Competence For Natural Transformation Research Articles

Pneumococcal competence is a populational health sensor driving multilevel heterogeneity in response to antibiotics

Competence for natural transformation is a central driver of genetic diversity in bacteria. In the human pathogen Streptococcus pneumoniae, competence exhibits a populational character mediated by the stress-induced ComABCDE quorum-sensing (QS) system. Here, we explore how this cell-to-cell communication mechanism proceeds and the functional properties acquired by competent cells grown under lethal stress. We show that populational competence development depends on self-induced cells stochastically emerging in response to stresses, including antibiotics. Competence then propagates through the population from a low threshold density of self-induced cells, defining a biphasic Self-Induction and Propagation (SI&P) QS mechanism. We also reveal that a competent population displays either increased sensitivity or improved tolerance to lethal doses of antibiotics, dependent in the latter case on the competence-induced ComM division inhibitor. Remarkably, these surviving competent cells also display an altered transformation potential. Thus, the unveiled SI&P QS mechanism shapes pneumococcal competence as a health sensor of the clonal population, promoting a bet-hedging strategy that both responds to and drives cells towards heterogeneity.

Natural transformation and cell division delay in competent Staphylococcus aureus.

Genetic competence for natural transformation, considered one of the three main mechanisms leading to horizontal gene transfer in bacteria, is able to promote evolution, through genomic plasticity, and foster antibiotic resistance and virulence factors spreading. Conserved machinery and actors required to perform natural transformation have been shown to accumulate at different cellular localizations depending on the model organism considered. In this study, we investigate the transformation apparatus composition, localization, and dynamics in the human pathogen Staphylococcus aureus. We particularly show that most of the natural transformation actors co-localize in clusters. We also reveal that the localization of natural transformation proteins is dynamic, following the cell cycle. Ultimately, the natural transformation apparatus is preferentially established in the vicinity of the division septum. All these results demonstrate that DNA binding, uptake, and recombination are spatially and temporally coordinated to ensure S. aureus natural transformation. Finally, we hypothesize that S. aureus competent cells would initiate and then block cell division to ensure the success of natural transformation before the final constriction of the cytokinetic ring. IMPORTANCE Natural transformation, considered one of the three main mechanisms leading to horizontal gene transfer in bacteria, is able to promote genomic plasticity and foster antibiotic resistance spreading. Conserved machinery and actors required to perform natural transformation have been shown to accumulate at different cellular localizations depending on the model organism considered. Here, we show in the human pathogen Staphylococcus aureus that DNA binding, uptake, and recombination are spatially and temporally coordinated to ensure S. aureus natural transformation. We also reveal that localization of natural transformation proteins occurs in the vicinity of the division septum allowing S. aureus competent cells to block cell division to ensure the success of natural transformation before the final constriction of the cytokinetic ring.

Fever-like temperature bursts promote competence development via an HtrA-dependent pathway in Streptococcus pneumoniae.

Streptococcus pneumoniae (the pneumococcus) is well known for its ability to develop competence for natural DNA transformation. Competence development is regulated by an autocatalytic loop driven by variations in the basal level of transcription of the comCDE and comAB operons. These genes are part of the early gene regulon that controls expression of the late competence genes known to encode the apparatus of transformation. Several stressful conditions are known to promote competence development, although the induction pathways are remain poorly understood. Here we demonstrate that transient temperature elevation induces an immediate increase in the basal expression level of the comCDE operon and early genes that, in turn, stimulates its full induction, including that of the late competence regulon. This thermal regulation depends on the HtrA chaperone/protease and its proteolytic activity. We find that other competence induction stimulus, like norfloxacin, is not conveyed by the HtrA-dependent pathway. This finding strongly suggests that competence can be induced by at least two independent pathways and thus reinforces the view that competence is a general stress response system in the pneumococcus.

DNA modifications impact natural transformation of Acinetobacter baumannii.

Acinetobacter baumannii is a dangerous nosocomial pathogen, especially due to its ability to rapidly acquire new genetic traits, including antibiotic resistance genes (ARG). In A. baumannii, natural competence for transformation, one of the primary modes of horizontal gene transfer (HGT), is thought to contribute to ARG acquisition and has therefore been intensively studied. However, knowledge regarding the potential role of epigenetic DNA modification(s) on this process remains lacking. Here, we demonstrate that the methylome pattern of diverse A. baumannii strains differs substantially and that these epigenetic marks influence the fate of transforming DNA. Specifically, we describe a methylome-dependent phenomenon that impacts intra- and inter-species DNA exchange by the competent A. baumannii strain A118. We go on to identify and characterize an A118-specific restriction-modification (RM) system that impairs transformation when the incoming DNA lacks a specific methylation signature. Collectively, our work contributes towards a more holistic understanding of HGT in this organism and may also aid future endeavors towards tackling the spread of novel ARGs. In particular, our results suggest that DNA exchanges between bacteria that share similar epigenomes are favored and could therefore guide future research into identifying the reservoir(s) of dangerous genetic traits for this multi-drug resistant pathogen.

A Genome-Wide CRISPR Interference Screen Reveals an StkP-Mediated Connection between Cell Wall Integrity and Competence in Streptococcus salivarius.

Competence is one of the most efficient bacterial evolutionary and adaptative strategies by synchronizing production of antibacterial compounds and integration of DNA released by dead cells. In most streptococci, this tactic is orchestrated by the ComRS system, a pheromone communication device providing a short time window of activation in which only part of the population is responsive. Understanding how this developmental process integrates multiple inputs to fine-tune the adequate response is a long-standing question. However, essential genes involved in the regulation of ComRS have been challenging to study. In this work, we built a conditional mutant library using CRISPR interference and performed three complementary screens to investigate competence genetic regulation in the human commensal Streptococcus salivarius. We show that initiation of competence increases upon cell wall impairment, suggesting a connection between cell envelope stress and competence activation. Notably, we report a key role for StkP, a serine-threonine kinase known to regulate cell wall homeostasis. We show that StkP controls competence by a mechanism that reacts to peptidoglycan fragments. Together, our data suggest a key cell wall sensing mechanism coupling competence to cell envelope integrity. IMPORTANCE Survival of human commensal streptococci in the digestive tract requires efficient strategies which must be tightly and collectively controlled for responding to competitive pressure and drastic environmental changes. In this context, the autocrine signaling system ComRS controlling competence for natural transformation and predation in salivarius streptococci could be seen as a multi-input device integrating a variety of environmental stimuli. In this work, we revealed novel positive and negative competence modulators by using a genome-wide CRISPR interference strategy. Notably, we highlighted an unexpected connection between bacterial envelope integrity and competence activation that involves several cell wall sensors. Together, these results showcase how commensal streptococci can fine-tune the pheromone-based competence system by responding to multiple inputs affecting their physiological status in order to calibrate an appropriate collective behavior.

Markerless gene deletion in Ralstonia solanacearum based on its natural transformation competence.

Ralstonia solanacearum species complex (RSSC) is a group of Gram-negative bacterial pathogen capable of infecting numerous plants and crops, causing severe vascular wilt diseases. Functional analysis of the genes associated with bacterial virulence is critical for elucidating the molecular mechanisms that govern the bacterial pathogenicity. To this end, an efficient gene deletion method would be of great help. In this study, we set to develop an efficient and simple markerless gene deletion method by exploiting its natural transformation competence and the FLP/FRT recombination system. We found that natural transformation using PCR products provided much higher transformation frequency than the plasmid-based triparental mating and electroporation. We thus generated the gene deletion fusion PCR fragments by incorporating the upstream and downstream DNA fragments of the target gene and an antibiotic resistance gene flanked by FRT sites, and delivered the PCR products into R. solanacearum cells through natural transformation. Using this method, we knocked out the epsB and phcA genes, which are associated with exopolysaccharide (EPS) biosynthesis and regulation, respectively, in several R. solanacearum strains isolated from different host plants at a frequency from 5 (1E-08) to 45 (1E-08). To remove the antibiotic marker gene, the plasmid expressing the FLP enzyme was introduced into the above knockout mutants, which enabled removal of the marker gene. The effective combination of natural transformation and the FLP/FRT recombination system thus offers a simple and efficient method for functional study of putative virulence genes and for elucidation of R. solanacearum pathogenic mechanisms.

Simultaneous Single-Cell Genome and Transcriptome Sequencing of Termite Hindgut Protists Reveals Metabolic and Evolutionary Traits of Their Endosymbionts.

ABSTRACTSome of the protist species which colonize the hindguts of wood-feeding Reticulitermes termites are associated with endosymbiotic bacteria belonging to the genus Endomicrobium. In this study, we focused on the endosymbionts of three protist species from Reticulitermes flavipes, as follows: Pyrsonympha vertens, Trichonympha agilis, and Dinenympha species II. Since these protist hosts represented members of different taxa which colonize separate niches within the hindguts of their termite hosts, we investigated if these differences translated to differential gene content and expression in their endosymbionts. Following assembly and comparative genome and transcriptome analyses, we discovered that these endosymbionts differed with respect to some possible niche-specific traits, such as carbon metabolism. Our analyses suggest that species-specific genes related to carbon metabolism were acquired by horizontal gene transfer (HGT) and may have come from taxa which are common in the termite hind gut. In addition, our analyses suggested that these endosymbionts contain and express genes related to natural transformation (competence) and recombination. Taken together, the presence of genes acquired by HGT and a putative competence pathway suggest that these endosymbionts are not cut off from gene flow and that competence may be a mechanism by which members of Endomicrobium can acquire new traits.IMPORTANCE The composition and structure of wood, which contains cellulose, hemicellulose, and lignin, prevent most organisms from using this common food source. Termites are a rare exception among animals, and they rely on a complex microbiota housed in their hindguts to use wood as a source of food. The lower termite, Reticulitermes flavipes, houses a variety of protists and prokaryotes that are the key players in the disassembly of lignocellulose. Here, we describe the genomes and the gene expression profiles of five Endomicrobium endosymbionts living inside three different protist species from R. flavipes. Data from these genomes suggest that these Endomicrobium species have different mechanisms for using carbon. In addition, they harbor genes that may be used to import DNA from their environment. This process of DNA uptake may contribute to the high levels of horizontal gene transfer noted previously in Endomicrobium species.

Coevolution of the bacterial pheromone ComS and sensor ComR fine-tunes natural transformation in streptococci

Competence for natural transformation extensively contributes to genome evolution and the rapid adaptability of bacteria dwelling in challenging environments. In most streptococci, this process is tightly controlled by the ComRS signaling system, which is activated through the direct interaction between the (R)RNPP-type ComR sensor and XIP pheromone (mature ComS). The overall mechanism of activation and the basis of pheromone selectivity have been previously reported in Gram-positive salivarius streptococci; however, detailed 3D-remodeling of ComR leading up to its activation remains only partially understood. Here, we identified using a semirational mutagenesis approach two residues in the pheromone XIP that bolster ComR sensor activation by interacting with two aromatic residues of its XIP-binding pocket. Random and targeted mutagenesis of ComR revealed that the interplay between these four residues remodels a network of aromatic–aromatic interactions involved in relaxing the sequestration of the DNA-binding domain. Based on these data, we propose a comprehensive model for ComR activation based on two major conformational changes of the XIP-binding domain. Notably, the stimulation of this newly identified trigger point by a single XIP substitution resulted in higher competence and enhanced transformability, suggesting that pheromone-sensor coevolution counter-selects for hyperactive systems in order to maintain a trade-off between competence and bacterial fitness. Overall, this study sheds new light on the ComRS activation mechanism and how it could be exploited for biotechnological and biomedical purposes.

Natural Competence for Transformation in Lactobacili

Natural Competence for Transformation in Lactobacili

Strategies to Attenuate the Competence Regulon in Streptococcus pneumoniae.

Streptococcus pneumoniae is an opportunistic respiratory human pathogen that poses a continuing threat to human health. Natural competence for genetic transformation in S. pneumoniae plays an important role in aiding pathogenicity and it is the best-characterized feature to acquire antimicrobial resistance genes by a frequent process of recombination. In S. pneumoniae, competence, along with virulence factor production, is controlled by a cell-density communication mechanism termed the competence regulon. In this review, we present the recent advances in the development of alternative methods to attenuate the pathogenicity of S. pneumoniae by targeting the various stages of the non-essential competence regulon communication system. We mainly focus on new developments related to competitively intercepting the competence regulon signaling through the introduction of promising dominant-negative Competence Stimulating Peptide (dnCSP) scaffolds. We also discuss recent reports on antibiotics that can block CSP export by disturbing the proton motive force (PMF) across the membrane and various ways to control the pneumococcal pathogenicity by activating the counter signaling circuit and targeting the pneumococcal proteome.

Pilus Production in Acinetobacter baumannii Is Growth Phase Dependent and Essential for Natural Transformation.

Acinetobacter baumannii is a severe threat to human health as a frequently multidrug-resistant hospital-acquired pathogen. Part of the danger from this bacterium comes from its genome plasticity and ability to evolve quickly by taking up and recombining external DNA into its own genome in a process called natural competence for transformation. This mode of horizontal gene transfer is one of the major ways that bacteria can acquire new antimicrobial resistances and toxic traits. Because these processes in A. baumannii are not well studied, we herein characterized new aspects of natural transformability in this species that include the species' competence window. We uncovered a strong correlation with a growth phase-dependent synthesis of a type IV pilus (TFP), which constitutes the central part of competence-induced DNA uptake machinery. We used bacterial genetics and microscopy to demonstrate that the TFP is essential for the natural transformability and surface motility of A. baumannii, whereas pilus-unrelated proteins of the DNA uptake complex do not affect the motility phenotype. Furthermore, TFP biogenesis and assembly is subject to input from two regulatory systems that are homologous to Pseudomonas aeruginosa, namely, the PilSR two-component system and the Pil-Chp chemosensory system. We demonstrated that these systems affect not only the piliation status of cells but also their ability to take up DNA for transformation. Importantly, we report on discrepancies between TFP biogenesis and natural transformability within the same genus by comparing data for our work on A. baumannii to data reported for Acinetobacter baylyi, the latter of which served for decades as a model for natural competence.IMPORTANCE Rapid bacterial evolution has alarming negative impacts on animal and human health which can occur when pathogens acquire antimicrobial resistance traits. As a major cause of antibiotic-resistant opportunistic infections, A. baumannii is a high-priority health threat which has motivated renewed interest in studying how this pathogen acquires new, dangerous traits. In this study, we deciphered a specific time window in which these bacteria can acquire new DNA and correlated that with its ability to produce the external appendages that contribute to the DNA acquisition process. These cell appendages function doubly for motility on surfaces and for DNA uptake. Collectively, we showed that A. baumannii is similar in its TFP production to Pseudomonas aeruginosa, though it differs from the well-studied species A. baylyi.

The type IV pilus protein PilU functions as a PilT-dependent retraction ATPase.

Type IV pili are dynamic cell surface appendages found throughout the bacteria. The ability of these structures to undergo repetitive cycles of extension and retraction underpins their crucial roles in adhesion, motility and natural competence for transformation. In the best-studied systems a dedicated retraction ATPase PilT powers pilus retraction. Curiously, a second presumed retraction ATPase PilU is often encoded immediately downstream of pilT. However, despite the presence of two potential retraction ATPases, pilT deletions lead to a total loss of pilus function, raising the question of why PilU fails to take over. Here, using the DNA-uptake pilus and mannose-sensitive haemagglutinin (MSHA) pilus of Vibrio cholerae as model systems, we show that inactivated PilT variants, defective for either ATP-binding or hydrolysis, have unexpected intermediate phenotypes that are PilU-dependent. In addition to demonstrating that PilU can function as a bona fide retraction ATPase, we go on to make the surprising discovery that PilU functions exclusively in a PilT-dependent manner and identify a naturally occurring pandemic V. cholerae PilT variant that renders PilU essential for pilus function. Finally, we show that Pseudomonas aeruginosa PilU also functions as a PilT-dependent retraction ATPase, providing evidence that the functional coupling between PilT and PilU could be a widespread mechanism for optimal pilus retraction.

Conserved Pheromone Production, Response and Degradation by Streptococcus mutans.

Streptococcus mutans, a bacterium with high cariogenic potential, coordinates competence for natural transformation and bacteriocin production via the XIP and CSP pheromones. CSP is effective in inducing bacteriocin responses but not competence in chemically defined media (CDM). This is in contrast to XIP, which is a strong inducer of competence in CDM but can also stimulate bacteriocin genes as a late response. Interconnections between the pathways activated by the two pheromones have been characterized in certain detail in S. mutans UA159, but it is mostly unknown whether such findings are representative for the species. In this study, we used bioassays based on luciferase reporters for the bacteriocin gene cipB and the alternative sigma factor sigX to investigate various S. mutans isolates for production and response to CSP and XIP pheromones in CDM. Similar to S. mutans UA159, endogenous CSP was undetectable in the culture supernatants of all tested strains. During optimization of the bioassay using the cipB reporter, we discovered that the activity of exogenous CSP used as a standard was reduced over time during S. mutans growth. Using a FRET-CSP reporter peptide, we found that S. mutans UA159 was able to degrade CSP, and that such activity was not significantly different in isogenic mutants with deletion of the protease gene htrA or the competence genes sigX, oppD, and comR. CSP cleavage was also detected in all the wild type strains, indicating that this is a conserved feature in S. mutans. For the XIP pheromone, endogenous production was observed in the supernatants of all 34 tested strains at peak concentrations in culture supernatants that varied between 200 and 26000 nM. Transformation in the presence of exogenous XIP was detected in all but one of the isolates. The efficiency of transformation varied, however, among the different strains, and for those with the highest transformation rates, endogenous XIP peak concentrations in the supernatants were above 2000 nM XIP. We conclude that XIP production and inducing effect on transformation, as well as the ability to degrade CSP, are conserved functions among different S. mutans isolates. Understanding the functionality and conservation of pheromone systems in S. mutans may lead to novel strategies to prevent or treat unbalances in oral microbiomes that may favor diseases.

Neighbor predation linked to natural competence fosters the transfer of large genomic regions in Vibrio cholerae.

Natural competence for transformation is a primary mode of horizontal gene transfer. Competent bacteria are able to absorb free DNA from their surroundings and exchange this DNA against pieces of their own genome when sufficiently homologous. However, the prevalence of non-degraded DNA with sufficient coding capacity is not well understood. In this context, we previously showed that naturally competent Vibrio cholerae use their type VI secretion system (T6SS) to actively acquire DNA from non-kin neighbors. Here, we explored the conditions of the DNA released through T6SS-mediated killing versus passive cell lysis and the extent of the transfers that occur due to these conditions. We show that competent V. cholerae acquire DNA fragments with a length exceeding 150 kbp in a T6SS-dependent manner. Collectively, our data support the notion that the environmental lifestyle of V. cholerae fosters the exchange of genetic material with sufficient coding capacity to significantly accelerate bacterial evolution.

Amino acids as nutritional factors and (p)ppGpp as an alarmone of the stringent response regulate natural transformation in Micrococcus luteus

Natural competence for genetic transformation refers to the natural ability of various bacteria to take up exogenous DNA from their surroundings and to incorporate internalized genetic information into their genomes. By promoting bacterial diversification and adaptability, this process represents a major driving force in bacterial evolution. Micrococcus luteus was one of the first organisms used to study natural transformation in bacteria. Since then, however, only very little information about this phenomenon has been reported in M. luteus or in any member of the Actinobacteria phylum (low-GC Gram-positive bacteria). Previous work in our group indicated major differences between the transformation apparatus of M. luteus and the transformation machinery described for various Gram-negative and Gram-positive model bacteria belonging to the phyla Proteobacteria and Firmicutes (high-GC Gram-positive bacteria). This prompted us to initiate a study concerning the regulation mechanism of competence development in M. luteus. In this report, we identify amino acids as a nutritional factor that influences competence in a concentration-dependent manner. By using a transcriptional reporter strain for one of the late competence genes, we demonstrate how increasing concentrations of both amino acids mixtures and single amino acids supplemented to the growth medium affect transformability on transcriptional and post-transcriptional level. Furthermore, we revisit previously generated auxotrophic mutants to show that the transformation machinery is turned down during a state of extreme hunger for amino acids presumably as a part of a general response to auxotrophy. Finally, by generating and analysing knockout mutants for two predicted stringent response enzymes, we provide evidence for the involvement of the alarmone (p)ppGpp as a putative mediator of the effects on transformation development caused by amino acids. As a member of the Actinobacteria phylum, M. luteus could serve as a model for other representatives of the phylum, including a number of important human pathogens.

DNA-uptake pili of Vibrio cholerae are required for chitin colonization and capable of kin recognition via sequence-specific self-interaction.

How bacteria colonise surfaces and how they distinguish the individuals around them are fundamental biological questions. Type IV pili are a widespread and multi-purpose class of cell surface polymers. Here we directly visualise the DNA-uptake pilus of Vibrio cholerae, which is produced specifically during growth upon its natural habitat - chitinous surfaces. As predicted, these pili are highly dynamic and retract prior to DNA-uptake during competence for natural transformation. Interestingly, DNA-uptake pili can also self-interact to mediate auto-aggregation. This capability is conserved in disease-causing pandemic strains, which typically encode the same major pilin subunit, PilA. Unexpectedly, however, we discovered that extensive strain-to-strain variability in PilA, present in environmental isolates, creates a set of highly specific interactions, enabling cells producing pili composed of different PilA subunits to distinguish between one another. We go on to show that DNA-uptake pili bind to chitinous surfaces, are required for chitin colonisation under flow, and that pili capable of self-interaction connect cells on chitin within dense pili networks. Our results suggest a model whereby DNA-uptake pili function to promote inter-bacterial interactions during surface colonisation. Moreover, they provide evidence that type IV pili could offer a simple and potentially widespread mechanism for bacterial kin recognition.

Competence for Natural Transformation Is Common among Clinical Strains of Resistant Acinetobacter spp.

Horizontal gene transfer events provide the basis for extensive dissemination of antimicrobial resistance traits between bacterial populations. Conjugation is considered to be the most frequent mechanism behind new resistance acquisitions in clinical pathogens but does not fully explain the resistance patterns seen in some bacterial genera. Gene transfer by natural transformation has been described for numerous clinical isolates, including some Acinetobacter species. The main aim of this study was to determine to what extent clinical, resistant Acinetobacter spp. isolates, express competence for natural transformation. Twenty-two clinical Acinetobacter spp. isolates collected over a 16-year time period, from five different geographical separated and/or distinct Portuguese Hospitals were tested for natural transformability. Fourteen isolates, including 11 A. baumannii, 2 A. nosocomialis and 1 Acinetobacter sp., were identified as competent on semisolid media facilitating surface-motility. Competent Acinetobacter isolates were found in all the hospitals tested. Furthermore, osmolarity was shown to influence the uptake of exogenous DNA by competent A. baumannii A118. Our study demonstrates that natural competence is common among clinical isolates of Acinetobacter spp., and hence likely an important trait for resistance acquisition.

Methods for Natural Transformation in Acinetobacter baumannii.

The genomes of Acinetobacter baumannii tell us stories about horizontal gene transfer (HGT) events that steadily drive the evolution of this nosocomial pathogen toward multidrug resistance. Natural transformation competence constitutes one of the several possible pathways that mediate HGT in A. baumannii. Here, we describe and discuss the methods for studying DNA uptake in A. baumannii via natural transformation.

Intensive targeting of regulatory competence genes by transposable elements in streptococci.

Competence for natural transformation is a widespread developmental process of streptococci. By allowing the uptake and recombination of exogenous naked DNA into the genome, natural transformation, as transposable elements, plays a key role in the plasticity of bacterial genomes. We previously analysed the insertion sites of IS1548, an insertion sequence present in Streptococcus agalactiae and S. pyogenes, and showed that some targeted loci are involved in competence induction. In this work, we investigated on a large scale if loci coding for early competence factors (ComX and the two pheromone-dependent signalling systems ComCDE and ComRS) of streptococci are especially targeted by transposable elements. The transposable elements inserted in regions surrounding these genes and housekeeping genes used for Multilocus Sequence Typing (MLST) were systematically searched for. We found numerous insertion events in the close vicinity of early competence genes, but only very few into the MLST loci. The incidence of transposable elements, mainly insertion sequences, is particularly high in the intergenic regions surrounding comX alleles in numerous species belonging to most streptococcal groups. The identification of scarce disruptive insertions inside early competence genes indicates that the maintenance of competence is essential for streptococci. The specific association of transposable elements with intergenic regions bordering the main regulatory genes of competence may impact on the induction of transformability and so, on the genome plasticity and adaptive evolution of streptococci. This widespread phenomenon brings new perspectives on our understanding of competence regulation and its role in the bacterial life cycle.

Pull in and Push Out: Mechanisms of Horizontal Gene Transfer in Bacteria.

Horizontal gene transfer (HGT) plays an important role in bacterial evolution. It is well accepted that DNA is pulled/pushed into recipient cells by conserved membrane-associated DNA transport systems, which allow the entry of only single-stranded DNA (ssDNA). However, recent studies have uncovered a new type of natural bacterial transformation in which double-stranded DNA (dsDNA) is taken up into the cytoplasm, thus complementing the existing methods of DNA transfer among bacteria. Regulated by the stationary-phase regulators RpoS and cAMP receptor protein (CRP), Escherichia coli establishes competence for natural transformation with dsDNA, which occurs in agar plates. To pass across the outer membrane, a putative channel, which may compete for the substrate with the porin OmpA, may mediate the transfer of exogenous dsDNA into the cell. To pass across the inner membrane, dsDNA may be bound to the periplasmic protein YdcS, which delivers it into the inner membrane channel formed by YdcV. The discovery of cell-to-cell contact-dependent plasmid transformation implies the presence of additional mechanism(s) of transformation. This review will summarize the current knowledge about mechanisms of HGT with an emphasis on recent progresses regarding non-canonical mechanisms of natural transformation. Fully understanding the mechanisms of HGT will provide a foundation for monitoring and controlling multidrug resistance.