The amount of cells plated should produce a sufficient and also not too numerous number of individual, distinct colonies for further screening. Cells cultured in S. If very few colonies are anticipated, the entire cell suspension may be plated. However, if a very high number of colonies is expected, the cell suspension may be diluted up to in S. Even distribution of the cells on the agar plate is critical for analysis of the colonies. A sterile hockey-stick or L-shaped cell spreader is commonly used to spread the cell suspension while gently rotating the plate Figures 6, 7A.
Avoid puncturing the agar surface while spreading the cells. Alternatively, autoclaved glass beads 4 mm diameter may be used to spread the cells. In this approach, 10 to 20 beads are placed on the plate after applying the cell suspension, and the plate is gently swirled so that the cell suspension is spread by the beads Figure 7B. Cells must be spread quickly before the liquid suspension dries. The culture plates are examined the next day for colony formation. Prolonged incubation should be avoided, as it often results in fusion of large colonies and the appearance of smaller, antibiotic-sensitive surrounding colonies called satellite colonies due to antibiotic breakdown around large colonies.
To calculate the transformation efficiency, divide the number of transformants by the amount of DNA added, and factor in cell dilution if performed , using the following formula:. Colonies need to be further screened for the presence of the desired plasmid and correct sequence as necessary see colony screening methods.
Once confirmed, desired colonies may be employed in downstream applications such as plasmid isolation , subcloning , transfection , and protein expression. Don't have an account? Create Account. Sign in Quick Order. Search Thermo Fisher Scientific. Search All. Bacterial Transformation Workflow—4 Main Steps.
See Navigation. The four key steps in bacterial transformation are:. Preparation of competent cells Transformation Cell recovery period Cell plating. Figure 1. Key steps in the process of bacterial transformation: 1 competent cell preparation, 2 transformation of cells, 3 cell recovery, and 4 cell plating. Heat-shock transformation: Competent cells are chemically prepared by incubating the cells in calcium chloride CaCl 2 to make the cell membrane more permeable [1,2]. Electroporation: The harvested cells are washed with ice-cold deionized water several times by repeated pelleting and resuspension to remove salts and other components that may interfere with electroporation.
Figure 2. Preparation of chemically competent and electrocompetent cells. Overview of chemical transformation. Figure 3. Bacterial transformation using A chemically competent cells and heat shock, and B electrocompetent cells and electroporation.
Figure 4. A Exponential decay of electric pulse. B Electroporation process. The growth arrest activity was not the result of cell killing because dead cells by PI stained did not increase on addition of the conditioned medium data not shown. These results suggest that CAG produces and releases a soluble factor that can promote cell-to-cell transformation and arrest growth of other E.
To examine whether the soluble factor in medium conditioned with CAG is a protein or peptide, we performed the following three experiments. Furthermore, size fractionation Fig. These results suggest that the factor responsible for both promotion of cell-to-cell transformation and growth arrest is a protein or a polypeptide.
The minimum active concentration of CAGconditioned medium was examined by diluting this medium. Surprisingly, the medium was effective up to the dilutions of 10 —5 —10 —6 in promoting cell-to-cell transformation Fig.
Growth arrest activity also occurred at low concentrations but it was weaker and almost lost at the dilution of 10 —5 Fig. These results suggest that the factor present in CAGconditioned medium acts as a bioactive signal factor like a pheromone, which can transduce specific signals from certain cells to other cell populations at extremely low concentrations.
From the above results, we drew the following two conclusions: 1 spontaneous lateral plasmid transfer in mixed E. The occurrence of cell-to-cell transformation was deduced from the following three lines of evidence: 1 lateral plasmid transfer was decreased by degrading extracellular DNA in culture with DNase I treatment Fig. Although natural transformation in E. Despite the growing numbers of examples of natural transformation in E.
At the culture level, several reports [20] , [24] including ours [23] , [30] , [31] suggested that natural transformation in E.
This study showed for the first time that natural transformation in E. Therefore, it was revealed that solid culture is not essential for efficient natural transformation in E. However, some results cannot be explained solely based on the action of such ions [19] , [22] , [23] , [25] , [31]. At the genetic level, recently, the com gene homologues, which are reported to be involved in natural transformation in other Gram-negative bacteria [10] , were also found in E.
However, our results were obtained under nutrient-rich conditions and we therefore think that the involvement of com -like genes in cell-to-cell transformation is unlikely. The type IV secretion system T4SS is also known to be involved in natural transformation in Gram-negative bacteria [10] , [45] , [46]. However, the E. Therefore, the involvement of T4SS in cell-to-cell transformation is unlikely.
Besides, as shown in Fig. Therefore, we postulate that cell-to-cell transformation may occur through a mechanism different from artificial transformations. It is possible that natural transformation in E. This possibility has also been proposed by Sun et al. We postulated that plasmid DNA was probably supplied naturally from dead cells through cell disruption because this is the most natural manner of DNA supply in culture.
Consistent with this idea, a small amount of extracellular plasmid DNA and dead cells was detected in the culture medium Table 5 and Fig. We performed a preliminary test of the ability of dead cells as the source of transformed DNA, and found that the plasmid DNA included in dead cells could be transformed by cells in culture, at least in colony biofilm culture data not shown.
However, since artificial cell-killing manipulations often damage DNA and proteins, a clear demonstration of the involvement of dead cell DNA would require further carefully planned experiments. Regarding the requirement for cell-derived DNA for cell-to-cell transformation to occur, the results of Tsen et al. They reported that natural transformation in E. Their result suggests that E. A variety of materials can be released from dead cells, and some of them such as DNA-binding proteins and lipopolysaccharide have the abilities to associate with DNA [48] , [49].
The preferred transfer of pHSG Table 2 may suggest the presence of specific sequence s that promotes cell-to-cell transformation by binding of such DNA-associating molecule s. Alternatively or additionally, a DNA conformation specific to living cells, such as supercoiling, may also be a requirement for the substrate of cell-to-cell transformation.
DNA secretion as a physiological process of living cells has been proposed in several bacterial systems [53]. This may be another possible DNA-supply mechanism in cell-to-cell transformation. Presently, we have no additional evidence to clarify the mechanism of DNA release from donor cells or the role of dead or living donor cells.
Including these points, the detailed molecular mechanism of cell-to-cell transformation should be investigated further. In this study, we also suggested for the first time the presence of a novel pheromone-like factor that promotes cell-to-cell natural transformation in E. Bacterial pheromones such as AHLs are generally known to work at nM concentrations [54].
Although presently we do not know the exact concentration of the promoting factor in our experimental system, our detection of this activity in conditioned medium diluted to 10 —6 Fig.
Our data on heat sensitivity, estimated molecular mass and protease sensitivity Figs. No peptide factors showing similar activity have been identified in E. Because of the presence of an outer membrane in Gram-negative bacteria, it is believed that polypeptide-type factors cannot transmit signals easily from the outside of cells. However, a few reports postulate the presence of peptide pheromones in Gram-negative bacteria [55] , [56]. In Gram-positive bacteria, several competence factors or pheromones were found to be peptide factors [12] — [15].
These data support an idea that a peptide-type competence pheromone may also be present in E. If the effect is on donor cells, the factor may promote the release of plasmid DNA from donor cells. However, since we did not find a cell-killing activity for the putative factor, such a scenario is unlikely to be involved in cell-to-cell transformation.
Alternatively, in donor cells the factor may up-regulate unknown DNA-associating molecule s that can promote uptake by recipient cells when they are released together with plasmid DNA. This idea seems to be consistent with our finding of a requirement for cell-derived DNA in cell-to-cell transformation. Besides, we found growth arrest activity in medium conditioned with CAG Fig.
Although this activity and the activity promoting cell-to-cell transformation behaved similarly toward physical and biochemical treatments Figs. The transformation-promoting activity appears to be effective on CAG itself because CAG can exhibit high activity as both donor and recipient Tables 2 and 3.
However, the growth-arrest activity appears to be ineffective on CAG itself because CAG growth in sole culture is apparently normal. Therefore, these two effects may act on cells independently. The target s , the ranges of actions and the working mechanisms of this putative pheromone are to be investigated further. Based on the results in Table 7 , unidentified mutation s in CAG may cause expression of pheromone activity.
Our preliminary study in progress suggests that a few other E. Therefore, production of this pheromone may not be specific to CAG The identity and the gene for this pheromone as well as the responsible mutation in the CAG chromosome should be investigated. It is noteworthy that, under optimal conditions, cell-to-cell transformation occurred as frequently as artificial transformations Fig.
This means that cell-to-cell transformation in E. In this respect, further experiments using natural strains of E. Furthermore to our results, other recent results [18] — [20] , [24] , [58] suggest that non-conjugative plasmids are more mobile than was previously believed. Reevaluation of plasmid dynamics in various environments is needed to confirm this possibility [4] — [6]. The E. The following E. Distilled water DNase- and RNase-free, molecular biology grade and kanamycin kan were from Invitrogen.
Nylonmembrane filter pore size: 0. Syringe filters for sterilisation pore size: 0. Agar powder guaranteed-reagent grade , proteinase K, trypsin, and other general reagents were from Wako. Lateral plasmid transfer experiments in a colony-biofilm system were performed as described previously [30] , [31]. However, the protocol was modified slightly based on tentative data from studies of the experimental conditions. The colony biofilms that formed were collected and spread onto LB agar plates containing two antibiotics to select recipient cells that had acquired plasmids.
The occurrence of lateral plasmid transfer was detected by the appearance of double-resistant transformants. Transformation frequency was calculated as the ratio of the transformant number to the estimated recipient cell number, which was regarded as half of the total cell number in each sample. The total cell number in each sample was deduced from the OD value of the cell suspension just before plating.
Lateral plasmid transfer experiments in liquid culture were performed by following the same protocol as that for colony biofilm experiments, except that cell-mixed culture was performed in 1 mL TSB with shaking. The cell mixture after culture was diluted and plated on antibiotic-free LB agar.
This ratio was used in calculating the recipient cell number; the resultant value was used for calculation of plasmid-transfer frequency described above. Exceptionally, in the case of co-culture of MG harbouring pHSG and MG harbouring pGBM1, the total cell number was regarded as the recipient cell number, because all the cells can act as recipient cells.
Filter-mediated plasmid transfer experiments in colony biofilm culture were performed using a protocol similar to that used for simple colony biofilm experiments.
The recipient cells were then recovered from the filter and plated on LB agar plates containing two antibiotics to select recipient cells that had acquired plasmids. Dead cell numbers, which were stained with PI, and total cell numbers were counted by phase-contrast microscopy and fluorescent microscopy excitation, nm; emission, nm , respectively. Cultured medium samples each 1 mL were centrifuged and filtered using the same protocol as that used for conditioned medium described below.
Natural transformation experiments in liquid culture were performed as follows: E. The PEG method was performed as described by Chung et al. The general mechanism underlying DNA transfer during natural transformation is summarized in Figure 1.
Classical DNA uptake during natural transformation. Exogenous DNA is pulled into the cytoplasm by the extension and retraction of pseudopili, as a consequence of the assembly and disassembly of pseudopilin multimers PilA.
The assembly and disassembly of a type IV pilus causes a fiber-like pseudopilus to be extruded out of and hauled back into the pore-forming OM proteins Chen and Dubnau, The pore that accommodates exogenous DNA is 6—6. In this process, one strand of the dsDNA is translocated into the cytoplasm, simultaneously the degradation of the other strand occurs Barouki and Smith, ; Berge et al. Bacterial conjugation was first discovered in E. Relying on cell-to-cell contact, DNA can be pushed out of a donor cell and transported into a recipient cell during bacterial conjugation.
A group of modular mobile genetic elements, known as integrative and conjugative elements ICEs or conjugative transposons Franke and Clewell, , has been found in many bacterial genomes Wozniak and Waldor, ; Bi et al.
The transfer of conjugative DNA across the membrane of the donor bacterium relies on a large membrane-associated protein complex, that belongs to the type IV secretion system T4SS Goessweiner-Mohr et al. The mechanism of DNA transfer is summarized in Figure 2. DNA transfer during bacterial conjugation. Inside the channel, the conjugative pilus formed by pilins is responsible for pushing ssDNA out of membrane.
The mechanism of DNA transfer via conjugation has been best exemplified by the Vir system. To export DNA out of the donor cell, a conjugative plasmid encodes a complicated membrane protein complex. During conjugation, a plasmid- or ICE-encoded relaxase creates a nick in one strand of the conjugative DNA at the oriT site, followed by ssDNA translocation across the channel formed by components of the T4SS and replication of the remaining strand, either independently from or in concert with conjugation Ilangovan et al.
In natural transformation and conjugation, different types of pili participate in the movement of DNA. Competence pili or pseudopili mediate the transfer of dsDNA across the membrane during natural transformation, whereas conjugative pili mediates the transfer of ssDNA across the membrane during conjugation Cabezon et al. It remains unclear how conjugative DNA is further transported in the recipient cell.
In this century, the natural transformation of E. Although natural plasmid transformation of E. First, natural transformation is promoted by an increased concentration of agar, whereas chemical transformation relies on high concentrations of divalent ions i. Third, exponentially growing E. Natural transformation of E. Nevertheless, attempts to confirm these observations in three independent laboratories have not succeeded Sun, ; Johnston et al.
DNA, which has been thought to serve as the sole carbon source could not account for cell growth, implying that other nutrient sources should be present in the culture Sun, ; Johnston et al.
It is possible that degraded DNA in the minimal culture serves as a source of building blocks for the synthesis of new DNA in bacteria.
During the natural transformation of E. These transporters are different from the known classical DNA uptake proteins that mediate natural bacterial transformation. The mechanism of this new type of DNA transfer is proposed in Figure 3. A new route for dsDNA transfer in Escherichia coli.
It remains unclear whether a plasmid enters E. Escherichia coli has a complete set of genes that potentially encode components of the classical DNA uptake machinery. These genes are homologous to the conserved DNA uptake genes in other naturally transformable bacteria. Comparative genomic analysis predicts that, in E. OmpA is unlikely to form an open gate under natural conditions, but can be switched to the open state with the molecular force of electrostatic interaction i.
The closed and open states of the gate are dependent on the formation of salt bridges of ArgGlu52 and LysGlu, respectively Hong et al. During natural transformation of E.
The putative channel may be consisted of OM components i. Based on a membrane topology study, the conserved IM protein called ComEC is predicted to mediate the translocation of ssDNA during classical natural transformation Chen and Dubnau, ; Claverys et al. The single-hit kinetics in natural plasmid transformation of E. Inactivation of ydcS and ydcV reduces natural transformation 6.
Chemical transformation is also reduced by Inactivation of ydcS , whereas the chemical transformation in a ydcV mutant is not reduced as compared to its wild-type counterpart Sun, Nonetheless, with respect to significantly reduced transformation frequency in the ydcV and the ydcS mutants, ydcT seems to have only a minor effect on DNA transport. It is likely that additional energy source is required for efficient transport of dsDNA across the IM.
Cell-to-cell contact is often required for bacterial conjugation. Of note, plasmid transformation of E. Cell-to-cell contact-dependent plasmid transformation occurs not only within the same genus but also across genera Wang et al.
During cell-to-cell contact-dependent plasmid transformation of E. Screening of the Keio collection Baba et al. Because the homologs of DNA uptake genes e. Recently, cell-to-cell contact-dependent transformation is discovered in B. Classical mechanisms of HGT i. Recent studies have uncovered new types of DNA transfer, which are independent of the classical DNA uptake or conjugation machineries, suggesting that other therapeutic targets should be considered in the fight against ARGs.
On the other hand, the discovery of non-classical HGT mechanisms suggests that controlling the spread of ARGs is more complicated than previously thought. LY16C, Y The author declares that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. Aas, F.
Competence for natural transformation in Neisseria gonorrhoeae : components of DNA binding and uptake linked to type IV pilus expression. Assalkhou, R. Microbiology , — Baba, T.
Construction of Escherichia coli K in-frame, single-gene knockout mutants: the Keio collection. Baker, J. Proteins 84, — Barouki, R. Reexamination of phenotypic defects in rec-1 and rec-2 mutants of Haemophilus influenzae Rd. PubMed Abstract Google Scholar. Berge, M. Berka, R. Microarray analysis of the Bacillus subtilis K-state: genome-wide expression changes dependent on ComK. Bi, D. ICEberg: a web-based resource for integrative and conjugative elements found in bacteria. Nucleic Acids Res.
Burkhardt, J. Burton, B. Membrane-associated DNA transport machines. Cold Spring Harb. Cabezon, E.
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