Dissecting an ancient stress resistance trait syndrome in the compost yeast Kluyveromyces marxianus

Abstract

In the search to understand how evolution builds new traits, ancient events are often the hardest to dissect. Species-unique traits pose a particular challenge for geneticists-cases in which a character arose long ago and, in the modern day, is conserved within a species, distinguishing it from reproductively isolated relatives. In this work, we have developed the budding yeast genus Kluyveromyces as a model for mechanistic dissection of trait variation across species boundaries. Phenotypic profiling revealed robust heat and chemical-stress tolerance phenotypes that distinguished the compost yeast K. marxianus from the rest of the clade. We used culture-based, transcriptomic, and genetic approaches to characterize the metabolic requirements of the K. marxianus trait syndrome. We then generated a population-genomic resource for K. marxianus and harnessed it in molecular-evolution analyses, which found hundreds of housekeeping genes with evidence for adaptive protein variation unique to this species. Our data support a model in which, in the distant past, K. marxianus underwent a vastly complex remodeling of its proteome to achieve stress resistance. Such a polygenic architecture, involving nucleotide-level allelic variation on a massive scale, is consistent with theoretical models of the mechanisms of long-term adaptation, and suggests principles of broad relevance for interspecies trait genetics.

Figure 1.. K. marxianus shows a unique heat and chemical stress resistance phenotype.

A, Phylogenetic tree of the Kluyveromyces genus with marine and terrestrial species delineated^,. Branch lengths are not to scale. B, Each panel reports the results of a growth profiling experiment across the genus in the indicated condition. In a given panel, each trace reports a growth timecourse for the indicated species, with the solid line displaying the mean across technical replicates and the faint outline showing the standard error. OD, optical density.

Figure 2.. The unique heat tolerance of K. marxianus only manifests during its growing stage.

A, Experimental workflow created with BioRender.com. From left, inoculum from a starting liquid culture is introduced into fresh liquid medium for regrowth at 28°C and then sampled at nutrient exhaustion (Stationary) or in log phase (Exponential). The liquid sample is subjected to a heat shock of 47°C for 30 minutes and then spotted as serial dilutions onto solid medium. Viable cells after heat shock grow into colonies after incubation at 28°C. B-C, Each row reports viability after heat shock of a sample of the indicated species in the indicated liquid growth phase. See Figure S3 for unshocked controls.

Figure 3.. Overall regulatory volatility by K. lactis relative to K. marxianus and stress-evoked repression of glycolysis in both species.

A, Shown are results of principal component (PC) analysis of K. lactis and K. marxianus transcriptomes across treatments. Each point reports the values of the top two PCs for one replicate transcriptome of the indicated species subjected to the indicated stress treatment or a 28°C unstressed control. In a given axis label, the value in parentheses reports the proportion of variance across the set of transcriptomes explained by the indicated PC. B,C, Each panel reports the effect of stress on expression of genes of the indicated Gene Ontology term in K. marxianus and K. lactis. In a given panel, each cell reports, for the indicated gene, the log₂ fold-change between expression levels in the indicated stress treatment and an unstressed control.

Figure 4.. Genetic or culture-based block of respiration compromises stress tolerance in K. marxianus and K. lactis.

A, B, Each panel reports cell viability assays after culture in liquid medium with stress, or an unstressed control, of K. marxianus or K. lactis in normoxia or hypoxia. In a given panel, each row shows serial dilutions of the liquid culture spotted onto solid medium and incubated without stress at 28°C. C, Data are as in A,B except that panels report wild-type K. marxianus or an isogenic strain lacking the respiration factor COX15.

Figure 5.. K. marxianus has many genes with independently evolving sites.

A, Each bar shows the number of genes with significant evidence for positive selection on the indicated lineage (adjusted p > 0.05) in a phylogenetic PAML branch-site test. B, The set of bars of each color reports the distribution, across the genes from A, of the number of sites per gene inferred by PAML to be targets of positive selection on the indicated lineage. C, Amino acid alignment of an example region of a gene, KLLA0B02607g (S. cerevisiae ortholog FAT3/YKL187C), with evidence for positive selection from the PAML test in A and from the McDonald-Kreitman test of Table 1. Highlighted columns show inferred targets of positive selection from the PAML test in B. Highlighting indicates the amino acid’s BLOSUM 62 matrix score with respect to the consensus amino acid.