seqA

Bacterial chromosome, the nucleoid, is traditionally modeled as a rosette of DNA mega-loops, organized around proteinaceous central scaffold by nucleoid-associated proteins (NAPs), and mixed with the cytoplasm by transcription and translation. Electron microscopy of fixed cells confirms dispersal of the cloud-like nucleoid within the ribosome-filled cytoplasm. Here, I discuss evidence that the nucleoid in live cells forms DNA phase separate from riboprotein phase, the "riboid." I argue that the nucleoid-riboid interphase, where DNA interacts with NAPs, transcribing RNA polymerases, nascent transcripts, and ssRNA chaperones, forms the transcription zone...

In this work we analyzed protein-protein interactions (PPIs) formed by E. coli replication proteins under three disparate bacterial growth conditions. The chosen conditions corresponded to fast exponential growth, slow exponential growth and growth cessation at the stationary phase. We performed affinity purification coupled with mass spectrometry (AP-MS) of chromosomally expressed proteins (DnaA, DnaB, Hda, SeqA, DiaA, DnaG, HolD, NrdB), tagged with sequential peptide affinity (SPA) tag. Composition of protein complexes was characterized using MaxQuant software...

Biofilm-associated foodborne Salmonella infections in poultry have become increasingly challenging for veterinarians, particularly in developing countries, and warrant thorough investigation. We assessed the biofilm-forming tendency of poultry isolates of Salmonella enterica , namely Salmonella Typhimurium ( n  = 23), Salmonella Infantis ( n  = 28), and Salmonella Heidelberg ( n  = 18), in nutrient-rich Rappaport-Vassiliadis Soya (RVS) peptone broth and nutrient-deficient diluted Tryptone Soya Broth (TSB)...

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...

Soil erosion is a vector of disturbances to agricultural productivity and economic development in the western highlands of Ethiopia. Yet, tough vegetation cover loss swapped to other land uses could have amplified the soil loss rate at which land cover change preceded, but little is known about their effects on soil loss in the Limu-Seqa watershed. This study was designed to evaluate the historical trends of the effects of land use-land cover change on soil erosion dynamics as a threshold for potential monitoring of soil loss...

In this study, comparative transcriptomic analyzes (mRNA and miRNA) were performed on the biofilm forms of S. Typhimurium ATCC 14028 wild-type strain and its seqA gene mutant in order to determine the regulation characteristics of the seqA gene in detail. The results of global gene expression analyses showed an increase in the expression level of 54 genes and a decrease in the expression level of 155 genes (p<0.05) in the seqA mutant compared to the wild-type strain. 10 of the 48 miRNAs identified on behalf of sequence analysis are new miRNA records for Salmonella...

Directed evolution is a powerful technique for identifying beneficial mutations in defined DNA sequences with the goal of improving desired phenotypes. Recent methodological advances have made the evolution of short DNA sequences quick and easy. However, the evolution of DNA sequences >5kb in length, notably gene clusters, is still a challenge for most existing methods. Since many important microbial phenotypes are encoded by multigene pathways, they are usually improved via adaptive laboratory evolution (ALE), which while straightforward to implement can suffer from off-target and hitchhiker mutations that can adversely affect the fitness of the evolved strain...

DnaA, the initiator of Escherichia coli chromosomal replication, has in its adenosine triphosphatase (ATPase) domain residues required for adenosine 5'-triphosphate (ATP) binding and membrane attachment. Here, we show that D118Q substitution in the DnaA linker domain, a domain known to be without major regulatory functions, influences ATP binding of DnaA and replication initiation in vivo. Although initiation defective by itself, overexpression of DnaA(D118Q) caused overinitiation of replication in dnaA46 ts cells and prevented cell growth...

About 10% of bacteria have a multichromosome genome with a primary replicon of bacterial origin, called the chromosome, and other replicons of plasmid origin, the chromids. Studies on multichromosome bacteria revealed potential points of coordination between the replication/segregation of chromids and the progression of the cell cycle. For example, replication of the chromid of Vibrionales (called Chr2) is initiated upon duplication of a sequence carried by the primary chromosome (called Chr1), in such a way that replication of both replicons is completed synchronously...

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 ...

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. There exists a short time lag of about 10-20 min between replication and segregation in which the daughter chromosomes are intertwined. Exactly where TopoIV binds during the cell cycle has been the subject of much debate...

Using droplet sequencing and full-length mRNA information in parallel has become an emerging requirement to help generate and better understand rich single-cell datasets. To address this need, we developed the SMART-Seq® Single Cell Kit (SSsc) using new chemistry with unparalleled sensitivity and a highly scalable and easily automatable workflow. These features make SSsc chemistry extremely useful for difficult cells-e.g., clinical research samples that often have very low RNA content-making it ideal for highly detailed characterization of precious samples...

The aim of this study was to compare the acute effects of four resistance-training (RT) exercise orders on rate of perceived exertion (RPE) and RT variables with exercise load properly adjusted according to its position within the sequence in older women. That is, the load was adjusted so that it was possible that the sets were performed within the repetition-zone established. Fifteen trained older women (67.4 ± 5.3 years) participated in a crossover-design, combining single-joint (SJ) and multi-joint (MJ) exercises for upper-(UB) and lower-body (LB) in the following exercise orders: SEQA = UBMJ-UBSJ-LBMJ-LBMJ; SEQB = UBSJ-UBMJ-LBSJ-LBMJ; SEQC = LBMJ-LBSJ-UBMJ-UBSJ; SEQD = LBSJ-LBMJ-UBSJ-UBMJ...

The stringent response enables bacteria to respond to a variety of environmental stresses, especially various forms of nutrient limitation. During the stringent response, the cell produces large quantities of the nucleotide alarmone ppGpp, which modulates many aspects of cell physiology, including reprogramming transcription, blocking protein translation, and inhibiting new rounds of DNA replication. The mechanism by which ppGpp inhibits DNA replication initiation in Escherichia coli remains unclear. Prior work suggested that ppGpp blocks new rounds of replication by inhibiting transcription of the essential initiation factor dnaA , but we found that replication is still inhibited by ppGpp in cells ectopically producing DnaA...

The Escherichia coli CrfC protein is an important regulator of nucleoid positioning and equipartition. Previously we revealed that CrfC homo-oligomers bind the clamp, a DNA-binding subunit of the DNA polymerase III holoenzyme, promoting colocalization of the sister replication forks, which ensures the nucleoid equipartition. In addition, CrfC localizes at the cell pole-proximal loci via an unknown mechanism. Here, we demonstrate that CrfC localizes to the distinct subnucleoid structures termed nucleoid poles (the cell pole-proximal nucleoid-edges) even in elongated cells as well as in wild-type cells...

The SeqA protein of Escherichia coli is required to prevent immediate re-initiation of chromosome replication from oriC . The SeqA protein is phosphorylated at the serine-36 (Ser36) residue by the HipA kinase. The role of phosphorylation was addressed by mutating the Ser36 residue to alanine, which cannot be phosphorylated and to aspartic acid, which mimics a phosphorylated serine residue. Both mutant strains were similar to wild-type with respect to origin concentration and initiation synchrony. The minimal time between successive initiations was also unchanged...

In this study, the effects of dam and seqA genes on the formation of pellicle and biofilm was determined using five different Salmonella serovars S. Group C1 (DMC2 encoded), S. Typhimurium (DMC4 encoded), S. Virchow (DMC11 encoded), S. Enteritidis (DMC22 encoded), and S. Montevideo (DMC89 encoded). dam and seqA mutants in Salmonella serovars were performed by the single step lambda red recombination method. The mutants obtained were examined according to the properties of biofilm on the polystyrene surfaces and the pellicle formation on the liquid medium...

This study compared the effect of a strength training session performed at different exercise orders and rest intervals on blood pressure and heart rate variability (HRV). Fifteen trained men performed different upper body exercise sequences [large to small muscle mass (SEQA) and small to large muscle mass (SEQB)] in randomized order with rest intervals between sets and exercises of 40 or 90 seconds. Fifteen repetition maximum loads were tested to control the training intensity and the total volume load. The results showed, significant reductions for systolic blood pressure (SBP) for all sequences compared to baseline and, post-exercise: SEQA90 at 20, 30, 40, 50 and 60 minutes; SEQA40 and SEQB40 at 20 minutes and SEQB90 at 10, 20, 30, 40, 50 and 60 minutes...

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...