scholarly journals Dynamic landscape of protein occupancy across the Escherichia coli chromosome

2020 ◽  
Author(s):  
Peter L. Freddolino ◽  
Thomas J. Goss ◽  
Haley M. Amemiya ◽  
Saeed Tavazoie
Keyword(s):  
Genetic Manipulation ◽  
Dynamic Condition ◽  
Bacterial Genome ◽  
Structural Features ◽  
Sequence Specificity ◽  
Bacterial Physiology ◽  
E Coli ◽  
Transcriptional Regulatory ◽  
Protein Occupancy

AbstractFree living bacteria adapt to environmental change by reprogramming gene expression through precise interactions of hundreds of DNA-binding proteins. A predictive understanding of bacterial physiology requires us to globally monitor all such protein-DNA interactions across a range of environmental and genetic perturbations. Here, we show that such global observations are possible using a modification of in vivo protein occupancy display technology (IPOD-HR) applied to E. coli. We observe that the E. coli protein-DNA interactome organizes into two distinct prototypic features: (1) highly dynamic condition-dependent transcription factor occupancy by dedicated transcriptional regulators, and (2) condition-invariant kilobase scale occupancy by nucleoid factors, forming silencing domains analogous to eukaryotic heterochromatin. We show that occupancy dynamics across a range of conditions can rapidly reveal the global transcriptional regulatory organization of a bacterium. Beyond discovery of previously hidden regulatory logic, we show that these observations can be utilized to computationally determine sequence-specificity models for the majority of active transcription factors. Our study demonstrates that global observations of protein occupancy combined with statistical inference can rapidly and systematically reveal the transcriptional regulatory and structural features of a bacterial genome. This capacity is particularly crucial for non-model bacteria which are not amenable to routine genetic manipulation.

scholarly journals Dynamic landscape of protein occupancy across the Escherichia coli chromosome

PLoS Biology ◽  
2021 ◽  
Vol 19 (6) ◽  
pp. e3001306
Author(s):  
Peter L. Freddolino ◽  
Haley M. Amemiya ◽  
Thomas J. Goss ◽  
Saeed Tavazoie
Keyword(s):  
Escherichia Coli ◽  
Genetic Manipulation ◽  
Dynamic Condition ◽  
Bacterial Genome ◽  
Structural Features ◽  
Sequence Specificity ◽  
Bacterial Physiology ◽  
Transcriptional Regulatory ◽  
Protein Occupancy

Free-living bacteria adapt to environmental change by reprogramming gene expression through precise interactions of hundreds of DNA-binding proteins. A predictive understanding of bacterial physiology requires us to globally monitor all such protein–DNA interactions across a range of environmental and genetic perturbations. Here, we show that such global observations are possible using an optimized version of in vivo protein occupancy display technology (in vivo protein occupancy display—high resolution, IPOD-HR) and present a pilot application to Escherichia coli. We observe that the E. coli protein–DNA interactome organizes into 2 distinct prototypic features: (1) highly dynamic condition-dependent transcription factor (TF) occupancy; and (2) robust kilobase scale occupancy by nucleoid factors, forming silencing domains analogous to eukaryotic heterochromatin. We show that occupancy dynamics across a range of conditions can rapidly reveal the global transcriptional regulatory organization of a bacterium. Beyond discovery of previously hidden regulatory logic, we show that these observations can be utilized to computationally determine sequence specificity models for the majority of active TFs. Our study demonstrates that global observations of protein occupancy combined with statistical inference can rapidly and systematically reveal the transcriptional regulatory and structural features of a bacterial genome. This capacity is particularly crucial for non-model bacteria that are not amenable to routine genetic manipulation.

scholarly journals Targeted Transposition by the V(D)J Recombinase

2002 ◽  
Vol 22 (7) ◽  
pp. 2068-2077 ◽  
Author(s):  
Gregory S. Lee ◽  
Matthew B. Neiditch ◽  
Richard R. Sinden ◽  
David B. Roth
Keyword(s):  
Signal Sequence ◽  
Target Selection ◽  
Structural Features ◽  
Sequence Specificity ◽  
Recombination Signal ◽  
Chromosome Translocations ◽  
Hybrid Joints ◽  
Sequence Elements ◽  
Rag Proteins

ABSTRACT Cleavage by the V(D)J recombinase at a pair of recombination signal sequences creates two coding ends and two signal ends. The RAG proteins can integrate these signal ends, without sequence specificity, into an unrelated target DNA molecule. Here we demonstrate that such transposition events are greatly stimulated by—and specifically targeted to—hairpins and other distorted DNA structures. The mechanism of target selection by the RAG proteins thus appears to involve recognition of distorted DNA. These data also suggest a novel mechanism for the formation of alternative recombination products termed hybrid joints, in which a signal end is joined to a hairpin coding end. We suggest that hybrid joints may arise by transposition in vivo and propose a new model to account for some recurrent chromosome translocations found in human lymphomas. According to this model, transposition can join antigen receptor loci to partner sites that lack recombination signal sequence elements but bear particular structural features. The RAG proteins are capable of mediating all necessary breakage and joining events on both partner chromosomes; thus, the V(D)J recombinase may be far more culpable for oncogenic translocations than has been suspected.

scholarly journals Genetic Manipulation of Prochlorococcus Strain MIT9313: Green Fluorescent Protein Expression from an RSF1010 Plasmid and Tn5 Transposition

2006 ◽  
Vol 72 (12) ◽  
pp. 7607-7613 ◽  
Author(s):  
Andrew C. Tolonen ◽  
Gregory B. Liszt ◽  
Wolfgang R. Hess
Keyword(s):  
Green Fluorescent Protein ◽  
Fluorescent Protein ◽  
Genetic Manipulation ◽  
E Coli ◽  
Phage T7 ◽  
Ecological Importance ◽  
Green Fluorescent ◽  
Oceanic Gyres ◽  
Global Carbon

ABSTRACT Prochlorococcus is the smallest oxygenic phototroph yet described. It numerically dominates the phytoplankton community in the mid-latitude oceanic gyres, where it has an important role in the global carbon cycle. The complete genomes of several Prochlorococcus strains have been sequenced, revealing that nearly half of the genes in each genome are of unknown function. Genetic methods, such as reporter gene assays and tagged mutagenesis, are critical to unveiling the functions of these genes. Here, we describe conditions for the transfer of plasmid DNA into Prochlorococcus strain MIT9313 by interspecific conjugation with Escherichia coli. Following conjugation, E. coli bacteria were removed from the Prochlorococcus cultures by infection with E. coli phage T7. We applied these methods to show that an RSF1010-derived plasmid will replicate in Prochlorococcus strain MIT9313. When this plasmid was modified to contain green fluorescent protein, we detected its expression in Prochlorococcus by Western blotting and cellular fluorescence. Further, we applied these conjugation methods to show that a mini-Tn5 transposon will transpose in vivo in Prochlorococcus. These genetic advances provide a basis for future genetic studies with Prochlorococcus, a microbe of ecological importance in the world's oceans.

scholarly journals Genetic Footprinting in Bacteria

2001 ◽  
Vol 183 (5) ◽  
pp. 1694-1706 ◽  
Author(s):  
Roberta S. Hare ◽  
Scott S. Walker ◽  
Thomas E. Dorman ◽  
Jonathan R. Greene ◽  
Luz-Maria Guzman ◽  
...  
Keyword(s):  
Recombinant Proteins ◽  
High Frequency ◽  
Growth Conditions ◽  
Sequence Specificity ◽  
Bacteriophage Λ ◽  
E Coli ◽  
Genetic Footprinting ◽  
Transposon Insertions ◽  
Selection Of

ABSTRACT In vivo genetic footprinting was developed in the yeastSaccharomyces cerevisiae to simultaneously assess the importance of thousands of genes for the fitness of the cell under any growth condition. We have developed in vivo genetic footprinting forEscherichia coli, a model bacterium and pathogen. We further demonstrate the utility of this technology for rapidly discovering genes that affect the fitness of E. coli under a variety of growth conditions. The definitive features of this system include a conditionally regulated Tn10 transposase with relaxed sequence specificity and a conditionally regulated replicon for the vector containing the transposase and mini-Tn10transposon with an outwardly oriented promoter. This system results in a high frequency of randomly distributed transposon insertions, eliminating the need for the selection of a population containing transposon insertions, stringent suppression of transposon mutagenesis, and few polar effects. Successful footprints have been achieved for most genes longer than 400 bp, including genes located in operons. In addition, the ability of recombinant proteins to complement mutagenized hosts has been evaluated by genetic footprinting using a bacteriophage λ transposon delivery system.

scholarly journals Comparison of Escherichia coli surface attachment methods for single-cell microscopy

2019 ◽  
Vol 9 (1) ◽  
Author(s):  
Yao-Kuan Wang ◽  
Ekaterina Krasnopeeva ◽  
Ssu-Yuan Lin ◽  
Fan Bai ◽  
Teuta Pilizota ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Single Cell ◽  
Super Resolution ◽  
Bacterial Cells ◽  
Surface Attachment ◽  
Bacterial Physiology ◽  
E Coli ◽  
Single Cell Imaging ◽  
Atomic Force

AbstractFor in vivo, single-cell imaging bacterial cells are commonly immobilised via physical confinement or surface attachment. Different surface attachment methods have been used both for atomic force and optical microscopy (including super resolution), and some have been reported to affect bacterial physiology. However, a systematic comparison of the effects these attachment methods have on the bacterial physiology is lacking. Here we present such a comparison for bacterium Escherichia coli, and assess the growth rate, size and intracellular pH of cells growing attached to different, commonly used, surfaces. We demonstrate that E. coli grow at the same rate, length and internal pH on all the tested surfaces when in the same growth medium. The result suggests that tested attachment methods can be used interchangeably when studying E. coli physiology.

scholarly journals The yefM-yoeB Toxin-Antitoxin Systems of Escherichia coli and Streptococcus pneumoniae: Functional and Structural Correlation

2006 ◽  
Vol 189 (4) ◽  
pp. 1266-1278 ◽  
Author(s):  
Concha Nieto ◽  
Izhack Cherny ◽  
Seok Kooi Khoo ◽  
Mario García de Lacoba ◽  
Wai Ting Chan ◽  
...  
Keyword(s):  
Escherichia Coli ◽  
Streptococcus Pneumoniae ◽  
Structural Features ◽  
E Coli ◽  
Modeled Structure ◽  
The Family ◽  
Bona Fide ◽  
K 12

ABSTRACT Toxin-antitoxin loci belonging to the yefM-yoeB family are located in the chromosome or in some plasmids of several bacteria. We cloned the yefM-yoeB locus of Streptococcus pneumoniae, and these genes encode bona fide antitoxin (YefM Spn ) and toxin (YoeB Spn ) products. We showed that overproduction of YoeB Spn is toxic to Escherichia coli cells, leading to severe inhibition of cell growth and to a reduction in cell viability; this toxicity was more pronounced in an E. coli B strain than in two E. coli K-12 strains. The YoeB Spn -mediated toxicity could be reversed by the cognate antitoxin, YefM Spn , but not by overproduction of the E. coli YefM antitoxin. The pneumococcal proteins were purified and were shown to interact with each other both in vitro and in vivo. Far-UV circular dichroism analyses indicated that the pneumococcal antitoxin was partially, but not totally, unfolded and was different than its E. coli counterpart. Molecular modeling showed that the toxins belonging to the family were homologous, whereas the antitoxins appeared to be specifically designed for each bacterial locus; thus, the toxin-antitoxin interactions were adapted to the different bacterial environmental conditions. Both structural features, folding and the molecular modeled structure, could explain the lack of cross-complementation between the pneumococcal and E. coli antitoxins.

Streptonigrin toxicity in Escherichia coli: oxygen dependence and the role of the intracellular oxidation–reduction state

1982 ◽  
Vol 28 (5) ◽  
pp. 545-552 ◽  
Author(s):  
John B. Harley ◽  
Caroline J. Fetterolf ◽  
Cesar A. Bello ◽  
Joel G. Flaks
Keyword(s):  
Escherichia Coli ◽  
Superoxide Dismutase ◽  
Superoxide Dismutase Activity ◽  
Lethal Concentration ◽  
Bacterial Physiology ◽  
E Coli ◽  
Escherichia Coli K12 ◽  
Oxidation Reduction

The bacterial physiology of streptonigrin toxicity was further investigated. An optimal oxygen concentration for toxicity was inferred from data showing that steptonigrin at 5 µg/mL was rapidly lethal to aerobic cultures of Escherichia coli K12 JF361, but was without effect on anaerobic cultures and was bacteriostatic to cultures incubated in 5 atm of oxygen plus 1 atm of air (5 atm O2 plus air) (1 atm = 101.325 kPa). Escherichia coli were protected from a potentially lethal concentration of streptonigrin during anaerobic incubation, whether previously grown anaerobically, aerobically, or in 5 atm O2 plus air. Superoxide dismutase activity increased with increasing oxygen tension in the medium, but was not significantly changed by a lethal concentration of streptonigrin. Although the superoxide dismutase activity was four times greater in E. coli grown in 5 atm O2 plus air than those grown in air alone, the aerobic survival in 5 µg/mL streptonigrin was identical, which suggested that superoxide dismutase was not rate limiting for toxicity. Escherichia coli K12 strains deficient in glutathione (KMBL54-129, AB1157-821, and AB1157-830) were protected from streptonigrin poisoning. Dithiothreotol (5.0 mM), diamide (1 mM), methyl viologen (1 mM), and cyanide (10 mM) protected aerobic E. coli from 5 µg/mL streptonigrin.These data are also consistent with a model of in vivo streptonigrin toxicity that requires a favorable intracellular oxidation–reduction state and an optimal concentration of molecular oxygen.

scholarly journals Isolation of secreted proteins fromDrosophilaovaries and embryos throughin vivoBirA-mediated biotinylation

2019 ◽  
Author(s):  
Leslie M. Stevens ◽  
Yuan Zhang ◽  
Yuri Volnov ◽  
Geng Chen ◽  
David S. Stein
Keyword(s):  
Transcriptional Control ◽  
Ovarian Follicle ◽  
Structural Features ◽  
Secreted Proteins ◽  
Follicle Cells ◽  
Covalent Interaction ◽  
E Coli ◽  
Experimental Approaches ◽  
Female Germline

AbstractThe extraordinarily strong non-covalent interaction between biotin and avidin (kD = 10-14-10-16) has permitted this interaction to be used in a wide variety of experimental contexts. The Biotin Acceptor Peptide (BAP), a 15 amino acid motif that can be biotinylated by theE. coliBirA protein, has been fused to proteins of interest, making them substrates forin vivobiotinylation. Here we report on the construction and characterization of a modified BirA bearing signals for secretion and endoplasmic reticulum (ER) retention, for use in experimental contexts requiring biotinylation of secreted proteins. When expressed in theDrosophilafemale germline or ovarian follicle cells under Gal4-mediated transcriptional control, the modified BirA protein could be detected and shown to be enzymatically active in ovaries and progeny embryos. Surprisingly, however, it was not efficiently retained in the ER, and instead appeared to be secreted. To determine whether this secreted protein, now designated secBirA, could biotinylate secreted proteins, we generated BAP-tagged versions of two secretedDrosophilaproteins, Torsolike (Tsl) and Gastrulation Defective (GD), which are normally expressed maternally and participate in embryonic pattern formation. Both Tsl-BAP and GD-BAP were shown to exhibit normal patterning activity. Co-expression of Tsl-BAP together with secBirA in ovarian follicle cells resulted in its biotinylation, which permitted its isolation from both ovaries and progeny embryos using Avidin-coupled affinity matrix. In contrast, co-expression with secBirA in the female germline did not result in detectable biotinylation of GD-BAP, possibly because the C-terminal location of the BAP tag made it inaccessible to BirAin vivo. Our results indicate that secBirA directs biotinylation of proteins bound for secretionin vivo, providing access to powerful experimental approaches for secreted proteins of interest. However, efficient biotinylation of target proteins may vary depending upon the location of the BAP tag or other structural features of the protein.

scholarly journals Automated Design and Implementation of a NOR Gate in Pseudomonas Putida

2021 ◽  
Author(s):  
Huseyin Tas ◽  
Lewis Grozinger ◽  
Angel Goñi-Moreno ◽  
Victor de Lorenzo
Keyword(s):  
Pseudomonas Putida ◽  
Design Tool ◽  
Genetic Circuits ◽  
Automated Generation ◽  
Inducible Promoters ◽  
E Coli ◽  
Transcriptional Regulatory ◽  
Cross Platform ◽  
Nor Gate

ABSTRACT Boolean NOR gates have been widely implemented in Escherichia coli as transcriptional regulatory devices for building complex genetic circuits. Yet, their portability to other bacterial hosts/chassis is generally hampered by frequent changes in the parameters of the INPUT/OUTPUT response functions brought about by new genetic and biochemical contexts. Here, we have used the circuit design tool CELLO for assembling a NOR gate in the soil bacterium and metabolic engineering platform Pseudomonas putida with components tailored for E. coli. To this end, we capitalized on the functional parameters of 20 genetic inverters for each host and the resulting compatibility between NOT pairs. Moreover, we added to the gate library 3 inducible promoters that are specific to P. putida, thus expanding cross-platform assembly options. While the number of potential connectable inverters decreased drastically when moving the library from E. coli to P. putida, the CELLO software was still able to find an effective NOR gate in the new chassis. Automated generation of the corresponding DNA sequence and in vivo experimental verification accredited that some genetic modules initially optimized for E. coli can indeed be reused to deliver NOR logic in P. putida as well. Furthermore, the results highlight the value of creating host-specific collections of well-characterized regulatory inverters for quick assembly of genetic circuits to meet complex specifications.

scholarly journals The yeast alpha2 and Mcm1 proteins interact through a region similar to a motif found in homeodomain proteins of higher eukaryotes.

1996 ◽  
Vol 16 (5) ◽  
pp. 2135-2143 ◽  
Author(s):  
J Mead ◽  
H Zhong ◽  
T B Acton ◽  
A K Vershon
Keyword(s):  
Dna Binding ◽  
Transcriptional Repression ◽  
Direct Interaction ◽  
Homeodomain Protein ◽  
Sequence Specificity ◽  
Homeodomain Proteins ◽  
Yeast Saccharomyces Cerevisiae ◽  
Transcriptional Regulatory

Homeodomain proteins are transcriptional regulatory factors that, in general, bind DNA with relatively low sequence specificity and affinity. One mechanism homeodomain proteins use to increase their biological specificity is through interactions with other DNA-binding proteins. We have examined how the yeast (Saccharomyces cerevisiae) homeodomain protein alpha2 specifically interacts with Mcm1, a MADS box protein, to bind DNA specifically and repress transcription. A patch of predominantly hydrophobic residues within a region preceding the homeodomain of alpha2 has been identified that specifies direct interaction with Mcm1 in the absence of DNA. This hydrophobic patch is required for cooperative DNA binding with Mcm1 in vitro and for transcriptional repression in vivo. We have also found that a conserved motif, termed YPWM, frequently found in homeodomain proteins of insects and mammals, partially functions in place of the patch in alpha2 to interact with Mcm1. These findings suggest that homeodomain proteins from diverse organisms may use analogous interaction motifs to associate with other proteins to achieve high levels of DNA binding affinity and specificity.

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