How can I change the payload of a bacteriophage used to transform E. coli?

How can I change the payload of a bacteriophage used to transform E. coli?

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I was looking at bacteriophages and how they're used to transform E.coli. While the whole process of how a bacteriophage works makes sense theoretically, I wanted to know how one goes about changing the default payload of a bacteriophage with the payload of interest (engineered plasmid) in practice?


payload - Genetic material that the phage inserts into the bacteria

default payload - Whatever genetic material the phage inserts into the bacteria naturally

payload of interest - Engineered plasmid that I want to insert into the bacteria

I am currently using a 'phage integrase' system to insert my gene of interest into P. putida and it is definitely a seamless process with high efficiency so I highly recommend it!

As for your question, the method through which we integrate the gene of interest using the phage integrase system is simpler than you think. We do not change the payload of a bacteriophage. We simply electroporate with two plasmids, one that expresses the BxB1 integrase and another plasmid that has our gene of interest that we want to integrate into the genome of P. putida. This integration is highly efficient, and it operates through a serine recombinase that recognizes specific attP and attB sequences in the plasmid that contains my gene of interest and native genome of my engineered strain. This method is significantly more efficient for engineering certain strains (typically non-model organisms) for genome integration purposes.

References for additional information on the system for your review:

Here is a paper that describes how we use the phage integrase system to effectively edit the genome.

Here is the paper that discovered and characterized the mechanism this system operates through.

Bacteriophage genes that inactivate the CRISPR/Cas bacterial immune system

A widespread system used by bacteria for protection against potentially dangerous foreign DNA molecules consists of the clustered regularly interspaced short palindromic repeats (CRISPR) coupled with cas (CRISPR-associated) genes 1 . Similar to RNA interference in eukaryotes 2 , these CRISPR/Cas systems use small RNAs for sequence-specific detection and neutralization of invading genomes 3 . Here we describe the first examples of genes that mediate the inhibition of a CRISPR/Cas system. Five distinct ‘anti-CRISPR’ genes were found in the genomes of bacteriophages infecting Pseudomonas aeruginosa. Mutation of the anti-CRISPR gene of a phage rendered it unable to infect bacteria with a functional CRISPR/Cas system, and the addition of the same gene to the genome of a CRISPR/Cas-targeted phage allowed it to evade the CRISPR/Cas system. Phage-encoded anti-CRISPR genes may represent a widespread mechanism for phages to overcome the highly prevalent CRISPR/Cas systems. The existence of anti-CRISPR genes presents new avenues for the elucidation of CRISPR/Cas functional mechanisms and provides new insight into the co-evolution of phages and bacteria.


Bacteriophages (phages) are viral parasites of bacteria and are the most abundant biological entities on Earth, with an estimated global population of over 10 30 , 10-fold more than their bacterial hosts (Wommack and Colwell, 2000 Dy et al., 2014 ). Phages are near-ubiquitous, having been isolated from soil, water, plants and animals (Elbreki et al., 2014 ). Global phage infections have been estimated at approximately 10 25 per second and are of profound biological, ecological and evolutionary importance (Wommack and Colwell, 2000 ). The phage–host interaction starts with adsorption, where the phage receptor-binding protein attaches to the specific receptor on the host cell surface (Lindbergl, 1973 ). In Gram-negative bacteria, a variety of surface-exposed components can be exploited as phage receptors, including outer membrane proteins (OMPs), lipopolysaccharide (LPS), exopolysaccharide, capsular polysaccharide, flagella and pili (Bertozzi Silva et al., 2016 ). Phages target these receptors with exquisite molecular specificity, and this can be one important parameter contributing to the narrow host range of many phages (Nobrega et al., 2018 ). Knowledge of the molecular mechanisms and temporal dynamics of this targeting system not only provides new insights into phage biology but also improves the utility of phage-derived applications (Bertozzi Silva et al., 2016 ). Nonetheless, over past decades, most fundamental research on this initial phage–host interaction has been conducted on a very restricted group of well-characterized phages and their receptors in cognate hosts (Chatterjee and Rothenberg, 2012 ). Therefore, given the sheer global abundance of phages, the fundamental basis of the bacterial host range and its ecological plasticity remains largely ill-defined.

Difficulty in isolating phages on certain bacterial species, and the repetitive isolation of similar phages that target the same bacterial receptor(s), has limited wider analysis of phage–receptor interactions, despite the abundance and diversity of environmental phages (Hyman, 2019 ). The isolation of a variety of phages which target different receptors of any particular bacterial host is desirable for (i) studies of fundamental phage biology, ecology and molecular biology and (ii) phage therapy, where a collection of phages which target different receptors of a host bacterium are useful in phage cocktails to circumvent the evolution of potential bacterial resistance (Chan et al., 2013 ). Some phage receptors, such as OMPs, can have simple structures and may also play roles in antibiotic resistance or virulence (Seed et al., 2014 ). The phage-resistant mutants of such bacteria may be less virulent and more susceptible to antibiotics. Consequently, for various reasons, it would be useful to have a simple method to isolate a collection of phages that target a specific receptor of a particular bacterium.

The conventional route for phage isolation is to enrich for phages against a specific bacterial host, then the nature of the receptor can be elucidated using mutant screens (Charbit and Hofnung, 1985 Li et al., 2019 ). To then isolate phages targeting a ‘new’ receptor, bacterial mutants lacking the ‘old’ receptor(s) can be used first for enrichment (Beher and Pugsley, 1981 ). However, these methods are comparatively inefficient in that the isolation of phages that target any particular receptor may necessitate screening of very large numbers of phages (Beher and Pugsley, 1981 Charbit and Hofnung, 1985 ). In this study, we decided to use simple enrichment methods to investigate the impacts on phage isolation of heterologous surface receptor expression. The aim was to test the hypothesis that engineering the nature of the receptor expressed on the bacterial cell surface would bias facile enrichments leading to the isolation and discovery of new environmental phages adsorbing in a predictable way.

The study focused on a well-characterized bacterial receptor for a well-studied phage. During the past decades, phage λ has been intensively studied as an important research tool for investigating fundamental molecular microbiology processes and because it can be exploited for various molecular tools including as a vehicle for transposon delivery and for cosmid cloning and engineering (Palva et al., 1987 Ellard et al., 1989 ). Phage λ is a Siphoviridae family member phage with a long, non-contractile tail (Ackermann, 2003 ). The gpJ protein at the end of the phage tail recognizes and interacts with the cognate receptor, the Escherichia coli LamB (EcLamB) protein, which is a trimeric OMP embedded in the outer membrane (OM) with surface-exposed domains (Schirmer et al., 1995 Chatterjee and Rothenberg, 2012 ). The natural physiological role of LamB in E. coli is as a porin, permissive for the transport of maltose and maltodextrins from the environment into the bacterial cells (Ishii et al., 1981 ). Previous research revealed that the C-terminal region of the trimeric gpJ protein of the phage tail determines host specificity by directly interacting with EcLamB when phage λ attaches to the host cell surface (Wang et al., 2000 ). Moreover, 12 residues of EcLamB involved in phage λ recognition have been identified by screening E. coli K12 strains with missense mutations in lamB gene (Gehring et al., 1987 Charbit et al., 1994 ). Perhaps as expected, given the topology of the protein in the OM, most of the key residues are located on the extracellular surface of the EcLamB porin and are therefore accessible to the phage (Charbit et al., 1994 ). In addition to phage λ, the EcLamB protein is also recognized by a small selection of other phages, such as K10, TP1, SS1 and AB48 (Wandersman and Schwartz, 1978 Roa, 1979 Beher and Pugsley, 1981 Charbit and Hofnung, 1985 ). Most of these phages are λ-like Siphoviridae family members in terms of morphology. However, other information about these EcLamB-targeting phages is very limited in the literature that dates back to the last century. Consequently, little is known about the abundance, distribution, or diversity of environmental phages that can target EcLamB as their receptor.

In this study, we designed a simple method to isolate a group of novel environmental phages that target the EcLamB protein as their receptor. Three enterobacteria, namely Citrobacter rodentium (Citrobacter), Yersinia enterocolitica (Yersinia) and Serratia ATCC 39006 (Serratia) were transformed previously with a plasmid (pMUT13) carrying the E. coli lamB gene. Several phages that target EcLamB were isolated via enrichment with one of these pMUT13-containing strains. Although these newly isolated phages target the same receptor as phage λ, they share no similarity with phage λ in terms of morphology or genomic DNA. Furthermore, in addition to the EcLamB protein, we found that these new phages also have the capacity to target an alternative receptor as a route to the viral invasion of their respective bacterial host cells. Our results suggest a potentially simple enrichment approach to bias isolation of phages that target any defined receptor exposed on the bacterial cell surface. This approach enables future studies on the molecular nature and evolution phages, and the bases of host range specificity. It is also obvious that this strategy could have biotechnological utility.

Metal Biology Associated with Huntington’s Disease

Terry Jo V. Bichell , . Aaron B. Bowman , in Biometals in Neurodegenerative Diseases , 2017

MRE-11—Meiotic Recombination 11, and FAN1—Fanconi’s Associated Nuclease 1

Like ATM, MRE-11, and FAN1 are part of the DNA repair pathway, and both are Mn-dependent enzymes linked with DNA damage in HD. MRE-11 associates with ATM and complexes with Ras-Proximate 1 (RAP1) to maintain telomere length, 269 while FAN1 functions to remove the interstrand DNA crosslinks that impede double-stranded repair. 270,271 Pathological double-stranded DNA repair is found in HD and in other polyQ expansion diseases. 272 Recently, a genome wide association study of 1462 patients with HD and other polyQ spinocerebellar ataxias (SCAs) revealed that a single-nucleotide polymorphisms (SNP) from FAN1 was a genetic modifier for the age of onset. 273 Both Mn and Mg can serve as cofactors for FAN1, 271 but MRE-11 is not active when Mg is substituted for Mn. 274

All of the evidence linking abnormalities in Mn-dependent enzymes to HD mentioned earlier strongly supports the data showing that there is reduced Mn bioavailability contributing to pathologies seen in the disease.

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Ch 6 Bacterial growth, nutrition, and differentiation

Stationary phase- when viable cell numbers stop rising owning to a lack of key nutrients or buildup of waste products. growth of individual cells slows and the number of cells dividing approximately equals the number of cells dying. resistance to antibiotics and host defenses develops during this time.

A chemostat is a continuous culture system in which the diluting medium contains a growth-limiting amount of an essential nutrient.

strict anaerobes- die in the least bit of oxygen. They do not use oxygen as an electron acceptor and die because they are vulnerable to the highly toxic, chemically reactive oxygen species produced by their own metabolism when exposed to oxygen. Reactive Oxygen Species (ROS) are oxygen molecules or ions with one too few or too many electrons.

Also contain resourceful enzyme systems that detect and repair macromolecules damaged by oxidation.

Aerotolerant anaerobes- use only fermentation to provide energy but contain superoxide dismutase and catalase or peroxidase to protect them from ROS. Aerotolerant anaerobes grow in both the presence and absence of oxygen-although they grow better without it

spores can exist in the soil for at least 50-100 years and some last thousands and even millions of years.

antisepsis- similar to disinfection but applies to removing pathogens from the surface of LIVING tissues, such as skin

Disinfection- the killing or removal of DISEASE-PRODUCING organisms from inanimate surfaces it does not necessarily result in sterilization

Cidal agents: kill microbes
---- Bactericidal, algicidal, fungicidal, virucidal, depending on what type of microbe is killed

today, pasteurization involves heating a particular food (such as milk) to a moderately high temperature long enough to kill Coxiella burnetii, the causative agent of Q fever and the most heat resistant spore-forming pathogen known

-LTLT (low temperature, long time) process involves bringing the temperature to 63 C (146 F) for 30 minutes. This is the original way it was done.

-HTST (high temperature, short time) process (also called flash pasteurization) brings the temperature to 72 C (162 F) for only 15 seconds. This is STANDARD PROCEDURE.

-long-term storage of bacteria usually requires placing solutions in glycerol at very low temperatures (-70C or -94F)
- this deep freezing suspends growth altogether and keeps cells from dying.

Lyophilization (freeze-drying) is another method used for long-term storage. Cultures are quickly frozen at very low temperatures and placed under a vacuum, which causes the water to sublimate, removing all water from the cells

Cold doesnt kill well. We use cold temp to preserve microbes. WHAT KILLS THEM is if ice forms inside the cells, the glycerol protects them from that- and freeze-drying removes the water and thus keeps it from killing.

Laminar flow biological safety cabinets- elaborate and effective ventilated workbenches in which air is forced through high-efficiency particulate air filters (HEPA filters) to remove more than 99.9% airborne particulate material 0.3 micrometers or larger.

Usually gamma rays are used for foods or plastics that cant be sterilized or disinfected by any other method.

Gamma rays, electron beams, X-rays- Radiation dosage is measured in a unit called the Gray (Gy), which is the amount of energy transferred to the food, microbe, or other substance being irradiated.

one chest x-ray delivers about one half a miliGray.
to kill Salmonella, a freshly slaughtered chicken can be irradiated at up to 4.5 kiloGray (kGy)

It takes more radiation to kill bacteria and even more to kill a bacterial spore. Viral pathogens have the smallest amount of nucleic acid, making them resistant to irradiation doses approved for foods.

prion particles do not contain any nucleic acid and are inactivated by irradiation at extremely high doses

Size of the target and cell structure is important when it comes to using irradiation

****CDC levels of disinfection based on range of microbes affected.

highly reactive compounds that damage proteins, lipids, and DNA

help in the mechanical removal of microbes from surfaces

-no single chemotherapeutic agent affects all microbes.
- antimicrobial drugs are classified based by the type of organism they affect (ex: antibacterial, antifungal, etc.)
- even within a group, one agent may have a narrow spectrum of activity, whereas another may affect many species.

-Penicillin has a relatively narrow spectrum while ampicillin has a much broader spectrum due to its slightly different structure.

-Many drugs do not kill the organism but simply prevent its growth while letting the body's immune system take care of the invader

-the in vitro effectiveness of an agent is determined by how little of it is needed to stop growth.
-this is measured in terms of the antibiotic's minimal inhibitory concentration (MIC)
-the MIC is defined as the lowest concentration of the drug that will prevent the growth of an organism.

the time can be reduced by doing a strip test (Etest) which avoids the need for dilutions.

-for an antibiotic to stop bacterial growth in the patient, the drug's concentration in tissue must remain higher than the MIC at all times.
-The tissue level of a drug over time (half-life) depends on how quickly the antibiotic is removed from the body via secretion by the kidney or destruction in the liver.
- usefulness also depends on whether side effects appear at tissue concentrations needed to affect the pathogen.

-the therapeutic dose is the minimum dose per kg of body weight that stops pathogen growth.

-the toxic dose is the maximum dose tolerated by the patient

-combinations of antibiotics can be either synergistic or antagonistic

-SYNERGISTIC drugs may work poorly when they are given individually but very well when combined (combined effect is greater than additive effect) Ex: aminoglycoside + vancomycin

-the mechanisms of action of ANTAGONISTIC drugs interfere with each other and diminish their effectiveness ex: penicillin + macrolides

SYNERGISM- Aminoglycosides and vancomycin work poorly on their own for some types of infections (such as Enterococcus). When combined, the vancomycin, a cell wall synthesis inhibitor, weakens the cell wall of the organism and facilitates the transport of the aminoglycoside into the cell.

-Mechanisms include:
Cell wall synthesis
DNA synthesis
RNA synthesis
protein synthesis
cell membrane integrity


505 ribosome subunit:
- Macrolides (erythromycin)
-Oxazolidinones (linezolid)
-Streptogramins (synercid)
-Aminoglycosides (gentamicin)
-Tetracyclines (doxycycline)



1) disaccharide units of NAG and NAM are first made in the cytoplasm and shuttled across the membrane by a lipid carrier molecule.

2) the disaccharide units are then assembled at the cell membrane to form long strands of peptidoglycan.

3) adjacent strands are then linked by a short peptide that comes from NAM molecules

Cell wall (peptidoglycan) synthesis.
NAG and NAM are made and joined as units in the cytoplasm (steps 1-3). Each unit is then transported across the cell membrane by a specialized lipid carrier molecule called bactoprenol (step 4). Enzymes then link the new unit to a preexisting chain of peptidoglycan and catalyze a cross-link between peptide chains sticking out from NAM molecules on parallel chains.
Penicillin, vancomycin, and cephalosporins inhibit the activity of these enzymes.

sulfonamide (sulfa) drugs act to inhibit the synthesis of nucleic acids by preventing the synthesis of folic acid, an important cofactor in the synthesis of nucleic acid precursors

all organisms use folic acid to synthesize nucleic acids. Bacteria make folic acid from the combination of PABA, glutamic acid, and pteridine. Mammals do not synthesize folic acid and must get it from the diet or microbes.

PABA, pteridine, and glutamic acid combine to make the vitamin folic acid.

Normal synthesis of folic acid requires that all three components engage the active site of the biosynthetic enzyme. The sulfa drugs replace PABA at the active site. The sulfur group, however, will not form a peptide bond with glutamic acid, and the size of sulfanilamide sterically hinders the binding of pteridine, so folic acid cannot be made.

DNA gyrase bound to and inactivated by a quinolone will block progression of a DNA replication fork

because bacterial DNA gyrases are structurally distinct from their mammalian counterparts, quinolone antibiotics will not affect mammalian DNA replication.

Ciprofloxacin and Levofloxacin

Rifampin is the best-known member of the rifamycin family of antibiotics that selectively binds to bacterial RNA polymerase and prevents transcription.

Rifampin is also used to treat tuberculosis and meningococcal meningitis

Drugs that affect the 30S ribosomal subunit:

aminoglycosides : (streptomycin, gentamicin, tobramycin)
---causing misreading of mRNA and inhibit peptidyl-tRNA translocation

tetracyclines (doxycycline)
--binds to the 30S subunit and prevent tRNAs carrying amino acids from entering the A site

The S is for a Svedberg unit which is a measurement of size and density, so as subunits combine they get smaller.

-chloramphenicol- prevents peptide bond formation by inhibiting peptidyltransferase in the 50S subunit

Macrolides (erythromycin, azithromycin, clarithromycin)- binds to 50S subunit and inhibit translocation of tRNA from the A site to the P site.

Lincosamides (clindamycin)- bind to peptidyltransferase and prevents peptide bond formation

oxazolidinones- bind to 50S subunit and prevent assembly of the 70S ribosomes

-Modify the target- so that it no longer binds the antibiotic. EX: altered DNA gyrase no longer binds to quinolones
(there are several ways this has happened-altered DNA gyrase, altered penicillin binding protein, or altered ribosome.)

-Destroy the antibiotic- before it gets into the cell. EX: the enzyme beta-lactamase (penicillinase)
(beta-lactamase destroying penicillin and cephalosporin)

Add modifying groups-that inactivate the antibiotic. EX: enzymes that modify and inactivate aminoglycosides.
(bacteria will have enzymes that add things like methyl groups, acetyl groups or others so the antibiotic can no longer attach to its target site. )

Pump the antibiotic out- of the cell using specific or nonspecific transport proteins. EX: Tetracycline resistance is due to an efflux pump.
(an efflux pump sends the antibiotic out of the cell before it can do its job. )

resistance can arise spontaneously through mutation or gene duplication followed by random mutations that "repurpose" the deplicated gene or genes.
--MDR efflux pumps evolved from genes encoding other transport mechanisms.

once a mechanism of resistance is developed, gene transfer mechanisms such as conjugation can move the gene from one organism to another and from one species to another.
-- transfer is particularly easy if the gene has been incorporated into a plasmid

dummy target compounds that inactivate resistance enzymes have been developed.
EX: Clavulanic acid binds and ties up beta-lactamases secreted from penicillin resistant bacteria.

Individuals can take the following actions to help:
-frequent hand washing
-avoiding use of antibiotics for viral infections
-refusing leftover antibiotics
- take full course of antibiotics prescribed.

Drug resistance has spread due to a number of reasons and now we have to try to combat those reasons mainly through education. Much of it developed because in the beginning we didn't understand the differences between bacteria and viruses, so doctors prescribed them for everything. In many countries, antibiotics are not regulated so anyone can go in and buy them, we've used them in food animals because they made the animals grow faster and bigger, people not taking them as prescribed, etc.


The colony-to-lawn transformation method for changing hosts of plasmids is illustrated in Fig. 1a. The first step is preparation of plasmid-containing cell lysate. A single colony from plasmid donor strain is suspended in 75% ethanol followed by centrifugation to get pellet, and then the cells are resuspended in CaCl2 solution and lysed by freeze–thaw cycles to obtain plasmid-containing cell lysate. The second step is preparation of recipient cells for transformation. Cells of E. coli recipient strain are scraped carefully from fresh lawn without gouging the agar and then suspended in ice-cold water. The third step is transformation. An aliquot of recipient cells and plasmid-containing cell lysate are mixed gently and we performed transformation by heat shock method. Then, transformed bacteria are grown and selected by standard methods. The colony-to-lawn transformation method is more convenient and rapid than current methods, because no plasmid extraction and competent cell preparation steps are needed (Fig. 1b). Using pETM11-P450-BM3 as a sample, we changed its hosts from E. coli JM109 to BL21(DE3) either by colony-to-lawn transformation or by chemical transformation. After IPTG induction, the similar expression levels of P450-BM3 protein were obtained (Fig. 1c), indicating that there is no fundamental difference between these transformants. We tested the colony-to-lawn transformation method by using it to change the hosts of various plasmids, including the low-copy-number plasmid pLysS. As a result, no less than 60 transformants were available after each transformation with a single colony of plasmid-containing donor strain (Fig. 1d). Additionally, the method is very convenient, because the LB agar plates with colonies of donor strains and recipient strain can be stocked at 4°C for at least 7 days without affecting the transformation obviously (data not shown). As a control, plasmids from the randomly selected transformants were successfully re-transformed into E. coli BL21(DE3) by chemical transformation, indicating that the antibiotic-resistant colonies after colony-based transformation were real transformants but not E. coli mutants or contaminants. In addition, no colony was found on the antibiotic-containing agar plates spread with the plasmid-containing cell lysate, indicating that 75% ethanol incubation and freeze–thaw cycles were efficient for sterilization, and no transformants obtained after transformation were mutants or contaminants.

The colony-to-lawn transformation method used for changing hosts of plasmid. (a) Outline of the colony-to-lawn transformation method. A single colony from plasmid donor strain is washed with 75% ethanol and air-dried, and then cells are suspended in CaCl2 solution and lysed by freeze–thaw cycles to obtain plasmid-containing cell lysate. At the same time, cells of plasmid recipient strain are scraped carefully from fresh lawn and suspended in ice-cold water. Then, the recipient cells and plasmid-containing cell lysate are mixed gently and performed transformation by heat shock method. The transformed bacteria are grown and selected by standard methods. (b) Comparison of the colony-to-lawn transformation method and the chemical transformation method. Plasmid extraction and competent cell preparation are essential steps for chemical transformation, but not necessary for colony-to-lawn transformation. (c) SDS-PAGE gel shows protein expression of P450-BM3 before and after changing hosts of pETM11-P450-BM3 either by colony-to-lawn transformation or by chemical transformation. Lane M: protein molecular weight marker lane 1, after host changing of pETM11-P450-BM3 with the chemical transformation method lanes 2, after host changing of pETM11-P450-BM3 with the colony-to-lawn transformation method lanes 3, before host changing of pETM11-P450-BM3 (i.e. protein expressed in Escherichia coli JM109). (d) The numbers of transformants obtained by changing hosts of various plasmids with the colony-to-lawn transformation method. Each value represents the mean of five independent experiments.

A process based on colony-to-lawn transformation and protein expression was designed and conveniently used to remove frameshift mutations during the construction of mutant library (Fig. 2a). Recombinant plasmids are constructed and transformed into E. coli cloning strain, followed by changing the hosts of plasmids from cloning strain to expression strain with the colony-to-lawn transformation method. Then, randomly selected transformants are cultured in auto-inducing media overnight. An aliquot of each culture is used to check protein expression by SDS-PAGE, and only the positive clones having expected protein expression are used to extract plasmids from their remaining cultures, and DNA sequencing or another round of mutagenesis was performed Although the current transformation methods can be used for the same purpose, the process should be less convenient, because more time and an additional experimental step (competent cell preparation) are needed (Fig. 2b). Additionally, more plasmids should to be extracted, because the clones used for plasmid extraction are before protein expression screening. In this work, the recombinant plasmids with random mutations of P450-BM3 gene introduced by error-prone PCR were used to test this method. The recombinant plasmids were changed hosts from cloning strain JM109 to expression strain BL21(DE3) with the colony-to-lawn transformation method. Then, ten randomly selected transformants were used to check protein expression levels, five of them were found to have expected protein expression. The plasmids were extracted and DNA sequencing was performed, and as a result, all the DNA sequences of positive clones were found to be in the correct open reading frames.

Protein expression in combination with the colony-to-lawn transformation method to screen in-frame clones from mutant library. (a) Outline of the experimental strategy. Plasmids from mutant library construction were changed hosts from cloning strain to expression strain with the colony-to-lawn transformation method. Then, the randomly selected transformants are checked for protein expression by SDS-PAGE. Plasmids are extracted for positive clones, and DNA sequencing or next round of mutagenesis was performed (shown as dotted line). (b) The chemical transformation method used for the same purpose. Plasmids are extracted from randomly selected clones after mutant library construction and transformed into competent cells of expression strain for protein expression and SDS-PAGE analysis. The plasmids extracted from the clones which have expected protein expression are used for DNA sequencing or next round of mutagenesis (shown as dotted line).


Recent studies have revealed that epigenetic genome methylation is associated with many aspects of life processes through effects on gene expression and other steps [1–3]. Especially, epigenetic methylation is involved in silencing of selfish genetic elements and other aspects of intragenomic conflicts. Experimental alteration of epigenetic DNA methylation systems can cause a wide variety of changes [4–8] for example, in Prokaryota, DNA methyltransferase action can change the transcriptome [7]. Horizontal gene transfer contributes considerably to the building up of prokaryotic genomes [9, 10]. In particular, the DNA methyltransferase genes frequently move between genomes [11–15] and could, therefore, present potential threats to prokaryotic genomes, although they can also be beneficial to bacteria in many ways, including in cell cycle regulation and cell differentiation [3, 8].

Prokaryotic DNA methyltransferases often form a restriction-modification (RM) system together with a restriction enzyme [16, 17]. Some RM systems behave as mobile elements, as suggested by their amplification, mobility, and involvement in genome rearrangements, as well as their mutual competition and regulation of gene expression [13–15, 18–21]. Some type II RM systems cleave chromosomes of their host cells when their genes are eliminated by a competitor genetic element [20, 22, 23], as illustrated in Figure 1a. Such host killing, called 'post-segregational killing' or 'genetic addiction', has been recognized to be involved in stable maintenance in many plasmids [24]. The RM systems have evolved regulatory systems to suppress their potential to kill the host. When they enter a new host, they prevent host cell killing by expressing their methyltransferase first and delaying expression of their restriction enzyme [19, 25–27].

Host killing by RM systems and by methyl-specific DNases (McrBC) in competition. (a) When a resident RM gene complex is replaced by a competitor genetic element, a decrease in the modification enzyme level results in exposure of newly replicated chromosomal restriction sites to lethal cleavage by the remaining restriction enzyme molecules. The intact genome copies will survive in uninfected neighboring clonal cells. (b) When a DNA methylation system enters a cell and begins to methylate chromosomal recognition sites, McrBC senses the change and triggers cell death by chromosomal cleavage. The intact genome copies will survive in uninfected neighboring clonal cells.

Host chromosome cleavage by RM systems is not trivial. In general, cleavage of chromosomes by cellular DNases is prevented in various ways: inhibitor binding, compartmentalization, proteolysis, DNA modification and DNA structure specificity. Indeed, host killing by RM systems after loss of their genes is not always obvious because hosts have apparently adapted to counteract it in various ways. Recombination repair of chromosomal breakage can reduce the lethal effects of chromosome cleavage [28]. Host killing by an RM gene complex is suppressed by a solitary methyltransferase recognizing the same sequence [29, 30]. Proteolytic digestion of restriction enzymes suppresses chromosome cleavage by EcoKI, a type I RM system, even in the absence of the cognate methyltransferase [31]. These host defense systems against RM systems cannot, however, avoid host genome methylation and its potentially deleterious effects.

In the present work, we provide evidence for the existence of a group of genetic elements that compete with epigenetic DNA methylation systems (for example, with DNA methyltransferases from RM systems) through host cell killing. These anti-methylation elements are methyl-specific endodeoxyribonuclease McrBC of Escherichia coli [32] and its homologs. McrBC cleaves DNA between two separate R m C (R = A or G, m C = m4 C or m5 C) sites in vitro [33], which are modified by many DNA methyltransferases from different RM systems [16, 17]. This activity was first recognized for restriction of incoming bacteriophage genomes carrying hydroxymethylcytosine instead of cytosine [34, 35]. McrBC may also protect cells against infection by methylated DNA elements, such as viral genomes and plasmids, through such direct cleavage. However, such methylated DNAs are not usually strongly restricted by McrBC [36, 37] therefore, we hypothesized that McrBC may mediate suicidal defense in response to epigenetic genome methylation systems, such as RM systems, as illustrated in Figure 1b. When such a system enters the cell and begins to methylate the host genome, McrBC would sense these epigenetic changes and trigger cell death through chromosomal cleavage. Intact (unmethylated) genomes with mcrBC genes would survive in the neighboring clonal cells.

Defense against invasion of genetic elements through cell death, as illustrated in Figure 1a,b, has been reported for multicellular eukaryotic cells, such as virus-infected mammalian cells and plant cells [38]. Similar phenomena against virus infection have been known for bacteria under the name of 'phage exclusion' or 'phage abortion' [39]. Bacteriophage reproduction is aborted by the action of a cell death gene. As a result, this gene would survive within the clonal cells that would, otherwise, all die by secondary infection. For example, the prr gene in some Escherichia coli strains senses bacteriophage T4 infection and triggers cell death by cleaving host tRNA Lys [40].

We first asked whether McrBC-mediated cell death through cleavage of methylated chromosomes takes place upon entry/induction of a methyltransferase gene and aborts its establishment/activation. After obtaining positive experimental results, we asked how important this role has been in the spread and maintenance of McrBC genes. Our analyses of their molecular evolution and genomic contexts support the hypothesis that, during evolution, they have behaved as mobile elements. Taken together, these results support our hypothesis that McrBCs have evolved as mobile elements that compete with specific genome methylation systems through host killing.


The most important conclusions from our results can be summarized as follows: (i) two new, small, early T4 proteins, RepEA and RepEB, encoded in the oriE region, are important for T4 DNA replication (ii) RepEB is specifically important for the priming of leading-strand DNA synthesis at oriE, whereas RepEA appears to have an auxiliary function, but neither protein is required for transcription from the early promoter (iii) neither expression of repEA or repEB nor initiation of DNA replication at oriE requires the activator of middle promoters, MotA protein (iv) the early transcripts encoding RepEA and RepEB, which can also serve as primers for leading-strand DNA synthesis, are short-lived.

These results suggested to us the following working hypothesis for initiation of DNA replication from oriE: we propose that one or both of the new repE proteins facilitates loading of helicases to oriE DNA (e.g., to the iteron DNA) and that tracking of these helicases modulates the status of some oriE transcripts (i.e., whether they are terminated or processed and whether they are base paired to DNA, as required for priming, or displaced from the DNA, as required for translation). Experimental tests of this working hypothesis will undoubtedly demand modifications and extensions.

OriE-specific transcripts and proteins are synthesized only briefly after infection.

T4 uses transcripts to prime leading-strand DNA synthesis at origins (4, 35, 39, 44, 48). Formation of primers must include steps by which the nascent RNA is kept base paired or is reinserted into the DNA duplex. Partial digestion of transcripts may occur, and it may or may not be obligatory for priming. Within the framework of our working hypothesis, one or both of the two new RepE proteins synthesized from oriE transcripts facilitates the access of helicases, which in turn facilitates the base pairing of a sister transcript with the DNA template, the priming of DNA synthesis, and perhaps transcription termination, at a distance.

Database searches provided no clue for the functions of RepEA and RepEB proteins. Moreover, RepEA and RepEB proteins, with or without His tags, turned out to be largely insoluble. However, RepEA and RepEB, when fused to maltose-binding protein, are soluble. They bind to single-stranded DNA, preferentially to the iterons marked in Fig. ​ Fig.1 1 and ​ and2 2 (70), suggesting that they might facilitate the opening of double-stranded DNA, like origin proteins of other replicons (32, 37). In contrast to origins of other replicons, in T4 DNA the primary targets of such proteins, the iterons, are far away from the major priming sites for leading-strand DNA synthesis (Fig. ​ (Fig.1 1 ).

The presence of iterons in T4 oriE (Fig. ​ (Fig.2) 2 ) was unexpected at first. Based on bacteria, phage, and plasmid paradigms (15, 17, 32), it is generally assumed that iterons are hallmarks of origins in which primers for leading-strand DNA synthesis are synthesized by primases, which require single-stranded segments for binding to DNA. As mentioned earlier, primers for T4 origin of initiation of leading DNA strands are, however, synthesized by RNA polymerase (4, 39, 48).

Within the framework of our working hypothesis, migration of helicases loaded at the iterons would modulate the status of oriE transcripts (i.e., whether the RNA is base paired to the DNA or to be displaced) far from their target sites. This might depend on competitions between several helicases, (e.g., replicative helicase gp41 孖, 57], the Rho RNA helicase 孥, 66], or the UvsW helicase which is, however, a late protein 嬐]).

Loading of the replicative T4 helicase gp41 by one or both of the RepE proteins can explain another puzzling observation. The gp41 protein, which associates with T4 primase, requires the helper protein gp59 in vitro (56). However, in vivo, gene 59 mutants are proficient in origin DNA replication, and they arrest DNA replication like recombination-deficient T4 mutants (47, 72), suggesting that other proteins load helicases at origins.

The early cessation of transcription through oriE must be a major reason for early cessation of origin-dependent DNA replication (39) at this origin. The small window of time when repEA and repEB are expressed is sufficient to allow a single or few initiations from oriE, but apparent lability of PE1-dependent transcripts and of the RepEB protein, as well as the synthesis of antisense RNA from the late promoters, are all bound to contribute to the apparent early demise of oriE function during phage T4 development.

The early cessation of transcription from PE1 is readily explained by the accumulation of the T4 AsiA protein. This protein sequesters the recognition motif for the � region of ς 70 , and, therefore, inhibits transcription from promoters containing consensus � sequences, such as PE1 (13, 64). Most other early T4 promoters have variant � sequences (71) and may not be subject to similarly strong asiA-dependent inhibition. The apparent lability of early oriE transcripts (Fig. ​ (Fig.3) 3 ) may be related, in part, to possible base pairing with the late antisense transcripts synthesized from the same region.

We do not know whether the DNA priming sites within oriE (examples are shown in Fig. ​ Fig.7A 7 A and B) correspond to transcription termination sites. Unlike oriA (40) and oriF (21, 22, 48), oriE does not contain a classical factor-independent transcription terminator. Transcription termination factor Rho (66) may be required, and the anomalous priming sites of repEA mutant DNA (Fig. ​ (Fig.7B) 7 B) might suggest the possibility that the T4 RepEA protein may also be involved. Distinguishing features of rho-dependent transcription termination sites are largely unknown apparent 3′ transcript ends near palindromes have been attributed to posttranscriptional decay that stops at RNA hairpins (59, 61).

The untranslated RNA segment between repEA and repEB is unusually large for early T4 transcripts. It is intriguing to note that this untranslated RNA corresponds to a DNA segment encoding the lysozyme segment of the base plate protein gp5 (53). The amino acid sequence of this segment resembles that of the soluble T4 lysozyme gpe, suggesting that a copy of this or a related gene was inserted into an ancestor of the base plate gene 5 during evolution. Such an insertion would have increased the distance between the repEA and repEB genes on the complementary DNA strand (Fig. ​ (Fig.2 2 ).

RepEA and repEB mutants have different phenotypes.

The residual replication of the rep motA double mutants at late times after infection and the residual progeny production may be due to residual initiation of transcription from motA-dependent promoters (12, 22, 35), initiation from other origins (31, 67, 73), and/or, most likely, from recombinational intermediates. Recombinational intermediates can be formed in the absence of prior DNA replication, albeit with a much longer delay than when DNA replication is allowed (8, 14, 44).

However, the different phenotypes of the repEA motA and repEB motA double mutants at different growth temperatures (Fig. ​ (Fig.6) 6 ) require additional comments. One possible scenario to explain why the repEA1 mutant is more leaky at high temperatures than at low temperatures (Fig. ​ (Fig.6) 6 ) postulates a role for RepEA protein, predicted to be hydrophobic, in membrane attachment of T4 DNA. Such membrane association has been observed by many investigators in T4 (18, 19, 29, 30, 41, 43) and is well demonstrated in other organisms (38, 58). This association may be important but not essential (33), since membrane-free in vitro systems can synthesize DNA under optimal conditions with speed similar to that of in vivo systems (2, 32, 56, 57). Another, and possibly related, explanation for the leaky phenotype of the repEA1 mutant at 42ଌ (Fig. ​ (Fig.5B) 5 B) is that in this mutant T4 primase and/or topoisomerase activities are impaired, and that this deficiency is bypassed by a temperature-dependent recombinational mechanism (50, 52, 54) that requires, among others, T4 endonuclease VII. The T4 topoisomerase is one of several membrane-bound T4 proteins (29, 30).

Different replication origins of T4 have different sequences, different structures, and different requirements for functioning.

The functioning of RepE proteins, the presence of iterons, and the absence of a motA requirement for oriE distinguish this origin from oriA and oriF, suggesting that T4 origins use different initiation mechanisms, which may allow functioning under different conditions. For example, oriE is preferentially used when torsional stress in the replicating T4 DNA is reduced by mutations in T4 topoisomerase and host gyrase or by excessive damage due to 32 P decay or oxidation (reference 55 and unpublished observations). oriE is also preferred when T4 infects certain host mutants (nusD) altered in transcription termination factor Rho (67).

These differences, as well as comparisons with T4-related phages (44), support the hypothesis that the T4 genome was assembled during evolution from modular components from several sources (9) and that such processes can generate novel and redundant origins of DNA replication, redundant replication proteins, and complex overlapping transcription patterns (26, 44, 49, 51). The redundancies, in turn, can provide selective advantages under different developmental and environmental conditions because they facilitate coordination of essential viral processes and adjustments of DNA replication and gene expression to different growth conditions (44, 48). Moreover, timed transcription from complementary strands of the same DNA can help to adjust DNA replication and other DNA transactions to optimal growth and progeny production.

Such redundancies are bound to enhance the viability of any organism under different environmental or developmental conditions. The multiple T4 origins may serve as models for the complexities of multiple origins in metazoan chromosomes (16), which may differ in structure and function for reasons similar to those of T4.

Watch the video: Make recombinant plasmids-transform bacteria (May 2022).