Transporter genes in biosynthetic gene clusters predict metabolite characteristics and siderophore activity

Abstract

Biosynthetic gene clusters (BGCs) are operonic sets of microbial genes that synthesize specialized metabolites with diverse functions, including siderophores and antibiotics, which often require export to the extracellular environment. For this reason, genes for transport across cellular membranes are essential for the production of specialized metabolites and are often genomically colocalized with BGCs. Here, we conducted a comprehensive computational analysis of transporters associated with characterized BGCs. In addition to known exporters, in BGCs we found many importer-specific transmembrane domains that co-occur with substrate binding proteins possibly for uptake of siderophores or metabolic precursors. Machine learning models using transporter gene frequencies were predictive of known siderophore activity, molecular weights, and a measure of lipophilicity (log P) for corresponding BGC-synthesized metabolites. Transporter genes associated with BGCs were often equally or more predictive of metabolite features than biosynthetic genes. Given the importance of siderophores as pathogenicity factors, we used transporters specific for siderophore BGCs to identify both known and uncharacterized siderophore-like BGCs in genomes from metagenomes from the infant and adult gut microbiome. We find that 23% of microbial genomes from premature infant guts have siderophore-like BGCs, but only 3% of those assembled from adult gut microbiomes do. Although siderophore-like BGCs from the infant gut are predominantly associated with Enterobacteriaceae and Staphylococcus, siderophore-like BGCs can be identified from taxa in the adult gut microbiome that have rarely been recognized for siderophore production. Taken together, these results show that consideration of BGC-associated transporter genes can inform predictions of specialized metabolite structure and function.

Figure 1.

Distributions of transporter classes in biosynthetic gene clusters. (A) Structures of characterized examples of major transporter classes often found in BGCs, colored and labeled by Pfam domains. The extracellular/periplasmic side of the membrane is shown as a red line, and the intracellular side is in blue. (B) The frequencies of common Pfam transporter domains across the bacterial BGCs in the MIBiG database. (C) The percentages of bacterial BGCs in MIBiG that do and do not contain transporter domains. Each square represents 1% of BGCs. (D) The counts of transporter domains per each bacterial BGC that contains at least one transporter gene across MIBiG.

Figure 2.

Presence of importer-specific domains and co-occurrence between transporters across BGCs. (A) Spearman's correlations between commonly occurring Pfam transporter domains across MIBiG BGCs—only correlations with P < 0.001 are shown. (B) Counts of Pfam transporter substrate binding domain families and corresponding substrate binding protein clusters described by Berntsson et al. (2010). (C) Counts of importer-specific CATH domains across MIBiG BGCs. The CATH functional family “Iron ABC permease” is essentially synonymous with the FecCD Pfam.

Figure 3.

Transporter domains are predictive of siderophore BGCs. (A) The frequencies of common transporter Pfam domains across siderophore BGCs and BGCs of other known activities in Gram-positive and Gram-negative bacteria. Bars in green were significantly different in frequency between the two classes (Fisher's exact test; Q < 0.05) (B) Precision-recall curves for two-layer decision trees classifying siderophore BGCs using Pfam transporter, CATH transporter, and Pfam biosynthetic gene features in Gram-negative and Gram-positive bacteria. (C) Examples of three siderophore BGCs without activity labels in MIBiG 2.0, which could be identified using transporter frequencies. Transporter genes are blue, core biosynthetic genes (NRPS and PKS) are dark red, accessory biosynthetic genes are light red, and regulatory genes are green.

Figure 4.

Presence of general and siderophore-specific transporters by biosynthetic class and bacterial genus across the antiSMASH database. (A) ATP-dependent and ATP-independent (MFS) transporters are commonly associated with a variety of BGCs in the antiSMASH database across a wide range of genera. Each point is the percentage of a BGC class with a transporter within a particular genus. Each genus is colored by its Gram status, and genera with fewer than 20 BGCs of a particular class are excluded. (B) Siderophore-specific transporters are associated with few BGC classes in the antiSMASH database. Each point is the percentage of a BGC class with a transporter within a particular genus. Each genus is colored by its Gram status, and genera with fewer than 20 BGCs of a particular class are excluded.

Figure 5.

Transporter domains associated with molecular size and partition coefficient. (A) The frequencies of common transporter Pfam domains in BGCs that synthesize metabolites >1000 Da (left) and <1000 Da (right). Bars in green were significantly different in frequency between the two classes (Fisher's exact test; Q < 0.05). (B) Precision-recall curves for two-layer decision trees and LASSO logistic regression models classifying BGCs producing metabolites >1000 Da using Pfam transporter, CATH transporter, and Pfam biosynthetic gene features. (C) The distribution of metabolite molecular weights synthesized by BGCs with at least one NBD-binding ABC transporter domain, at least one MFS domain, and the ABC2_membrane_3 transmembrane domain. (D) Predicted partition coefficients (log P) for metabolites synthesized by BGCs that contain at least one variant of two different ABC transporter transmembrane domains.

Figure 6.

BGCs with siderophore-like transporters from human gut microbiomes. (A) Concatenated ribosomal protein tree (collapsed to the genus level) for high-quality genomes from the infant and adult gut microbiomes that encode siderophore-like BGCs. On the right are counts of siderophore BGCs from infant gut genomes (blue) and adult gut genomes (red). (B) Gene Cluster Families of BGCs containing known siderophores (bright red) and human microbiome–derived BGCs with siderophore-like transporters. BGCs are connected by similarity to other BGCs in the same gene cluster family, calculated using BiG-SCAPE. (C) Families of siderophore-like BGCs without any similarity to existing known BGCs. BGCs in the network are colored by the taxonomy of the genome of origin and are grouped and labeled by the antiSMASH reported biosynthetic class: for NRPS gene clusters, the adenylation domain specificities are reported.