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

The Role of Viral Population Diversity in Adaptation of Bovine Coronavirus to New Host Environments

<div><p>The high mutation rate of RNA viruses enables a diverse genetic population of viral genotypes to exist within a single infected host. In-host genetic diversity could better position the virus population to respond and adapt to a diverse array of selective pressures such as host-switching events. Multiple new coronaviruses, including SARS, have been identified in human samples just within the last ten years, demonstrating the potential of coronaviruses as emergent human pathogens. Deep sequencing was used to characterize genomic changes in coronavirus quasispecies during simulated host-switching. Three bovine nasal samples infected with bovine coronavirus were used to infect human and bovine macrophage and lung cell lines. The virus reproduced relatively well in macrophages, but the lung cell lines were not infected efficiently enough to allow passage of non lab-adapted samples. Approximately 12 kb of the genome was amplified before and after passage and sequenced at average coverages of nearly 950×(454 sequencing) and 38,000×(Illumina). The consensus sequence of many of the passaged samples had a 12 nucleotide insert in the consensus sequence of the spike gene, and multiple point mutations were associated with the presence of the insert. Deep sequencing revealed that the insert was present but very rare in the unpassaged samples and could quickly shift to dominate the population when placed in a different environment. The insert coded for three arginine residues, occurred in a region associated with fusion entry into host cells, and may allow infection of new cell types via heparin sulfate binding. Analysis of the deep sequencing data indicated that two distinct genotypes circulated at different frequency levels in each sample, and support the hypothesis that the mutations present in passaged strains were “selected” from a pre-existing pool rather than through de novo mutation and subsequent population fixation.</p> </div

Structural model of the receptor binding domain from the BCoV passaged sample.

<p>Ribbons representation (left) and hydrophobic surface (right). Colors of the hydrophobic surface range from blue for the most polar residues to white to orange red for the most hydrophobic residues. Positions of non-synonymous mutations are colored in green (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052752#pone-0052752-t002" target="_blank">Table 2</a>).</p

Non-synonymous mutations associated with cell passage.

<p>UP = unpassaged, P = passaged, AA = amino acid, (ts) = transition, (tv) = transversion. Mutations located in N-terminal receptor binding domain in the spike protein (see <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052752#pone-0052752-g003" target="_blank">Figure 3</a>) are in italics.</p

Multiple sequence alignment of BCoV insert region compared with that of other coronaviruses.

<p>Sequence region (894–927) including insert SRRR from the passaged BCoV (B2.27.BO.P1) was aligned with other coronavirus spike protein amino acid sequences. The inserted amino acids are underlined; amino acids conserved among coronavirus species are in blue text; RR motifs observed in other sequences are in red. In green is highlighted 793-KPTKR-797 region in SARS-CoV S2 domain described as a furin cleavage site allowed trypsin-independent cell-cell fusion <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052752#pone.0052752-Belouzard1" target="_blank">[<b>23</b>]</a>.</p

Percent of consensus SNPs that occur as subconsensus variants in unpassaged samples.

<p>The consensus sequence for each unpassaged parent sample was compared to its passaged descendants. For each sample that clustered away from the unpassaged parent (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0052752#pone-0052752-g001" target="_blank">Figure 1</a>), the percentage of passaged consensus SNPs that were present as variants in the unpassaged sample is shown on the y- axis. The x-axis shows the name of the passaged descendant identified by sample (1, 27, or 59), host cell type (BO, THP or HRT) and passage number (1, 4 or 5).</p

Percentage of 454 reads containing the multibasic insert (Illumina data in parentheses).

<p>Columns are for the different samples: 27, 59, 1, and Nebraska strain. Samples are labeled using cell type, and passage number. na: sample data not available.</p