Bacterial adenine cross-feeding stems from a purine salvage bottleneck

Abstract Diverse ecosystems host microbial relationships that are stabilized by nutrient cross-feeding. Cross-feeding can involve metabolites that should hold value for the producer. Externalization of such communally valuable metabolites is often unexpected and difficult to predict. Previously, we discovered purine externalization by Rhodopseudomonas palustris by its ability to rescue an Escherichia coli purine auxotroph. Here we found that an E. coli purine auxotroph can stably coexist with R. palustris due to purine cross-feeding. We identified the cross-fed purine as adenine. Adenine was externalized by R. palustris under diverse growth conditions. Computational modeling suggested that adenine externalization occurs via diffusion across the cytoplasmic membrane. RNAseq analysis led us to hypothesize that adenine accumulation and externalization stem from a salvage pathway bottleneck at the enzyme encoded by apt. Ectopic expression of apt eliminated adenine externalization, supporting our hypothesis. A comparison of 49 R. palustris strains suggested that purine externalization is relatively common, with 16 strains exhibiting the trait. Purine externalization was correlated with the genomic orientation of apt, but apt orientation alone could not always explain purine externalization. Our results provide a mechanistic understanding of how a communally valuable metabolite can participate in cross-feeding. Our findings also highlight the challenge in identifying genetic signatures for metabolite externalization.


CROSS-FEEDING MODEL FOR TESTING THE VALIDITY OF ADENINE DIFFUSION
(1) E. coli growth rate (h −1 ): dRp/dt = Rp • µRp (8) change in E. coli population (cells/ml/h): where, µ is the specific growth rate of the indicated species (h −1 ).µMAX is the maximum specific growth rate of the indicated species (h −1 ) G is the extracellular glucose concentration (mM) P is the extracellular purine (adenine) concentration (mM) IP is the intracellular purine (adenine) concentration (mM) C is the extracellular organic acid concentration (mM) f is the extracellular formate concentration (mM) k is the half saturation constant (km) for the indicated substrate (mM).Ec is the E. coli cell density (cells/ml) Rp is the R. palustris cell density (cells/ml) b modulates sensitivity to inhibitory effects of accumulated organic acids (mM ).Y is the growth yield on the indicated substrate (cells / μmol).F is the excretion value for the indicated compound during growth (μmol / cell) r is the growth-independent excretion rate (μmol / cell / h) Perm is the permeability coefficient for adenine (cm / s) (1) SA is the cell surface area (cm 2 )  Growth of E. coli (Ec) ΔpurH in supernatant samples taken from stationary-phase CGA0092 monocultures grown under various growth conditions.Each data point represents the mean of three to six biological replicates +/-SD.Linear regression (gray solid line) +/-95% confidence intervals (dashed lines) was applied to all samples across all conditions.SN, supernatant.

Fig. S3 .Fig S4 .
Fig. S3.R. palustris (Rp) CGA4005 supernatants support E. coli (Ec) ΔpurH monoculture growth.Error bars = SD, n = 3.Some error bars are smaller than the symbols.A. E. coli ΔpurH monoculture growth curves in media supplemented with difference amounts of R. palustris CGA4005 monoculture supernatant B. Linear regression of E. coli ΔpurH monoculture growth and the amount of R. palustris CGA4005 supernatant provided.

Fig. S5 .
Fig. S5.Monoculture growth trends are similar for R. palustris (Rp) CGA0092 and TIE-1.Data points from all three biological replicates are shown.

Fig. S6 .
Fig. S6.CGA0092 exhibits higher lemA expression than TIE-1.Top, orientation of the lemA-apt cluster in CGA0092 and TIE-1.Bottom, sequencing reads (height of gray bars) across the lemA gene in each strain.See the supplementary RNAseq data for the corresponding differential expression values that estimate a 2.2-fold higher transcript levels in CGA0092.
Fig. S7.Gene neighborhoods for apt (red).Images are from the Joint Genome Institute's Integrative Microbial Genomes & Microbiomes Gene Neighborhoods tool.

Fig S8 .
Fig S8.Purine externalizing by R. palustris strains organized by apt orientation.Purple and blue shading indicates CGA0092 or TIE-1 apt gene orientation, respectively.Gray shading indicates unknown apt gene orientation.Purines were measured in R. palustris monoculture supernatants using the E. coli ΔpurH bioassay.Yellow shading indicates the CGA0092 standard deviation.*, significantly more purine than TIE-1 from One-way ANOVA with a Dunnett correction for multiple comparisons; p < 0.1.Error bars = SD, n = 3. Top.CGA0092 and TIE-1 apt-lemA clusters shown to scale.Other bacteria with the TIE-1 orientation do not have the transposon (Tn; Fig S7).

Table S6 . Intracellular concentrations of nucleobase-containing compounds in CGA0092 and TIE-1 supernatants.
ND, not detected.Values are means ± SD; n= 1-3.Triplicate samples were run in each case but not all compounds were detected in each sample.Values without SD indicate that a compound was only detected in one of the three replicates.