Vitamin auxotrophies shape microbial community assembly on model marine particles

Abstract

Microbial community assembly is governed by the flow of carbon sources and other primary metabolites between species. However, central metabolism represents only a small fraction of the biosynthetic repertoire of microbes: metabolites such as antimicrobial compounds, signaling molecules, and co-factors are underexplored in their potential to shape microbial communities. Here, we focus on B vitamin exchange in marine bacterial communities that degrade polysaccharides, a key component of particulate organic matter. We found that in a screen of 150 natural isolates, almost a third were auxotrophs for one or more B vitamins. By measuring physiological parameters such as uptake affinities and comparing those to ambient seawater concentrations, we showed that marine bacteria live at the edge of vitamin limitation in the environment. To understand how auxotrophs survive in the open oceans, we used our experimental data to model vitamin cross-feeding on particles through both secretion and lysis. Our results highlight the importance of vitamin auxotrophies in shaping microbial community assembly and succession, adding another layer of complexity to the trophic structure of particle-associated communities.

Keywords: B vitamins; auxotrophies; chitin; community assembly; cross-feeding; marine snow.

Figure 1

Coastal seawater communities are vitamin-limited and vitamin auxotrophies are prevalent. (A) Coastal seawater collected at Nahant, MA, was incubated with chitin particles, with and without a vitamin mix spike-in. The addition of vitamins led to differences in taxonomy at the order level. Each data point represents a taxon that reached at least 1% abundance in one or more time points. (B) Chitin degradation proteins families (Pfams) were depleted in the vitamin-supplemented seawater condition compared to the seawater condition. (C) 150 bacterial coastal marine isolates from Nahant, MA, were screened for vitamin auxotrophies in a high-throughput format. Top left, example of growth with and without vitamin mix over three daily transfers, due to auxotrophy for B1. Chitin degraders were prototrophic (marked with circles on tree). Abbreviations: Vibrio. = Vibrionales; Altero. = Alteromonadales; Pseudo. = Pseudomonadales; Campylo. = Campylobacterales; Rhodo. = Rhodobacterales; Flavo. = Flavobacteriales.; fibro. = Fibrobacterales.

Figure 2

Three evolutionary modes of auxotroph formation. (A) Partial pathway loss leading to formation of B1 pyrimidine auxotrophs in Rhodobacterales. Right, three critical genes in the thiamine biosynthesis pathway: thiC, which forms the pyrimidine precursor 4-amino-5-hydroxymethyl-2-methylpyrimidine pyrophosphate (HMP-PP); thiG, which forms the thiazole precursor 2-(2-carboxy-4-methylthiazol-5-yl) ethyl phosphate (Thz-P); and thiE, which couples the two moieties to form thiamine. (B) Total pathway loss. The genomic region of the thiamine biosynthesis cluster is lost in Flavobacteriales B1 auxotrophs compared to two closely related prototrophic isolates, with flanking regions conserved. (C) Loss of vitamin-independent metabolism. Both prototrophic and auxotrophic Alteromonadales isolates are missing B12 biosynthesis genes and can scavenge B12. B12 auxotrophs have lost metE, retaining only the B12-dependent pathway for the biosynthesis of methionine via metH.

Figure 3

Auxotroph growth rates are limited under environmental vitamin concentrations. Auxotrophs were grown on different vitamin or precursor concentrations: (A) Thiamine (B1) and the B1 vitamer 4-amino-5-hydroxymethyl-2-methylpyrimidine (HMP), (B) Niacin (B3), (C) Biotin (B7), and (D) Cobalamin (B12). Cultures were grown for three growth-dilution cycles, diluting 1:40 every 24 hours. After the last transfer, a kinetic growth measurement was run and growth rates calculated from the exponential phase. Literature values for vitamin concentrations measured in the surface oceans are shown as gray bars above each panel (n = 193 samples, Supplementary Dataset S2) [43, 59–85]. Abbreviations: Altero. = Alteromonadales; Pseudo. = Pseudomonadales; Rhodo. = Rhodobacterales; Flavo. = Flavobacteriales.

Figure 4

Auxotrophies are only partially alleviated in co-culture and by supernatants. (A) Eight auxotrophs were grown in co-culture, for a total of 28 pairs. Top, the auxotrophs were grown separately with vitamins, then combined 1:1 and transferred to fresh medium without vitamins. Growth was measured after three growth-dilution cycles. Bottom, only 4/17 co-cultures predicted to successfully cross-feed (circles) grew to detectable levels, as well as 1/11 of the co-cultures predicted not to grow. The co-cultures reached between 7% and 52% of the final optical density of co-cultures with vitamin supplementation (% maximum optical density, shading). The 8 monocultures appear on the diagonal. (B) Three auxotrophs (auxotrophs 3, 4, and 6) were further tested for growth with supernatants (left) and lysates containing both supernatant and lysed cells (right), collected from a Vibrio. 1A01 prototroph culture in late exponential phase. Growth was measured after 3 growth-dilution cycles. Data are presented as a percentage of the optical density obtained when the supernatant is supplemented by vitamins (% maximum optical density), to correct for the effect of the depletion or release of additional nutrients in each condition (see Fig. S9 for raw data). Abbreviations: Vibrio. = Vibrionales; Altero. = Alteromonadales; Pseudo. = Pseudomonadales; Rhodo. = Rhodobacterales.

Figure 5

Model of vitamin cross-feeding in a particle-associated community. (A) Scheme of model particle community. Degraders supply vitamins to auxotrophs through secretion (s) or lysis (μ). The concentration of substrates and vitamins depends on the rate of diffusion and the population size of degraders, based on their effective growth rate (r_deg). The auxotroph growth rate (r_auxo) depends on cellular vitamin requirements, i.e. inverse yield (molecules/cell; Y_vit⁻¹), and uptake efficiency, i.e. half-saturation constants (K_S). (B and C) Auxotroph growth rates via secretion (left) and via lysis (right) is shown in shaded areas (>90% of maximal growth rate, dark; >50%, medium; >10%, light). Values for measured K_S values and estimated vitamin yields for auxotrophs are marked by symbols. Growth under secretion (B) is characterized by two threshold values: a vertical line corresponding to the maximal half-saturation constant, and a horizontal line corresponding to the maximal yield. Growth under lysis (C) is approximately proportional to the product of the inverse vitamin yield and half-saturation constant. Abbreviations: Altero. = Alteromonadales; Pseudo. = Pseudomonadales; Rhodo. = Rhodobacterales; Flavo. = Flavobacteriales.