Diazotrophic Diversity and Distribution in the Tropical and Subtropical Atlantic Ocean

To understand the structure of marine diazotrophic communities in the tropical and subtropical Atlantic Ocean, the molecular diversity of the nifH gene was studied by nested PCR amplification using degenerate primers, followed by cloning and sequencing. Sequences of nifH genes were amplified from environmental DNA samples collected during three cruises (November-December 2000, March 2002, and October-November 2002) covering an area between 0 to 28.3°N and 56.6 to 18.5°W. A total of 170 unique sequences were recovered from 18 stations and 23 depths. Samples from the November-December 2000 cruise contained both unicellular and filamentous cyanobacterial nifH phylotypes, as well as γ-proteobacterial and cluster III sequences, so far only reported in the Pacific Ocean. In contrast, samples from the March 2002 cruise contained only phylotypes related to the uncultured group A unicellular cyanobacteria. The October-November 2002 cruise contained both filamentous and unicellular cyanobacterial and γ-proteobacterial sequences. Several sequences were identical at the nucleotide level to previously described environmental sequences from the Pacific Ocean, including group A sequences. The data suggest a community shift from filamentous cyanobacteria in surface waters to unicellular cyanobacteria and/or heterotrophic bacteria in deeper waters. With one exception, filamentous cyanobacterial nifH sequences were present within temperatures ranging between 26.5 and 30°C and where nitrate was undetectable. In contrast, nonfilamentous nifH sequences were found throughout a broader temperature range, 15 to 30°C, more often in waters with temperature of <26°C, and were sometimes recovered from waters with detectable nitrate concentrations.     

PCR Analysis of the Distribution of Unicellular Cyanobacterial Diazotrophs in the Arabian Sea

An oligonucleotide primer, NITRO821R, targeting the 16S rRNA gene of unicellular cyanobacterial N₂ fixers was developed based on newly derived sequences from Crocosphaera sp. strain WH 8501 and Cyanothece sp. strains WH 8902 and WH 8904 as well as several previously described sequences of Cyanothece sp. and sequences of intracellular cyanobacterial symbionts of the marine diatom Climacodium frauenfeldianum. This oligonucleotide is specific for the targeted organisms, which represent a well-defined phylogenetic lineage, and can detect as few as 50 cells in a standard PCR when it is used as a reverse primer together with the cyanobacterium- and plastid-specific forward primer CYA359F (U. Nübel, F. Garcia-Pichel, and G. Muyzer, Appl. Environ. Microbiol. 63:3327-3332, 1997). Use of this primer pair in the PCR allowed analysis of the distribution of marine unicellular cyanobacterial diazotrophs along a transect following the 67°E meridian from Victoria, Seychelles, to Muscat, Oman (0.5°S to 26°N) in the Arabian Sea. These organisms were found to be preferentially located in warm (>29°C) oligotrophic subsurface waters between 0 and 7°N, but they were also found at a station north of Oman at 26°N, 56°35′E, where similar water column conditions prevailed. Slightly cooler oligotrophic waters (<29°C) did not contain these organisms or the numbers were considerably reduced, suggesting that temperature is a key factor in dictating the abundance of this unicellular cyanobacterial diazotroph lineage in marine environments.     

High rates of denitrification and nitrous oxide emission in arid biological soil crusts from the Sultanate of Oman

Using a combination of process rate determination, microsensor profiling and molecular techniques, we demonstrated that denitrification, and not anaerobic ammonium oxidation (anammox), is the major nitrogen loss process in biological soil crusts from Oman. Potential denitrification rates were 584±101 and 58±20 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Complete denitrification to N₂ was further confirmed by an ¹⁵NO₃⁻ tracer experiment with intact crust pieces that proceeded at rates of 103±19 and 27±8 μmol N m⁻² h⁻¹ for cyanobacterial and lichen crust, respectively. Strikingly, N₂O gas was emitted at very high potential rates of 387±143 and 31±6 μmol N m⁻² h⁻¹ from the cyanobacterial and lichen crust, respectively, with N₂O accounting for 53–66% of the total emission of nitrogenous gases. Microsensor measurements revealed that N₂O was produced in the anoxic layer and thus apparently originated from incomplete denitrification. Using quantitative PCR, denitrification genes were detected in both the crusts and were expressed either in comparable (nirS) or slightly higher (narG) numbers in the cyanobacterial crusts. Although 99% of the nirS sequences in the cyanobacterial crust were affiliated to an uncultured denitrifying bacterium, 94% of these sequences were most closely affiliated to Paracoccus denitrificans in the lichen crust. Sequences of nosZ gene formed a distinct cluster that did not branch with known denitrifying bacteria. Our results demonstrate that nitrogen loss via denitrification is a dominant process in crusts from Oman, which leads to N₂O gas emission and potentially reduces desert soil fertility.     

Chemolithotrophic acetogenic H2/CO2 utilization in Italian rice field soil

Acetate oxidation in Italian rice field at 50 °C is achieved by uncultured syntrophic acetate oxidizers. As these bacteria are closely related to acetogens, they may potentially also be able to synthesize acetate chemolithoautotrophically. Labeling studies using exogenous H₂ (80%) and ¹³CO₂ (20%), indeed demonstrated production of acetate as almost exclusive primary product not only at 50 °C but also at 15 °C. Small amounts of formate, propionate and butyrate were also produced from ¹³CO₂. At 50 °C, acetate was first produced but later on consumed with formation of CH₄. Acetate was also produced in the absence of exogenous H₂ albeit to lower concentrations. The acetogenic bacteria and methanogenic archaea were targeted by stable isotope probing of ribosomal RNA (rRNA). Using quantitative PCR, ¹³C-labeled bacterial rRNA was detected after 20 days of incubation with ¹³CO₂. In the heavy fractions at 15 °C, terminal restriction fragment length polymorphism, cloning and sequencing of 16S rRNA showed that Clostridium cluster I and uncultured Peptococcaceae assimilated ¹³CO₂ in the presence and absence of exogenous H₂, respectively. A similar experiment showed that Thermoanaerobacteriaceae and Acidobacteriaceae were dominant in the ¹³C treatment at 50 °C. Assimilation of ¹³CO₂ into archaeal rRNA was detected at 15 °C and 50 °C, mostly into Methanocellales, Methanobacteriales and rice cluster III. Acetoclastic methanogenic archaea were not detected. The above results showed the potential for acetogenesis in the presence and absence of exogenous H₂ at both 15 °C and 50 °C. However, syntrophic acetate oxidizers seemed to be only active at 50 °C, while other bacterial groups were active at 15 °C.     

Characterization of microbial community structure during continuous anaerobic digestion of straw and cow manure

Responses of bacterial and archaeal communities to the addition of straw during anaerobic digestion of manure at different temperatures (37°C, 44°C and 52°C) were investigated using five laboratory-scale semi-continuous stirred tank reactors. The results revealed that including straw as co-substrate decreased the species richness for bacteria, whereas increasing the operating temperature decreased the species richness for both archaea and bacteria, and also the evenness of the bacteria. Taxonomic classifications of the archaeal community showed that Methanobrevibacter dominated in the manure samples, while Methanosarcina dominated in all digesters regardless of substrate. Increase of the operating temperature to 52°C led to increased relative abundance of Methanoculleus and Methanobacterium. Among the bacteria, the phyla Firmicutes and Bacteroidetes dominated within all samples. Compared with manure itself, digestion of manure resulted in a higher abundance of an uncultured class WWE1 and lower abundance of Bacilli. Adding straw to the digesters increased the level of Bacteroidia, while increasing the operating temperature decreased the level of this class and instead increased the relative abundance of an uncultured genus affiliated to order MBA08 (Clostridia). A considerable fraction of bacterial sequences could not be allocated to genus level, indicating that novel phylotypes are resident in these communities.     

Epilithic Cyanobacterial Communities of a Marine Tropical Beach Rock (Heron Island, Great Barrier Reef): Diversity and Diazotrophy

The diversity and nitrogenase activity of epilithic marine microbes in a Holocene beach rock (Heron Island, Great Barrier Reef, Australia) with a proposed biological calcification “microbialite” origin were examined. Partial 16S rRNA gene sequences from the dominant mat (a coherent and layered pink-pigmented community spread over the beach rock) and biofilms (nonstratified, differently pigmented microbial communities of small shallow depressions) were retrieved using denaturing gradient gel electrophoresis (DGGE), and a clone library was retrieved from the dominant mat. The 16S rRNA gene sequences and morphological analyses revealed heterogeneity in the cyanobacterial distribution patterns. The nonheterocystous filamentous genus Blennothrix sp., phylogenetically related to Lyngbya, dominated the mat together with unidentified nonheterocystous filaments of members of the Pseudanabaenaceae and the unicellular genus Chroococcidiopsis. The dominance and three-dimensional intertwined distribution of these organisms were confirmed by nonintrusive scanning microscopy. In contrast, the less pronounced biofilms were dominated by the heterocystous cyanobacterial genus Calothrix, two unicellular Entophysalis morphotypes, Lyngbya spp., and members of the Pseudanabaenaceae family. Cytophaga-Flavobacterium-Bacteroides and Alphaproteobacteria phylotypes were also retrieved from the beach rock. The microbial diversity of the dominant mat was accompanied by high nocturnal nitrogenase activities (as determined by in situ acetylene reduction assays). A new DGGE nifH gene optimization approach for cyanobacterial nitrogen fixers showed that the sequences retrieved from the dominant mat were related to nonheterocystous uncultured cyanobacterial phylotypes, only distantly related to sequences of nitrogen-fixing cultured cyanobacteria. These data stress the occurrence and importance of nonheterocystous epilithic cyanobacteria, and it is hypothesized that such epilithic cyanobacteria are the principal nitrogen fixers of the Heron Island beach rock.     

DNA-SIP Reveals That Syntrophaceae Play an Important Role in Methanogenic Hexadecane Degradation

The methanogenic degradation of linear alkanes is a common process in oil-impacted environments. However, little is known about the key players involved in this process. Here, the hexadecane-degrading organisms in a methanogenic, hexadecane-degrading consortium designated M82 obtained from Shengli oilfield and maintained at 35°C for over 4 years, were identified by DNA-stable isotope probing with UL-¹³C-hexadecane, followed by density-resolved terminal restriction fragment length polymorphism (T-RFLP) analysis, cloning and phylogenetic analysis of 16S rRNA gene fragments. Compared to the fractions of the ¹²C treatment, the relative abundance of two phylotypes significantly increased in the heavy fractions of the ¹³C-hexadecane incubated microcosm. One belongs to a uncultured member of the bacterial family Syntrophaceae, which show 95–97% rRNA sequence identity with Smithella propionica, and the other is affiliated with Methanoculleus receptaculi (>99% sequence identity). The results of the present study prove the significant role of uncultured Syntrophaceae in degradation of hexadecane, probably through syntrophic interactions with hydrogenotrophic methanogens.     

Identification of Cyanophage Ma-LBP and Infection of the Cyanobacterium Microcystis aeruginosa from an Australian Subtropical Lake by the Virus

Viruses can control the structure of bacterial communities in aquatic environments. The aim of this project was to determine if cyanophages (viruses specific to cyanobacteria) could exert a controlling influence on the abundance of the potentially toxic cyanobacterium Microcystis aeruginosa (host). M. aeruginosa was isolated, cultured, and characterized from a subtropical monomictic lake—Lake Baroon, Sunshine Coast, Queensland, Australia. The viral communities in the lake were separated from cyanobacterial grazers by filtration and chloroform washing. The natural lake viral cocktail was incubated with the M. aeruginosa host growing under optimal light and nutrient conditions. The specific growth rate of the host was 0.023 h⁻¹; generation time, 30.2 h. Within 6 days, the host abundance decreased by 95%. The density of the cyanophage was positively correlated with the rate of M. aeruginosa cell lysis (r² = 0.95). The cyanophage replication time was 11.2 h, with an average burst size of 28 viral particles per host cell. However, in 3 weeks, the cultured host community recovered, possibly because the host developed resistance (immunity) to the cyanophage. The multiplicity of infection was determined to be 2,890 virus-like particles/cultured host cell, using an undiluted lake viral population. Transmission electron microscopy showed that two types of virus were likely controlling the host cyanobacterial abundance. Both viruses displayed T7-like morphology and belonged to the Podoviridiae group (short tails) of viruses that we called cyanophage Ma-LBP. In Lake Baroon, the number of the cyanophage Ma-LBP was 5.6 × 10⁴ cyanophage·ml⁻¹, representing 0.23% of the natural viral population of 2.46 × 10⁷·ml⁻¹. Our results showed that this cyanophage could be a major natural control mechanism of M. aeruginosa abundance in aquatic ecosystems like Lake Baroon. Future studies of potentially toxic cyanobacterial blooms need to consider factors that influence cyanophage attachment, infectivity, and lysis of their host alongside the physical and chemical parameters that drive cyanobacterial growth and production.     

Soil Bacterial Community Shift Correlated with Change from Forest to Pasture Vegetation in a Tropical Soil

The change in vegetative cover of a Hawaiian soil from forest to pasture led to significant changes in the composition of the soil bacterial community. DNAs were extracted from both soil habitats and compared for the abundance of guanine-plus-cytosine (G+C) content, by analysis of abundance of phylotypes of small-subunit ribosomal DNA (SSU rDNA) amplified from fractions with 63 and 35% G+C contents, and by phylogenetic analysis of the dominant rDNA clones in the 63% G+C content fraction. All three methods showed differences between the forest and pasture habitats, providing evidence that vegetation had a strong influence on microbial community composition at three levels of taxon resolution. The forest soil DNA had a peak in G+C content of 61%, while the DNA of the pasture soil had a peak in G+C content of 67%. None of the dominant phylotypes found in the forest soil were detected in the pasture soil. For the 63% G+C fraction SSU rDNA sequence analysis of the three most dominant members revealed that their phyla changed from Fibrobacter and Syntrophomonas assemblages in the forest soil to Burkholderia and Rhizobium–Agrobacterium assemblages in the pasture soil.     

Distribution of Crenarchaeota Representatives in Terrestrial Hot Springs of Russia and Iceland

Culture-independent (PCR with Crenarchaeota-specific primers and subsequent denaturing gradient gel electrophoresis) and culture-dependent approaches were used to study the diversity of Crenarchaeota in terrestrial hot springs of the Kamchatka Peninsula and the Lake Baikal region (Russia) and of Iceland. Among the phylotypes detected there were relatives of both cultured (mainly hyperthermophilic) and uncultured Crenarchaeota. It was found that there is a large and diverse group of uncultured Crenarchaeota that inhabit terrestrial hot springs with moderate temperatures (55 to 70°C). Two of the lineages of this group were given phenotypic characterization, one as a result of cultivation in an enrichment culture and another one after isolation of a pure culture, “Fervidococcus fontis,” which proved to be a moderately thermophilic, neutrophilic (optimum pH of 6.0 to 7.5), anaerobic organotroph.     

Global distribution and vertical patterns of a prymnesiophyte–cyanobacteria obligate symbiosis

A marine symbiosis has been recently discovered between prymnesiophyte species and the unicellular diazotrophic cyanobacterium UCYN-A. At least two different UCYN-A phylotypes exist, the clade UCYN-A1 in symbiosis with an uncultured small prymnesiophyte and the clade UCYN-A2 in symbiosis with the larger Braarudosphaera bigelowii. We targeted the prymnesiophyte–UCYN-A1 symbiosis by double CARD-FISH (catalyzed reporter deposition-fluorescence in situ hybridization) and analyzed its abundance in surface samples from the MALASPINA circumnavigation expedition. Our use of a specific probe for the prymnesiophyte partner allowed us to verify that this algal species virtually always carried the UCYN-A symbiont, indicating that the association was also obligate for the host. The prymnesiophyte–UCYN-A1 symbiosis was detected in all ocean basins, displaying a patchy distribution with abundances (up to 500 cells ml⁻¹) that could vary orders of magnitude. Additional vertical profiles taken at the NE Atlantic showed that this symbiosis occupied the upper water column and disappeared towards the Deep Chlorophyll Maximum, where the biomass of the prymnesiophyte assemblage peaked. Moreover, sequences of both prymnesiophyte partners were searched within a large 18S rDNA metabarcoding data set from the Tara-Oceans expedition around the world. This sequence-based analysis supported the patchy distribution of the UCYN-A1 host observed by CARD-FISH and highlighted an unexpected homogeneous distribution (at low relative abundance) of B. bigelowii in the open ocean. Our results demonstrate that partners are always in symbiosis in nature and show contrasted ecological patterns of the two related lineages.     

Identification, Cloning, and Characterization of a Novel Ketoreductase from the Cyanobacterium Synechococcus sp. Strain PCC 7942

A new ketoreductase useful for asymmetric synthesis of chiral alcohols was identified in the cyanobacterium Synechococcus sp. strain PCC 7942. Mass spectrometry of trypsin-digested peptides identified the protein as 3-ketoacyl-[acyl-carrier-protein] reductase (KR) (EC 1.1.1.100). The gene, referred to as fabG, was cloned, functionally expressed in Escherichia coli, and subsequently purified to homogeneity. The enzyme displayed a temperature optimum at 44°C and a broad pH optimum between pH 7 and pH 9. The NADPH-dependent KR was able to asymmetrically reduce a variety of prochiral ketones with good to excellent enantioselectivities (>99.8%). The KR showed particular high specific activity for asymmetric reduction of ethyl 4-chloroacetoacetate (38.29 ± 2.15 U mg⁻¹) and 2′,3′,4′,5′,6′-pentafluoroacetophenone (8.57 ± 0.49 U mg⁻¹) to the corresponding (S)-alcohols. In comparison with an established industrial enzyme like the alcohol dehydrogenase from Lactobacillus brevis, the KR showed seven-times-higher activity toward 2′,3′,4′,5′,6′-pentafluoroacetophenone, with a remarkably higher enantiomeric excess (>99.8% [S] versus 43.3% [S]).     

Desiccation induced changes in osmolytes production and the antioxidative defence in the cyanobacterium Anabaena sp. PCC 7120

Cells of Anabaena sp. PCC 7120, a low desiccation tolerant cyanobacterium, was subjected to prolonged desiccation and effect of loss of water was examined on production of osmolytes, and antioxidant response as well as on overall viability in terms of photosynthetic activity. During dehydration (22 h), the organism maintained about 98.5 % loss of cellular water, yet cells remained viable as about 30 % of photosynthetic O₂-evolution activity resumed upon hydrating (1 h) such cells. In desiccated state, cyanobacterial cells accumulated osmolytes within 1 h though their contents decreased thereafter. The highest levels of trehalose (179 nmol mg⁻¹ protein), sucrose (805 nmol mg⁻¹ protein) and proline (23.2 nmol mg⁻¹ protein) were attained within 1 h. Chlorophyll a and carotenoid contents also increased within 1 h but phycocyanin level showed opposite trend. The oxygen-evolving activity declined in desiccated cyanobacterial biomass while rehydration led to instant recovery, indicating that cells protect the photosynthetic machinery against desiccation. Notwithstanding, activities of antioxidant enzymes (catalase, peroxidase and superoxide dismutase) attained their peaks after 3 h of desiccation, though within 10 min of rehydration, their levels returned back close to basal activities of the cultured cells. We propose that onset of osmolyte production in conjunction with upshift of antioxidant enzymes apparently protects the cyanobacterial cells from desiccation stress.     

Nitrogen Fuelling of the Pelagic Food Web of the Tropical Atlantic

We estimated the relative contribution of atmosphere (ic Nitrogen (N) input (wet and dry deposition and N fixation) to the epipelagic food web by measuring N isotopes of different functional groups of epipelagic zooplankton along 23°W (17°N-4°S) and 18°N (20-24°W) in the Eastern Tropical Atlantic. Results were related to water column observations of nutrient distribution and vertical diffusive flux as well as colony abundance of Trichodesmium obtained with an Underwater Vision Profiler (UVP5). The thickness and depth of the nitracline and phosphocline proved to be significant predictors of zooplankton stable N isotope values. Atmospheric N input was highest (61% of total N) in the strongly stratified and oligotrophic region between 3 and 7°N, which featured very high depth-integrated Trichodesmium abundance (up to 9.4×10⁴ colonies m^-2), strong thermohaline stratification and low zooplankton δ¹⁵N (~2‰). Relative atmospheric N input was lowest south of the equatorial upwelling between 3 and 5°S (27%). Values in the Guinea Dome region and north of Cape Verde ranged between 45 and 50%, respectively. The microstructure-derived estimate of the vertical diffusive N flux in the equatorial region was about one order of magnitude higher than in any other area (approximately 8 mmol m^-2 d ¹). At the same time, this region received considerable atmospheric N input (35% of total). In general, zooplankton δ¹⁵N and Trichodesmium abundance were closely correlated, indicating that N fixation is the major source of atmospheric N input. Although Trichodesmium is not the only N fixing organism, its abundance can be used with high confidence to estimate the relative atmospheric N input in the tropical Atlantic (r² = 0.95). Estimates of absolute N fixation rates are two- to tenfold higher than incubation-derived rates reported for the same regions. Our approach integrates over large spatial and temporal scales and also quantifies fixed N released as dissolved inorganic and organic N. In a global analysis, it may thus help to close the gap in oceanic N budgets.     

Nitrate Transport and Not Photoinhibition Limits Growth of the Freshwater Cyanobacterium Synechococcus Species PCC 6301 at Low Temperature

The effect of low temperature on cell growth, photosynthesis, photoinhibition, and nitrate assimilation was examined in the cyanobacterium Synechococcus sp. PCC 6301 to determine the factor that limits growth. Synechococcus sp. PCC 6301 grew exponentially between 20°C and 38°C, the growth rate decreased with decreasing temperature, and growth ceased at 15°C. The rate of photosynthetic oxygen evolution decreased more slowly with temperature than the growth rate, and more than 20% of the activity at 38°C remained at 15°C. Oxygen evolution was rapidly inactivated at high light intensity (3 mE m⁻² s⁻¹) at 15°C. Little or no loss of oxygen evolution was observed under the normal light intensity (250 μE m⁻² s⁻¹) for growth at 15°C. The decrease in the rate of nitrate consumption by cells as a function of temperature was similar to the decrease in the growth rate. Cells could not actively take up nitrate or nitrite at 15°C, although nitrate reductase and nitrite reductase were still active. These data demonstrate that growth at low temperature is not limited by a decrease in the rate of photosynthetic electron transport or by photoinhibition, but that inactivation of the nitrate/nitrite transporter limits growth at low temperature.