Multilayered horizontal operon transfers from bacteria reconstruct a thiamine salvage pathway in yeasts Carla Gonçalvesa and Paula Gonçalvesa,1 aApplied Molecular Biosciences Unit-UCIBIO, Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa, 2829-516 Caparica, Portugal Edited by Edward F. DeLong, University of Hawaii at Manoa, Honolulu, HI, and approved September 22, 2019 (received for review June 14, 2019) Horizontal acquisition of bacterial genes is presently recognized as nisms presumed to have facilitated a transition from bacterial an important contribution to the adaptation and evolution of operon transcription to eukaryotic-style gene expression were eukaryotic genomes. However, the mechanisms underlying ex- proposed, such as gene fusion giving rise to multifunctional pro- pression and consequent selection and fixation of the prokaryotic teins (6, 23, 24), increase in intergenic distances between genes to genes in the new eukaryotic setting are largely unknown. Here we generate room for eukaryotic promoters, and independent tran- show that genes composing the pathway for the synthesis of the scription producing mRNAs with poly(A) tails have been dem- essential vitamin B1 (thiamine) were lost in an ancestor of a yeast onstrated (22). In the best documented study, which concerns a lineage, the Wickerhamiella/Starmerella (W/S) clade, known to bacterial siderophore biosynthesis operon acquired by yeasts be- harbor an unusually large number of genes of alien origin. The longing to the Wickerhamiella/Starmerella (W/S) clade, the bacte- thiamine pathway was subsequently reassembled, at least twice, rial genes acquired as an operon were shown to be functional (22). by multiple HGT events from different bacterial donors involving Thiamine, commonly known as vitamin B1, is essential for all both single genes and entire operons. In the W/S-clade species living organisms because its active form, thiamine pyrophosphate Starmerella bombicola we obtained direct genetic evidence that (TPP), is an indispensable cofactor of enzymes participating in all bacterial genes of the thiamine pathway are functional. The amino acid and carbohydrate metabolism (26–30). Some or- reconstructed pathway is composed by yeast and bacterial genes ganisms that are unable to synthesize thiamine de novo are operating coordinately to scavenge thiamine derivatives from the nevertheless capable of using a salvage pathway to rescue the EVOLUTION environment. The adaptation of the newly acquired operons to pyrimidine (hydroxymethylpyrimidine or HMP) and thiazole the eukaryotic setting involved a repertoire of mechanisms until (hydroxyethylthiazole or HET) precursors and similar compounds now only sparsely documented, namely longer intergenic regions, that result from natural thiamine degradation in the surrounding post-horizontal gene transfer (HGT) gene fusions fostering coordi- nated expression, gene relocation, and possibly recombination environment (31, 32). In the present study we describe a composite thiamine salvage generating mosaic genes. The results provide additional evidence that HGT occurred recurrently in this yeast lineage and was crucial pathway made up of yeast and bacterial genes found in several for the reestablishment of lost functions and that similar mecha- species of the yeast W/S clade. Our recent work (5) revealed that nisms are used across a broad range of eukaryotic microbes to pro- the W/S-yeast clade harbors an unusual large number of HGT mote adaptation of prokaryotic genes to their new environment. events in yeasts, mostly as single genes, a finding independently confirmed in a large study in which ∼300 yeast genomes were horizontal gene transfer | horizontal operon transfer | yeast metabolism | examined (9). Here we show that in W/S-clade species most of gene fusion | thiamine Significance orizontal transfer of genes (HGT) from bacteria to various Heukaryotic settings is well documented over the entire scope Food is the only source of the essential vitamin B1 for humans, of eukaryotes (1–4), including eukaryotic microbes comprising but many microorganisms such as yeast and bacteria can syn- fungi, protists, and algae (5–9). Notwithstanding this, the events thetize it themselves. Here we report on a group of yeasts that leading to the selection and fixation of xenologous genes in their have lost part of the vitamin B1 biosynthetic pathway in the new environment are still relatively poorly understood. One past but have managed to rebuild it by capturing multiple important hurdle that needs to be overcome for protein coding genes from bacteria through horizontal gene transfer (HGT). genes is the fact that while expression is a prerequisite for se- We show a mosaic pathway composed of yeast and bacterial lection, mechanisms of gene expression are widely distinct be- genes working coordinately to accomplish the synthesis of an tween eukaryotes and prokaryotes. This is particularly relevant essential nutrient. This involved adaptation of the bacterial for horizontally acquired traits that require the action of more genes to the very different expression rules in their new en- than one gene product, because this means that all genes in- vironment using several different mechanisms. Our results volved must be functional in order for the selectable phenotype endorse HGT as an important mechanism for evolutionary ad- aptation in eukaryotes. to be expressed. Functionally related genes are often located in genomic Author contributions: C.G. and P.G. designed research; C.G. and P.G. performed research; proximity. In bacteria, they are organized in operons and are P.G. contributed new reagents/analytic tools; C.G. analyzed data; and C.G. and P.G. wrote transcribed as polycistronic mRNAs, while in eukaryotes they the paper. may be found in clusters of independently transcribed genes (10– The authors declare no competing interest. 12). This is thought to facilitate horizontal transfer of function This article is a PNAS Direct Submission. related genes between similar organisms in a single event (13– Published under the PNAS license. 21). Remarkably, this also seems to apply to horizontal trans- Data deposition: All the alignment files and complete phylogenies and can be accessed in fers of operons from bacteria to eukaryotes according to pre- figshare (DOI: 10.6084/m9.figshare.9800636). vious reports (6, 22–25). Bacterial operons transferred to 1To whom correspondence may be addressed. Email: [email protected]. eukaryotic organisms have been proposed to fulfill important This article contains supporting information online at www.pnas.org/lookup/suppl/doi:10. roles such as changes in the nutritional status of the host (22) or 1073/pnas.1909844116/-/DCSupplemental. adaptation to extreme environments (23, 25). Different mecha- First published October 14, 2019. www.pnas.org/cgi/doi/10.1073/pnas.1909844116 PNAS | October 29, 2019 | vol. 116 | no. 44 | 22219–22228 Downloaded by guest on September 25, 2021 the genes in the thiamine salvage pathway were originally ac- Saccharomycotina yeasts (SI Appendix,Fig.S1). Thi20 seems to be quired from bacteria as part of an operon and we use genetic encoded by 2 separate genes, while for Thi6 a single gene was dissection to link each of the transferred genes with an observ- predicted in most species. A subsequent BLASTp search using able phenotype in the yeast setting. Moreover, we present evi- the 3 predicted proteins from St. bombicola (2 corresponding to dence for the occurrence of 2 independent horizontal operon different domains of THI20 and 1 corresponding to THI6) re- transfer (HOT) events in distinct subclades within the W/S lineage trieved bacterial proteins as top hits (Fig. 1B and Dataset S1) that, complemented by the independent horizontal acquisition of and no yeast proteins among the first 1,000 hits. The same result single functionally related genes, endow the yeast host with the was obtained for all W/S-clade THI6 or THI20 homologs, sug- ability to salvage a wide range of thiamine precursors. gesting that these genes, when present, always have bacterial origin in the W/S clade. The proteins exhibiting the Thi6 and Results Thi20 enzymatic activities are both encoded by 2 genes each in Thiamine Genes in the W/S-Yeast Clade. Many organisms lack the most bacteria (thiE and thiM, and tenA and thiD, respectively), as genes encoding enzymes of the 2 upper branches of the pathway depicted in Fig. 1A. It was not possible to ascertain the origin of used to generate the pyrimidine and thiazole precursors for de THI6 and THI20 in Wickerhamiella hasegawae, because pre- novo biosynthesis of thiamine, while maintaining only the genes liminary BLASTx searches using the C-terminal and N-terminal composing the salvage pathway for thiamine or its precursors domains of the Thi6 and Thi20 from this species showed a (31–33). Thi5 and Thi4 are the most important enzymes in each mosaic-like pattern for both genes, where part of the protein of the 2 pathway branches required for de novo thiamine syn- exhibited high sequence similarity with bacterial proteins while thesis (34) (Fig. 1A). THI5 is absent in all W/S clade species the other domain presented homology with fungal proteins (Fig. examined and THI4 is missing in about half of the species in the 1B and Dataset S2). clade. They maintain however the indispensable genes encoding a thiamine transporter, Dur31 (35), and the kinase Thi80, re- Independent Acquisitions of Bacterial Thiamine Operons by W/S-Clade sponsible for