MGFfinder (a bioinformatic toolbox that identifies integrative MGEs and their insertion sites using short-read sequencing data)
Introduction/Definitions:
Conjugation — DNA transfer through direct cell-to-cell contact.
Transformation — uptake of extracellular DNA.
Transduction — bacteriophage-mediated DNA transfer.
Mobile genetic elements (MGEs) are segments of genetic material capable of moving from one location to another within a genome or between different cells, contributing to genetic variation and horizontal gene transfer.
Plasmid is an extrachromosomal DNA molecule capable of autonomous replication, typically independent of the bacterial chromosome.
Acquisition of foreign DNA material through horizontal gene transfer (HGT) is one of the most important drivers of bacterial evolution and it is frequently responsible for the development of antimicrobial resistance. Most antimicrobial agents used in clinical practice are (or derive from) products naturally found in the environmment (mostly soil). Bacteria sharing the environment with these molecules harbor intrinsic genetic determinants of resistance and there is robust evidence suggesting that such environmental resitome is a prolific source for the acquisition of antibiotic resistance genes in clinically relevant bacteria. Classically, bacteria acquire external genetic material through three main strategies (i) transformation (incorporation of naked DNA), ii) transduction (phage mediated) and, (iii) conjugation (bacterial “sex”). (Arias, “Mechanisms of Antibiotic Resistance” Microbiol Spectr, 2016, 4(2)).
The ecological role of naturally produced antibiotics should be to inhibit competitors. Given this role, resistance genes should have evolved to counteract antimicrobial action, so that the function of each of these determinants is to avoid the activity of one specific antibotic (or a family of antibiotics with similar structures). Work with genes acquired by horizonatal gene transfer (HGT) confirm this idea. Plasmid-encoded beta-lactamases serve to resist beta-lactam antibiotics and are not active agaisnt other kinds of drugs. The same applies for mutation-drived resistance; quinolone reistance is the consequence of mutations in topoisoerases genes, which do not alter bacterial susceptibility to other antibotics. The only impact that hte acquisition of resistance is usually supposed to have on bacterial physiology is to produce a general metabolic burden. (Rojo, “Metabolic regulation of antibiotic resistance” FEMS Microbiol Rev 35 (2011) 768-789).
Plasmids, bacteriophages, and extracellular DNA are the three primary drivers of HGT through the processes of conjugation, transduction, and natural transformation, respectively. See Cameron Mobile genetic celments (MGEs), such as bacteriophages, plasmids, and integrative and conjugative elements (ICEs), play a central role in shaping the structure and function of microbioal communities. (Zhao, “Jumbo phage-mediated transduction of genomic islands” 2025)
Conjugation:
Conjugation through plasmids is considered the main recognized mechanism responsible for genetic material transfer in bacteria and for the emergence of multi-drug resistance in hospital environments and aquaculture, amongst others. Conjugation is one of the most active ways of gene transfer, and it is responsible for the propagation of different antibiotic resistance genes in the Enterobacteriaceae family, the conjugative plasmids being the most studied mobile genetic elements.
Bacterial conjugation: Bacterial conjugation is the transfer of genetic material, including plasmid or chromosomal DNA, from a donor bacterium to a recipient bacterium through direct cell-to-cell contact.
Bacterial conjugation also referred to as bacterial sex, is a major horizontal gene transfer mechanism through which DNA is transferred from a donor to a recipient bacterium by direct contact. Conjugation is universally conserved among bacteria and occurs in a wide range of environments (soil, plant surfaces, water, sewage, biofilms, and host-associated bacterial communities). Within these habitats, conjugation drives the rapid evolution and adaptation of bacterial strains by mediating the propagation of various metabolic properties, including symbiotic lifestyle, virulence, biofilm formation, resistance to heavy metals, and, most importantly, resistance to antibiotics.
Conjugation was first discovered in 1946 by Edward Tatum and Joshua Lederberg, who showed that bacteria could exchange genetic information through the unidirectional transfer of DNA, mediated by a so-called F (Fertility) factor. It was later realized that the F factor is a replicative extra-chromosomal genetic element, for which they later coined the term plasmid, that can be transferred across the cell membranes of the parental strains. Since this seminal discovery, the identification of a plethora of conjugative elements, including plasmids, conjugative transposons, and integrative conjugative elements (ICEs), has revealed that conjugation is a universally conserved DNA transfer mechanism among Gram-negative and Gram-positive bacteria. See Lesterlin
Conjugative plasmids generally carry all the genes required for their maintenance during the vertical transfer from the mother to the daughter cells, as well as the genes necessary for horizontal transfer during conjugation from the donor to the recipient cell. These functions are encoded by different regions or modules that compose what is generally referred to as the plasmid backbone. Isolation and sequence analysis of an increasing number of conjugative plasmids has revealed considerable diversity in terms of genetic properties and organization. This diversity also indicates that different plasmids might use various regulations, molecular reactions, and strategies to achieve productive conjugational transfer and maintenance.
In contrast to transformation and transduction, conjugation requires direct cell–cell contact, with the interaction of a highly specialized structure called pilus, which is formed by a protein encoded in the plasmid termed pro-pilin that is part of the tubular structure of the pilus. This cell–cell interaction results in the unidirectional transfer of genetic material (from the donor cell to the recipient cell). This transfer is carried out with the formation of a bridge or conjugative pore that allows the communication between cellular cytoplasm of both cells. Once the recipient cell has acquired the new genetic material, it also acquires the genetic and phenotypic characteristics (encoded in the plasmid) including the transfer characteristics (tra genes).
The typical conjugative apparatus consists of four components: an origin of transfer (oriT), a relaxase, a type IV coupling protein, and a type IV secretion system (a tube-like structure called pilus that allows donors to contact recipients). Only a very small fraction of cells
carrying conjugative plasmids express the conjugation machinery, but if the plasmids provide a fitness advantage, the transconjugants can rapidly expand in the population. Between 35% and over 80% of E. coli isolates collected from different habitats have conjugative plasmids with genes that confer resistance to at least one antibiotic. Mating experiments conducted in laboratory culture have shown that the conjugation frequency is affected by multiple factors, including the nature of the recipient cells, growth phase, cell density, donor-to-recipient ratio, whether conjugation is conducted in liquid or solid media, carbon, oxygen, bile salts, metal concentrations, presence of mammalian cells, temperature, pH, and mating time. See Cortez-Cortez
Plasmids
Plasmids are self-replicating mobile genetic elements. They are separate from the chromosome and contain a specific subset of genes from the bacterial genetic pool. Many plasmids conjugate between different bacteria, especially related ones, leading to intra- and inter-specific dissemination of plasmid-specific genes, for instance, antibiotic resistance genes. As a result, virtually identical plasmids are isolated repetitively in different bacterial species.
Plasmids are extrachromosomal genetic elements that replicate independently of chromosomes. Persistence of these selfish genetic elements is improved when they carry genes that are useful to the host cell, such as ARGs in the presence of antibiotics. Consequently, many different ARGs circulate on plasmids.
Plasmids disseminate through bacterial populations primarily through the process of conjugation. Conjugation requires physical contact between two cells in the same environment, followed by the formation of a bridge that enables the transfer of a plasmid from a donor to a recipient cell
Transformation:
Bacterial transformation is the genetic alteration in a cell as a result of the direct absorption,
incorporation, and expression of exogenous DNA between closely related bacteria, and it is mediated chromosomally by encoded proteins. This foreign genetic material is ‘naked’ and can be present in the environment in which the bacterium thrives, and it can penetrate the bacterial cell membrane when the bacterium is in a ‘competent’ state, either due to lack of nutrients or elevated cell density. In order for the transformation to happen, the DNA must be transferred from the surface to the cytoplasmic membrane and then cross the cytoplasmic membrane through a highly conserved membrane channel.
This natural transformation is distinct from artificial transformation techniques involving electroporation and heat-shock treatments with chemically modified (e.g. CaCl 2 and MgCl2) cells that are routinely used for molecular biology applications. Transformation competence can be naturally or artificially induced, but not all bacterial species develop natural competence. In naturally transformable bacteria, competence is usually transient and induced by alterations in the growth state of organism. A group of “competence genes” has been identified, and general mechanistic models have been proposed. In general, Escherichia coli is not believed to be naturally transformable; it develops high genetic competence only under artificial conditions, including exposure to high Ca2+ concentrations and temperature shock. However, reportedly, E. coli can express modest competence under certain conditions that are feasible in its natural environments.
In many naturally competent bacterial species type-IV pili (T4P) are required for natural transformation. While the exact role of T4P in natural transformation is unclear, the generally accepted model is that DNA binds to the pilus structure, which retracts and pulls the DNA to the cell surface. It is unclear whether or not DNA is translocated across the outer membrane through the PilQ secretin pore.
In Gram-positive bacterial species that do not produce T4P, natural transformation involves a number of proteins with homology to T4P proteins, which are thought to form a pseudopilus
structure that spans the cell wall and is coupled to the DNA translocation complex at the cytoplasmic membrane.
Once exogenous DNA has been taken up by the cell it can be stably incorporated into the genome via recombination or transposition, or be maintained as an independent replicon if
plasmid DNA is taken up by an appropriate host.
Transduction through bacteriophages:
Another HGT mechanism is transduction, in which DNA transfer is mediated by independently replicating bacteriophages, bacterial viruses that can package segments of host DNA in their capsid, and inject it into a new host when an environmental stimulus triggers cell lysis. When this happens, the new injected genetic material in the cell infected by the virus can be recombined with the chromosomal DNA, generating either a lytic or lysogenic cycle.
Bacteriophages (or phages) are viruses that infect bacteria. The phage–host relationship is specific and complex. Receptor-binding proteins (RBPs) of phages such as tail fibers and tail spikes are the first phage proteins interacting with the host, initiating the infection process. These proteins can specifically bind outer cell wall structures of bacteria such as capsular polysaccharides (CPS), or lipopolysaccharides (LPS), (lipo)teichoic acids, outer membrane proteins, flagella, or pili. Whereas most phages encode a single or two RBPs, some polyvalent phages express multiple RBPs, forming a branched RBP structure. Each of these RBPs
recognizes a different receptor, allowing the phage to infect multiple hosts.
Transduction occurs when viral particles transfer bacterial genes. After infection with a bacteriophage, bacterial DNA is sometimes accidentally packaged in a bacteriophage capsid. A capsid containing bacterial DNA is fully capable of binding to a recipient cell and injecting the foreign DNA. If the transferred bacterial DNA is recombined into the genome of the recipient cell, transduction has occurred.