Mechanism Of Horizontal Gene Transfer Research Articles

Horizontal gene transfer: Implications in human health and diseases

Horizontal gene transfer (HGT) between the different organisms of the same or different species can cause both beneficial and adverse biological consequences. HGT has been demonstrated among all the phylogenetic groups including human beings. There is rising evidence that prokaryotic HGT is a dominant evolutionary force. Integration of microbes with human beings and their interaction influences genetic, physiological, and biotic sustainability of host. In the present era, this mechanism in one end is used for constructing desired genome for gene therapy, and in other hand, it is decisive in progression and surfacing many human diseases because of rapid evolution of microorganism and resistant of these microorganism to the treated antibiotics. Transformation, transduction, and conjugation are the principal HGT mechanisms in prokaryote, whereas exosomes, apoptotic bodies, cell free DNA acts as mediators in eukaryotes. Conducive environment and diversified microbiome in human gut and their interaction with host facilitate HGT in gastrointestinal microbiome, which can modulate human health and diseases. Integration of viral and bacterial genome by HGT into human genomic DNA can lead to cancers such as human papillomavirus, acute myeloid leukemia, adenocarcinoma of stomach, and many more. Many metabolic, immunological, and neurodegenerative diseases are also emerging because of HGT. The development of resistance to antibiotics or multidrug in human beings are the consequences of HGT among microbiome and simultaneously clinicians therapeutic concern. Hence, the way of understanding different mediators of HGT and their integration may facilitate better diagnostic and therapeutic modalities in control of diseases.

Resistome Study in Aquatic Environments.

Since the first discovery of antibiotics, introduction of new antibiotics has been coupled with the occurrence of antibiotic resistant bacteria (ARB) and antibiotic resistance genes (ARGs). Rapid dissemination of ARB and ARGs in the aquatic environments has become a global concern. ARB and ARGs have been already disseminated in the aquatic environments via various routes. Main hosts of most of ARGs were found to belong to Gammaproteobacteria class, including clinically important potential pathogens. Transmission of ARGs also occurs by horizontal gene transfer (HGT) mechanisms between bacterial strains in the aquatic environments, resulting in ubiquity of ARGs. Thus, a few of ARGs and MGEs (e.g., strA, sul1, int1) have been suggested as indicators for global comparability of contamination level in the aquatic environments. With ARB and ARGs contamination, the occurrence of critical pathogens has been globally issued due to their widespread in the aquatic environments. Thus, active surveillance systems have been launched worldwide. In this review, we described advancement of methodologies for ARGs detection, and occurrence of ARB and ARGs and their dissemination in the aquatic environments. Even though numerous studies have been conducted for ARB and ARGs, there is still no clear strategy to tackle antibiotic resistance (AR) in the aquatic environments. At least, for consistent surveillance, a strict framework should be established for further research in the aquatic environments.

The Use of a Multimodal Case Study To Illustrate Microbial Genetics, Metabolism, and Evolution: The Emergence of VRSA-1.

Antibiotic Resistance (ABR) is a global concern and while many students are aware of this issue, many of them are unclear on the mechanisms by which ABR may emerge. The mechanism of horizontal gene transfer is something many students are not familiar with. In this curriculum contribution we present 2 versions of an 'interrupted case study' that is designed as an introduction to horizontal gene transfer for early major students and as a review case for advanced major students in biology and life sciences. The case is based on an authentic patient who developed infections with both methicillin resistant Staphylococcus aureus and vancomycin resistant S. aureus. The interrupted case study is appropriate for small and large groups and engages students while content is introduced in a highly structured way. This type of case study can be done by novice and seasoned instructors and lead to considerable learning gains in both introductory and intermediate microbiology courses.

Prophage-like gene transfer agents promote Caulobacter crescentus survival and DNA repair during stationary phase.

Gene transfer agents (GTAs) are prophage-like entities found in many bacterial genomes that cannot propagate themselves and instead package approximately 5 to 15 kbp fragments of the host genome that can then be transferred to related recipient cells. Although suggested to facilitate horizontal gene transfer (HGT) in the wild, no clear physiological role for GTAs has been elucidated. Here, we demonstrate that the α-proteobacterium Caulobacter crescentus produces bona fide GTAs. The production of Caulobacter GTAs is tightly regulated by a newly identified transcription factor, RogA, that represses gafYZ, the direct activators of GTA synthesis. Cells lacking rogA or expressing gafYZ produce GTAs harboring approximately 8.3 kbp fragment of the genome that can, after cell lysis, be transferred into recipient cells. Notably, we find that GTAs promote the survival of Caulobacter in stationary phase and following DNA damage by providing recipient cells a template for homologous recombination-based repair. This function may be broadly conserved in other GTA-producing organisms and explain the prevalence of this unusual HGT mechanism.

Distribution of fitness effects of cross-species transformation reveals potential for fast adaptive evolution

Bacterial transformation, a common mechanism of horizontal gene transfer, can speed up adaptive evolution. How its costs and benefits depend on the growth environment is poorly understood. Here, we characterize the distributions of fitness effects (DFE) of transformation in different conditions and test whether they predict in which condition transformation is beneficial. To determine the DFEs, we generate hybrid libraries between the recipient Bacillus subtilis and different donor species and measure the selection coefficient of each hybrid strain. In complex medium, the donor Bacillus vallismortis confers larger fitness effects than the more closely related donor Bacillus spizizenii. For both donors, the DFEs show strong effect beneficial transfers, indicating potential for fast adaptive evolution. While some transfers of B. vallismortis DNA show pleiotropic effects, various transfers are beneficial only under a single growth condition, indicating that the recipient can benefit from a variety of donor genes to adapt to varying growth conditions. We scrutinize the predictive value of the DFEs by laboratory evolution under different growth conditions and show that the DFEs correctly predict the condition at which transformation confers a benefit. We conclude that transformation has a strong potential for speeding up adaptation to varying environments by profiting from a gene pool shared between closely related species.

Unravelling the DNA sequences carried by Streptomyces coelicolor membrane vesicles

Membrane vesicles (MVs) are spherical particles with nanoscale dimensions and characterized by the presence of diverse cargos, such as nucleic acids, proteins, lipids, and cellular metabolites. Many examples of (micro)organisms producing MVs are reported in literature. Among them, bacterial MVs are of particular interest because they are now considered as the fourth mechanism of horizontal gene transfer. Streptomyces bacteria are well-known for their ecological roles and ability to synthesize bioactive compounds, with Streptomyces coelicolor being the model organism. It was previously demonstrated that it can produce distinct populations of MVs characterized by different protein and metabolite cargos. In this work we demonstrated for the first time that MVs of S. coelicolor carry both DNA and RNA and that their DNA content represents the entire chromosome of the bacterium. These findings suggest that MV DNA could have a role in the evolution of Streptomyces genomes and that MVs could be exploited in new strain engineering strategies.

Plasmids as Key Players in Acinetobacter Adaptation.

This review briefly summarizes the data on the mechanisms of development of the adaptability of Acinetobacters to various living conditions in the environment and in the clinic. A comparative analysis of the genomes of free-living and clinical strains of A. lwoffii, as well as the genomes of A. lwoffii and A. baumannii, has been carried out. It has been shown that plasmids, both large and small, play a key role in the formation of the adaptability of Acinetobacter to their living conditions. In particular, it has been demonstrated that the plasmids of various strains of Acinetobacter differ from each other in their structure and gene composition depending on the lifestyle of their host bacteria. Plasmids of modern strains are enriched with antibiotic-resistant genes, while the content of genes involved in resistance to heavy metals and arsenic is comparable to plasmids from modern and ancient strains. It is concluded that Acinetobacter plasmids may ensure the survival of host bacteria under conditions of various types of environmental and clinical stresses. A brief overview of the main mechanisms of horizontal gene transfer on plasmids inherent in Acinetobacter strains is also given.

Acinetobacter baylyi Strain BD413 Can Acquire an Antibiotic Resistance Gene by Natural Transformation on Lettuce Phylloplane and Enter the Endosphere.

Antibiotic resistance spread must be considered in a holistic framework which comprises the agri-food ecosystems, where plants can be considered a bridge connecting water and soil habitats with the human microbiome. However, the study of horizontal gene transfer events within the plant microbiome is still overlooked. Here, the environmental strain Acinetobacter baylyi BD413 was used to study the acquisition of extracellular DNA (exDNA) carrying an antibiotic resistance gene (ARG) on lettuce phylloplane, performing experiments at conditions (i.e., plasmid quantities) mimicking those that can be found in a water reuse scenario. Moreover, we assessed how the presence of a surfactant, a co-formulant widely used in agriculture, affected exDNA entry in bacteria and plant tissues, besides the penetration and survival of bacteria into the leaf endosphere. Natural transformation frequency in planta was comparable to that occurring under optimal conditions (i.e., temperature, nutrient provision, and absence of microbial competitors), representing an entrance pathway of ARGs into an epiphytic bacterium able to penetrate the endosphere of a leafy vegetable. The presence of the surfactant determined a higher presence of culturable transformant cells in the leaf tissues but did not significantly increase exDNA entry in A. baylyi BD413 cells and lettuce leaves. More research on HGT (Horizontal Gene Transfer) mechanisms in planta should be performed to obtain experimental data on produce safety in terms of antibiotic resistance.

LINE-1 retrotransposons facilitate horizontal gene transfer into poxviruses.

There is ample phylogenetic evidence that many critical virus functions, like immune evasion, evolved by the acquisition of genes from their hosts through horizontal gene transfer (HGT). However, the lack of an experimental system has prevented a mechanistic understanding of this process. We developed a model to elucidate the mechanisms of HGT into vaccinia virus, the prototypic poxvirus. All identified gene capture events showed signatures of long interspersed nuclear element-1 (LINE-1)-mediated retrotransposition, including spliced-out introns, polyadenylated tails, and target site duplications. In one case, the acquired gene integrated together with a polyadenylated host U2 small nuclear RNA. Integrations occurred across the genome, in some cases knocking out essential viral genes. These essential gene knockouts were rescued through a process of complementation by the parent virus followed by nonhomologous recombination during serial passaging to generate a single, replication-competent virus. This work links multiple evolutionary mechanisms into one adaptive cascade and identifies host retrotransposons as major drivers for virus evolution.

Extracellular vesicles as a horizontal gene transfer mechanism in Leishmania

Douanne et al. recently reported horizontal gene transfer (HGT) mediated by extracellular vesicles (EVs) in Leishmania; this constitutes the first report of this phenomenon in parasites. They showed that EVs facilitate the transmission of drug-resistance genes and increased cell fitness in stressful environments, indicating potential clinical application of these findings.

Leishmania parasites exchange drug-resistance genes through extracellular vesicles

Leishmania are eukaryotic parasites that have retained the ability to produce extracellular vesicles (EVs) through evolution. To date, it has been unclear if different DNA entities could be associated with Leishmania EVs and whether these could constitute a mechanism of horizontal gene transfer (HGT). Herein, we investigate the DNA content of EVs derived from drug-resistant parasites, as well as the EVs' potential to act as shuttles for DNA transfer. Next-generation sequencing and PCR assays confirm the enrichment of amplicons carrying drug-resistance genes associated with EVs. Transfer assays of drug-resistant EVs highlight a significant impact on the phenotype of recipient parasites induced by the expression of the transferred DNA. Recipient parasites display an enhanced growth and better control of oxidative stress. We provide evidence that eukaryotic EVs function as efficient mediators in HGT, thereby facilitating the transmission of drug-resistance genes and increasing the fitness of cells when encountering stressful environments.

Ferrate-enhanced electrocoagulation/ultrafiltration system on municipal secondary effluent treatment: Identify synergistic contribution of coagulant and oxidation

Herein, we presented a ferrate-enhanced electrocoagulation/ultrafiltration (fECUF) system with three pathways of contaminants removal during the oxidation of Fe(II), realizing superior water production, excellent fouling control, and outstanding environmental risk reduction in municipal wastewater reuse. The system addressed the issue of the rapid inorganic membrane fouling caused by the traditional electrocoagulation processes. The formation of a unique cake layer with a bilayer structure in fECUF was favored to decrease the accumulation of organics and inorganics in membrane pores and maintain the membrane flux. Compared with the UF control system, the fECUF system decreased the transmembrane pressure by 63.13%. Redundancy analysis confirmed that oxidation dominated the removal of humic-like substances and UV254, while oxidation and coagulation synergistically promoted the reduction of DOC, protein-like substances, and trace organics. Also, coagulation contributed to the removal of antibiotics resistance genes (ARGs), which reduced their ability to be widely transmuted by a horizontal gene transfer mechanism. Compared to the UF control system, 4.48–39.76 times higher reduction of four ARGs were obtained in fECUF. Overall, this study has verified the potential advantages of the fECUF system in membrane fouling control and contaminants reduction for water reclamation.

Bacteriophages vehiculate a high amount of antibiotic resistance determinants of bacterial origin in the Orne River ecosystem.

Aquatic environments are important dissemination routes of antibiotic resistance genes (ARGs) from and to pathogenic bacteria. Nevertheless, in these complex matrices, identifying and characterizing the driving microbial actors and ARG dissemination mechanisms they are involved in remain difficult. We here explored the distribution/compartmentalization of a panel of ARGs and mobile genetic elements (MGEs) in bacteria and bacteriophages collected in the water, suspended material and surface sediments from the Orne River ecosystem (France). By using a new bacteriophage DNA extraction method, we showed that, when packaging bacterial DNA, bacteriophages rather encapsidate both ARGs and MGEs than 16S rRNA genes, i.e. chromosomal fragments. We also show that the bacteria and bacteriophage capsid contents in ARGs/MGEs were similarly influenced by seasonality but that the distribution of ARGs/MGEs between the river physical compartments (water vs. suspended mater vs. sediment) is more impacted when these markers were carried by bacteria. These demonstrations will likely modify our understanding of the formation and fate of transducing viral particles in the environment. Consequently, they will also likely modify our estimations of the relative frequencies of the different horizontal gene transfer mechanisms in disseminating antibiotic resistance by reinforcing the roles played by environmental bacteriophages and transduction.

Mechanism for Reducing the Horizontal Transfer Risk of the Airborne Antibiotic-Resistant Genes of Escherichia coli Species through Microwave or UV Irradiation.

Antibiotic-resistant bacteria (ARBs) and antibiotic-resistant genes (ARGs) as new types of contaminants are discharged into the environment, increasing the risk of horizontal gene transfer (HGT). However, few researchers have examined the impacts of airborne ARB deactivation on HGT risk. The deactivation of airborne Escherichia coli 10667 (carrying sul genes) and the emission and removal of ARGs were mainly investigated in this study. Moreover, the potential mechanisms of HGT and transfer frequencies under microwave (MW) and ultraviolet (UV) irradiation were investigated using the nonresistant E. coli GMCC 13373 and E. coli DH5α with plasmid RP4 as the recipient and donor, respectively. E. coli CICC 10667 and E. coli DH5α with RP4 plasmid achieve log inactivation values as high as 5.5-log and 5.0-log, respectively, which were quite different from the antibiotic-sensitive strain E. coli CGMCC 13373 (3.4-log) subjected to MW irradiation. For UV disinfection, E. coli DH5α with the RP4 plasmid was reduced at 4.4-log, E. coli CGMCC 13373 was reduced at 2.3-log, and E. coli CICC 10667 was inactivated at 2.1-log. The removal rates of ARGs and HGT frequencies under MW irradiation were compared with those under UV irradiation. The ARGs removal efficiency (85.5%) obtained by MW was higher than that obtained by UV (48.2%). Consequently, the HGT frequency (0.008) of airborne ARGs released to the recipient (forward transfer) decreased and was lower than that under UV irradiation (0.014). Moreover, the plasmid RP4 was transferred from the donor to the surviving damaged E. coli 10667 as cell permeability (reverse transfer) was increased at a high HGT frequency (0.003) by MW, which was close to the value by UV (0.002). Additionally, sul1 and sul2 genes were confirmed to be more resistant to MW than the sul3 gene. These findings reveal the mechanism of HGT between damaged E. coli 10667 and surrounding environmental microbes. Microwave is a promising technology for disinfecting airborne microbes and preventing the spread of antibiotic resistance.

Conjugative transfer of naturally occurring plasmid in Mycolicibacterium sp.

Conjugation is considered the main horizontal gene transfer mechanism in bacterial adaptation and evolution. In the Mycobacteriaceae family, Mycolicibacterium smegmatis has been used as the model organism for the conjugative transfer of hybrid plasmids. However, the natural conjugation process in any bacteria would involve the transfer of naturally occurring plasmids. Currently, there is a gap in this regard about this abundant environmental genus of Mycobacteriaceae. Here, we performed conjugation experiments between wild Mycolicibacterium sp. strains involving naturally occurring plasmids, and interestingly, evidence of conjugative transfer was obtained. Thus, it is likely that conjugation occurs in Mycolicibacterium in the natural environment, representing a source of diversification and evolution in this genus of bacteria.

Dead but Not Forgotten: How Extracellular DNA, Moisture, and Space Modulate the Horizontal Transfer of Extracellular Antibiotic Resistance Genes in Soil.

Antibiotic-resistant bacteria and the spread of antibiotic resistance genes (ARGs) pose a serious risk to human and veterinary health. While many studies focus on the movement of live antibiotic-resistant bacteria to the environment, it is unclear whether extracellular ARGs (eARGs) from dead cells can transfer to live bacteria to facilitate the evolution of antibiotic resistance in nature. Here, we use eARGs from dead, antibiotic-resistant Pseudomonas stutzeri cells to track the movement of eARGs to live P. stutzeri cells via natural transformation, a mechanism of horizontal gene transfer involving the genomic integration of eARGs. In sterile, antibiotic-free agricultural soil, we manipulated the eARG concentration, soil moisture, and proximity to eARGs. We found that transformation occurred in soils inoculated with just 0.25 μg of eDNA g-1 soil, indicating that even low concentrations of soil eDNA can facilitate transformation (previous estimates suggested ∼2 to 40 μg eDNA g-1 soil). When eDNA was increased to 5 μg g-1 soil, there was a 5-fold increase in the number of antibiotic-resistant P. stutzeri cells. We found that eARGs were transformed under soil moistures typical of terrestrial systems (5 to 30% gravimetric water content) but inhibited at very high soil moistures (>30%). Overall, this work demonstrates that dead bacteria and their eARGs are an overlooked path to antibiotic resistance. More generally, the spread of eARGs in antibiotic-free soil suggests that transformation allows genetic variants to establish in the absence of antibiotic selection and that the soil environment plays a critical role in regulating transformation. IMPORTANCE Bacterial death can release eARGs into the environment. Agricultural soils can contain upwards of 109 ARGs g-1 soil, which may facilitate the movement of eARGs from dead to live bacteria through a mechanism of horizontal gene transfer called natural transformation. Here, we track the spread of eARGs from dead, antibiotic-resistant Pseudomonas stutzeri cells to live antibiotic-susceptible P. stutzeri cells in sterile agricultural soil. Transformation increased with the abundance of eARGs and occurred in soils ranging from 5 to 40% gravimetric soil moisture but was lowest in wet soils (>30%). Transformants appeared in soil after 24 h and persisted for up to 15 days even when eDNA concentrations were only a fraction of those found in field soils. Overall, our results show that natural transformation allows eARGs to spread and persist in antibiotic-free soils and that the biological activity of eDNA after bacterial death makes environmental eARGs a public health concern.

Antimicrobial Use and Resistance in Plant Agriculture: A One Health Perspective

Bactericides, fungicides, and other pesticides play an important role in the management of plant diseases. However, their use can result in residues on plants and in the environment, with potentially detrimental consequences. The use of streptomycin, oxytetracycline, copper-based products, and some fungicides is correlated with increased resistance among plant pathogens to these agents. Likewise, the recent rise in the incidence of environmental triazole fungicide-resistant Aspergillus fumigatus, the cause of aspergillosis in humans, has caused concern, particularly in Europe. Through horizontal gene transfer, genes can be exchanged among a variety of bacteria in the plant production environment, including phytopathogens, soil bacteria, and zoonotic bacteria that are occasionally present in that environment and in the food chain. Through mechanisms of horizontal gene transfer, co-resistance, cross-resistance, and gene up-regulation, resistance to one compound may confer resistance and multi-drug resistance to other similar, or even very dissimilar, compounds. Given the global rise in antimicrobial-resistant (AMR) organisms, and their effects on plant, animal, and human health, the prudent use of pesticides is required to maintain their effectiveness for food security and sustainable production, and to minimize the emergence and transmission of AMR organisms from horticultural sources.

The Complete Mitogenome of Elymus sibiricus and Insights Into Its Evolutionary Pattern Based on Simple Repeat Sequences of Seed Plant Mitogenomes.

The most intriguing characteristics of plant mitochondrial genomes (mitogenomes) include their high variation in both sequence and structure, the extensive horizontal gene transfer (HGT), and the important role they play in hypoxic adaptation. However, the investigation of the mechanisms of hypoxic adaptation and HGT in plant mitochondria remains challenging due to the limited number of sequenced mitogenomes and non-coding nature of the transferred DNA. In this study, the mitogenome of Elymus sibiricus (Gramineae, Triticeae), a perennial grass species native to the Qinghai-Tibet plateau (QTP), was de novo assembled and compared with the mitogenomes of eight Gramineae species. The unique haplotype composition and higher TE content compared to three other Triticeae species may be attributed to the long-term high-altitude plateau adaptability of E. sibiricus. We aimed to discover the connection between mitogenome simple sequence repeats (SSRs) (mt-SSRs) and HGT. Therefore, we predicted and annotated the mt-SSRs of E. sibiricus along with the sequencing of 87 seed plants. The clustering result based on all of the predicted compound mitogenome SSRs (mt-c-SSRs) revealed an expected synteny within systematic taxa and also inter-taxa. The mt-c-SSRs were annotated to 11 genes, among which “(ATA)3agtcaagtcaag (AAT)3” occurred in the nad5 gene of 8 species. The above-mentioned results further confirmed the HGT of mitogenomes sequences even among distant species from the aspect of mt-c-SSRs. Two genes, nad4 and nad7, possessed a vast number of SSRs in their intron regions across the seed plant mitogenomes. Furthermore, five pairs of SSRs developed from the mitogenome of E. sibiricus could be considered as potential markers to distinguish between the species E. sibiricus and its related sympatric species E. nutans.

Interbacterial Transfer of Carbapenem Resistance and Large Antibiotic Resistance Islands by Natural Transformation in Pathogenic Acinetobacter.

ABSTRACTAcinetobacter baumannii infection poses a major health threat, with recurrent treatment failure due to antibiotic resistance, notably to carbapenems. While genomic analyses of clinical strains indicate that homologous recombination plays a major role in the acquisition of antibiotic resistance genes, the underlying mechanisms of horizontal gene transfer often remain speculative. Our understanding of the acquisition of antibiotic resistance is hampered by the lack of experimental systems able to reproduce genomic observations. We here report the detection of recombination events occurring spontaneously in mixed bacterial populations and which can result in the acquisition of resistance to carbapenems. We show that natural transformation is the main driver of intrastrain but also interstrain recombination events between A. baumannii clinical isolates and pathogenic species of Acinetobacter. We observed that interbacterial natural transformation in mixed populations is more efficient at promoting the acquisition of large resistance islands (AbaR4 and AbaR1) than when the same bacteria are supplied with large amounts of purified genomic DNA. Importantly, analysis of the genomes of the recombinant progeny revealed large recombination tracts (from 13 to 123 kb) similar to those observed in the genomes of clinical isolates. Moreover, we highlight that transforming DNA availability is a key determinant of the rate of recombinants and results from both spontaneous release and interbacterial predatory behavior. In the light of our results, natural transformation should be considered a leading mechanism of genome recombination and horizontal gene transfer of antibiotic resistance genes in Acinetobacter baumannii.

Natural Transformation in Acinetobacter baumannii W068: A Genetic Analysis Reveals the Involvements of the CRP, XcpV, XcpW, TsaP, and TonB2.

Acinetobacter baumannii is a serious threat to public health, and there is increasing attention to the development of antibiotic resistance in this bacterium. Natural transformation is a major horizontal gene transfer mechanism that can lead to antibiotic resistance. To better understand the mechanism of natural transformation in A. baumannii, we selected a clinical isolate that was transformable but had no visible extracellular type IV pili (T4P) filaments and then examined the effects of multiple single-gene knockouts on natural plasmid transformation. Among 33 candidate genes, 28 knockout mutants had severely or completely impaired transformability. Some of these genes had established roles in T4P biogenesis; DNA transfer across the outer membrane, periplasm, or inner membrane; and protection of intracellular single-stranded DNA (ssDNA). Other genes had no previously reported roles in natural transformation of A. baumannii, including competence activator cAMP receptor protein (CRP), a periplasmic protein that may function in T4P assembly (TonB2), a T4P secretin-associated protein (TsaP), and two type II secretion system (T2SS) minor pseudopilus assembly prime complex competent proteins (XcpV and XcpW). The deletion of the T2SS assembly platform protein X had no effect on transformation, and the minor pseudopilins were capable of initiating major pilin assembly. Thus, we speculate that XcpV and XcpW may function in DNA uptake with major pilin assembly, a non-T2SS-dependent mechanism and that a competence pseudopilus similar to T4P constituted the central part of the DNA uptake complex. These results may help guide future research on the alarming increase of antibiotic resistance in this pathogen.