Active nitrogen-fixing heterotrophic bacteria at and below the chemocline of the central Baltic Sea (original) (raw)
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Cultivation and isolation of N2-fixing bacteria from suboxic waters in the Baltic Sea
Nitrogenase genes (nifH) from heterotrophic dinitrogen (N 2 )-fixing bacteria appear ubiquitous in marine bacterioplankton, but the significance of these bacteria for N cycling is unknown. Quantitative data on the N 2 -fixation potential of marine and estuarine heterotrophs are scarce, and the shortage of cultivated specimens currently precludes ecophysiological characterization of these bacteria. Through the cultivation of diazotrophs from suboxic (1.79 lmol O 2 L À1 ) Baltic Sea water in an artificial seawater medium devoid of combined N, we report the cultivability of a considerable fraction of the diazotrophic community in the Gotland Deep. Two nifH clades were present both in situ and in enrichment cultures showing gene abundances of up to 4.6 9 10 5 and 5.8 9 10 5 nifH gene copies L À1 within two vertical profiles in the Baltic Sea. The distributions of the two clades suggested a relationship with the O 2 concentrations in the water column as abundances increased in the suboxic and anoxic waters. It was possible to cultivate and isolate representatives from one of these prevalent clades, and preliminary analysis of their ecophysiology demonstrated growth optima at 0.5-15 lmol O 2 L À1 and 186-194 lmol O 2 L À1 in the absence of combined N.
Cultivation and isolation of N2-fixing bacteria from suboxic waters in the Baltic Sea
FEMS Microbiology Ecology, 2014
Nitrogenase genes (nifH) from heterotrophic dinitrogen (N 2)-fixing bacteria appear ubiquitous in marine bacterioplankton, but the significance of these bacteria for N cycling is unknown. Quantitative data on the N 2-fixation potential of marine and estuarine heterotrophs are scarce, and the shortage of cultivated specimens currently precludes ecophysiological characterization of these bacteria. Through the cultivation of diazotrophs from suboxic (1.79 lmol O 2 L À1) Baltic Sea water in an artificial seawater medium devoid of combined N, we report the cultivability of a considerable fraction of the diazotrophic community in the Gotland Deep. Two nifH clades were present both in situ and in enrichment cultures showing gene abundances of up to 4.6 9 10 5 and 5.8 9 10 5 nifH gene copies L À1 within two vertical profiles in the Baltic Sea. The distributions of the two clades suggested a relationship with the O 2 concentrations in the water column as abundances increased in the suboxic and anoxic waters. It was possible to cultivate and isolate representatives from one of these prevalent clades, and preliminary analysis of their ecophysiology demonstrated growth optima at 0.5-15 lmol O 2 L À1 and 186-194 lmol O 2 L À1 in the absence of combined N.
Plos One, 2011
Cyanobacteria are thought to be the main N 2 -fixing organisms (diazotrophs) in marine pelagic waters, but recent molecular analyses indicate that non-cyanobacterial diazotrophs are also present and active. Existing data are, however, restricted geographically and by limited sequencing depths. Our analysis of 79,090 nitrogenase (nifH) PCR amplicons encoding 7,468 unique proteins from surface samples (ten DNA samples and two RNA samples) collected at ten marine locations worldwide provides the first in-depth survey of a functional bacterial gene and yield insights into the composition and diversity of the nifH gene pool in marine waters. Great divergence in nifH composition was observed between sites. Cyanobacteria-like genes were most frequent among amplicons from the warmest waters, but overall the data set was dominated by nifH sequences most closely related to non-cyanobacteria. Clusters related to Alpha-, Beta-, Gamma-, and Delta-Proteobacteria were most common and showed distinct geographic distributions. Sequences related to anaerobic bacteria (nifH Cluster III) were generally rare, but preponderant in cold waters, especially in the Arctic. Although the two transcript samples were dominated by unicellular cyanobacteria, 42% of the identified non-cyanobacterial nifH clusters from the corresponding DNA samples were also detected in cDNA. The study indicates that non-cyanobacteria account for a substantial part of the nifH gene pool in marine surface waters and that these genes are at least occasionally expressed. The contribution of noncyanobacterial diazotrophs to the global N 2 fixation budget cannot be inferred from sequence data alone, but the prevalence of non-cyanobacterial nifH genes and transcripts suggest that these bacteria are ecologically significant.
Isolation and gene quantification of heterotrophic N 2 -fixing bacterioplankton in the Baltic Sea
Environmental Microbiology, 2007
Cyanobacteria are regarded as the main N2-fixing organisms in marine waters. However, recent clone libraries from various oceans show a wide distribution of the dinitrogenase reductase gene (nifH) originating from heterotrophic bacterioplankton. We isolated heterotrophic N2-fixing bacteria from Baltic Sea bacterioplankton using low-nitrogen plates and semi-solid diazotroph medium (SSDM) tubes. Isolates were analysed for the nitrogenase (nifH) gene and active N2 fixation by nested polymerase chain reaction (PCR) and acetylene reduction respectively. A primer-probe set targeting the nifH gene from a γ-proteobacterial isolate, 97% 16S rDNA similarity to Pseudomonas stutzeri, was designed for measuring in situ dynamics using quantitative real-time PCR. This nifH gene sequence was detected at two of 11 stations in a Baltic Proper transect at abundances of 3 × 104 and 0.8 × 103 copies per litre seawater respectively. Oxygen requirements of isolates were examined by cultivation in SSDM tubes where oxygen gradients were determined with microelectrodes. Growth, and thereby N2 fixation, was observed as horizontal bands formed at oxygen levels of 0–6% air saturation. The apparent microaerophilic or facultative anaerobic nature of the isolates explains why the SSDM approach is the most appropriate isolation method. Our study illustrates how combined isolation, functional analyses and in situ quantification yielded insights into the oxygen requirements of heterotrophic N2-fixing bacterioplankton isolates, which were confirmed to be present in situ.
FEMS Microbiology Ecology, 2012
The Arabian Sea oxygen minimum zone (OMZ), the largest suboxic region in the world's oceans, is responsible for up to half of the global mesopelagic fixed nitrogen (N) loss from the ocean via denitrification and anammox. Dinitrogen (N2) fixation is usually attributed to cyanobacteria in the surface ocean. Model prediction and physiological inhibition of N2 fixation by oxygen, however, suggest that N2 fixation should be enhanced near the oxygen-deficient zone (ODZ) of the Arabian Sea. N2 fixation and cyanobacterial nifH genes (the gene encoding dinitrogenase reductase) have been reported in surface waters overlying the Arabian Sea ODZ. Here, water samples from depths above and within the Arabian Sea ODZ were examined to explore the distribution, diversity, and expression of nifH genes. In surface waters, nifH DNA and cDNA sequences related to Trichodesmium, a diazotroph known to occur and fix N2 in the Arabian Sea, were detected. Proteobacterial nifH phylotypes (DNA but not cDNA) were also detected in surface waters. Proteobacterial nifH DNA and cDNA sequences, as well as nifH DNA and cDNA sequences related to strictly anaerobic N-fixers, were obtained from oxygen-deficient depths. This first report of nifH gene expression in subsurface low-oxygen waters suggests that there is potential for active N2 fixation by several phylogenetically and potentially metabolically diverse microorganisms in pelagic OMZs.
Aquat Microb Ecol, 2010
Marine waters are generally considered to be nitrogen (N) limited and are therefore favourable environments for diazotrophs, i.e. organisms converting atmospheric N 2 into ammonium or nitrogen oxides available for growth. In some regions, this import of N supports up to half of the primary productivity. Diazotrophic Cyanobacteria appear to be the major contributors to marine N 2 fixation in surface waters, whereas the contribution of heterotrophic or chemoautotrophic diazotrophs to this process is usually regarded inconsequential. Culture-independent studies reveal that non-cyanobacterial diazotrophs are diverse, widely distributed, and actively expressing the nitrogenase gene in marine and estuarine environments. The detection of nifH genes and nifH transcripts, even in N-replete marine waters, suggests that N 2 fixation is an ecologically important process throughout the oceans. Because this process is highly sensitive to and inhibited by molecular oxygen (O 2 ), diazotrophy requires efficient scavenging of intracellular O 2 or growth in environments with low ambient O 2 concentration. Particles with interior low-O 2 micro-zones and oceanic oxygen minimum zones are just 2 potential habitats suitable for N 2 fixation by non-cyanobacterial diazotrophs. Our ignorance about the regulation of N 2 fixation by non-Cyanobacteria in their natural marine environments currently prevents an evaluation of their importance in marine N cycling and budgets. A review of the molecular data on distribution and expression of nifH genes in non-Cyanobacteria suggests that further study of the role of these Bacteria in N cycling at local, regional and global scales is needed.
mBio, 2015
The ability to reduce atmospheric nitrogen (N2) to ammonia, known as N2 fixation, is a widely distributed trait among prokaryotes that accounts for an essential input of new N to a multitude of environments. Nitrogenase reductase gene (nifH) composition suggests that putative N2-fixing heterotrophic organisms are widespread in marine bacterioplankton, but their autecology and ecological significance are unknown. Here, we report genomic and ecophysiology data in relation to N2 fixation by three environmentally relevant heterotrophic bacteria isolated from Baltic Sea surface water: Pseudomonas stutzeri strain BAL361 and Raoultella ornithinolytica strain BAL286, which are gammaproteobacteria, and Rhodopseudomonas palustris strain BAL398, an alphaproteobacterium. Genome sequencing revealed that all were metabolically versatile and that the gene clusters encoding the N2 fixation complex varied in length and complexity between isolates. All three isolates could sustain growth by N2 fixati...
Applied and environmental microbiology, 1998
Oligotrophic oceanic waters of the central ocean gyres typically have extremely low dissolved fixed inorganic nitrogen concentrations, but few nitrogen-fixing microorganisms from the oceanic environment have been cultivated. Nitrogenase gene (nifH) sequences amplified directly from oceanic waters showed that the open ocean contains more diverse diazotrophic microbial populations and more diverse habitats for nitrogen fixers than previously observed by classical microbiological techniques. Nitrogenase genes derived from unicellular and filamentous cyanobacteria, as well as from the alpha and gamma subdivisions of the class Proteobacteria, were found in both the Atlantic and Pacific oceans. nifH sequences that cluster phylogenetically with sequences from sulfate reducers or clostridia were found associated with planktonic crustaceans. Nitrogenase sequence types obtained from invertebrates represented phylotypes distinct from the phylotypes detected in the picoplankton size fraction. T...