Günter Jost

Epsilonproteobacteria Represent the Major Portion of Chemoautotrophic Bacteria in Sulfidic Waters of Pelagic Redoxclines of the Baltic and Black Seas

ABSTRACT Recent studies have indicated that chemoautotrophic Epsilonproteobacteria might play an important role, especially as anaerobic or microaerophilic dark CO2-fixing organisms, in marine pelagic redoxclines. However, knowledge of their distribution and abundance as actively CO2-fixing microorganisms in pelagic redoxclines is still deficient. We determined the contribution of Epsilonproteobacteria to dark CO2 fixation in the sulfidic areas of central Baltic Sea and Black Sea redoxclines by combining catalyzed reporter deposition-fluorescence in situ hybridization with microautoradiography using [14C]bicarbonate and compared it to the total prokaryotic chemoautotrophic activity. In absolute numbers, up to 3 × 10514CO2-fixing prokaryotic cells ml−1 were enumerated in the redoxcline of the central Baltic Sea and up to 9 × 10414CO2-fixing cells ml−1 were enumerated in the Black Sea redoxcline, corresponding to 29% and 12%, respectively, of total cell abundance. 14CO2-incorporating cells belonged exclusively to the domain Bacteria. Among these, members of the Epsilonproteobacteria were approximately 70% of the cells in the central Baltic Sea and up to 100% in the Black Sea. For the Baltic Sea, the Sulfurimonas subgroup GD17, previously assumed to be involved in autotrophic denitrification, was the most dominant CO2-fixing group. In conclusion, Epsilonproteobacteria were found to be mainly responsible for chemoautotrophic activity in the dark CO2 fixation maxima of the Black Sea and central Baltic Sea redoxclines. These Epsilonproteobacteria might be relevant in similar habitats of the worlds oceans, where high dark CO2 fixation rates have been measured.

Genome and physiology of a model Epsilonproteobacterium responsible for sulfide detoxification in marine oxygen depletion zones

Eutrophication and global climate change lead to expansion of hypoxia in the ocean, often accompanied by the production of hydrogen sulfide, which is toxic to higher organisms. Chemoautotrophic bacteria are thought to buffer against increased sulfide concentrations by oxidizing hydrogen sulfide before its diffusion to oxygenated surface waters. Model organisms from such environments have not been readily available, which has contributed to a poor understanding of these microbes. We present here a detailed study of “Sulfurimonas gotlandica” str. GD1, an Epsilonproteobacterium isolated from the Baltic Sea oxic-anoxic interface, where it plays a key role in nitrogen and sulfur cycling. Whole-genome analysis and laboratory experiments revealed a high metabolic flexibility, suggesting a considerable capacity for adaptation to variable redox conditions. S. gotlandica str. GD1 was shown to grow chemolithoautotrophically by coupling denitrification with oxidation of reduced sulfur compounds and dark CO2 fixation. Metabolic versatility was further suggested by the use of a range of different electron donors and acceptors and organic carbon sources. The number of genes involved in signal transduction and metabolic pathways exceeds those of other Epsilonproteobacteria. Oxygen tolerance and environmental-sensing systems combined with chemotactic responses enable this organism to thrive successfully in marine oxygen-depletion zones. We propose that S. gotlandica str. GD1 will serve as a model organism in investigations that will lead to a better understanding how members of the Epsilonproteobacteria are able to cope with water column anoxia and the role these microorganisms play in the detoxification of sulfidic waters.

Manganese cycling in the Gotland Deep, Baltic Sea

Abstract Manganese plays an important role as both an electron donor and acceptor in redox processes of stratified marine environments. Here we present results on Mn cycling in the water column of the Gotland Deep, Baltic Sea—a basin with periodically observed anoxic conditions in bottom waters. In the period 1999–2001, the deeper part of the Gotland Basin was permanently anoxic, and no inflows of significant amounts of oxygenated North Sea water were recorded below the halocline. The upward Mn(II) flux at the chemocline varied between 28 and 77 μmol m−2 day−1 (avg. 53 μmol m−2 day−1) and it was balanced by the downward flux of oxidized Mn over the entire year. The vertical flux of Mn(II) in the water column was mostly regulated by the flux of settling Mn oxides with a minor contribution of Mn(II) diffusing from bottom sediments (7.1–8.2 μmol m−2 day−1). The potential Mn(II) oxidation contributed no more than 2% to the total transferable electron flux (potential chemosynthesis) from the anoxic into the oxic zone, whereas the flux of settling Mn oxides presumably accounted for 3–30% of sulfide oxidation. Two different morphotypes of Mn-rich particles, formed during Mn(II) oxidation in the Gotland Basin chemocline, were identified using SEM-EDX: amorphous particles (92%) and Metallogenium-like particles. About 68% of all Mn-rich particles were associated with large aggregates containing an organic matrix. Manganese represented more than 60% of the total elemental composition of the particles; calcium, iron and silica were also detected.

13C‐isotope analyses reveal that chemolithoautotrophic Gamma‐ and Epsilonproteobacteria feed a microbial food web in a pelagic redoxcline of the central Baltic Sea

Marine pelagic redoxclines are zones of high dark CO(2) fixation rates, which can correspond up to 30% of the surface primary production. However, despite this significant contribution to the pelagic carbon cycle, the identity of most chemolithoautotrophic organisms is still unknown. Therefore, the aim of this study was to directly link the dark CO(2) fixation capacity of a pelagic redoxcline in the central Baltic Sea (Landsort Deep) with the identity of the main chemolithoautotrophs involved. Our approach was based on the analysis of natural carbon isotope signatures in fatty acid methyl esters (FAMEs) and on measurements of CO(2) incorporation in (13)C-bicarbonate pulse experiments. The incorporation of (13)C into chemolithoautotrophic cells was investigated by rRNA-based stable isotope probing (RNA-SIP) and FAME analysis after incubation for 24 and 72 h under in situ conditions. Our results demonstrated that fatty acids indicative of Proteobacteria were significantly enriched in (13)C slightly below the chemocline. RNA-SIP analyses revealed that two different Gammaproteobacteria and three closely related Epsilonproteobacteria of the Sulfurimonas cluster were active dark CO(2)-fixing microorganisms, with a time-dependent community shift between these groups. Labelling of Archaea was not detectable, but after 72 h of incubation the (13)C-label had been transferred to a potentially bacterivorous ciliate related to Euplotes sp. Thus, RNA-SIP provided direct evidence for the contribution of chemolithoautotrophic production to the microbial food web in this marine pelagic redoxcline, emphasizing the importance of dark CO(2)-fixing Proteobacteria within this habitat.

Active nitrogen-fixing heterotrophic bacteria at and below the chemocline of the central Baltic Sea

The Baltic Sea receives large nitrogen inputs by diazotrophic (N2-fixing) heterocystous cyanobacteria but the significance of heterotrophic N2 fixation has not been studied. Here, the diversity, abundance and transcription of the nifH fragment of the nitrogenase enzyme in two basins of the Baltic Sea proper was examined. N2 fixation was measured at the surface (5 m) and in anoxic water (200 m). Vertical sampling profiles of >10 and <10 μm size fractions were collected in 2007, 2008 and 2011 at the Gotland Deep and in 2011 in the Bornholm Basin. Both of these stations are characterized by permanently anoxic bottom water. The 454-pyrosequencing nifH analysis revealed a diverse assemblage of nifH genes related to alpha-, beta- and gammaproteobacteria (nifH cluster I) and anaerobic bacteria (nifH cluster III) at and below the chemocline. Abundances of genes and transcripts of seven diazotrophic phylotypes were investigated using quantitative polymerase chain reaction revealing abundances of heterotrophic nifH phylotypes of up to 2.1 × 107 nifH copies l−1. Abundant nifH transcripts (up to 3.2 × 104 transcripts l−1) within nifH cluster III and co-occurring N2 fixation (0.44±0.26 nmol l−1 day−1) in deep water suggests that heterotrophic diazotrophs are fixing N2 in anoxic ammonium-rich waters. Our results reveal that N2 fixation in the Baltic Sea is not limited to illuminated N-deplete surface waters and suggest that N2 fixation could also be of importance in other suboxic regions of the world’s oceans.

Impact of different in vitro electron donor/acceptor conditions on potential chemolithoautotrophic communities from marine pelagic redoxclines.

ABSTRACT 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.

Measuring unbiased metatranscriptomics in suboxic waters of the central Baltic Sea using a new in situ fixation system

An analysis of the microbial metabolism is fundamental to understanding globally important element transformations. One culture-independent approach to deduce those prokaryotic metabolic functions is to analyze metatranscriptomes. Unfortunately, since mRNA is extremely labile, it is unclear whether the abundance patterns detected in nature are vulnerable to considerable modification in situ simply due to sampling procedures. Exemplified on comparisons of metatranscriptomes retrieved from pelagic suboxic zones of the central Baltic Sea (70–120 m depth), earlier identified as areas of high aerobic ammonium oxidation activity, and quantification of specific transcripts in them, we show that different sampling techniques significantly influence the relative abundance of transcripts presumably diagnostic of the habitat. In situ fixation using our newly developed automatic flow injection sampler resulted in an abundance of thaumarchaeal ammonia monooxygenase transcripts that was up to 30-fold higher than that detected in samples obtained using standard oceanographic sampling systems. By contrast, the abundance of transcripts indicative of cellular stress was significantly greater in non-fixed samples. Thus, the importance of in situ fixation in the reliable evaluation of distinct microbial activities in the ecosystem based on metatranscriptomics is obvious. In consequence, our data indicate that the significance of thaumarchaeota to aerobic ammonium oxidation could yet have been considerably underestimated. Taken these results, this could in general also be the case in attempts aimed at an unbiased gene expression analysis of areas below the epipelagic zone, which cover 90% of the worlds oceans.

Sulfurimonas gotlandica sp. nov., a chemoautotrophic and psychrotolerant epsilonproteobacterium isolated from a pelagic redoxcline, and an emended description of the genus Sulfurimonas

A psychro- and aerotolerant bacterium was isolated from the sulfidic water of a pelagic redox zone of the central Baltic Sea. The slightly curved rod- or spiral-shaped cells were motile by one polar flagellum or two bipolar flagella. Growth was chemolithoautotrophic, with nitrate or nitrite as electron acceptor and either a variety of sulfur species of different oxidation states or hydrogen as electron donor. Although the bacterium was able to utilize organic substances such as acetate, pyruvate, peptone and yeast extract for growth, these compounds yielded considerably lower cell numbers than obtained with reduced sulfur or hydrogen; in addition, bicarbonate supplementation was necessary. The cells also had an absolute requirement for NaCl. Optimal growth occurred at 15 °C and at pH 6.6–8.0. The predominant fatty acid of this organism was 16 : 1ω7c, with 3-OH 14 : 0, 16 : 0, 16 : 1ω5c+t and 18 : 1ω7c present in smaller amounts. The DNA G+C content was 33.6 mol%. As determined in 16S rRNA gene sequence phylogeny analysis, the isolate belongs to the genus Sulfurimonas, within the class Epsilonproteobacteria, with 93.7 to 94.2 % similarity to the other species of the genus Sulfurimonas, Sulfurimonas autotrophica, Sulfurimonas paralvinellae and Sulfurimonas denitrificans. However, the distinct physiological and genotypic differences from these previously described taxa support the description of a novel species, Sulfurimonas gotlandica sp. nov. The type strain is GD1T ( = DSM 19862T = JCM 16533T). Our results also justify an emended description of the genus Sulfurimonas.

Quantitative Distributions of Epsilonproteobacteria and a Sulfurimonas Subgroup in Pelagic Redoxclines of the Central Baltic Sea

ABSTRACT Members of the class Epsilonproteobacteria are known to be of major importance in biogeochemical processes at oxic-anoxic interfaces. In pelagic redoxclines of the central Baltic Sea, an uncultured epsilonproteobacterium related to Sulfurimonas denitrificans was proposed to play a key role in chemolithotrophic denitrification (I. Brettar, M. Labrenz, S. Flavier, J. Bötel, H. Kuosa, R. Christen, and M. G. Höfle, Appl. Environ. Microbiol. 72:1364-1372, 2006). In order to determine the abundance, activity, and vertical distribution of this bacterium in high-resolution profiles, 16S rRNA cloning and catalyzed reporter deposition and fluorescence in situ hybridization (CARD-FISH) and quantitative PCR measurements were carried out. The results showed that 21% of the derived clone sequences, which in the present study were grouped together under the name GD17, had >99% similarity to the uncultured epsilonproteobacterium. A specific gene probe against GD17 (S-*-Sul-0090-a-A-18) was developed and used for enumeration by CARD-FISH. In different pelagic redoxclines sampled during August 2003, May 2005, and February 2006, GD17 cells were always detected from the lower oxic area to the sulfidic area. Maximal abundance was detected around the chemocline, where sulfide and nitrate concentrations were close to the detection limit. The highest GD17 numbers (2 × 105 cells ml−1), representing up to 15% of the total bacteria, were comparable to those reported for Epsilonproteobacteria in pelagic redoxclines of the Black Sea and the Cariaco Trench (X. Lin, S. G. Wakeham, I. F. Putnam, Y. M. Astor, M. I. Scranton, A. Y. Chistoserdov, and G. T. Taylor, Appl. Environ. Microbiol. 72:2679-2690, 2006). However, in the Baltic Sea redoxclines, Epsilonproteobacteria consisted nearly entirely of cells belonging to the distinct GD17 group. This suggested that GD17 was the best-adapted epsilonproteobacterium within this ecological niche.

Diversity of active chemolithoautotrophic prokaryotes in the sulfidic zone of a Black Sea pelagic redoxcline as determined by rRNA-based stable isotope probing

Marine pelagic redoxclines are characterized by pronounced activities of chemolithoautotrophic microorganisms. As evidenced by the high dark CO(2) 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 CO(2) 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.