Anitra E. Ingalls

Interaction and signalling between a cosmopolitan phytoplankton and associated bacteria

Interactions between primary producers and bacteria impact the physiology of both partners, alter the chemistry of their environment, and shape ecosystem diversity. In marine ecosystems, these interactions are difficult to study partly because the major photosynthetic organisms are microscopic, unicellular phytoplankton. Coastal phytoplankton communities are dominated by diatoms, which generate approximately 40% of marine primary production and form the base of many marine food webs. Diatoms co-occur with specific bacterial taxa, but the mechanisms of potential interactions are mostly unknown. Here we tease apart a bacterial consortium associated with a globally distributed diatom and find that a Sulfitobacter species promotes diatom cell division via secretion of the hormone indole-3-acetic acid, synthesized by the bacterium using both diatom-secreted and endogenous tryptophan. Indole-3-acetic acid and tryptophan serve as signalling molecules that are part of a complex exchange of nutrients, including diatom-excreted organosulfur molecules and bacterial-excreted ammonia. The potential prevalence of this mode of signalling in the oceans is corroborated by metabolite and metatranscriptome analyses that show widespread indole-3-acetic acid production by Sulfitobacter-related bacteria, particularly in coastal environments. Our study expands on the emerging recognition that marine microbial communities are part of tightly connected networks by providing evidence that these interactions are mediated through production and exchange of infochemicals.

Nonmarine Crenarchaeol in Nevada Hot Springs

ABSTRACT Glycerol dialkyl glycerol tetraethers (GDGTs) are core membrane lipids of the Crenarchaeota. The structurally unusual GDGT crenarchaeol has been proposed as a taxonomically specific biomarker for the marine planktonic group I archaea. It is found ubiquitously in the marine water column and in sediments. In this work, samples of microbial community biomass were obtained from several alkaline and neutral-pH hot springs in Nevada, United States. Lipid extracts of these samples were analyzed by high-performance liquid chromatography-mass spectrometry and by gas chromatography-mass spectrometry. Each sample contained GDGTs, and among these compounds was crenarchaeol. The distribution of archaeal lipids in Nevada hot springs did not appear to correlate with temperature, as has been observed in the marine environment. Instead, a significant correlation with the concentration of bicarbonate was observed. Archaeal DNA was analyzed by denaturing gradient gel electrophoresis. All samples contained 16S rRNA gene sequences which were more strongly related to thermophilic crenarchaeota than to Cenarchaeum symbiosum, a marine nonthermophilic crenarchaeon. The occurrence of crenarchaeol in environments containing sequences affiliated with thermophilic crenarchaeota suggests a wide phenotypic distribution of this compound. The results also indicate that crenarchaeol can no longer be considered an exclusive biomarker for marine species.

The role of biominerals in the sinking flux and preservation of amino acids in the Southern Ocean along 170°W

Abstract In a study of the transport and transformation of particulate amino acids in the Southern Ocean, we found that silicate and calcium carbonate biominerals play an important role in the preservation of amino acids throughout the water column and surface sediments. Plankton, sinking particle and sediment samples were collected during the USJGOFS AESOPS transect across the Antarctic Polar Front (APF) along 170°W. Total hydrolyzable amino acids (THAA) made up of 17–27% of total organic carbon (Corg) in sinking particles and 6–23% of Corg in surface sediments. In addition to THAA, we measured amino acids bound in silicate (SiTHAA) and calcium carbonate (CaTHAA) biominerals. Although the fraction of biomineral bound to total amino acids in plankton was small, Amino acid composition has been used to develop a Degradation Index (sensu Dauwe and Middelburg, 1998). The unusual amino acid composition of Southern Ocean plankton, i.e., dominated by diatom cell walls, resulted in an apparent mismatch between the absolute value of the Degradation Index and the presumed extent of degradation. However, changes in amino acid composition that accompanied degradation were similar to those found in previous studies. Principal components analysis suggests that the greatest change in THAA composition occurred between the sediment surface floc layer and deeper sediments where particles had the longest residence time. Compositional changes observed in the water column suggested that degradation processes resulted in complete removal of amino acids, whereas changes in sediments were consistent both with selective degradation of plankton amino acids with depth and with the conversion of primarily phytoplankton biomass to that of bacterial biomass.

Marine ammonia-oxidizing archaeal isolates display obligate mixotrophy and wide ecotypic variation

Significance Ammonia-oxidizing archaea (AOA) influence the form and availability of nitrogen in marine environments and are a major contributor to N2O release and plausible indirect source of methane in the upper ocean. Thus, their sensitivity to ocean acidification and other physicochemical changes associated with climate change has global significance. Here, we report on the physiological response of marine AOA isolates to key environmental variables. Although reported as highly sensitive to reduction in ocean pH, we now show that some coastal marine AOA can remain active with increasing acidification of the oceans. All AOA isolates assimilate fixed carbon and two are obligate mixotrophs, suggesting this globally significant assemblage serves a significant function in coupling chemolithotrophy with organic matter assimilation in marine food webs. Ammonia-oxidizing archaea (AOA) are now implicated in exerting significant control over the form and availability of reactive nitrogen species in marine environments. Detailed studies of specific metabolic traits and physicochemical factors controlling their activities and distribution have not been well constrained in part due to the scarcity of isolated AOA strains. Here, we report the isolation of two new coastal marine AOA, strains PS0 and HCA1. Comparison of the new strains to Nitrosopumilus maritimus strain SCM1, the only marine AOA in pure culture thus far, demonstrated distinct adaptations to pH, salinity, organic carbon, temperature, and light. Strain PS0 sustained nearly 80% of ammonia oxidation activity at a pH as low as 5.9, indicating that coastal strains may be less sensitive to the ongoing reduction in ocean pH. Notably, the two novel isolates are obligate mixotrophs that rely on uptake and assimilation of organic carbon compounds, suggesting a direct coupling between chemolithotrophy and organic matter assimilation in marine food webs. All three isolates showed only minor photoinhibition at 15 µE⋅m−2⋅s−1 and rapid recovery of ammonia oxidation in the dark, consistent with an AOA contribution to the primary nitrite maximum and the plausibility of a diurnal cycle of archaeal ammonia oxidation activity in the euphotic zone. Together, these findings highlight an unexpected adaptive capacity within closely related marine group I Archaea and provide new understanding of the physiological basis of the remarkable ecological success reflected by their generally high abundance in marine environments.

Holocene Southern Ocean surface temperature variability west of the Antarctic Peninsula

The disintegration of ice shelves, reduced sea-ice and glacier extent, and shifting ecological zones observed around Antarctica highlight the impact of recent atmospheric and oceanic warming on the cryosphere. Observations and models suggest that oceanic and atmospheric temperature variations at Antarcticas margins affect global cryosphere stability, ocean circulation, sea levels and carbon cycling. In particular, recent climate changes on the Antarctic Peninsula have been dramatic, yet the Holocene climate variability of this region is largely unknown, limiting our ability to evaluate ongoing changes within the context of historical variability and underlying forcing mechanisms. Here we show that surface ocean temperatures at the continental margin of the western Antarctic Peninsula cooled by 3–4 °C over the past 12,000 years, tracking the Holocene decline of local (65° S) spring insolation. Our results, based on TEX86 sea surface temperature (SST) proxy evidence from a marine sediment core, indicate the importance of regional summer duration as a driver of Antarctic seasonal sea-ice fluctuations. On millennial timescales, abrupt SST fluctuations of 2–4 °C coincide with globally recognized climate variability. Similarities between our SSTs, Southern Hemisphere westerly wind reconstructions and El Niño/Southern Oscillation variability indicate that present climate teleconnections between the tropical Pacific Ocean and the western Antarctic Peninsula strengthened late in the Holocene epoch. We conclude that during the Holocene, Southern Ocean temperatures at the western Antarctic Peninsula margin were tied to changes in the position of the westerlies, which have a critical role in global carbon cycling.

The radiocarbon signature of microorganisms in the mesopelagic ocean

Several lines of evidence indicate that microorganisms in the meso- and bathypelagic ocean are metabolically active and respiring carbon. In addition, growing evidence suggests that archaea are fixing inorganic carbon in this environment. However, direct quantification of the contribution from deep ocean carbon sources to community production in the dark ocean remains a challenge. In this study, carbon flow through the microbial community at 2 depths in the mesopelagic zone of the North Pacific Subtropical Gyre was examined by exploiting the unique radiocarbon signatures (Δ14C) of the 3 major carbon sources in this environment. The radiocarbon content of nucleic acids, a biomarker for viable cells, isolated from size-fractionated particles (0.2–0.5 μm and >0.5 μm) showed the direct incorporation of carbon delivered by rapidly sinking particles. Most significantly, at the 2 mesopelagic depths examined (670 m and 915 m), carbon derived from in situ autotrophic fixation supported a significant fraction of the free-living microbial community (0.2–0.5 μm size fraction), but the contribution of chemoautotrophy varied markedly between the 2 depths. Results further showed that utilization of the oceans largest reduced carbon reservoir, 14C-depleted, dissolved organic carbon, was negligible in this environment. This isotopic portrait of carbon assimilation by the in situ, free-living microbial community, integrated over >50,000 L of seawater, implies that recent, photosynthetic carbon is not always the major carbon source supporting microbial community production in the mesopelagic realm.

Ammonia oxidation kinetics and temperature sensitivity of a natural marine community dominated by Archaea

Archaeal ammonia oxidizers (AOAs) are increasingly recognized as prominent members of natural microbial assemblages. Evidence that links the presence of AOA with in situ ammonia oxidation activity is limited, and the abiotic factors that regulate the distribution of AOA natural assemblages are not well defined. We used quantitative PCR to enumerate amoA (encodes α-subunit of ammonia monooxygenase) abundances; AOA amoA gene copies greatly outnumbered ammonia-oxidizing bacteria and amoA transcripts were derived primarily from AOA throughout the water column of Hood Canal, Puget Sound, WA, USA. We generated a Michaelis–Menten kinetics curve for ammonia oxidation by the natural community and found that the measured Km of 98±14 nmol l−1 was close to that for cultivated AOA representative Nitrosopumilus maritimus SCM1. Temperature did not have a significant effect on ammonia oxidation rates for incubation temperatures ranging from 8 to 20 °C, which is within the temperature range for depths of measurable ammonia oxidation at the site. This study provides substantial evidence, through both amoA gene copies and transcript abundances and the kinetics response, that AOA are the dominant active ammonia oxidizers in this marine environment. We propose that future ammonia oxidation experiments use a Km for the natural community to better constrain ammonia oxidation rates determined with the commonly used 15NH4+ dilution technique.

Preservation of organic matter in mound-forming coral skeletons

This study demonstrates that intracrystalline organic matter in coral skeletons is well preserved over century timescales. The extent of preservation of organic matter in coral skeletons was investigated by measuring total organic carbon (TOC), total hydrolyzable amino acid (THAA), chloropigment, and lipid concentrations in 0–300 year old annual growth bands from Montastraea annularis (Florida Keys) and Porites lutea (Red Sea). Organic matter intrinsic to the calcium carbonate mineral (intracrystalline) was analyzed separately from total skeletal organic matter. The Red Sea coral had less TOC (0.02–0.04 wt%) than the Florida Keys coral (0.04–0.11 wt%), but a higher percent of intracrystalline organic matter in all annual bands measured. Carbon in the form of THAA, most likely from mineral-precipitating proteins, contributed 30–45% of the TOC in both corals. Carbon in lipids represented about 3% of the TOC in the coral skeletons. Chlorophyll-a and b were present in annual bands where endolithic algae were present, but these compounds were minor contributors to TOC. The distribution of specific organic compounds showed that organic matter was well preserved throughout the time period sampled in both the total and intracrystalline pools. Variations in THAA were not correlated with TOC over time, suggesting that organic matter that is involved in biomineralization, like amino acids, may be deposited in response to different environmental factors than are other components of skeletal organic matter. Differences in the quantity and composition of organic matter between the two corals investigated here were assessed using principal components analysis and suggest that location, species and skeletal structure may all influence organic matter content and possibly the degree of physical protection of organic matter by the coral skeleton. Further, our study suggests that intracrystalline organic matter may be better protected from diagenesis than non-intracrystalline organic matter and may therefore be a more reliable source of organic matter for paleoceanographic studies than total skeletal organic matter.

The production of nitric oxide by marine ammonia-oxidizing archaea and inhibition of archaeal ammonia oxidation by a nitric oxide scavenger

Nitrification is a critical process for the balance of reduced and oxidized nitrogen pools in nature, linking mineralization to the nitrogen loss processes of denitrification and anammox. Recent studies indicate a significant contribution of ammonia-oxidizing archaea (AOA) to nitrification. However, quantification of the relative contributions of AOA and ammonia-oxidizing bacteria (AOB) to in situ ammonia oxidation remains challenging. We show here the production of nitric oxide (NO) by Nitrosopumilus maritimus SCM1. Activity of SCM1 was always associated with the release of NO with quasi-steady state concentrations between 0.05 and 0.08 μM. NO production and metabolic activity were inhibited by the nitrogen free radical scavenger 2-phenyl-4,4,5,5,-tetramethylimidazoline-1-oxyl-3-oxide (PTIO). Comparison of marine and terrestrial AOB strains with SCM1 and the recently isolated marine AOA strain HCA1 demonstrated a differential sensitivity of AOB and AOA to PTIO and allylthiourea (ATU). Similar to the investigated AOA strains, bulk water column nitrification at coastal and open ocean sites with sub-micromolar ammonia/ammonium concentrations was inhibited by PTIO and insensitive to ATU. These experiments support predictions from kinetic, molecular and biogeochemical studies, indicating that marine nitrification at low ammonia/ammonium concentrations is largely driven by archaea and suggest an important role of NO in the archaeal metabolism.

An interlaboratory study of TEX86 and BIT analysis of sediments, extracts, and standard mixtures

Two commonly used proxies based on the distribution of glycerol dialkyl glycerol tetraethers (GDGTs) are the TEX86 (TetraEther indeX of 86 carbon atoms) paleothermometer for sea surface temperature reconstructions and the BIT (Branched Isoprenoid Tetraether) index for reconstructing soil organic matter input to the ocean. An initial round-robin study of two sediment extracts, in which 15 laboratories participated, showed relatively consistent TEX86 values (reproducibility +/- 3-4 degrees C when translated to temperature) but a large spread in BIT measurements (reproducibility +/- 0.41 on a scale of 0-1). Here we report results of a second round-robin study with 35 laboratories in which three sediments, one sediment extract, and two mixtures of pure, isolated GDGTs were analyzed. The results for TEX86 and BIT index showed improvement compared to the previous round-robin study. The reproducibility, indicating interlaboratory variation, of TEX86 values ranged from 1.3 to 3.0 degrees C when translated to temperature. These results are similar to those of other temperature proxies used in paleoceanography. Comparison of the results obtained from one of the three sediments showed that TEX86 and BIT indices are not significantly affected by interlaboratory differences in sediment extraction techniques. BIT values of the sediments and extracts were at the extremes of the index with values close to 0 or 1, and showed good reproducibility (ranging from 0.013 to 0.042). However, the measured BIT values for the two GDGT mixtures, with known molar ratios of crenarchaeol and branched GDGTs, had intermediate BIT values and showed poor reproducibility and a large overestimation of the true (i.e., molar-based) BIT index. The latter is likely due to, among other factors, the higher mass spectrometric response of branched GDGTs compared to crenarchaeol, which also varies among mass spectrometers. Correction for this different mass spectrometric response showed a considerable improvement in the reproducibility of BIT index measurements among laboratories, as well as a substantially improved estimation of molar-based BIT values. This suggests that standard mixtures should be used in order to obtain consistent, and molar-based, BIT values.