scholarly journals λ Recombineering Used to Engineer the Genome of Phage T7

Antibiotics ◽  
2020 ◽  
Vol 9 (11) ◽  
pp. 805
Author(s):  
Jordan D. Jensen ◽  
Adam R. Parks ◽  
Sankar Adhya ◽  
Alison J. Rattray ◽  
Donald L. Court
Keyword(s):  
Rapid Development ◽  
Bacteriophage T7 ◽  
Lytic Phage ◽  
Dna Transfection ◽  
E Coli ◽  
Recombination Proteins ◽  
Phage T7 ◽  
Technical Advances ◽  
Red Recombination

Bacteriophage T7 and T7-like bacteriophages are valuable genetic models for lytic phage biology that have heretofore been intractable with in vivo genetic engineering methods. This manuscript describes that the presence of λ Red recombination proteins makes in vivo recombineering of T7 possible, so that single base changes and whole gene replacements on the T7 genome can be made. Red recombination functions also increase the efficiency of T7 genome DNA transfection of cells by ~100-fold. Likewise, Red function enables two other T7-like bacteriophages that do not normally propagate in E. coli to be recovered following genome transfection. These results constitute major technical advances in the speed and efficiency of bacteriophage T7 engineering and will aid in the rapid development of new phage variants for a variety of applications.

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 Matrix-trapped viruses can prevent invasion of bacterial biofilms by colonizing cells

eLife ◽  
2021 ◽  
Vol 10 ◽  
Author(s):  
Matthew C Bond ◽  
Lucia Vidakovic ◽  
Praveen K Singh ◽  
Knut Drescher ◽  
Carey D Nadell
Keyword(s):  
Escherichia Coli ◽  
Bacterial Biofilms ◽  
Lytic Phage ◽  
Prior Work ◽  
Polymer Layers ◽  
E Coli ◽  
The Matrix ◽  
Phage T7 ◽  
Biofilm Matrix

Bacteriophages can be trapped in the matrix of bacterial biofilms, such that the cells inside them are protected. It is not known whether these phages are still infectious and whether they pose a threat to newly arriving bacteria. Here we address these questions using Escherichia coli and its lytic phage T7. Prior work has demonstrated that T7 phages are bound in the outermost curli polymer layers of the E. coli biofilm matrix. We show that these phages do remain viable and can kill colonizing cells that are T7-susceptible. If cells colonize a resident biofilm before phages do, we find that they can still be killed by phage exposure if it occurs soon thereafter. However, if colonizing cells are present on the biofilm long enough before phage exposure, they gain phage protection via envelopment within curli-producing clusters of the resident biofilm cells.

scholarly journals Evaluation of a Cocktail of Three Bacteriophages for Biocontrol of Escherichia coli O157:H7

2004 ◽  
Vol 70 (6) ◽  
pp. 3417-3424 ◽  
Author(s):  
G. O'Flynn ◽  
R. P. Ross ◽  
G. F. Fitzgerald ◽  
A. Coffey
Keyword(s):  
Escherichia Coli ◽  
Biocontrol Agents ◽  
Lytic Phage ◽  
O157 Strain ◽  
E Coli ◽  
Phage Cocktail ◽  
Phage Sensitivity ◽  
Unit Reduction

ABSTRACT Escherichia coli O157:H7 is an endemic pathogen causing a variety of human diseases including mild diarrhea, hemorrhagic colitis, hemolytic-uremic syndrome, and thrombotic thrombocytopenic purpura. This study concerns the exploitation of bacteriophages as biocontrol agents to eliminate the pathogen E. coli O157:H7. Two distinct lytic phages (e11/2 and e4/1c) isolated against a human strain of E. coli O157:H7, a previously isolated lytic phage (pp01), and a cocktail of all three phages were evaluated for their ability to lyse the bacterium in vivo and in vitro. Phage e11/2, pp01, and the cocktail of all three virulent phages resulted in a 5-log-unit reduction of pathogen numbers in 1 h at 37�C. However, bacteriophage-insensitive mutants (BIMs) emerged following the challenge. All tested BIMs had a growth rate which approximated that of the parental O157 strain, although many of these BIMs had a smaller, more coccoid cellular morphology. The frequency of BIM formation (10−6 CFU) was similar for e11/2, pp01, and the phage cocktail, while BIMs insensitive to e4/1c occurred at the higher frequency (10−4 CFU). In addition, BIMs commonly reverted to phage sensitivity within 50 generations. In an initial meat trial experiment, the phage cocktail completely eliminated E. coli O157:H7 from the beef meat surface in seven of nine cases. Given that the frequency of BIM formation is low (10−6 CFU) for two of the phages, allied to the propensity of these mutants to revert to phage sensitivity, we expect that BIM formation should not hinder the use of these phages as biocontrol agents, particularly since low levels of the pathogen are typically encountered in the environment.

Observation of DNA by negative staining: phage t7 DNA-capsid complexes

1978 ◽  
Vol 36 (2) ◽  
pp. 228-229
Author(s):  
Philip Serwer
Keyword(s):  
Cytochrome C ◽  
Uranyl Acetate ◽  
Bacteriophage T7 ◽  
E Coli ◽  
Negative Stain ◽  
Carbon Coated ◽  
Phage T7 ◽  
Transparent Region ◽  
Dna 2 ◽  
T7 Phage

A technique has been developed for observing extended nucleic acids in specimens negatively stained with uranyl acetate. This technique has been used to characterize bacteriophage T7 DNA-capsid complexes obtained by: a) disruption of T7 phage using glutaraldehyde treatment; b) isolation from Xysates of bacteriophage T7-infected E. coli. The latter complexes may be in the DNA packaging pathway of T7.A sample of circular, duplex bacteriophage ØX174 DNA (.2 μg/ml) in. 1 M NaCl, .01 M Tris-Cl,. 001 M EDTA, pH 7.4, was mixed with an equal volume of cytochrome c (200 μg/ml) and was incubated for five minutes. This mixture was incubated with a carbon-coated grid for one minute; the grid was washed and was negatively stained with 1% uranyl acetate. An accumulation of negative stain around the DNA-bound cytochrome c reveals the presence of DNA circles (Figure 1). The cytochrome c bound to DNA is observed as an electron transparent region, 40-65 Å in diameter, within the negative stain.

Inability Of Agrobacterium Tumefaciens Ribosomes To Translate In Vivo Mrnas Containing Non-Shine-Dalgarno Translational Initiators

2002 ◽  
Vol 57 (3-4) ◽  
pp. 307-312 ◽  
Author(s):  
Ashkan Golshani ◽  
John Xu ◽  
Vihren Kolev ◽  
Mounir G. AbouHaidar ◽  
Ivan G. Ivanov
Keyword(s):  
Escherichia Coli ◽  
Agrobacterium Tumefaciens ◽  
Mosaic Virus ◽  
Translational Control ◽  
Yellow Mosaic Virus ◽  
Viral Origin ◽  
E Coli ◽  
Gene Coding ◽  
Phage T7

Numerous data accumulated during the last decade have shown that the Shine-Dalgarno (SD) sequence is not a unique initiator of translation for Escherichia coli. Several other sequences, mostly of viral origin, have demonstrated their capability of either enhancing or initiating translation in vivo. A phage T7 gene 10 sequence, called “epsilon” (ε), has shown its high enhancing activity on translation in both Escherichia coli and Agrobacterium tumefaciens cells. In this study the ε, together with three other nucleotide sequences derived from the 5′ non-translated regions of tobacco mosaic virus (TMV), papaya mosaic virus (PMV) and clover yellow mosaic virus (CYMV) RNAs are tested for translation initiation activity in A. tumefaciens cells. The obtained results indicate that none of them was capable of initiating translation in vivo of chloramphenicol acetyltransferase (CAT) mRNA. To determine whether their inactivity was related with structural differences in the ribosomal protein S1, the rpsA gene (coding for S1 protein in E. coli) was co-expressed in A. tumefaciens together with the cat gene placed under the translational control of the above sequences. Our results showed that the rpsA gene product did not make any of the four viral enhancers active in A. tumefaciens cells. The inability of A. tumefaciens ribosomes to translate mRNAs devoid of SDsequences indicates for a substantial difference in the ribosome structure of the two Gram negative bacteria E. coli and A. tumefaciens

Local bacteriophage isolates showed anti-Escherichia coliO157:H7 potency in an experimental ligated rabbit ileal loop model

2011 ◽  
Vol 57 (5) ◽  
pp. 408-415 ◽  
Author(s):  
Muntasir Alam ◽  
Marufa Zerin Akhter ◽  
Mahmuda Yasmin ◽  
Chowdhury Rafiqul Ahsan ◽  
Jamalun Nessa
Keyword(s):  
Bacterial Growth ◽  
Lytic Phage ◽  
Loop Model ◽  
Ileal Loop ◽  
E Coli ◽  
Combined Application ◽  
Food Borne ◽  
In Vivo Testing

Escherichia coli O157:H7 is considered among the most important recently emerged food-borne bacteria causing severe hemorrhagic diarrhea. Antibiotic treatment is not recommended as a prospective curative agent against this pathogen. Therefore, potency assessment of the local lytic phage isolates infecting E. coli O157:H7 as an alternate remedy to antibiotics was the principal concern of this study. Phage isolates against E. coli O157:H7 were checked by polymerase chain reaction for the presence of the virulence genes stx1 and stx2, and the safe phages were further screened in vitro for their capacity as biocontrol agents. Two bacteriophage strains, namely PAH6 and P2BH2, that had expressed potential antibacterial activity (P < 0.05) in vitro were selected for in vivo testing in ligated rabbit ileal loop models. Both phage isolates were capable of decreasing fluid accumulation in rabbit ileal loops along with reducing bacterial growth (r = 0.992). Combined application of the phages was found most satisfactory, reducing seven log cycles of bacterial growth. Consistent results in both in vivo and in vitro experiments demonstrate the applicability of bacteriophages as a rapid response tool against E. coli O157:H7. To our knowledge, this is the first successful application of the rabbit ileal loop test for therapeutic evaluation of bacteriophages.

scholarly journals A Frameshift Mutation in wcaJ Associated with Phage Resistance in Klebsiella pneumoniae

2020 ◽  
Vol 8 (3) ◽  
pp. 378 ◽  
Author(s):  
Demeng Tan ◽  
Yiyuan Zhang ◽  
Jinhong Qin ◽  
Shuai Le ◽  
Jingmin Gu ◽  
...  
Keyword(s):  
Klebsiella Pneumoniae ◽  
Phage Therapy ◽  
Rapid Development ◽  
Phage Resistance ◽  
Comparative Genomic ◽  
Lytic Phage ◽  
Wild Type ◽  
Coding Region

Phage therapy is a potential and promising avenue for controlling the emergence and spread of multidrug-resistant (MDR) Klebsiella pneumoniae, however, the rapid development of anti-phage resistance has been identified as an obstacle to the development of phage therapy. Little is known about the mechanism employed by MDR K. pneumoniae strains and how they protect themselves from lytic phage predation in vitro and in vivo. In this study, comparative genomic analysis shows undecaprenyl-phosphate glucose-1-phosphate transferase (WcaJ), the initial enzyme catalyzing the biosynthesis of colanic acid, is necessary for the adsorption of phage 117 (Podoviridae) to the host strain Kp36 to complete its lytic life cycle. In-frame deletion of wcaJ alone was sufficient to provide phage 117 resistance in the Kp36 wild-type strain. Complementation assays demonstrated the susceptibility of phage 117, and the mucoid phenotype could be restored in the resistant strain Kp36-117R by expressing the wild-type version of wcaJ. Remarkably, we found that bacterial mobile genetic elements (insA and insB) block phage 117 infections by disrupting the coding region of wcaJ, thus preventing phage adsorption to its phage receptor. Further, we revealed that the wcaJ mutation likely occurred spontaneously rather than adapted by phage 117 predation under unfavorable environments. Taken together, our results address a crucial evolutionary question around the mechanisms of phage–host interactions, increasing our current understandings of anti-phage defense mechanisms in this important MDR pathogen.

scholarly journals Biological Characterization and Evolution of Bacteriophage T7-△holin During the Serial Passage Process

2021 ◽  
Vol 12 ◽  
Author(s):  
Hai Xu ◽  
Xi Bao ◽  
Weiming Hong ◽  
Anping Wang ◽  
Kaimin Wang ◽  
...  
Keyword(s):  
Electron Microscopy ◽  
High Titer ◽  
Burst Size ◽  
Serial Passage ◽  
Bacteriophage T7 ◽  
Potential Contribution ◽  
Sequencing Analysis ◽  
Mrna Levels ◽  
E Coli ◽  
Phage T7

Bacteriophage T7 gene 17.5 coding for the only known holin is one of the components of its lysis system, but the holin activity in T7 is more complex than a single gene, and evidence points to the existence of additional T7 genes with holin activity. In this study, a T7 phage with a gene 17.5 deletion (T7-△holin) was rescued and its biological characteristics and effect on cell lysis were determined. Furthermore, the genomic evolution of mutant phage T7-△holin during serial passage was assessed by whole-genome sequencing analysis. It was observed that deletion of gene 17.5 from phage T7 delays lysis time and enlarges the phage burst size; however, this biological characteristic recovered to normal lysis levels during serial passage. Scanning electron microscopy showed that the two opposite ends of E. coli BL21 cells swell post-T7-△holin infection rather than drilling holes on cell membrane when compared with T7 wild-type infection. No visible progeny phage particle accumulation was observed inside the E. coli BL21 cells by transmission electron microscopy. Following serial passage of T7-△holin from the 1st to 20th generations, the mRNA levels of gene 3.5 and gene 19.5 were upregulated and several mutation sites were discovered, especially two missense mutations in gene 19.5, which indicate a potential contribution to the evolution of the T7-△holin. Although the burst size of T7-△holin increased, high titer cultivation of T7-△holin was not achieved by optimizing the culture process. Accordingly, these results suggest that gene 19.5 is a potential lysis-related component that needs to be studied further and that the T7-△holin strain with its gene 17.5 deletion is not adequate to establish the high-titer phage cultivation process.

Development of a novel bacteriophage based biomagnetic separation method as an aid for sensitive detection of viable Escherichia coli

The Analyst ◽  
2016 ◽  
Vol 141 (3) ◽  
pp. 1009-1016 ◽  
Author(s):  
Ziyuan Wang ◽  
Danhui Wang ◽  
Juhong Chen ◽  
David A. Sela ◽  
Sam R. Nugen
Keyword(s):  
Escherichia Coli ◽  
Genetic Engineering ◽  
Magnetic Beads ◽  
Separation Method ◽  
Sensitive Detection ◽  
Bacteriophage T7 ◽  
E Coli ◽  
Liquid Samples ◽  
Oriented Immobilization

Genetic engineering of bacteriophage T7 allowed thein vivobiotinylation of capsid proteins. Oriented immobilization of the phage on magnetic beads then enabled the adsorption and separation ofE. colifrom liquid samples.

scholarly journals Matrix-trapped viruses can protect bacterial biofilms from invasion by colonizing cells

2020 ◽  
Author(s):  
Matthew C. Bond ◽  
Lucia Vidakovic ◽  
Praveen K. Singh ◽  
Knut Drescher ◽  
Carey D. Nadell
Keyword(s):  
Escherichia Coli ◽  
Extracellular Matrix ◽  
Bacterial Biofilms ◽  
Lytic Phage ◽  
Prior Work ◽  
E Coli ◽  
Phage T7

AbstractBacteria often live in the context of spatially restricted groups held together by a self-secreted, adhesive extracellular matrix. These groups, termed biofilms, are likely where many phage-bacteria encounters occur. A number of recent studies have documented that phages can be trapped in the outer matrix layers of biofilms, such that the bacteria inside are protected from exposure. It is not known, however, what might happen after this: are the trapped phages still viable on the biofilm exterior? If so, do they pose a threat to newly arriving cells that might otherwise colonize the existing biofilm? Here we set out to address these questions using a biofilm-producing strain of Escherichia coli and its lytic phage T7. Prior work has demonstrated that T7 phages are trapped in the outermost layers of curli polymers within the E. coli matrix. We show that these phages do remain viable and kill incoming colonizing cells so long as they are T7-susceptible. If colonizing cells arrive to the outside of a resident biofilm before phages do, they can still be killed by phage exposure if it occurs soon thereafter. However, if colonizing cells are present on the biofilm long enough before phage exposure, they gain phage protection via envelopment within curli-producing clusters of the resident biofilm cells. This work establishes that phages trapped in the outer matrix layers of a resident biofilm can be incidentally weaponized as a mode of protection from competition by newly arriving cells that might otherwise colonize the biofilm exterior.

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