A DnaA-dependent riboswitch for transcription attenuation of the operon.

Transcription attenuation in response to the availability of a specific amino acid is believed to be controlled by alternative configurations of RNA secondary structures that lead to the arrest of translation or the release of the arrested ribosome from the leader mRNA molecule. In this study, we first report a possible example of the DnaA-dependent riboswitch for transcription attenuation in . We show that (i) DnaA regulates the transcription of the structural genes but not that of the leader gene; (ii) DnaA might bind to rDnaA boxes present in the HisL-SL RNA, and subsequently attenuate the transcription of the operon; (iii) the HisL-SL RNA and rDnaA boxes are phylogenetically conserved and evolutionarily important; and (iv) the translating ribosome is required for deattenuation of the operon, whereas tRNA strengthens attenuation.

Antimicrobial resistance: A challenge awaiting the post-COVID-19 era.

Microbe exposure to pharmaceutical and non-pharmaceutical agents plays a role in the development of antibiotic resistance. The risks and consequences associated with extensive disinfectant use during the COVID-19 pandemic remain unclear. Some disinfectants, like sanitizers, contain genotoxic chemicals that damage microbial DNA, like phenol and hydrogen peroxide.

Topoisomerase IV tracks behind the replication fork and the SeqA complex during DNA replication in Escherichia coli.

Topoisomerase IV (TopoIV) is a vital bacterial enzyme which disentangles newly replicated DNA and enables segregation of daughter chromosomes. In bacteria, DNA replication and segregation are concurrent processes. This means that TopoIV must continually remove inter-DNA linkages during replication.

Antibiotic-induced DNA damage results in a controlled loss of pH homeostasis and genome instability.

Extracellular pH has been assumed to play little if any role in how bacteria respond to antibiotics and antibiotic resistance development. Here, we show that the intracellular pH of Escherichia coli equilibrates to the environmental pH following treatment with the DNA damaging antibiotic nalidixic acid. We demonstrate that this allows the environmental pH to influence the transcription of various DNA damage response genes and physiological processes such as filamentation.

Peptides containing the PCNA interacting motif APIM bind to the β-clamp and inhibit bacterial growth and mutagenesis.

In the fight against antimicrobial resistance, the bacterial DNA sliding clamp, β-clamp, is a promising drug target for inhibition of DNA replication and translesion synthesis. The β-clamp and its eukaryotic homolog, PCNA, share a C-terminal hydrophobic pocket where all the DNA polymerases bind. Here we report that cell penetrating peptides containing the PCNA-interacting motif APIM (APIM-peptides) inhibit bacterial growth at low concentrations in vitro, and in vivo in a bacterial skin infection model in mice.

Multiple links connect central carbon metabolism to DNA replication initiation and elongation in Bacillus subtilis.

DNA replication is coupled to growth by an unknown mechanism. Here, we investigated this coupling by analyzing growth and replication in 15 mutants of central carbon metabolism (CCM) cultivated in three rich media. In about one-fourth of the condition tested, defects in replication resulting from changes in initiation or elongation were detected.

Phenotypes of Mutant Cells Indicate that the Escherichia coli Clamp Loader Has a Role in the Restart of Stalled Replication Forks.

The mutation was discovered in connection with a screen for multicopy suppressors of the temperature-sensitive topoisomerase IV mutation The gene for the clamp loader subunits τ and γ, , but not the mutant , was found to suppress (Ts) when overexpressed. Purified mutant protein was found to be functional , and few phenotypes were found apart from problems with partitioning of DNA in rich medium. We show here that a large number of the replication forks that initiate at never reach the terminus in mutant cells.

SeqA structures behind Escherichia coli replication forks affect replication elongation and restart mechanisms.

The SeqA protein binds hemi-methylated GATC sites and forms structures that sequester newly replicated origins and trail the replication forks. Cells that lack SeqA display signs of replication fork disintegration. The broken forks could arise because of over-initiation (the launching of too many forks) or lack of dynamic SeqA structures trailing the forks.

Lack of the H-NS Protein Results in Extended and Aberrantly Positioned DNA during Chromosome Replication and Segregation in Escherichia coli.

Unlabelled: The architectural protein H-NS binds nonspecifically to hundreds of sites throughout the chromosome and can multimerize to stiffen segments of DNA as well as to form DNA-protein-DNA bridges. H-NS has been suggested to contribute to the orderly folding of the Escherichia coli chromosome in the highly compacted nucleoid. In this study, we investigated the positioning and dynamics of the origins, the replisomes, and the SeqA structures trailing the replication forks in cells lacking the H-NS protein.

The DnaA Protein Is Not the Limiting Factor for Initiation of Replication in Escherichia coli.

The bacterial replication cycle is driven by the DnaA protein which cycles between the active ATP-bound form and the inactive ADP-bound form. It has been suggested that DnaA also is the main controller of initiation frequency. Initiation is thought to occur when enough ATP-DnaA has accumulated.

Dynamic Escherichia coli SeqA complexes organize the newly replicated DNA at a considerable distance from the replisome.

The Escherichia coli SeqA protein binds to newly replicated, hemimethylated DNA behind replication forks and forms structures consisting of several hundred SeqA molecules bound to about 100 kb of DNA. It has been suggested that SeqA structures either direct the new sister DNA molecules away from each other or constitute a spacer that keeps the sisters together. We have developed an image analysis script that automatically measures the distance between neighboring foci in cells.

Escherichia coli SeqA structures relocalize abruptly upon termination of origin sequestration during multifork DNA replication.

The Escherichia coli SeqA protein forms complexes with new, hemimethylated DNA behind replication forks and is important for successful replication during rapid growth. Here, E. coli cells with two simultaneously replicating chromosomes (multifork DNA replication) and YFP tagged SeqA protein was studied.

DNA compaction in the early part of the SOS response is dependent on RecN and RecA.

The nucleoids of undamaged Escherichia coli cells have a characteristic shape and number, which is dependent on the growth medium. Upon induction of the SOS response by a low dose of UV irradiation an extensive reorganization of the nucleoids occurred. Two distinct phases were observed by fluorescence microscopy.

The Escherichia coli datA site promotes proper regulation of cell division.

In Escherichia coli inhibition of replication leads to a block of cell division. This checkpoint mechanism ensures that no cell divides without having two complete copies of the genome to pass on to the two daughter cells. The chromosomal datA site is a 1 kb region that contains binding sites for the DnaA replication initiator protein, and which contributes to the inactivation of DnaA.

The Fis protein has a stimulating role in initiation of replication in Escherichia coli in vivo.

The Fis protein is a nucleoid associated protein that has previously been reported to act negatively in initiation of replication in Escherichia coli. In this work we have examined the influence of this protein on the initiation of replication under different growth conditions using flow cytometry. The Fis protein was found to be increasingly important with increasing growth rate.

Regulating DNA replication in bacteria.

The replication origin and the initiator protein DnaA are the main targets for regulation of chromosome replication in bacteria. The origin bears multiple DnaA binding sites, while DnaA contains ATP/ADP-binding and DNA-binding domains. When enough ATP-DnaA has accumulated in the cell, an active initiation complex can be formed at the origin resulting in strand opening and recruitment of the replicative helicase.

Single transmembrane peptide DinQ modulates membrane-dependent activities.

The functions of several SOS regulated genes in Escherichia coli are still unknown, including dinQ. In this work we characterize dinQ and two small RNAs, agrA and agrB, with antisense complementarity to dinQ. Northern analysis revealed five dinQ transcripts, but only one transcript (+44) is actively translated.

Depletion of acidic phospholipids influences chromosomal replication in Escherichia coli.

In Escherichia coli, coordinated activation and deactivation of DnaA allows for proper timing of the initiation of chromosomal synthesis at the origin of replication (oriC) and assures initiation occurs once per cell cycle. In vitro, acidic phospholipids reactivate DnaA, and in vivo depletion of acidic phospholipids, results in growth arrest. Growth can be restored by the expression of a mutant form of DnaA, DnaA(L366K), or by oriC-independent DNA synthesis, suggesting acidic phospholipids are required for DnaA- and oriC-dependent replication.

Replication fork movement and methylation govern SeqA binding to the Escherichia coli chromosome.

In Escherichia coli, the SeqA protein binds specifically to GATC sequences which are methylated on the A of the old strand but not on the new strand. Such hemimethylated DNA is produced by progression of the replication forks and lasts until Dam methyltransferase methylates the new strand. It is therefore believed that a region of hemimethylated DNA covered by SeqA follows the replication fork.

An easy-to-use simulation program demonstrates variations in bacterial cell cycle parameters depending on medium and temperature.

Many studies are performed on chromosome replication and segregation in Escherichia coli and other bacteria capable of complex replication with C phases spanning several generations. For such investigations an understanding of the replication patterns, including copy numbers of origins and replication forks, is crucial for correct interpretation of the results.Flow cytometry is an important tool for generation of experimental DNA distributions of cell populations.

Hydrophilic interaction chromatography of nucleoside triphosphates with temperature as a separation parameter.

Eight deoxynucleoside triphosphates (dNTPs) and nucleoside triphosphates (NTPs): ATP, CTP, GTP, UTP, dATP, dCTP, dGTP and dTTP, were separated with two 15 cm ZIC-pHILIC columns coupled in series, using LC-UV instrumentation. The polymer-based ZIC-pHILIC column gave significantly better separations and peak shape than a silica-based ZIC-HILIC column. Better separations were obtained with isocratic elution as compared to gradient elution.

The G157C mutation in the Escherichia coli sliding clamp specifically affects initiation of replication.

Escherichia coli cells with a point mutation in the dnaN gene causing the amino acid change Gly157 to Cys, were found to underinitiate replication and grow with a reduced origin and DNA concentration. The mutant β clamp also caused excessive conversion of ATP-DnaA to ADP-DnaA. The DnaA protein was, however, not the element limiting initiation of replication.

Replication patterns and organization of replication forks in Vibrio cholerae.

We have investigated the replication patterns of the two chromosomes of the bacterium Vibrio cholerae grown in four different media. By combining flow cytometry and quantitative real-time PCR with computer simulations, we show that in rich media, V. cholerae cells grow with overlapping replication cycles of both the large chromosome (ChrI) and the small chromosome (ChrII).

ChIP on Chip: surprising results are often artifacts.

Background: The method of chromatin immunoprecipitation combined with microarrays (ChIP-Chip) is a powerful tool for genome-wide analysis of protein binding. However, a high background signal is a common phenomenon.

Results: Reinvestigation of the chromatin immunoprecipitation procedure led us to discover four causes of high background: i) non-unique sequences, ii) incomplete reversion of crosslinks, iii) retention of protein in spin-columns and iv) insufficient RNase treatment.

A novel DNA gyrase inhibitor rescues Escherichia coli dnaAcos mutant cells from lethal hyperinitiation.

Objectives: In order to search for novel antibacterial compounds we used a previously developed screening strain designed specifically to discover inhibitors of the bacterial initiator protein, DnaA. This strain (SF53) is not viable at 30 degrees C due to overinitiation. Therefore, compounds that are able to restore growth to SF53 cells are likely to cause either partial or complete inhibition of DnaA function.