Wenjun Guan

Relaxation of yeast mitochondrial functions after whole-genome duplication

Mitochondria are essential for cellular energy production in most eukaryotic organisms. However, when glucose is abundant, yeast species that underwent whole-genome duplication (WGD) mostly conduct fermentation even under aerobic conditions, and most can survive without a functional mitochondrial genome. In this study, we show that the rate of evolution for the nuclear-encoded mitochondrial genes was greater in post-WGD species than pre-WGD species. Furthermore, codon usage bias was relaxed for these genes in post-WGD yeast species. The codon usage pattern and the distribution of a particular transcription regulatory element suggest that the change to an efficient aerobic fermentation lifestyle in this lineage might have emerged after WGD between the divergence of Kluyveromyces polysporus and Saccharomyces castellii from their common ancestor. This new energy production strategy could have led to the relaxation of mitochondrial function in the relevant yeast species.

Tinkering Evolution of Post-Transcriptional RNA Regulons: Puf3p in Fungi as an Example

Genome-wide studies of post-transcriptional mRNA regulation in model organisms indicate a “post-transcriptional RNA regulon” model, in which a set of functionally related genes is regulated by mRNA–binding RNAs or proteins. One well-studied post-transcriptional regulon by Puf3p functions in mitochondrial biogenesis in budding yeast. The evolution of the Puf3p regulon remains unclear because previous studies have shown functional divergence of Puf3p regulon targets among yeast, fruit fly, and humans. By analyzing evolutionary patterns of Puf3p and its targeted genes in forty-two sequenced fungi, we demonstrated that, although the Puf3p regulon is conserved among all of the studied fungi, the dedicated regulation of mitochondrial biogenesis by Puf3p emerged only in the Saccharomycotina clade. Moreover, the evolution of the Puf3p regulon was coupled with evolution of codon usage bias in down-regulating expression of genes that function in mitochondria in yeast species after genome duplication. Our results provide a scenario for how evolution like a tinker exploits pre-existing materials of a conserved post-transcriptional regulon to regulate gene expression for novel functional roles.

Detecting positive selection in the budding yeast genome

Available abundant genomic data allows us to study the evolution of the yeast genome at a fine scale. In this study, we examined the adaptive evolution of coding and promoter regions in three Saccharomyces cerevisiae strains. First, using a maximum‐likelihood approach, we identified 76 positively selected genes (PSG) whose coding regions likely have undergone positive selection in the recent past. These genes show significant bias in terms of biological function and they show over‐representation of charged amino acids at positively selected sites. Next, using recent data on yeast transcription‐start sites to define core‐promoter regions, we identified 31 positively selected promoters, and their corresponding genes are significantly enriched in transmembrane transporter function. We found PSG show no correlation with promoter adaption or expression variation, suggesting that positive selection on coding regions and positive selection on promoter regions are not coupled. Together, our study provides insights into the evolution of S. cerevisiae strains from different environments.

Reciprocal Regulation between SigK and Differentiation Programs in Streptomyces coelicolor

Here we reported that deletion of SigK (SCO6520), a sigma factor in Streptomyces coelicolor, caused an earlier switch from vegetative mycelia to aerial mycelia and higher expression of chpE and chpH than that in the wild type. Loss of SigK also resulted in accelerated and enhanced production of antibiotics, actinorhodin, and undecylprodigiosin and increased expression of actII-orf4 and redD. These results suggested that SigK had a negative role in morphological transition and secondary metabolism. Furthermore, the sigK promoter (sigKp) activity gradually increased and sigK expression was partially dependent on SigK, but this dependence decreased during the developmental course of substrate mycelia. Meanwhile, two potentially nonspecific cleavages occurred between SigK and green fluorescent protein, and the SigK fusion proteins expressed under the constitutive promoter ermEp* sharply decreased and disappeared when aerial mycelia emerged. If expressed under sigKp, 3FLAG-SigK showed similar dynamic patterns but did not decrease as sharply as SigK expressed under ermEp*. These data suggested that the climbing expression of sigK might reduce the prompt degradation of SigK during vegetative hypha development for the proper timing of morphogenesis and that SigK vanished to remove the block for the emergence of aerial mycelia. Thus, we proposed that SigK had inhibitory roles on developmental events and that these inhibitory effects may be released by SigK degradation.

Involvement of SigT and RstA in the differentiation of Streptomyces coelicolor

MINT‐7262574: RstA (uniprotkb:Q9S6U2) physically interacts (MI:0915) with sigT (uniprotkb:O86856) by anti tag coimmunoprecipitation (MI:0007)

Antagonistic Changes in Sensitivity to Antifungal Drugs by Mutations of an Important ABC Transporter Gene in a Fungal Pathogen

Fungal pathogens can be lethal, especially among immunocompromised populations, such as patients with AIDS and recipients of tissue transplantation or chemotherapy. Prolonged usage of antifungal reagents can lead to drug resistance and treatment failure. Understanding mechanisms that underlie drug resistance by pathogenic microorganisms is thus vital for dealing with this emerging issue. In this study, we show that dramatic sequence changes in PDR5, an ABC (ATP-binding cassette) efflux transporter protein gene in an opportunistic fungal pathogen, caused the organism to become hypersensitive to azole, a widely used antifungal drug. Surprisingly, the same mutations conferred growth advantages to the organism on polyenes, which are also commonly used antimycotics. Our results indicate that Pdr5p might be important for ergosterol homeostasis. The observed remarkable sequence divergence in the PDR5 gene in yeast strain YJM789 may represent an interesting case of adaptive loss of gene function with significant clinical implications.

FtsY Affects Sporulation and Antibiotic Production by whiH in Streptomyces coelicolor

FtsY, the Signal Recognition Particle (SRP) receptor in bacteria, is known to facilitate the cotranslational protein targeting by recruiting SRP-protein complex to secYEG. We show in this work that deletion of the ftsY gene in Streptomyces coelicolor would also lead to complete blockage of sporulation process and reduced production of antibiotic actinorhodin. These defects cannot be complemented by only the NG domain of FtsY, while full-length ftsY was able to restore spore generation and increase production of actinorhodin in ftsY-disrupted strains. Further transcriptional analysis on sporulation controlling genes, i.e., whiG, whiB, whiH, and prox, indicated that the regulation of sporulation by ftsY is likely to take effect through whiH.

A Mutation in Intracellular Loop 4 Affects the Drug-Efflux Activity of the Yeast Multidrug Resistance ABC Transporter Pdr5p

Multidrug resistance protein Pdr5p is a yeast ATP-binding cassette (ABC) transporter in the plasma membrane. It confers multidrug resistance by active efflux of intracellular drugs. However, the highly polymorphic Pdr5p from clinical strain YJM789 loses its ability to expel azole and cyclohexmide. To investigate the role of amino acid changes in this functional change, PDR5 chimeras were constructed by segmental replacement of homologous BY4741 PDR5 fragments. Functions of PDR5 chimeras were evaluated by fluconazole and cycloheximide resistance assays. Their expression, ATPase activity, and efflux efficiency for other substrates were also analyzed. Using multiple lines of evidence, we show that an alanine-to-methionine mutation at position 1352 located in the predicted short intracellular loop 4 significantly contributes to the observed transport deficiency. The degree of impairment is likely correlated to the size of the mutant residue.

Mutations adjacent to the end of transmembrane helices 6 and 7 independently affect drug efflux capacity of yeast ABC transporter Pdr5p.

As a mammalian p-glycoprotein homolog, Pdr5p is a major ATP-binding cassette transporter for cellular detoxification in the yeast Saccharomyces cerevisiae. In this study, two novel loss-of-function mutations located adjacent to the ends of the predicted transmembrane helices of Pdr5p were identified. C793F and S1230L mutations considerably impaired the transport activity of Pdr5p without affecting the ATPase activity and the expression level of the protein. Our results demonstrate that the size of residue 793 and the hydrophobicity of residue 1230 are important for Pdr5p efflux function. It reveals that amino acid residues located near the end of transmembrane helix play an important role in drug efflux of Pdr5p. Molecular docking results further suggest that these two single mutations might have disturbed interactions between the drugs and Pdr5p, preventing the drugs from approaching the intracellular or extracellular portal and subsequently from being exported by Pdr5p.

Multiple transporters are involved in natamycin efflux in Streptomyces chattanoogensis L10

Antibiotic‐producing microorganisms have evolved several self‐resistance mechanisms to prevent auto‐toxicity. Overexpression of specific transporters to improve the efflux of toxic antibiotics has been found one of the most important and intrinsic resistance strategies used by many Streptomyces strains. In this work, two ATP‐binding cassette (ABC) transporter‐encoding genes located in the natamycin biosynthetic gene cluster, scnA and scnB, were identified as the primary exporter genes for natamycin efflux in Streptomyces chattanoogensis L10. Two other transporters located outside the cluster, a major facilitator superfamily transporter Mfs1 and an ABC transporter NepI/II were found to play a complementary role in natamycin efflux. ScnA/ScnB and Mfs1 also participate in exporting the immediate precursor of natamycin, 4,5‐de‐epoxynatamycin, which is more toxic to S. chattanoogensis L10 than natamycin. As the major complementary exporter for natamycin efflux, Mfs1 is up‐regulated in response to intracellular accumulation of natamycin and 4,5‐de‐epoxynatamycin, suggesting a key role in the stress response for self‐resistance. This article discusses a novel antibiotic‐related efflux and response system in Streptomyces, as well as a self‐resistance mechanism in antibiotic‐producing strains.