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The Fungus Candida albicans Tolerates Ambiguity at Multiple Codons.

Simões J, Bezerra AR, Moura GR, Araújo H, Gut I, Bayes M, Santos MA - Front Microbiol (2016)

Bottom Line: In order to determine whether such flexibility also exists at other codons, we have constructed several serine tRNAs that decode various non-cognate codons.Parallel evolution of the recombinant strains (100 generations) followed by full genome resequencing identified various strain specific single nucleotide polymorphisms (SNP) and one SNP in the deneddylase (JAB1) gene in all strains.Since JAB1 is a subunit of the COP9 signalosome complex, which interacts with cullin (Cdc53p) to mediate degradation of a variety of cellular proteins, our data suggest that neddylation plays a key role in tolerance and adaptation to codon ambiguity in C. albicans.

View Article: PubMed Central - PubMed

Affiliation: Health Sciences Program, Department of Medical Sciences, Institute of Biomedicine - iBiMED, University of Aveiro Aveiro, Portugal.

ABSTRACT
The ascomycete Candida albicans is a normal resident of the gastrointestinal tract of humans and other warm-blooded animals. It occurs in a broad range of body sites and has high capacity to survive and proliferate in adverse environments with drastic changes in oxygen, carbon dioxide, pH, osmolarity, nutrients, and temperature. Its biology is unique due to flexible reassignment of the leucine CUG codon to serine and synthesis of statistical proteins. Under standard growth conditions, CUG sites incorporate leucine (3% of the times) and serine (97% of the times) on a proteome wide scale, but leucine incorporation fluctuates in response to environmental stressors and can be artificially increased up to 98%. In order to determine whether such flexibility also exists at other codons, we have constructed several serine tRNAs that decode various non-cognate codons. Expression of these tRNAs had minor effects on fitness, but growth of the mistranslating strains at different temperatures, in medium with different pH and nutrients composition was often enhanced relatively to the wild type (WT) strain, supporting our previous data on adaptive roles of CUG ambiguity in variable growth conditions. Parallel evolution of the recombinant strains (100 generations) followed by full genome resequencing identified various strain specific single nucleotide polymorphisms (SNP) and one SNP in the deneddylase (JAB1) gene in all strains. Since JAB1 is a subunit of the COP9 signalosome complex, which interacts with cullin (Cdc53p) to mediate degradation of a variety of cellular proteins, our data suggest that neddylation plays a key role in tolerance and adaptation to codon ambiguity in C. albicans.

No MeSH data available.


Related in: MedlinePlus

Phenotypic assays. Data represent the average size of the colonies obtained for all the transformed cells after 5 days of growth under the indicated conditions normalized with growth at 30°C and compared with the normalized control pUA552 (considered 0%) for that condition (relative growth score). The assay was performed with two independent growth cultures and three independent clones. Brick color intensity represents the strength of the phenotype. White squares represent a phenotype that is indistinguishable from the control strain pUA552. Green and red squares represent either a growth reduction or enhancement, respectively.
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Figure 2: Phenotypic assays. Data represent the average size of the colonies obtained for all the transformed cells after 5 days of growth under the indicated conditions normalized with growth at 30°C and compared with the normalized control pUA552 (considered 0%) for that condition (relative growth score). The assay was performed with two independent growth cultures and three independent clones. Brick color intensity represents the strength of the phenotype. White squares represent a phenotype that is indistinguishable from the control strain pUA552. Green and red squares represent either a growth reduction or enhancement, respectively.

Mentions: Previous studies also showed that yeast strains mistranslating the CUG codon have a competitive edge under specific stress conditions, namely high temperature, salts, heavy metals, and oxidants (Santos et al., 1999). To clarify whether mistranslation of different codons would also produce positive growth outcomes, a phenotypic screen assay testing 21 different conditions was carried out. These tests probed a broad spectrum of metabolic networks, including growth in different carbon sources, pH values (pH 5–8.6) and different temperatures (25–42°C) on agar plates supplemented with elevated concentrations of cations or antifungal agents (Supplementary Table 2). For drug and nutrient conditions the concentrations were calibrated to determine whether impairment or enhancement of growth relative to control could be observed. Independent isolates of each mistranslating strain were plated as a series of 10 fold dilutions on solid media, using a 96-pin bolt replicator (Caliper). After 3–5 days of incubation, plates were photographed using Axio Vision Software from Zeiss. All images were imported and processed using ImageJ software and scored for growth phenotypes by comparison to the control strains (pUA552) included in the test plates. This approach generated individual growth scores for each mutant on each growth medium (Figure 2). Hence, the scoring system measured the reduction or enhancement of growth relative to control (pUA552). The majority of the strains showed growth disadvantages at 37°C, except strains misreading the Leu CTA and Ala GCC codons. At 42°C the strains misreading the Lys AAG and Tyr TAC codons showed a slight growth advantage. Strains misreading Leu CTC and CTA and Thr ACC codons displayed advantageous growth in media containing CaCl2, CuSO4 (Figure 3), and galactose as single carbon source. Medium with pH 5.0 was deleterious to strains misreading Leu CTC and CTT, Ile ATC and Ala GCC codons. Although the changes in growth were idiosyncratic with each strain exhibiting a unique pattern of phenotypes, all strains had increased resistance to sorbitol (1.5 M) and guanidine hydro chloride (5 mM). When exposed to urea (25 mM), strains misreading Leu, Thr and Tyr codons also displayed advantageous growth. Finally, the sensitivity to oxidative stress was tested in media containing hydrogen peroxide (H2O2), and the strains misreading Leu CTC, Gly GGA, Lys AAG, and Thr ACC codons, grew faster than the control. Overall, there was variability between clones and stress conditions, nevertheless, strains expressing the misreading tRNAs performed better than the control strain in various growth conditions, supporting previous data from our laboratory on selective advantages of tRNA misreading under stress (Bezerra et al., 2013).


The Fungus Candida albicans Tolerates Ambiguity at Multiple Codons.

Simões J, Bezerra AR, Moura GR, Araújo H, Gut I, Bayes M, Santos MA - Front Microbiol (2016)

Phenotypic assays. Data represent the average size of the colonies obtained for all the transformed cells after 5 days of growth under the indicated conditions normalized with growth at 30°C and compared with the normalized control pUA552 (considered 0%) for that condition (relative growth score). The assay was performed with two independent growth cultures and three independent clones. Brick color intensity represents the strength of the phenotype. White squares represent a phenotype that is indistinguishable from the control strain pUA552. Green and red squares represent either a growth reduction or enhancement, respectively.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4814463&req=5

Figure 2: Phenotypic assays. Data represent the average size of the colonies obtained for all the transformed cells after 5 days of growth under the indicated conditions normalized with growth at 30°C and compared with the normalized control pUA552 (considered 0%) for that condition (relative growth score). The assay was performed with two independent growth cultures and three independent clones. Brick color intensity represents the strength of the phenotype. White squares represent a phenotype that is indistinguishable from the control strain pUA552. Green and red squares represent either a growth reduction or enhancement, respectively.
Mentions: Previous studies also showed that yeast strains mistranslating the CUG codon have a competitive edge under specific stress conditions, namely high temperature, salts, heavy metals, and oxidants (Santos et al., 1999). To clarify whether mistranslation of different codons would also produce positive growth outcomes, a phenotypic screen assay testing 21 different conditions was carried out. These tests probed a broad spectrum of metabolic networks, including growth in different carbon sources, pH values (pH 5–8.6) and different temperatures (25–42°C) on agar plates supplemented with elevated concentrations of cations or antifungal agents (Supplementary Table 2). For drug and nutrient conditions the concentrations were calibrated to determine whether impairment or enhancement of growth relative to control could be observed. Independent isolates of each mistranslating strain were plated as a series of 10 fold dilutions on solid media, using a 96-pin bolt replicator (Caliper). After 3–5 days of incubation, plates were photographed using Axio Vision Software from Zeiss. All images were imported and processed using ImageJ software and scored for growth phenotypes by comparison to the control strains (pUA552) included in the test plates. This approach generated individual growth scores for each mutant on each growth medium (Figure 2). Hence, the scoring system measured the reduction or enhancement of growth relative to control (pUA552). The majority of the strains showed growth disadvantages at 37°C, except strains misreading the Leu CTA and Ala GCC codons. At 42°C the strains misreading the Lys AAG and Tyr TAC codons showed a slight growth advantage. Strains misreading Leu CTC and CTA and Thr ACC codons displayed advantageous growth in media containing CaCl2, CuSO4 (Figure 3), and galactose as single carbon source. Medium with pH 5.0 was deleterious to strains misreading Leu CTC and CTT, Ile ATC and Ala GCC codons. Although the changes in growth were idiosyncratic with each strain exhibiting a unique pattern of phenotypes, all strains had increased resistance to sorbitol (1.5 M) and guanidine hydro chloride (5 mM). When exposed to urea (25 mM), strains misreading Leu, Thr and Tyr codons also displayed advantageous growth. Finally, the sensitivity to oxidative stress was tested in media containing hydrogen peroxide (H2O2), and the strains misreading Leu CTC, Gly GGA, Lys AAG, and Thr ACC codons, grew faster than the control. Overall, there was variability between clones and stress conditions, nevertheless, strains expressing the misreading tRNAs performed better than the control strain in various growth conditions, supporting previous data from our laboratory on selective advantages of tRNA misreading under stress (Bezerra et al., 2013).

Bottom Line: In order to determine whether such flexibility also exists at other codons, we have constructed several serine tRNAs that decode various non-cognate codons.Parallel evolution of the recombinant strains (100 generations) followed by full genome resequencing identified various strain specific single nucleotide polymorphisms (SNP) and one SNP in the deneddylase (JAB1) gene in all strains.Since JAB1 is a subunit of the COP9 signalosome complex, which interacts with cullin (Cdc53p) to mediate degradation of a variety of cellular proteins, our data suggest that neddylation plays a key role in tolerance and adaptation to codon ambiguity in C. albicans.

View Article: PubMed Central - PubMed

Affiliation: Health Sciences Program, Department of Medical Sciences, Institute of Biomedicine - iBiMED, University of Aveiro Aveiro, Portugal.

ABSTRACT
The ascomycete Candida albicans is a normal resident of the gastrointestinal tract of humans and other warm-blooded animals. It occurs in a broad range of body sites and has high capacity to survive and proliferate in adverse environments with drastic changes in oxygen, carbon dioxide, pH, osmolarity, nutrients, and temperature. Its biology is unique due to flexible reassignment of the leucine CUG codon to serine and synthesis of statistical proteins. Under standard growth conditions, CUG sites incorporate leucine (3% of the times) and serine (97% of the times) on a proteome wide scale, but leucine incorporation fluctuates in response to environmental stressors and can be artificially increased up to 98%. In order to determine whether such flexibility also exists at other codons, we have constructed several serine tRNAs that decode various non-cognate codons. Expression of these tRNAs had minor effects on fitness, but growth of the mistranslating strains at different temperatures, in medium with different pH and nutrients composition was often enhanced relatively to the wild type (WT) strain, supporting our previous data on adaptive roles of CUG ambiguity in variable growth conditions. Parallel evolution of the recombinant strains (100 generations) followed by full genome resequencing identified various strain specific single nucleotide polymorphisms (SNP) and one SNP in the deneddylase (JAB1) gene in all strains. Since JAB1 is a subunit of the COP9 signalosome complex, which interacts with cullin (Cdc53p) to mediate degradation of a variety of cellular proteins, our data suggest that neddylation plays a key role in tolerance and adaptation to codon ambiguity in C. albicans.

No MeSH data available.


Related in: MedlinePlus