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Abstract
The Burkholderia cepacia complex (Bcc) consists of 17 closely related species of opportunistic bacterial pathogens, which are particularly problematic for cystic fibrosis patients and immunocompromised individuals. Bcc genomes consist of multiple replicons, and each strain sequenced to date has three chromosomes. In addition to genes thought to be essential for survival, each chromosome carries at least one rRNA operon. We isolated three mutants during a transposon mutagenesis screen that were non-pathogenic in a Caenorhabditis elegans infection model. It was demonstrated that ...
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Abstract
Chromatin immunoprecipitation (ChIP-chip and ChIP-seq) assays identify where proteins bind throughout a genome. However, DNA contamination and DNA fragmentation heterogeneity produce false positives (erroneous calls) and imprecision in mapping. Consequently, stringent data filtering produces false negatives (missed calls). Here we describe ChIP-exo, where an exonuclease trims ChIP DNA to a precise distance from the crosslinking site. Bound locations are detectable as peak pairs by deep ...
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Abstract
We describe a one-step, isothermal assembly method for synthesizing DNA molecules from overlapping oligonucleotides. The method cycles between in vitro recombination and amplification until the desired length is reached. As a demonstration of its simplicity and robustness, we synthesized the entire 16.3-kilobase mouse mitochondrial genome from 600 overlapping 60-mers. ...
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by Benjamin Lang, Nicolas Blot, Emeline Bouffartigues, et al.Malcolm Buckle, Marcel Geertz, Claudio O. Gualerzi, Ramesh Mavathur, Georgi Muskhelishvili, Cynthia L. Pon, Sylvie Rimsky, Stefano Stella, Madan M. Babu, Andrew Travers
Abstract
The global transcriptional regulator H-NS selectively silences bacterial genes associated with pathogenicity and responses to environmental insults. Although there is ample evidence that H-NS binds preferentially to DNA containing curved regions, we show here that a major basis for this selectivity is the presence of a conserved sequence motif in H-NS target transcriptons. We further show that there is a strong tendency for the H-NS binding sites to be clustered, both within operons and in genes contained in the pathogenicity-associated islands. ...
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Abstract
A transcriptional regulatory network encompasses sets of genes (regulons) whose expression states are directly altered in response to an activating signal, mediated by trans-acting regulatory proteins and cis-acting regulatory sequences. Enumeration of these network components is an essential step toward the creation of a framework for systems-based analysis of biological processes. Profile-based methods for the detection of cis-regulatory elements are often applied to predict regulon members, but they suffer from poor specificity. In this report we describe Regulogger, a novel computational ...
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Abstract
During cell division, chromosomes are segregated to nascent daughter cells by attaching to the microtubules of the mitotic spindle through the kinetochore. Kinetochores are assembled on a specialized chromatin domain called the centromere, which is characterized by the replacement of nucleosomal histone H3 with the histone H3 variant centromere protein A (CENP-A). CENP-A is essential for centromere and kinetochore formation in all eukaryotes but it is unknown how CENP-A chromatin directs centromere and kinetochore assembly. Here we generate synthetic CENP-A chromatin ...
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Abstract
O-Phosphoserine (Sep), the most abundant phosphoamino acid in the eukaryotic phosphoproteome, is not encoded in the genetic code, but synthesized posttranslationally. Here, we present an engineered system for specific cotranslational Sep incorporation (directed by UAG) into any desired position in a protein by an Escherichia coli strain that harbors a Sep-accepting transfer RNA (tRNASep), its cognate Sep–tRNA synthetase (SepRS), and an engineered EF-Tu (EF-Sep). Expanding the genetic code rested on reengineering EF-Tu to relax its quality-control function and permit Sep-tRNASep binding. ...
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by Ankur Mutreja, Dong Wook W. Kim, Nicholas R. Thomson, et al.Thomas R. Connor, Je Hee H. Lee, Samuel Kariuki, Nicholas J. Croucher, Seon Young Y. Choi, Simon R. Harris, Michael Lebens, Swapan Kumar K. Niyogi, Eun Jin J. Kim, T. Ramamurthy, Jongsik Chun, James L. Wood, John D. Clemens, Cecil Czerkinsky, G. Balakrish Nair, Jan Holmgren, Julian Parkhill, Gordon Dougan
Abstract
Vibrio cholerae is a globally important pathogen that is endemic in many areas of the world and causes 3-5 million reported cases of cholera every year. Historically, there have been seven acknowledged cholera pandemics; recent outbreaks in Zimbabwe and Haiti are included in the seventh and ongoing pandemic. Only isolates in serogroup O1 (consisting of two biotypes known as 'classical' and 'El Tor') and the ...
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Abstract
Science, technology, engineering, and mathematics (STEM) graduate students are often encouraged to maximize their engagement with supervised research and minimize teaching obligations. However, the process of teaching students engaged in inquiry provides practice in the application of important research skills. Using a performance rubric, we compared the quality of methodological skills demonstrated in written research proposals for two groups of early career graduate students (those with both teaching and research responsibilities and those with only research responsibilities) at the beginning and ...
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Abstract
Vibrio cholerae carries homologs of plasmid-borne parA and parB genes on both of its chromosomes. The par genes help to segregate many plasmids and chromosomes. Here we have studied the par genes of V. cholerae chromosome I. Earlier studies suggested that ParBI binds to the centromeric site parSI near the origin of replication (oriI), and parSI-ParBI complexes are placed at the cell poles by ParAI. Deletion of parAI and parSI caused the origin-proximal DNA to be less polar. Here we found ...
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Proceedings of the National Academy of Sciences of the United States of America, Vol. 108, No. 15. (12 April 2011), pp. 6199-6204, doi:10.1073/pnas.1013244108
Abstract
Plasmid origins of replication are rare in bacterial chromosomes, except in multichromosome bacteria. The replication origin of Vibrio cholerae chromosome II (chrII) closely resembles iteron-bearing plasmid origins. Iterons are repeated initiator binding sites in plasmid origins and participate both in replication initiation and its control. The control is mediated primarily by coupling of iterons via the bound initiators ("handcuffing"), which causes steric hindrance to the origin. The control in chrII must be different, since the timing of its replication is cell ...
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Abstract
There is little knowledge of factors and mechanisms for coordinating bacterial chromosome replication and segregation. Previous studies have revealed that genes (and their products) that surround the origin of replication (oriCII) of Vibrio cholerae chromosome II (chrII) are critical for controlling the replication and segregation of this chromosome. rctB, which flanks one side of oriCII, encodes a protein that initiates chrII replication; rctA, which flanks the other side of oriCII, inhibits rctB activity. The chrII parAB2 operon, which is essential for ...
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Abstract
We describe a protocol, DNA sampling, for the rapid isolation of specific segments of DNA, together with bound proteins, from Escherichia coli K-12. The DNA to be sampled is generated as a discrete fragment within cells by the yeast I-SceI meganuclease, and is purified using FLAG-tagged LacI repressor and beads carrying anti-FLAG antibody. We illustrate the method by investigating the proteins bound to the colicin K gene regulatory region, either before or after induction of the colicin K gene promoter. ...
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Abstract
Condensins play a central role in global chromatin organization. In bacteria, two families of condensins have been identified, the MukBEF and SMC-ScpAB complexes. Only one of the two complexes is usually found in a given species, giving rise to a paradigm that a single condensin organizes bacterial chromosomes. Using sequence analysis, we identified a third family of condensins, MksBEF (MukBEF-like SMC proteins), which is broadly ...
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Abstract
BACKGROUND:Since publication in 1977 of plasmid pBR322, many breakthroughs in Biology have depended on increasingly sophisticated vector platforms for analysis and engineering of given bacterial strains. Although restriction sites impose a certain format in the procedures for assembling cloned genes, every attempt thus far to standardize vector architecture and nomenclature has ended up in failure. While this state of affairs may still be tolerable for traditional one-at-a-time studies of single genes, the onset of systems and synthetic biology calls for a ...
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Abstract
An insertional mutagenesis system that uses transposons carrying unique DNA sequence tags was developed for the isolation of bacterial virulence genes. The tags from a mixed population of bacterial mutants representing the inoculum and bacteria recovered from infected hosts were detected by amplification, radiolabeling, and hybridization analysis. When applied to a murine model of typhoid fever caused by Salmonella typhimurium, mutants with attenuated virulence were revealed by use of tags that were present in the inoculum but not in bacteria recovered ...
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Abstract
Replicon architecture in bacteria is commonly comprised of one indispensable chromosome and several dispensable plasmids. This view has been enriched by the discovery of additional chromosomes, identified mainly by localization of rRNA and/or tRNA genes, and also by experimental demonstration of their requirement for cell growth. The genome of Rhizobium etli CFN42 is constituted by one chromosome and six large plasmids, ranging in size from 184 to 642 kb. Five of the six plasmids are dispensable for cell viability, but plasmid ...
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Abstract
ABSTRACT: BACKGROUND: repABC operons are present on large, low copy-number plasmids and on some secondary chromosomes in at least 19 alpha-proteobacterial genera, and are responsible for the replication and segregation properties of these replicons. These operons consist, with some variations, of three genes: repA, repB, and repC. RepA and RepB are involved in plasmid partitioning and in the negative regulation of their own transcription, and RepC is the limiting factor for replication. An antisense RNA encoded between the repB-repC genes modulates ...
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Abstract
A triazole mimic of a DNA phosphodiester linkage has been produced by templated chemical ligation of oligonucleotides functionalized with 5′-azide and 3′-alkyne. The individual azide and alkyne oligonucleotides were synthesized by standard phosphoramidite methods and assembled using a straightforward ligation procedure. This highly efficient chemical equivalent of enzymatic DNA ligation has been used to assemble a 300-mer from three 100-mer oligonucleotides, demonstrating the total chemical synthesis of very long oligonucleotides. The base sequences of the DNA strands containing this artificial linkage ...
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Abstract
We recently reported on CFP-Epac-YFP, an Epac-based single polypeptide FRET reporter to resolve cAMP levels in living cells. In this study, we compared and optimized the fluorescent protein donor/acceptor pairs for use in biosensors such as CFP-Epac-YFP. Our strategy was to prepare a wide range of constructs consisting of different donor and acceptor fluorescent proteins separated by a short linker. Constructs were expressed in HEK293 cells and tested for FRET and other relevant properties. The most promising pairs were subsequently used ...
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Abstract
Bimolecular fluorescence complementation (BiFC) has been widely used in the analysis of protein-protein interactions (PPIs) in recent years. There are many notable advantages of BiFC such as convenience and direct visualization of PPI in cells. However, BiFC has one common limitation: the separated non-fluorescent fragments can be spontaneously self-assembled into an intact protein, which leads to false-positive results. In this study, a pair of complementary fragments (sfGFPN and sfGFPC) was constructed by splitting superfolder GFP (sfGFP) between the 214 and 215 ...
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Abstract
Numerous technologies based on utilizing fluorescent proteins have been developed for biological research, and fluorescence complementation (FC) is a recent application for visualization of molecular events in living cells and organisms. Currently, ten fluorescent proteins have been demonstrated to support FC. Over the past five years, FC-based technologies have been developed to visualize a variety of molecular events, such as protein–protein interactions, post-translational modifications, protein folding, conformational changes, RNA–protein interactions, mRNA localization and DNA hybridization. In addition, FC has also been ...
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Abstract
The specificity of biological regulatory mechanisms relies on selective interactions between different proteins in different cell types and in response to different extracellular signals. We describe a bimolecular fluorescence complementation (BiFC) approach for the simultaneous visualization of multiple protein interactions in the same cell. This approach is based on complementation between fragments of fluorescent proteins with different spectral characteristics. We have identified 12 bimolecular fluorescent complexes that correspond to 7 different spectral classes. Bimolecular complex formation between fragments of different fluorescent ...
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Abstract
Protein interactions integrate stimuli from different signaling pathways and developmental programs. Bimolecular fluorescence complementation (BiFC) analysis has been developed for visualization of protein interactions in living cells. This approach is based on complementation between two fragments of a fluorescent protein when they are brought together by an interaction between proteins fused to the fragments, and it enables visualization of the subcellular locations of protein interactions in the normal cellular environment. It can be used for the analysis of many protein interactions ...
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Abstract
Protein interactions integrate stimuli from different signaling pathways and developmental programs. Bimolecular fluorescence complementation (BiFC) analysis has been developed for visualization of protein interactions in living cells. This approach is based on complementation between two fragments of a fluorescent protein when they are brought together by an interaction between proteins fused to the fragments, and it enables visualization of the subcellular locations of protein interactions in the normal cellular environment. It can be used for the analysis of many protein interactions ...
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Abstract
Summary Transformable (competent) cells of Bacillus subtilis are blocked in cell division because the traffic ATPase ComGA prevents the formation of FtsZ rings. Although ComGA-deficient cells elongate and form FtsZ rings, cell division remains blocked at a later stage and the cells become mildly filamented. Here we show that the highly conserved protein Maf is synthesized predominantly in competent cells under the direct control of the transcription factor ComK and is solely responsible for the later block in cell division. In ...
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Abstract
Compartmentalization is an important process, since it allows the segregation of metabolic activities and, in the era of synthetic biology, represents an important tool by which defined microenvironments can be created for specific metabolic functions. Indeed, some bacteria make specialized proteinaceous metabolic compartments called bacterial microcompartments (BMCs) or metabolosomes. Here we demonstrate that the shell of the metabolosome (representing an empty BMC) can be produced within E. coli cells by the coordinated expression of genes encoding structural proteins. A plethora of diverse ...
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Abstract
Replication timing profiles are cell type-specific and reflect genome organization changes during differentiation. In this protocol, we describe how to analyze genome-wide replication timing (RT) in mammalian cells. Asynchronously cycling cells are pulse labeled with the nucleotide analog 5-bromo-2-deoxyuridine (BrdU) and sorted into S-phase fractions on the basis of DNA content using flow cytometry. BrdU-labeled DNA from each fraction is immunoprecipitated, amplified, differentially labeled and ...
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Abstract
The development of the technology to synthesize new genomes and to introduce them into hosts with inactivated wild-type chromosome opens the door to new horizons in synthetic biology. Here it is of outmost importance to harness the ability of using computational design to predict and optimize a synthetic genome before attempting its synthesis. The methodology to computationally design a genome is based on an optimization that computationally mimics genome evolution. The biggest bottleneck lies on the use of an appropriate fitness ...
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Abstract
We describe an in situ technique for studying the chromatin binding of proteins in the fission yeast Schizosaccharomyces pombe. After tagging the protein of interest with green fluorescent protein (GFP), chromatin-associated protein is detected by GFP fluorescence following cell permeabilization and washing with a non-ionic detergent. Cell morphology and nuclear structure are preserved in this procedure, allowing structures such as the mitotic spindle to be detected by indirect immunofluorescence. Cell cycle changes in the chromatin association of proteins can therefore be ...
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Abstract
An in situ technique for studying the chromatin binding of proteins in single fission yeast cells (Schizosaccharomyces pombe) is described. Cells are permeabilized by enzymatic digestion and extracted with a detergent-containing buffer. This procedure removes soluble proteins, but proteins that are bound to insoluble cell structures such as chromatin are retained, and overall cell morphology is maintained. Extraction of proteins is monitored by fluorescence microscopy, either using fluorescently tagged proteins or by indirect immunofluorescence. This method allows the chromatin association of ...
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Abstract
DNA polymerases of the Y-family, such as Escherichia coli UmuC and DinB, are specialized enzymes induced by the SOS response, which bypass lesions allowing the continuation of DNA replication. umuDC orthologs are absent in Caulobacter crescentus and other bacteria, raising the question about the existence of SOS mutagenesis in these organisms. Here, we report that the C.crescentus dinB ortholog is not involved in damage-induced mutagenesis. However, an operon composed of two hypothetical genes and dnaE2, encoding a second copy of the ...
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Abstract
Bacterial-based interaction trap systems provide a powerful method to identify interacting macromolecules. When carried out in the context of a genetic selection, interacting pairs can be rapidly isolated from large combinatorial libraries. This technology has been adapted to allow the identification of DNA-binding sequences for a transcription factor (TF) from a large randomized library. This procedure uses a library of randomized binding sites upstream of a cocistronic HIS3-URA3 reporter cassette. The URA3 reporter allows self-activating sequences to be removed from the ...
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Abstract
We examine the use of high-throughput sequencing on binding sites recovered using a bacterial one-hybrid (B1H) system and find that improved models of transcription factor (TF) binding specificity can be obtained compared to standard methods of sequencing a small subset of the selected clones. We can obtain even more accurate binding models using a modified version of B1H selection method with constrained variation (CV-B1H). However, achieving these improved models using CV-B1H data required the development of a new method of analysis—GRaMS ...
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Abstract
Numerous protocols for the isolation of bacterial nucleoids have been described based on treatment of cells with sucrose-lysozyme-EDTA and subsequent lysis with detergents in the presence of counterions (e.g., NaCl, spermidine). Depending on the lysis conditions both envelope-free and envelope-bound nucleoids could be obtained, often in the same lysate. To investigate the mechanism(s) involved in compacting bacterial DNA in the living cell, we wished to isolate intact nucleoids in the absence of detergents and high concentrations of counterions. Here, we compare ...
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Abstract
Plasmids are autonomously replicating extrachromosomal DNA molecules that often impart key phenotypes to their bacterial hosts. Plasmids are abundant in marine bacteria, but there is scant knowledge of the mechanisms that control their replication in these hosts. Here, we identified and characterized the factors governing replication of a new family of plasmids from marine bacteria, typified by the virulence-linked plasmid pB1067 of Vibrio nigripulchritudo. Members of this family are prevalent among, yet restricted to, the Vibrionaceae. Unlike almost all plasmid families ...
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Molecular microbiology, Vol. 41, No. 4. (August 2001), pp. 885-896
Abstract
Many well-known transcriptional regulatory proteins are composed of at least two independently folding domains and, typically, only one of these is a DNA-binding domain. However, some transcriptional regulators have been described that have more than one DNA-binding domain. Regulators with a single DNA-binding domain often bind co-operatively to the DNA in homotypic or heterotypic combinations, and two or more DNA-binding domains of a single regulatory protein can also bind co-operatively to suitably positioned recognition sequences. Here, we examine the behaviour of ...
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Abstract
Signal recognition particle (SRP)-dependent protein targeting is a universally conserved process that delivers proteins to the bacterial cytoplasmic membrane or to the endoplasmic reticulum membrane in eukaryotes. Crucial during targeting is the transfer of the ribosome-nascent chain complex (RNC) from SRP to the Sec translocon. In eukaryotes, this step is co-ordinated by the SRβ subunit of the SRP receptor (SR), which probably senses a vacant translocon by direct interaction with the translocon. Bacteria lack the SRβ subunit and how they co-ordinate ...
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by Asako Sakaue-Sawano, Hiroshi Kurokawa, Toshifumi Morimura, et al.Aki Hanyu, Hiroshi Hama, Hatsuki Osawa, Saori Kashiwagi, Kiyoko Fukami, Takaki Miyata, Hiroyuki Miyoshi, Takeshi Imamura, Masaharu Ogawa, Hisao Masai, Atsushi Miyawaki
Abstract
The cell-cycle transition from G1 to S phase has been difficult to visualize. We have harnessed antiphase oscillating proteins that mark cell-cycle transitions in order to develop genetically encoded fluorescent probes for this purpose. These probes effectively label individual G1 phase nuclei red and those in S/G2/M phases green. We were able to generate cultured cells and transgenic mice constitutively expressing the cell-cycle probes, in ...
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Abstract
We purified an inhibitor of oriC plasmid replication and determined that it is a truncated form of ribosomal protein L2 evidently lacking 59 amino acid residues from the C-terminal region encoded by rplB. We show that this truncated form of L2 or mature L2 physically interacts with the N-terminal region of DnaA to inhibit initiation from oriC by apparently interfering with DnaA oligomer formation, and the subsequent assembly of the prepriming complex on an oriC plasmid. Both forms of L2 also ...
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Abstract
Summary Non-replicating Escherichia coli chromosomes are organized as sausage-shaped structures with the left (L) and the right (R) chromosome arms (replichores) on opposite cell halves and the replication origin (oriC) close to midcell. The replication termination region (ter) therefore passes between the two outer edges of the nucleoid. Four alignment patterns of the two <LR> sister chromosomes within a cell have been detected in an asynchronous population, with the <LRLR> pattern predominating. We test the hypothesis that the minority <LRRL> and ...
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Abstract
The stochasticity of chromosome organization was investigated by fluorescently labeling genetic loci in live Escherichia coli cells. In spite of the common assumption that the chromosome is well modeled by an unstructured polymer, measurements of the locus distributions reveal that the E. coli chromosome is precisely organized into a nucleoid filament with a linear order. Loci in the body of the nucleoid show a precision of positioning within the cell of better than 10% of the cell length. The precision of ...
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by Gareth Butland, Jose M. Peregrin-Alvarez, Joyce Li, et al.Wehong Yang, Xiaochun Yang, Veronica Canadien, Andrei Starostine, Dawn Richards, Bryan Beattie, Nevan Krogan, Michael Davey, John Parkinson, Jack Greenblatt, Andrew Emili
Abstract
Proteins often function as components of multi-subunit complexes. Despite its long history as a model organism1, no large-scale analysis of protein complexes in Escherichia coli has yet been reported. To this end, we have targeted DNA cassettes into the E. coli chromosome to create carboxy-terminal, affinity-tagged alleles of 1,000 open reading frames (~ 23% of the genome). A total of 857 proteins, including 198 of the most highly conserved, soluble non-ribosomal proteins essential in at least one bacterial species, were tagged ...
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Abstract
Motivation: The Dam methyltransferase (DamMT) activity, broadly distributed in association with restriction endonucleases, as part of the restriction-modification defense systems, has evolved to become intimately associated with essential biological functions in a few organisms. In Escherichia coli, DamMT is involved in multiple aspects of DNA maintenance, replication initiation, daughter chromosome segregation, DNA mismatch repair, gene expression control, etc.The participation of DamMT in such a diverse set of functions required that other genes adapted, or emerged through evolution, in response to the ...
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Molecular & general genetics : MGG, Vol. 162, No. 3. (4 July 1978), pp. 269-275
Abstract
We have isolated plasmids by linking the 5.9 MD EcoRI fragment of E. coli that carries the origin of replication to an EcoRI fragment that carries an amplicillin resistance determinant, but lacks an origin of replication. 3 plasmids of this type, pOC1, pOC2, and pOC3, are described in detail in this report. Although the plasmids have some adverse effect on the growth properties of the host strain, their existence shows that two functioning chromosomal origins can coexist in one cell. Deletions ...
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Abstract
Cellular decision making is the process whereby cells assume different, functionally important and heritable fates without an associated genetic or environmental difference. Such stochastic cell fate decisions generate nongenetic cellular diversity, which may be critical for metazoan development as well as optimized microbial resource utilization and survival in a fluctuating, frequently stressful environment. Here, we review several examples of cellular decision making from viruses, bacteria, yeast, lower metazoans, and mammals, highlighting the role of regulatory network structure and molecular noise. We ...
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Abstract
Abstract Formerly regarded as small ‘bags’ of nucleic acids with randomly diffusing enzymes, bacteria are organized by a sophisticated and tightly regulated molecular machinery. Here, we review qualitative and quantitative data on the intracellular organization of bacteria and provide a detailed inventory of macromolecular structures such as the divisome, the degradosome and the bacterial ‘nucleolus’. We discuss how these metabolically active structures manage the spatial organization of the cell and how macromolecular crowding influences them. We present for the first time ...
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