Biotechnological applications of mobile group II introns and their reverse transcriptases: gene targeting, RNA-seq, and non-coding RNA analysis
© Enyeart et al.; licensee BioMed Central Ltd. 2014
Received: 19 September 2013
Accepted: 19 November 2013
Published: 13 January 2014
Mobile group II introns are bacterial retrotransposons that combine the activities of an autocatalytic intron RNA (a ribozyme) and an intron-encoded reverse transcriptase to insert site-specifically into DNA. They recognize DNA target sites largely by base pairing of sequences within the intron RNA and achieve high DNA target specificity by using the ribozyme active site to couple correct base pairing to RNA-catalyzed intron integration. Algorithms have been developed to program the DNA target site specificity of several mobile group II introns, allowing them to be made into ‘targetrons.’ Targetrons function for gene targeting in a wide variety of bacteria and typically integrate at efficiencies high enough to be screened easily by colony PCR, without the need for selectable markers. Targetrons have found wide application in microbiological research, enabling gene targeting and genetic engineering of bacteria that had been intractable to other methods. Recently, a thermostable targetron has been developed for use in bacterial thermophiles, and new methods have been developed for using targetrons to position recombinase recognition sites, enabling large-scale genome-editing operations, such as deletions, inversions, insertions, and ‘cut-and-pastes’ (that is, translocation of large DNA segments), in a wide range of bacteria at high efficiency. Using targetrons in eukaryotes presents challenges due to the difficulties of nuclear localization and sub-optimal magnesium concentrations, although supplementation with magnesium can increase integration efficiency, and directed evolution is being employed to overcome these barriers. Finally, spurred by new methods for expressing group II intron reverse transcriptases that yield large amounts of highly active protein, thermostable group II intron reverse transcriptases from bacterial thermophiles are being used as research tools for a variety of applications, including qRT-PCR and next-generation RNA sequencing (RNA-seq). The high processivity and fidelity of group II intron reverse transcriptases along with their novel template-switching activity, which can directly link RNA-seq adaptor sequences to cDNAs during reverse transcription, open new approaches for RNA-seq and the identification and profiling of non-coding RNAs, with potentially wide applications in research and biotechnology.
KeywordsGenome engineering Metabolic engineering Next-generation RNA sequencing Ribozyme Synthetic biology Systems biology Targetron
Mobile group II introns are bacterial retrotransposons that perform a remarkable ribozyme-based, site-specific DNA integration reaction (‘retrohoming’) and encode an equally remarkable reverse transcriptase (RT), both of which have been harnessed for biotechnological applications [1–3]. Retrohoming occurs by a mechanism in which the group II intron RNA uses its ribozyme activity to insert directly into a DNA strand, where it is reverse transcribed by the intron-encoded RT (also referred to as the intron-encoded protein or IEP), yielding a cDNA copy of the intron that is integrated into the genome . Because mobile group II introns recognize DNA target sequences largely by base pairing of sequence motifs within the intron RNA, they can be programmed to insert into desired DNA sites by simply modifying the intron sequences so as to base pair to the new target site. This feature allows mobile group II introns to be made into gene targeting vectors, or ‘targetrons’, which combine high DNA integration efficiency with readily programmable and reliable DNA target specificity [5–7]. Targetrons are widely used for genetic engineering of bacteria, and efforts continue to adapt them for function in eukaryotes.
Group II intron RTs function in retrohoming by synthesizing a full-length cDNA of the highly structured intron RNA with high processivity and fidelity [8–10], properties that are useful for biotechnological applications involving cDNA synthesis, such as qRT-PCR and next-generation RNA sequencing (RNA-seq). The RTs also have a novel template-switching activity that enables facile attachment of adaptor sequences containing primer-binding sites and barcodes to cDNAs. These properties, combined with the availability of naturally occurring thermostable group II intron RTs [11, 12] open new approaches for RNA-seq and the profiling and discovery of miRNAs and other non-coding RNAs [10, 13].
Here, we describe how the novel biochemical activities of mobile group II introns and their RTs, which were acquired during the evolution of group II introns as mobile genetic elements, have been adapted for biotechnological applications. We then review how group II intron-derived targetrons have been used for the genetic engineering of diverse bacteria, as well as recent advances in targetron technology. The latter include the development of a thermotargetron for gene targeting in thermophiles, methods for using targetrons to position recombinase recognition sites for large-scale genome rearrangements, and progress in developing targetrons for gene targeting in eukaryotes. Finally, we discuss the development of thermostable group II intron RTs from bacterial thermophiles as new tools for cDNA synthesis, with potentially wide applications in research and biotechnology.
Mobile group II introns
Mobile group II introns are found in bacteria, archaea, and the mitochondrial and chloroplast DNAs of some eukaryotes, and are thought to be evolutionary ancestors of spliceosomal introns, the spliceosome, retrotransposons, and retroviruses in higher organisms [3, 14, 15]. They are especially prevalent and widespread in bacteria, with hundreds of bacterial group II introns having been identified by genome sequencing .
The folded group II intron RNA contains an active site that uses specifically bound Mg2+ ions to catalyze RNA splicing via two sequential transesterification reactions that yield ligated exons and an excised intron lariat RNA, the same reaction mechanism used for the splicing of nuclear spliceosomal introns in eukaryotes (Figure 1C) . Because the transesterification reactions used for splicing are reversible, the intron RNA can also catalyze reverse splicing of the intron into RNA or DNA sites containing the ligated exon sequence, with reverse splicing into DNA playing a key role in intron mobility. Both steps of reverse splicing (referred to as complete reverse splicing) result in the insertion of the excised intron RNA between the 5’ and 3’ exons, while the first step (referred to as partial reverse splicing) results in the attachment of the 3’ end of the intron RNA to the 5’ end of the downstream exon, leaving a strand break.
Some key regions of group II intron RNAs are DI, which contains the motifs that base pair with the DNA target site; DIV, which contains the ORF encoding the RT; DV, a metal-ion-binding domain that comprises most of the active site; and DVI, which contains the branch-point nucleotide . Three subclasses of group II introns, denoted IIA, IIB, and IIC, have been distinguished by variations of the conserved RNA structure . Crystal structures of a group IIC intron at different stages of reaction have been determined, providing insight into the nature of the active site and the mechanisms of RNA splicing and reverse splicing (Figure 1B) [19–21].
Group II intron retrohoming
The cDNA copy of the reverse-spliced intron RNA is integrated into the host genome by common cellular DNA recombination or repair mechanisms, a feature that contributes to the wide host range of group II introns. Recent findings have further elucidated late steps in group II intron integration in Escherichia coli, in which a cellular RNase H degrades the intron RNA, and replication restart proteins then recruit the host replicative DNA polymerase, which synthesizes DNA corresponding to the sense strand of the intron . Host nucleases trim DNA overhangs, and ligases repair remaining nicks .
Some group II introns splice via hydrolysis rather than branching and thus excise a linear rather than a lariat intron RNA [32, 33]. During retrohoming, linear group II intron RNAs can carry out only the first step of reverse splicing, attaching the 3’ end of the linear intron to the downstream DNA exon, which, combined with En cleavage of the opposite strand, yields a double-strand break that can be repaired by homologous recombination with exogenous DNA . This double-strand break-stimulated recombination provides an alternative gene targeting mechanism for group II introns, analogous to that used by Zn-finger nucleases, TALENs, and CRISPR-based systems . In some hosts, the linear group II intron RNA inserted at a target site is reverse-transcribed to yield a cDNA that can be integrated into the genome by non-homologous end joining [36, 37].
DNA-target site recognition
In group IIA introns, like the Ll.LtrB intron, the intron RNA contains three sequence motifs in DI that recognize DNA target sites by base pairing. These are denoted EBS1, EBS2, and δ, and they base pair to complementary sequences in the DNA target site denoted IBS1, IBS2, and δ’ (where EBS stands for ‘exon-binding site’ and IBS stands for ‘intron-binding site’; these same interactions also occur upon splicing out of a larger RNA molecule). The Ll.LtrB RT (denoted LtrA protein) recognizes nucleotides both upstream and downstream of the IBS/δ’ sequences (colored purple and blue, respectively, in Figure 4). Binding of the RT promotes DNA melting , enabling the intron RNA to base pair to the DNA target sequence, and DNA bending, which positions the target DNA properly for cleavage and priming of reverse transcription .
Group IIB introns, like EcI5 and RmInt1, also contain three sequence elements that recognize the DNA target site by base pairing. Specifically, EBS1, EBS2, and EBS3 base pair to corresponding IBS sequences in the target. The EBS3 sequence is located in a different part of the DI structure than the corresponding δ sequence in group IIA introns . The RT again recognizes flanking sequences. In EcI5, a relatively well-studied example of this class , the RT recognizes a similar number of residues as the RT of Ll.LtrB, although the identities and locations of these residues differ. RmInt1, whose RT lacks the En domain, inserts into the single-stranded DNA formed during replication and thus has no requirement for DNA melting . The RmInt1 RT recognizes only two critical nucleotide residues, but additional sequences may contribute .
Group IIC introns recognize short IBS1 and IBS3 sequences. A DNA hairpin, such as those found in gene terminators or phage attachment sites, is also a key recognition determinant and seems to take the place of the IBS2 sequence for these introns, although the mechanism of recognition is as yet unknown [44–46]. Group IIC introns can thus integrate into multiple sites, and specificity is limited.
Group IIA and IIB introns have high DNA-target specificity and integrate only rarely into ectopic sites (for example, retrotransposition of the Ll.LtrB intron into ectopic sites in the E. coli chromosome occurs at a frequency of 0.1 to 30 × 10-6) [3, 47]. This high specificity reflects, in part, the fact that group II introns use both the RT and base pairing of the intron RNA to recognize their DNA target sequences, with the RTs of the Ll.LtrB and EcI5 introns most stringently recognizing 4 to 5 nts and intron RNA base pairing extending over 11 to 14 nts spanning the intron-insertion site. Additionally, because the heteroduplex between the intron RNA and DNA target strand must bind to the intron RNA’s active site for reverse splicing, mismatches in base pairing strongly affect the kcat as well as Km of the targeting reaction, providing greater discrimination against mispairings than can be obtained by binding affinity alone .
This intertwining of DNA target binding and catalysis differs from CRISPR-based systems, which have been used in bacteria and eukaryotes and also rely on base-pairing between RNA and DNA to provide specificity [49–55]. CRISPR systems use a guide RNA bound by a protein endonuclease (Cas9 being the canonical example) and can in theory target any stretch of twenty base pairs that is followed by a specific ‘protospacer adjacent motif’ (PAM), which in currently utilized systems is a stretch of two to five nts recognized by the endonuclease. However, the guide RNA does not play a catalytic role and thus specificity appears to be governed solely by its binding affinity to the DNA target site, with the protein endonuclease cutting anytime the RNA/protein complex tarries long enough at a given site. Indeed, concerns have been raised about the high off-targeting rate of these systems, with off-target sites having up to five mismatches found to be targeted at efficiencies similar to the intended site . A further limitation for wide use in bacteria is that, unlike group II introns, CRISPR-based systems function only to introduce a double-strand break, and integration of exogenous DNA at the break site is dependent upon homologous recombination at a higher efficiency than is found in most bacterial species .
Because mobile group II introns recognize their DNA target sites by a combination of base-pairing interactions and site-specific binding of the RT, the target site recognized by the RNP can be modified by finding other sites compatible with RT recognition and then changing the EBS/δ sequences of the intron as necessary to match the new site . Such retargeted mobile group II introns are called ‘targetrons.’ Group II introns that have been made into targetrons include both group IIA introns (Ll.LtrB ) and group IIB introns (EcI5  and RmInt1 ). Group IIC introns are less appealing as candidates for retargeting because they recognize hairpin structures via as yet unknown mechanisms. The Ll.LtrB targetron is commercially available through Sigma-Aldrich, and both the Ll.LtrB and EcI5 targetrons are available through Targetronics.
Although group II introns can and have been retargeted by the method mentioned above, in which the closest match to the native recognition site in a sequence to be targeted is identified, and the base-pairing sequences of the intron are modified to accommodate discrepancies, the rules by which introns recognize their target sites are actually more complex. For instance, the RT recognizes different residues at the target site with different stringencies, and none of these recognition events are absolutely required for retrohoming to occur [5, 58, 59]. If only the wild-type recognition sequence is used, then new targeting sites may be hard to come by, but knowing which bases can be varied and how is not a simple matter. The EBS/δ sequences may also differ in the stringency of required base-pairing interactions at different positions. Algorithms have therefore been developed for retargeting the Ll.LtrB  and EcI5  introns. These algorithms were developed by examining libraries of inserted mobile group II introns with randomized base-pairing motifs for the most frequently conserved residues and base-pairing interactions, and using these frequencies to generate weighting schemes for the various interactions. Potential target sites are then assessed using the weighted criteria and assigned a score. Although the algorithms have limitations and do not always correctly predict insertion frequency, typically a targetron efficient enough to be screened for site-specific insertion via colony PCR without selection can be found for any given stretch of 1,000 base pairs of DNA. Off-target integrations by the Ll.LtrB and EcI5 targetrons are rare and can generally be avoided by the prudent step of scanning the genome for closely matching target sites. However, the specificity of targetrons could vary for different target sites, making it important to confirm desired single integrations by Southern hybridization.
The actual retargeting process is carried out by using PCRs that modify the EBS/δ sequences within the intron to base pair to the DNA target site and simultaneously modify the IBS sequences upstream of the intron to base pair to the retargeted EBS sequences to allow the intron to splice out of a precursor RNA [6, 7]. The PCR product corresponding to a segment of the intron and upstream exon is then cloned into a targetron expression vector (see below). Alternatively, the entire region covering the IBS1 and 2 and the EBS1, 2 and δ sequences can be commercially synthesized in a single DNA molecule (for example, as a gBlock sold by IDT) that can be cloned directly into the vector . The outlying δ’ or EBS3/IBS3 positions are typically adjusted by cloning the PCR product into one of four parallel targetron vectors already containing the correct bases for these interactions.
Targetron use in bacteria
Bacteria in which targetrons have been used successfully
It is relatively simple to tailor the commercially available Ll.LtrB or EcI5 targetron cassettes for use in different bacterial hosts. This typically requires re-cloning the targetron cassette from the provided donor plasmid into an established host-specific or broad-host-range expression plasmid with a strong promoter. Continuous targetron expression, which can lead to off-target integrations, can be avoided by using an inducible promoter or a donor plasmid that is readily curable in the absence of selection. A RAM capable of functioning in the desired bacteria can also be introduced into the intron, but targeting frequencies are typically high enough to screen for targetron insertions by colony PCR, making such a marker dispensable. The ClosTron, which has made possible gene targeting in a wide range of notoriously difficult Clostridum spp., is a highly successful example of adaptation of the Ll.LtrB targetron from a commercial kit [63, 80], and similar adaptations of the Ll.LtrB targetron have been made for a variety of other bacteria (for example, [62, 64, 71, 81]). Because the initial reverse splicing and target-DNA-primed reverse transcription reactions are catalyzed by group II intron RNPs, and because the late steps of second-strand synthesis and cDNA integration are performed by common host factors [30, 31, 37, 82, 83], there are in principle no limitations to the number of bacterial species in which targetrons might function. As mobile group II introns are present in the genomes of some archaea , it seems likely that targetrons will prove useful in archaea, as well.
Applications of targetrons in bacteria
Targetrons have most frequently been used to generate knock-outs in bacteria. A great deal of work has been done using this method, with examples including identifying virulence factors [70, 72, 74, 85–88] and potential drug targets [89, 90], and examining the combinatorial effect of different genomic loci on protein expression .
Targetrons have been particularly widely used in strains of the genus Clostridium. Suicide plasmids were previously the only method of utility in these strains , but since Clostridia typically have very low transformations frequencies (for instance, more than a milligram of plasmid is required to transform Clostridium acetobutylicum), suicide plasmids are difficult to use in these organisms. Targetrons have thus greatly increased our understanding of and ability to engineer Clostridia, many of which are of medical and industrial importance. For instance, Clostridia include a number of biofuel-producing strains, and targetrons have come into frequent use to aid in understanding the metabolism of these strains and to engineer them for higher yields [92–110]. Targetron-mediated knockouts have been used in a large number of studies on sporulation, germination, and other aspects of the biology of Clostridium difficile, a leading cause of diarrhea in hospitals [88, 111–143]. Targetron technology has also benefitted the study of toxin production, sporulation, and other biological processes in Clostridium botulinum[144–153], Clostridium perfringens[69, 85, 154–164], and Clostridium sordellii[87, 165]. Work on developing targetrons for the thermophilic bacterium Clostridium thermocellum is discussed in more detail below.
Many bacteria of interest are difficult to transform due to restriction-modification systems. In Staphylococcus aureus, Clostridium acetobutylicum, and Clostridium cellulolyticum, targetrons were used to knock out restriction enzymes, thereby opening clinical and environmental isolates to systematic mutational analysis. Besides S. aureus and the Clostridium species mentioned previously, targetrons have been developed for use in other pathogenic bacteria, such as Francisella tularensis, Bacillus anthracis[68, 168], Listeria monocytogenes, Pasteurella multocida, Vibrio cholerae, and Ehrlichia chaffeensis, opening up the possibility of using targetrons to develop vaccine strains of these organisms.
Targetrons have also been used to deliver cargo genes, including genes for fluorescent proteins , phage resistance , and antigens for release into a host’s digestive system as a live vaccine . Unstructured sequences of less than 100 nts in length can usually be carried without impacting intron mobility. Longer sequences may impair functionality, and sequences above 1,000 nts can drastically decrease efficiency. DIV, particularly the DIVb loop, has been shown to be the best location to insert cargo genes for minimal impact on intron mobility . Targetrons have also been used to induce targeted genomic deletions via homologous recombination, albeit at much lower efficiencies than are possible in tandem use with recombinases .
Finally, the relative simplicity of targetron retargeting, combined with the falling costs of gene synthesis  and the increasing ability to automate the laboratory techniques involved [173, 174], opens the door to high-throughput construction of targetrons for simultaneous integration into a multiplicity of loci. Applications could include rapidly generating whole-genome knock-out libraries for novel organisms and testing in parallel different combinations of mutants discovered in random screens in order to, for example, improve the yield of a target metabolite or develop a suitable vaccine strain for a pathogenic organism. Two other recent extensions of targetron technology in bacteria are discussed below.
A thermotargetron for gene targeting in thermophiles
This TeI3c/4c thermotargetron was used for efficient gene targeting in Clostridium thermocellum, an organism used in the consolidated bioprocessing of lignocellulose biomass [178, 179]. Like many species of Clostridia, C. thermocellum has low, variable transformation frequencies. An important feature of the thermotargetron is its high integration efficiency, 67 to 100% without selection for seven successful gene disruptions, making it possible to identify disruptants by colony PCR of only a small number of transformants. Gene disruptions that block pathways leading to by-products of cellulose degradation increased cellulolytic ethanol production in C. thermocellum.
Another notable feature of the thermotargetron is that it recognizes DNA target sites almost entirely by base pairing of the intron RNA (11-bp), while the RT recognizes only two bases (Figure 7B). The contribution of the RT to DNA melting appears to be largely dispensable at higher temperatures. This feature is advantageous because it increases the number of potential target sites and should facilitate the targeting of short ORFs and small non-coding RNAs, not only in thermophiles but also potentially in mesophiles that can tolerate short times at elevated temperatures (45 to 48°C). A downside of the more limited protein recognition, however, is that it decreases DNA target specificity, thus requiring greater attention to targetron design to avoid integration into closely matching off-target sites. The decreased target specificity may also contribute to the lower success rate for gene disruptions (7 of 25 targetrons in initial tests), which could be due in part to deleterious off-target integrations. This situation should be ameliorated by the development of algorithms to minimize off target integrations, as done for other targetrons. The TeI3c/4c thermotargetron functions in both Gram-negative and Gram-positive bacteria and should be adaptable to a wide variety of thermophiles.
Use of targetrons for large-scale genome engineering
Targetrons have recently been adapted for carrying lox sites to facilitate large-scale genome engineering . While recombinase sites have been previously included in targetrons, they had rarely been used for any purpose other than removing selectable markers after integration [59, 80]. Lox sites and other recombinase recognition motifs with palindromic sequences can form stable hairpin structures upon transcription into RNA. In the absence of a selectable marker, such hairpin structures can significantly impair the functionality of both the Ll.LtrB and EcI5 targetrons. This effect was mitigated by adding non-base-pairing regions to the base of the hairpin structures, which presumably made the hairpins more flexible, such that they no longer interfered with the catalytic structures of the intron. These results point out the importance of considering structure when designing targetrons to carry cargo.
These examples are likely but the first in a series of innovations that will allow targetrons to be used for large-scale genomic engineering. There are currently few alternatives that allow the facile, site-specific introduction of genetic material into microorganisms. While some organisms, such as Streptococcus pneumoniae and Acinetobacter, have relatively robust systems for homologous recombination, most others do not. Similarly, while methods such as recombineering [183, 184] and MAGE  have been developed that allow PCR products and oligonucleotides to be readily introduced into E. coli in a site-specific manner, these methods do not scale to most other microorganisms. Targetrons are essentially the only tool that can be used to site-specifically ‘punctuate’ the genomes of a wide array of bacteria, as has previously been observed for recalcitrant thermophilic strains and Clostridia, discussed elsewhere in this review. While lox sites have been introduced to promote site-specific recombination, the option also exists to introduce a wide variety of other short genetic elements that can impact phenotype, including promoters, terminators, leader sequences, affinity tags, and even origins of replication. The use of targetron libraries [59, 66] to seek out sites that lead to improved functionality, combined with the use of efficient targetron insertion to rapidly introduce multiple targetrons into a single strain, either serially or in parallel, makes targetrons the tool of choice for the engineering of industrially-relevant microorganisms.
Prospects for targetron use in eukaryotes
Although efficient eukaryotic gene targeting technologies have been developed, including Zn-finger nucleases, TALENS, and CRISPR-based systems, targetrons offer the advantages of greater ease of retargeting than Zn-finger nucleases or TALENS and potentially higher DNA target specificity than any of the other methods. However, the barriers to targetron use in eukaryotes include the requirement for delivering RNPs containing a large, structured group II intron RNA to the nucleus, as well as the relatively high Mg2+ concentrations required for group II intron RNA function. Group II introns evolved to function in bacteria, whose free Mg2+ concentrations are generally 1 to 4 mM , whereas in eukaryotes, Mg2+ concentrations are <1 mM and possibly lower in nuclei, where Mg2+ is sequestered by binding to large amounts of DNA [186, 187]. These lower Mg2+ concentrations constitute a barrier to group II intron invasion of nuclear genomes and limit their efficiency for gene targeting in eukaryotes. Additional host defense and innate immunity mechanisms could also come into play.
Initial studies showed that Ll.LtrB targetron RNPs introduced into mammalian cells by transfection could integrate into separately transfected plasmid target sites albeit at low efficiency  and envisioned methods that might be used for targeted repair of human genes . In a later systematic study testing the feasibility of using targetrons in eukaryotes, Ll.LtrB targetron RNPs were microinjected directly into Xenopus laevis oocyte nuclei and tested for retrohoming and gene targeting via double-strand-break-stimulated homologous recombination in plasmid assays . These studies showed that retrohoming and targeting via group II intron-stimulated homologous recombination occurred efficiently (up to 38% and 4.8% of plasmid target sites, respectively), but required the injection of additional Mg2+, sufficient to obtain an intracellular concentration of 5 to 10 mM. A similar requirement for the injection of additional Mg2+ for retrohoming was found for targetron RNPs injected into Drosophila and zebrafish embryos . Injection of targetron RNPs plus Mg2+ have given targeted integration into the chromosomal yellow gene in flies at frequencies up to 0.12% in pooled embryos and 0.021% in pooled adult files , and in X. laevis, a different approach, using group II intron RNPs for site-specific DNA modification in sperm nuclei followed by in vitro fertilization, gave targeted integration at frequencies sufficiently high to detect knockouts in a single copy mitF gene by PCR screening of tail clippings (M Mastroianni, J Yao, and AM Lambowitz, unpublished data). However, the frequencies were variable and further improvements in efficiency and reliability are needed for these to become routine methods.
There is some prospect that more active group II introns with enhanced retrohoming in eukaryotes can be selected by directed evolution approaches. Recent work showed that Ll.LtrB introns that retrohome more efficiently at lower Mg2+ could be selected in an E. coli mutant deficient in Mg2+-transport , laying the groundwork for direct selections of group II introns that function more efficiently in eukaryotic cells. The recent group II intron RNA X-ray crystal structures [19–21] may also enable rational design approaches to enhancing group II intron function. If these efforts prove successful, the same rationales that are driving the use of targetrons for genomic engineering in bacteria will extend to genomic engineering in eukaryotes.
Thermostable group II intron reverse transcriptases
Reverse transcriptases are widely used in biotechnology for applications involving cDNA synthesis, such as qRT-PCR and RNA-seq. Most if not all of these applications would benefit from using RTs that synthesize cDNAs with high processivity and fidelity. However, the retroviral RTs that are commonly used for these methods have inherently low fidelity and processivity, reflecting that these enzymes evolved to help retroviruses evade host defenses by introducing sequence variations and rapidly propagating successful ones by RNA recombination . Vast sums have been expended to engineer variants of retroviral RTs that overcome these inherent deficiencies.
By contrast, group II intron RTs evolved to have high processivity and fidelity, reflecting their function in retrohoming, which requires synthesis of an accurate, full-length cDNA copy of a highly structured group II intron RNA [8, 9]. Other advantageous characteristics of group II intron RTs are their lack of RNase H activity, which enables reuse of RNA templates, and their difficulty in initiating on DNA templates, which preserves RNA strand information by minimizing recopying of cDNAs [23, 31].
By applying the above methods to group II intron RTs from the bacterial thermophiles Thermosynechococcus elongatus and Geobacillus stearothermophilus (Figure 2B), it was possible to obtain thermostable group II intron RT fusion proteins that synthesize cDNAs at temperatures up to 81°C with much higher processivity and two- to four-fold higher fidelity than retroviral RTs . The high processivity of group II intron RTs is advantageous for synthesizing long cDNAs that preserve information about alternatively spliced RNAs and for RNA footprinting and structure mapping using RNA modification reagents, where premature terminations by retroviral RTs at unmodified sites result in high background and loss of information. The higher fidelity of group II intron RT should benefit applications, such as tumor profiling, that require the analysis of sequence variants.
We are still at the early stages of developing methods and applications utilizing the novel properties of these enzymes. Group II intron RTs behave differently from retroviral RTs, both in terms of optimal conditions for different applications and their tighter binding to nucleic acids, which necessitates different types of clean-up procedures for cDNA products. Consequently, group II intron RTs cannot simply be substituted into protocols developed for retroviral RTs and must be optimized for each application. In a published application, a thermostable group II intron RT was used to generate RNA-seq libraries of human mRNAs, using an oligo(dT)42 primer . The resulting libraries showed relatively uniform 5’ to 3’ coverage of all size classes of human mRNAs, including those >7 kb, whereas parallel libraries prepared using the thermostable retroviral RT, SuperScript III, showed a strong bias for reads near the 3’ ends of mRNAs, reflecting premature terminations. The ability to obtain RNA-seq libraries with uniform 5’ to 3’ coverage using an oligo(dT) primer avoids steps such as ribodepletion and RNA fragmentation, which are needed to minimize rRNA contamination and obtain uniform coverage in libraries prepared using retroviral RTs. The minimal manipulation needed to prepare whole cell RNA-seq libraries using group II intron RTs may be useful for procedures that start with small amounts of RNA, such as transcriptome analysis from single cells.
Like other DNA and RNA polymerases, group II intron RTs are prone to add extra non-templated nucleotides to DNA upon reaching the end of an RNA template (ref.  and references therein), which could lead to non-seamless junctions and biases during template switching. This problem is avoided by using an initial template/primer substrate consisting of a synthetic RNA oligonucleotide annealed to a DNA primer that leaves a single nucleotide 3’ overhang. This 3’ overhang nucleotide base pairs with the 3’ terminal nucleotide of the second RNA template, resulting in a seamless switch to the second template. A specific 3’ overhang nucleotide can be used to direct the RT to a specific target RNA, while a mixture of 3’ overhang nucleotides is used to minimize bias for mixtures of templates having different 3’ RNA ends.
After template switching, the resulting cDNA linked to adapter sequences is circularized with CircLigase and PCR amplified to generate an RNA-seq library (Figure 10). By incorporating an additional step for removal of a 3’ phosphate of the target RNAs, the method can also be applied to protein- and ribosome-bound RNA fragments in procedures such as HITS-CLIP, CRAC, RIP-Seq, and ribosome profiling. Because thermostable group II intron RTs can template-switch to RNAs with modified 3’ ends and reverse transcribe through highly structured RNAs, the method can potentially lead to the identification of novel miRNAs and other non-coding RNAs that cannot be cloned by current methods using retroviral RTs.
The biotechnological applications of mobile group II introns and their RTs are an example of how basic research into biochemical mechanisms and evolution can lead to unexpected practical applications. Targetrons, which arose from studies of the mechanism of group II intron mobility, now provide a broad-host-range solution to knock-outs and, when combined with other technologies, such as site-specific recombinases, can be employed to make a wide variety of changes in almost any bacteria, including previously recalcitrant medically and industrially important species. Together with the prospect of targetron-mediated mutagenesis in archaea and the possibility of adapting targetrons for use in eukaryotes, targetrons are well-positioned to play an expanding role in the analysis and engineering of genomes for biotechnological and medical applications. The unique properties of group II RTs, enzymes that were discovered solely as a consequence of basic research, may ameliorate many of the problems of current in vitro methodologies for RNA analysis, qRT-PCR, and RNA-seq, with potentially wide applications in biomedical research, diagnostics, and biotechnology.
Cross-linking and analysis of cDNA
Group II intron RNA domains I-VI
DNA-binding domain of group II intron reverse transcriptases
- E1 and E2:
5’ and 3’ exons
DNA endonuclease domain of group II intron reverse transcriptases
High-throughput sequencing by cross-linking immunoprecipitation
Group II intron with ORF encoding the RT deleted
Protospacer adjacent motif
RNA immunoprecipitation and sequencing
We thank Dr. Gregory Davis (Sigma-Aldrich) for comments on the manuscript. Research on targetrons and group II intron reverse transcriptases in AML’s laboratory is supported by NIH grants GM037949 and GM037951 and Welch Foundation grant F-1607. Research on targetrons in ADE’s laboratory has been supported by a National Science Foundation Graduate Research Fellowship for PJE under Grant No. DGE-1110007, a National Security Science and Engineering Faculty Fellowship (FA9550-10-1-0169), and the Welch Foundation (F-1654).
- Pyle AM, Lambowitz AM: Group II introns: ribozymes that splice RNA and invade DNA. In The RNA World, Third Edition. Edited by: Gesteland RF, Cech T, Atkins JF. Cold Spring Harbor, N.Y: Cold Spring Harbor Laboratory Press; 2006:469-505.Google Scholar
- Toro N, Jiménez-Zurdo JI, García-Rodríguez FM: Bacterial group II introns: not just splicing. FEMS Microbiol Rev 2007, 31: 342-358. 10.1111/j.1574-6976.2007.00068.xPubMedGoogle Scholar
- Lambowitz AM, Zimmerly S: Group II introns: mobile ribozymes that invade DNA. Cold Spring Harb Perspect Biol 2011, 3: a003616.PubMedPubMed CentralGoogle Scholar
- Lambowitz AM, Zimmerly S: Mobile group II introns. Annu Rev Genet 2004, 38: 1-35. 10.1146/annurev.genet.38.072902.091600PubMedGoogle Scholar
- Guo H, Karberg M, Long M, Jones JP 3rd, Sullenger B, Lambowitz AM: Group II introns designed to insert into therapeutically relevant DNA target sites in human cells. Science 2000, 289: 452-457. 10.1126/science.289.5478.452PubMedGoogle Scholar
- Karberg M, Guo H, Zhong J, Coon R, Perutka J, Lambowitz AM: Group II introns as controllable gene targeting vectors for genetic manipulation of bacteria. Nat Biotechnol 2001, 19: 1162-1167. 10.1038/nbt1201-1162PubMedGoogle Scholar
- Perutka J, Wang W, Goerlitz D, Lambowitz AM: Use of computer-designed group II introns to disrupt Escherichia coli DExH/D-box protein and DNA helicase genes. J Mol Biol 2004, 336: 421-439. 10.1016/j.jmb.2003.12.009PubMedGoogle Scholar
- Cousineau B, Smith D, Lawrence-Cavanagh S, Mueller JE, Yang J, Mills D, Manias D, Dunny G, Lambowitz AM, Belfort M: Retrohoming of a bacterial group II intron: mobility via complete reverse splicing, independent of homologous DNA recombination. Cell 1998, 94: 451-462. 10.1016/S0092-8674(00)81586-XPubMedGoogle Scholar
- Conlan LH, Stanger MJ, Ichiyanagi K, Belfort M: Localization, mobility and fidelity of retrotransposed group II introns in rRNA genes. Nucleic Acids Res 2005, 33: 5262-5270. 10.1093/nar/gki819PubMedPubMed CentralGoogle Scholar
- Mohr S, Ghanem E, Smith W, Sheeter D, Qin Y, King O, Polioudakis D, Iyer VR, Hunicke-Smith S, Swamy S, Kuersten S, Lambowitz AM: Thermostable group II intron reverse transcriptase fusion proteins and their use in cDNA synthesis and next-generation RNA sequencing. RNA 2013, 19: 958-970. 10.1261/rna.039743.113PubMedPubMed CentralGoogle Scholar
- Vellore J, Moretz SE, Lampson BC: A group II intron-type open reading frame from the thermophile Bacillus (Geobacillus) stearothermophilus encodes a heat-stable reverse transcriptase. Appl Environ Microbiol 2004, 70: 7140-7147. 10.1128/AEM.70.12.7140-7147.2004PubMedPubMed CentralGoogle Scholar
- Ng B, Nayak S, Gibbs MD, Lee J, Bergquist PL: Reverse transcriptases: intron-encoded proteins found in thermophilic bacteria. Gene 2007, 393: 137-144. 10.1016/j.gene.2007.02.003PubMedGoogle Scholar
- Collins K, Nilsen TW: Enzyme engineering through evolution: thermostable recombinant group II intron reverse transcriptases provide new tools for RNA research and biotechnology. RNA 2013, 19: 1017-1018. 10.1261/rna.040451.113PubMedPubMed CentralGoogle Scholar
- Martin W, Koonin EV: Introns and the origin of nucleus-cytosol compartmentalization. Nature 2006, 440: 41-45. 10.1038/nature04531PubMedGoogle Scholar
- Keating KS, Toor N, Perlman PS, Pyle AM: A structural analysis of the group II intron active site and implications for the spliceosome. RNA 2010, 16: 1-9. 10.1261/rna.1791310PubMedPubMed CentralGoogle Scholar
- Candales MA, Duong A, Hood KS, Li T, Neufeld RA, Sun R, McNeil BA, Wu L, Jarding AM, Zimmerly S: Database for bacterial group II introns. Nucleic Acids Res 2012, 40: D187-D190. 10.1093/nar/gkr1043PubMedPubMed CentralGoogle Scholar
- Michel F, Ferat JL: Structure and activities of group II introns. Annu Rev Biochem 1995, 64: 435-461. 10.1146/annurev.bi.64.070195.002251PubMedGoogle Scholar
- Qin PZ, Pyle AM: The architectural organization and mechanistic function of group II intron structural elements. Curr Opin Struct Biol 1998, 8: 301-308. 10.1016/S0959-440X(98)80062-6PubMedGoogle Scholar
- Toor N, Keating KS, Taylor SD, Pyle AM: Crystal structure of a self-spliced group II intron. Science 2008, 320: 77-82. 10.1126/science.1153803PubMedPubMed CentralGoogle Scholar
- Toor N, Keating KS, Fedorova O, Rajashankar K, Wang J, Pyle AM: Tertiary architecture of the Oceanobacillus iheyensis group II intron. RNA 2010, 16: 57-69. 10.1261/rna.1844010PubMedPubMed CentralGoogle Scholar
- Marcia M, Somarowthu S, Pyle AM: Now on display: a gallery of group II intron structures at different stages of catalysis. Mob DNA 2013, 4: 14. 10.1186/1759-8753-4-14PubMedPubMed CentralGoogle Scholar
- Muñoz-Adelantado E, San Filippo J, Martínez-Abarca F, García-Rodríguez FM, Lambowitz AM, Toro N: Mobility of the Sinorhizobium meliloti group II intron RmInt1 occurs by reverse splicing into DNA, but requires an unknown reverse transcriptase priming mechanism. J Mol Biol 2003, 327: 931-943. 10.1016/S0022-2836(03)00208-0PubMedGoogle Scholar
- Blocker FJ, Mohr G, Conlan LH, Qi L, Belfort M, Lambowitz AM: Domain structure and three-dimensional model of a group II intron-encoded reverse transcriptase. RNA 2005, 11: 14-28. 10.1261/rna.7181105PubMedPubMed CentralGoogle Scholar
- Zimmerly S, Guo H, Perlman PS, Lambowitz AM: Group II intron mobility occurs by target DNA-primed reverse transcription. Cell 1995, 82: 545-554. 10.1016/0092-8674(95)90027-6PubMedGoogle Scholar
- Zimmerly S, Guo H, Eskes R, Yang J, Perlman PS, Lambowitz AM: A group II intron RNA is a catalytic component of a DNA endonuclease involved in intron mobility. Cell 1995, 83: 529-538. 10.1016/0092-8674(95)90092-6PubMedGoogle Scholar
- Yang J, Zimmerly S, Perlman PS, Lambowitz AM: Efficient integration of an intron RNA into double-stranded DNA by reverse splicing. Nature 1996, 381: 332-335. 10.1038/381332a0PubMedGoogle Scholar
- Zhong J, Lambowitz AM: Group II intron mobility using nascent strands at DNA replication forks to prime reverse transcription. EMBO J 2003, 22: 4555-4565. 10.1093/emboj/cdg433PubMedPubMed CentralGoogle Scholar
- Martínez-Abarca F, Barrientos-Durán A, Fernández-López M, Toro N: The RmInt1 group II intron has two different retrohoming pathways for mobility using predominantly the nascent lagging strand at DNA replication forks for priming. Nucleic Acids Res 2004, 32: 2880-2888. 10.1093/nar/gkh616PubMedPubMed CentralGoogle Scholar
- Toro N, Martínez-Abarca F: Comprehensive phylogenetic analysis of bacterial group II intron-encoded ORFs lacking the DNA endonuclease domain reveals new varieties. PLoS One 2013, 8: e55102. 10.1371/journal.pone.0055102PubMedPubMed CentralGoogle Scholar
- Yao J, Truong DM, Lambowitz AM: Genetic and biochemical assays reveal a key role for replication restart proteins in group II intron retrohoming. PLoS Genet 2013, 9: e1003469. 10.1371/journal.pgen.1003469PubMedPubMed CentralGoogle Scholar
- Smith D, Zhong J, Matsuura M, Lambowitz AM, Belfort M: Recruitment of host functions suggests a repair pathway for late steps in group II intron retrohoming. Genes Dev 2005, 19: 2477-2487. 10.1101/gad.1345105PubMedPubMed CentralGoogle Scholar
- Podar M, Chu VT, Pyle AM, Perlman PS: Group II intron splicing in vivo by first-step hydrolysis. Nature 1998, 391: 915-918. 10.1038/36142PubMedGoogle Scholar
- Vogel J, Börner T: Lariat formation and a hydrolytic pathway in plant chloroplast group II intron splicing. EMBO J 2002, 21: 3794-3803. 10.1093/emboj/cdf359PubMedPubMed CentralGoogle Scholar
- Mastroianni M, Watanabe K, White TB, Zhuang F, Vernon J, Matsuura M, Wallingford J, Lambowitz AM: Group II intron-based gene targeting reactions in eukaryotes. PLoS One 2008, 3: e3121. 10.1371/journal.pone.0003121PubMedPubMed CentralGoogle Scholar
- Gaj T, Gersbach CA, Barbas CF 3rd: ZFN, TALEN, and CRISPR/Cas-based methods for genome engineering. Trends Biotechnol 2013, 31: 397-405. 10.1016/j.tibtech.2013.04.004PubMedPubMed CentralGoogle Scholar
- Zhuang F, Mastroianni M, White TB, Lambowitz AM: Linear group II intron RNAs can retrohome in eukaryotes and may use nonhomologous end-joining for cDNA ligation. Proc Natl Acad Sci USA 2009, 106: 18189-18194. 10.1073/pnas.0910277106PubMedPubMed CentralGoogle Scholar
- White TB, Lambowitz AM: The retrohoming of linear group II intron RNAs in Drosophila melanogaster occurs by both DNA ligase 4-dependent and -independent mechanisms. PLoS Genet 2012, 8: e1002534. 10.1371/journal.pgen.1002534PubMedPubMed CentralGoogle Scholar
- Guo H, Zimmerly S, Perlman PS, Lambowitz AM: Group II intron endonucleases use both RNA and protein subunits for recognition of specific sequences in double-stranded DNA. EMBO J 1997, 16: 6835-6848. 10.1093/emboj/16.22.6835PubMedPubMed CentralGoogle Scholar
- Singh NN, Lambowitz AM: Interaction of a group II intron ribonucleoprotein endonuclease with its DNA target site investigated by DNA footprinting and modification interference. J Mol Biol 2001, 309: 361-386. 10.1006/jmbi.2001.4658PubMedGoogle Scholar
- Noah JW, Park S, Whitt JT, Perutka J, Frey W, Lambowitz AM: Atomic force microscopy reveals DNA bending during group II intron ribonucleoprotein particle integration into double-stranded DNA. Biochemistry 2006, 45: 12424-12435. 10.1021/bi060612hPubMedPubMed CentralGoogle Scholar
- Costa M, Michel F, Westhof E: A three-dimensional perspective on exon binding by a group II self-splicing intron. EMBO J 2000, 19: 5007-5018. 10.1093/emboj/19.18.5007PubMedPubMed CentralGoogle Scholar
- Zhuang F, Karberg M, Perutka J, Lambowitz AM: EcI5, a group IIB intron with high retrohoming frequency: DNA target site recognition and use in gene targeting. RNA 2009, 15: 432-449. 10.1261/rna.1378909PubMedPubMed CentralGoogle Scholar
- Jiménez-Zurdo JI, García-Rodríguez FM, Barrientos-Durán A, Toro N: DNA target site requirements for homing in vivo of a bacterial group II intron encoding a protein lacking the DNA endonuclease domain. J Mol Biol 2003, 326: 413-423. 10.1016/S0022-2836(02)01380-3PubMedGoogle Scholar
- Toor N, Robart AR, Christianson J, Zimmerly S: Self-splicing of a group IIC intron: 5’ exon recognition and alternative 5’ splicing events implicate the stem-loop motif of a transcriptional terminator. Nucleic Acids Res 2006, 34: 6461-6471. 10.1093/nar/gkl820PubMedPubMed CentralGoogle Scholar
- Robart AR, Seo W, Zimmerly S: Insertion of group II intron retroelements after intrinsic transcriptional terminators. Proc Natl Acad Sci USA 2007, 104: 6620-6625. 10.1073/pnas.0700561104PubMedPubMed CentralGoogle Scholar
- Léon G, Roy PH: Group IIC intron mobility into attC sites involves a bulged DNA stem-loop motif. RNA 2009, 15: 1543-1553. 10.1261/rna.1649309PubMedPubMed CentralGoogle Scholar
- Coros CJ, Landthaler M, Piazza CL, Beauregard A, Esposito D, Perutka J, Lambowitz AM, Belfort M: Retrotransposition strategies of the Lactococcus lactis Ll.LtrB group II intron are dictated by host identity and cellular environment. Mol Microbiol 2005, 56: 509-524. 10.1111/j.1365-2958.2005.04554.xPubMedGoogle Scholar
- Xiang Q, Qin PZ, Michels WJ, Freeland K, Pyle AM: Sequence specificity of a group II intron ribozyme: multiple mechanisms for promoting unusually high discrimination against mismatched targets. Biochemistry 1998, 37: 3839-3849. 10.1021/bi972661nPubMedGoogle Scholar
- Jinek M, Chylinski K, Fonfara I, Hauer M, Doudna JA, Charpentier E: A programmable dual-RNA-guided DNA endonuclease in adaptive bacterial immunity. Science 2012, 337: 816-821. 10.1126/science.1225829PubMedGoogle Scholar
- Cho SW, Kim S, Kim JM, Kim JS: Targeted genome engineering in human cells with the Cas9 RNA-guided endonuclease. Nat Biotechnol 2013, 31: 230-232. 10.1038/nbt.2507PubMedGoogle Scholar
- Cong L, Ran FA, Cox D, Lin S, Barretto R, Habib N, Hsu PD, Wu X, Jiang W, Marraffini LA, Zhang F: Multiplex genome engineering using CRISPR/Cas systems. Science 2013, 339: 819-823. 10.1126/science.1231143PubMedPubMed CentralGoogle Scholar
- Hwang WY, Fu Y, Reyon D, Maeder ML, Tsai SQ, Sander JD, Peterson RT, Yeh JR, Joung JK: Efficient genome editing in zebrafish using a CRISPR-Cas system. Nat Biotechnol 2013, 31: 227-229. 10.1038/nbt.2501PubMedPubMed CentralGoogle Scholar
- Jiang W, Bikard D, Cox D, Zhang F, Marraffini LA: RNA-guided editing of bacterial genomes using CRISPR-Cas systems. Nat Biotechnol 2013, 31: 233-239. 10.1038/nbt.2508PubMedPubMed CentralGoogle Scholar
- Mali P, Yang L, Esvelt KM, Aach J, Guell M, DiCarlo JE, Norville JE, Church GM: RNA-guided human genome engineering via Cas9. Science 2013, 339: 823-826. 10.1126/science.1232033PubMedPubMed CentralGoogle Scholar
- Hou Z, Zhang Y, Propson NE, Howden SE, Chu LF, Sontheimer EJ, Thomson JA: Efficient genome engineering in human pluripotent stem cells using Cas9 from Neisseria meningitidis . Proc Natl Acad Sci USA 2013, 110: 15644-15649. 10.1073/pnas.1313587110PubMedPubMed CentralGoogle Scholar
- Fu Y, Foden JA, Khayter C, Maeder ML, Reyon D, Joung JK, Sander JD: High-frequency off-target mutagenesis induced by CRISPR-Cas nucleases in human cells. Nat Biotechnol 2013, 31: 822-826. 10.1038/nbt.2623PubMedPubMed CentralGoogle Scholar
- García-Rodríguez FM, Barrientos-Durán A, Díaz-Prado V, Fernández-López M, Toro N: Use of RmInt1, a group IIB intron lacking the intron-encoded protein endonuclease domain, in gene targeting. Appl Environ Microbiol 2011, 77: 854-861. 10.1128/AEM.02319-10PubMedPubMed CentralGoogle Scholar
- Mohr G, Smith D, Belfort M, Lambowitz AM: Rules for DNA target-site recognition by a lactococcal group II intron enable retargeting of the intron to specific DNA sequences. Genes Dev 2000, 14: 559-573.PubMedPubMed CentralGoogle Scholar
- Zhong J, Karberg M, Lambowitz AM: Targeted and random bacterial gene disruption using a group II intron (targetron) vector containing a retrotransposition-activated selectable marker. Nucleic Acids Res 2003, 31: 1656-1664. 10.1093/nar/gkg248PubMedPubMed CentralGoogle Scholar
- Enyeart PJ, Chirieleison SM, Dao MN, Perutka J, Quandt EM, Yao J, Whitt JT, Keatinge-Clay AT, Lambowitz AM, Ellington AD: Generalized bacterial genome editing using mobile group II introns and Cre- lox . Mol Syst Biol 2013, 9: 685.PubMedPubMed CentralGoogle Scholar
- Frazier CL, San Filippo J, Lambowitz AM, Mills DA: Genetic manipulation of Lactococcus lactis by using targeted group II introns: generation of stable insertions without selection. Appl Environ Microbiol 2003, 69: 1121-1128. 10.1128/AEM.69.2.1121-1128.2003PubMedPubMed CentralGoogle Scholar
- Yao J, Zhong J, Fang Y, Geisinger E, Novick RP, Lambowitz AM: Use of targetrons to disrupt essential and nonessential genes in Staphylococcus aureus reveals temperature sensitivity of Ll.LtrB group II intron splicing. RNA 2006, 12: 1271-1281. 10.1261/rna.68706PubMedPubMed CentralGoogle Scholar
- Heap JT, Pennington OJ, Cartman ST, Carter GP, Minton NP: The ClosTron: a universal gene knock-out system for the genus Clostridium . J Microbiol Methods 2007, 70: 452-464. 10.1016/j.mimet.2007.05.021PubMedGoogle Scholar
- Yao J, Lambowitz AM: Gene targeting in Gram-negative bacteria by use of a mobile group II intron (“targetron”) expressed from a broad-host-range vector. Appl Environ Microbiol 2007, 73: 2735-2743. 10.1128/AEM.02829-06PubMedPubMed CentralGoogle Scholar
- Ichiyanagi K, Beauregard A, Lawrence S, Smith D, Cousineau B, Belfort M: Retrotransposition of the Ll.LtrB group II intron proceeds predominantly via reverse splicing into DNA targets. Mol Microbiol 2002, 46: 1259-1272.PubMedGoogle Scholar
- Yao J, Zhong J, Lambowitz AM: Gene targeting using randomly inserted group II introns (targetrons) recovered from an Escherichia coli gene disruption library. Nucleic Acids Res 2005, 33: 3351-3362. 10.1093/nar/gki649PubMedPubMed CentralGoogle Scholar
- Malhotra M, Srivastava S: An ipdC gene knock-out of Azospirillum brasilense strain SM and its implications on indole-3-acetic acid biosynthesis and plant growth promotion. Antonie Van Leeuwenhoek 2008, 93: 425-433. 10.1007/s10482-007-9207-xPubMedGoogle Scholar
- Akhtar P, Khan SA: Two independent replicons can support replication of the anthrax toxin-encoding plasmid pXO1 of Bacillus anthracis . Plasmid 2012, 67: 111-117. 10.1016/j.plasmid.2011.12.012PubMedPubMed CentralGoogle Scholar
- Chen Y, McClane BA, Fisher DJ, Rood JI, Gupta P: Construction of an alpha toxin gene knockout mutant of Clostridium perfringens type A by use of a mobile group II intron. Appl Environ Microbiol 2005, 71: 7542-7547. 10.1128/AEM.71.11.7542-7547.2005PubMedPubMed CentralGoogle Scholar
- Cheng C, Nair AD, Indukuri VV, Gong S, Felsheim RF, Jaworski D, Munderloh UG, Ganta RR: Targeted and random mutagenesis of Ehrlichia chaffeensis for the identification of genes required for in vivo infection. PLoS Pathog 2013, 9: e1003171. 10.1371/journal.ppat.1003171PubMedPubMed CentralGoogle Scholar
- Rodriguez SA, Yu JJ, Davis G, Arulanandam BP, Klose KE: Targeted inactivation of Francisella tularensis genes by group II introns. Appl Environ Microbiol 2008, 74: 2619-2626. 10.1128/AEM.02905-07PubMedPubMed CentralGoogle Scholar
- Alonzo F 3rd, Port GC, Cao M, Freitag NE: The posttranslocation chaperone PrsA2 contributes to multiple facets of Listeria monocytogenes pathogenesis. Infect Immun 2009, 77: 2612-2623. 10.1128/IAI.00280-09PubMedPubMed CentralGoogle Scholar
- Zarschler K, Janesch B, Zayni S, Schäffer C, Messner P: Construction of a gene knockout system for application in Paenibacillus alvei CCM 2051 T, exemplified by the S-layer glycan biosynthesis initiation enzyme WsfP. Appl Environ Microbiol 2009, 75: 3077-3085. 10.1128/AEM.00087-09PubMedPubMed CentralGoogle Scholar
- Steen JA, Steen JA, Harrison P, Seemann T, Wilkie I, Harper M, Adler B, Boyce JD: Fis is essential for capsule production in Pasteurella multocida and regulates expression of other important virulence factors. PLoS Pathog 2010, 6: e1000750. 10.1371/journal.ppat.1000750PubMedPubMed CentralGoogle Scholar
- Pearson MM, Mobley HL: The type III secretion system of Proteus mirabilis HI4320 does not contribute to virulence in the mouse model of ascending urinary tract infection. J Med Microbiol 2007, 56: 1277-1283. 10.1099/jmm.0.47314-0PubMedGoogle Scholar
- Park JM, Jang YS, Kim TY, Lee SY: Development of a gene knockout system for Ralstonia eutropha H16 based on the broad-host-range vector expressing a mobile group II intron. FEMS Microbiol Lett 2010, 309: 193-200.PubMedGoogle Scholar
- Smith CL, Weiss BL, Aksoy S, Runyen-Janecky LJ: Characterization of the achromobactin iron acquisition operon in Sodalis glossinidius . Appl Environ Microbiol 2013, 79: 2872-2881. 10.1128/AEM.03959-12PubMedPubMed CentralGoogle Scholar
- Kumar S, Smith KP, Floyd JL, Varela MF: Cloning and molecular analysis of a mannitol operon of phosphoenolpyruvate-dependent phosphotransferase (PTS) type from Vibrio cholerae O395. Arch Microbiol 2011, 193: 201-208. 10.1007/s00203-010-0663-8PubMedPubMed CentralGoogle Scholar
- Palonen E, Lindstrom M, Karttunen R, Somervuo P, Korkeala H: Expression of signal transduction system encoding genes of Yersinia pseudotuberculosis IP32953 at 28°C and 3°C. PLoS One 2011, 6: e25063. 10.1371/journal.pone.0025063PubMedPubMed CentralGoogle Scholar
- Heap JT, Kuehne SA, Ehsaan M, Cartman ST, Cooksley CM, Scott JC, Minton NP: The ClosTron: mutagenesis in Clostridium refined and streamlined. J Microbiol Methods 2010, 80: 49-55. 10.1016/j.mimet.2009.10.018PubMedGoogle Scholar
- Corvaglia AR, Francois P, Hernandez D, Perron K, Linder P, Schrenzel J: A type III-like restriction endonuclease functions as a major barrier to horizontal gene transfer in clinical Staphylococcus aureus strains. Proc Natl Acad Sci USA 2010, 107: 11954-11958. 10.1073/pnas.1000489107PubMedPubMed CentralGoogle Scholar
- Eskes R, Yang J, Lambowitz AM, Perlman PS: Mobility of yeast mitochondrial group II introns: engineering a new site specificity and retrohoming via full reverse splicing. Cell 1997, 88: 865-874. 10.1016/S0092-8674(00)81932-7PubMedGoogle Scholar
- Eskes R, Liu L, Ma H, Chao MY, Dickson L, Lambowitz AM, Perlman PS: Multiple homing pathways used by yeast mitochondrial group II introns. Mol Cell Biol 2000, 20: 8432-8446. 10.1128/MCB.20.22.8432-8446.2000PubMedPubMed CentralGoogle Scholar
- Rest JS, Mindell DP: Retroids in archaea: phylogeny and lateral origins. Mol Biol Evol 2003, 20: 1134-1142. 10.1093/molbev/msg135PubMedGoogle Scholar
- Sayeed S, Uzal FA, Fisher DJ, Saputo J, Vidal JE, Chen Y, Gupta P, Rood JI, McClane BA: Beta toxin is essential for the intestinal virulence of Clostridium perfringens type C disease isolate CN3685 in a rabbit ileal loop model. Mol Microbiol 2008, 67: 15-30.PubMedGoogle Scholar
- Buchan BW, McCaffrey RL, Lindemann SR, Allen LA, Jones BD: Identification of migR , a regulatory element of the Francisella tularensis live vaccine strain iglABCD virulence operon required for normal replication and trafficking in macrophages. Infect Immun 2009, 77: 2517-2529. 10.1128/IAI.00229-09PubMedPubMed CentralGoogle Scholar
- Carter GP, Awad MM, Hao Y, Thelen T, Bergin IL, Howarth PM, Seemann T, Rood JI, Aronoff DM, Lyras D: TcsL is an essential virulence factor in Clostridium sordellii ATCC 9714. Infect Immun 2011, 79: 1025-1032. 10.1128/IAI.00968-10PubMedPubMed CentralGoogle Scholar
- Francis MB, Allen CA, Shrestha R, Sorg JA: Bile acid recognition by the Clostridium difficile germinant receptor, CspC, is important for establishing infection. PLoS Pathog 2013, 9: e1003356. 10.1371/journal.ppat.1003356PubMedPubMed CentralGoogle Scholar
- Zoraghi R, See RH, Gong H, Lian T, Swayze R, Finlay BB, Brunham RC, McMaster WR, Reiner NE: Functional analysis, overexpression, and kinetic characterization of pyruvate kinase from methicillin-resistant Staphylococcus aureus . Biochemistry 2010, 49: 7733-7747. 10.1021/bi100780tPubMedGoogle Scholar
- Zoraghi R, Worrall L, See RH, Strangman W, Popplewell WL, Gong H, Samaai T, Swayze RD, Kaur S, Vuckovic M, Finlay BB, Brunham RC, McMaster WR, Davies-Coleman MT, Strynadka NC, Andersen RJ, Reiner NE: Methicillin-resistant Staphylococcus aureus (MRSA) pyruvate kinase as a target for bis-indole alkaloids with antibacterial activities. J Biol Chem 2011, 286: 44716-44725. 10.1074/jbc.M111.289033PubMedPubMed CentralGoogle Scholar
- Rawsthorne H, Turner KN, Mills DA: Multicopy integration of heterologous genes, using the lactococcal group II intron targeted to bacterial insertion sequences. Appl Environ Microbiol 2006, 72: 6088-6093. 10.1128/AEM.02992-05PubMedPubMed CentralGoogle Scholar
- Shao L, Hu S, Yang Y, Gu Y, Chen J, Yang Y, Jiang W, Yang S: Targeted gene disruption by use of a group II intron (targetron) vector in Clostridium acetobutylicum . Cell Res 2007, 17: 963-965. 10.1038/cr.2007.91PubMedGoogle Scholar
- Jiang Y, Xu C, Dong F, Yang Y, Jiang W, Yang S: Disruption of the acetoacetate decarboxylase gene in solvent-producing Clostridium acetobutylicum increases the butanol ratio. Metab Eng 2009, 11: 284-291. 10.1016/j.ymben.2009.06.002PubMedGoogle Scholar
- Tolonen AC, Chilaka AC, Church GM: Targeted gene inactivation in Clostridium phytofermentans shows that cellulose degradation requires the family 9 hydrolase Cphy3367. Mol Microbiol 2009, 74: 1300-1313. 10.1111/j.1365-2958.2009.06890.xPubMedPubMed CentralGoogle Scholar
- Cai G, Jin B, Saint C, Monis P: Genetic manipulation of butyrate formation pathways in Clostridium butyricum . J Biotechnol 2011, 155: 269-274. 10.1016/j.jbiotec.2011.07.004PubMedGoogle Scholar
- Lehmann D, Lütke-Eversloh T: Switching Clostridium acetobutylicum to an ethanol producer by disruption of the butyrate/butanol fermentative pathway. Metab Eng 2011, 13: 464-473. 10.1016/j.ymben.2011.04.006PubMedGoogle Scholar
- Cooksley CM, Zhang Y, Wang H, Redl S, Winzer K, Minton NP: Targeted mutagenesis of the Clostridium acetobutylicum acetone-butanol-ethanol fermentation pathway. Metab Eng 2012, 14: 630-641. 10.1016/j.ymben.2012.09.001PubMedGoogle Scholar
- Jang YS, Lee JY, Lee J, Park JH, Im JA, Eom MH, Lee SH, Song H, Cho JH, Seung Do Y, Lee SY: Enhanced butanol production obtained by reinforcing the direct butanol-forming route in Clostridium acetobutylicum . mBio 2012, 3: 12.Google Scholar
- Jia K, Zhang Y, Li Y: Identification and characterization of two functionally unknown genes involved in butanol tolerance of Clostridium acetobutylicum . PLoS One 2012, 7: e38815. 10.1371/journal.pone.0038815PubMedPubMed CentralGoogle Scholar
- Kuit W, Minton NP, López-Contreras AM, Eggink G: Disruption of the acetate kinase (ack) gene of Clostridium acetobutylicum results in delayed acetate production. Appl Microbiol Biotechnol 2012, 94: 729-741. 10.1007/s00253-011-3848-4PubMedPubMed CentralGoogle Scholar
- Lehmann D, Hönicke D, Ehrenreich A, Schmidt M, Weuster-Botz D, Bahl H, Lütke-Eversloh T: Modifying the product pattern of Clostridium acetobutylicum : physiological effects of disrupting the acetate and acetone formation pathways. Appl Microbiol Biotechnol 2012, 94: 743-754. 10.1007/s00253-011-3852-8PubMedGoogle Scholar
- Li Y, Tschaplinski TJ, Engle NL, Hamilton CY, Rodriguez M Jr, Liao JC, Schadt CW, Guss AM, Yang Y, Graham DE: Combined inactivation of the Clostridium cellulolyticum lactate and malate dehydrogenase genes substantially increases ethanol yield from cellulose and switchgrass fermentations. Biotechnol Biofuels 2012, 5: 2. 10.1186/1754-6834-5-2PubMedPubMed CentralGoogle Scholar
- Steiner E, Scott J, Minton NP, Winzer K: An agr quorum sensing system that regulates granulose formation and sporulation in Clostridium acetobutylicum . Appl Environ Microbiol 2012, 78: 1113-1122. 10.1128/AEM.06376-11PubMedPubMed CentralGoogle Scholar
- Wietzke M, Bahl H: The redox-sensing protein Rex, a transcriptional regulator of solventogenesis in Clostridium acetobutylicum . Appl Microbiol Biotechnol 2012, 96: 749-761. 10.1007/s00253-012-4112-2PubMedGoogle Scholar
- Cai G, Jin B, Monis P, Saint C: A genetic and metabolic approach to redirection of biochemical pathways of Clostridium butyricum for enhancing hydrogen production. Biotechnol Bioeng 2013, 110: 338-342. 10.1002/bit.24596PubMedGoogle Scholar
- Celik H, Blouzard JC, Voigt B, Becher D, Trotter V, Fierobe HP, Tardif C, Pagès S, de Philip P: A two-component system (XydS/R) controls the expression of genes encoding CBM6-containing proteins in response to straw in Clostridium cellulolyticum . PLoS One 2013, 8: e56063. 10.1371/journal.pone.0056063PubMedPubMed CentralGoogle Scholar
- Fendri I, Abdou L, Trotter V, Dedieu L, Maamar H, Minton NP, Tardif C: Regulation of cel genes of C. cellulolyticum : identification of GlyR2, a transcriptional regulator regulating cel5D gene expression. PLoS One 2013, 8: e44708. 10.1371/journal.pone.0044708PubMedPubMed CentralGoogle Scholar
- Ferdinand PH, Borne R, Trotter V, Pagès S, Tardif C, Fierobe HP, Perret S: Are cellulosome scaffolding protein CipC and CBM3-containing protein HycP, involved in adherence of Clostridium cellulolyticum to cellulose? PLoS One 2013, 8: e69360. 10.1371/journal.pone.0069360PubMedPubMed CentralGoogle Scholar
- Jang YS, Woo HM, Im JA, Kim IH, Lee SY: Metabolic engineering of Clostridium acetobutylicum for enhanced production of butyric acid. Appl Microbiol Biotechnol 2013, 97: 9355-9363. 10.1007/s00253-013-5161-xPubMedGoogle Scholar
- Wang Y, Li X, Milne CB, Janssen H, Lin W, Phan G, Hu H, Jin Y-S, Price ND, Blaschek HP: Development of a gene knockout system using mobile group II introns (targetron) and genetic disruption of acid production pathways in Clostridium beijerinckii . Appl Environ Microbiol 2013, 79: 5853-5863. 10.1128/AEM.00971-13PubMedPubMed CentralGoogle Scholar
- Emerson JE, Reynolds CB, Fagan RP, Shaw HA, Goulding D, Fairweather NF: A novel genetic switch controls phase variable expression of CwpV, a Clostridium difficile cell wall protein. Mol Microbiol 2009, 74: 541-556. 10.1111/j.1365-2958.2009.06812.xPubMedPubMed CentralGoogle Scholar
- Kirby JM, Ahern H, Roberts AK, Kumar V, Freeman Z, Acharya KR, Shone CC: Cwp84, a surface-associated cysteine protease, plays a role in the maturation of the surface layer of Clostridium difficile . J Biol Chem 2009, 284: 34666-34673. 10.1074/jbc.M109.051177PubMedPubMed CentralGoogle Scholar
- Underwood S, Guan S, Vijayasubhash V, Baines SD, Graham L, Lewis RJ, Wilcox MH, Stephenson K: Characterization of the sporulation initiation pathway of Clostridium difficile and its role in toxin production. J Bacteriol 2009, 191: 7296-7305. 10.1128/JB.00882-09PubMedPubMed CentralGoogle Scholar
- Burns DA, Heap JT, Minton NP: SleC is essential for germination of Clostridium difficile spores in nutrient-rich medium supplemented with the bile salt taurocholate. J Bacteriol 2010, 192: 657-664. 10.1128/JB.01209-09PubMedPubMed CentralGoogle Scholar
- Antunes A, Martin-Verstraete I, Dupuy B: CcpA-mediated repression of Clostridium difficile toxin gene expression. Mol Microbiol 2011, 79: 882-899. 10.1111/j.1365-2958.2010.07495.xPubMedGoogle Scholar
- Barketi-Klai A, Hoys S, Lambert-Bordes S, Collignon A, Kansau I: Role of fibronectin-binding protein A in Clostridium difficile intestinal colonization. J Med Microbiol 2011, 60: 1155-1161. 10.1099/jmm.0.029553-0PubMedGoogle Scholar
- Dawson LF, Donahue EH, Cartman ST, Barton RH, Bundy J, McNerney R, Minton NP, Wren BW: The analysis of para -cresol production and tolerance in Clostridium difficile 027 and 012 strains. BMC Microbiol 2011, 11: 86. 10.1186/1471-2180-11-86PubMedPubMed CentralGoogle Scholar
- Dingle TC, Mulvey GL, Armstrong GD: Mutagenic analysis of the Clostridium difficile flagellar proteins, FliC and FliD, and their contribution to virulence in hamsters. Infect Immun 2011, 79: 4061-4067. 10.1128/IAI.05305-11PubMedPubMed CentralGoogle Scholar
- Ho TD, Ellermeier CD: PrsW is required for colonization, resistance to antimicrobial peptides, and expression of extracytoplasmic function sigma factors in Clostridium difficile . Infect Immun 2011, 79: 3229-3238. 10.1128/IAI.00019-11PubMedPubMed CentralGoogle Scholar
- Kuehne SA, Cartman ST, Minton NP: Both, toxin A and toxin B, are important in Clostridium difficile infection. Gut Microbes 2011, 2: 252-255. 10.4161/gmic.2.4.16109PubMedPubMed CentralGoogle Scholar
- McBride SM, Sonenshein AL: The dlt operon confers resistance to cationic antimicrobial peptides in Clostridium difficile . Microbiology 2011, 157: 1457-1465. 10.1099/mic.0.045997-0PubMedPubMed CentralGoogle Scholar
- de la Riva L, Willing SE, Tate EW, Fairweather NF: Roles of cysteine proteases Cwp84 and Cwp13 in biogenesis of the cell wall of Clostridium difficile . J Bacteriol 2011, 193: 3276-3285. 10.1128/JB.00248-11PubMedPubMed CentralGoogle Scholar
- Saujet L, Monot M, Dupuy B, Soutourina O, Martin-Verstraete I: The key sigma factor of transition phase, SigH, controls sporulation, metabolism, and virulence factor expression in Clostridium difficile . J Bacteriol 2011, 193: 3186-3196. 10.1128/JB.00272-11PubMedPubMed CentralGoogle Scholar
- Bakker D, Smits WK, Kuijper EJ, Corver J: TcdC does not significantly repress toxin expression in Clostridium difficile 630ΔErm. PLoS One 2012, 7: e43247. 10.1371/journal.pone.0043247PubMedPubMed CentralGoogle Scholar
- Dawson LF, Valiente E, Faulds-Pain A, Donahue EH, Wren BW: Characterisation of Clostridium difficile biofilm formation, a role for Spo0A. PLoS One 2012, 7: e50527. 10.1371/journal.pone.0050527PubMedPubMed CentralGoogle Scholar
- Deakin LJ, Clare S, Fagan RP, Dawson LF, Pickard DJ, West MR, Wren BW, Fairweather NF, Dougan G, Lawley TD: The Clostridium difficile spo0A gene is a persistence and transmission factor. Infect Immun 2012, 80: 2704-2711. 10.1128/IAI.00147-12PubMedPubMed CentralGoogle Scholar
- Govind R, Dupuy B: Secretion of Clostridium difficile toxins A and B requires the holin-like protein TcdE. PLoS Pathog 2012, 8: e1002727. 10.1371/journal.ppat.1002727PubMedPubMed CentralGoogle Scholar
- Olling A, Seehase S, Minton NP, Tatge H, Schröter S, Kohlscheen S, Pich A, Just I, Gerhard R: Release of TcdA and TcdB from Clostridium difficile cdi 630 is not affected by functional inactivation of the tcdE gene. Microb Pathog 2012, 52: 92-100. 10.1016/j.micpath.2011.10.009PubMedGoogle Scholar
- Adams CM, Eckenroth BE, Putnam EE, Doublié S, Shen A: Structural and functional analysis of the CspB protease required for Clostridium spore germination. PLoS Pathog 2013, 9: e1003165. 10.1371/journal.ppat.1003165PubMedPubMed CentralGoogle Scholar
- Bouillaut L, Self WT, Sonenshein AL: Proline-dependent regulation of Clostridium difficile Stickland metabolism. J Bacteriol 2013, 195: 844-854. 10.1128/JB.01492-12PubMedPubMed CentralGoogle Scholar
- Ðapa T, Leuzzi R, Ng YK, Baban ST, Adamo R, Kuehne SA, Scarselli M, Minton NP, Serruto D, Unnikrishnan M: Multiple factors modulate biofilm formation by the anaerobic pathogen Clostridium difficile . J Bacteriol 2013, 195: 545-555. 10.1128/JB.01980-12PubMedPubMed CentralGoogle Scholar
- Martin MJ, Clare S, Goulding D, Faulds-Pain A, Barquist L, Browne HP, Pettit L, Dougan G, Lawley TD, Wren BW: The agr locus regulates virulence and colonization genes in Clostridium difficile 027. J Bacteriol 2013, 195: 3672-3681. 10.1128/JB.00473-13PubMedPubMed CentralGoogle Scholar
- Putnam EE, Nock AM, Lawley TD, Shen A: SpoIVA and SipL are Clostridium difficile spore morphogenetic proteins. J Bacteriol 2013, 195: 1214-1225. 10.1128/JB.02181-12PubMedPubMed CentralGoogle Scholar
- Permpoonpattana P, Phetcharaburanin J, Mikelsone A, Dembek M, Tan S, Brisson MC, La Ragione R, Brisson AR, Fairweather N, Hong HA, Cutting SM: Functional characterization of Clostridium difficile spore coat proteins. J Bacteriol 2013, 195: 1492-1503. 10.1128/JB.02104-12PubMedPubMed CentralGoogle Scholar
- Ammam F, Meziane-cherif D, Mengin-Lecreulx D, Blanot D, Patin D, Boneca IG, Courvalin P, Lambert T, Candela T: The functional vanG Cd cluster of Clostridium difficile does not confer vancomycin resistance. Mol Microbiol 2013, 89: 612-625. 10.1111/mmi.12299PubMedGoogle Scholar
- Baban ST, Kuehne SA, Barketi-Klai A, Cartman ST, Kelly ML, Hardie KR, Kansau I, Collignon A, Minton NP: The role of flagella in Clostridium difficile pathogenesis: comparison between a non-epidemic and an epidemic strain. PLoS One 2013, 8: e73026. 10.1371/journal.pone.0073026PubMedPubMed CentralGoogle Scholar
- Ng KM, Ferreyra JA, Higginbottom SK, Lynch JB, Kashyap PC, Gopinath S, Naidu N, Choudhury B, Weimer BC, Monack DM, Sonnenburg JL: Microbiota-liberated host sugars facilitate post-antibiotic expansion of enteric pathogens. Nature 2013, 502: 96-99. 10.1038/nature12503PubMedPubMed CentralGoogle Scholar
- Tulli L, Marchi S, Petracca R, Shaw HA, Fairweather NF, Scarselli M, Soriani M, Leuzzi R: CbpA: a novel surface exposed adhesin of Clostridium difficile targeting human collagen. Cell Microbiol 2013, 15: 1674-1687.PubMedGoogle Scholar
- Saujet L, Pereira FC, Serrano M, Soutourina O, Monot M, Shelyakin PV, Gelfand MS, Dupuy B, Henriques AO, Martin-Verstraete I: Genome-wide analysis of cell type-specific gene transcription during spore formation in Clostridium difficile . PLoS Genet 2013, 9: e1003756. 10.1371/journal.pgen.1003756PubMedPubMed CentralGoogle Scholar
- McKee RW, Mangalea MR, Purcell EB, Borchardt EK, Tamayo R: The second messenger cyclic di-GMP regulates Clostridium difficile toxin production by controlling expression of sigD . J Bacteriol 2013, 195: 5174-5185. 10.1128/JB.00501-13PubMedPubMed CentralGoogle Scholar
- Pereira FC, Saujet L, Tome AR, Serrano M, Monot M, Couture-Tosi E, Martin-Verstraete I, Dupuy B, Henriques AO: The spore differentiation pathway in the enteric pathogen Clostridium difficile . PLoS Genet 2013, 9: e1003782. 10.1371/journal.pgen.1003782PubMedPubMed CentralGoogle Scholar
- Janoir C, Denève C, Bouttier S, Barbut F, Hoys S, Caleechum L, Chapetón-Montes D, Pereira FC, Henriques AO, Collignon A, Monot M, Dupuy B: Adaptive strategies and pathogenesis of Clostridium difficile from in vivo transcriptomics. Infect Immun 2013, 81: 3757-3769. 10.1128/IAI.00515-13PubMedPubMed CentralGoogle Scholar
- Fimlaid KA, Bond JP, Schutz KC, Putnam EE, Leung JM, Lawley TD, Shen A: Global analysis of the sporulation pathway of Clostridium difficile . PLoS Genet 2013, 9: e1003660. 10.1371/journal.pgen.1003660PubMedPubMed CentralGoogle Scholar
- Bradshaw M, Marshall KM, Heap JT, Tepp WH, Minton NP, Johnson EA: Construction of a nontoxigenic Clostridium botulinum strain for food challenge studies. Appl Environ Microbiol 2010, 76: 387-393. 10.1128/AEM.02005-09PubMedPubMed CentralGoogle Scholar
- Cooksley CM, Davis IJ, Winzer K, Chan WC, Peck MW, Minton NP: Regulation of neurotoxin production and sporulation by a putative agrBD signaling system in proteolytic Clostridium botulinum . Appl Environ Microbiol 2010, 76: 4448-4460. 10.1128/AEM.03038-09PubMedPubMed CentralGoogle Scholar
- Marshall KM, Bradshaw M, Johnson EA: Conjugative botulinum neurotoxin-encoding plasmids in Clostridium botulinum . PLoS One 2010, 5: e11087. 10.1371/journal.pone.0011087PubMedPubMed CentralGoogle Scholar
- Selby K, Lindström M, Somervuo P, Heap JT, Minton NP, Korkeala H: Important role of class I heat shock genes hrcA and dnaK in the heat shock response and the response to pH and NaCl stress of group I Clostridium botulinum strain ATCC 3502. Appl Environ Microbiol 2011, 77: 2823-2830. 10.1128/AEM.02633-10PubMedPubMed CentralGoogle Scholar
- Söderholm H, Lindström M, Somervuo P, Heap J, Minton N, Linden J, Korkeala H: cspB encodes a major cold shock protein in Clostridium botulinum ATCC 3502. Int J Food Microbiol 2011, 146: 23-30. 10.1016/j.ijfoodmicro.2011.01.033PubMedGoogle Scholar
- Kirk DG, Dahlsten E, Zhang Z, Korkeala H, Lindström M: Involvement of Clostridium botulinum ATCC 3502 sigma factor K in early-stage sporulation. Appl Environ Microbiol 2012, 78: 4590-4596. 10.1128/AEM.00304-12PubMedPubMed CentralGoogle Scholar
- Lindström M, Dahlsten E, Söderholm H, Selby K, Somervuo P, Heap JT, Minton NP, Korkeala H: Involvement of two-component system CBO0366/CBO0365 in the cold shock response and growth of group I (proteolytic) Clostridium botulinum ATCC 3502 at low temperatures. Appl Environ Microbiol 2012, 78: 5466-5470. 10.1128/AEM.00555-12PubMedPubMed CentralGoogle Scholar
- Dahlsten E, Kirk D, Lindström M, Korkeala H: Alternative sigma factor SigK has a role in stress tolerance of group I Clostridium botulinum strain ATCC 3502. Appl Environ Microbiol 2013, 79: 3867-3869. 10.1128/AEM.04036-12PubMedPubMed CentralGoogle Scholar
- Zhang Z, Korkeala H, Dahlsten E, Sahala E, Heap JT, Minton NP, Lindström M: Two-component signal transduction system CBO0787/CBO0786 represses transcription from botulinum neurotoxin promoters in Clostridium botulinum ATCC 3502. PLoS Pathog 2013, 9: e1003252. 10.1371/journal.ppat.1003252PubMedPubMed CentralGoogle Scholar
- Derman Y, Isokallio M, Lindström M, Korkeala H: The two-component system CBO2306/CBO2307 is important for cold adaptation of Clostridium botulinum ATCC 3502. Int J Food Microbiol 2013, 167: 87-91. 10.1016/j.ijfoodmicro.2013.06.004PubMedGoogle Scholar
- Paredes-Sabja D, Torres JA, Setlow P, Sarker MR: Clostridium perfringens spore germination: characterization of germinants and their receptors. J Bacteriol 2008, 190: 1190-1201. 10.1128/JB.01748-07PubMedPubMed CentralGoogle Scholar
- Vidal JE, Ohtani K, Shimizu T, McClane BA: Contact with enterocyte-like Caco-2 cells induces rapid upregulation of toxin production by Clostridium perfringens type C isolates. Cell Microbiol 2009, 11: 1306-1328. 10.1111/j.1462-5822.2009.01332.xPubMedPubMed CentralGoogle Scholar
- Cheung JK, Keyburn AL, Carter GP, Lanckriet AL, Van Immerseel F, Moore RJ, Rood JI: The VirSR two-component signal transduction system regulates NetB toxin production in Clostridium perfringens . Infect Immun 2010, 78: 3064-3072. 10.1128/IAI.00123-10PubMedPubMed CentralGoogle Scholar
- Li J, McClane BA: Evaluating the involvement of alternative sigma factors SigF and SigG in Clostridium perfringens sporulation and enterotoxin synthesis. Infect Immun 2010, 78: 4286-4293. 10.1128/IAI.00528-10PubMedPubMed CentralGoogle Scholar
- Chen J, Rood JI, McClane BA: Epsilon-toxin production by Clostridium perfringens type D strain CN3718 is dependent upon the agr operon but not the VirS/VirR two-component regulatory system. mBio 2011, 2: e00275. 11PubMedPubMed CentralGoogle Scholar
- Fujita M, Tsuchida A, Hirata A, Kobayashi N, Goto K, Osumi K, Hirose Y, Nakayama J, Yamanoi T, Ashida H, Mizuno M: Glycoside hydrolase family 89 α-N-acetylglucosaminidase from Clostridium perfringens specifically acts on GlcNAcα1,4Galβ1R at the non-reducing terminus of O-glycans in gastric mucin. J Biol Chem 2011, 286: 6479-6489. 10.1074/jbc.M110.206722PubMedPubMed CentralGoogle Scholar
- Li J, Chen J, Vidal JE, McClane BA: The Agr-like quorum-sensing system regulates sporulation and production of enterotoxin and beta2 toxin by Clostridium perfringens type A non-food-borne human gastrointestinal disease strain F5603. Infect Immun 2011, 79: 2451-2459. 10.1128/IAI.00169-11PubMedPubMed CentralGoogle Scholar
- Li J, Sayeed S, Robertson S, Chen J, McClane BA: Sialidases affect the host cell adherence and epsilon toxin-induced cytotoxicity of Clostridium perfringens type D strain CN3718. PLoS Pathog 2011, 7: e1002429. 10.1371/journal.ppat.1002429PubMedPubMed CentralGoogle Scholar
- Chen J, McClane BA: Role of the Agr-like quorum-sensing system in regulating toxin production by Clostridium perfringens type B strains CN1793 and CN1795. Infect Immun 2012, 80: 3008-3017. 10.1128/IAI.00438-12PubMedPubMed CentralGoogle Scholar
- Banawas S, Paredes-Sabja D, Korza G, Li Y, Hao B, Setlow P, Sarker MR: The Clostridium perfringens germinant receptor protein GerKC is located in the spore inner membrane and is crucial for spore germination. J Bacteriol 2013, 195: 5084-5091. 10.1128/JB.00901-13PubMedPubMed CentralGoogle Scholar
- Li J, Ma M, Sarker MR, McClane BA: CodY is a global regulator of virulence-associated properties for Clostridium perfringens type D strain CN3718. mBio 2013, 4: e00770-13.PubMedPubMed CentralGoogle Scholar
- Sirigi Reddy AR, Girinathan BP, Zapotocny R, Govind R: Identification and characterization of Clostridium sordellii toxin gene regulator. J Bacteriol 2013, 195: 4246-4254. 10.1128/JB.00711-13PubMedPubMed CentralGoogle Scholar
- Dong H, Zhang Y, Dai Z, Li Y: Engineering Clostridium strain to accept unmethylated DNA. PLoS One 2010, 5: e9038. 10.1371/journal.pone.0009038PubMedPubMed CentralGoogle Scholar
- Cui GZ, Hong W, Zhang J, Li WL, Feng Y, Liu YJ, Cui Q: Targeted gene engineering in Clostridium cellulolyticum H10 without methylation. J Microbiol Methods 2012, 89: 201-208. 10.1016/j.mimet.2012.02.015PubMedGoogle Scholar
- Saldanha RJ, Pemberton A, Shiflett P, Perutka J, Whitt JT, Ellington A, Lambowitz AM, Kramer R, Taylor D, Lamkin TJ: Rapid targeted gene disruption in Bacillus anthracis . BMC Biotechnol 2013, 13: 72. 10.1186/1472-6750-13-72PubMedPubMed CentralGoogle Scholar
- Chen Y, Caruso L, McClane B, Fisher D, Gupta P: Disruption of a toxin gene by introduction of a foreign gene into the chromosome of Clostridium perfringens using targetron-induced mutagenesis. Plasmid 2007, 58: 182-189. 10.1016/j.plasmid.2007.04.002PubMedPubMed CentralGoogle Scholar
- Plante I, Cousineau B: Restriction for gene insertion within the Lactococcus lactis Ll.LtrB group II intron. RNA 2006, 12: 1980-1992. 10.1261/rna.193306PubMedPubMed CentralGoogle Scholar
- Jia K, Zhu Y, Zhang Y, Li Y: Group II intron-anchored gene deletion in Clostridium . PLoS One 2011, 6: e16693. 10.1371/journal.pone.0016693PubMedPubMed CentralGoogle Scholar
- Carr PA, Church GM: Genome engineering. Nat Biotechnol 2009, 27: 1151-1162. 10.1038/nbt.1590PubMedGoogle Scholar
- King RD, Rowland J, Oliver SG, Young M, Aubrey W, Byrne E, Liakata M, Markham M, Pir P, Soldatova LN, Sparks A, Whelan KE, Clare A: The automation of science. Science 2009, 324: 85-89. 10.1126/science.1165620PubMedGoogle Scholar
- Wang HH, Isaacs FJ, Carr PA, Sun ZZ, Xu G, Forest CR, Church GM: Programming cells by multiplex genome engineering and accelerated evolution. Nature 2009, 460: 894-898. 10.1038/nature08187PubMedPubMed CentralGoogle Scholar
- Haki GD, Rakshit SK: Developments in industrially important thermostable enzymes: a review. Bioresource Technol 2003, 89: 17-34. 10.1016/S0960-8524(03)00033-6Google Scholar
- Turner P, Mamo G, Karlsson EN: Potential and utilization of thermophiles and thermostable enzymes in biorefining. Microb Cell Fact 2007, 6: 9. 10.1186/1475-2859-6-9PubMedPubMed CentralGoogle Scholar
- Taylor MP, van Zyl L, Tuffin IM, Leak DJ, Cowan DA: Genetic tool development underpins recent advances in thermophilic whole-cell biocatalysts. Microb Biotechnol 2011, 4: 438-448. 10.1111/j.1751-7915.2010.00246.xPubMedPubMed CentralGoogle Scholar
- Mohr G, Hong W, Zhang J, Cui GZ, Yang Y, Cui Q, Liu YJ, Lambowitz AM: A targetron system for gene targeting in thermophiles and its application in Clostridium thermocellum . PLoS One 2013, 8: e69032. 10.1371/journal.pone.0069032PubMedPubMed CentralGoogle Scholar
- Lynd LR, Grethlein HE, Wolkin RH: Fermentation of cellulosic substrates in batch and continuous culture by Clostridium thermocellum . Appl Environ Microbiol 1989, 55: 3131-3139.PubMedPubMed CentralGoogle Scholar
- Nagy A: Cre recombinase: the universal reagent for genome tailoring. Genesis 2000, 26: 99-109. 10.1002/(SICI)1526-968X(200002)26:2<99::AID-GENE1>3.0.CO;2-BPubMedGoogle Scholar
- Claverys JP, Prudhomme M, Mortier-Barrière I, Martin B: Adaptation to the environment: Streptococcus pneumoniae , a paradigm for recombination-mediated genetic plasticity? Mol Microbiol 2000, 35: 251-259. 10.1046/j.1365-2958.2000.01718.xPubMedGoogle Scholar
- Metzgar D, Bacher JM, Pezo V, Reader J, Döring V, Schimmel P, Marlière P, de Crécy-Lagard V: Acinetobacter sp. ADP1: an ideal model organism for genetic analysis and genome engineering. Nucleic Acids Res 2004, 32: 5780-5790. 10.1093/nar/gkh881PubMedPubMed CentralGoogle Scholar
- Ellis HM, Yu D, DiTizio T, Court DL: High efficiency mutagenesis, repair, and engineering of chromosomal DNA using single-stranded oligonucleotides. Proc Natl Acad Sci USA 2001, 98: 6742-6746. 10.1073/pnas.121164898PubMedPubMed CentralGoogle Scholar
- Costantino N, Court DL: Enhanced levels of λ Red-mediated recombinants in mismatch repair mutants. Proc Natl Acad Sci USA 2003, 100: 15748-15753. 10.1073/pnas.2434959100PubMedPubMed CentralGoogle Scholar
- Lusk JE, Williams RJ, Kennedy EP: Magnesium and the growth of Escherichia coli . J Biol Chem 1968, 243: 2618-2624.PubMedGoogle Scholar
- Horowitz SB, Tluczek LJ: Gonadotropin stimulates oocyte translation by increasing magnesium activity through intracellular potassium-magnesium exchange. Proc Natl Acad Sci USA 1989, 86: 9652-9656. 10.1073/pnas.86.24.9652PubMedPubMed CentralGoogle Scholar
- Günther T: Concentration, compartmentation and metabolic function of intracellular free Mg 2+ . Magnes Res 2006, 19: 225-236.PubMedGoogle Scholar
- Jones JP 3rd, Kierlin MN, Coon RG, Perutka J, Lambowitz AM, Sullenger BA: Retargeting mobile group II introns to repair mutant genes. Mol Ther 2005, 11: 687-694. 10.1016/j.ymthe.2005.01.014PubMedGoogle Scholar
- White TB PhD thesis. In Group II intron retrohoming and gene targeting reactions in Drosophila melanogaster. University of Texas: Cell and Molecular Biology; 2011.Google Scholar
- Truong DM, Sidote DJ, Russell R, Lambowitz AM: Enhanced group II intron retrohoming in magnesium-deficient Escherichia coli via selection of mutations in the ribozyme core. Proc Natl Acad Sci USA 2013, 110: E3800-E3809. 10.1073/pnas.1315742110PubMedPubMed CentralGoogle Scholar
- Hu WS, Hughes SH: HIV-1 reverse transcription. Cold Spring Harb Perspect Med 2012, 2: a006882.PubMedPubMed CentralGoogle Scholar
This article is published under license to BioMed Central Ltd. This is an Open Access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/2.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.