Replication Initiator Protein Research Articles

Influence and mechanism of typical transition metal ions on the denitrification performance of heterotrophic nitrification-aerobic denitrification bacteria

To investigate the inhibitory effects of various transition metal ions on nitrogen removal and their underlying mechanisms, the single and combined effects of Cu2+ Ni2+ and Zn2+ on Heterotrophic nitrification-aerobic denitrification (HN-AD) bacteria Acinetobacter sp. TAC-1 were studied in a batch experiment system. The results revealed that increasing concentrations of Cu2+ and Ni2+ had a detrimental effect on the removal of ammonium nitrogen (NH4+-N) and total nitrogen (TN). Specifically, Cu2+ concentration of 10 mg/L, the TN degradation rate was 55.09%, compared to 77.60% in the control group. Cu2+ exhibited a pronounced inhibitory effect. In contrast, Zn2+ showed no apparent inhibitory effect on NH4+-N removal and even enhanced TN removal at lower concentrations. However, when the mixed ion concentration of Zn2++Ni2+ exceeded 5 mg/L, the removal rates of NH4+-N and TN were significantly reduced. Moreover, transition metal ions did not significantly impact the removal rates of chemical oxygen demand (COD). The inhibition model fitting results indicated that the inhibition sequence was Cu2+ > Zn2+ > Ni2+. Transcriptome analysis demonstrated that metal ions influence TAC-1 activity by modulating the expression of pivotal genes, including zinc ABC transporter substrate binding protein (znuA), ribosomal protein (rpsM), and chromosome replication initiation protein (dnaA) and DNA replication of TAC-1 under metal ion stress, leading to disruptions in transcription, translation, and cell membrane structure. Finally, a conceptual model was proposed by us to summarize the inhibition mechanism and possible response strategies of TAC-1 bacteria under metal ion stress, and to address the lack of understanding regarding the influence mechanism of TAC-1 on nitrogen removal in wastewater co-polluted by metal and ammonia nitrogen. The results provided practical guidance for the management of transition metal and ammonia nitrogen co-polluted water bodies, as well as the removal of high nitrogen.

Specificities and commonalities of the Planctomycetes plasmidome.

Plasmids, despite their critical role in antibiotic resistance and modern biotechnology, are understood in only a few bacterial groups in terms of their natural ecological dynamics. The bacterial phylum Planctomycetes, known for its unique molecular and cellular biology, has a largely unexplored plasmidome. This study offers a thorough exploration of the diversity of natural plasmids within Planctomycetes, which could serve as a foundation for developing various genetic research tools for this phylum. Planctomycetes plasmids encode a broad range of biological functions and appear to have coevolved significantly with their host chromosomes, sharing many homologues. Recent transfer events of insertion sequences between cohabiting chromosomes and plasmids were also observed. Interestingly, 64% of plasmid genes are distantly related to either chromosomally encoded genes or have homologues in plasmids from other bacterial groups. The planctomycetal plasmidome is composed of 36% exclusive proteins. Most planctomycetal plasmids encode a replication initiation protein from the Replication Protein A family near a putative iteron-containing replication origin, as well as active type I partition systems. The identification of one conjugative and three mobilizable plasmids suggests the occurrence of horizontal gene transfer via conjugation within this phylum. This comprehensive description enhances our understanding of the plasmidome of Planctomycetes and its potential implications in antibiotic resistance and biotechnology.

Targeting synthesis of the Chromosome Replication Initiator Protein DnaA by antisense PNA-peptide conjugates in Escherichia coli

Initiation of chromosome replication is an essential stage of the bacterial cell cycle that is controlled by the DnaA protein. With the aim of developing novel antimicrobials, we have targeted the initiation of DNA replication, using antisense peptide nucleic acids (PNAs), directed against DnaA translation. A series of anti-DnaA PNA conjugated to lysine-rich bacterial penetrating peptides (PNA-BPPs) were designed to block DnaA translation. These anti-DnaA PNA-BPPs inhibited growth of wild-type Escherichia coli cells at low micromolar concentrations, and cells exposed to anti-DnaA PNA-BPPs exhibited characteristic hallmarks of chromosome replication inhibition. These results present one of very few compounds successfully targeting initiation of chromosome replication, an essential step in the bacterial cell cycle.

A New Inovirus from the Human Blood Encodes Proteins with Nuclear Subcellular Localization.

Viruses infecting bacteria (bacteriophages) represent the most abundant viral particles in the human body. They participate in the control of the human-associated bacterial communities and play an important role in the dissemination of virulence genes. Here, we present the identification of a new filamentous single-stranded DNA phage of the family Inoviridae, named Ralstonia Inoviridae Phage 1 (RIP1), in the human blood. Metagenomics and PCR analyses detected the RIP1 genome in blood serum, in the absence of concomitant bacterial infection or contamination, suggesting inovirus persistence in the human blood. Finally, we have experimentally demonstrated that the RIP1-encoded rolling circle replication initiation protein and serine integrase have functional nuclear localization signals and upon expression in eukaryotic cells both proteins were translocated into the nucleus. This observation adds to the growing body of data suggesting that phages could have an overlooked impact on the evolution of eukaryotic cells.

Toward an understanding of the DNA replication initiation in bacteria.

Although the mechanism of DNA replication initiation has been investigated for over 50 years, many important discoveries have been made related to this process in recent years. In this mini-review, we discuss the current state of knowledge concerning the structure of the origin region in bacterial chromosomes and plasmids, recently discovered motifs recognized by replication initiator proteins, and proposed in the literature models describing initial origin opening. We review structures of nucleoprotein complexes formed by replication initiators at chromosomal and plasmid replication origins and discuss their functional implications. We also discuss future research challenges in this field.

A Directed Evolution Protocol for Engineering Minimal Transcription Factors, Based on CIS Display.

Directed evolution is an efficient strategy for obtaining desired biomolecules. Since the 1990s, the emergence of display techniques has enabled high-throughput screening of functional proteins. However, classical methods require library construction by plasmid cloning and are limited by transformation efficiencies, typically limiting library sizes to ~106-107 variants. More recently, in vitro techniques have emerged that avoid cloning, allowing library sizes of >1012 members. One of these, CIS display, is a DNA-based display technique which allows high-throughput selection of biomolecules in vitro. CIS display creates the genotype-phenotype link required for selection by a DNA replication initiator protein, RepA, that binds exclusively to the template from which it has been expressed. This method has been successfully used to evolve new protein-protein interactions but has not been used before to select DNA-binding proteins, which are major components in mammalian synthetic biology. In this chapter, we describe a directed evolution method using CIS display to efficiently select functional DNA-binding proteins from pools of nonbinding proteins. The method is illustrated by enriching the minimal transcription factor Cro from a low starting frequency (1 in 109). This protocol is also applicable to engineering other DNA-binding proteins or transcription factors from combinatorial libraries.

Molecular characterization of a tetra segmented ssDNA virus infecting Botrytis cinerea worldwide

BackgroundFamily Genomoviridae was recently established, and only a few mycoviruses have been described and characterized, and almost all of them (Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1, Fusarium graminearum gemyptripvirus 1 and Botrytis cinerea gemydayirivirus 1) induced hypovirulence in their host. Botrytis cinerea ssDNA virus 1 (BcssDV1), a tetrasegmented single-stranded DNA virus infecting the fungus Botrytis cinerea, has been molecularly characterized in this work.MethodsBcssDV1 was detected in Spanish and Italian B. cinerea field isolates obtained from grapevine. BcssDV1 variants genomes were molecularly characterized via NGS and Sanger sequencing. Nucleotide and amino acid sequences were used for diversity and phylogenetic analysis. Prediction of protein tertiary structures and putative associated functions were performed by AlphaFold2 and DALI.ResultsBcssDV1 is a tetrasegmented single-stranded DNA virus. The mycovirus was composed by four genomic segments of approximately 1.7 Kb each, which are DNA-A, DNA-B, and DNA-C and DNA-D, that coded, respectively, for the rolling-circle replication initiation protein (Rep), capsid protein (CP) and two hypothetical proteins. BcssDV1 was present in several Italian and Spanish regions with high incidence and low variability among the different viral variants. DNA-A and DNA-D were found to be the more conserved genomic segments among variants, while DNA-B and DNA-C segments were shown to be the most variable ones. Tertiary structures of the proteins encoded by each segment suggested specific functions associated with each of them.ConclusionsThis study presented the first complete sequencing and characterization of a tetrasegmented ssDNA mycovirus, its incidence in Spain and Italy, its presence in other countries and its high conservation among regions.

A new genus of alphasatellites associated with banana bunchy top virus in Southeast Asia.

Autonomously replicating alphasatellites (family Alphasatellitidae) are frequently associated with plant single-stranded (ss)DNA viruses of the families Geminiviridae, Metaxyviridae, and Nanoviridae. Alphasatellites encode a single replication-initiator protein (Rep) similar to Rep proteins of helper viruses and depend on helper viruses for encapsidation, movement, and transmission. Costs versus benefits of alphasatellite-helper virus association are poorly understood. Our surveys in Southeast Asia (SEA) for wild and cultivated banana plants infected with banana bunchy top virus (BBTV, Nanoviridae) and Illumina sequencing reconstruction of their viromes revealed, in addition to a six-component BBTV genome, one to three distinct alphasatellites present in sixteen of twenty-four BBTV-infected plants. Comparative nucleotide and Rep protein sequence analyses classified these alphasatellites into four distinct species: two known species falling into the genus Muscarsatellite (subfamily Petromoalphasatellitinae) previously identified in SEA and two novel species falling into the tentative genus Banaphisatellite (subfamily Nanoalphasatellitinae) so far containing a single species recently identified in Africa. The banaphisatellites were found to be most related to members of the genus Fabenesatellite of subfamily Nanoalphasatellitinae and the genus Gosmusatellite of subfamily Geminialphasatellitinae, both infecting dicots. This suggests a dicot origin of banaphisatellites that got independently associated with distinct strains of monocot-infecting BBTV in Africa and SEA. Analysis of conserved sequence motifs in the common regions driving replication and gene expression of alphasatellites and BBTV strains revealed both differences and similarities, pointing at their ongoing co-evolution. An impact of alphasatellites on BBTV infection and evasion of RNA interference-based antiviral defences was evaluated by measuring relative abundance of BBTV genome components and alphasatellites and by profiling BBTV- and alphasatellite-derived small interfering RNAs. Taken together, our findings shed new light on the provenance of alphasatellites, their co-evolution with helper viruses, and potential mutual benefits of their association.

The crystal structure of the human smacovirus 1 Rep domain.

Replication initiator proteins (Reps) from the HUH endonuclease family process specific single-stranded DNA sequences to initiate rolling-circle replication in viruses. Here, the first crystal structure of the apo state of a Rep domain from the smacovirus family is reported. The structure of the human smacovirus 1 Rep domain was obtained at 1.33 Å resolution and represents an expansion of the HUH endonuclease superfamily, allowing greater diversity in bioconjugation-tag applications.

Propidium Monoazide-Treated, Cell-Direct, Quantitative PCR for Detecting Viable Chloramphenicol-Resistant Escherichia coli and Corynebacterium glutamicum Cells.

With the rapid development and commercialization of industrial genetically modified microorganisms (GMMs), public concerns regarding their potential effects are on the rise. It is imperative to promptly monitor the unintended release of viable GMMs into wastewater, the air, and the surrounding ecosystems to prevent the risk of horizontal gene transfer to native microorganisms. In this study, we have developed a method that combines propidium monoazide (PMA) with a dual-plex quantitative PCR (qPCR) approach based on TaqMan probes. This method targets the chloramphenicol-resistant gene (CmR) along with the endogenous genes D-1-deoxyxylulose 5-phosphate synthase (dxs) and chromosomal replication initiator protein (dnaA). It allows for the direct quantitative detection of viable genetically modified Escherichia coli and Corynebacterium glutamicum cells, eliminating the requirement for DNA isolation. The dual-plex qPCR targeting CmR/dxs and CmR/dnaA demonstrated excellent performance across various templates, including DNA, cultured cells, and PMA-treated cells. Repeatability and precision, defined as RSDr% and bias%, respectively, were calculated and found to fall within the acceptable limits specified by the European Network of GMO Laboratories (ENGL). Through PMA-qPCR assays, we determined the detection limits for viable chloramphenicol-resistant E. coli and C. glutamicum strains to be 20 and 51 cells, respectively, at a 95% confidence level. Notably, this method demonstrated superior sensitivity compared to Enzyme-Linked Immunosorbent Assay (ELISA), which has a detection limit exceeding 1000 viable cells for both GM bacterial strains. This approach offers the potential to accurately and efficiently detect viable cells of GMMs, providing a time-saving and cost-effective solution.

ITRAQ-based quantitative proteomic analysis of the antibacterial mechanism of silver nanoparticles against multidrug-resistant Streptococcus suis.

The increase in antibiotic resistance of bacteria has become a major concern in clinical treatment. Silver nanoparticles (AgNPs) have significant antibacterial effects against Streptococcus suis. Therefore, this study aimed to investigate the antibacterial activity and mechanism of action of AgNPs against multidrug-resistant S. suis. The effect of AgNPs on the morphology of multidrug-resistant S. suis was observed using scanning electron microscopy (SEM). Differentially expressed proteins were analyzed by iTRAQ quantitative proteomics, and the production of reactive oxygen species (ROS) was assayed by H2DCF-DA staining. SEM showed that AgNPs disrupted the normal morphology of multidrug-resistant S. suis and the integrity of the biofilm structure. Quantitative proteomic analysis revealed that a large number of cell wall synthesis-related proteins, such as penicillin-binding protein and some cell cycle proteins, such as the cell division protein FtsZ and chromosomal replication initiator protein DnaA, were downregulated after treatment with 25 μg/mL AgNPs. Significant changes were also observed in the expression of the antioxidant enzymes glutathione reductase, alkyl hydroperoxides-like protein, α/β superfamily hydrolases/acyltransferases, and glutathione disulfide reductases. ROS production in S. suis positively correlated with AgNP concentration. The potential antibacterial mechanism of AgNPs may involve disrupting the normal morphology of bacteria by inhibiting the synthesis of cell wall peptidoglycans and inhibiting the growth of bacteria by inhibiting the cell division protein FtsZ and Chromosomal replication initiator protein DnaA. High oxidative stress may be a significant cause of bacterial death. The potential mechanism by which AgNPs inhibit S. suis biofilm formation may involve affecting bacterial adhesion and interfering with the quorum sensing system.

Exogenous delivery of dsRNA for management of mungbean yellow mosaic virus on blackgram.

Exogenous application of dsRNA molecules targeting MYMV genes offers a promising approach to effectively mitigate yellow mosaic disease in blackgram, demonstrating potential for sustainable plant viral disease management. The exogenous application of double-stranded RNA (dsRNA) molecules to control plant viral diseases is gaining traction due to its advantages over conventional methods, such as target specificity, non-polluting nature, and absence of residue formation. Furthermore, this approach does not involve genome modification. In this study, dsRNA molecules targeting the coat protein gene (dsCP) and replication initiator protein gene (dsRep) of mungbean yellow mosaic virus (MYMV) were synthesised using an in vitro transcription method. To evaluate the effectiveness of dsRNA treatment, blackgram plants exhibiting MYMV symptoms at the first trifoliate stage were subjected to exogenous application of dsRNA. Second, third, and fourth trifoliate leaves, which emerged at 7, 15, and 21days after dsRNA application, respectively, were monitored for MYMV symptoms. Remarkably, a significant reduction in yellow mosaic disease (YMD) symptoms was observed in the newly emerged trifoliate leaves of MYMV-infected blackgram plants after treatment with dsRNA targeting both gene regions. This reduction was evident as a decrease in the intensity of yellow mosaic coverage on the leaf lamina compared to control. dsCP effectively reduced the MYMV titre in the treated plants for up to 15days. However, dsRep demonstrated greater efficiency in conferring resistance to MYMV at 15days post-application. These findings were supported by quantitative real-time PCR analysis, where the observed Ct values for DNA extracted from dsRep-treated plants were significantly higher compared to the Ct values of DNA from dsCP-treated plants at 15days post-application. Similarly, higher viral copy numbers were observed in dsCP-treated plants 15days after dsRNA treatment, in contrast to plants treated with dsRep.

Australasian Pigeon Circoviruses Demonstrate Natural Spillover Infection.

Pigeon circovirus (PiCV) is considered to be genetically diverse, with a relatively small circular single-stranded DNA genome of 2 kb that encodes for a capsid protein (Cap) and a replication initiator protein (Rep). Australasia is known to be the origin of diverse species of the Order Columbiformes, but limited data on the PiCV genome sequence has hindered phylogeographic studies in this species. To fill this gap, this study was conducted to investigate PiCV in 118 characteristic samples from different birds across Australia using PCR and sequencing. Eighteen partial PiCV Rep sequences and one complete PiCV genome sequence were recovered from reservoir and aberrant hosts. Phylogenetic analyses revealed that PiCV circulating in Australia was scattered across three different subclades. Importantly, one subclade dominated within the PiCV sequenced from Australia and Poland, whereas other PiCV sequenced in this study were more closely related to the PiCV sequenced from China, USA and Japan. In addition, PiCV Rep sequences obtained from clinically affected plumed whistling duck, blue billed duck and Australian magpie demonstrated natural spillover of PiCV unveiled host generalist characteristics of the pigeon circovirus. These findings indicate that PiCV genomes circulating in Australia lack host adapted population structure but demonstrate natural spillover infection.

Rep protein accommodates together dsDNA and ssDNA which enables a loop-back mechanism to plasmid DNA replication initiation.

For DNA replication initiation in Bacteria, replication initiation proteins bind to double-stranded DNA (dsDNA) and interact with single-stranded DNA (ssDNA) at the replication origin. The structural-functional relationship of the nucleoprotein complex involving initiator proteins is still elusive and different models are proposed. In this work, based on crosslinking combined with mass spectrometry (MS), the analysis of mutant proteins and crystal structures, we defined amino acid residues essential for the interaction between plasmid Rep proteins, TrfA and RepE, and ssDNA. This interaction and Rep binding to dsDNA could not be provided in trans, and both are important for dsDNA melting at DNA unwinding element (DUE). We solved two crystal structures of RepE: one in a complex with ssDNA DUE, and another with both ssDNA DUE and dsDNA containing RepE-specific binding sites (iterons). The amino acid residues involved in interaction with ssDNA are located in the WH1 domain in stand β1, helices α1 and α2 and in the WH2 domain in loops preceding strands β1' and β2' and in these strands. It is on the opposite side compared to RepE dsDNA-recognition interface. Our data provide evidence for a loop-back mechanism through which the plasmid replication initiator molecule accommodates together dsDNA and ssDNA.

Phylogenetic analysis and molecular characterization of BBTV DNA-R of wild and cultivated banana isolates from East Java, Indonesia

Banana bunchy top virus (BBTV) molecular detection and understanding its origin are important issues for mitigating future spread. The aim of this study was to molecularly detect BBTV infection and analyze the characteristic also phylogenetic of banana isolates from East Java Indonesia. Two BBTV asymptomatic wild bananas and two BBTV symptomatic banana cultivars were examined. PCR amplifications were accomplished using BBTV DNA-R primers for master replication initiation protein. Sequences evaluations were conducted in SeqScanner. Sequences identification was performed in nucleotide BLAST. Translation of ORFs was determined using ORF Finder server tool. Protein identification was conducted in protein BLAST. Sequences polymorphisms were analyzed using DnaSP6. Phylogenetic analysis was employed using Neighbor-Joining algorithm with Kimura two-parameter (K2P) substitution model in MEGA7. Results showed that BBTV DNA-R components were detected in all isolates and confirmed as Rep protein. The sequences length were varied from 616 to 1,074 bp, low GC content (42.90%) and low conservation (56.47%). Asymptomatic wild bananas generated shorter length and more variable sequences, presumably related to the resistance mechanism. Phylogenetic analysis of BBTV DNA-R East Java with other 38 homolog sequences worldwide were found clustered in Asian Group, closely related to Vietnam, Thailand, and China. Hence, it presumably originated from China mainland via Malay Peninsula route.

PSK41/pGO1-family conjugative plasmids of Staphylococcus aureus encode a cryptic repressor of replication

The majority of large multiresistance plasmids of Staphylococcus aureus utilise a RepA_N-type replication initiation protein, the expression of which is regulated by a small antisense RNA (RNAI) that overlaps the rep mRNA leader. The pSK41/pGO1-family of conjugative plasmids additionally possess a small (86 codon) divergently transcribed ORF (orf86) located upstream of the rep locus. The product of pSK41 orf86 was predicted to have a helix-turn-helix motif suggestive of a likely function in transcriptional repression. In this study, we investigated the effect of Orf86 on transcription of thirteen pSK41 backbone promoters. We found that Orf86 only repressed transcription from the rep promoter, and hence now redesignate the product as Cop. Over-expression of Cop in trans reduced the copy number of pSK41 mini-replicons, both in the presence and absence of rnaI. in vitro protein-DNA binding experiments with purified 6 × His-Cop demonstrated specific DNA binding, adjacent to, and partially overlapping the −35 hexamer of the rep promoter. The crystal structure of Cop revealed a dimeric structure similar to other known transcriptional regulators. Cop mRNA was found to result from “read-through” transcription from the strong RNAI promoter that escapes the rnaI terminator. Thus, PrnaI is responsible for transcription of two distinct negative regulators of plasmid copy number; the antisense RNAI that primarily represses Rep translation, and Cop protein that can repress rep transcription. Deletion of cop in a native plasmid did not appear to impact copy number, indicating a cryptic auxiliary role.

Sensitive Detection of Tumor Cells Using Protein Nanoparticles with Multiple Displays of DNA Aptamers and Bioluminescent Reporters.

Simple and effective detection methods for circulating tumor cells are essential for early detection and progression monitoring of tumors. The use of DNA aptamer and bioluminescence is expected to be a key tool for the simple, effective, and sensitive detection of tumor cells. Herein, we designed multifunctional protein nanoparticles for the detection of tumor cells using DNA aptamer and bioluminescence. Fusion proteins (ELP-poly(d)-POIs), composed of elastin-like polypeptide (ELP) fused with protein of interests (POIs) via poly(aspartic acid) (poly(d)), formed the protein nanoparticles based on the temperature responsivity of ELP sequences, leading to multiply displayed POIs on the protein nanoparticles. In the present study, we focused on porcine circovirus type 2 replication initiation protein (Rep), which covalently conjugated with DNA aptamers, and NanoLuc luciferase (Nluc), which emitted a strong bioluminescence, as POIs. ELP-poly(d)-Rep and ELP-poly(d)-Nluc were constructed and formed the protein nanoparticles with multiply displayed Nluc and Rep (DNA aptamer) that amplified the bioluminescence signal and tumor recognition ability. Mucin-1 (MUC1)-overexpressing human breast tumor MCF7 cells and MUC1-recognizing aptamer (MUC1 aptamer) were selected as models. The MUC1 aptamer-conjugated protein nanoparticles exhibited a 13.7-fold higher bioluminescence signal to MCF-7 cells than to human embryonic kidney 293 (HEK293) cells, which express low levels of MUC1. Furthermore, the protein nanoparticles could detect up to 70.7 cells/mL of MCF-7 cells from a cell suspension containing HEK-293. The protein nanoparticles with multiple Rep and Nluc show a great potential as a material for detecting CTCs.

Betasatellite-encoded βC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of geminivirus-encoded replication initiator protein.

Geminivirus-betasatellite disease complexes are an epidemic threat to the majority of economically important crops across the world. Plant virus satellites including betasatellites are maintained by their associated helper virus. Geminivirus-betasatellites influence viral pathogenesis by substantially increasing or decreasing their helper virus accumulation. In the present study, we attempted to understand the mechanistic details of the geminivirus-betasatellite interaction. Here, we used tomato leaf curl Gujarat virus (ToLCGV) and tomato leaf curl Patna betasatellite (ToLCPaB) as a model system. This study reveals that ToLCGV can efficiently trans-replicate ToLCPaB in Nicotiana benthamiana plants, but ToLCPaB greatly reduced the accumulation of its helper virus DNA. For the first time, we have identified that the ToLCPaB-encoded βC1 protein is able to interact with ToLCGV-encoded replication initiator protein (Rep). In addition, we demonstrate that the C-terminal region of βC1 interacts with the C-terminus of Rep (RepC) protein. Our previous study had established that βC1 proteins encoded by diverse betasatellites possess a novel ATP hydrolysis activity and the conserved lysine/arginine residues at positions 49 and 91 are necessary for this function. Here, we show that mutating lysine at positions 49 to alanine of βC1 (βC1K49A) protein did not affect its ability to interact with RepC protein. Biochemical studies performed with ATP hydrolysis activity-deficient K49A mutated βC1 (βC1K49A) and RepC proteins revealed that Rep-βC1 interaction interferes with the ATP hydrolysis activity of Rep protein. Further, we demonstrate that βC1 protein is able to interact with D227A and D289A mutated RepC proteins but not with D262A, K272A or D286A mutated RepC proteins, suggesting that the βC1-interacting region of Rep protein encompasses its Walker-B and B' motifs. The results of docking studies supported that the βC1-interacting region of Rep protein encompasses its motifs associated with ATP binding and ATP hydrolysis activities. Docking studies also provided evidence that the Rep-βC1 interaction interferes with the ATP binding activity of Rep protein. Together, our findings suggest that βC1 protein regulates helper virus accumulation by interfering with the ATP hydrolysis activity of helper virus Rep protein.

Inhibition of Replication Fork Formation and Progression: Targeting the Replication Initiation and Primosomal Proteins.

Over 1.2 million deaths are attributed to multi-drug-resistant (MDR) bacteria each year. Persistence of MDR bacteria is primarily due to the molecular mechanisms that permit fast replication and rapid evolution. As many pathogens continue to build resistance genes, current antibiotic treatments are being rendered useless and the pool of reliable treatments for many MDR-associated diseases is thus shrinking at an alarming rate. In the development of novel antibiotics, DNA replication is still a largely underexplored target. This review summarises critical literature and synthesises our current understanding of DNA replication initiation in bacteria with a particular focus on the utility and applicability of essential initiation proteins as emerging drug targets. A critical evaluation of the specific methods available to examine and screen the most promising replication initiation proteins is provided.

Rhizobium Phage-Like Microvirus Genome Sequence Identified in Wastewater in Arizona, USA, in November 2020 Encodes an Endolysin and a Putative Multiheme Cytochrome c-like Protein.

We describe the genome (4,696 nucleotides [GC content, 56%; coverage, 3,641×) of MAZ-Nov-2020, a microvirus identified from municipal wastewater in Maricopa County, Arizona, USA, in November 2020. The MAZ-Nov-2020 genome encodes major capsid protein, endolysin, replication initiator protein, and two hypothetical proteins, one of which was predicted to likely be a membrane-associated multiheme cytochrome c.