Eukaryotic Acquisition of a Bacterial Operon - PubMed (original) (raw)

. 2019 Mar 7;176(6):1356-1366.e10.

doi: 10.1016/j.cell.2019.01.034. Epub 2019 Feb 21.

Drew T Doering 2, Dana A Opulente 1, Xing-Xing Shen 3, Xiaofan Zhou 4, Jeremy DeVirgilio 5, Amanda B Hulfachor 6, Marizeth Groenewald 7, Mcsean A Mcgee 8, Steven D Karlen 9, Cletus P Kurtzman 5, Antonis Rokas 3, Chris Todd Hittinger 10

Affiliations

Eukaryotic Acquisition of a Bacterial Operon

Jacek Kominek et al. Cell. 2019.

Abstract

Operons are a hallmark of bacterial genomes, where they allow concerted expression of functionally related genes as single polycistronic transcripts. They are rare in eukaryotes, where each gene usually drives expression of its own independent messenger RNAs. Here, we report the horizontal operon transfer of a siderophore biosynthesis pathway from relatives of Escherichia coli into a group of budding yeast taxa. We further show that the co-linearly arranged secondary metabolism genes are expressed, exhibit eukaryotic transcriptional features, and enable the sequestration and uptake of iron. After transfer, several genetic changes occurred during subsequent evolution, including the gain of new transcription start sites that were sometimes within protein-coding sequences, acquisition of polyadenylation sites, structural rearrangements, and integration of eukaryotic genes into the cluster. We conclude that the genes were likely acquired as a unit, modified for eukaryotic gene expression, and maintained by selection to adapt to the highly competitive, iron-limited environment.

Keywords: Saccharomycotina; Starmerella; Wickerhamiella; budding yeasts; central dogma of biology; enterobactin; horizontal gene transfer; operon; siderophore biosynthesis.

Copyright © 2019 Elsevier Inc. All rights reserved.

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Conflict of interest statement

Declaration of Interests:

The authors declare no competing interests.

Figures

Figure 1.

Figure 1.. Distribution of the iron uptake and storage systems among fungi.

Plus (green) and minus (orange) signs indicate the presence and absence of iron uptake and storage systems in specific taxonomic groups. The numbers in parentheses (green) indicate the number of species in a taxonomic group that possess a specific system, if it is not ubiquitous in that group. The blue box indicates the budding yeasts. RIA - Reductive Iron Assimilation. IRGF - Iron-Responsive GATA Factor. Asterisks (*) mark paraphyletic groups. Note that only Wickerhamiella/Starmerella (W/S) clade fungi contain the bacterial or catecholate-class siderophore biosynthesis pathway, whereas many other dikaryon fungi contain hydroxamate-class siderophore biosynthesis pathways. See also Figure S1 and Table S1.

Figure 2.

Figure 2.. Yeast siderophore biosynthesis originated from an Enterobacteriales lineage.

(A) ML phylogeny from the super-alignment of entABCDE genes from 207 Gammaproteobacteria and 12 yeasts, rooted at the midpoint. Bootstrap support values are shown for relevant branches within the Enterobacteriales (red). Other Gammaproteobacteria are blue.(B) Detailed view of the yeast clade from the main phylogeny, with bootstrap support values. (C) Alternative scenarios for the horizontal operon transfer. (D) P-values of the AU tests of different evolutionary hypotheses; EO - Enterobacteriales origin; non-EO - non-Enterobacteriales origin; 12-mono - 12 yeast sequences are monophyletic, 11-mono - 11 yeast sequences monophyletic and one unconstrained (12 alternatives tested, lowest p-value shown, full details in Table S2); 5G - topology of the yeast clade constrained to the one inferred from the super-alignment of entABCDE genes. Shimodaira-Hasegawa (SH) tests had less statistical power but produced fully concordant results. See also Table S1 and Table S2.

Figure 3.

Figure 3.. Evolution of the siderophore biosynthesis genes in yeasts.

(A) ML phylogeny reconstructed from the concatenated alignment of 661 conserved, single copy genes (834,750 sites), with branch support values below 100 shown. Strains in bold denote genomes sequenced in this study, while strains in red denote genomes containing the siderophore biosynthesis genes. Black diamonds indicate secondary losses in yeast lineages, accompanied by losses of the siderophore importer ARN genes, which are often found in close proximity. (1) Horizontal operon transfer from an Enterobacteriales lineage. (2) Rearrangement and integration of genes encoding ferric reductase (FRE) and an uncharacterized transmembrane protein (TM). (3) Disruption by integration of the SNZ-SNO gene pair and translocation. (B) Species-specific data on presence/absence of the siderophore biosynthesis genes and experimental evidence for the presence of enterobactin in the yeast cultures as determined by an O-CAS assay (not specific to enterobactin) and direct chemical detection by HPLC-MS/MS (enterobactin produced by E. coli was used as the standard; nt - not tested). Note that culture conditions between assays were not identical, and siderophore expression is often condition-dependent (Machuca and Milagres, 2003). (C) Genetic structure of the siderophore biosynthesis operon in E. coli and yeasts, drawn to scale. Individual colors represent homologous genes, and gray marks bacterial genes not found in yeasts. Black circles represent contig termini within 25kb. See also Figure S4 and Table S4.

Figure 4.

Figure 4.. Transcriptomics of the siderophore biosynthesis genes in W. versatilis.

(A, B, D) Diagram of siderophore biosynthesis genes as present in the genomes of St. bombicola (A), St. apicola (B), and W. versatilis (D), drawn to scale. Counts above the diagram indicate read-pairs that map to both predicted protein-coding sequences (low, non-zero read counts are likely DNA contamination). Counts below indicate the size of intergenic regions between adjacent protein-coding sequences, in base pairs. (C) The orange area indicates per-base coverage by RNA-Seq reads (read coverage). The blue area indicates per-base cumulative coverage by RNA-Seq reads and inserts between read-pairs (span coverage). The black line indicates the ratio of the read coverage over the span coverage, which is expected to remain ~50% in the middle of gene transcripts and rise towards 100% at transcript termini. Thus, transcript boundaries are visualized as a coverage trough between two spikes that approach 100% ratios. Ratios below 100% at the putative 5’ or 3’ ends of annotated transcripts, coupled with non-zero coverage of their intergenic regions, suggest overlapping or potentially bicistronic transcripts. The expected 3’ coverage bias can be observed for individual transcripts in the raw coverage data. (E) Results of 5′ and 3′ RACE experiments, depicting the positions of all detected m7G caps (green vertical lines) and poly(A) tails (red vertical lines) in the entB-entD (left) and entA-entH (right) gene pairs in W. versatilis. The outer and inner gene-specific primers are marked by diagonal black lines and were used along with outer and inner primers specific to the 5’ or 3’ RACE adapters provided in the kit (see Materials and Methods), which were adjacent to either the 5’ m7G cap or the 3’ poly(A) tail, respectively. Dotted lines indicate sequences amplified only during the outer nested RACE PCR step, while solid lines indicate the portions of the transcripts that were amplified during the inner nested RACE PCR step, and which were subsequently cloned for sequencing (F) Diagram of siderophore biosynthesis genes as present in the E. coli genome drawn to scale. Counts above the diagram indicate read-pairs crossmapping between genes (based on data from Seo et al., 2014, complete coverage maps shown in Figure S6). Counts below indicate the size of intergenic regions between adjacent protein-coding sequences, in base pairs (negative numbers indicate overlap). The f prefix (fA-fG) indicates the fepA-fepG genes. See also Figure S2, Figure S3 and Table S3.

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