scholarly journals Unraveling Protein Interactions between the Temperate Virus Bam35 and Its Bacillus Host Using an Integrative Yeast Two Hybrid–High Throughput Sequencing Approach

2021 ◽  
Vol 22 (20) ◽  
pp. 11105
Author(s):  
Ana Lechuga ◽  
Cédric Lood ◽  
Mónica Berjón-Otero ◽  
Alicia del Prado ◽  
Jeroen Wagemans ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Sequencing ◽  
Genomic Library ◽  
Structural Protein ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Novel Approach ◽  
Host Membrane ◽  
Two Hybrid

Bacillus virus Bam35 is the model Betatectivirus and member of the family Tectiviridae, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. Interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, which are known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein–protein interactions (PPIs) network for a tectivirus–host system by studying the Bam35–Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and high-throughput sequencing (Y2H-HTS). We generated and thoroughly analyzed a genomic library of Bam35′s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait–prey couples. Initial analysis of the raw data enabled the identification of over 4000 candidate interactions, which were sequentially filtered to produce 182 high-confidence interactions that were defined as part of the core virus–host interactome. Overall, host metabolism proteins and peptidases were particularly enriched within the detected interactions, distinguishing this host–phage system from the other reported host–phage PPIs. Our approach also suggested biological roles for several Bam35 proteins of unknown function, including the membrane structural protein P25, which may be a viral hub with a role in host membrane modification during viral particle morphogenesis. This work resulted in a better understanding of the Bam35–B. thuringiensis interaction at the molecular level and holds great potential for the generalization of the Y2H-HTS approach for other virus–host models.

scholarly journals Unraveling protein interactions between the temperate virus Bam35 and its Bacillus host using an integrative yeast two hybrid-high throughput sequencing approach

2021 ◽  
Author(s):  
Ana Lechuga ◽  
Cédric Lood ◽  
Mónica Berjón-Otero ◽  
Alicia Del Prado ◽  
Jeroen Wagemans ◽  
...  
Keyword(s):  
High Throughput ◽  
Protein Interactions ◽  
High Throughput Sequencing ◽  
Genomic Library ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Protein Protein Interaction ◽  
Novel Approach ◽  
Diverse Groups ◽  
Two Hybrid

Bacillus virus Bam35 is the model Betatectivirus and member of the Tectiviridae family, which is composed of tailless, icosahedral, and membrane-containing bacteriophages. The interest in these viruses has greatly increased in recent years as they are thought to be an evolutionary link between diverse groups of prokaryotic and eukaryotic viruses. Additionally, betatectiviruses infect bacteria of the Bacillus cereus group, known for their applications in industry and notorious since it contains many pathogens. Here, we present the first protein-protein interactions network for a tectivirus-host system by studying the Bam35- Bacillus thuringiensis model using a novel approach that integrates the traditional yeast two-hybrid system and Illumina high-throughput sequencing. We generated and thoroughly analyzed a genomic library of Bam35’s host B. thuringiensis HER1410 and screened interactions with all the viral proteins using different combinations of bait-prey couples. In total, this screen resulted in the detection of over 4,000 potential interactions, of which 183 high-confidence interactions were defined as part of the core virus-host interactome. Overall, host metabolism proteins and peptidases are particularly enriched within the detected interactions, distinguishing this host-phage system from the other reported host-phage protein-protein interaction networks (PPIs). Our approach also suggests biological roles for several Bam35 proteins of unknown function, resulting in a better understanding of the Bam35- B. thuringiensis interaction at the molecular level.

Analysing protein–protein interactions of the Myxococcus xanthus Dif signalling pathway using the yeast two-hybrid system

Microbiology ◽  
2005 ◽  
Vol 151 (5) ◽  
pp. 1535-1541 ◽  
Author(s):  
Hope L. Lancero ◽  
Schryl Castaneda ◽  
Nora B. Caberoy ◽  
Xiaoyuan Ma ◽  
Anthony G. Garza ◽  
...  
Keyword(s):  
Protein Interactions ◽  
Genomic Library ◽  
Myxococcus Xanthus ◽  
Signalling Pathway ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Fruiting Body Formation ◽  
New Information ◽  
Chemotaxis Proteins ◽  
Two Hybrid

The dif operon is essential for fruiting body formation, fibril (exopolysaccharide) production and social motility of Myxococcus xanthus. The dif locus contains a gene cluster homologous to chemotaxis genes such as mcp (difA), cheW (difC), cheY (difD), cheA (difE) and cheC (difF), as well as an unknown ORF called difB. This study used yeast two-hybrid analysis to investigate possible interactions between Dif proteins, and determined that DifA, C, D and E interact in a similar fashion to chemotaxis proteins of Escherichia coli and Bacillus subtilis. It also showed that DifF interacted with DifD, and that the novel protein DifB did not interact with Dif proteins. Furthermore, DifA–F proteins were used to determine other possible protein–protein interactions in the M. xanthus genomic library. The authors not only confirmed the specific interactions among known Dif proteins, but also discovered two novel interactions between DifE and Nla19, and DifB and YidC, providing some new information about the Dif signalling pathway. Based on these findings, a model for the Dif signalling pathway is proposed.

scholarly journals Yeast Two-Hybrid Systems and Protein Interaction Mapping Projects for Yeast and Worm

Yeast ◽  
2000 ◽  
Vol 1 (2) ◽  
pp. 88-94 ◽  
Author(s):  
Albertha J. M. Walhout ◽  
Simon J. Boulton ◽  
Marc Vidal
Keyword(s):  
Hybrid Systems ◽  
High Throughput ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Large Scale ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Systematic Analysis ◽  
Technical Features ◽  
Two Hybrid

The availability of complete genome sequences necessitates the development of standardized functional assays to analyse the tens of thousands of predicted gene products in high-throughput experimental settings. Such approaches are collectively referred to as ‘functional genomics’. One approach to investigate the properties of a proteome of interest is by systematic analysis of protein–protein interactions. So far, the yeast two-hybrid system is the most commonly used method for large-scale, high-throughput identification of potential protein–protein interactions. Here, we discuss several technical features of variants of the two-hybrid systems in light of data recently obtained from different protein interaction mapping projects for the budding yeastSaccharomyces cerevisiaeand the nematodeCaenorhabditis elegans.

scholarly journals A High-ThroughputCandida albicansTwo-Hybrid System

mSphere ◽  
2018 ◽  
Vol 3 (4) ◽  
Author(s):  
Floris Schoeters ◽  
Carol A. Munro ◽  
Christophe d’Enfert ◽  
Patrick Van Dijck
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Candida Albicans ◽  
High Throughput ◽  
Protein Interactions ◽  
Fungal Pathogen ◽  
Model Organism ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Content Type ◽  
Two Hybrid

ABSTRACTCandida albicansis a human fungal pathogen that does not follow the universal codon usage, as it translates the CUG codon into serine rather than leucine. This makes it difficult to study protein-protein interactions using the standard yeast two-hybrid (Y2H) system in the model organismSaccharomyces cerevisiae. Due to the lack of adapted tools, only a small number of protein-protein interactions (PPIs) have been detected or studied usingC. albicans-optimized tools despite the importance of PPIs to understand cell biology. However, with the sequencing of the whole genome ofC. albicans, the availability of an ORFeome collection containing 5,099 open reading frames (ORFs) in Gateway-adapted donor vectors, and the creation of a Gateway-compatibleC. albicans-specific two-hybrid (C2H) system, it became possible to study protein-protein interactions on a larger scale usingC. albicansitself as the model organism. Erroneous translations are hereby eliminated compared to using theS. cerevisiaeY2H system. Here, we describe the technical adaptations and the first application of the C2H system for a high-throughput screen, thus making it possible to screen thousands of PPIs at once inC. albicansitself. This first, small-scale high-throughput screen, using Pho85 as a bait protein against 1,646 random prey proteins, yielded one interacting partner (Pcl5). The interaction found with the high-throughput setup was further confirmed with a low-throughput C2H experiment and with a coimmunoprecipitation (co-IP) experiment.IMPORTANCECandida albicansis a major fungal pathogen, and due to the rise of fungal infections and emerging resistance to the limited antifungals available, it is important to develop novel and more specific antifungals. Protein-protein interactions (PPIs) can be applied as very specific drug targets. However, because of the aberrant codon usage ofC. albicans, the traditional yeast two-hybrid system inSaccharomyces cerevisiaeis difficult to use, and only a limited number of PPIs have been described inC. albicans. To overcome this, aC. albicanstwo-hybrid (C2H) system was developed in 2010. The current work describes, for the first time, the application of the C2H system in a high-throughput setup. We hereby show the usefulness of the C2H system to investigate and detect PPIs inC. albicans, making it possible to further elucidate protein networks inC. albicans, which has the potential to lead to the development of novel antifungals which specifically disrupt PPIs important for virulence.

scholarly journals Yeast Two-Hybrid Systems and Protein Interaction Mapping Projects for Yeast and Worm

Yeast ◽  
2000 ◽  
Vol 1 (2) ◽  
pp. 88-94 ◽  
Author(s):  
Albertha J. M. Walhout ◽  
Simon J. Boulton ◽  
Marc Vidal
Keyword(s):  
Hybrid Systems ◽  
High Throughput ◽  
Protein Interaction ◽  
Protein Interactions ◽  
Large Scale ◽  
Protein Protein Interactions ◽  
Yeast Two Hybrid ◽  
Systematic Analysis ◽  
Technical Features ◽  
Two Hybrid

The availability of complete genome sequences necessitates the development of standardized functional assays to analyse the tens of thousands of predicted gene products in high-throughput experimental settings. Such approaches are collectively referred to as ‘functional genomics’. One approach to investigate the properties of a proteome of interest is by systematic analysis of protein–protein interactions. So far, the yeast two-hybrid system is the most commonly used method for large-scale, high-throughput identification of potential protein–protein interactions. Here, we discuss several technical features of variants of the two-hybrid systems in light of data recently obtained from different protein interaction mapping projects for the budding yeast Saccharomyces cerevisiae and the nematode Caenorhabditis elegans.

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