BiLSTM-5mC: A Bidirectional Long Short-Term Memory-Based Approach for Predicting 5-Methylcytosine Sites in Genome-Wide DNA Promoters

An important reason of cancer proliferation is the change in DNA methylation patterns, characterized by the localized hypermethylation of the promoters of tumor-suppressor genes together with an overall decrease in the level of 5-methylcytosine (5mC). Therefore, identifying the 5mC sites in the promoters is a critical step towards further understanding the diverse functions of DNA methylation in genetic diseases such as cancers and aging. However, most wet-lab experimental techniques are often time consuming and laborious for detecting 5mC sites. In this study, we proposed a deep learning-based approach, called BiLSTM-5mC, for accurately identifying 5mC sites in genome-wide DNA promoters. First, we randomly divided the negative samples into 11 subsets of equal size, one of which can form the balance subset by combining with the positive samples in the same amount. Then, two types of feature vectors encoded by the one-hot method, and the nucleotide property and frequency (NPF) methods were fed into a bidirectional long short-term memory (BiLSTM) network and a full connection layer to train the 22 submodels. Finally, the outputs of these models were integrated to predict 5mC sites by using the majority vote strategy. Our experimental results demonstrated that BiLSTM-5mC outperformed existing methods based on the same independent dataset.

Structure–function studies of Rgg binding to pheromones and target promoters reveal a model of transcription factor interplay

Regulator gene of glucosyltransferase (Rgg) family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in Streptococcus species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg–SHP and Rgg–target DNA promoter specificity was unknown. To close this gap, we determined the cryoelectron microscopy (cryo-EM) structure of Streptococcus thermophilus Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures with that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both Streptococcus dysgalactiae Rgg2 and S. thermophilus Rgg3 in complex with their target DNA promoters. The physiological importance of observed Rgg–DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure–function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci.

Structural basis of Rgg protein binding to their regulatory pheromones and target DNA promoters

Rgg family proteins, such as Rgg2 and Rgg3, have emerged as primary quorum-sensing regulated transcription factors in Streptococcus species, controlling virulence, antimicrobial resistance, and biofilm formation. Rgg2 and Rgg3 function is regulated by their interaction with oligopeptide quorum-sensing signals called short hydrophobic peptides (SHPs). The molecular basis of Rgg-SHP and Rgg-target DNA promoter specificity was unknown. To close this gap, we determined the cryo-EM structure of Streptococcus thermophilus Rgg3 bound to its quorum-sensing signal, SHP3, and the X-ray crystal structure of Rgg3 alone. Comparison of these structures to that of an Rgg in complex with cyclosporin A (CsA), an inhibitor of SHP-induced Rgg activity, reveals the molecular basis of CsA function. Furthermore, to determine how Rgg proteins recognize DNA promoters, we determined X-ray crystal structures of both S. dysgalactiae Rgg2 and S. thermophilus Rgg3 in complex with their target DNA promoters. The physiological importance of the observed Rgg-DNA interactions was dissected using in vivo genetic experiments and in vitro biochemical assays. Based on these structure-function studies, we present a revised unifying model of Rgg regulatory interplay. In contrast to existing models, where Rgg2 proteins are transcriptional activators and Rgg3 proteins are transcriptional repressors, we propose that both are capable of transcriptional activation. However, when Rgg proteins with different activation requirements compete for the same DNA promoters, those with more stringent activation requirements function as repressors by blocking promoter access of the SHP-bound conformationally active Rgg proteins. While a similar gene expression regulatory scenario has not been previously described, in all likelihood it is not unique to streptococci.Significance StatementSecreted peptide pheromones regulate critical biological processes in Gram-positive bacteria. In streptococci such as the human pathogen S. pyogenes, oligopeptide pheromones, like the short hydrophobic peptides (SHPs), regulate virulence, antimicrobial resistance, and biofilm formation. SHPs directly regulate the activity of transcription factors called Rgg2 and Rgg3. We present the cryo-EM structure of Rgg3 in complex with SHP3, as well as X-ray crystal structures of Rgg2 bound to target promoter DNA, Rgg3 bound to target promoter DNA, and Rgg3 alone. Based on the cryo-EM, X-ray crystallographic, biochemical, and genetic studies presented here, we provide not only detailed mechanistic insight into the molecular basis of Rgg3-SHP3, Rgg2-DNA, and Rgg3-DNA binding specificity, but also a new model of transcription factor regulatory interplay.

Isolating promoters from Corynebacterium ammoniagenes ATCC 6871 and application in CoA synthesis

Background Corynebacterium ammoniagenes is an important industrial organism that is widely used to produce nucleotides and the potential for industrial production of coenzyme A by C. ammoniagenes ATCC 6871 has been shown. However, the yield of coenzyme A needs to be improved, and the available constitutive promoters are rather limited in this strain. Results In this study, 20 putative DNA promoters derived from genes with high transcription levels and 6 promoters from molecular chaperone genes were identified. To evaluate the activity of each promoter, red fluorescence protein (RFP) was used as a reporter. We successfully isolated a range of promoters with different activity levels, and among these a fragment derived from the upstream sequence of the 50S ribosomal protein L21 (Prpl21) exhibited the strongest activity among the 26 identified promoters. Furthermore, type III pantothenate kinase from Pseudomonas putida (PpcoaA) was overexpressed in C. ammoniagenes under the control of Prpl21, CoA yield increased approximately 4.4 times. Conclusions This study provides a paradigm for rational isolation of promoters with different activities and their application in metabolic engineering. These promoters will enrich the available promoter toolkit for C. ammoniagenes and should be valuable in current platforms for metabolic engineering and synthetic biology for the optimization of pathways to extend the product spectrum or improve the productivity in C. ammoniagenes ATCC 6871 for industrial applications.

Isolating promoters from Corynebacterium ammoniagenes ATCC 6871 and application in CoA synthesis

Background ： Corynebacterium ammoniagenes is an important industrial organism that is widely used to produce nucleotides and the potential for industrial production of coenzyme A by C. ammoniagenes ATCC 6871 has been shown. However, the yield of coenzyme A needs to be improved, and the available constitutive promoters are rather limited in this strain. Results ： In this study, 20 putative DNA promoters derived from genes with high transcription levels and 6 promoters from molecular chaperone genes were identified. To evaluate the activity of each promoter, red fluorescence protein (RFP) was used as a reporter. We successfully isolated a range of promoters with different activity levels, and among these a fragment derived from the upstream sequence of the 50S ribosomal protein L21 (P rpl21 ) exhibited the strongest activity among the 26 identified promoters. Furthermore, type III pantothenate kinase from Pseudomonas putida ( Pp coaA) was overexpressed in C. ammoniagenes under the control of P rpl21 , CoA yield increased approximately 4.4 times. Conclusions ： This study provides a paradigm for rational isolation of promoters with different activities and their application in metabolic engineering. These promoters will enrich the available promoter toolkit for C. ammoniagenes and should be valuable in current platforms for metabolic engineering and synthetic biology for the optimization of pathways to extend the product spectrum or improve the productivity in C. ammoniagenes ATCC 6871 for industrial applications.

Isolating promoters from Corynebacterium ammoniagenes ATCC 6871 and application in CoA synthesis

Background ： Corynebacterium ammoniagenes is an important industrial organism that is widely used to produce nucleotides and the potential for industrial production of coenzyme A by C. ammoniagenes ATCC 6871 has been shown. However, the yield of coenzyme A needs to be improved, and the available constitutive promoters are rather limited in this strain. Results ： In this study, 20 putative DNA promoters derived from genes with high transcription levels and 6 promoters from molecular chaperone genes were identified. To evaluate the activity of each promoter, red fluorescence protein (RFP) was used as a reporter. We successfully isolated a range of promoters with different activity levels, and among these a fragment derived from the upstream sequence of the 50S ribosomal protein L21 (P rpl21 ) exhibited the strongest activity among the 26 identified promoters. Furthermore, type III pantothenate kinase from Pseudomonas putida ( Pp coaA) was overexpressed in C. ammoniagenes under the control of P rpl21 , CoA yield increased approximately 4.4 times. Conclusions ： This study provides a paradigm for rational isolation of promoters with different activities and their application in metabolic engineering. These promoters will enrich the available promoter toolkit for C. ammoniagenes and should be valuable in current platforms for metabolic engineering and synthetic biology for the optimization of pathways to extend the product spectrum or improve the productivity in C. ammoniagenes ATCC 6871 for industrial applications.

Isolating promoters from Corynebacterium ammoniagenes ATCC 6871 and application in CoA synthesis

Background：Corynebacterium ammoniagenes is an important industrial organism that is widely used to produce nucleotides and the potential for industrial production of coenzyme A by Corynebacterium ammoniagenes ATCC 6871 has been shown. However, the yield of coenzyme A needs to be improved and the available constitutive promoters are rather limited in this strain. Results：In this study, 20 putative DNA promoters derived from genes with high transcription levels and 6 promoters from molecular chaperone genes were identified. To evaluate the activity of each promoter, red fluorescence protein (RFP) was used as a reporter. We successfully isolated a range of promoters with different activity levels resulting in different fluorescent intensities. A fragment derived from the upstream sequence of the 50S ribosomal protein L21 (Prpl21) exhibited the strongest activity among the 26 identified promoters. Furthermore, type III pantothenate kinase from Pseudomonas putida (PpcoaA) was overexpressed in C. ammoniagenes under the control of Prpl21, CoA yield increased approximately 4.1 times. Conclusions：This study provides a paradigm for rational isolation of promoters with different activities and their application in metabolic engineering. These promoters will enrich the available promoter toolkit for C. ammoniagenes and should be valuable in current platforms for metabolic engineering and synthetic biology for the optimization of pathways to extend the product spectrum or improve the productivity in C. ammoniagenes ATCC 6871 for industrial applications.