Escherichia coli persisters in biofilm can perform horizontal gene transfer by transformation.

Persisters represent a subset of cells that exhibit transient tolerance to antimicrobials. These persisters can withstand sudden exposure to antimicrobials, even as the majority of normal cells perish. In this study, we have demonstrated the capacity of ampicillin-tolerant and alkali-tolerant persisters to execute horizontal gene transfer via in situ transformation within biofilms. Air-solid biofilms, comprising two Escherichia coli populations each with a distinct plasmid, were formed on agar media. They were treated with lethal doses of ampicillin or NaOH for 24 h, followed by a 1-min glass-ball roll. This process led to a high frequency of horizontal plasmid transfer (10-7-10-6 per cell) from dead cells to surviving persisters within the biofilms. Plasmid transfer was DNase-sensitive and also occurred by adding purified plasmid DNA to plasmid-free biofilms, demonstrating a transformation mechanism. This marks the first evidence of persisters' novel ability for horizontal gene transfer, via transformation.

Subminimal inhibitory concentrations of ampicillin and mechanical stimuli cooperatively promote cell-to-cell plasmid transformation in Escherichia coli.

Horizontal gene transfer (HGT) is a bacterial evolution tool for improved survival. Although several environmental stimuli induce or promote HGT, the diversity and complexity of the environmental factors have not been sufficiently elucidated. In this study, we showed that the biofilm culture of Escherichia coli at the air-solid interface in the presence of a subminimal inhibitory concentration (sub-MIC) of ampicillin (∼0.5-4 µg/mL) and subsequent mechanical stimulation (rolling small glass balls, ø = 5-8 mm) cooperatively promoted horizontal plasmid transfer without the usual competence-inducing conditions. Either of the two treatments promoted plasmid transfer at a lower frequency than when the treatments were combined. The effect of several parameters on the two treatments was tested and then optimized, achieving a high frequency of plasmid transfer (up to 10-6 per cell) under optimal conditions. Plasmid transfer was DNase-sensitive for both treatments, demonstrating its mechanism of transformation. Plasmid transfer occurred using various E. coli strains, plasmids, ball materials, shaking conditions, and even the mechanical stimulation of brushing the biofilm with a toothbrush, indicating the conditional flexibility of this phenomenon. This is the first demonstration of the promoting effect of the combination of a sub-MIC antibiotic and mechanical stimulation on horizontal plasmid transfer between E. coli cells via transformation. Regarding environmental bacterial physiology, the aggregations or biofilms of bacterial cells may encounter both sub-MIC antibiotics and mechanical stimuli in some specific environments, therefore, this type of HGT could also occur naturally.

Bovine serum promotes the formation and phenotype memory retention of persister cells in Escherichia coli liquid cultures.

Persister cells, or persisters, are a fraction of bacterial cells that have become temporarily tolerant to antibiotics despite their lack of typical antibiotic-resistant genes. In a previous study, we found that colony-biofilm culture (i.e., biofilm formed at an air-solid interface) promoted the formation and phenotype memory retention of persisters of Escherichia coli and other bacteria. To assess whether these same effects are caused by other types of stimuli that bacterial cells encounter in the environment, we examined the effects of bovine serum on the formation and phenotype retention of ampicillin-tolerant persisters in E. coli liquid culture. Bovine serum did indeed exert these effects significantly, and its effects were negated by heat treatment. Similar effects were observed with bovine serum albumin, albeit weaker than those of BS. Given that serum is a component of blood and lymph and is thus a general substance within animal and human bodies, our findings suggest that bacteria encountering these body fluids may enhance their abilities for persister formation and phenotype memory retention to allow their longer survival in antibiotic-containing environments.

Natural Transformation in Escherichia coli.

Escherichia coli is well known for its ability to undergo artificial transformation. This bacterium is not generally recognized as naturally transformable. However, several past studies have demonstrated that laboratory and natural strains of E. coli can express modest natural competence under feasible environmental conditions. Here we describe a protocol outlining natural transformation in E. coli in environmental media such as natural freshwater.

High temperatures promote cell-to-cell plasmid transformation in Escherichia coli.

Bacteria continuously change their genetic characteristics to adapt to the changing environment by means of horizontal gene transfer. Although three conventional mechanisms of horizontal gene transfer are well known (transformation, transduction, and conjugation), new variations of these mechanisms have also been described. We previously reported that DNase-sensitive cell-to-cell transfer of non-conjugative plasmids, termed as "cell-to-cell transformation," occurs between the cells of two Escherichia coli strains in a co-culture. In this study, to further investigate the mechanism of cell-to-cell transformation, we constructed a new experimental system for cell-to-cell transformation. By using this system, we found that high temperatures of approximately 41ºC-45 °C significantly promote cell-to-cell plasmid transformation. This transfer was much more frequent in solid-air biofilms than in liquid culture, suggesting an importance of biofilm environment. Plasmid transfer frequency reached over 10-7/cell under the optimal strain-plasmid combination and conditions tested. DNase sensitivity test and plasmid isolation from the transformants confirmed the horizontal transfer of full-length plasmids via transformation. Comparative natural transformation experiments, which used similar strains and plasmids under equivalent culture conditions, revealed that cell-to-cell transformation occurs approximately 103 times more frequently than natural transformation, indicating the uniqueness and effectiveness of the cell-to-cell transformation mechanism. As temperatures of approximately 41ºC-45 °C are common in the avian intestines and under some other environmental situations, the phenomenon demonstrated here can occur efficiently in such locations. To the best of our knowledge, this is the first study to demonstrate the enhancing effect of high temperatures on cell-to-cell plasmid transformation in E. coli.

Horizontal Plasmid Transfer by Transformation in Escherichia coli: Environmental Factors and Possible Mechanisms.

Transformation is one mode of horizontal gene transfer (HGT) in bacteria, wherein extracellular naked DNA is taken up by cells that have developed genetic competence. Sensitivity to DNase, which degrades naked DNA, is the key to distinguishing transformation from the DNase-resistant HGT mechanisms. In general, Escherichia coli is not believed to be naturally transformable; it develops high competence only under artificial conditions, including exposure to high Ca2+ concentrations. However, E. coli can reportedly express modest competence under certain conditions that are feasible in natural environments outside laboratory. In addition, recent data suggest that environmental factors influence multiple routes of transformation. In this mini review, we (1) summarize our studies on transformation-based HGT using E. coli experimental systems and (2) discuss the possible occurrence of transformation via multiple mechanisms in the environment and its possible impact on the spread of antibiotic resistance genes.

Bacterial Memory of Persisters: Bacterial Persister Cells Can Retain Their Phenotype for Days or Weeks After Withdrawal From Colony-Biofilm Culture.

Persister cells, or persisters, are a specific subpopulation of bacterial cells that have acquired temporary antibiotic-resistant phenotypes. In this study, we showed that Escherichia coli produces many more persister cells in colony-biofilm culture than in the usual liquid culture and that these persisters can be maintained in higher numbers than those from liquid culture for up to 4 weeks at 37°C in a fresh, nutrient-rich, antibiotic-containing medium, even after complete withdrawal from the colony-biofilm culture. This suggests the presence of a long-retention effect, or "memory effect", in the persister cell state of E. coli cells. We also discovered that such increases in persisters during colony-biofilm culture and their memory effects are common, to a greater or lesser degree, in other bacterial species. This is true not only for gram-negative bacteria (Acinetobacter and Salmonella) but also for gram-positive bacteria (Staphylococcus and Bacillus). This is the first report to suggest the presence of a common memory mechanism for the persister cell state, which is inscribed during colony-biofilm culture, in a wide variety of bacteria.

Bacteriophage P1vir-induced cell-to-cell plasmid transformation in Escherichia coli.

Bacteria undergo horizontal gene transfer via various mechanisms. We recently reported that cell-to-cell transfer of nonconjugative plasmids occurs between strains of Escherichia coli in co-cultures, and that a specific strain (CAG18439) causes frequent plasmid transfer involving a DNase-sensitive mechanism, which we termed "cell-to-cell transformation". Here we found that CAG18439 is a type of P1 bacteriophage lysogen that continuously releases phages. We tested the ability of P1vir bacteriophage to induce horizontal plasmid transfer and demonstrated that such a horizontal plasmid transfer was caused by adding culture supernatants of P1vir-infected cells harboring plasmids to other plasmid-free cells. This plasmid transfer system also reproduced the major features of plasmid transfer involving CAG18439, suggesting that P1vir-induced plasmid transfer is equivalent or very similar to plasmid transfer involving CAG18439. We further revealed that approximately two-thirds of the P1vir-induced plasmid transfer was DNase-sensitive, but that complete abolition of plasmid transfer was observed when proteins were denatured or removed, despite the presence or absence of DNase. Therefore, we concluded that P1vir-induced plasmid transfer is largely due to the occurrence of cell-to-cell transformation, which involves the assistance of some proteinaceous factor, and partly due to the occurrence of plasmid transduction, which is mediated by phage virions. This is the first demonstration of the P1-phage-induced cell-to-cell transformation.

Natural Escherichia coli strains undergo cell-to-cell plasmid transformation.

Horizontal gene transfer is a strong tool that allows bacteria to adapt to various environments. Although three conventional mechanisms of horizontal gene transfer (transformation, transduction, and conjugation) are well known, new variations of these mechanisms have also been observed. We recently reported that DNase-sensitive cell-to-cell transfer of nonconjugative plasmids occurs between laboratory strains of Escherichia coli in co-culture. We termed this phenomenon "cell-to-cell transformation." In this report, we found that several combinations of Escherichia coli collection of reference (ECOR) strains, which were co-cultured in liquid media, resulted in DNase-sensitive cell-to-cell transfer of antibiotic resistance genes. Plasmid isolation of these new transformants demonstrated cell-to-cell plasmid transfer between the ECOR strains. Natural transformation experiments, using a combination of purified plasmid DNA and the same ECOR strains, revealed that cell-to-cell transformation occurs much more frequently than natural transformation under the same culture conditions. Thus, cell-to-cell transformation is both unique and effective. In conclusion, this study is the first to demonstrate cell-to-cell plasmid transformation in natural E. coli strains.

Genome-wide screen for Escherichia coli genes involved in repressing cell-to-cell transfer of non-conjugative plasmids.

Acquiring new genetic traits by lateral gene transfer is a bacterial strategy for environment adaptation. We previously showed that Escherichia coli could laterally transmit non-conjugative plasmids in co-cultures containing strains with and without the plasmid. In this study, using the Keio collection, a comprehensive library of E. coli knock-out mutants for non-essential genes, we screened for genes responsible for repressing cell-to-cell plasmid transfer in recipient cells. By stepwise screening, we identified 55 'transfer-up' mutants that exhibited approximately 2- to 30-fold increased activities. We confirmed plasmid acquisition by these 'up' mutants and revealed that there were no significant changes in antibiotic resistance in the original Keio strains. The presumed functions of these gene products covered a wide range of activities, including metabolism and synthesis, transport, transcription or translation and others. Two competence-gene homologues (ybaV and yhiR) were identified from among these genes. The presumed localizations of these 55 gene products were estimated to be 34 cytoplasmic proteins, 20 in or around the cell surface and 1 unknown location. Our results suggest that these 55 genes may be involved in repressing plasmid uptake during cell-to-cell plasmid transfer.

Genome-wide screening of Escherichia coli genes involved in execution and promotion of cell-to-cell transfer of non-conjugative plasmids: rodZ (yfgA) is essential for plasmid acceptance in recipient cells.

Acquisition of new genetic traits by horizontal gene transfer is a bacterial strategy for adaptation to the environment. We previously showed that Escherichia coli can transmit non-conjugative plasmids laterally in a co-culture containing strains with and without the plasmid. In this study, using the Keio collection, a comprehensive library of E. coli knock-out mutants for non-essential genes, we screened for genes responsible for the execution and promotion of cell-to-cell plasmid transfer in recipient cells. By stepwise screening of 'transfer-down' mutants, two essential genes and six promoting genes were obtained. One of the essential genes was priA, which is involved in DNA replication. This priA mutant was also unable to be transformed by artificial transformation methods, probably due to the deficiency of the plasmid maintenance function. The other essential gene was rodZ (yfgA), a gene involved in the regulation of rod-shaped structure of E. coli cells. This rodZ mutant was transformable by all three methods of artificial transformation tested, suggesting that this gene is essential for cell-to-cell plasmid transfer but not for artificial transformation. These are the first data that suggest that rodZ plays an essential role in DNA acquisition.

Identification of a novel DNA element that promotes cell-to-cell transformation in Escherichia coli.

Recently, we discovered a novel phenomenon, "cell-to-cell transformation" by which non-conjugative plasmids are transmitted horizontally in co-cultures of Escherichia coli F(-) strains. In this study, we aimed to identify the DNA element responsible for the high cell-to-cell transformability of pHSG299. By transplanting pHSG299 DNA fragments into pHSG399, a plasmid showing low transformability, we discovered that a specific 88 bp fragment of pHSG299 significantly promoted pHSG399 transformability. Although several short motif-like repetitive sequences (6-10 bp) were present in the 88 bp sequence, no known DNA motifs were recognized, suggesting that this 88 bp sequence (cell-to-cell transformation promoting sequence, CTPS; Accession number: AB634455) is a novel DNA element.

Cell-to-cell transformation in Escherichia coli: a novel type of natural transformation involving cell-derived DNA and a putative promoting pheromone.

Escherichia coli is not assumed to be naturally transformable. However, several recent reports have shown that E. coli can express modest genetic competence in certain conditions that may arise in its environment. We have shown previously that spontaneous lateral transfer of non-conjugative plasmids occurs in a colony biofilm of mixed E. coli strains (a set of a donor strain harbouring a plasmid and a plasmid-free recipient strain). In this study, with high-frequency combinations of strains and a plasmid, we constructed the same lateral plasmid transfer system in liquid culture. Using this system, we demonstrated that this lateral plasmid transfer was DNase-sensitive, indicating that it is a kind of transformation in which DNase-accessible extracellular naked DNA is essential. However, this transformation did not occur with purified plasmid DNA and required a direct supply of plasmid from co-existing donor cells. Based on this feature, we have termed this transformation type as 'cell-to-cell transformation'. Analyses using medium conditioned with the high-frequency strain revealed that this strain released a certain factor(s) that promoted cell-to-cell transformation and arrested growth of the other strains. This factor is heat-labile and protease-sensitive, and its roughly estimated molecular mass was between ∼9 kDa and ∼30 kDa, indicating that it is a polypeptide factor. Interestingly, this factor was effective even when the conditioned medium was diluted 10(-5)-10(-6), suggesting that it acts like a pheromone with high bioactivity. Based on these results, we propose that cell-to-cell transformation is a novel natural transformation mechanism in E. coli that requires cell-derived DNA and is promoted by a peptide pheromone. This is the first evidence that suggests the existence of a peptide pheromone-regulated transformation mechanism in E. coli and in Gram-negative bacteria.

Horizontal transfer of nonconjugative plasmids in a colony biofilm of Escherichia coli.

We tested the possibility of nonconjugative lateral DNA transfer in a colony biofilm of mixed Escherichia coli strains. By simply coculturing a plasmid-free F(-) strain and another F(-) strain harboring a nonconjugative plasmid in a colony biofilm on antibiotic-free agar media, transformed cells were produced within 24-48 h at the frequency of 10(-10)-10(-9) per recipient cell. PCR analysis of the transformed cells demonstrated the occurrence of lateral plasmid transfer. These cells survived until at least day 7 under antibiotic-free conditions. Liquid cultures of the same strains in Luria-Bertani broth produced no or few transformants, suggesting the importance of colony-biofilm formation for plasmid transfer. This is a novel line of evidence indicating that nonconjugative, nonviral horizontal gene transfer can occur between E. coli cells.

Transformation of colonial Escherichia coli on solid media.

We report that colonial Escherichia coli cells on various solid media can develop modest genetic competence. Using an on-filter culture system, we found that E. coli colonies on CaCl2-containing agar were transformed in the presence of plasmid DNA. Interestingly, transformation also occurred on LB-agar, various moist foods and even on H2O-agar. These results suggest that some populations of colonial E. coli in various environments could become transformable regardless of the surrounding Ca2+ concentration.

Spatial distribution of mDLG6 mRNA in embryonic and adult mouse brain.

We have isolated mouse DLG6 (mDLG6) cDNA clones by RT-PCR and then by using the RT-PCR products to screen a mouse brain cDNA library. The deduced amino acid sequence of mDLG6 shows 79.2% and 82.7% overall identity to human (hDLG6) and rat DLG6 (rDLG6), respectively. In situ hybridization revealed that mDLG6 mRNA is predominantly expressed in embryonic and adult brain.