scholarly journals The Symmetrical Wave Pattern of Base-Pair Substitution Rates across theEscherichia coliChromosome Has Multiple Causes

mBio ◽  
2019 ◽  
Vol 10 (4) ◽  
Author(s):  
Brittany A. Niccum ◽  
Heewook Lee ◽  
Wazim MohammedIsmail ◽  
Haixu Tang ◽  
Patricia L. Foster
Keyword(s):  
Escherichia Coli ◽  
Error Correction ◽  
Base Pair ◽  
Three Dimensional ◽  
Wave Pattern ◽  
Mutation Rates ◽  
Dimensional Structure ◽  
Origin Of Replication ◽  
Content Type ◽  
Eukaryotic Genomes

ABSTRACTMutation accumulation experiments followed by whole-genome sequencing have revealed that, for several bacterial species, the rate of base-pair substitutions (BPSs) is not constant across the chromosome but varies in a wave-like pattern that is symmetrical about the origin of replication. The experiments reported here demonstrated that, inEscherichia coli, several interacting factors determine the wave. The origin is a major driver of BPS rates. When it is relocated, the BPS rates in a 1,000-kb region surrounding the new origin reproduce the pattern that surrounds the normal origin. However, the pattern across distant regions of the chromosome is unaltered and thus must be determined by other factors. Increasing the deoxynucleoside triphosphate (dNTP) concentration shifts the wave pattern away from the origin, supporting the hypothesis that fluctuations in dNTP pools coincident with replication firing contribute to the variations in the mutation rate. The nucleoid binding proteins (HU and Fis) and the terminus organizing protein (MatP) are also major factors. These proteins alter the three-dimensional structure of the DNA, and results suggest that mutation rates increase when highly structured DNA is replicated. Biases in error correction by proofreading and mismatch repair, both of which may be responsive to dNTP concentrations and DNA structure, also are major determinants of the wave pattern. These factors should apply to most bacterial and, possibly, eukaryotic genomes and suggest that different areas of the genome evolve at different rates.IMPORTANCEIt has been found in several species of bacteria that the rate at which single base pairs are mutated is not constant across the genome but varies in a wave-like pattern that is symmetrical about the origin of replication. UsingEscherichia colias our model system, we show that this pattern is the result of several interconnected factors. First, the timing and progression of replication are important in determining the wave pattern. Second, the three-dimensional structure of the DNA is also a factor, and the results suggest that mutation rates increase when highly structured DNA is replicated. Finally, biases in error correction, which may be responsive both to the progression of DNA synthesis and to DNA structure, are major determinants of the wave pattern. These factors should apply to most bacterial and, possibly, eukaryotic genomes and suggest that different areas of the genome evolve at different rates.

scholarly journals The symmetrical pattern of base-pair substitutions rates across the chromosome in Escherichia coli has multiple causes

2019 ◽  
Author(s):  
Brittany A. Niccum ◽  
Heewook Lee ◽  
Wazim MohammedIsmail ◽  
Haixu Tang ◽  
Patricia L. Foster
Keyword(s):  
Escherichia Coli ◽  
Base Pair ◽  
Bacterial Species ◽  
Replication Timing ◽  
Mutation Rates ◽  
Whole Genome ◽  
Origin Of Replication ◽  
Symmetrical Pattern ◽  
Major Factors ◽  
Eukaryotic Genomes

AbstractMutation accumulation experiments followed by whole-genome sequencing have revealed that for several bacterial species the rate of base-pair substitutions is not constant across the chromosome but varies in a wave-like pattern symmetrical about the origin of replication. The experiments reported here demonstrate that in Escherichia coli several interacting factors determine the wave. Perturbing replication timing, progression, or the structure of the terminus disrupts the pattern. Biases in error-correction by proofreading and mismatch repair are major factors. The activities of the nucleoid binding proteins, HU and Fis, are important, suggesting that mutation rates increase when highly structured DNA is replicated. These factors should apply to most bacterial, and possibly eukaryotic, genomes, and imply that different areas of the genome evolve at different rates.

scholarly journals PBP1B Glycosyltransferase and Transpeptidase Activities Play Different Essential Roles during the De Novo Regeneration of Rod Morphology in Escherichia coli

2017 ◽  
Vol 199 (7) ◽  
Author(s):  
Dev K. Ranjit ◽  
Matthew A. Jorgenson ◽  
Kevin D. Young
Keyword(s):  
Escherichia Coli ◽  
Cell Wall ◽  
Cell Walls ◽  
De Novo ◽  
Spatial Interaction ◽  
Three Dimensional ◽  
Recovery Process ◽  
Dimensional Structure ◽  
Content Type ◽  
Specific Shape

ABSTRACT Peptidoglycan is a vital component of nearly all cell wall-bearing bacteria and is a valuable target for antibacterial therapy. However, despite decades of work, there remain important gaps in understanding how this macromolecule is synthesized and molded into a three-dimensional structure that imparts specific morphologies to individual cells. Here, we investigated the particularly enigmatic area of how peptidoglycan is synthesized and shaped during the first stages of creating cell shape de novo, that is, in the absence of a preexisting template. We found that when lysozyme-induced (LI) spheroplasts of Escherichia coli were allowed to resynthesize peptidoglycan, the cells divided first and then elongated to recreate a normal rod-shaped morphology. Penicillin binding protein 1B (PBP1B) was critical for the first stage of this recovery process. PBP1B synthesized peptidoglycan de novo, and this synthesis required that PBP1B interact with the outer membrane lipoprotein LpoB. Surprisingly, when LpoB was localized improperly to the inner membrane, recovering spheroplasts synthesized peptidoglycan and divided but then propagated as amorphous spheroidal cells, suggesting that the regeneration of a normal rod shape depends on a particular spatial interaction. Similarly, spheroplasts carrying a PBP1B variant lacking transpeptidase activity or those in which PBP1A was overproduced could synthesize new peptidoglycan and divide but then grew as oddly shaped spheroids. We conclude that de novo cell wall synthesis requires the glycosyltransferase activity of PBP1B but that PBP1B transpeptidase activity is needed to assemble cell walls with wild-type morphology. IMPORTANCE Bacterial cell wall peptidoglycan is synthesized and modified by penicillin binding proteins (PBPs), which are targeted by about half of all currently prescribed antibiotics, including penicillin and its derivatives. Because antibiotic resistance is rising, it has become increasingly urgent that we fill the gaps in our knowledge about how PBPs create and assemble this protective wall. We report here that PBP1B plays an essential role in synthesizing peptidoglycan in the absence of a preexisting template: its glycosyltransferase activity is responsible for de novo synthesis, while its transpeptidase activity is required to construct cell walls of a specific shape. These results highlight the importance of this enzyme and distinguish its biological roles from those of other PBPs and peptidoglycan synthases.

scholarly journals An Intramolecular Salt Bridge in Bacillus thuringiensis Cry4Ba Toxin Is Involved in the Stability of Helix α-3, Which Is Needed for Oligomerization and Insecticidal Activity

2017 ◽  
Vol 83 (20) ◽  
Author(s):  
Sabino Pacheco ◽  
Isabel Gómez ◽  
Jorge Sánchez ◽  
Blanca-Ines García-Gómez ◽  
Mario Soberón ◽  
...  
Keyword(s):  
Bacillus Thuringiensis ◽  
Double Point ◽  
Single Point ◽  
Three Dimensional ◽  
Point Mutations ◽  
Salt Bridge ◽  
Dimensional Structure ◽  
Cry Toxins ◽  
Content Type ◽  
Domain I

ABSTRACT Bacillus thuringiensis three-domain Cry toxins kill insects by forming pores in the apical membrane of larval midgut cells. Oligomerization of the toxin is an important step for pore formation. Domain I helix α-3 participates in toxin oligomerization. Here we identify an intramolecular salt bridge within helix α-3 of Cry4Ba (D111-K115) that is conserved in many members of the family of three-domain Cry toxins. Single point mutations such as D111K or K115D resulted in proteins severely affected in toxicity. These mutants were also altered in oligomerization, and the mutant K115D was more sensitive to protease digestion. The double point mutant with reversed charges, D111K-K115D, recovered both oligomerization and toxicity, suggesting that this salt bridge is highly important for conservation of the structure of helix α-3 and necessary to promote the correct oligomerization of the toxin. IMPORTANCE Domain I has been shown to be involved in oligomerization through helix α-3 in different Cry toxins, and mutations affecting oligomerization also elicit changes in toxicity. The three-dimensional structure of the Cry4Ba toxin reveals an intramolecular salt bridge in helix α-3 of domain I. Mutations that disrupt this salt bridge resulted in changes in Cry4Ba oligomerization and toxicity, while a double point reciprocal mutation that restored the salt bridge resulted in recovery of toxin oligomerization and toxicity. These data highlight the role of oligomer formation as a key step in Cry4Ba toxicity.

scholarly journals DUF3380 Domain from a Salmonella Phage Endolysin Shows PotentN-Acetylmuramidase Activity

2016 ◽  
Vol 82 (16) ◽  
pp. 4975-4981 ◽  
Author(s):  
Lorena Rodríguez-Rubio ◽  
Hans Gerstmans ◽  
Simon Thorpe ◽  
Stéphane Mesnage ◽  
Rob Lavigne ◽  
...  
Keyword(s):  
Enzymatic Activity ◽  
Thermal Resistance ◽  
High Performance ◽  
Antimicrobial Agents ◽  
Biochemical Characterization ◽  
Specific Activity ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Gram Negative ◽  
Content Type

ABSTRACTBacteriophage-encoded endolysins are highly diverse enzymes that cleave the bacterial peptidoglycan layer. Current research focuses on their potential applications in medicine, in food conservation, and as biotechnological tools. Despite the wealth of applications relying on the use of endolysin, little is known about the enzymatic properties of these enzymes, especially in the case of endolysins of bacteriophages infecting Gram-negative species. Automated genome annotations therefore remain to be confirmed. Here, we report the biochemical analysis and cleavage site determination of a novelSalmonellabacteriophage endolysin, Gp110, which comprises an uncharacterizeddomain ofunknownfunction (DUF3380; pfam11860) in its C terminus and shows a higher specific activity (34,240 U/μM) than that of 14 previously characterized endolysins active against peptidoglycan from Gram-negative bacteria (corresponding to 1.7- to 364-fold higher activity). Gp110 is a modular endolysin with an optimal pH of enzymatic activity of pH 8 and elevated thermal resistance. Reverse-phase high-performance liquid chromatography (RP-HPLC) analysis coupled to mass spectrometry showed that DUF3380 hasN-acetylmuramidase (lysozyme) activity cleaving the β-(1,4) glycosidic bond betweenN-acetylmuramic acid andN-acetylglucosamine residues. Gp110 is active against directly cross-linked peptidoglycans with various peptide stem compositions, making it an attractive enzyme for developing novel antimicrobial agents.IMPORTANCEWe report the functional and biochemical characterization of theSalmonellaphage endolysin Gp110. This endolysin has a modular structure with an enzymatically active domain and a cell wall binding domain. The enzymatic activity of this endolysin exceeds that of all other endolysins previously characterized using the same methods. A domain of unknown function (DUF3380) is responsible for this high enzymatic activity. We report that DUF3380 hasN-acetylmuramidase activity against directly cross-linked peptidoglycans with various peptide stem compositions. This experimentally verified activity allows better classification and understanding of the enzymatic activities of endolysins, which mostly are inferred by sequence similarities. Three-dimensional structure predictions for Gp110 suggest a fold that is completely different from that of known structures of enzymes with the same peptidoglycan cleavage specificity, making this endolysin quite unique. All of these features, combined with increased thermal resistance, make Gp110 an attractive candidate for engineering novel endolysin-based antibacterials.

Hospitality situations, consumer expertise, and perceptions of wine attributes: three empirical studies

2019 ◽  
Vol 31 (1) ◽  
pp. 68-88 ◽  
Author(s):  
Dale F. Duhan ◽  
Shannon B. Rinaldo ◽  
Natalia Velikova ◽  
Tim Dodd ◽  
Brent Trela
Keyword(s):  
Empirical Studies ◽  
Three Dimensional ◽  
Total Sample ◽  
Dimensional Structure ◽  
Product Knowledge ◽  
Product Attributes ◽  
Content Type ◽  
Wine Consumption ◽  
Consumer Expertise ◽  
Academic Researchers

PurposeWine choices are not always fully understood by academic researchers or the industry. This paper aims to outline and test a theoretical model proposing that wine consumption may be dependent on differences in consumer expertise, the hospitality situation, characteristics of the wine itself and an interaction of these variables.Design/methodology/approachThree empirical studies (total sample size = 356) tested these theoretical propositions. Consumers with varying levels of wine knowledge were presented with experimental vignettes showing videos of wine opening and pouring and were asked to pair wines with hospitality situations.FindingsStudy 1 found that consumers with low product knowledge were more sensitive to hospitality situations and extrinsic product attributes (closures) than were the experts. Study 2 found that wine hospitality situations fall into three predicted categories, namely, food, friends and formality, although contrary to prediction, the presence of food was the weakest predictors. Study 3 demonstrated the robustness of the three-dimensional structure of wine hospitality situations.Practical implicationsThese studies provided important practical information because targeting various market segments requires the industry to know what product attributes are favored by different groups of consumers different situations.Originality/valuePrevious researchers have discussed the difficulty of measuring consumption situations. By limiting these studies to wine consumption within hospitality situations, the authors learned much about how consumers’ characteristics, product attributes and the situations interact to influence not only product assessments but also choices.

Sequence-specific1H n.m.r. assignments and determination of the three-dimensional structure of reduced escherichia coli glutaredoxin

1991 ◽  
Vol 221 (4) ◽  
pp. 1311-1324 ◽  
Author(s):  
Patrick Sodano ◽  
Tai-he Xia ◽  
John H. Bushweller ◽  
Olof Björnberg ◽  
Arne Holmgren ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Three Dimensional Structure

Three-Dimensional Structure of Escherichia coli Dihydrodipicolinate Reductase

Biochemistry ◽  
1995 ◽  
Vol 34 (11) ◽  
pp. 3502-3512 ◽  
Author(s):  
Giovanna Scapin ◽  
John S. Blanchard ◽  
James C. Sacchettini
Keyword(s):  
Escherichia Coli ◽  
Three Dimensional ◽  
Dimensional Structure ◽  
Three Dimensional Structure

scholarly journals Membrane Protein Complex ExbB4-ExbD1-TonB1from Escherichia coli Demonstrates Conformational Plasticity

2015 ◽  
Vol 197 (11) ◽  
pp. 1873-1885 ◽  
Author(s):  
Aleksandr Sverzhinsky ◽  
Jacqueline W. Chung ◽  
Justin C. Deme ◽  
Lucien Fabre ◽  
Kristian T. Levey ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Iron Acquisition ◽  
Proton Translocation ◽  
Three Dimensional ◽  
Structural Data ◽  
Structural Features ◽  
Gram Negative Bacteria ◽  
Gram Negative ◽  
Content Type

ABSTRACTIron acquisition at the outer membrane (OM) of Gram-negative bacteria is powered by the proton motive force (PMF) of the cytoplasmic membrane (CM), harnessed by the CM-embedded complex of ExbB, ExbD, and TonB. Its stoichiometry, ensemble structural features, and mechanism of action are unknown. By panning combinatorial phage libraries, periplasmic regions of dimerization between ExbD and TonB were predicted. Using overexpression of full-length His6-taggedexbB-exbDand S-taggedtonB, we purified detergent-solubilized complexes of ExbB-ExbD-TonB fromEscherichia coli. Protein-detergent complexes of ∼230 kDa with a hydrodynamic radius of ∼6.0 nm were similar to previously purified ExbB4-ExbD2complexes. Significantly, they differed in electronegativity by native agarose gel electrophoresis. The stoichiometry was determined to be ExbB4-ExbD1-TonB1. Single-particle electron microscopy agrees with this stoichiometry. Two-dimensional averaging supported the phage display predictions, showing two forms of ExbD-TonB periplasmic heterodimerization: extensive and distal. Three-dimensional (3D) particle classification showed three representative conformations of ExbB4-ExbD1-TonB1. Based on our structural data, we propose a model in which ExbD shuttles a proton across the CM via an ExbB interprotein rearrangement. Proton translocation would be coupled to ExbD-mediated collapse of extended TonB in complex with ligand-loaded receptors in the OM, followed by repositioning of TonB through extensive dimerization with ExbD. Here we present the first report for purification of the ExbB-ExbD-TonB complex, molar ratios within the complex (4:1:1), and structural biology that provides insights into 3D organization.IMPORTANCEReceptors in the OM of Gram-negative bacteria allow entry of iron-bound siderophores that are necessary for pathogenicity. Numerous iron-acquisition strategies rely upon a ubiquitous and unique protein for energization: TonB. Complexed with ExbB and ExbD, the Ton system links the PMF to OM transport. Blocking iron uptake by targeting a vital nanomachine holds promise in therapeutics. Despite much research, the stoichiometry, structural arrangement, and molecular mechanism of the CM-embedded ExbB-ExbD-TonB complex remain unreported. Here we demonstratein vitroevidence of ExbB4-ExbD1-TonB1complexes. Using 3D EM, we reconstructed the complex in three conformational states that show variable ExbD-TonB heterodimerization. Our structural observations form the basis of a model for TonB-mediated iron acquisition.

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