Cell Transformation

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Introduction

Chang LS, Shenk T The adenovirus DNA-binding protein stimulates the rate of transcription directed by adenovirus and adeno-associated virus promoters. Chauvin C, Suh M, Remy C, Benabid AL Failure to detect viral genomic sequences of three viruses herpes simplex, simian virus 40 and adenovirus in human and rat brain tumors. Oncogene 6: — Google Scholar. Esche H Viral gene products in adenovirus type 2-transformed hamster cells. Esche H, Siegman B Expression of early viral gene products in adenovirus type infected and -transformed cells.

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Cell Absorbs #17 - Cell's First Transformation

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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].

Table of Contents

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. Although this activity and the activity promoting cell-to-cell transformation behaved similarly toward physical and biochemical treatments Figs.

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. Our preliminary study in progress suggests that a few other E. 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. Transformation frequency was calculated as the ratio of the transformant number to the recipient competent cell number.

Conditioned medium was prepared as follows. This culture solution was centrifuged g , 10 min , and the supernatant was filtered with a membrane filter pore size: 0. For size fractionation of the conditioned medium by ultrafiltration, the prepared conditioned medium was centrifuged in Amicon Ultra-4 3 K g , 30 min or Microcon Ultracel YM 10 K g , 30 min. According to the manufacturer's information, molecule sizes in flow-through fractions were regarded as 3-times larger than those described on the product labels. After centrifugation, the ultrafiltration membrane was washed onece with fresh TSB, and the residual materials retained on the membrane was withdrawn by pipetting with fresh TSB.

Competing Interests: The authors have declared that no competing interests exist. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript. National Center for Biotechnology Information , U. PLoS One. Published online Jan Jose Alejandro Chabalgoity, Editor. Author information Article notes Copyright and License information Disclaimer. Received Aug 2; Accepted Dec Copyright Etchuuya et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are properly credited.

This article has been cited by other articles in PMC. Abstract Escherichia coli is not assumed to be naturally transformable. Introduction Lateral gene transfer between bacterial cells contributes to bacterial adaptation to various environments and, in the long term, to bacterial evolution [1] — [3]. Results Comparison of lateral plasmid transfer in colony biofilm with various combinations of E. Table 1 E. Open in a separate window. Table 2 Lateral plasmid transfer with various combinations of strains and plasmids in colony biofilm culture.

Lateral plasmid transfer in liquid culture with high-frequency combinations of E. Table 3 Lateral plasmid transfer with various combinations of strains and plasmids in liquid culture. Demonstration of transformation mechanism for lateral plasmid transfer The most important criterion of transformation is the uptake of extracellular DNA by cells.

Figure 1. Effect of DNase I activity on lateral plasmid transfer and detection of plasmid in culture medium. Table 4 Lateral plasmid transfer through nylon membrane filter in colony biofilm culture. Detection of dead cells and free plasmid DNA in cultured medium An important premise of our hypothesis is that the transformed plasmid DNA source in cell-mixed culture is dead cells. Table 5 Measurement of the dead cell ratio in liquid and colony biofilm cultures. Natural transformation with purified plasmid DNA in liquid culture To further investigate the transformation mechanism, we performed an experiment in which purified plasmid DNA was added.

Table 6 Natural transformation with purified plasmid in liquid culture. Comparison of cell-to-cell transformation with artificial transformation To further examine the features of cell-to-cell transformation, we compared this transformation type with conventional artificial transformation Fig. Figure 2.


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Comparison of cell-to-cell transformation with artificial transformation. Figure 3. Effects of media conditioned with various strains on cell-to-cell transformation and on cell growth. Figure 4. Figure 5. Figure 6. Table 7 Effects of Tn10 and lacI mutations on cell-to-cell transformation. Discussion From the above results, we drew the following two conclusions: 1 spontaneous lateral plasmid transfer in mixed E.