Response to Cold: A Comparative Transcriptomic Analysis in Eight Cold-Adapted Yeasts

Abstract

Microorganisms have evolved to colonize all biospheres, including extremely cold environments, facing several stressor conditions, mainly low/freezing temperatures. In general, terms, the strategies developed by cold-adapted microorganisms include the synthesis of cryoprotectant and stress-protectant molecules, cold-active proteins, especially enzymes, and membrane fluidity regulation. The strategy could differ among microorganisms and concerns the characteristics of the cold environment of the microorganism, such as seasonal temperature changes. Microorganisms can develop strategies to grow efficiently at low temperatures or tolerate them and grow under favorable conditions. These differences can be found among the same kind of microorganisms and from the same cold habitat. In this work, eight cold-adapted yeasts isolated from King George Island, subAntarctic region, which differ in their growth properties, were studied about their response to low temperatures at the transcriptomic level. Sixteen ORFeomes were assembled and used for gene prediction and functional annotation, determination of gene expression changes, protein flexibilities of translated genes, and codon usage bias. Putative genes related to the response to all main kinds of stress were found. The total number of differentially expressed genes was related to the temperature variation that each yeast faced. The findings from multiple comparative analyses among yeasts based on gene expression changes and protein flexibility by cellular functions and codon usage bias raise significant differences in response to cold among the studied Antarctic yeasts. The way a yeast responds to temperature change appears to be more related to its optimal temperature for growth (OTG) than growth velocity. Yeasts with higher OTG prepare to downregulate their metabolism to enter the dormancy stage. In comparison, yeasts with lower OTG perform minor adjustments to make their metabolism adequate and maintain their growth at lower temperatures.

Keywords: Antarctic yeasts; codon bias; cold adaptation; cold-adapted yeasts; stress genes; transcriptomes.

FIGURE 1

Box plot for the log₂ fold change of putative genes between yeasts cultivated at high temperature and 4°C. IQR, Interquartile range.

FIGURE 2

Differentially expressed putative genes classified by metabolic pathways between yeasts cultivated at high temperature (HT) and 4°C. The number of differentially expressed genes (DEGs) is shown in panel (A), and the average of Log₂-fold change (log₂FC) by pathway is shown in panel (B). Down, downregulated DEGs; Up, upregulated DEGs. Genet. Inf. Proc, Genetic Information Processing; Env. Inf. Proc, Environmental Information Processing; Cel. Proc., Cellular Processes.

FIGURE 3

Log₂-fold change of putative genes classified in different kinds of stress responses (A) and ribosomal subunits (B).

FIGURE 4

Comparisons among yeasts of the flexibility of translated proteins classified in cellular pathways. The flexibility was estimated as percentages of very flexible (Vf) and very flexible plus moderately flexible (VMf) amino acids and M2 and M1 + 2 levels, calculated by MEDUSA. The proteins grouped in cellular pathways were compared, and those with significant differences (Tukey post hoc tests, p < 0.05) are shown. The comparisons were performed among all yeasts individually (A) and grouped according to their growth rates (B) (h-1) and their optimal temperature for growth (C) (°C).

FIGURE 5

Clustering of yeasts according to codon frequencies. The codon frequencies were calculated using highly expressed ORFs (≥1,000) in the eight Antarctic yeasts studied here, from the Kazusa coding usage database and from (Baeza et al., 2015). Scer, S. cerevisiae; Ppas, Pichia pastoris; Cant, Candida antarctica; Cthe, Candida thermophila; Calb, Candida albicans; Cneof, Cryptococcus neoformans var. Grubii; Cwic, Candida wickerhamii; Cdub, Candida dubliniensis; Cint, Candida intermedia; Ctro, Candida tropicalis; Clusi, Clavispora lusitania; DhanCBS767, Debaryomyces hansenii CBS767; Huva, Hanseniaspora uvarum; Prhod, Phaffia rhodozyma; Mfur, Malassezia furfur; Mpach, Malassezia pachydermatis; Cglab, Candida glabrata; Ptsuk, Pseudozyma tsukubaensis; Cposa, Coccidioides posadasii; KlacNRRL Y-1141, Kluyveromyces lactis NRRL Y-1141; Xd385, Xanthophyllomyces dendrorhous UCD67-385.