pufM gene clone libraries. In addition, numbers of CFU containing the pufM gene were determined, and representative strains were isolated. Both approaches indicated that AAP reached maximal abundance in the euphotic zone. Maximal AAP abundance was 2.5 × 105 cells ml-1 (11% of total prokaryotes) or 1.0 × 10³ CFU ml-1 (9 to 10% of total CFU). Environmental pufM clone sequences were grouped into 11 operational taxonomic units phylogenetically related to cultivated members of the Alpha-, Beta-, and Gammaproteobacteria. In spite of varying pufM compositions, five clones were present in all libraries. Of these, Jannaschia-related clones were always found in relative abundances representing 25 to 30% of the total AAP clones. The abundances of the other clones varied. Clones potentially affiliated with typical freshwater Betaproteobacteria sequences were present at three Baltic Sea stations, whereas clones grouping with Loktanella represented 40% of the total cell numbers in the Gotland Basin. For three alphaproteobacterial clones, probable pufM phylogenetic relationships were supported by 16S rRNA gene analyses of Baltic AAP isolates, which showed nearly identical pufM sequences. Our data indicate that the studied AAP assemblages represented a mixture of marine and freshwater taxa, thus characterizing the Baltic Sea as a "melting pot" of abundant, polyphyletic aerobic photoheterotrophic bacteria. [ABSTRACT FROM AUTHOR]

We determined the abundance and distribution of chemolithoautotrophic prokaryotes in the redoxcline in two basins (Gotland Deep, Landsort Deep) of the central Baltic Sea by combining dark CO2 fixation measurements with flow cytometric cell sorting. Maximum CO2 fixation rates were recorded in sulfidic waters about 20 m below the chemocline. Flow cytometric analyses of deoxyribonucleic acid (DNA)-stained bacterioplankton revealed the existence of at least five different prokaryotic clusters in water samples collected below the chemocline. Dark CO2 fixation in these clusters was determined by flow cytometric sorting after anoxic incubations with NaH14CO3 tracer. Two clusters, representing about 30% of total prokaryotes, were responsible for 65% to 100% of the total dark fixation. Calculated cell-specific CO2 fixation rates in the two basins ranged from 3.5 to 24.7 fg C cell-1 d-1 and suggested that these clusters are dominated by chemolithoautotrophic prokaryotes. Mean cell-specific fixation rates reached more than 10 fg C cell-1 d-1 in most cases, indicating relatively high growth rates (doubling times 1-2 d) of chemolithoautotrophic prokaryotes. Our results provide the first evidence of such high cell-specific CO2 uptake and abundance of chemolithoautotrophic prokaryotes in a pelagic marine environment. However, the identity of the organisms as well as the mechanisms fueling CO2 dark fixation in the anoxic zone remain unknown. [ABSTRACT FROM AUTHOR]