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The positions of DNA regions close to the chromosome replication origin and terminus in growing cells of Escherichia coli have been visualized simultaneously, using new widely applicable reagents. Furthermore, the positions of these regions with respect to a replication factory-associated protein have been analysed. Time-lapse analysis has allowed the fate of origins, termini and the FtsZ ring to be followed in a lineage-specific manner during the formation of microcolonies. These experiments reveal new aspects of the E. coli cell cycle and demonstrate that the replication terminus region is frequently located asymmetrically, on the new pole side of mid-cell. This asymmetry could provide a mechanism by which the chromosome segregation protein FtsK, located at the division septum, can act directionally to ensure that the septal region is free of DNA before the completion of cell division. [ABSTRACT FROM AUTHOR]

Assembly of cell division protein FtsZ into the Z‐ring at the division site is a key step in bacterial cell division. The Min proteins can restrict the Z‐ring to the middle of the cell. MinC is the main protein that obstructs Z‐ring formation by inhibiting FtsZ assembly. Its N‐terminal domain (MinCN) regulates the localization of the Z‐ring by inhibiting FtsZ polymerization, while its C‐terminal domain (MinCC) binds to MinD as well as to FtsZ. Previous studies have shown that MinC and MinD form copolymers in vitro. This copolymer may greatly enhance the binding of MinC to FtsZ, and/or prevent FtsZ filaments from diffusing to the ends of the cell. Here, we investigated the assembly properties of MinCC–MinD of Pseudomonas aeruginosa. We found that MinCC is sufficient to form the copolymers. Although MinCC–MinD assembles into larger bundles, most likely because MinCC is spatially more readily bound to MinD, its copolymerization has similar dynamic properties: the concentration of MinD dominates their copolymerization. The critical concentration of MinD is around 3 μm and when MinD concentration is high enough, a low concentration MinCC could still be copolymerized. We also found that MinCC–MinD can still rapidly bind to FtsZ protofilaments, providing direct evidence that MinCC also interacts directly with FtsZ. However, although the presence of minCC can slightly improve the division defect of minC‐knockout strains and shorten the cell length from an average of 12.2 ± 6.7 to 6.6 ± 3.6 μm, it is still insufficient for the normal growth and division of bacteria. [ABSTRACT FROM AUTHOR]

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. [ABSTRACT FROM AUTHOR]

Summary: Spray‐induced gene silencing (SIGS) using topical dsRNA applications has risen as a promising, target‐specific, and environmentally friendly disease management strategy against phytopathogenic fungi. However, dsRNA stability, efficacy, and scalability are still the main constraints facing SIGS broader application. Here we show that Escherichia coli‐derived anucleated minicells can be utilized as a cost‐effective, scalable platform for dsRNA production and encapsulation. We demonstrated that minicell‐encapsulated dsRNA (ME‐dsRNA) was shielded from RNase degradation and stabilized on strawberry surfaces, allowing dsRNA persistence in field‐like conditions. ME‐dsRNAs targeting chitin synthase class III (Chs3a, Chs3b) and DICER‐like proteins (DCL1 and DCL2) genes of Botryotinia fuckeliana selectively knocked‐down the target genes and led to significant fungal growth inhibition in vitro. We also observed a compensatory relationship between DCL1 and DCL2 gene transcripts, where the silencing of one gene upregulated the expression of the other. Contrary to naked‐dsRNAs, ME‐dsRNAs halted disease progression in strawberries for 12 days under greenhouse conditions. These results elucidate the potential of ME‐dsRNAs to enable the commercial application of RNAi‐based, species‐specific biocontrols comparable in efficacy to conventional synthetics. ME‐dsRNAs offer a platform that can readily be translated to large‐scale production and deployed in open‐field applications to control grey mould in strawberries. [ABSTRACT FROM AUTHOR]

The synthesis and antibacterial studies of polysubstituted pyrrolo[3,4‐b]pyridine derivatives have been described. The preparation of pyrrolo[3,4‐b]pyridine derivatives was carried out by the reaction of enamino imides, aromatic aldehydes and malononitrile/ethyl cyanoacetate using 10 mol % of DBU (1,8‐diazabicyclo[5.4.0]undec‐7‐ene) in ethanol at 78 °C in good yields. The compounds were characterized by standard spectroscopic techniques including IR, 1H & 13C NMR and elemental analysis and also final confirmation was done by single crystal X‐ray. The antibacterial activity of all the synthesized compounds was tested against two Gram positive (S. pneumoniae MTCC 655 and E. faecalis MTCC 439) and three Gram negative (E. coli ATCC 25922, S. typhimurium MTCC 3224, and P. aeruginosa MTCC 2453) bacterial strains. Most of the tested compounds showed moderate to good antibacterial activity. Compounds pyrrolo[3,4‐b]pyridine derivatives (4 j and 4 l) were the most potent and displayed bactericidal activities against E. coli strain with MIC (minimum inhibitory concentration) values of 62.5 μg/mL and 125.0 μg/mL respectively. Growth kinetic studies against E. coli, toxicity studies using human RBCs (red blood cells) and also docking studies of the selected compounds 4 j and 4 l supported that these compounds inhibit the growth of bacterial cells, non‐toxic in nature and interact with key amino residues of DNA (deoxyribonucleic acid) duplex (PDBID: 1BNA) and have drug‐like properties. [ABSTRACT FROM AUTHOR]

Initiation of Escherichia coli chromosome replication is controlled by the DnaA initiator protein. Both rifampicin‐mediated inhibition of transcription and ppGpp‐induced changes in global transcription stops replication at the level of initiation. Here, we show that continued DnaA protein synthesis allows for replication initiation both during the rifampicin treatment and during the stringent response when the ppGpp level is high. A reduction in or cessation of de novo DnaA synthesis, therefore, causes the initiation arrest in both cases. In accordance with this, inhibition of translation with chloramphenicol also stops initiations. The initiation arrest caused by rifampicin was faster than that caused by chloramphenicol, despite of the latter inhibiting DnaA accumulation immediately. During chloramphenicol treatment transcription is still ongoing and we suggest that transcriptional events in or near the origin, that is, transcriptional activation, can allow for a few extra initiations when DnaA becomes limiting. We suggest, for both rifampicin treated cells and for cells accumulating ppGpp, that a turn‐off of initiation from oriC requires a stop in de novo DnaA synthesis and that an additional lack of transcriptional activation enhances this process, that is, leads to a faster initiation stop. [ABSTRACT FROM AUTHOR]

The Sra protein is a component of the 30S ribosomal subunit while RimJ is a ribosome‐associated protein that plays a role in the maturation of the 30S ribosomal subunit. Here we found that Δsra and ΔrimJ cells showed a delayed initiation of DNA replication, prolonged doubling time, decreased cell size, and decreased amounts of total protein and DnaA per cell compared with these observed for wild‐type cells. A temperature sensitivity test demonstrated that absence of the Sra or RimJ protein did not change the temperature sensitivity of the dnaA46, dnaB252, or dnaC2 mutants. Moreover, ectopic expression of Sra reversed the mutant phenotype while cells carrying the pACYC177‐rimJ plasmid did not reverse the rimJ mutant phenotype. The results indicate that deletion of sra or rimJ cause defects in ribosomal function and affect the translation process, leading to a decrease in synthesis of proteins including DnaA. Therefore, we conclude that Sra‐ and RimJ‐mediated ribosomal function is required for precise timing of initiation of chromosome replication. [ABSTRACT FROM AUTHOR]

With the realization that bacteria display phenotypic variability among cells and exhibit complex subcellular organization critical for cellular function and behavior, microscopy has re-emerged as a primary tool in bacterial research during the last decade. However, the bottleneck in today's single-cell studies is quantitative image analysis of cells and fluorescent signals. Here, we address current limitations through the development of Oufti, a stand-alone, open-source software package for automated measurements of microbial cells and fluorescence signals from microscopy images. Oufti provides computational solutions for tracking touching cells in confluent samples, handles various cell morphologies, offers algorithms for quantitative analysis of both diffraction and non-diffraction-limited fluorescence signals and is scalable for high-throughput analysis of massive datasets, all with subpixel precision. All functionalities are integrated in a single package. The graphical user interface, which includes interactive modules for segmentation, image analysis and post-processing analysis, makes the software broadly accessible to users irrespective of their computational skills. [ABSTRACT FROM AUTHOR]

The regulators of M ycobacterium tuberculosis DNA replication are largely unknown. Here, we demonstrate that in synchronously replicating M . tuberculosis, MtrA access to origin of replication ( ori C) is enriched in the post-replication ( D) period. The increased ori C binding results from elevated MtrA phosphorylation ( MtrA∼ P) as evidenced by reduced expression of dna N, dna A and increased expression of select cell division targets. Overproduction of gain-of-function MtrAY102C advanced the MtrA ori C access to the C period, reduced dna A and dna N expression, interfered with replication synchrony and compromised cell division. Overproduction of wild-type ( MtrA+) or phosphorylation-defective MtrAD56N did not promote ori C access in the C period, nor affected cell cycle progression. MtrA interacts with DnaA signaling a possibility that DnaA helps load MtrA on ori C. Therefore, ori C sequestration by MtrA∼ P in the D period may normally serve to prevent untimely initiations and that DnaA- MtrA interactions may facilitate regulated ori C replication. Finally, despite the near sequence identity of MtrA in M . smegmatis and M . tuberculosis, the M . smegmatis ori C is not MtrA-target. We conclude that M . tuberculosis ori C has evolved to be regulated by MtrA and that cell cycle progression in this organisms are governed, at least in part, by oscillations in the MtrA∼ P levels. [ABSTRACT FROM AUTHOR]

Bacterial growth is often difficult to estimate beyond classical cultivation approaches. Low cell numbers, particles or coloured and dense media may disturb reliable growth assessment. Further difficulties appear when cells are attached to surfaces and detachment is incomplete. Therefore, flow cytometry was tested and used for analysis of bacterial growth on the single-cell level. Shewanella putrefaciens was cultivated as a model organism in planktonic or biofilm culture. Materials of smooth and rough surfaces were used for biofilm cultivation. Both aerobic and anaerobic as well as feast and famine conditions were applied. Visualization of growth was also done using Environmental Scanning and Phase Contrast Microscopy. Bioinformatic tools were applied for data interpretation. Cytometric proliferation patterns based on distributions of DNA contents per cell corresponded distinctly to the various lifestyles, electron acceptors and substrates tested. Therefore, cell cycling profiles of S. putrefaciens were found to mirror growth conditions. The cytometric patterns were consistently detectable with exception of some biofilm types whose resolution remained challenging. Corresponding heat maps proved to be useful for clear visualization of growth behaviour under all tested conditions. Therefore, flow cytometry in combination with bioinformatic tools proved to be powerful means to determine various growth states of S. putrefaciens, even in constrained environments. The approach is universal and will also be applicable for other bacterial species. [ABSTRACT FROM AUTHOR]

Subcellular biomolecular localization is critical for the metabolic and structural properties of the cell. The functional implications of the spatiotemporal distribution of protein complexes during the bacterial cell cycle have long been acknowledged; however, the molecular mechanisms for generating and maintaining their dynamic localization in bacteria are not completely understood. Here we demonstrate that the trans-envelope Tol- Pal complex, a widely conserved component of the cell envelope of Gram-negative bacteria, is required to maintain the polar positioning of chemoreceptor clusters in E scherichia coli. Localization of the chemoreceptors was independent of phospholipid composition of the membrane and the curvature of the cell wall. Instead, our data indicate that chemoreceptors interact with components of the Tol- Pal complex and that this interaction is required to polarly localize chemoreceptor clusters. We found that disruption of the Tol- Pal complex perturbs the polar localization of chemoreceptors, alters cell motility, and affects chemotaxis. We propose that the E . coli Tol- Pal complex restricts mobility of the chemoreceptor clusters at the cell poles and may be involved in regulatory mechanisms that co-ordinate cell division and segregation of the chemosensory machinery. [ABSTRACT FROM AUTHOR]

On 19 January 2014 Rolf (' Roffe') Bernander passed away unexpectedly. Rolf was a dedicated scientist; his research aimed at unravelling the cell biology of the archaeal domain of life, especially cell cycle-related questions, but he also made important contributions in other areas of microbiology. Rolf had a professor position in the Molecular Evolution programme at Uppsala University, Sweden for about 8 years, and in January 2013 he became chair professor at the Department of Molecular Biosciences, The Wenner- Gren Institute at Stockholm University in Sweden. Rolf was an exceptional colleague and will be deeply missed by his family and friends, and the colleagues and co-workers that he leaves behind in the scientific community. He will be remembered for his endless enthusiasm for science, his analytical mind, and his quirky sense of humour. [ABSTRACT FROM AUTHOR]

Synthetic single-strand oligonucleotides (oligos) with homology to genomic DNA have proved to be highly effective for constructing designed mutations in targeted genomes, a process referred to as recombineering. The cellular functions important for this type of homologous recombination have yet to be determined. Towards this end, we have identified Escherichia coli functions that process the recombining oligo and affect bacteriophage λ Red-mediated oligo recombination. To determine the nature of oligo processing during recombination, each oligo contained multiple nucleotide changes: a single base change allowing recombinant selection, and silent changes serving as genetic markers to determine the extent of oligo processing during the recombination. Such oligos were often not incorporated into the host chromosome intact; many were partially degraded in the process of recombination. The position and number of these silent nucleotide changes within the oligo strongly affect both oligo processing and recombination frequency. Exonucleases, especially those associated with DN A Polymerases I and III, affect inheritance of the silent nucleotide changes in the oligos. We demonstrate for the first time that the major DNA polymerases ( Pol I and Pol III) and DNA ligase are directly involved with oligo recombination. [ABSTRACT FROM AUTHOR]

The process of Sister Chromosome Cohesion (SCC), which holds together sister chromatids upon replication, is essential for chromosome segregation and DNA repair in eukaryotic cells. Although cohesion at the molecular level has never been described in E. coli, previous studies have reported that sister sequences remain co-localized for a period after their replication. Here, we have developed a new genetic recombination assay that probes the ability of newly replicated chromosome loci to interact physically. We show that Sister Chromatid Interaction (SCI) occurs exclusively within a limited time frame after replication. Importantly, we could differentiate sister cohesion and co-localization since factors such as MatP and MukB that reduced the co-localization of markers had no effect on molecular cohesion. The frequency of sister chromatid interactions were modulated by the activity of Topo-IV, revealing that DNA topology modulates cohesion at the molecular scale in bacteria. [ABSTRACT FROM AUTHOR]

In vivo site-directed mutagenesis by single-stranded deoxyribonucleic acid recombineering is a facile method to change the color of fluorescent proteins (FPs) without cloning. Two different starting alleles of GFP were targeted for mutagenesis: gfpmut3* residing in the Escherichia coli genome and egfp carried by a bacterial/mammalian dual expression lentiviral plasmid vector. Fluorescent protein spectra were shifted by subtle modification of the chromophore region and residues interacting with the chromophore of the FP. Eight different FPs (Violeta, Azure, Aqua, Mar, Celeste, Amarillo, Mostaza, and Bronze) were isolated and shown to be useful in multicolor imaging and flow cytometry of bacteria and transgenic human stem cells. To make in vivo site-directed mutagenesis more efficient, the recombineering method was optimized using the fluorescence change as a sensitive quantitative assay for recombination. A set of rules to simplify mutant isolation by recombineering is provided. © 2012 IUBMB IUBMB Life, 64(8): 684-689, 2012 [ABSTRACT FROM AUTHOR]

The Caulobacter crescentus chromosome replication origin ( Cori) has five binding sites for CtrA, an OmpR/PhoB family ‘response regulator’. CtrA is degraded in replicating ‘stalked’ cells but is abundant in the non-replicating ‘swarmer’ cells, where it was proposed to repress replication by binding to Cori. We systematically mutated all Cori CtrA binding sites, and examined their consequences in the contexts of autonomous Cori-plasmid replication and in the natural chromosome locus. Remarkably, the C. crescentus chromosome tolerates severe mutations in all five CtrA binding sites, demonstrating that CtrA is not essential for replication. Further physiological and cell cycle experiments more rigorously supported the original hypothesis that CtrA represses replication. However, our experiments argued against another hypothesis that residual and/or replenished CtrA protein in stalked cells might prevent extra or unscheduled chromosome replication before cell division. Surprisingly, we also demonstrated that Cori CtrA binding sites are very advantageous and can become essential when cells encounter nutrients and antibiotics. Therefore, the CtrA cell cycle regulator co-ordinates replication with viable cell growth in stressful and rapidly changing environments. We argue that this new role for CtrA provided the primary selective pressure for evolving control by CtrA. [ABSTRACT FROM AUTHOR]

The initiation of replication in Escherichia coli is negatively controlled by a mechanism referred to as ‘initiator titration’, a process by which the initiator protein, DnaA, is titrated to newly replicated binding sequences on the chromosome to reduce the initiation potential for replication. Initiator titration occurs predominantly at the datA locus that binds exceptionally large amounts of DnaA molecules to prevent aberrant initiations. We found that this was enabled by integration host factor (IHF). Within datA, there is a consensus IHF recognition sequence between the two DnaA recognition sequences (DnaA boxes) essential for its function. Binding of IHF to this site was demonstrated both in vitro and in vivo. Disruption of the core sequence in the consensus of the IHF-binding resulted in increased origin concentration as observed in ΔdatA cells. Furthermore, the number of DnaA molecules bound to datA was reduced in cells carrying a disruption in the IHF-binding core sequence. The IHF-binding site and the essential DnaA boxes had to be located at a proper distance and orientation to maintain the accurate initiation timing. Therefore, IHF is a unique element in the control of replication initiation that acts negatively at datA, while known to act as a positive regulator at oriC. [ABSTRACT FROM AUTHOR]

Various events involving partitioning of sister chromosomes were precisely analyzed in asynchronously growing Escherichia coli cells in various conditions. To examine the cohesion between sister chromosomes, we analyzed living cells growing under various conditions for the number of the replication origin ( oriC) copies by flow cytometry and the foci of oriC by fluorescence microscopy. The average number of the oriC foci per cell was significantly smaller than the number of oriC copies per cell with few exceptions, suggesting cohesion of oriC sister copies. Cohesion phenomenon of oriC sister copies was also observed in a mukB null mutant cells under some growth conditions. Sister copies of the terminal region ( ter) were also found to be cohesive. Immunofluorescence microscopy for nascent DNA pulse-labeled with 5-bromo-2′-deoxyuridine (BrdU) indicated that paired replication forks acting on bidirectional replication were able to migrate toward opposite directions during ongoing replication in poor medium; however, some of them were closely associated in rich media. Analysis of the foci of MukB–GFP indicates that the number of MukB foci was always larger than the number of replication forks. The number of MukB–GFP foci increased together with cell length. The sequence of these chromosomal events in the growing cells has been depicted. [ABSTRACT FROM AUTHOR]

Prior to initiating DNA synthesis, Escherichia coli oriC switches from ORC, comprising initiator DnaA bound at three high-affinity sites, to pre-RC, when additional DnaA molecules interact with low-affinity sites. Two types of low-affinity sites exist: R boxes that bind DnaA-ATP and DnaA-ADP with equal affinity, and I-sites with a three- to fourfold preference for DnaA-ATP. To assess the regulatory role of weak DnaA interactions during pre-RC assembly in vivo, we compared the behaviour of plasmid-borne wild-type oriC with mutants having an increased or decreased number of DnaA-ATP discriminatory I-sites. Increasing the number of discriminatory sites by replacing R5M with I2 inactivated extrachromosomal oriC function. Mutants with no discriminatory sites perturbed host growth and rapidly replaced wild-type chromosomal oriC, but normal function returned if one I-site was restored at either the I2, I3 or R5M position. These observations are consistent with assembly of E. coli pre-RC in vivo from mixtures of DnaA-ATP and DnaA-ADP, with I-site interactions coupling pre-RC assembly to DnaA-ATP levels. [ABSTRACT FROM AUTHOR]

The homeostatic system that sets the copy number, and corrects over-replication and under-replication, seems to be different for chromosomes and plasmids in bacteria. Whereas plasmid replication is random in time, chromosome replication is tightly coordinated with the cell cycle such that all origins are initiated synchronously at the same cell mass per origin once per cell cycle. In this review, we propose that despite their apparent differences, the copy-number control of the Escherichia coli chromosome is similar to that of plasmids. The basic mechanism that is shared by both systems is negative-feedback control of the availability of a protein or RNA positive initiator. Superimposed on this basic mechanism are at least three systems that secure the synchronous initiation of multiple origins; however, these mechanisms are not essential for maintaining the copy number. [ABSTRACT FROM AUTHOR]

Negative feedback loops have been invoked as a way to control and decrease transcriptional noise. Here, we have built three circuits to test the effect of negative feedback loops on transcriptional noise of an autoregulated gene encoding a transcription factor (TF) and a downstream gene (DG), regulated by this TF. Experimental analysis shows that self-repression decreases noise compared to expression from a non-regulated promoter. Interestingly enough, we find that noise minimization by negative feedback loop is optimal within a range of repression strength. Repression values outside this range result in noise increase producing a U-shaped behaviour. This behaviour is the result of external noise probably arising from plasmid fluctuations as shown by simulation of the network. Regarding the target gene of a self-repressed TF (sTF), we find a strong decrease of noise when repression by the sTF is strong and a higher degree of noise anti-correlation between sTF and its target. Simulations of the circuits indicate that the main source of noise in these circuits could come from plasmid variation and therefore that negative feedback loops play an important role in suppressing both external and internal noise. An important observation is that DG expression without negative feedback exhibits bimodality at intermediate TF repression values. This bimodal behaviour seems to be the result of external noise as it can only be found in those simulations that include plasmid variation. [ABSTRACT FROM AUTHOR]

Cell division proteins FtsZ (FtsA, ZipA, ZapA), FtsE/X, FtsK, FtsQ, FtsL/B, FtsW, PBP3, FtsN and AmiC localize at mid cell inEscherichia coliin an interdependent order as listed. To investigate whether this reflects a time dependent maturation of the divisome, the average cell age at which FtsZ, FtsQ, FtsW, PBP3 and FtsN arrive at their destination was determined by immuno- and GFP-fluorescence microscopy of steady state grown cells at a variety of growth rates. Consistently, a time delay of 14–21 min, depending on the growth rate, between Z-ring formation and the mid cell recruitment of proteins down stream of FtsK was found. We suggest a two-step model for bacterial division in which the Z-ring is involved in the switch from cylindrical to polar peptidoglycan synthesis, whereas the much later localizing cell division proteins are responsible for the modification of the envelope shape into that of two new poles. [ABSTRACT FROM AUTHOR]

Genetic recombination is central to DNA metabolism. It promotes sequence diversity and maintains genome integrity in all organisms. However, it can have perverse effects and profoundly influence the cell cycle. In bacteria harbouring circular chromosomes, recombination frequently has an unwanted outcome, the formation of chromosome dimers. Dimers form by homologous recombination between sister chromosomes and are eventually resolved by the action of two site-specific recombinases, XerC and XerD, at their target site,dif, located in the replication terminus of the chromosome. Studies of the Xer system and of the modalities of dimer formation and resolution have yielded important knowledge on how both homologous and site-specific recombination are controlled and integrated in the cell cycle. Here, we briefly review these advances and highlight the important questions they raise. [ABSTRACT FROM AUTHOR]

Summary In Escherichia coli , the level of the initiator protein DnaA is limiting for initiation of replication at oriC . A high-affinity binding site for DnaA, datA , plays an important role. Here, the effect of extra datA sites was studied. A moderate increase in datA dosage (≈ fourfold) delayed initiation of replication and cell division, but increased the rate of replication fork movement about twofold. At a further increase in the datA gene dosage, the SOS response was induced, and incomplete rounds of chromosome replication were detected. Overexpression of DnaA protein suppressed the SOS response and restored normal replication timing and rate of fork movement. In the presence of extra datA sites, cells showed a dependency on PriA and RecA proteins, indicating instability of the replication fork. The results suggest that wild-type replication fork progression normally includes controlled pausing, and that this is a prerequisite for normal replication fork function. [ABSTRACT FROM AUTHOR]

Abstract Background: During the cell cycle, the initiation of chromosomal replication is strictly controlled. In Escherichia coli , the initiator DnaA and the replication origin oriC are major targets for this regulation. Here, we assessed the role of transcription of the mioC gene, which reads through the adjacent oriC region. This mioC-oriC transcription is regulated in coordination with the replication cycle so that it is activated after initiation and repressed before initiation. Results: We isolated a strain bearing a mioC promoter mutation that causes constitutive mioC-oriC transcription from the chromosome. A quantitative S1 nuclease assay indicated that in this mutant, the level of transcription does not fluctuate. Introduction of this mutation suppressed the growth defect of an overinitiation-type dnaAcos mutant, and severely inhibited the growth of initiation-defective dnaA mutants at semipermissive temperatures in a dnaA allele-specific manner. These results suggest that mioC-oriC transcription inhibits initiation at oriC . Indeed, flow cytometry analysis and quantification of DNA replication in synchronized cultures revealed that the mioC promoter mutation alters the control of the initiation of chromosomal replication, for instance by delaying replication within the cell cycle. Conclusions: These results suggest that the transcriptional regulation of the mioC gene is required for cell cycle-coordinated initiation of chromosomal replication. [ABSTRACT FROM AUTHOR]

Summary The antigen 43 surface protein of Escherichia coli is expressed in a phase-variable manner by a mechanism involving alternative activation and repression of transcription of the agn43 gene. The repressor is the OxyR DNA-binding protein, and its binding site was found to be located downstream of the agn43 transcription start site in a region of DNA that encompasses three 5′ -GATC-3′ sequences that are subject to Dam-mediated DNA methylation. It has been suggested previously that the phase-variable expression of antigen 43 results from a competition between Dam methylase and the OxyR repressor for these sites. The 5′ -GATC-3′ sequences were inactivated for methylation by site-directed mutagenesis, and all possible combinations of inactive and active sites were assessed for effects on phase-variable expression of the agn43 gene. Inactivation of any 5′ -GATC-3′ site individually had no effect; at least two sites had to be inactivated to disrupt the normal pattern of expression. Studies of OxyR interaction with agn43 DNA showed that methylation of any two 5′ -GATC-3′ sites was necessary and sufficient to block binding of the repressor. It was also found that the adenines of the second and third 5′ -GATC-3′ sites are required for OxyR binding, demonstrating that the sites for Dam methylation and for repressor binding are intimately associated. This is consistent with a competition model in which Dam and OxyR share a preference for specific DNA sequences in the regulatory region of the agn43 gene. [ABSTRACT FROM AUTHOR]

Summary Cultures of Escherichia coli could be separated into more than 15 cell populations, each forming a discrete band after Percoll gradient centrifugation. The cell separation was found to result from the difference in buoyant density but not the size difference. The cell density increases upon transition from exponential growth to stationary phase. Exponential phase cultures formed at least five discrete bands with lower densities, whereas stationary phase cultures formed more than 10 bands with higher densities. Two molecular markers characterizing each cell population were identified: the functioning promoter species, as identified by measuring the expression of green fluorescent protein under the control of test promoters; and the expressed protein species, as monitored by quantitative immunoblotting. These findings together suggest that the growth phase-coupled transition of E. coli phenotype is discontinuous. [ABSTRACT FROM AUTHOR]

We have used ethidium bromide titration for direct measurement of the changes in the negative supercoiling of Esdieridiia coli chromosome caused by mutations inactivating the cell cycle functions mukB and seqA. The amounts of the intercalative agent required to relax the supercoiled chromosome in mukB and seqA mutants were lower and higher, respectively, than for the wild-type parent, confirming that these cell cycle genes modulate the topology of the L coli chromosome. Plasmid superhelicity measured in these mutant strains showed similar effects albeit of reduced magnitude. As the effects of mukB and segA mutations were not restricted to the chromosome alone, MukB and SeqA proteins possibly interact with factors involved in the maintenance of intracellular DNA topology. To our knowledge, this is the first direct demonstration of the influence of mukB and segA genes on the superhelicity of the L coli chromosome. [ABSTRACT FROM AUTHOR]

The process of partitioning bacterial sister chromosomes into daughter cells seems to be distinct from chromatid segregation during eukaryotic mitosis. In Escherichia coli, partitioning starts soon after initiation of replication, when the two newly replicated oriCs move from the cell centre to quarter positions within the cell. As replication proceeds, domains of the compact, supercoiled chromosome are locally decondensed ahead of the replication fork. The nascent daughter chromosomes are recondensed and moved apart through the concerted activities of topoisomerases and the SeqA (sequestration) and MukB (chromosome condensation) proteins, all of which modulate nucleoid superhelicity. Thus, genes involved in chromosome topology, once set aside as 'red herrings' in the search for 'true' partition functions, are again recognized as being important for chromosome partitioning in E. coli. [ABSTRACT FROM AUTHOR]

Temperature sensitivity of DNA polymerization and growth, resulting from mutation of the τ and γ subunits of Escherichia coli DNA polymerase III, are suppressed by Cs,Sx mutations of the initiator gene, dnaA. These mutations simultaneously cause defective initiation at 20°C. Efficient suppression, defined as restoration of normal growth rate at 39°C to essentially all the cells, depends on functional oriC. Increasing DnaA activity in a strain capable of suppression, by introducing a copy of the wild-type allele, increasing the suppressor gene dosage or introducing a seqA mutation, reversed the suppression. This suggests that the suppression mechanism depends on reduced activity of DnaACs,Sx. Models that assume that suppression results from an initiation defect or from DnaACs,Sx interaction with polymerization proteins during nascent strand synthesis are proposed. [ABSTRACT FROM AUTHOR]

In Escherichia coli, initiation of chromosome replication requires that DnaA binds to R boxes (9-mer repeats) in oriC, the unique chromosomal replication origin. At the time of initiation, integration host factor (IHF) also binds to a specific site in oriC. IHF stimulates open complex formation by DnaA on supercoiled oriC in cell-free replication systems, but it is unclear whether this stimulation involves specific changes in the oriC nucleoprotein complex. Using dimethylsulphate (DMS) footprinting on supercoiled oriC plasmids, we observed that IHF redistributed prebound DnaA, stimulating binding to sites R2, R3 and R5(M), as well as to three previously unidentified non-R sites with consensus sequence (A/T)G(G/C) (A/T)N(G/C)G(A/T)(A/T)(T/C)A. Redistribution was dependent on IHF binding to its cognate site and also required a functional R4 box. By reducing the DnaA level required to separate DNA strands and trigger initiation of DNA replication at each origin, IHF eliminates competition between strong and weak sites for free DnaA and enhances the precision of initiation synchrony during the cell cycle. [ABSTRACT FROM AUTHOR]

A hybrid assay, based on the properties of the λ repressor, was developed to detect FtsZ dimerization in Escherichia coli in vivo. A gene fusion comprising the N-terminal end of the λ cI repressor gene and the complete E. coli ftsZ gene was constructed. The fused protein resulted in a functional λ repressor and was able to complement the thermosensitive mutant ftsZ84. Using the same strategy, a series of 10 novel mutants of FtsZ that are unable to dimerize was selected, and a deletion analysis of the protein was carried out. Characterization of these mutants allowed the identification of three separate FtsZ portions: the N-terminal of about 150 amino acids; the C-terminal of about 60 amino acids, which corresponds to the less conserved portion of the protein; and a central region of about 150 residues. Mutants belonging to this region would define the dimerization domain of FtsZ. [ABSTRACT FROM AUTHOR]

The DNA-membrane complex has been the subject of intensive investigation for over 35 years as the possible site for DNA replication in the prokaryotic cell and the site through which newly synthesized chromosomes are segregated into daughter cells. However, the molecular mechanisms which control these phenomena are, for the most part, poorly understood despite genetic, biochemical, and morphologic evidence in favour of their existence. This is probably due to the transient nature and non-covalent interactions that occur between DNA and the membrane. In addition, there is a paucity of knowledge concerning the nature of the membrane receptors for DNA and whether the membrane plays simply a structural or metabolic role in the two processes. Plasmids can provide important insights into the role of the membrane in replication and partitioning because the plasmid life cycle is relatively simple, with replication occurring during the cell cycle and partitioning during cell division. The replicon model of Jacob et al. (1963, Cold Spring Harbor Symp Quant Biol 28: 329-348) still represents a good conceptual framework (with modifications) to explain how plasmid replication and partitioning are linked by the membrane. In its simplest form, the model focuses on specific membrane binding sites (possibly along the equator of the cell) for plasmid (or bacterial) replication, with the membrane acting as a motive force to separate the newly synthesized replicons and their attached sites into daughter cells. Indeed, proteins involved in both plasmid replication and partitioning have been found in membrane fractions and some plasmids require membrane binding for initiation and an active partitioning. We propose that several factors are critical for both plasmid DNA replication and partitioning. One factor is the extent of negative supercoiling (brought about by an interplay of various topoisomerases, but most importantly by DNA gyrase). Supercoiling is known to be critical for initiation of DNA replication but may also be important for the formation of a partition complex in contact with the cell membrane. Another factor is the presence of specific subdomains of the membrane which can interact specifically with origin DNA and possibly other regions involved in partitioning. Such domains may be induced transiently or be present at all times during the cell cycle. [ABSTRACT FROM AUTHOR]

We report the construction of intP1 and intFs strains, in which the basic replicon from either plasmid P1 or plasmid F (oriS) has been integrated in both orientations into the origin of replication, oriC, of the Escherichia coli chromosome. In these strains, oriC is no longer functional and chromosome-replication is instead controlled by the integrated plasmid replicon. The strains were viable, showing that the deviation from normal chromosome-replication control was not large enough to prohibit cell survival. The strains showed a broader cell-size distribution than a wild-type strain and were more filamentous in rich than in minimal media, although cells of wild-type size were also present. Celts which contained aberrantly shaped or aberrantly distributed nucleoids were also observed. Marker-frequency analysis indicated that chromosome replication was predominantly bidirectional in both intFs strains. In the intP1 strains, the degree of bidirectionality depended upon the orientation of the integrated replicon. [ABSTRACT FROM AUTHOR]

The nature of the structure of the bacterial cell is becoming clearer. The envelope contains periseptal annuli, a discontinuous periplasm and adhesion sites, whilst the cytoplasmic membrane is probably organized into distinct proteolipid domains by the coupled transcription-translation-insertion (transertion) of membrane proteins. The structure of the nucleoid is determined by proteins which self-associate and by attachment to membrane, which is achieved in part by transertion. Metabolic pathways form multi-enzyme complexes which channel substrates and which connect membranes and nucleic acids to create the extensive, cross-linked, intracellular structure we term the 'nzoskeleton'. This enzoskeleton includes eukaryotic-like cytoskeletal structures and elements such as the MukB and FtsZ proteins. We propose that the enzoskeleton is regulated by calcium and by protein phosphorylation during adaptation to different environments and during the cell cycle. [ABSTRACT FROM AUTHOR]

Escherichia coli mutants, (verA, dilA) specifically resistant to the Ca2+ channel inhibitors verapamil and diltiazem, respectively, are hypersensitive to EGTA and BAPTA. We have shown, using 1-D and 2-D gel electrophoresis, that the synthesis of at least 25 polypeptides in the mutants was enhanced by treatment with Ca2+ chelators and the synthesis of at least 11 polypeptides was repressed. This pattern of induction was not observed in heat- or SDS-treated cells and therefore does not appear to be a general stress response. The majority of the induced proteins are low molecular weight, extremely heat stable and acidic, characteristic properties of caimodulin. Moreover, of the major induced species, three with apparent molecular masses of 12, 18, and 34kDa all cross-reacted with polydonal and monoclonal anti-bodies to eukaryote calmodulins or calerythrin, a heat-resistant Ca2+-binding protein from Saccharo-polyspora erythraea. The verA, dilA mutants. In being hypersensitive to EGTA and to the Ca2+ ionophore A23187 + Ca2+, may be defective in the regulation of the level of free intracellular Ca2+. [ABSTRACT FROM AUTHOR]

Transcriptional levels of the Escherichia coli mioC and gidA genes, which flank the chromosomal origin of replication (oriC) and the dnaA gene, were correlated with the time of initiation of chromosome replication. The transcripts were measured either in dnaC2(ts) mutants that had been aligned for initiation of chromosome replication by a temperature shift or in synchronous cultures of cells obtained using the baby machine technique. In both types of experiments, mioC transcription was inhibited prior to initiation of chromosome replication and resumed several minutes after initiation. Conversely, gidA and dnaA transcription were both inhibited after initiation of replication, coincident with the period of hemimethylation of oriC DNA. It is proposed that mioC transcription prevents initiation of chromosome replication, and must terminate before replication can begin. It is further proposed that the eclipse period between rounds of replication, i.e. the minimum interval between successive initiations, encompasses the time required to methylate GATC sequences in newly replicated oriC plus the time required to terminate mioC transcription. Conversely, the active transcription of gidA and dna prior to initiation is consistent with their positive effects on initiation, and their shutdown after initiation could serve to limit premature reinitiation. [ABSTRACT FROM AUTHOR]

In Escherichia coli minB mutants, cell division can take place at the cell poles as well as non-polarly in the cell. We have examined growth, division patterns, and nucleoid distribution in individual cells of a minC point mutant and a minBdeletion mutant, and compared them to the corresponding wild-type strain and an intR1 strain in which the chromosome is over-replicated. The main findings were as follows. In the minBmutants, polar and non-polar divisions appeared to occur independently of each other. Furthermore, the timing of cell division in the cell cycle was found to be severely affected. In addition, nucleold conformation and distribution were considerably disturbed. The results obtained call for a reevaluation of the role of the MinB system in the E. coli cell cycle, and of the concept that limiting quanta of cell division factors are regularly produced during the cell cycle. [ABSTRACT FROM AUTHOR]