Bacteriophage T4 Gene Research Articles

C-terminal Domain of T4 gene 32 Protein Enables Rapid Filament Reorganization and Dissociation

Bacteriophage T4 gene 32 protein (gp32) is a single-stranded DNA (ssDNA) binding protein essential for DNA replication. gp32 forms stable protein filaments on ssDNA through cooperative interactions between its core and N-terminal domain. gp32′s C-terminal domain (CTD) is believed to primarily help coordinate DNA replication via direct interactions with constituents of the replisome. However, the exact mechanisms of these interactions are not known, and it is unclear how tightly-bound gp32 filaments are readily displaced from ssDNA as required for genomic processing. Here, we utilized truncated gp32 variants to demonstrate a key role of the CTD in regulating gp32 dissociation. Using optical tweezers, we probed the binding and dissociation dynamics of CTD-truncated gp32, *I, to an 8.1 knt ssDNA molecule and compared these measurements with those for full-length gp32. The *I-ssDNA helical filament becomes progressively unwound with increased protein concentration but remains significantly more stable than that of full-length, wild-type gp32. Protein oversaturation, concomitant with filament unwinding, facilitates rapid dissociation of full-length gp32 from across the entire ssDNA segment. In contrast, *I primarily unbinds slowly from only the ends of the cooperative clusters, regardless of the protein density and degree of DNA unwinding. Our results suggest that the CTD may constrain the relative twist angle of proteins within the ssDNA filament such that upon critical unwinding the cooperative interprotein interactions largely vanish, facilitating prompt removal of gp32. We propose a model of CTD-mediated gp32 displacement via internal restructuring of its filament, providing a mechanism for rapid ssDNA clearing during genomic processing.

A real-time fluorescent gp32 probe-based assay for monitoring single-stranded DNA-dependent DNA processing enzymes

Single-stranded DNA (ssDNA) generated during DNA replication, recombination and damage repair reactions is an important intermediate and ssDNA-binding proteins that binds these intermediates coordinate various DNA metabolic processes. Mechanistic details of these ssDNA-dependent processes can be explored by monitoring the generation and consumption of ssDNA in real time. In this work, a fluorescein-labeled gp32-based sensor was employed to continuously monitor various aspects of ssDNA-dependent DNA replication and recombination processes in real time. The gp32 protein probe displayed high sensitivity and specificity to a variety of ssDNA-dependent processes of T4 phage. Several applications of the probe are illustrated here: the solution dynamics of ssDNA-binding protein, protein-protein and protein-DNA interactions involving gp32 protein and its mode of interaction, ssDNA translocation and protein displacement activities of helicases, primer extension activity of DNA polymerase holoenzyme and nucleoprotein filament formation during DNA recombination. The assay has identified new protein-protein interactions of gp32 during T4 replication and recombination. The fluorescent probe described here can thus be used as a universal probe for monitoring in real time various ssDNA-dependent processes, which is based on a well-characterized and easy-to-express bacteriophage T4 gene 32 protein, gp32.

T4 gp32 forms dynamic, compacting filaments on ssDNA that reorganize through interprotein interactions.

Bacteriophage T4 gene 32 protein (gp32) is a model single-stranded DNA (ssDNA) binding protein, essential for DNA replication, recombination, and repair. gp32 forms cooperative filaments on ssDNA through interprotein interactions mediated by its N-terminal domain. Detailed understanding of gp32 filament structure and organization remains incomplete, particularly with respect to longer, biologically-relevant DNA lengths. We use optical tweezers and atomic force microscopy (AFM) to probe the structure and binding dynamics of gp32 on long (∼7-8 knt) ssDNA substrates. We find that cooperative binding of gp32 reduces the contour length of ssDNA by ∼40%, suggesting that gp32 filaments helically wind ssDNA, a binding conformation that may be favorable for the synthesis of dsDNA on the ssDNA template during replication. Additionally, gp32 binding gives rise to multiphasic DNA extension changes which reflect competition between increases in ssDNA persistence length and changes in its contour length. Notably, under conditions of high force or high protein concentration, cooperative gp32 clusters are able to modulate their binding footprint to drive elongation of the DNA molecule relative to its compacted state. This binding mode is unstable and marked by an additional phase of rapid protein dissociation not seen at lower concentrations, suggesting a possible mechanism for prompt gp32 dissociation during DNA replication.

Setting Up a Better Infection: Overexpression of the Early Bacteriophage T4 Gene motB During Infection Results in a More Favorable tRNA Pool for the Phage.

: Although many bacteriophage T4 early genes are nonessential with unknown functions, they are believed to aid in the takeover of the Escherichia coli host. Understanding the functions of these genes could be helpful to develop novel antibacterial strategies. MotB, encoded by a previously uncharacterized T4 early gene, is a DNA-binding protein that compacts the host nucleoid and alters host gene expression. : MotB structure was predicted by AlphaFold 2. RNA-seq and mass spectrometry (MS) analyses were performed to determine RNA and protein changes when motB was overexpressed in E. coli BL21(DE3) ±5 min T4 infection. : MotB structure is predicted to be a two-domain protein with N-terminal Kyprides-Onzonis-Woese and C-terminal oligonucleotide/oligosaccharide-fold domains. In E. coli B, motB overexpression during infection does not affect T4 RNAs, but affects the expression of host genes, including the downregulation of 21 of the 84 chargeable host tRNAs. Many of these tRNAs are used less frequently by T4 or have a counterpart encoded within the T4 genome. The MS analyses indicate that the levels of multiple T4 proteins are changed by motB overexpression. : Our results suggest that in this E. coli B host, motB is involved in establishing a more favorable tRNA pool for the phage during infection.

Biological Characterization and Evolution of Bacteriophage T7-△holin During the Serial Passage Process.

Bacteriophage T7 gene 17.5 coding for the only known holin is one of the components of its lysis system, but the holin activity in T7 is more complex than a single gene, and evidence points to the existence of additional T7 genes with holin activity. In this study, a T7 phage with a gene 17.5 deletion (T7-△holin) was rescued and its biological characteristics and effect on cell lysis were determined. Furthermore, the genomic evolution of mutant phage T7-△holin during serial passage was assessed by whole-genome sequencing analysis. It was observed that deletion of gene 17.5 from phage T7 delays lysis time and enlarges the phage burst size; however, this biological characteristic recovered to normal lysis levels during serial passage. Scanning electron microscopy showed that the two opposite ends of E. coli BL21 cells swell post-T7-△holin infection rather than drilling holes on cell membrane when compared with T7 wild-type infection. No visible progeny phage particle accumulation was observed inside the E. coli BL21 cells by transmission electron microscopy. Following serial passage of T7-△holin from the 1st to 20th generations, the mRNA levels of gene 3.5 and gene 19.5 were upregulated and several mutation sites were discovered, especially two missense mutations in gene 19.5, which indicate a potential contribution to the evolution of the T7-△holin. Although the burst size of T7-△holin increased, high titer cultivation of T7-△holin was not achieved by optimizing the culture process. Accordingly, these results suggest that gene 19.5 is a potential lysis-related component that needs to be studied further and that the T7-△holin strain with its gene 17.5 deletion is not adequate to establish the high-titer phage cultivation process.

Improved Dynamic Range of a Rhamnose-Inducible Promoter for Gene Expression in Burkholderia spp.

A diverse genetic toolkit is critical for understanding bacterial physiology and genotype-phenotype relationships. Inducible promoter systems are an integral part of this toolkit. In Burkholderia and related species, the l-rhamnose-inducible promoter is among the first choices due to its tight control and the lack of viable alternatives. To improve upon its maximum activity and dynamic range, we explored the effect of promoter system modifications in Burkholderia cenocepacia with a LacZ-based reporter. By combining the bacteriophage T7 gene 10 stem-loop and engineered rhaI transcription factor-binding sites, we obtained a rhamnose-inducible system with a 6.5-fold and 3.0-fold increases in maximum activity and dynamic range, respectively, compared to the native promoter. We then added the modified promoter system to pSCrhaB2 and pSC201, common genetic tools used for plasmid-based and chromosome-based gene expression, respectively, in Burkholderia, creating pSCrhaB2plus and pSC201plus. We demonstrated the utility of pSCrhaB2plus for gene expression in B. thailandensis, B. multivorans, and B. vietnamiensis and used pSC201plus to control highly expressed essential genes from the chromosome of B. cenocepacia. The utility of the modified system was demonstrated as we recovered viable mutants to control ftsZ, rpoBC, and rpsF, whereas the unmodified promoter was unable to control rpsF. The modified expression system allowed control of an essential gene depletion phenotype at lower levels of l-rhamnose, the inducer. pSCRhaB2plus and pSC201plus are expected to be valuable additions to the genetic toolkit for Burkholderia and related species. IMPORTANCE Species of Burkholderia are dually recognized as being of attractive biotechnological potential but also opportunistic pathogens for immunocompromised individuals. Understanding the genotype-phenotype relationship is critical for synthetic biology approaches in Burkholderia to disentangle pathogenic from beneficial traits. A diverse genetic toolkit, including inducible promoters, is the foundation for these investigations. Thus, we sought to improve on the commonly used rhamnose-inducible promoter system. Our modifications resulted in both higher levels of heterologous protein expression and broader control over highly expressed essential genes in B. cenocepacia. The significance of our work is in expanding the genetic toolkit to enable more comprehensive studies into Burkholderia and related bacteria.

Microdroplet-based one-step RT-PCR for ultrahigh throughput single-cell multiplex gene expression analysis and rare cell detection

Gene expression analysis of individual cells enables characterization of heterogeneous and rare cell populations, yet widespread implementation of existing single-cell gene analysis techniques has been hindered due to limitations in scale, ease, and cost. Here, we present a novel microdroplet-based, one-step reverse-transcriptase polymerase chain reaction (RT-PCR) platform and demonstrate the detection of three targets simultaneously in over 100,000 single cells in a single experiment with a rapid read-out. Our customized reagent cocktail incorporates the bacteriophage T7 gene 2.5 protein to overcome cell lysate-mediated inhibition and allows for one-step RT-PCR of single cells encapsulated in nanoliter droplets. Fluorescent signals indicative of gene expressions are analyzed using a probabilistic deconvolution method to account for ambient RNA and cell doublets and produce single-cell gene signature profiles, as well as predict cell frequencies within heterogeneous samples. We also developed a simulation model to guide experimental design and optimize the accuracy and precision of the assay. Using mixtures of in vitro transcripts and murine cell lines, we demonstrated the detection of single RNA molecules and rare cell populations at a frequency of 0.1%. This low cost, sensitive, and adaptable technique will provide an accessible platform for high throughput single-cell analysis and enable a wide range of research and clinical applications.

Gp4 is a nuclease required for morphogenesis of T4-like bacteriophages

An essential step in the morphogenesis of tailed bacteriophages is the joining of heads and tails to form infectious virions. Our understanding of the maturation of complete virus particles remains incomplete. Through an unknown mechanism, phage T4 gene product 4 (gp4) plays an essential role in the head-tail joining step of T4-like phages. Alignment of T4 gp4 homologs identified a type II restriction endonuclease motif. Purified gp4 from both T4 and a marine T4-like bacteriophage, YC, have non-specific nuclease activity in vitro. Mutation of a single conserved amino acid residue in the endonuclease fold of T4 and YC gp4 abrogates nuclease activity. When expressed in trans, the wild type T4 gp4, but neither the mutated T4 protein nor the YC homolog, rescues a T4 gene 4 amber mutant phage. Thus the nuclease activity appears essential for morphogenesis, potentially by cleaving packaged DNA to enable the joining of heads to tails.

The Many Roles of Binding Cooperativity in the Control of DNA Replication

The Many Roles of Binding Cooperativity in the Control of DNA Replication

Single-Molecule Movies of the Interplay between DNA Polymerase and Single Strand DNA Binding (SSB) Protein

Reliable DNA replication is completed by the orchestrated activation of a multi-protein machinery, the replisome, in a highly coordinated and dynamic fashion. The bacteriophage T7 gene 2.5 protein (gp2.5) works as a single strand DNA(ssDNA) binding protein and has been demonstrated to play a key role in the maintenance of ssDNA stability as well as the suppression of hairpin formation during each cycle of Okazaki fragment synthesis. After gp2.5 binding to ssDNA, gp2.5 might act as a road block for DNA polymerase to proceed. Bulk biochemical assays, however, demonstrate that the presence of gp2.5 increases the processivity and rate of template extension, while a mutant gp2.5 without c-terminus decreases the activity. Here we present movies of the single-molecule dynamics of T7 DNA polymerase and gp2.5 using optical trapping combined with confocal fluorescence microscopy. First, we investigated at single molecule level the effect of presence of wide type and mutant gp2.5 on DNA polymerase activity under various conditions. Then, we observed in real-time the labelled gp2.5 binding and unbinding dynamics interacting with DNA polymerase. It demonstrates that gp2.5 binds with ssDNA at low force (<30pN) but that its binding to ssDNA decreases significantly at higher force (>30pN). Beside, fluorescence data shows labelled DNA polymerase appearing at multiple places on the DNA which indicates the DNA polymerase might bind to DNA at multiple sites. These results combined with further visualization of the two labeled proteins will help us better understand the interplay between DNA polymerase and gp2.5.

BNO—An ontology for understanding the transittability of complex biomolecular networks

Analysis of biological systems is being progressively facilitated by computational tools. Most of these tools are based on qualitative and numerical methods. However, they are not always evident, and there is an increasing need to provide an additional semantic layer. Semantic technologies, especially ontologies, are one of the tools frequently used for this purpose. Indeed, they are indispensable for understanding the semantic knowledge about the operation of cells at a molecular level. We describe here the biomolecular network ontology (BNO) created specially to address the needs of analysing the complex biomolecular network’s behaviour. A biomolecular network consists of nodes, denoting cellular entities, and edges, representing interactions among cellular components. The BNO ontology provides a foundation for qualitative simulation of complex biomolecular networks. We test the performance of the proposed BNO ontology by using a real example of a biomolecular network, the bacteriophage T4 gene 32. We illustrate the proposed BNO ontology for reasoning and inferring new knowledge with sets of rules expressed in SWRL. Results demonstrate that the BNO ontology allows to precisely interpret the corresponding semantic context and intelligently model biomolecular networks and their state changes. The Biomolecular Network Ontology (BNO) is freely available at https://github.com/AliAyadi/BNO-ontology-version-1.0.

BNO - An Ontology for Understanding the Transittability of Complex Biomolecular Networks

Analysis of biological systems is being progressively facilitated by computational tools. Most of these tools are based on qualitative and numerical methods. However, they are not always evident, and there is an increasing need to provide an additional semantic layer. Semantic technologies, especially ontologies, are one of the tools frequently used for this purpose. Indeed, they are indispensable for understanding the semantic knowledge about the operation of cells at a molecular level. We describe here the biomolecular network ontology (BNO) created specially to address the needs of analysing the complex biomolecular network's behaviour. A biomolecular network consists of nodes, denoting cellular entities, and edges, representing interactions among cellular components. The BNO ontology provides a foundation for qualitative simulation of complex biomolecular networks. We test the performance of the proposed BNO ontology by using a real example of a biomolecular network, the bacteriophage T4 gene 32. We illustrate the proposed BNO ontology for reasoning and inferring new knowledge with sets of rules expressed in SWRL. Results demonstrate that the BNO ontology allows to precisely interpret the corresponding semantic context and intelligently model biomolecular networks and their state changes. The Biomolecular Network Ontology (BNO) is freely available at https://github.com/AliAyadi/BNO-ontology-version-1.0.

The role of the C-domain of bacteriophage T4 gene 32 protein in ssDNA binding and dsDNA helix-destabilization: Kinetic, single-molecule, and cross-linking studies.

The model single-stranded DNA binding protein of bacteriophage T4, gene 32 protein (gp32) has well-established roles in DNA replication, recombination, and repair. gp32 is a single-chain polypeptide consisting of three domains. Based on thermodynamics and kinetics measurements, we have proposed that gp32 can undergo a conformational change where the acidic C-terminal domain binds internally to or near the single-stranded (ss) DNA binding surface in the core (central) domain, blocking ssDNA interaction. To test this model, we have employed a variety of experimental approaches and gp32 variants to characterize this conformational change. Utilizing stopped-flow methods, the association kinetics of wild type and truncated forms of gp32 with ssDNA were measured. When the C-domain is present, the log-log plot of k vs. [NaCl] shows a positive slope, whereas when it is absent (*I protein), there is little rate change with salt concentration, as expected for this model.A gp32 variant lacking residues 292–296 within the C-domain, ΔPR201, displays kinetic properties intermediate between gp32 and *I. The single molecule force-induced DNA helix-destabilizing activitiesas well as the single- and double-stranded DNA affinities of ΔPR201 and gp32 truncated at residue 295 also fall between full-length protein and *I. Finally, chemical cross-linking of recombinant C-domain and gp32 lacking both N- and C-terminal domains is inhibited by increasing concentrations of a short single-stranded oligonucleotide, and the salt dependence of cross-linking mirrors that expected for the model. Taken together, these results provide the first evidence in support of this model that have been obtained through structural probes.

The Interaction between Bacteriophage T7 DNA Polymerase and Gene 2.5 Protein at the Single-Molecule Level

The Interaction between Bacteriophage T7 DNA Polymerase and Gene 2.5 Protein at the Single-Molecule Level

BNO: An ontology for describing the behaviour of complex biomolecular networks

The use of semantic technologies, such as ontologies, to describe and analyse biological systems is at the heart of systems biology. Indeed, understanding the behaviour of cells requires a large amount of context information. In this paper, we propose an ontology entitled ”Biomolecular Network ontology” using the OWL language. The BNO ontology standardises the terminology used by biologists experts to address issues including semantic behaviour representation, reasoning and knowledge sharing. The main benefit of this proposed ontology is the ability to reason about dynamical behaviour of complex biomolecular networks over time. We demonstrate our proposed ontology with a detailed example, the bacteriophage T4 gene 32 use case.

CBNSimulator: a simulator tool for understanding the behaviour of complex biomolecular networks using discrete time simulation

Because of the lack of satisfactory solutions to explain biological systems, biologists usually focus on modelling and simulation tools to understand the behaviour of these complex organisms. Indeed, computational modelling and simulation of cells plays a pivotal role in systems biology. In this paper, we tackle the problem of studying the behaviour of human cells by reproducing the behaviour of complex biomolecular networks. To this end, we present in this paper an approach for simulating complex biomolecular networks inspired by the discrete-event simulation model (DEVS), a formalism developed for supporting the modelling of complex systems. In this paper, we propose a simulation tool, named ”CBNSimulator”, based on a logical model of the biomolecular network and taking advantage of the performance of a discrete-time simulation model for understanding the evolution and the behaviour of complex biomolecular networks as a discrete sequence of events in time. The proposed tool has been applied to the case study of a ribosomal protein regulation network, named ”the bacteriophage T4 gene 32”, and results given by this simulation tool are in agreement with the expert’s judgement. Moreover, the graphical user interface of CBNSimulator allows biologists to easily reproduce, analyse and understand behaviour of complex biomolecular networks through discrete simulation.

Cooperation between the chloroplast psbA 5'-untranslated region and coding region is important for translational initiation: the chloroplast translation machinery cannot read a human viral gene coding region.

Chloroplast mRNA translation is regulated by the 5'-untranslated region (5'-UTR). Chloroplast 5'-UTRs also support translation of the coding regions of heterologous genes. Using an in vitro translation system from tobacco chloroplasts, we detected no translation from a human immunodeficiency virus tat coding region fused directly to the tobacco chloroplast psbA 5'-UTR. This lack of apparent translation could have been due to rapid degradation of mRNA templates or synthesized protein products. Replacing the psbA 5'-UTR with the E. coli phage T7 gene 10 5'-UTR, a highly active 5'-UTR, and substituting synonymous codons led to some translation of the tat coding region. The Tat protein thus synthesized was stable during translation reactions. No significant degradation of the added tat mRNAs was observed after translation reactions. These results excluded the above two possibilities and confirmed that the tat coding region prevented its own translation. The tat coding region was then fused to the psbA 5'-UTR with a cognate 5'-coding segment. Significant translation was detected from the tat coding region when fused after 10 or more codons. That is, translation could be initiated from the tat coding region once translation had started, indicating that the tat coding region inhibits translational initiation but not elongation. Hence, cooperation/compatibility between the 5'-UTR and its coding region is important for translational initiation.

Homologous DNA strand exchange activity of the human mitochondrial DNA helicase TWINKLE.

A crucial component of the human mitochondrial DNA replisome is the ring-shaped helicase TWINKLE—a phage T7-gene 4-like protein expressed in the nucleus and localized in the human mitochondria. Our previous studies showed that despite being a helicase, TWINKLE has unique DNA annealing activity. At the time, the implications of DNA annealing by TWINKLE were unclear. Herein, we report that TWINKLE uses DNA annealing function to actively catalyze strand-exchange reaction between the unwinding substrate and a homologous single-stranded DNA. Using various biochemical experiments, we demonstrate that the mechanism of strand-exchange involves active coupling of unwinding and annealing reactions by the TWINKLE. Unlike strand-annealing, the strand-exchange reaction requires nucleotide hydrolysis and greatly stimulated by short region of homology between the recombining DNA strands that promote joint molecule formation to initiate strand-exchange. Furthermore, we show that TWINKLE catalyzes branch migration by resolving homologous four-way junction DNA. These four DNA modifying activities of TWINKLE: strand-separation, strand-annealing, strand-exchange and branch migration suggest a dual role of TWINKLE in mitochondrial DNA maintenance. In addition to playing a major role in fork progression during leading strand DNA synthesis, we propose that TWINKLE is involved in recombinational repair of the human mitochondrial DNA.

DNA Binding Proteins and Drug Delivery Vehicles: Tales of Elephants and Snakes.

We compare the DNA-interactive properties of bacteriophage T4 gene 32 protein (gp32) with those of crotamine, a component of the venom of the South American rattlesnake. Gene 32 protein is a classical single-stranded DNA binding protein that has served as a model for this class of proteins. We discuss its biological functions, structure, binding specificities, and how it controls its own expression. In addition, we delineate the roles of the structural domains of gp32 and how they regulate the protein's various activities. Crotamine, a component of the venom of the South American rattlesnake, is probably not a DNA binding protein in nature, but clearly shows significant DNA binding in vitro. Crotamine has been shown to selectively disrupt rapidly dividing cells and this specificity has been demonstrated for crotamine-facilitated delivery of plasmid DNA Thus, crotamine, or a variant of the protein, could have important clinical and/or diagnostic roles. Understanding its DNA binding properties may therefore lead to more effective drug delivery vehicles.

Unraveling the Interplay between Single-Stranded DNA-Binding Protein, DNA Polymerase and Single-Stranded DNA

Bacteriophage T7 gene 2.5 protein (gp2.5), a single-stranded DNA binding (SSB) protein, contains two main structural elements which confer it essential functions. First, an oligosaccharide/oligonucleotide binding fold which can interact with ssDNA and thus provide a protective role while removing secondary structure impediments. Second, an acidic C-terminal tail capable to interact with other gp2.5 units and with other replisome proteins, such as the T7 DNA primase-helicase (gp4) and polymerase (gp5), allowing for organizational roles within the replisome. The impact of SSBs on the replisome dynamics is hard to study using conventional biochemistry tools. Here, we present data obtained from a unique combination of optical tweezers and confocal fluorescence microscopy, which offers real-time high spatial and temporal resolution to study protein-DNA interactions. Our results show that the SSB gp2.5 binds efficiently to ssDNA, forming highly static protein-DNA complexes. We also observed a decrease in the ssDNA end-to-end length indicating that the protein binds by bending or wrapping ssDNA, resulting in a highly tension-sensitive binding mode. Next, we studied to what degree the presence of wild type (wt) gp2.5 or a mutant lacking the terminal phenylalanine (ΔF), the interaction motif with DNA polymerase, affects replication activity. We show that wt gp2.5 alters the rate, duration and probability of occurrence of replicative and proofreading polymerase events in contrast to the ΔF mutant or the absence of the protein. Thus, our findings indicate that the presence of gp2.5 strongly stimulates the function of DNA polymerase within the replisome.