scholarly journals Genome of Bacteriophage P1

2004 ◽  
Vol 186 (21) ◽  
pp. 7032-7068 ◽  
Author(s):  
Małgorzata B. Łobocka ◽  
Debra J. Rose ◽  
Guy Plunkett ◽  
Marek Rusin ◽  
Arkadiusz Samojedny ◽  
...  
Keyword(s):  
Dna Methyltransferase ◽  
Enteric Bacteria ◽  
Origin Of Replication ◽  
Protein Coding ◽  
Bacteriophage P1 ◽  
E Coli ◽  
Base Content ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Rna Polymerase Holoenzyme

ABSTRACT P1 is a bacteriophage of Escherichia coli and other enteric bacteria. It lysogenizes its hosts as a circular, low-copy-number plasmid. We have determined the complete nucleotide sequences of two strains of a P1 thermoinducible mutant, P1 c1-100. The P1 genome (93,601 bp) contains at least 117 genes, of which almost two-thirds had not been sequenced previously and 49 have no homologs in other organisms. Protein-coding genes occupy 92% of the genome and are organized in 45 operons, of which four are decisive for the choice between lysis and lysogeny. Four others ensure plasmid maintenance. The majority of the remaining 37 operons are involved in lytic development. Seventeen operons are transcribed from σ70 promoters directly controlled by the master phage repressor C1. Late operons are transcribed from promoters recognized by the E. coli RNA polymerase holoenzyme in the presence of the Lpa protein, the product of a C1-controlled P1 gene. Three species of P1-encoded tRNAs provide differential controls of translation, and a P1-encoded DNA methyltransferase with putative bifunctionality influences transcription, replication, and DNA packaging. The genome is particularly rich in Chi recombinogenic sites. The base content and distribution in P1 DNA indicate that replication of P1 from its plasmid origin had more impact on the base compositional asymmetries of the P1 genome than replication from the lytic origin of replication.

scholarly journals Cryo-EM reconstruction of AlfA fromBacillus subtilisreveals the structure of a simplified actin-like filament at 3.4 Å resolution

2017 ◽  
Author(s):  
Andrzej Szewczak-Harris ◽  
Jan Löwe
Keyword(s):  
Atomic Structure ◽  
Adaptor Protein ◽  
Nucleotide Hydrolysis ◽  
E Coli ◽  
Left Handed ◽  
Copy Number Plasmid ◽  
Resolution Electron ◽  
Centromeric Sequence ◽  
Low Copy Number ◽  
Almost All

AbstractLow copy-number plasmid pLS32 ofBacillus subtilissubsp.nattocontains a partitioning system that ensures segregation of plasmid copies during cell division. The partitioning locus comprises actin-like protein AlfA, adaptor protein AlfB and the centromeric sequenceparN. Similar to the ParMRC partitioning system fromE. coliplasmid R1, AlfA filaments form actin-like double helical filaments that arrange into an antiparallel bipolar spindle, which attaches its growing ends to sister plasmids, through interactions with AlfB andparN. Since, compared with ParM and other actin-like proteins, AlfA is highly diverged in sequence, we determined the atomic structure of non-bundling AlfA filaments to 3.4 Å resolution by cryo-EM. The structure reveals how the deletion of subdomain IIB of the canonical actin-fold has been accommodated by unique longitudinal and lateral contacts, whilst still enabling formation of left-handed, double helical, polar and staggered filaments that are architecturally similar to ParM. Through cryo-EM reconstruction of bundling AlfA filaments we obtained a pseudo-atomic model of AlfA doublets: the assembly of two filaments. The filaments are antiparallel, as required by the segregation mechanism, and exactly anti-phasic with 8-fold integer helical symmetry, to enable efficient doublet formation. The structure of AlfA filaments and doublets shows, in atomic detail, signs of the strong evolutionary pressure for simplicity, placed on plasmids: deletion of an entire domain of the actin fold is compensated by changes to all interfaces so that the required properties of polymerisation, nucleotide hydrolysis and antiparallel doublet formation are retained to fulfil the system's biological raison d'être.Significance StatementProtein filaments perform a vast array of functions inside almost all living cells. Actin-like proteins in archaea and bacteria have previously been found to form a surprising diversity of filament architectures, reflecting their divergent cellular roles. Actin-like AlfA is unique in that it is much smaller than all other filament forming actin-like proteins. With an atomic structure of the AlfA filament, obtained by high-resolution electron cryo-microscopy, we have revealed—at atomic level of detail—how AlfA filaments form dynamic filaments capable of transporting plasmid DNA in cells and how these filaments arrange into antiparallel bundles required for the segregation mechanism.

scholarly journals The Toxin-Antitoxin System of the Streptococcal Plasmid pSM19035

2005 ◽  
Vol 187 (17) ◽  
pp. 6094-6105 ◽  
Author(s):  
Urszula Zielenkiewicz ◽  
Piotr Cegłowski
Keyword(s):  
Streptococcus Pyogenes ◽  
Copy Number ◽  
Bacterial Species ◽  
Erythromycin Resistance ◽  
Gram Positive ◽  
E Coli ◽  
Gram Positive Bacteria ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Function Expression

ABSTRACT pSM19035 of the pathogenic bacterium Streptococcus pyogenes is a low-copy-number plasmid carrying erythromycin resistance, stably maintained in a broad range of gram-positive bacteria. We show here that the ω-ε-ζ operon of this plasmid constitutes a novel proteic plasmid addiction system in which the ε and ζ genes encode an antitoxin and toxin, respectively, while ω plays an autoregulatory function. Expression of toxin Zeta is bactericidal for the gram-positive Bacillus subtilis and bacteriostatic for the gram-negative Escherichia coli. The toxic effects of ζ gene expression in both bacterial species are counteracted by proper expression of ε. The ε-ζ toxin-antitoxin cassette stabilizes plasmids in E. coli less efficiently than in B. subtilis.

Complete sequence of low-copy-number plasmid MccC7-H22 of probiotic Escherichia coli H22 and the prevalence of mcc genes among human E. coli

Plasmid ◽  
2008 ◽  
Vol 59 (1) ◽  
pp. 1-10 ◽  
Author(s):  
David Šmajs ◽  
Michal Strouhal ◽  
Petra Matějková ◽  
Darina Čejková ◽  
Luciana Cursino ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Copy Number ◽  
Complete Sequence ◽  
E Coli ◽  
Copy Number Plasmid ◽  
Low Copy Number

Existence and Decontamination of HVC, Infectious Enteric Bacteria and Parasites in Sewaged Soils

2014 ◽  
Vol 3 (1) ◽  
pp. 150-158 ◽  
Author(s):  
Mohey A. Hassanain ◽  
Nawal A. Hassanain ◽  
Esam A. Hobballa ◽  
Fatma H. Abd- El Zaher ◽  
Mohamed Saber M. Saber
Keyword(s):  
Soil Sample ◽  
Potassium Permanganate ◽  
Enteric Bacteria ◽  
Sewage Effluent ◽  
Calcium Hypochlorite ◽  
Sand Soil ◽  
E Coli ◽  
Surface Sample ◽  
Loamy Sand Soil ◽  
Sewage Farm

A surface sample representing a high contaminated loamy sand soil irrigated with sewage effluent since 30 years and was cultivated with artichoke was collected from Abu-Rawash sewage farm. The existence of HVC, enteric infectious bacteria and parasites in sewaged soil found to be negative for the forward and positive for the latter's. Out of the 30 samples separated from the sewaged soil sample, only 3 samples contained parasitic fauna of developed and undeveloped Ascaris (10%) and five samples contained Entamoeba coli. Results showed that the number of Ascaris eggs/gm soil was 0.017 and the number of E. coli/gm was 0.26. Decontamination of soil parasites was effective using either calcium hypochlorite or potassium permanganate. Salmonella, Vibrio and Campelobacter were detected in the high contaminated sewaged soil and survived for 120 days in the sewaged soil under all control and bioremediated treatments irrigated with either sewage effluent or water.

Use of gene probes for the detection of quiescent enteric bacteria in marine and fresh waters

1995 ◽  
Vol 31 (5-6) ◽  
pp. 291-298
Author(s):  
Sally A. Anderson ◽  
Gillian D. Lewis ◽  
Michael N. Pearson
Keyword(s):  
Membrane Filtration ◽  
Enteric Bacteria ◽  
Probe Method ◽  
Detection Methods ◽  
23S Rrna ◽  
Specific Gene ◽  
Gene Probe ◽  
Selective Media ◽  
Selective Enrichment ◽  
E Coli

Specific gene probe detection methods that utilise a non-selective culturing step were tested for the ability to recognise the presence of quiescent enteric bacteria (Escherichia coli and Enterococcus faecalis ) within illuminated freshwater and seawater microcosms. An E. coli specific uidA gene probe and a 23S rRNA oligonucleotide probe for Enterococci were compared with recoveries using membrane filtration and incubation on selective media (mTEC and mE respectively). From these microcosm experiments a greater initial detection (from 4 hours to 1 day) of E. coli and Ent. faecalis using gene probe methods was observed. Additionally, a comparison of E. coli direct viable counts (DVC) in sunlight exposed microcosms with recoveries by selective media and gene probe methods revealed a large number of viable non-culturable cells. This suggests that enumeration of E. coli by a gene probe method is limited by the replication of the bacteria during the initial non-selective enrichment step. The detection of stressed Ent. faecalis by the oligonucleotide gene probe method was significantly greater than recovery on selective mE agar, indicating an Enterococci non-growth phase.

scholarly journals The Location of Substitutions and Bacterial Genome Arrangements

2020 ◽  
Author(s):  
Daniella F Lato ◽  
G Brian Golding
Keyword(s):  
Sinorhizobium Meliloti ◽  
Bacterial Genome ◽  
Evolutionary Analysis ◽  
Origin Of Replication ◽  
Ancestral Reconstruction ◽  
Molecular Change ◽  
E Coli ◽  
A Genome ◽  
The Impact ◽  
Molecular Evolutionary Analysis

Abstract Increasing evidence supports the notion that different regions of a genome have unique rates of molecular change. This variation is particularly evident in bacterial genomes where previous studies have reported gene expression and essentiality tend to decrease, while substitution rates usually increases with increasing distance from the origin of replication. Genomic reorganization such as rearrangements occur frequently in bacteria and allow for the introduction and restructuring of genetic content, creating gradients of molecular traits along genomes. Here, we explore the interplay of these phenomena by mapping substitutions to the genomes of Escherichia coli, Bacillus subtilis, Streptomyces, and Sinorhizobium meliloti, quantifying how many substitutions have occurred at each position in the genome. Preceding work indicates that substitution rate significantly increases with distance from the origin. Using a larger sample size and accounting for genome rearrangements through ancestral reconstruction, our analysis demonstrates that the correlation between the number of substitutions and distance from the origin of replication is often significant but small and inconsistent in direction. Some replicons had a significantly decreasing trend (E. coli and the chromosome of S. meliloti), while others showed the opposite significant trend (B. subtilis, Streptomyces, pSymA and pSymB in S. meliloti). dN, dS and ω were examined across all genes and there was no significant correlation between those values and distance from the origin. This study highlights the impact that genomic rearrangements and location have on molecular trends in some bacteria, illustrating the importance of considering spatial trends in molecular evolutionary analysis. Assuming that molecular trends are exclusively in one direction can be problematic.

scholarly journals Lowering DNA binding affinity of SssI DNA methyltransferase does not enhance the specificity of targeted DNA methylation in E. coli

2021 ◽  
Vol 11 (1) ◽  
Author(s):  
Krystyna Ślaska-Kiss ◽  
Nikolett Zsibrita ◽  
Mihály Koncz ◽  
Pál Albert ◽  
Ákos Csábrádi ◽  
...  
Keyword(s):  
Dna Methylation ◽  
Dna Binding ◽  
Binding Affinity ◽  
Dna Methyltransferase ◽  
Restriction Enzymes ◽  
Dna Binding Protein ◽  
E Coli ◽  
Dna Binding Affinity ◽  
Higher Eukaryotes

AbstractTargeted DNA methylation is a technique that aims to methylate cytosines in selected genomic loci. In the most widely used approach a CG-specific DNA methyltransferase (MTase) is fused to a sequence specific DNA binding protein, which binds in the vicinity of the targeted CG site(s). Although the technique has high potential for studying the role of DNA methylation in higher eukaryotes, its usefulness is hampered by insufficient methylation specificity. One of the approaches proposed to suppress methylation at unwanted sites is to use MTase variants with reduced DNA binding affinity. In this work we investigated how methylation specificity of chimeric MTases containing variants of the CG-specific prokaryotic MTase M.SssI fused to zinc finger or dCas9 targeting domains is influenced by mutations affecting catalytic activity and/or DNA binding affinity of the MTase domain. Specificity of targeted DNA methylation was assayed in E. coli harboring a plasmid with the target site. Digestions of the isolated plasmids with methylation sensitive restriction enzymes revealed that specificity of targeted DNA methylation was dependent on the activity but not on the DNA binding affinity of the MTase. These results have implications for the design of strategies of targeted DNA methylation.

scholarly journals Backbone assignments, and effect of Asn deamidation, of the N-terminal region of the partitioning protein IncC1 from the plasmid RK2

2021 ◽  
Author(s):  
M. Fayyaz Rehman ◽  
M. Jeeves ◽  
E. I. Hyde
Keyword(s):  
Amino Acids ◽  
Chemical Shift Assignments ◽  
Backbone Assignments ◽  
Intrinsically Disordered ◽  
Nmr Chemical Shift Assignments ◽  
Protein Partner ◽  
Copy Number Plasmid ◽  
Low Copy Number ◽  
Plasmid Rk2

AbstractIncC from the low-copy number plasmid RK2, is a member of the ParA family of proteins required for partitioning DNA in many bacteria and plasmids. It is an ATPase that binds DNA and its ParB protein partner, KorB. Together, the proteins move replicated DNA to appropriate cellular positions, so that each daughter cell inherits a copy on cell division. IncC from RK2 is expressed in two forms. IncC2 is homologous to bacterial ParA proteins, while IncC1 has an N-terminal extension of 105 amino acids and is similar in length to ParA homologues in other plasmids. We have been examining the role of this extension, here called IncC NTD. We present its backbone NMR chemical shift assignments and show that it is entirely intrinsically disordered. The assignments were achieved using C-detected, CON-based spectra, complemented by HNN spectra to obtain connectivities from three adjacent amino acids. We also observed evidence of deamidation of the protein at a GNGG sequence, to give isoAsp, giving 2 sets of peaks for residues up to 5 amino acids on either side of the modification. We have assigned resonances from around the position of modification for this form of the protein.

scholarly journals Chromosomal Lesion Suppression and Removal in Escherichia coli via Linear DNA Degradation

Genetics ◽  
2003 ◽  
Vol 163 (4) ◽  
pp. 1255-1271 ◽  
Author(s):  
Anabel Miranda ◽  
Andrei Kuzminov
Keyword(s):  
Dna Degradation ◽  
Dna Helicase ◽  
Structural Element ◽  
E Coli ◽  
Linear Dna ◽  
Replication Factory ◽  
Low Copy Number ◽  
Chromosomal Lesion ◽  
Recbcd Enzyme

Abstract RecBCD is a DNA helicase/exonuclease implicated in degradation of foreign linear DNA and in RecA-dependent recombinational repair of chromosomal lesions in E. coli. The low viability of recA recBC mutants vs. recA mutants indicates the existence of RecA-independent roles for RecBCD. To distinguish among possible RecA-independent roles of the RecBCD enzyme in replication, repair, and DNA degradation, we introduced wild-type and mutant combinations of the recBCD chromosomal region on a low-copy-number plasmid into a ΔrecA ΔrecBCD mutant and determined the viability of resulting strains. Our results argue against ideas that RecBCD is a structural element in the replication factory or is involved in RecA-independent repair of chromosomal lesions. We found that RecBCD-catalyzed DNA degradation is the only activity important for the recA-independent viability, suggesting that degradation of linear tails of σ-replicating chromosomes could be one of the RecBCD’s roles. However, since the weaker DNA degradation capacity due a combination of the RecBC helicase and ssDNA-specific exonucleases restores viability of the ΔrecA ΔrecBCD mutant to a significant extent, we favor suppression of chromosomal lesions via linear DNA degradation at reversed replication forks as the major RecA-independent role of the RecBCD enzyme.

scholarly journals Global versus Local Regulatory Roles for Lrp-Related Proteins: Haemophilus influenzae as a Case Study

2001 ◽  
Vol 183 (13) ◽  
pp. 4004-4011 ◽  
Author(s):  
Devorah Friedberg ◽  
Michael Midkiff ◽  
Joseph M. Calvo
Keyword(s):  
Amino Acid ◽  
Haemophilus Influenzae ◽  
Regulatory Protein ◽  
Enteric Bacteria ◽  
Regulatory Role ◽  
Regulatory Function ◽  
Ecological Niches ◽  
E Coli ◽  
Sequence Identity ◽  
Related Proteins

ABSTRACT Lrp (leucine-responsive regulatory protein) plays a global regulatory role in Escherichia coli, affecting expression of dozens of operons. Numerous lrp-related genes have been identified in different bacteria and archaea, includingasnC, an E. coli gene that was the first reported member of this family. Pairwise comparisons of amino acid sequences of the corresponding proteins shows an average sequence identity of only 29% for the vast majority of comparisons. By contrast, Lrp-related proteins from enteric bacteria show more than 97% amino acid identity. Is the global regulatory role associated withE. coli Lrp limited to enteric bacteria? To probe this question we investigated LrfB, an Lrp-related protein fromHaemophilus influenzae that shares 75% sequence identity with E. coli Lrp (highest sequence identity among 42 sequences compared). A strain of H. influenzae having anlrfB null allele grew at the wild-type growth rate but with a filamentous morphology. A comparison of two-dimensional (2D) electrophoretic patterns of proteins from parent and mutant strains showed only two differences (comparable studies withlrp + and lrp E. coli strains by others showed 20 differences). The abundance of LrfB in H. influenzae, estimated by Western blotting experiments, was about 130 dimers per cell (compared to 3,000 dimers per E. colicell). LrfB expressed in E. coli replaced Lrp as a repressor of the lrp gene but acted only to a limited extent as an activator of the ilvIH operon. Thus, although LrfB resembles Lrp sufficiently to perform some of its functions, its low abundance is consonant with a more local role in regulating but a few genes, a view consistent with the results of the 2D electrophoretic analysis. We speculate that an Lrp having a global regulatory role evolved to help enteric bacteria adapt to their ecological niches and that it is unlikely that Lrp-related proteins in other organisms have a broad regulatory function.

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