Initiation Of Chromosome Replication Research Articles

Establishing a biochemical understanding of the initiation of chromosome replication in bacteria

In the mid-1950s, Arthur Kornberg elucidated the enzymatic synthesis of DNA by DNA polymerase, for which he was recognized with the 1959 Nobel Prize in Physiology or Medicine. He then identified many of the proteins that cooperate with DNA polymerase to replicate duplex DNA of small bacteriophages. However, one major unanswered problem was understanding the mechanism and control of the initiation of chromosome replication in bacteria. In a seminal paper in 1981, Fuller, Kaguni, and Kornberg reported the development of a cell-free enzyme system that could replicate DNA that was dependent on the bacterial origin of DNA replication, oriC. This advance opened the door to a flurry of discoveries and important papers that elucidated the process and control of initiation of chromosome replication in bacteria.

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.

Three factors ParA, TipN, and DnaA-mediated chromosome replication initiation are contributors of centromere segregation in Caulobacter crescentus.

The ability of bacteria to maintain chromosomal integrity throughout their life cycle is crucial for survival. In Caulobacter crescentus, the polar factor TipN has been proposed to be involved with the partitioning system ParABS. Cells with tipN knocked out display subtle segregation defects of the centromere-like region parS. We hypothesized that TipN's role with parS segregation is obscured by other forces that are ParABS-independent. To test our hypothesis, we removed one of those forces - chromosome replication - and analyzed the role of TipN with ParA. We first confirm that ParA retains its ability to transport the centromeric region parS from the stalked pole to the opposite pole in the absence of chromosome replication. Our data revealed that in the absence of chromosome replication, TipN becomes essential for ParA's ability to transport parS. Furthermore, we identify a potential connection between the replication initiator DnaA and TipN. Although TipN is not essential for viability, tipN knockout cells lose viability when the regulation of DnaA levels is altered. Our data suggest that the DnaA-dependent susceptibility of tipN knockout cells is connected to parS segregation. Collectively, this work provides insights into the complex regulation involved in the coordination of chromosome replication and segregation in bacteria.

Read-through transcription of tRNA underlies the cell cycle-dependent dissociation of IHF from the DnaA-inactivating sequence datA.

Timely initiation of chromosomal DNA replication in Escherichia coli is achieved by cell cycle-coordinated regulation of the replication origin, oriC, and the replication initiator, ATP-DnaA. Cellular levels of ATP-DnaA increase and peak at the time for initiation at oriC, after which hydrolysis of DnaA-bound ATP causes those to fall, yielding initiation-inactive ADP-DnaA. This hydrolysis is facilitated by the chromosomal locus datA located downstream of the tRNA-Gly (glyV-X-Y) operon, which possesses a cluster of DnaA-binding sequences and a single binding site (IBS) for the DNA bending protein IHF (integration host factor). While IHF binding activates the datA function and is regulated to occur specifically at post-initiation time, the underlying regulatory mechanisms remain obscure. Here, we demonstrate that datA-IHF binding at pre-initiation time is down-regulated depending on the read-through transcription of datA IBS initiated at the glyV-X-Y promoter. During the cell cycle, the level of read-through transcription, but not promoter activity, fluctuated in a manner inversely related to datA-IHF binding. Transcription from the glyV-X-Y promoter was predominantly interrupted at datA IBS by IHF binding. The terminator/attenuator sequence of the glyV-X-Y operon, as well as DnaA binding within datA overall, contributed to attenuation of transcription upstream of datA IBS, preserving the timely fluctuation of read-through transcription. These findings provide a mechanistic insight of tRNA transcription-dependent datA-IHF regulation, in which an unidentified factor is additionally required for the timely datA-IHF dissociation, and support the significance of datA for controlling the cell cycle progression as a connecting hub of tRNA production and replication initiation.

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.

Chromosome Structure and DNA Replication Dynamics During the Life Cycle of the Predatory Bacterium Bdellovibrio bacteriovorus.

Bdellovibrio bacteriovorus, an obligate predatory Gram-negative bacterium that proliferates inside and kills other Gram-negative bacteria, was discovered more than 60 years ago. However, we have only recently begun to understand the detailed cell biology of this proficient bacterial killer. B. bacteriovorus exhibits a peculiar life cycle and bimodal proliferation, and thus represents an attractive model for studying novel aspects of bacterial cell biology. The life cycle of B. bacteriovorus consists of two phases: a free-living non-replicative attack phase and an intracellular reproductive phase. During the reproductive phase, B. bacteriovorus grows as an elongated cell and undergoes binary or nonbinary fission, depending on the prey size. In this review, we discuss: (1) how the chromosome structure of B. bacteriovorus is remodeled during its life cycle; (2) how its chromosome replication dynamics depends on the proliferation mode; (3) how the initiation of chromosome replication is controlled during the life cycle, and (4) how chromosome replication is spatiotemporally coordinated with the proliferation program.

Open Questions about the Roles of DnaA, Related Proteins, and Hyperstructure Dynamics in the Cell Cycle.

The DnaA protein has long been considered to play the key role in the initiation of chromosome replication in modern bacteria. Many questions about this role, however, remain unanswered. Here, we raise these questions within a framework based on the dynamics of hyperstructures, alias large assemblies of molecules and macromolecules that perform a function. In these dynamics, hyperstructures can (1) emit and receive signals or (2) fuse and separate from one another. We ask whether the DnaA-based initiation hyperstructure acts as a logic gate receiving information from the membrane, the chromosome, and metabolism to trigger replication; we try to phrase some of these questions in terms of DNA supercoiling, strand opening, glycolytic enzymes, SeqA, ribonucleotide reductase, the macromolecular synthesis operon, post-translational modifications, and metabolic pools. Finally, we ask whether, underpinning the regulation of the cell cycle, there is a physico-chemical clock inherited from the first protocells, and whether this clock emits a single signal that triggers both chromosome replication and cell division.

Thermotoga maritima oriC involves a DNA unwinding element with distinct modules and a DnaA-oligomerizing region with a novel directional binding mode

Initiation of chromosomal replication requires dynamic nucleoprotein complexes. In most eubacteria, the origin oriC contains multiple DnaA box sequences to which the ubiquitous DnaA initiators bind. In Escherichia coli oriC, DnaA boxes sustain construction of higher-order complexes via DnaA-DnaA interactions, promoting the unwinding of the DNA unwinding element (DUE) within oriC and concomitantly binding the single-stranded (ss) DUE to install replication machinery. Despite the significant sequence homologies among DnaA proteins, oriC sequences are highly diverse. The present study investigated the design of oriC (tma-oriC) from Thermotoga maritima, an evolutionarily ancient eubacterium. The minimal tma-oriC sequence includes a DUE and a flanking region containing five DnaA boxes recognized by the cognate DnaA (tmaDnaA). This DUE was comprised of two distinct functional modules, an unwinding module and a tmaDnaA-binding module. Three direct repeats of the trinucleotide TAG within DUE were essential for both unwinding and ssDUE binding by tmaDnaA complexes constructed on the DnaA boxes. Its surrounding AT-rich sequences stimulated only duplex unwinding. Moreover, head-to-tail oligomers of ATP-bound tmaDnaA were constructed within tma-oriC, irrespective of the directions of the DnaA boxes. This binding mode was considered to be induced by flexible swiveling of DnaA domains III and IV, which were responsible for DnaA-DnaA interactions and DnaA box binding, respectively. Phasing of specific tmaDnaA boxes in tma-oriC was also responsible for unwinding. These findings indicate that a ssDUE recruitment mechanism was responsible for unwinding and would enhance understanding of the fundamental molecular nature of the origin sequences present in evolutionarily divergent bacteria.

Regulatory elements coordinating initiation of chromosome replication to the Escherichia coli cell cycle

Escherichia coli coordinates replication and division cycles by initiating replication at a narrow range of cell sizes. By tracking replisomes in individual cells through thousands of division cycles in wild-type and mutant strains, we were able to compare the relative importance of previously described control systems. We found that accurate triggering of initiation does not require synthesis of new DnaA. The initiation size increased only marginally as DnaA was diluted by growth after dnaA expression had been turned off. This suggests that the conversion of DnaA between its active ATP- and inactive ADP-bound states is more important for initiation size control than the total free concentration of DnaA. In addition, we found that the known ATP/ADP converters DARS and datA compensate for each other, although the removal of them makes the initiation size more sensitive to the concentration of DnaA. Only disruption of the regulatory inactivation of DnaA mechanism had a radical impact on replication initiation. This result was corroborated by the finding that termination of one round of replication correlates with the next initiation at intermediate growth rates, as would be the case if RIDA-mediated conversion from DnaA-ATP to DnaA-ADP abruptly stops at termination and DnaA-ATP starts accumulating.

Binary or Nonbinary Fission? Reproductive Mode of a Predatory Bacterium Depends on Prey Size.

Most bacteria, including model organisms such as Escherichia coli, Bacillus subtilis, and Caulobacter crescentus, reproduce by binary fission. However, some bacteria belonging to various lineages, including antibiotic-producing Streptomyces and predatory Bdellovibrio, proliferate by nonbinary fission, wherein three or more chromosome copies are synthesized and the resulting multinucleoid filamentous cell subdivides into progeny cells. Here, we demonstrate for the first time that the predatory bacterium Bdellovibrio bacteriovorus reproduces through both binary and nonbinary fission inside different prey bacteria. Switching between the two modes correlates with the prey size. In relatively small prey cells, B. bacteriovorus undergoes binary fission; the FtsZ ring assembles in the midcell, and the mother cell splits into two daughter cells. In larger prey cells, B. bacteriovorus switches to nonbinary fission and creates multiple asynchronously assembled FtsZ rings to produce three or more daughter cells. Completion of bacterial cell cycle critically depends on precise spatiotemporal coordination of chromosome replication with other cell cycle events, including cell division. We show that B. bacteriovorus always initiates chromosome replication at the invasive pole of the cell, but the spatiotemporal choreography of subsequent steps depends on the fission mode and/or the number of progeny cells. In nonbinary dividing filaments producing five or more progeny cells, the last round(s) of replication may also be initiated at the noninvasive pole. Altogether, we find that B. bacteriovorus reproduces through bimodal fission and that extracellular factors, such as the prey size, can shape replication choreography, providing new insights about bacterial life cycles. IMPORTANCE Most eukaryotic and bacterial cells divide by binary fission, where one mother cell produces two progeny cells, or, rarely, by nonbinary fission. All bacteria studied to date use only one of these two reproduction modes. We demonstrate for the first time that a predatory bacterium, Bdellovibrio bacteriovorus, exhibits bimodal fission and the mode of division depends on the size of the prey bacterium inside which B. bacteriovorus grows. This work provides key insights into the mode and dynamics of B. bacteriovorus proliferation in different pathogens that pose a major threat to human health due to their emerging antibiotic resistance (Proteus mirabilis, Salmonella enterica, and Shigella flexneri). The use of predatory bacteria such as B. bacteriovorus is currently regarded as a promising strategy to kill antibiotic-resistant pathogens. We find that B. bacteriovorus employs different chromosome replication choreographies and division modes when preying on those pathogens. Our findings may facilitate the design of efficient pathogen elimination strategies.

Establishment and function of chromatin organization at replication origins.

The origin recognition complex (ORC) is essential for initiation of eukaryotic chromosome replication as it loads the replicative helicase-the minichromosome maintenance (MCM) complex-at replication origins1. Replication origins display a stereotypic nucleosome organization with nucleosome depletion at ORC-binding sites and flanking arrays of regularly spaced nucleosomes2-4. However, how this nucleosome organization is established and whether this organization is required for replication remain unknown. Here, using genome-scale biochemical reconstitution with approximately 300 replication origins, we screened 17 purified chromatin factors from budding yeast and found that the ORC established nucleosome depletion over replication origins and flanking nucleosome arrays by orchestrating the chromatin remodellers INO80, ISW1a, ISW2 and Chd1. The functional importance of the nucleosome-organizing activity of the ORC was demonstrated by orc1 mutations that maintained classical MCM-loader activity but abrogated the array-generation activity of ORC. These mutations impaired replication through chromatin in vitro and were lethal in vivo. Our results establish that ORC, in addition to its canonical role as the MCM loader, has a second crucial function as a master regulator of nucleosome organization at the replication origin, a crucial prerequisite for efficient chromosome replication.

★Track: Inferred counting and tracking of replicating DNA loci

Fluorescent microscopy is the primary method to study DNA organization within cells. However, the variability and low signal/noise commonly associated with live-cell time-lapse imaging challenges quantitative measurements. In particular, obtaining quantitative or mechanistic insight often depends on the accurate tracking of fluorescent particles. Here, we present ★Track, an inference method that determines the most likely temporal tracking of replicating intracellular particles such DNA loci while accounting for missing, merged, and spurious detections. It allows the accurate prediction of particle copy numbers as well as the timing of replication events. We demonstrate ★Track’s abilities and gain new insight into plasmid copy number control and the volume dependence of bacterial chromosome replication initiation. By enabling the accurate tracking of DNA loci, ★Track can help to uncover the mechanistic principles of chromosome organization and dynamics across a range of systems.

Prophage-encoded small protein YqaH counteracts the activities of the replication initiator DnaA in Bacillus subtilis.

Bacterial genomes harbour cryptic prophages that are mostly transcriptionally silent with many unannotated genes. Still, cryptic prophages may contribute to their host fitness and phenotypes. In Bacillus subtilis, the yqaF-yqaN operon belongs to the prophage element skin, and is tightly repressed by the Xre-like repressor SknR. This operon contains several small ORFs (smORFs) potentially encoding small-sized proteins. The smORF-encoded peptide YqaH was previously reported to bind to the replication initiator DnaA. Here, using a yeast two-hybrid assay, we found that YqaH binds to the DNA binding domain IV of DnaA and interacts with Spo0A, a master regulator of sporulation. We isolated single amino acid substitutions in YqaH that abolished the interaction with DnaA but not with Spo0A. Then, using a plasmid-based inducible system to overexpress yqaH WT and mutant derivatives, we studied in B. subtilis the phenotypes associated with the specific loss-of-interaction with DnaA (DnaA_LOI). We found that expression of yqaH carrying DnaA_LOI mutations abolished the deleterious effects of yqaH WT expression on chromosome segregation, replication initiation and DnaA-regulated transcription. When YqaH was induced after vegetative growth, DnaA_LOI mutations abolished the drastic effects of YqaH WT on sporulation and biofilm formation. Thus, YqaH inhibits replication, sporulation and biofilm formation mainly by antagonizing DnaA in a manner that is independent of the cell cycle checkpoint Sda.

Concerted actions of DnaA complexes with DNA-unwinding sequences within and flanking replication origin oriC promote DnaB helicase loading

Unwinding of the replication origin and loading of DNA helicases underlie the initiation of chromosomal replication. In Escherichia coli, the minimal origin oriC contains a duplex unwinding element (DUE) region and three (Left, Middle, and Right) regions that bind the initiator protein DnaA. The Left/Right regions bear a set of DnaA-binding sequences, constituting the Left/Right-DnaA subcomplexes, while the Middle region has a single DnaA-binding site, which stimulates formation of the Left/Right-DnaA subcomplexes. In addition, a DUE-flanking AT-cluster element (TATTAAAAAGAA) is located just outside of the minimal oriC region. The Left-DnaA subcomplex promotes unwinding of the flanking DUE exposing TT[A/G]T(T) sequences that then bind to the Left-DnaA subcomplex, stabilizing the unwound state required for DnaB helicase loading. However, the role of the Right-DnaA subcomplex is largely unclear. Here, we show that DUE unwinding by both the Left/Right-DnaA subcomplexes, but not the Left-DnaA subcomplex only, was stimulated by a DUE-terminal subregion flanking the AT-cluster. Consistently, we found the Right-DnaA subcomplex–bound single-stranded DUE and AT-cluster regions. In addition, the Left/Right-DnaA subcomplexes bound DnaB helicase independently. For only the Left-DnaA subcomplex, we show the AT-cluster was crucial for DnaB loading. The role of unwound DNA binding of the Right-DnaA subcomplex was further supported by in vivo data. Taken together, we propose a model in which the Right-DnaA subcomplex dynamically interacts with the unwound DUE, assisting in DUE unwinding and efficient loading of DnaB helicases, while in the absence of the Right-DnaA subcomplex, the AT-cluster assists in those processes, supporting robustness of replication initiation.

In Silico Core Proteomics and Molecular Docking Approaches for the Identification of Novel Inhibitors against Streptococcus pyogenes.

Streptococcus pyogenes is a significant pathogen that causes skin and upper respiratory tract infections and non-suppurative complications, such as acute rheumatic fever and post-strep glomerulonephritis. Multidrug resistance has emerged in S. pyogenes strains, making them more dangerous and pathogenic. Hence, it is necessary to identify and develop therapeutic methods that would present novel approaches to S. pyogenes infections. In the current study, a subtractive proteomics approach was employed to core proteomes of four strains of S. pyogenes using several bioinformatic software tools and servers. The core proteome consists of 1324 proteins, and 302 essential proteins were predicted from them. These essential proteins were analyzed using BLASTp against human proteome, and the number of potential targets was reduced to 145. Based on subcellular localization prediction, 46 proteins with cytoplasmic localization were chosen for metabolic pathway analysis. Only two cytoplasmic proteins, i.e., chromosomal replication initiator protein DnaA and two-component response regulator (TCR), were discovered to have the potential to be novel drug target candidates. Three-dimensional (3D) structure prediction of target proteins was carried out via the Swiss Model server. Molecular docking approach was employed to screen the library of 1000 phytochemicals against the interacting residues of the target proteins through the MOE software. Further, the docking studies were validated by running molecular dynamics simulation and highly popular binding free energy approaches of MM-GBSA and MM-PBSA. The findings revealed a promising candidate as a novel target against S. pyogenes infections.

Riboregulation in bacteria: From general principles to novel mechanisms of the trp attenuator and its sRNA and peptide products.

Gene expression strategies ensuring bacterial survival and competitiveness rely on cis- and trans-acting RNA-regulators (riboregulators). Among the cis-acting riboregulators are transcriptional and translational attenuators, and antisense RNAs (asRNAs). The trans-acting riboregulators are small RNAs (sRNAs) that bind proteins or base pairs with other RNAs. This classification is artificial since some regulatory RNAs act both in cis and in trans, or function in addition as small mRNAs. A prominent example is the archetypical, ribosome-dependent attenuator of tryptophan (Trp) biosynthesis genes. It responds by transcription attenuation to two signals, Trp availability and inhibition of translation, and gives rise to two trans-acting products, the attenuator sRNA rnTrpL and the leader peptide peTrpL. In Escherichia coli, rnTrpL links Trp availability to initiation of chromosome replication and in Sinorhizobium meliloti, it coordinates regulation of split tryptophan biosynthesis operons. Furthermore, in S. meliloti, peTrpL is involved in mRNA destabilization in response to antibiotic exposure. It forms two types of asRNA-containing, antibiotic-dependent ribonucleoprotein complexes (ARNPs), one of them changing the target specificity of rnTrpL. The posttranscriptional role of peTrpL indicates two emerging paradigms: (1) sRNA reprograming by small molecules and (2) direct involvement of antibiotics in regulatory RNPs. They broaden our view on RNA-based mechanisms and may inspire new approaches for studying, detecting, and using antibacterial compounds. This article is categorized under: RNA Interactions with Proteins and Other Molecules > Small Molecule-RNA Interactions RNA Interactions with Proteins and Other Molecules > RNA-Protein Complexes Regulatory RNAs/RNAi/Riboswitches > Regulatory RNAs.

Proteome based mapping and molecular docking revealed DnaA as a potential drug target against Shigella sonnei

Shigella sonnei is one of the major causes of diarrhea and remained a critical microbe responsible for higher morbidity and mortality rates resulting from dysentery every year across the world. Antibiotic therapy of Shigella diseases plays a critical role in decreasing the prevalence as well as the fatality rate of this infection. However, the management of these diseases remains challenging, owing to the overall increase in resistance against many antimicrobials. The situation necessitates the rapid development of effective and feasible S. sonnei treatments. In the present study, the subtractive genomics approach was utilized to find the potential drug targets for S. sonnei strain Ss046. Various tools of bioinformatics were implemented to remove the human-specific homologous and pathogen-specific paralogous sequences from the bacterial proteome. Then, metabolic pathway and subcellular location analysis were performed of essential bacterial proteins to describe their role in various cellular processes. Only one essential protein i-e Chromosomal replication initiator protein DnaA was found in the proteome of the pathogen that could be used as a potent target for designing new drugs. 3D structure prediction of DnaA protein was carried out using Phyre 2. Molecular docking of 5000 phytochemicals was performed against DnaA to identify four top-ranked phytochemicals (Riccionidin A, Dothistromin, Fustin, and Morin) based on scoring functions and interaction with the active site. This study suggests that these phytochemicals could be used as antibacterial drugs to treat S. sonnei infections in the future. To confirm their efficacy and evaluate their drug potency, further in vitro analyses are required.

Blocking, Bending, and Binding: Regulation of Initiation of Chromosome Replication During the Escherichia coli Cell Cycle by Transcriptional Modulators That Interact With Origin DNA.

Genome duplication is a critical event in the reproduction cycle of every cell. Because all daughter cells must inherit a complete genome, chromosome replication is tightly regulated, with multiple mechanisms focused on controlling when chromosome replication begins during the cell cycle. In bacteria, chromosome duplication starts when nucleoprotein complexes, termed orisomes, unwind replication origin (oriC) DNA and recruit proteins needed to build new replication forks. Functional orisomes comprise the conserved initiator protein, DnaA, bound to a set of high and low affinity recognition sites in oriC. Orisomes must be assembled each cell cycle. In Escherichia coli, the organism in which orisome assembly has been most thoroughly examined, the process starts with DnaA binding to high affinity sites after chromosome duplication is initiated, and orisome assembly is completed immediately before the next initiation event, when DnaA interacts with oriC’s lower affinity sites, coincident with origin unwinding. A host of regulators, including several transcriptional modulators, targets low affinity DnaA-oriC interactions, exerting their effects by DNA bending, blocking access to recognition sites, and/or facilitating binding of DnaA to both DNA and itself. In this review, we focus on orisome assembly in E. coli. We identify three known transcriptional modulators, SeqA, Fis (factor for inversion stimulation), and IHF (integration host factor), that are not essential for initiation, but which interact directly with E. coli oriC to regulate orisome assembly and replication initiation timing. These regulators function by blocking sites (SeqA) and bending oriC DNA (Fis and IHF) to inhibit or facilitate cooperative low affinity DnaA binding. We also examine how the growth rate regulation of Fis levels might modulate IHF and DnaA binding to oriC under a variety of nutritional conditions. Combined, the regulatory mechanisms mediated by transcriptional modulators help ensure that at all growth rates, bacterial chromosome replication begins once, and only once, per cell cycle.

YfiF, an unknown protein, affects initiation timing of chromosome replication in Escherichia coli.

The Escherichia coli YfiF protein is functionally unknown, being predicted as a transfer RNA/ribosomal RNA (tRNA/rRNA) methyltransferase. We find that absence of the yfiF gene delays initiation of chromosome replication and the delay is reversed by ectopic expression of YfiF, whereas excess YfiF causes an early initiation. A slight decrease in both cell size and number of origin per mass is observed in ΔyfiF cells. YfiF does not genetically interact with replication proteins such as DnaA, DnaB, and DnaC. Interestingly, YfiF is associated with ribosome modulation factor (RMF), hibernation promotion factor (HPF), and the tRNA methyltransferase TrmL. Defects in replication initiation of Δrmf, Δhpf, and ΔtrmL can be rescued by overexpression of YfiF, indicating that YfiF is functionally identical to RMF, HPF, and TrmL in terms of replication initiation. Also, YfiF interacts with the rRNA methyltransferase RsmC. Moreover, the total amount of proteins and DnaA content per cell decreases or increases in the absence of YfiF or the presence of excess YfiF. These facts suggest that YfiF is a ribosomal dormancy-like factor, affecting ribosome function. Thus, we propose that YfiF is involved in the correct timing of chromosome replication by changing the DnaA content per cell as a result of affecting ribosome function.