Adaptive radiation by waves of gene transfer leads to fine-scale resource partitioning in marine microbes

Abstract

Adaptive radiations are important drivers of niche filling, since they rapidly adapt a single clade of organisms to ecological opportunities. Although thought to be common for animals and plants, adaptive radiations have remained difficult to document for microbes in the wild. Here we describe a recent adaptive radiation leading to fine-scale ecophysiological differentiation in the degradation of an algal glycan in a clade of closely related marine bacteria. Horizontal gene transfer is the primary driver in the diversification of the pathway leading to several ecophysiologically differentiated Vibrionaceae populations adapted to different physical forms of alginate. Pathway architecture is predictive of function and ecology, underscoring that horizontal gene transfer without extensive regulatory changes can rapidly assemble fully functional pathways in microbes.

Figure 1. Evolutionary history and ecological occurrence of alginate lyases.

(a) Relative timed maximum-likelihood phylogeny of Vibrionaceae populations co-occurring in the same water samples. Species names are assigned if a previously described type strain falls within the population; otherwise, the designation Vibrio sp. is given. (b) Maximum copy number of alginate lyase families within members of a population identified by presence of a single enzymatic domain are represented by coloured rectangles. ND indicates that Alys and Olys were not detected. ND* indicates that while no Alys or Olys were detected in V. alginolyticus isolates from our collection, it is present in other V. alginolyticus isolates from geographically distant locations (Supplementary Fig. 1). (c) Normalized distribution of isolates obtained from algal detritus particles and zooplankton handpicked under a dissecting microscope and phylogenetically categorized by multilocus gene analysis for two seasonal samples. (d,e) Phylogenetic reconciliation (Methods) by comparison of pathway-specific gene trees (Supplementary Figs 6–9) and a timed ‘species' tree showing the history of each of four lyase gene families embedded in the reference species phylogeny: (d) Oal domains PL15 and PL17, and Aly domain PL6; (e) Aly domain PL7. Acquisition represents an independent entry of a subfamily into a clade within our collection. Solid and dashed lines on the phylogenetic tree indicate clades represented in our collection or obtained from Genbank, respectively. Numbers within symbols indicate multiple independent occurrences of the represented event. Within-population HGT and duplication are not depicted. Lowercase Roman numeral i indicates the crown group consisting of seven closely related populations.

Figure 2. Alginate lyase activity is modulated by gene dosage.

Isolates were grown in marine broth with added alginate oligosaccharides to induce enzyme expression. The cells were lysed to determine the total, cell associated alginate lyase activity by measuring the increase of absorption at 235 nm with alginate as enzyme substrate. The alginate lyase activity of each strain was normalized against the optical density of the respective cell culture measured at 600 nm. The experiment was carried out in triplicate and the error bars display the standard deviation of the mean.

Figure 3. Growth rates on alginate substrates of different chain length and solubility.

(a) Alginate lyase (PL6, orange; PL7, green) and oligoalginate lyase (PL15, red; PL17, blue) copy number for individual strains within populations of Vibrionaceae. (b) Isolates representing different populations were grown in seawater minimal medium containing low- (Dp∼3–4, aqua), medium- (Dp∼20, blue), or high- (degree of polymerization, Dp>50, dark purple) molecular weight alginate as the sole carbon source. Since alginate is a heteropolymer of guluronate and mannuronate, the low and medium molecular weight alginate was further purified into guluronate (G)- or mannuronate (M)-enriched fractions. Each dot represents the average growth rate of an isolate from the denoted population on the designated carbon source across three technical replicates. The number of isolates assayed per population (n) is indicated.

Figure 4. Membrane-bound versus broadcasted alginate lyases dictate growth lag time.

(a) Growth curves of isolates representing distinct pathway architectures on high- (degree of polymerization, Dp>50), medium- (Dp∼20), or low- (Dp∼3-4) molecular weight alginate. Low and medium molecular weight alginate was further purified into mannuronate (M)- or guluronate (G)- enriched fractions. (b) Quantified lag time differences between strains. (c) The cellular milieu was fractionated into extracellular (secreted), membrane-bound, and intracellular components. For each fraction, alginate lyase activity was measured using a bulk enzymatic activity assay (Methods). Among isolates assayed, those with longer lag phases displayed reduced broadcasted alginate lyase activity, despite similar levels of intracellular- and membrane-bound alginate lyase activity. Bar diagrams represent averaged technical replicates (n=3). Error bars represent standard deviations of the mean. One unit of activity defines an increase of 1.0 in absorbance at 235 nm per min. (d) Broadcasted alginate lyase activity measured independently with a plate-based assay (Methods). The size of the halo indicates the degree of broadcasted alginate lyase activity after a fixed period of time. Bar diagrams represent means of technical replicates (n=5) and error bars represent s.d.

Figure 5. Alginate degradation cascade of substrates varying in solubility and chain length.

Marine vibrios diversified into different populations characterized by their ability to consume insoluble alginate polysaccharide and soluble alginate oligosaccharides of different chain lengths. Pioneers are specialized in consumption of native, insoluble alginate due to their endowment with broadcast alginate lyases. These enzymes can diffuse freely into the alginate gel and depolymerize the alginate into soluble oligosaccharides. Harvester populations with secreted but tethered alginate lyases can exploit the range of soluble alginate substrates including medium and small oligosaccharides liberated by pioneers. Scavenger populations devoid of any alginate lyases can only use the smallest alginate oligosaccharides.