How to characterize a strain? Clonal heterogeneity in industrial Saccharomyces influences both phenotypes and heterogeneity in phenotypes

Abstract

Populations of microbes are constantly evolving heterogeneity that selection acts upon, yet heterogeneity is nontrivial to assess methodologically. The necessary practice of isolating single‐cell colonies and thus subclone lineages for establishing, transferring, and using a strain results in single‐cell bottlenecks with a generally neglected effect on the characteristics of the strain itself. Here, we present evidence that various subclone lineages for industrial yeasts sequenced for recent genomic studies show considerable differences, ranging from loss of heterozygosity to aneuploidies. Subsequently, we assessed whether phenotypic heterogeneity is also observable in industrial yeast, by individually testing subclone lineages obtained from products. Phenotyping of industrial yeast samples and their newly isolated subclones showed that single‐cell bottlenecks during isolation can indeed considerably influence the observable phenotype. Next, we decoupled fitness distributions on the level of individual cells from clonal interference by plating single‐cell colonies and quantifying colony area distributions. We describe and apply an approach using statistical modeling to compare the heterogeneity in phenotypes across samples and subclone lineages. One strain was further used to show how individual subclonal lineages are remarkably different not just in phenotype but also in the level of heterogeneity in phenotype. With these observations, we call attention to the fact that choosing an initial clonal lineage from an industrial yeast strain may vastly influence downstream performances and observations on karyotype, on phenotype, and also on heterogeneity.

Virulence Factors and in-Host Selection on Phenotypes in Infectious Probiotic Yeast Isolates (Saccharomyces ‘boulardii’)

“…All samples were void of extensive subculturing to prevent accumulation of mutations and genome structure variations during lab propagation. Furthermore, as clonal heterogeneity even in single batches of commercial yeasts may be prevalent [35], we used two different subclone isolates of each of the probiotic yeast products available in the country. Detailed patient and isolation data enabled us to compare non-mycosis and mycosis clinical samples as well.…”

“…Stocks of the clinical and commercial isolates were saved at −70 • C in YPD broth (VWR Chemicals, Solon, OH, USA, pH 5.8) supplemented with 30% glycerol. These commercial and clinical isolates were subcultured only once (multiple single-cell bottlenecks were avoided) to prevent accumulation of potential geno-and phenotypic changes in the samples due to the phenomenon of clonal heterogeneity that was recently demonstrated for yeast strains [35]. A list of the isolates used in this study is presented in Table 1.…”

“…In other words, we do not know whether S. 'boulardii' can evolve novel characteristics under selection in the host environment, either during benign colonization or in the cases of infection. The S. 'boulardii' subclade contains very closely related isolates, but it is not entirely genetically uniform [12], and it was also shown that probiotic products, among other commercial yeasts, may contain phenotypically diverse subclonal lineages [35]. This standing genetic and phenotypic variation in the probiotic yeast may plausibly lead to in-host selection and evolution (compare with [34]) that needs to be studied.…”

Heme Oxygenase-1 (HMX1) Loss of Function Increases the In-Host Fitness of the Saccharomyces ‘boulardii’ Probiotic Yeast in a Mouse Fungemia Model

“…Stocks of the commercial and clinical isolates and deletion mutants were saved at −70 °C in YPD broth (VWR Chemicals, Solon, OH, USA, pH 5.8) supplemented with 30% glycerol. Subculturing was minimalized in order to prevent the geno- and phenotypic changes that might be a result of clonal heterogeneity [ 45 ]. Isolates were divided into three groups according to Imre et al [ 10 ]: a commercial (C; PY0001, PY0002), a non-mycosis (NM; 465/2018, 2251/2018), and a mycosis group (M; DE6507, DE35762).…”

“…Next, median coverages of all genes in the annotation of the PY0001 assembly were calculated with BEDTools. Gene copy number variations were inferred from this data as follows (adapted from [ 45 ]). Medians of the median coverages of the upstream and downstream 20 genes along with the genes in question (altogether 41 genes) were calculated for each gene (for the first and last 20 genes of each PY0001 chromosome, medians of the median coverages of the first and last 41 genes of the chromosome, respectively, were used).…”