Yu-Dong Li

The rapid evolution of signal peptides is mainly caused by relaxed selection on non-synonymous and synonymous sites

The precursor of a secretory protein usually contains an N-terminal signal peptide (SP), which directs the protein to cross the membrane. We performed a genome-wide analysis of secretory proteins in prokaryotes and eukaryotes, and found that signal peptides evolved faster than mature proteins. To determine whether the evolutionary pattern could be explained by selective pressure changes, we studied the amino acid replacements in signal peptides. We found that they tend to be more conserved than those in mature regions of the proteins, suggesting relaxed selective pressure acting on non-synonymous sites. This is potentially explained by similar biochemical requirements of signal peptides. We also observed a decreased codon adaptation index (CAI), suggesting a relaxed purifying selection on synonymous sites of signal peptides. In addition, the evolutionary rate of signal sequences increases with codon usage bias, suggesting that increased rare codon frequency in signal peptides is a result of natural selection to improve secretion efficiency. Evidence also suggests signal peptides might have undergone positive selection. In summary, the evolution of signal peptides may be caused by a mixture of selection forces, primarily relaxation of purifying selection.

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.

New Approach to Achieve High-Level Secretory Expression of Heterologous Proteins by Using Tat Signal Peptide

The twin-arginine translocation (Tat) pathway is an attractive route for secretory production of heterologous proteins in E. coli. In this study, we investigated the potential use of Tat signal peptide from S. coelicolor to improve secretory expression. The results showed that Tat signal peptide (ssDagA) could effectively secrete active Green fluorescent protein (GFP) to periplasm. When the rare codons of signal sequence were optimized, the expression and secretion yield of GFP improved by about 2-3 folds as detected qualitatively by western blotting and fluorescent analysis. The increase of translation rate could be explained by the unstability of mRNA secondary structure. In summary, our strategy could provide a new approach for high-level secretory expression of heterologous proteins in E. coli.

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.

Function and Evolution of Two Forms of SecDF Homologs in Streptomyces coelicolor

The general secretion (Sec) pathway plays a prominent role in bacterial protein export, and the accessory component SecDF has been shown to improve transportation efficiency. Inspection of Streptomyces coelicolor genome reveals the unexpected presence of two different forms of secDF homologous genes: one in fused form (secDF) and the other in separated form (secD and secF). However, the functional role of two SecDF homologs in S. coelicolor has not yet been determined. Transcriptional analysis of secDF homologs reveals that these genes are constitutively expressed. However, the transcript levels of secD and secF are much higher than that of secDF in S. coelicolor. Deletion of secDF or/and secD/secF in S. coelicolor did result in reduced secretion efficiency of Xylanase A and Amylase C, suggesting that they may have redundant functions for Sec-dependent translocation pathway. Moreover, our results also indicate that SecD/SecF plays a more prominent role than SecDF in protein translocation. Evolutionary analysis suggests that the fused and separated SecDF homologs in Streptomyces may have disparate evolutionary ancestries. SecD/SecF may be originated from vertical transmission of existing components from ancestor of Streptomyces species. However, SecDF may be derived from bacterial ancestors through horizontal gene transfer. Alternately, it is also plausible that SecDF may have arisen through additional gene duplication and fusion events. The acquisition of a second copy may confer a selective benefit to Streptomyces by enhancing protein transport capacity. Taken together, our results provide new insights into the potential biological function and evolutionary aspects of the prokaryotic SecDF complex.

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.

Characterization and evolutionary implications of the triad Asp-Xxx-Glu in group II phosphopantetheinyl transferases.

Phosphopantetheinyl transferases (PPTases), which play an essential role in both primary and secondary metabolism, are magnesium binding enzymes. In this study, we characterized the magnesium binding residues of all known group II PPTases by biochemical and evolutionary analysis. Our results suggested that group II PPTases could be classified into two subgroups, two-magnesium-binding-residue-PPTases containing the triad Asp-Xxx-Glu and three-magnesium-binding-residue-PPTases containing the triad Asp-Glu-Glu. Mutations of two three-magnesium-binding-residue-PPTases and one two-magnesium-binding-residue-PPTase indicate that the first and the third residues in the triads are essential to activities; the second residues in the triads are non-essential. Although variations of the second residues in the triad Asp-Xxx-Glu exist throughout the whole phylogenetic tree, the second residues are conserved in animals, plants, algae, and most prokaryotes, respectively. Evolutionary analysis suggests that: the animal group II PPTases may originate from one common ancestor; the plant two-magnesium-binding-residue-PPTases may originate from one common ancestor; the plant three-magnesium-binding-residue-PPTases may derive from horizontal gene transfer from prokaryotes.

Complete Genome Sequence of Bifidobacterium actinocoloniiforme Type Strain DSM 22766T, Isolated from Bumblebee Digestive Tracts

ABSTRACT Bifidobacteria are one of the most important beneficial bacteria in the gut of mammals and insects. We sequenced the genome of B. actinocoloniiforme DSM 22766, which was isolated from the digestive tracts of bumblebees. The genome contains 1,548 protein-coding genes, 49 RNAs and two CRISPR repeats.

Two Bacterial Group II Phosphopantetheinyl Transferases Involved in Both Primary Metabolism and Secondary Metabolism

It is known that bacterial group II phosphopantetheinyl transferases (PPTases) usually phosphopantetheinylate acyl carrier proteins (ACPs) involved in the secondary metabolism. For example, a bacterial group II PPTase SchPPT has been known to phosphopantetheinylate only ACPs involved in secondary metabolism, such as scn ACP0-2 and scn ACP7. In this study, we found two bacterial group II PPTases, Hppt and Sppt, could phosphopantetheinylate not only scn ACP0-2 and scn ACP7, but also sch FAS ACP, an ACP involved in primary metabolism. Swapping of the N terminus and C terminus of PPTases showed that (i) both the hybrids Hppt-Sppt and Sppt-Hppt could phosphopantetheinylate sch FAS ACP but not scn ACP0-2; (ii) both the hybrids Sppt-SchPPT and SchPPT-Sppt lost abilities to phosphopantetheinylate sch FAS ACP and scn ACP0-2. Hppt and Sppt represent group II PPTases which phosphopantetheinylate both ACPs involved in primary metabolism and ACPs involved in secondary metabolism.

Erratum for Chen et al., Complete Genome Sequence of Bifidobacterium actinocoloniiforme Type Strain DSM 22766T, Isolated from Bumblebee Digestive Tracts

Volume 3, no. 5, [e01084-15][1], 2015. Page 1: The byline and affiliation line should read as given above. [1]: /lookup/doi/10.1128/genomeA.01084-15