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Major advances in understanding the diversity, distribution, and activity of marine N2-fixing microorganisms (diazotrophs) have been made in the past decades, however, large gaps in knowledge remain about the environmental controls on growth and mortality rates. In order to measure diazotroph net growth rates and microzooplankton grazing rates on diazotrophs, nutrient perturbation experiments and dilution grazing experiments were conducted using free-floating in situ incubation arrays in the vicinity of Station ALOHA in March 2016. Net growth rates for targeted diazotroph taxa as well as Prochlorococcus, Synechococcus and photosynthetic picoeukaryotes were determined under high (H) and low (L) nitrate:phosphate (NP) ratio conditions at four depths in the photic zone (25, 45, 75, and 100 m) using quantitative PCR and flow cytometry. Changes in the prokaryote community composition in response to HNP and LNP treatments were characterized using 16S rRNA variable region tag sequencing. Microzooplankton grazing rates on diazotrophs were measured using a modified dilution technique at two depths in the photic zone (15 and 125 m). Net growth rates for most of the targeted diazotrophs after 48 h were not stimulated as expected by LNP conditions, rather enhanced growth rates were often measured in HNP treatments. Interestingly, net growth rates of the uncultivated prymnesiophyte symbiont UCYN-A1 were stimulated in HNP treatments at 75 and 100 m, suggesting that N used for growth was acquired through continuing to fix N2 in the presence of nitrate. Net growth rates for UCYN-A1, UCYN-C, Crocosphaera sp. (UCYN-B) and the diatom symbiont Richelia (associated with Rhizosolenia) were uniformly high at 45 m (up to 1.6 ± 0.5 d-1), implying that all were growing optimally at the onset of the experiment at that depth. Differences in microzooplankton grazing rates on UCYN-A1 and UCYN-C in 15 m waters indicate that the grazer assemblage preyed preferentially on UCYN-A1. Deeper in the water column (125 m), both diazotrophs were grazed at substantial rates, suggesting grazing pressure may increase with depth in the photic zone. Constraining in situ diazotroph growth and mortality rates are important steps for improving parameterization for diazotrophs in global ecosystem models.

The VAHINE mesocosm experiment, conducted in the low-nutrient low-chlorophyll waters of the Noumea lagoon (coastal New Caledonia) was designed to trace the incorporation of nitrogen (N) fixed by diazotrophs into the food web, using large volume (50 m3) mesocosms. This experiment provided a unique opportunity to study the succession of different N2-fixing microorganisms (diazotrophs) and calculate in situ net growth and mortality rates in response to fertilization with dissolved inorganic phosphate (DIP) over a 23-day period, using quantitative polymerase chain reaction (qPCR) assays targeting widely distributed marine diazotroph lineages. Inside the mesocosms, the most abundant diazotroph was the heterocyst-forming Richelia associated with Rhizosolenia (Het-1) in the first half of the experiment, while unicellular cyanobacterial Group C (UCYN-C) became abundant during the second half of the experiment. Decreasing DIP concentrations following the fertilization event and increasing temperatures were significantly correlated with increasing abundances of UCYN-C. Maximum net growth rates for UCYN-C were calculated to range between 1.23 ± 0.07 and 2.16 ± 0.07 d−1 in the mesocosms, which are among the highest growth rates reported for diazotrophs. Outside the mesocosms in the New Caledonia lagoon, UCYN-C abundances remained low, despite increasing temperatures, suggesting that the microbial community response to the DIP fertilization created conditions favorable for UCYN-C growth inside the mesocosms. Diazotroph community composition analysis using PCR targeting a component of the nitrogenase gene (nifH) verified that diazotrophs targeted in qPCR assays were collectively among the major lineages in the lagoon and mesocosm samples, with the exception of Crocosphaera-like phylotypes, where sequence types not typically seen in the oligotrophic ocean grew in the mesocosms. Maximum net growth and mortality rates for nine diazotroph phylotypes throughout the 23-day experiment were variable between mesocosms, and repeated fluctuations between periods of net growth and mortality were commonly observed. The field population of diazotrophs in the New Caledonian lagoon waters appeared to be dominated by Het-1 over the course of the study period. However, results from both qPCR and PCR analysis indicated a diverse field population of diazotrophs was present in the lagoon at the time of sampling. Two ecotypes of the Braarudosphaera bigelowii symbiont unicellular group A (UCYN-A) were present simultaneously in the lagoon, with the recently described B. bigelowii/UCYN-A2 association present at higher abundances than the B. bigelowii/UCYN-A1 association.

The effects of ocean acidification on nitrogen (N2) fixation rates and on the community composition of N2-fixing microbes (diazotrophs) were examined in coastal waters of the North-Western Mediterranean Sea. Nine experimental mesocosm enclosures of ∼50 m3 each were deployed for 20 days during June–July 2012 in the Bay of Calvi, Corsica, France. Three control mesocosms were maintained under ambient conditions of carbonate chemistry. The remainder were manipulated with CO2 saturated seawater to attain target amendments of pCO2 of 550, 650, 750, 850, 1000 and 1250 μatm. Rates of N2 fixation were elevated up to 10 times relative to control rates (2.00 ± 1.21 nmol L−1d−1) when pCO2 concentrations were >1000 μatm and pHT (total scale)

Summary Mechanisms of dimethyl sulphide (DMS) and methanethiol (MT) production and consumption were determined in moderately hypersaline mats, Guerrero Negro, Mexico. Biological pathways regulated the net flux of DMS and MT as revealed by increases in flux resulting from decreased salinity, increased temperature and the removal of oxygen. Dimethylsulphoniopropionate (DMSP) was not present in these microbial mats and DMS and MT are probably formed by the reaction of photosynthetically produced low-molecular weight organic carbon and biogenic hydrogen sulphide derived from sulphate reduction. These observations provide an alternative to the notion that DMSP or S-containing amino acids are the dominant precursors of DMS in intertidal sediment systems. The major sink for DMS in the microbial mats was biological consumption, whereas photochemical oxidation to dimethylsulphoxide was the major sink for DMS in the overlying water column. Diel flux measurements demonstrated that significantly more DMS is released from the system during the night than during the day. The major consumers of DMS in the presence of oxygen were monooxygenase-utilizing bacteria, whereas under anoxic conditions, DMS was predominantly consumed by sulphate-reducing bacteria and methanethiol was consumed by methanogenic bacteria. Aerobic and anaerobic consumption rates of DMS were nearly identical. Mass balance estimates suggest that the consumption in the water column is likely to be smaller than net the flux from the mats. Volatile organic sulphur compounds are thus indicators of high rates of carbon fixation and sulphate reduction in these laminated sediment ecosystems, and atmospheric sulphur can be generated as a biogenic signature of the microbial mat community.

Acclimation of a marine sediment microbial community to the herbicide 2,4-dichlorophenoxyacetic acid (2,4-D) was examined by comparing its ability to metabolize 2,4-D before and after exposure to the herbicide. The experimental treatments (control: 2-week exposure, seawater only; test: 2-week exposure, seawater plus 100 ppm 2,4-D) took place under simulated in situ conditions, in an incubation chamber, which maintained the physical and chemical gradient structure of the sediment community. The surface of the sediment was exposed to recirculating seawater on a tidal cycle. 2,4-D (100 ppm) was added to the seawater so that its availability to the sediment microbes mimicked the natural situation. Before and after treatment, bacterial abundance, productivity, and transformation of 2,4-D were determined. After 2 weeks, bacterial populations were similar in the 2,4-D treatment and the initial sediment sample, but higher in the seawater-only treatment. Bacterial productivity was higher in both 2-week treatments, compared to productivity measured before treatment, but rates were the same in 2,4-D and seawater treatments. In contrast, 2,4-D transformation rates increased 28% in the 2-week 100-ppm 2,4-D treatment, compared to the average of the seawater control and the initial sample. This increased transformation rate indicates that the sediment community acclimated to the selective pressure of 2,4-D treatment by increasing its ability to utilize this compound as a substrate.

A synthetic 27-base-long oligodeoxyribonucleotide, termed PU1, has been shown to bind to duplex DNA to form a triplex at a single site within the human c-myc P1 promoter. PU1 has been administered to HeLa cells in culture to examine the feasibility of influencing transcription of the c-myc gene in vivo. It is shown that uptake of PU1 into the nucleus of HeLa cells is efficient and that the compound remains intact for at least 4 hr. In nuclei extracted from PU1-treated cells, inhibition of DNase I cleavage is detected within the c-myc P1 promoter at the target site for triplex formation. The inhibition is shown to be both site and oligodeoxyribonucleotide specific. After cellular uptake of PU1, it is shown that steady-state mRNA arising from the c-myc P1 initiation site is selectively reduced relative to total mRNA, relative to mRNA from the alternative c-myc P2 initiation site, and relative to mRNA derived from the beta-actin promoter. Significant mRNA repression is not seen upon treating cells with oligodeoxyribonucleotides that fail to bind to the P1 promoter target. Taken together, these data suggest that triplex formation can occur between an exogenous oligodeoxyribonucleotide and duplex DNA in the nucleus of treated cells.

Light-dependent reduction of p -nitro blue tetrazolium chloride (NBT) and 2,6-dichlorophenol-indophenol (DCPIP) was stimulated by nitrofen (2,4-dichlorophenyl p -nitrophenyl ether) in the presence or absence of the photosensitizer riboflavin. Enhancement of NBT reduction occurred at a concentration as low as 17 μ M nitrofen. Nitrofen had no effect on dark reduction of NBT by dithionite, ascorbate, or reduced phenazine methosulfate. NBT reduction proceeded in solution saturated with either air or nitrogen. Stimulation by nitrofen in the presence or absence of riboflavin also occurred independently of oxygen. Superoxide dismutase (superoxide:superoxide oxidoreductase; EC 1.15.1.1) inhibited light-dependent, aerobic reduction of NBT and DCPIP with and without riboflavin. The nitrogen-stimulated component was also eliminated. Thus, it appears nitrofen can effect reduction of NBT (and DCPIP) anaerobically by transferring electrons directly to NBT (and DCPIP) through an oxygen-independent mechanism or aerobically via superoxide radicals.

A 27-base-long DNA oligonucleotide was designed that binds to duplex DNA at a single site within the 5' end of the human c-myc gene, 115 base pairs upstream from the transcription origin P1. On the basis of the physical properties of its bound complex, it was concluded that the oligonucleotide forms a colinear triplex with the duplex binding site. By means of an in vitro assay system, it was possible to show a correlation between triplex formation at -115 base pairs and repression of c-myc transcription. The possibility is discussed that triplex formation (site-specific RNA binding to a DNA duplex) could serve as the basis for an alternative program of gene control in vivo.

Fructans synthesized from newly formed assimilates accumulate in wheat stems as nonstructural carbohydrates. Experiments performed tested the hypothesis that the fructose moiety from translocated sucrose is used preferentially in biosynthesis of these fructans. Results indicated: (a) a large percentage of labeled sucrose was translocated and unloaded in an unaltered state; and (b) sucrose contributed its fructose moiety to fructan synthesis in stems.