Integrons are genetic mechanisms that allow bacteria to adapt and evolve rapidly through the stockpiling and expression of new genes. [1] These genes are embedded in a specific genetic structure called gene cassette (a term that is lately changing to integron cassette) that generally carries one promoterless open reading frame (ORF) together with a recombination site (attC). Integron cassettes are incorporated to the attI site of the integron platform by site-specific recombination reactions mediated by the integrase.
Integrons were initially discovered on conjugative plasmids through their role in antibiotic resistance. [2] Indeed, these mobile integrons, as they are now known, can carry a variety of cassettes containing genes that are almost exclusively related to antibiotic resistance. Further studies have come to the conclusion that integrons are chromosomal elements, and that their mobilisation onto plasmids has been fostered by transposons and selected by the intensive use of antibiotics. The function of the majority of cassettes found in chromosomal integrons remains unknown.
Cassette maintenance requires that they be integrated within a replicative element (chromosome, plasmids). The integrase encoded by the integron preferentially catalyses two types of recombination reaction: 1) attC x attC, which results in cassette excision, 2) attI x attC, which allows integration of the cassette at the attI site of the integron. Once inserted, the cassette is maintained during cell division. [3] Successive integrations of gene cassettes result in the formation of a series of cassettes. The cassette integrated last is then the one closest to the Pc promoter at the attI site. The IntI-catalysed mode of recombination involves structured single-stranded DNA and gives the attC site recognition mode unique characteristics. [4] The integration of gene cassettes within an integron also provides a Pc promoter that allows expression of all cassettes in the array, much like an operon. [3] The level of gene expression of a cassette is then a function of the number and nature of the cassettes that precede it. In 2009, Didier Mazel and his team showed that the expression of the IntI integrase was controlled by the bacterial SOS response, thus coupling this adaptive apparatus to the stress response in bacteria. [5]
An integron is minimally composed of: [6] [7]
Additionally, an integron will usually contain one or more gene cassettes that have been incorporated into it. The gene cassettes may encode genes for antibiotic resistance, although most genes in integrons are uncharacterized. An attC sequence (also called 59-be) is a repeat that flanks cassettes and enables cassettes to be integrated at the attI site, excised and undergo horizontal gene transfer.
Integrons may be found as part of mobile genetic elements such as plasmids and transposons. Integrons can also be found in chromosomes.
The term super-integron was first applied in 1998 (but without definition) to the integron with a long cassette array on the small chromosome of Vibrio cholerae . [9] [10] The term has since been used for integrons of various cassette array lengths or for integrons on bacterial chromosomes (versus, for example, plasmids). Use of "super-integron" is now discouraged since its meaning is unclear. [9]
In more modern usage, an integron located on a bacterial chromosome is termed a sedentary chromosomal integron, and one associated with transposons or plasmids is called a mobile integron. [11]
Bacterial conjugation is the transfer of genetic material between bacterial cells by direct cell-to-cell contact or by a bridge-like connection between two cells. This takes place through a pilus. It is a parasexual mode of reproduction in bacteria.
A plasmid is a small, extrachromosomal DNA molecule within a cell that is physically separated from chromosomal DNA and can replicate independently. They are most commonly found as small circular, double-stranded DNA molecules in bacteria; however, plasmids are sometimes present in archaea and eukaryotic organisms. Plasmids often carry useful genes, such as for antibiotic resistance. While chromosomes are large and contain all the essential genetic information for living under normal conditions, plasmids are usually very small and contain additional genes for special circumstances.
Horizontal gene transfer (HGT) or lateral gene transfer (LGT) is the movement of genetic material between organisms other than by the ("vertical") transmission of DNA from parent to offspring (reproduction). HGT is an important factor in the evolution of many organisms. HGT is influencing scientific understanding of higher-order evolution while more significantly shifting perspectives on bacterial evolution.
Pathogenicity islands (PAIs), as termed in 1990, are a distinct class of genomic islands acquired by microorganisms through horizontal gene transfer. Pathogenicity islands are found in both animal and plant pathogens. Additionally, PAIs are found in both gram-positive and gram-negative bacteria. They are transferred through horizontal gene transfer events such as transfer by a plasmid, phage, or conjugative transposon. Therefore, PAIs contribute to microorganisms' ability to evolve.
A transposase is any of a class of enzymes capable of binding to the end of a transposon and catalysing its movement to another part of a genome, typically by a cut-and-paste mechanism or a replicative mechanism, in a process known as transposition. The word "transposase" was first coined by the individuals who cloned the enzyme required for transposition of the Tn3 transposon. The existence of transposons was postulated in the late 1940s by Barbara McClintock, who was studying the inheritance of maize, but the actual molecular basis for transposition was described by later groups. McClintock discovered that some segments of chromosomes changed their position, jumping between different loci or from one chromosome to another. The repositioning of these transposons allowed other genes for pigment to be expressed. Transposition in maize causes changes in color; however, in other organisms, such as bacteria, it can cause antibiotic resistance. Transposition is also important in creating genetic diversity within species and generating adaptability to changing living conditions.
A tumour inducing (Ti) plasmid is a plasmid found in pathogenic species of Agrobacterium, including A. tumefaciens, A. rhizogenes, A. rubi and A. vitis.
Lacticaseibacillus casei is an organism that belongs to the largest genus in the family Lactobacillaceae, a lactic acid bacteria (LAB), that was previously classified as Lactobacillus casei. This bacteria has been identified as facultatively anaerobic or microaerophilic, acid-tolerant, non-spore-forming bacteria.
Site-specific recombinase technologies are genome engineering tools that depend on recombinase enzymes to replace targeted sections of DNA.
In biology, a gene cassette is a type of mobile genetic element that contains a gene and a recombination site. Each cassette usually contains a single gene and tends to be very small; on the order of 500–1,000 base pairs. They may exist incorporated into an integron or freely as circular DNA. Gene cassettes can move around within an organism's genome or be transferred to another organism in the environment via horizontal gene transfer. These cassettes often carry antibiotic resistance genes. An example would be the kanMX cassette which confers kanamycin resistance upon bacteria.
Mobile genetic elements (MGEs), sometimes called selfish genetic elements, are a type of genetic material that can move around within a genome, or that can be transferred from one species or replicon to another. MGEs are found in all organisms. In humans, approximately 50% of the genome is thought to be MGEs. MGEs play a distinct role in evolution. Gene duplication events can also happen through the mechanism of MGEs. MGEs can also cause mutations in protein coding regions, which alters the protein functions. These mechanisms can also rearrange genes in the host genome generating variation. These mechanism can increase fitness by gaining new or additional functions. An example of MGEs in evolutionary context are that virulence factors and antibiotic resistance genes of MGEs can be transported to share genetic code with neighboring bacteria. However, MGEs can also decrease fitness by introducing disease-causing alleles or mutations. The set of MGEs in an organism is called a mobilome, which is composed of a large number of plasmids, transposons and viruses.
The mobilome is the entire set of mobile genetic elements in a genome. Mobilomes are found in eukaryotes, prokaryotes, and viruses. The compositions of mobilomes differ among lineages of life, with transposable elements being the major mobile elements in eukaryotes, and plasmids and prophages being the major types in prokaryotes. Virophages contribute to the viral mobilome.
Artificial gene synthesis, or simply gene synthesis, refers to a group of methods that are used in synthetic biology to construct and assemble genes from nucleotides de novo. Unlike DNA synthesis in living cells, artificial gene synthesis does not require template DNA, allowing virtually any DNA sequence to be synthesized in the laboratory. It comprises two main steps, the first of which is solid-phase DNA synthesis, sometimes known as DNA printing. This produces oligonucleotide fragments that are generally under 200 base pairs. The second step then involves connecting these oligonucleotide fragments using various DNA assembly methods. Because artificial gene synthesis does not require template DNA, it is theoretically possible to make a completely synthetic DNA molecule with no limits on the nucleotide sequence or size.
Plant transformation vectors are plasmids that have been specifically designed to facilitate the generation of transgenic plants. The most commonly used plant transformation vectors are T-DNA binary vectors and are often replicated in both E. coli, a common lab bacterium, and Agrobacterium tumefaciens, a plant-virulent bacterium used to insert the recombinant DNA into plants.
A toxin-antitoxin system consists of a "toxin" and a corresponding "antitoxin", usually encoded by closely linked genes. The toxin is usually a protein while the antitoxin can be a protein or an RNA. Toxin-antitoxin systems are widely distributed in prokaryotes, and organisms often have them in multiple copies. When these systems are contained on plasmids – transferable genetic elements – they ensure that only the daughter cells that inherit the plasmid survive after cell division. If the plasmid is absent in a daughter cell, the unstable antitoxin is degraded and the stable toxic protein kills the new cell; this is known as 'post-segregational killing' (PSK).
Plasmid-mediated resistance is the transfer of antibiotic resistance genes which are carried on plasmids. Plasmids possess mechanisms that ensure their independent replication as well as those that regulate their replication number and guarantee stable inheritance during cell division. By the conjugation process, they can stimulate lateral transfer between bacteria from various genera and kingdoms. Numerous plasmids contain addiction-inducing systems that are typically based on toxin-antitoxin factors and capable of killing daughter cells that don't inherit the plasmid during cell division. Plasmids often carry multiple antibiotic resistance genes, contributing to the spread of multidrug-resistance (MDR). Antibiotic resistance mediated by MDR plasmids severely limits the treatment options for the infections caused by Gram-negative bacteria, especially family Enterobacteriaceae. The global spread of MDR plasmids has been enhanced by selective pressure from antimicrobial medications used in medical facilities and when raising animals for food.
Ruth Milne Hall, OAM, FAA, FAAM is an Australian microbiologist whose research on mobile genetic elements in bacteria has provided deep insight into the transfer and evolution of antibiotic resistance in bacteria.
DNA transposons are DNA sequences, sometimes referred to "jumping genes", that can move and integrate to different locations within the genome. They are class II transposable elements (TEs) that move through a DNA intermediate, as opposed to class I TEs, retrotransposons, that move through an RNA intermediate. DNA transposons can move in the DNA of an organism via a single-or double-stranded DNA intermediate. DNA transposons have been found in both prokaryotic and eukaryotic organisms. They can make up a significant portion of an organism's genome, particularly in eukaryotes. In prokaryotes, TE's can facilitate the horizontal transfer of antibiotic resistance or other genes associated with virulence. After replicating and propagating in a host, all transposon copies become inactivated and are lost unless the transposon passes to a genome by starting a new life cycle with horizontal transfer. It is important to note that DNA transposons do not randomly insert themselves into the genome, but rather show preference for specific sites.
Polintons are large DNA transposons which contain genes with homology to viral proteins and which are often found in eukaryotic genomes. They were first discovered in the mid-2000s and are the largest and most complex known DNA transposons. Polintons encode up to 10 individual proteins and derive their name from two key proteins, a DNA polymerase and a retroviral-like integrase.
Integrative and conjugative elements (ICEs) are mobile genetic elements present in both gram-positive and gram-negative bacteria. In a donor cell, ICEs are located primarily on the chromosome, but have the ability to excise themselves from the genome and transfer to recipient cells via bacterial conjugation.
CRISPR-associated transposons or CASTs are mobile genetic elements (MGEs) that have evolved to make use of minimal CRISPR systems for RNA-guided transposition of their DNA. Unlike traditional CRISPR systems that contain interference mechanisms to degrade targeted DNA, CASTs lack proteins and/or protein domains responsible for DNA cleavage. Specialized transposon machinery, similar to that of the well characterized Tn7 transposon, complexes with the CRISPR RNA (crRNA) and associated Cas proteins for transposition. CAST systems have been characterized in a wide range of bacteria and make use of variable CRISPR configurations including Type I-F, Type I-B, Type I-C, Type I-D, Type I-E, Type IV, and Type V-K. MGEs remain an important part of genetic exchange by horizontal gene transfer and CASTs have been implicated in the exchange of antibiotic resistance and antiviral defense mechanisms, as well as genes involved in central carbon metabolism. These systems show promise for genetic engineering due to their programmability, PAM flexibility, and ability to insert directly into the host genome without double strand breaks requiring activation of host repair mechanisms. They also lack Cas1 and Cas2 proteins and so rely on other more complete CRISPR systems for spacer acquisition in trans.