Phylogenetic Patterns in the Microbial Response to Resource Availability: Amino Acid Incorporation in San Francisco Bay

<div><p>Aquatic microorganisms are typically identified as either oligotrophic or copiotrophic, representing trophic strategies adapted to low or high nutrient concentrations, respectively. Here, we sought to take steps towards identifying these and additional adaptations to nutrient availability with a quantitative analysis of microbial resource use in mixed communities. We incubated an estuarine microbial community with stable isotope labeled amino acids (AAs) at concentrations spanning three orders of magnitude, followed by taxon-specific quantitation of isotopic incorporation using NanoSIMS analysis of high-density microarrays. The resulting data revealed that trophic response to AA availability falls along a continuum between copiotrophy and oligotrophy, and high and low activity. To illustrate strategies along this continuum more simply, we statistically categorized microbial taxa among three trophic types, based on their incorporation responses to increasing resource concentration. The data indicated that taxa with copiotrophic-like resource use were not necessarily the most active, and taxa with oligotrophic-like resource use were not always the least active. Two of the trophic strategies were not randomly distributed throughout a 16S rDNA phylogeny, suggesting they are under selective pressure in this ecosystem and that a link exists between evolutionary relatedness and substrate affinity. The diversity of strategies to adapt to differences in resource availability highlights the need to expand our understanding of microbial interactions with organic matter in order to better predict microbial responses to a changing environment.</p></div

Ternary plot graphically depicting the ratios of the rRNA phylotype-specific incorporation to varying AA concentrations added to SF Bay water.

<p>Data are color-coded according to the trophic strategies identified in Fig. 3. The position of each data point in relation to the three corners represents the relative contribution of each concentration response.</p

Amino acid incorporation trophic strategies mapped onto a maximum parsimony unrooted 16S rRNA gene phylogeny of taxa from a SF Bay seawater sample.

<p>Ancestral states were identified by parsimony. Asterisks indicate strategies with a statistically clustered distribution indicating a phylogenetic signal.</p

Pairwise comparisons of isotopic incorporation of ¹⁵N labeled AAs by 107 16S rRNA phylotypes from SF Bay at two concentrations (high, 5 micromolar and low, 50 nanomolar).

<p>Each data point represents the HCE (hybridization corrected enrichment) for a probe set (the slope of delta permil divided by fluorescence). Error bars indicate two standard errors of the slope calculation. The black line represents the linear regression and the blue the 1 to l line.</p

High Initial Sputter Rate Found for Vaccinia Virions Using Isotopic Labeling, NanoSIMS, and AFM

High-lateral-resolution secondary ion mass spectrometry (SIMS) has the potential to provide functional and depth resolved information from small biological structures, such as viral particles (virions) and phage, but sputter rate and sensitivity are not characterized at shallow depths relevant to these structures. Here we combine stable isotope labeling of the DNA of vaccinia virions with correlated SIMS imaging depth profiling and atomic force microscopy (AFM) to develop a nonlinear, nonequilibrium sputter rate model for the virions and validate the model on the basis of reconstructing the location of the DNA within individual virions. Our experiments with a Cs⁺ beam show an unexpectedly high initial sputter rate (∼100 um²·nm·pA^–1·s^–1) with a rapid decline to an asymptotic rate of 0.7 um²·nm·pA^–1·s^–1 at an approximate depth of 70 nm. Correlated experiments were also conducted with glutaraldehyde-fixed virions, as well as O^– and Ga⁺ beams, yielding similar results. Based on our Cs⁺ sputter rate model, the labeled DNA in the virion was between 50 and 90 nm depth in the virion core, consistent with expectations, supporting our conclusions. Virion densification was found to be a secondary effect. Accurate isotopic ratios were obtained from the initiation of sputtering, suggesting that isotopic tracers could be successfully used for smaller virions and phage

Population growth rates.

<p>Population growth estimates of Chinook salmon (<i>Oncorhynchus tshawytscha</i>) on the Mokelumne River watershed from cohort reconstruction. Apparent growth rate estimates (open circles) show several years where cohort replacement values exceed 1 (solid line). Natural population growth rates (filled circles) remove the influence of immigration from hatchery fish. These results suggest that in-river populations are being supported by hatchery sources.</p

Frequency distribution of δ³⁴S values in salmon otoliths.

<p>Histogram of otolith δ³⁴S for the juvenile rearing portion of otoliths from unknown origin adult Chinook salmon (<i>Oncorhynchus tshawytscha</i>) spawning in-river on the Mokelumne River (USA). Fish assigned to natural origin (grey bars; N = 87) had δ³⁴S values less than 6‰ (dashed line) and did not overlap with δ³⁴S values from those identified as originating from a hatchery (open bars; N = 10). These results indicate that 90% of in-river spawners were produced in a hatchery.</p

Map of study region.

<p>The Mokelumne River and Mokelumne River Fish Hatchery in relation to the western United States, and the Sacramento-San Joaquin river system (shaded region) in California's Central Valley (insert). The in-river spawning habitat on the Mokelumne River consists of the area between its confluence with the Cosumnes River and upstream to the Camanche Dam (∼16 km).</p

Adult Chinook salmon population trend.

<p>Stacked bar graph of the total number of adult fall-run Chinook salmon (<i>Oncorhynchus tshawytscha</i>) spawning on the Mokelumne River (black bars), and in the Mokelumne River Fish Hatchery (grey bars) from 1940–2009 (East Bay Municipal Utility District, unpublished data). Graph shows adult spawning location and not rearing origin. Note: Mokelumne River Fish Hatchery was built in 1964.</p

Estimate of hatchery and natural-origin salmon.

<p>Proportion of adult Chinook salmon (<i>Oncorhynchus tshawytscha</i>) spawning in-river on the Mokelumne River (USA) or within the entire Mokelumne River watershed (river+hatchery) assigned to hatchery or natural origins based on δ³⁴S values in otoliths. Laplace point estimates and 95% Confidence Intervals (CI) were calculated using the Adjusted Wald estimate modified for small sample sizes.</p