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. 2019 Oct 29;116(44):22219-22228.
doi: 10.1073/pnas.1909844116. Epub 2019 Oct 14.

Multilayered horizontal operon transfers from bacteria reconstruct a thiamine salvage pathway in yeasts

Carla Gonçalves  1 , Paula Gonçalves  2
Affiliations

Multilayered horizontal operon transfers from bacteria reconstruct a thiamine salvage pathway in yeasts

Carla Gonçalves et al. Proc Natl Acad Sci U S A. .

Abstract

Horizontal acquisition of bacterial genes is presently recognized as an important contribution to the adaptation and evolution of eukaryotic genomes. However, the mechanisms underlying expression and consequent selection and fixation of the prokaryotic genes in the new eukaryotic setting are largely unknown. Here we show that genes composing the pathway for the synthesis of the essential vitamin B1 (thiamine) were lost in an ancestor of a yeast lineage, the Wickerhamiella/Starmerella (W/S) clade, known to harbor an unusually large number of genes of alien origin. The thiamine pathway was subsequently reassembled, at least twice, by multiple HGT events from different bacterial donors involving both single genes and entire operons. In the W/S-clade species Starmerella bombicola we obtained direct genetic evidence that all bacterial genes of the thiamine pathway are functional. The reconstructed pathway is composed by yeast and bacterial genes operating coordinately to scavenge thiamine derivatives from the environment. The adaptation of the newly acquired operons to the eukaryotic setting involved a repertoire of mechanisms until now only sparsely documented, namely longer intergenic regions, post-horizontal gene transfer (HGT) gene fusions fostering coordinated expression, gene relocation, and possibly recombination generating mosaic genes. The results provide additional evidence that HGT occurred recurrently in this yeast lineage and was crucial for the reestablishment of lost functions and that similar mechanisms are used across a broad range of eukaryotic microbes to promote adaptation of prokaryotic genes to their new environment.

Keywords: gene fusion; horizontal gene transfer; horizontal operon transfer; thiamine; yeast metabolism.

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Conflict of interest statement

The authors declare no competing interest.

Figures

Fig. 1.
Fig. 1.
(A) Thiamine biosynthetic pathway in bacteria and yeast. Genes involved in thiamine biosynthesis in B. subtilis are represented in blue while their counterparts in S. cerevisiae are represented in red. Yellow arrows represent the salvage pathways that can be present in both yeast and bacteria. The red stars represent thiamine degradation. (B) Presence and absence of the main genes involved in the de novo and salvage thiamine biosynthesis in the W/S clade. Gray squares denote missing genes; blue squares, genes of bacterial origin; and red squares, genes of fungal origin. The origin of the W. hasegawae THI6 and THI20 genes was unclear. Phylogenetic relationships between species are depicted based on a ML phylogeny constructed as described in Materials and Methods. Branches with 100% support are indicated by black dots while bootstrap values >75% are shown next to the respective branches.
Fig. 2.
Fig. 2.
Thiamine operon organization. (A) Organization of THI genes in the genomes of representative W/S-clade species. Thiamine metabolism-related genes are represented by different colors. Syntenic genes between species are represented in black while nonsyntenic genes are represented in white. Black vertical bars represent ends of scaffolds. (B) Thiamine operon organization in putative donor lineages. A representative species belonging to each order/phylum is shown. Genes and intergenic regions are drawn to scale. Arrows denote direction of transcription.
Fig. 3.
Fig. 3.
Maximum likelihood phylogenies of Thi proteins in the W/S clade. Pruned phylogenies showing the closest relatives to W/S-clade proteins are shown. Branches with support higher than 95% (ultrafast bootstrap) are indicated by black dots.
Fig. 4.
Fig. 4.
Maximum likelihood phylogenies of Thi proteins from W. galacta. Pruned phylogenies showing the closest relatives to W. galacta Thi proteins encoded in the operon are shown. Branches with support higher than 95% (ultrafast bootstrap) are indicated by black dots. The phylogenetic tree for TenA is shown in SI Appendix, Fig. S3.
Fig. 5.
Fig. 5.
Assimilation of thiamine derivatives by St. bombicola WT and deletion mutants. (A) Growth assays for St. bombicola strains (WT and mutants) cultivated on YNB without thiamine and supplemented with 0.2 µM of thiamine, 0.2 µM of HMP or 0.02 µM of aminoHMP, after 5 d of incubation at 25 °C. (B) Growth assays for other W/S-clade species in the presence of 0.2 µM of HET, 0.2 µM of HMP, or 0.02 µM of aminoHMP and different combinations of these compounds.
Fig. 6.
Fig. 6.
Expression profiles of THI genes in W/S-clade species. (A) Expression of THI genes (depicted as the log2 fold difference) in St. bombicola relative to the internal reference POL2 (black star) using only gene-specific primers. Expression levels of intergenic regions are also depicted. (B) Expression of the thiEM fusion gene in St. bombicola after preselection of poly(A)-tailed mRNAs. (C) Expression of THI genes in W. domercqiae and W. galacta after preselection of poly(A)-tailed mRNAs. Mean values are represented by the black horizontal lines. Raw data can be accessed in Dataset S3. The black stars indicate the gene that served as reference in each case.
Fig. 7.
Fig. 7.
Main events in the evolution of THI genes in the W/S clade. Schematic phylogenetic relationships between W/S species are depicted based on the ML phylogeny presented in Fig. 1B. Loss of native THI6 and THI20 genes in the most recent common ancestor (MRCA) of the W/S clade is indicated by a red cross. Putative horizontal gene transfer events are represented by boxes above/below which the putative donor lineage is indicated. The number of putative HGT events and fusion events are indicated in yellow and blue circles, respectively; numbers in gray represent cases where the number of HGT events could not be asserted with certainty. Operons are represented by a straight black line linking the genes, while the uncertainty surrounding the origin of thiE and thiM in the St. bombicola subclade is denoted by question marks. Horizontal acquisition of the putative N-formyl-4-amino-5-aminomethyl-2-methylpyrimidine deformylase (YlmB) is also shown. The origin of the W. hasegawae genes is elusive.
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