seqA

BACKGROUND: One of many strategies to overcome antibiotic resistance is the discovery of compounds targeting cellular processes, which have not yet been exploited. METHODS AND MATERIALS: Using various genetic tools, we constructed a novel high throughput, cell based, fluorescence screen for inhibitors of chromosome replication initiation in bacteria. RESULTS: The screen was validated by expression of an intra-cellular cyclic peptide interfering with the initiator protein DnaA and by over-expression of the negative initiation regulator SeqA...

OBJECTIVE: To verify the incidence of the G679A mutation in exon 2 of the gene inhibin alpha (INHA), in women with secondary amenorrhea and diagnosis of premature ovarian insufficiency, and in controls. METHODS: A 5mL sample of peripheral blood was collected from all study participants in an EDTA tube and was used for DNA extraction. For the patient group, 5mL of blood were also collected in a tube containing heparin for karyotype, and 5mL were collected in a dry tube for follicle stimulant hormone dosage...

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. To confirm one or both of these possible mechanisms, we compared two seqA mutants with the oriCm3 mutant. The oriCm3 mutant over-initiates because of a lack of origin sequestration but has wild-type SeqA protein...

The Escherichia coli origin of replication, oriC, comprises mostly binding sites of two proteins: DnaA, a positive regulator, and SeqA, a negative regulator. SeqA, although not essential, is required for timely initiation, and during rapid growth, synchronous initiation from multiple origins. Unlike DnaA, details of SeqA binding to oriC are limited. Here we have determined that SeqA binds to all its sites tested (9/11) and with variable efficiency. Titration of DnaA alters SeqA binding to two sites, both of which have overlapping DnaA sites...

A set of 3907 single-gene knockout (Keio collection) strains of Escherichia coli K-12 was examined for strains with increased susceptibility to killing by X- or UV-radiation. After screening with a high-throughput resazurin-based assay and determining radiation survival with triplicate clonogenic assays, we identified 76 strains (and associated deleted genes) showing statistically-significant increased radiation sensitivity compared to a control strain. To determine gene novelty, we constructed a reference database comprised of genes found in nine similar studies including ours...

The Ori region of bacterial genomes is segregated early in the replication cycle of bacterial chromosomes. Consequently, Ori region positioning plays a pivotal role in chromosome dynamics. The Ori region of the E. coli chromosome is organized as a macrodomain with specific properties concerning DNA mobility, segregation of loci and long distance DNA interactions. Here, by using strains with chromosome rearrangements and DNA mobility as a read-out, we have identified the MaoP/maoS system responsible for constraining DNA mobility in the Ori region and limiting long distance DNA interactions with other regions of the chromosome...

In this study, a simple TAMRA (tetramethyl-6-carboxyrhodamine) quenching-based aptasensing platform was designed for the detection of aflatoxin B1 (AFB1). Here, we compared the analytical performance of two aptamer sequences: seqA and seqB. The AFB1 detection was based on the interactions of FAM (carboxyfluorescein)-labeled aptamer with TAMRA-labeled DNA complementary strand in the presence and absence of target analyte. Under optimized experimental conditions, TAMRA-labeled strand quenched the fluorescence response of FAM-labeled aptamer due to the noncovalent interaction between the two DNA strands...

The initiation of bacterial chromosomal replication is regulated by multiple pathways. To explore novel regulators, we isolated multicopy suppressors for the cold-sensitive hda-185 ΔsfiA(sulA) mutant. Hda is crucial for the negative regulation of the initiator DnaA and the hda-185 mutation causes severe replication overinitiation at the replication origin oriC. The SOS-associated division inhibitor SfiA inhibits FtsZ ring formation, an essential step for cell division regulation during the SOS response, and ΔsfiA enhances the cold sensitivity of hda-185 cells in colony formation...

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...

Phase variation of the Salmonella enterica opvAB operon generates a bacterial lineage with standard lipopolysaccharide structure (OpvAB(OFF)) and a lineage with shorter O-antigen chains (OpvAB(ON)). Regulation of OpvAB lineage formation is transcriptional, and is controlled by the LysR-type factor OxyR and by DNA adenine methylation. The opvAB regulatory region contains four sites for OxyR binding (OBSA-D), and four methylatable GATC motifs (GATC1-4). OpvAB(OFF) and OpvAB(ON) cell lineages display opposite DNA methylation patterns in the opvAB regulatory region: (i) in the OpvAB(OFF) state, GATC1 and GATC3 are non-methylated, whereas GATC2 and GATC4 are methylated; (ii) in the OpvAB(ON) state, GATC2 and GATC4 are non-methylated, whereas GATC1 and GATC3 are methylated...

Escherichia coli (E. coli) cells replicate their genome once per cell cycle to pass on genetic information to the daughter cells. The SeqA protein binds the origin of replication, oriC, after DNA replication initiation and sequesters it from new initiations in order to prevent overinitiation. Conventional fluorescence microscopy studies of SeqA localization in bacterial cells have shown that the protein is localized to discrete foci. In this study we have used photo-activated localization microscopy (PALM) to determine the localization of SeqA molecules, tagged with fluorescent proteins, with a localization precision of 20-30 nm with the aim to visualize the SeqA subcellular structures in more detail than previously possible...

The goal of this work was the investigation of correlation between some peculiarities of membrane fatty acids composition, biofilm formation, and motility of dam and/or seqA mutants in Salmonella typhimurium bacterial cells and UV-C radiations. The exposure changed the fatty acids composition of dam and seqA/dam strains. Significant increase of unsaturated fatty acids was observed. Swarming and swimming were enhanced only in dam mutant and biofilm formation increased significantly in all tested strains after UV-C exposure...

The effect of N6-adenine methylation on the flexibility and shape of palindromic GATC sequences has been investigated by molecular dynamics simulations. Variations in DNA backbone geometry were observed, which were dependent on the degree of methylation and the identity of the bases. While the effect was small, more frequent BI to BII conversions were observed in the GA step of hemimethylated DNA. The increased BII population of the hemimethylated system positively correlated with increased stacking interactions between methylated adenine and guanine, while stacking interactions decreased at the TC step for the fully methylated strand...

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. Using this tool as well as direct stochastic optical reconstruction microscopy (dSTORM) we find that in cells with fluorescently tagged SeqA and replisome the sister SeqA structures were situated close together (less than about 30 nm apart) and relatively far from the replisome (on average 200-300 nm)...

Mechanical properties such as physical constraint and pushing of chromosomes are thought to be important for chromosome segregation in Escherichia coli and it could be mediated by a hypothetical molecular "tether." However, the actual tether that mediates these features is not known. We previously described that SecA (Secretory A) and Secretory Y (SecY), components of the membrane protein translocation machinery, and AcpP (Acyl carrier protein P) were involved in chromosome segregation and homeostasis of DNA topology...

In a fast-growing Escherichia coli cell, most RNA polymerase (RNAP) is allocated to rRNA synthesis forming transcription foci at clusters of rrn operons or bacterial nucleolus, and each of the several nascent nucleoids contains multiple pairs of replication forks. The composition of transcription foci has not been determined. In addition, how the transcription machinery is three-dimensionally organized to promote cell growth in concord with replication machinery in the nucleoid remains essentially unknown. Here, we determine the spatial and functional landscapes of transcription and replication machineries in fast-growing E...

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. Fluorescence microscopy showed that in the beginning of the cell cycle cells contained a single focus at midcell. The focus was found to remain relatively immobile at midcell for a period of time equivalent to the duration of origin sequestration...

This study was carried out to determine the effects of novobiocin, a gyrase inhibitor, on the growth, survival, motility and whole cell proteins of S. Typhimurium dam and/or seqA strains. Our results showed that the dam and seqA/dam mutants are the most sensitive to novobiocin, compared to wild type and seqA strains. Surprisingly, the motility of seqA mutants increased after exposure to novobiocin only in stationary phase cells. All the other strains showed a significant decrease in their motility. The analysis of protein profiles of all strains demonstrated several modifications as manifested by the alteration of the expression levels of certain bands...

SeqA protein negatively regulates replication initiation in Escherichia coli and is also proposed to organize maturation and segregation of the newly replicated DNA. The seqA mutants suffer from chromosomal fragmentation; since this fragmentation is attributed to defective segregation or nucleoid compaction, two-ended breaks are expected. Instead, we show that, in SeqA's absence, chromosomes mostly suffer one-ended DNA breaks, indicating disintegration of replication forks. We further show that replication forks are unexpectedly slow in seqA mutants...

To compare the hypotensive effect and performance responses between different resistance training intensities and different exercise orders in apparently healthy women, thirteen apparently healthy women performed four resistance training sessions in randomized order. One group performed the resistance training exercises with 60% of 1RM (SeqA60%): leg press (LG), chest press (CP), leg extension (LE), lat pull down (PD), leg curl (LC) and biceps curl (BC). Another group performed the resistance training exercises with 80% of 1RM (SeqA80%) with the same exercise order...