Bacteriophage Lambda Research Articles

H-NS is a Transcriptional Repressor of the CRISPR-Cas System in Acinetobacter baumannii ATCC 19606.

Acinetobacter baumannii is a multidrug-resistant opportunistic pathogen primarily associated with hospital-acquired infections. The bacterium can gain multidrug resistance through several mechanisms, including horizontal gene transfer. A CRISPR-Cas system including several Cas genes could restrict the horizontal gene transfer. However, the molecular mechanism of CRISPR- Cas transcriptional regulation remains unclear. We identified a type I-F CRISPR-Cas system in A. baumannii ATCC 19606T standard strain based on sequence analysis. We focused on the transcriptional regulation of Cas3, a key protein of the CRISPR-Cas system. We performed a DNA affinity chromatography-pulldown assay to identify transcriptional regulators of the Cas3 promoter. We identified several putative transcriptional factors, such as H-NS, integration host factor, and HU, that can bind to the promoter region of Cas3. We characterized AbH-NS using size exclusion chromatography and cross-linking experiments and demonstrated that the Cas3 promoter can be regulated by AbH-NS in a concentration-dependent manner via an in vitro transcription assay. CRISPR-Cas expression levels in wild-type and hns mutant strains in the early stationary phase were examined by qPCR and β-galactosidase assay. We found that H-NS can act as a repressor of Cas3. Our transformation efficiency results indicated that the hns mutation decreased the transformation efficiency, while the Cas3 mutation increased it. We report the existence and characterization of the CRISPR-Cas system in A. baumannii 19606T and demonstrate that AbH-NS is a transcriptional repressor of CRISPR-Cas-related genes in A. baumannii.

Fine-tuned spatiotemporal dynamics of DNA replication during phage lambda infection.

Temperate bacteriophages, such as lambda, incorporate environmental cues including host abundance and nutrient conditions to make optimal decisions between propagation and dormancy. A higher phage-to-host ratio or multiplicity of infection (MOI) during λ infection strongly biases toward lysogeny. However, a comprehensive understanding of this decision-making process and the impact of phage replication prior to the decision is yet to be achieved. Here, we used fluorescence microscopy to quantitatively track the spatiotemporal progression of viral DNA replication in individual cells with different cell fates. The implementation of this fluorescent reporter system and quantitative analysis workflow opens a new avenue for future studies to delve deeper into various types of virus-host interactions at a high resolution.

Second-Generation Phage Lambda Platform Employing SARS-CoV-2 Fusion Proteins as a Vaccine Candidate

The recent SARS-CoV-2 (COVID-19) pandemic exemplifies how newly emerging and reemerging viruses can quickly overwhelm and cripple global infrastructures. Coupled with synergistic factors such as increasing population densities, the constant and massive mobility of people across geographical areas and substantial changes to ecosystems worldwide, these pathogens pose serious health concerns on a global scale. Vaccines form an indispensable defense, serving to control and mitigate the impact of devastating outbreaks and pandemics. Towards these efforts, we developed a tunable vaccine platform that can be engineered to simultaneously display multiple viral antigens. Here, we describe a second-generation version wherein chimeric proteins derived from SARS-CoV-2 and bacteriophage lambda are engineered and used to decorate phage-like particles with defined surface densities and retention of antigenicity. This streamlines the engineering of particle decoration, thus improving the overall manufacturing potential of the system. In a prime-boost regimen, mice immunized with particles containing as little as 42 copies of the chimeric protein on their surface develop potent neutralizing antibody responses, and immunization protects mice against virulent SARS-CoV-2 challenge. The platform is highly versatile, making it a promising strategy to rapidly develop vaccines against a potentially broad range of infectious diseases.

Investigating the Quantification Capabilities of a Nanopore-Based Sequencing Platform for Food Safety Application via External Standards of Lambda DNA and Lambda Spiked Beef.

Six hundred million cases of disease and roughly 420,000 deaths occur globally each year due to foodborne pathogens. Current methods to screen and identify pathogens in swine, poultry, and cattle products include immuno-based techniques (e.g., immunoassay integrated biosensors), molecular methods (e.g., DNA hybridization and PCR assays), and traditional culturing. These methods are often used in tandem to screen, quantify, and characterize samples, prolonging real-time comprehensive analysis. Next-generation sequencing (NGS) is a relatively new technology that combines DNA-sequencing chemistry and bioinformatics to generate and analyze large amounts of short- or long-read DNA sequences and whole genomes. The goal of this project was to evaluate the quantitative capabilities of the real-time NGS Oxford Nanopore Technologies' MinION sequencer through a shotgun-based sequencing approach. This investigation explored the correlation between known amounts of the analyte (lambda DNA as a pathogenic bacterial surrogate) with data output, in both the presence and absence of a background matrix (Bos taurus DNA). A positive linear correlation was observed between the concentration of analyte and the amount of data produced, number of bases sequenced, and number of reads generated in both the presence and absence of a background matrix. In the presence of bovine DNA, the sequenced data were successfully mapped to the NCBI lambda reference genome. Furthermore, the workflow from pre-extracted DNA to target identification took less than 3 h, demonstrating the potential of long-read sequencing in food safety as a rapid method for screening, identification, and quantification.

Flat‐Field Lens Grating Microspectrometer for Spectroscopic Multiplex Bioassay

AbstractSpectroscopic multiplex bioassay allows the rapid and simultaneous detection of various biomarkers in biomedical applications. The high cost and bulky configuration of conventional spectrometers hinder the decentralization of biomedical instruments for point‐of‐care diagnostics. Here a flat‐field lens grating‐based microspectrometer (FLG‐µSPEC) is reported for continuous detection of multi‐target nucleic acid amplification. FLG‐µSPEC features the vertical integration of microslit, integrated flat‐field lens and transmission grating (FLG), concave mirror, and CMOS line sensor. FLG effectively corrects the field curvature by flattening the focal plane of dispersed wavelengths and also facilitates compact module packaging. FLG‐µSPEC module is precisely assembled into a dimension of 10 mm × 16 mm × 10 mm. The average spectral resolution exhibits 3.4 ± 0.9 nm over an operating range of 500 to 950 nm. A spectral detection system utilizing FLG‐µSPEC demonstrates the amplification of lambda DNA (𝜆‐DNA) for each target, exhibiting a high correlation with the obtained result from a conventional real‐time PCR (qPCR) machine. Furthermore, multiplex qPCR analysis using FLG‐µSPEC and spectral deconvolution successfully demonstrates simultaneous and accurate quantification of three different targets. The FLG‐µSPEC module offers diverse on‐demand analytical applications, including not only multiplex bioassay but also environmental monitoring.

Using bacterial population dynamics to count phages and their lysogens

Traditional assays for counting bacteriophages and their lysogens are labor-intensive and perturbative to the host cells. Here, we present a high-throughput infection method in a microplate reader, where the growth dynamics of the infected culture is measured using the optical density (OD). We find that the OD at which the culture lyses scales linearly with the logarithm of the initial phage concentration, providing a way of measuring phage numbers over nine orders of magnitude and down to single-phage sensitivity. Interpreting the measured dynamics using a mathematical model allows us to infer the phage growth rate, which is a function of the phage-cell encounter rate, latent period, and burst size. Adding antibiotic selection provides the ability to measure the rate of host lysogenization. Using this method, we found that when E. coli growth slows down, the lytic growth rate of lambda phages decreases, and the propensity for lysogeny increases, demonstrating how host physiology influences the viral developmental program.

Exploring the diversity of anti-defense systems across prokaryotes, phages, and mobile genetic elements.

The co-evolution of prokaryotes, phages, and mobile genetic elements (MGEs) over the past billions of years has driven the emergence and diversification of defense and anti-defense systems alike. Anti-defense proteins have diverse functional domains, sequences, and are typically small, creating a challenge to detect anti-defense homologs across the prokaryotic genomes. To date, no tools comprehensively annotate anti-defense proteins within a desired genome or MGE. Here, we developed "AntiDefenseFinder" - a free open-source tool and web service that detects 156 anti-defense systems (of one or more proteins) in any genomic sequence. Using this dataset, we identified 47,981 anti-defense systems distributed across prokaryotes, phage, and MGEs. We found that some genes co-localize in "anti-defense islands", including E. coli T4 and Lambda phages, although many are standalone. Out of the 112 systems detected in bacteria, 100 systems localize only or preferentially in prophages, plasmids, phage satellites, integrons, and integrative and conjugative elements. However, over 80% of anti-Pycsar protein 1 (Apyc1) resides in non-mobile regions of bacteria. Evolutionary and functional analyses revealed that Apyc1 likely originated in bacteria to regulate cNMP signaling, but was co-opted multiple times by phages to overcome cNMP-utilizing defenses. With the AntiDefenseFinder tool, we hope to facilitate the identification of the full repertoire of anti-defense systems in MGEs, the discovery of new protein functions, and a deeper understanding of host-pathogen arms race.

The Impact of Chloroform on Viability of Temperate Bacteriophage Lambda and Survival of its Host, Escherichia coli

The Impact of Chloroform on Viability of Temperate Bacteriophage Lambda and Survival of its Host, Escherichia coli

Metabolic flux regulates growth transitions and antibiotic tolerance in uropathogenic Escherichia coli.

Uropathogenic Escherichia coli (UPEC) are the leading cause of urinary tract infections (UTIs). Upon invasion into bladder epithelial cells, UPEC establish quiescent intracellular reservoirs that may lead to antibiotic tolerance and recurrent UTIs. Here, we demonstrate using an in vitro system that quiescent UPEC cells are tolerant to ampicillin and have decreased metabolism characterized by succinyl-CoA limitation. We identify the global regulator integration host factor complex and the cell division protein ZapE as critical modifiers of quiescence and antibiotic tolerance. Finally, we show that ZapE interacts with components of both the cell division machinery and the tricarboxylic acid cycle, and this interaction is conserved in non-pathogenic E. coli, establishing a novel link between cell division and metabolism.

Stability and gene strand bias of lambda prophages and chromosome organization in Escherichia coli.

Temperate phage-mediated horizontal gene transfer is a potent driver of genetic diversity in the evolution of bacteria. Most lambdoid prophages in Escherichia coli are integrated into the chromosome with the same orientation with respect to the direction of chromosomal replication, and their location on the chromosome is far from homogeneous. To better understand these features, we studied the interplay between lysogenic and lytic states of phage lambda in both native and inverted integration orientations at the wild-type integration site as well as at other sites on the bacterial chromosome. Measurements of free phage released by spontaneous induction showed that the stability of lysogenic states is affected by location and orientation along the chromosome, with stronger effects near the origin of replication. Competition experiments and range expansions between lysogenic strains with opposite orientations and insertion loci indicated that there are no major differences in growth. Moreover, measurements of the level of transcriptional bursts of the cI gene coding for the lambda phage repressor using single-molecule fluorescence in situ hybridization resulted in similar levels of transcription for both orientations and prophage location. We postulate that the preference for a given orientation and location is a result of a balance between the maintenance of lysogeny and the ability to lyse.IMPORTANCEThe integration of genetic material of temperate bacterial viruses (phages) into the chromosomes of bacteria is a potent evolutionary force, allowing bacteria to acquire in one stroke new traits and restructure the information in their chromosomes. Puzzlingly, this genetic material is preferentially integrated in a particular orientation and at non-random sites on the bacterial chromosome. The work described here reveals that the interplay between the maintenance of the stability of the integrated phage, its ability to excise, and its localization along the chromosome plays a key role in setting chromosomal organization in Escherichia coli.

Coinfecting phages impede each other’s entry into the cell

The developmental choice made by temperate phages, between cell death (lysis) and viral dormancy (lysogeny), is influenced by the relative abundance of viruses and hosts in the environment. The paradigm for this abundance-driven decision is phage lambda of E.coli, whose propensity to lysogenize increases with the number of viruses coinfecting the same bacterium. It is believed that lambda uses this number to infer whether phages or bacteria outnumber each other. However, this interpretation is premised on an accurate mapping between the extracellular phage-to-bacteria ratio and the intracellular multiplicity of infection (MOI). Here, we show this premise to be faulty. By simultaneously labeling phage capsids and genomes, we find that, while the number of phages landing on each cell reliably samples the population ratio, the number of phages entering the cell does not. Single-cell infections, performed in a microfluidic device and interpreted using a stochastic model, reveal that the probability and rate of phage entry decrease with the number of adsorbed phages. This decrease reflects an MOI-dependent perturbation to host physiology caused by phage attachment, as evidenced by compromised membrane integrity and loss of membrane potential. The dependence of entry dynamics on the surrounding medium results in a strong impact on the infection outcome, while the protracted entry of coinfecting phages increases the heterogeneity in infection outcome at a given MOI. Our findings in lambda, and similar results we obtained for phages T5 and P1, demonstrate the previously unappreciated role played by entry dynamics in determining the outcome of bacteriophage infection.

Molecular Insight into the Structural Dynamics of Holliday Junctions Modulated by Integration Host Factor.

The integration host factor (IHF) in Escherichia coli is a nucleoid-associated protein with multifaceted roles that encompass DNA packaging, viral DNA integration, and recombination. IHF binds to double-stranded DNA featuring a 13-base pair (bp) consensus sequence with high affinity, causing a substantial bend of approximately 160° upon binding. Although wild-type IHF (WtIHF) is principally involved in DNA bending to facilitate foreign DNA integration into the host genome, its engineered counterpart, single-chain IHF (ScIHF), was specifically designed for genetic engineering and biotechnological applications. Our study delves into the interactions of both IHF variants with Holliday junctions (HJs), pivotal intermediates in DNA repair, and homologous recombination. HJs are dynamic structures capable of adopting open or stacked conformations, with the open conformation facilitating processes such as branch migration and strand exchange. Using microscale thermophoresis, we quantitatively assessed the binding of IHF to four-way DNA junctions that harbor specific binding sequences H' and H1. Our findings demonstrate that both IHF variants exhibit a strong affinity for HJs, signifying a structure-based recognition mechanism. Circular dichroism (CD) experiments unveiled the impact of the protein on the junction's conformation. Furthermore, single-molecule Förster resonance energy transfer (smFRET) confirmed the influence of IHF on the junction's dynamicity. Intriguingly, our results revealed that WtIHF and ScIHF binding shifts the population toward the open conformation of the junction and stabilizes it in that state. In summary, our findings underscore the robust affinity of the IHF for HJs and its capacity to stabilize the open conformation of these junctions.

Control of a gene transfer agent cluster in Caulobacter crescentus by transcriptional activation and anti-termination

Gene Transfer Agents (GTAs) are phage-like particles that cannot self-multiply and be infectious. Caulobacter crescentus, a bacterium best known as a model organism to study bacterial cell biology and cell cycle regulation, has recently been demonstrated to produce bona fide GTA particles (CcGTA). Since C. crescentus ultimately die to release GTA particles, the production of GTA particles must be tightly regulated and integrated with the host physiology to prevent a collapse in cell population. Two direct activators of the CcGTA biosynthetic gene cluster, GafY and GafZ, have been identified, however, it is unknown how GafYZ controls transcription or how they coordinate gene expression of the CcGTA gene cluster with other accessory genes elsewhere on the genome for complete CcGTA production. Here, we show that the CcGTA gene cluster is transcriptionally co-activated by GafY, integration host factor (IHF), and by GafZ-mediated transcription anti-termination. We present evidence that GafZ is a transcription anti-terminator that likely forms an anti-termination complex with RNA polymerase, NusA, NusG, and NusE to bypass transcription terminators within the 14 kb CcGTA cluster. Overall, we reveal a two-tier regulation that coordinates the synthesis of GTA particles in C. crescentus.

Structural mechanism of bacteriophage lambda tail’s interaction with the bacterial receptor

Bacteriophage infection, a pivotal process in microbiology, initiates with the phage’s tail recognizing and binding to the bacterial cell surface, which then mediates the injection of viral DNA. Although comprehensive studies on the interaction between bacteriophage lambda and its outer membrane receptor, LamB, have provided rich information about the system’s biochemical properties, the precise molecular mechanism remains undetermined. This study revealed the high-resolution cryo-electron microscopy (cryo-EM) structures of the bacteriophage lambda tail complexed with its irreversible Shigella sonnei 3070 LamB receptor and the closed central tail fiber. These structures reveal the complex processes that trigger infection and demonstrate a substantial conformational change in the phage lambda tail tip upon LamB binding. Providing detailed structures of bacteriophage lambda infection initiation, this study contributes to the expanding knowledge of lambda-bacterial interaction, which holds significance in the fields of microbiology and therapeutic development.

An integrated in-silico approach for drug target identification in human pathogen Shigella dysenteriae.

Shigella dysenteriae, is a Gram-negative bacterium that emerged as the second most significant cause of bacillary dysentery. Antibiotic treatment is vital in lowering Shigella infection rates, yet the growing global resistance to broad-spectrum antibiotics poses a significant challenge. The persistent multidrug resistance of S. dysenteriae complicates its management and control. Hence, there is an urgent requirement to discover novel therapeutic targets and potent medications to prevent and treat this disease. Therefore, the integration of bioinformatics methods such as subtractive and comparative analysis provides a pathway to compute the pan-genome of S. dysenteriae. In our study, we analysed a dataset comprising 27 whole genomes. The S. dysenteriae strain SD197 was used as the reference for determining the core genome. Initially, our focus was directed towards the identification of the proteome of the core genome. Moreover, several filters were applied to the core genome, including assessments for non-host homology, protein essentiality, and virulence, in order to prioritize potential drug targets. Among these targets were Integration host factor subunit alpha and Tyrosine recombinase XerC. Furthermore, four drug-like compounds showing potential inhibitory effects against both target proteins were identified. Subsequently, molecular docking analysis was conducted involving these targets and the compounds. This initial study provides the list of novel targets against S. dysenteriae. Conclusively, future in vitro investigations could validate our in-silico findings and uncover potential therapeutic drugs for combating bacillary dysentery infection.

Beyond 16S sequencing for microbiome research: Nanopore sequencing resource at The University of Toledo Microbiome Consortium

Objective: The recognition that humans are holobionts has opened a new research area to examine the contributions of the microbiome to health and disease. This requires a full understanding of the identities of microbiota at the taxonomical level of the species. Introduction: The University of Toledo Microbiome Consortium currently offers the second-generation Illumina sequencing platform for 16S sequencing to determine microbiota composition. 16S sequencing of variable regions of the 16S operon, such as V3, V4, and the combination of V3 to V4, has long been the main stay for analyzing the composition of microbiota. The advantage of this approach is the ability to screen large numbers of samples at a lower cost per sample. However, the major drawback of this approach is a lower resolution whereby taxonomical identities of microbiota are limited to the genus level. To overcome this disadvantage, we introduced the third-generation sequencing technology from Nanopore into our pipeline. The known advantages of this approach are longer reads and the ability to sequence DNA in the native form without PCR-induced bias. Here we sought to conduct a comparative study of the relative effciencies of the 16S versus the nanopore technology for metagenomics. Methods: Genomic DNA was isolated from control and experimental rat fecal samples provided by an end-user. Libraries were prepared using Ligation sequencing DNA V14 (SqK-LSK114) on a Flow cell R10.4.1and Lambda DNA was used as a sequencing control. The native Libraries were run for 72hr, and high accuracy base calling was performed. The Fastq files (passed) generated by GridION were run on cloud-based CZ-ID pipeline with host and human read filtering, the results were normalized to one million bases sequenced from reads of each sample. Results and Conclusions: The 16S sequencing data highlighted the relative abundance of different taxa up to genus level such as Lactobacillus and Erysipelatoclostridium in the experimental group where as Stepcococcus and prevotellaceaeGa6A1group in the control group. However, Nanopore sequencing further identified twenty different species of Lactobacillus and , four different species of Erysipelatoclostridium. Similarly, nanopore sequencing was able to profile diverse species belonging to genera prevotellaceaeGa6A1group and Streptococcus. This species level resolution will greatly help in focusing on specific species and the pathways that a researcher intends to pursue. Therefore, the nanopore sequencing presents an opportunity to explore sequencing with a small footprint in terms of instrumentation and associated costs that greatly helps even a smaller laboratory to adopt and reap the benefits of species level resolution of metagenomic data. Summary: This study correlated well with 16S sequencing results at the genus level, while offering the added benefit of species level Identification and classification. Authors acknowledge the National Institutes of Health for funding support to Bina Joe (R01HL1430820). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Kinetics of inactivation of bacteria responsible for infections in hospitals using UV-LED

Controlling the microbial load in the environment is crucial to prevent the spread of organisms. The continuous spread of nosocomial infections in hospital facilities and the emergence of the coronavirus (COVID-19) highlighted the importance of disinfection processes in health safety. This work aimed to evaluate the effectiveness of LED-based disinfection lamps on bacteria from the ESKAPEE group and virus phage in vitro inactivation to be applied in hospital environments and health facilities disinfection.This study evaluated the effect of different UV wavelengths (275 nm, 280 nm (UVC), 310 nm (UVB) and 340 nm (UVA)) on the disinfection process of various microbial indicators including E. coli, S. aureus, P. aeruginosa, B. subtilis and Bacteriophage lambda DSM 4499. Exposure time (5 min–30 min), exposure distance (0.25 m and 0.5 m) and surface materials (glass, steel, and polished wood) were evaluated on the disinfection efficiency. Furthermore, the study determined the recovery capacity of each species after UV damage.UVC-LED lamps could inactivate 99.99 % of microbial indicators after 20 min exposures at a 0.5 m distance. The exposure time needed to completely inactivate E. coli, S. aureus, P. aeruginosa, B. subtilis and Bacteriophage lambda DSM 4499 can be decreased by reducing the exposure distance. UVB-LED and UVA-LED lamps were not able to promote a log reduction of 4 and were not effective on B. subtilis or bacteriophage lambda DSM 4499 inactivation. Thus, only UVC-LED lamps were tested on the decontamination of different surface materials, which was successful. P. aeruginosa showed the ability to recover from UV damage, but its inactivation rate remains 99.99 %, and spores from B. subtilis were not completely inactivated. Nevertheless, the inactivation rate of these indicators remained at 99.99 % with 24 h incubation after UVC irradiation. UVC-LED lamps emitting 280 nm were the most indicated to disinfect surfaces from microorganisms usually found in hospital environments.

Structural morphing in the viral portal vertex of bacteriophage lambda.

The tailed bacteriophages possess a distinct portal vertex that consists of a ring of 12 portal proteins associated with a 5-fold capsid shell. This portal protein is crucial in multiple stages of virus assembly and infection. Our research focused on examining the structures of the portal vertex in both its preliminary prohead state and the fully mature virion state of bacteriophage lambda. By analyzing these structures, we were able to understand how the portal protein undergoes conformational changes during maturation, the mechanism by which it prevents DNA from escaping, and the process of DNA spirally gliding.

Nucleoside-diphosphate kinase of uropathogenic Escherichia coli inhibits caspase-1-dependent pyroptosis facilitating urinary tract infection

Uropathogenic Escherichia coli (UPEC) is the most common causative agent of urinary tract infection (UTI). UPEC invades bladder epithelial cells (BECs) via fusiform vesicles, escapes into the cytosol, and establishes biofilm-like intracellular bacterial communities (IBCs). Nucleoside-diphosphate kinase (NDK) is secreted by pathogenic bacteria to enhance virulence. However, whether NDK is involved in UPEC pathogenesis remains unclear. Here, we find that the lack of ndk impairs the colonization of UPEC CFT073 in mouse bladders and kidneys owing to the impaired ability of UPEC to form IBCs. Furthermore, we demonstrate that NDK inhibits caspase-1-dependent pyroptosis by consuming extracellular ATP, preventing superficial BEC exfoliation, and promoting IBC formation. UPEC utilizes the reactive oxygen species (ROS) sensor OxyR to indirectly activate the regulator integration host factor, which then directly activates ndk expression in response to intracellular ROS. Here, we reveal a signaling transduction pathway that UPEC employs to inhibit superficial BEC exfoliation, thus facilitating acute UTI.

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.