High-Resolution Whole-Genome Analysis of Sister-Chromatid Contacts.

Sister-chromatid cohesion describes the orderly association of newly replicated DNA molecules behind replication forks. It plays an essential role in the maintenance and faithful transmission of genetic information. Cohesion is created by DNA topological links and proteinaceous bridges, whose formation and deposition could be potentially affected by many processes. Current knowledge on cohesion has been mainly gained by fluorescence microscopy observation. However, the resolution limit of microscopy and the restricted number of genomic positions that can be simultaneously visualized considerably hampered progress. Here, we present a high-throughput methodology to monitor sister-chromatid contacts (Hi-SC2). Using the multi-chromosomal Vibrio cholerae bacterium as a model, we show that Hi-SC2 permits to monitor local variations in sister-chromatid cohesion at a high resolution over a whole genome.

Bacteriophage lambda site-specific recombination.

The site-specific recombination pathway of bacteriophage λ encompasses isoenergetic but highly directional and tightly regulated integrative and excisive reactions that integrate and excise the vial chromosome into and out of the bacterial chromosome. The reactions require 240 bp of phage DNA and 21 bp of bacterial DNA comprising 16 protein binding sites that are differentially used in each pathway by the phage-encoded Int and Xis proteins and the host-encoded integration host factor and factor for inversion stimulation proteins. Structures of higher-order protein-DNA complexes of the four-way Holliday junction recombination intermediates provided clarifying insights into the mechanisms, directionality, and regulation of these two pathways, which are tightly linked to the physiology of the bacterial host cell. Here we review our current understanding of the mechanisms responsible for regulating and executing λ site-specific recombination, with an emphasis on key studies completed over the last decade.

Redefining the bacteriophage mv4 site-specific recombination system and the sequence specificity of its attB and core-attP sites.

Through their involvement in the integration and excision of a large number of mobile genetic elements, such as phages and integrative and conjugative elements (ICEs), site-specific recombination systems based on heterobivalent tyrosine recombinases play a major role in genome dynamics and evolution. However, despite hundreds of these systems having been identified in genome databases, very few have been described in detail, with none from phages that infect Bacillota (formerly Firmicutes). In this study, we reanalyzed the recombination module of Lactobacillus delbrueckii subsp. bulgaricus phage mv4, previously considered atypical compared with classical systems. Our results reveal that mv4 integrase is a 369 aa protein with all the structural hallmarks of recombinases from the Tn916 family and that it cooperatively interacts with its recombination sites. Using randomized DNA libraries, NGS sequencing, and other molecular approaches, we show that the 21-bp core-attP and attB sites have structural similarities to classical systems only if considering the nucleotide degeneracy, with two 7-bp inverted regions corresponding to mv4Int core-binding sites surrounding a 7-bp strand-exchange region. We also examined the different compositional constraints in the core-binding regions, which define the sequence space of permissible recombination sites.

Pdif-mediated antibiotic resistance genes transfer in bacteria identified by pdifFinder.

Modules consisting of antibiotic resistance genes (ARGs) flanked by inverted repeat Xer-specific recombination sites were thought to be mobile genetic elements that promote horizontal transmission. Less frequently, the presence of mobile modules in plasmids, which facilitate a pdif-mediated ARGs transfer, has been reported. Here, numerous ARGs and toxin-antitoxin genes have been found in pdif site pairs. However, the mechanisms underlying this apparent genetic mobility is currently not understood, and the studies relating to pdif-mediated ARGs transfer onto most bacterial genera are lacking. We developed the web server pdifFinder based on an algorithm called PdifSM that allows the prediction of diverse pdif-ARGs modules in bacterial genomes. Using test set consisting of almost 32 thousand plasmids from 717 species, PdifSM identified 481 plasmids from various bacteria containing pdif sites with ARGs. We found 28-bp-long elements from different genera with clear base preferences. The data we obtained indicate that XerCD-dif site-specific recombination mechanism may have evolutionary adapted to facilitate the pdif-mediated ARGs transfer. Through multiple sequence alignment and evolutionary analyses of duplicated pdif-ARGs modules, we discovered that pdif sites allow an interspecies transfer of ARGs but also across different genera. Mutations in pdif sites generate diverse arrays of modules which mediate multidrug-resistance, as these contain variable numbers of diverse ARGs, insertion sequences and other functional genes. The identification of pdif-ARGs modules and studies focused on the mechanism of ARGs co-transfer will help us to understand and possibly allow controlling the spread of MDR bacteria in clinical settings. The pdifFinder code, standalone software package and description with tutorials are available at https://github.com/mjshao06/pdifFinder.

In vivo cloning of PCR product via site-specific recombination in Escherichia coli.

Over the past years, several methods have been developed for gene cloning. Choosing a cloning strategy depends on various factors, among which simplicity and affordability have always been considered. The aim of this study, on the one hand, is to simplify gene cloning by skipping in vitro assembly reactions and, on the other hand, to reduce costs by eliminating relatively expensive materials. We investigated a cloning system using Escherichia coli harboring two plasmids, pLP-AmpR and pScissors-CmR. The pLP-AmpR contains a landing pad (LP) consisting of two genes (λ int and λ gam) that allow the replacement of the transformed linear DNA using site-specific recombination. After the replacement process, the inducible expressing SpCas9 and specific sgRNA from the pScissors-CmR (CRISPR/Cas9) vector leads to the removal of non-recombinant pLP-AmpR plasmids. The function of LP was explored by directly transforming PCR products. The pScissors-CmR plasmid was evaluated for curing three vectors, including the origins of pBR322, p15A, and pSC101. Replacing LP with a PCR product and fast-eradicating pSC101 origin-containing vectors was successful. Recombinant colonies were confirmed following gene replacement and plasmid curing processes. The results made us optimistic that this strategy may potentially be a simple and inexpensive cloning method. KEY POINTS: •The in vivo cloning was performed by replacing the target gene with the landing pad. •Fast eradication of non-recombinant plasmids was possible by adapting key vectors. •This strategy is not dependent on in vitro assembly reactions and expensive materials.

Simple and Rapid Site-Specific Integration of Multiple Heterologous DNAs into the Escherichia coli Chromosome.

Escherichia coli is the most studied and well understood microorganism, but research in this system can still be limited by available genetic tools, including the ability to rapidly integrate multiple DNA constructs efficiently into the chromosome. Site-specific, large serine-recombinases can be useful tools, catalyzing a single, unidirectional recombination event between 2 specific DNA sequences, attB and attP, without requiring host proteins for functionality. Using these recombinases, we have developed a system to integrate up to 12 genetic constructs sequentially and stably into in the E. coli chromosome. A cassette of attB sites was inserted into the chromosome and the corresponding recombinases were cloned onto temperature sensitive plasmids to mediate recombination between a non-replicating, attP-containing "cargo" plasmid and the corresponding attB site on the chromosome. The efficiency of DNA insertion into the E. coli chromosome was approximately 107 CFU/µg DNA for six of the recombinases when the competent cells already contained the recombinase-expressing plasmid and approximately 105 CFU/µg DNA or higher when the recombinase-expressing plasmid and "cargo" plasmid were co-transformed. The "cargo" plasmid contains ΦC31 recombination sites flanking the antibiotic gene, allowing for resistance markers to be removed and reused following transient expression of the ΦC31 recombinase. As an example of the utility of this system, eight DNA methyltransferases from Clostridium clariflavum 4-2a were inserted into the E. coli chromosome to methylate plasmid DNA for evasion of the C. clariflavum restriction systems, enabling the first demonstration of transformation of this cellulose-degrading species. IMPORTANCE More rapid genetic tools can help accelerate strain engineering, even in advanced hosts like Escherichia coli. Here, we adapt a suite of site-specific recombinases to enable simple, rapid, and highly efficient site-specific integration of heterologous DNA into the chromosome. This utility of this system was demonstrated by sequential insertion of eight DNA methyltransferases into the E. coli chromosome, allowing plasmid DNA to be protected from restriction in Clostridium clariflavum and enabling genetic transformation of this organism. This integration system should also be highly portable into non-model organisms.

Systematic Discovery of a New Catalogue of Tyrosine-Type Integrases in Bacterial Genomic Islands.

Site-specific recombinases (integrases) can mediate the horizontal transfer of genomic islands. The ability to integrate large DNA sequences into target sites is very important for genetic engineering in prokaryotic and eukaryotic cells. Here, we characterized an unprecedented catalogue of 530 tyrosine-type integrases by examining genes potentially encoding tyrosine integrases in bacterial genomic islands. The phylogeny of putative tyrosine integrases revealed that these integrases form an evolutionary clade that is distinct from those already known and are affiliated with novel integrase groups. We systematically searched for candidate integrase genes, and their integration activities were validated in a bacterial model. We verified the integration functions of six representative novel integrases by using a two-plasmid integration system consisting of a donor plasmid carrying the integrase gene and attP site and a recipient plasmid harboring an attB site in recA-deficient Escherichia coli. Further quantitative reverse transcription-PCR (qRT-PCR) assays validated that the six selected integrases can be expressed with their native promoters in E. coli. The attP region reductions showed that the extent of attP sites of integrases is approximately 200 bp for integration capacity. In addition, mutational analysis showed that the conserved tyrosine at the C terminus is essential for catalysis, confirming that these candidate proteins belong to the tyrosine-type recombinase superfamily, i.e., tyrosine integrases. This study revealed that the novel integrases from bacterial genomic islands have site-specific recombination functions, which is of physiological significance for their genomic islands in bacterial chromosomes. More importantly, our discovery expands the toolbox for genetic engineering, especially for efficient integration activity. IMPORTANCE Site-specific recombinases or integrases have high specificity for DNA large fragment integration, which is urgently needed for gene editing. However, known integrases are not sufficient for meeting multiple integrations. In this work, we discovered an array of integrases through bioinformatics analysis in bacterial genomes. Phylogeny and functional assays revealed that these new integrases belong to tyrosine-type integrases and have the ability to conduct site-specific recombination. Moreover, attP region extent and catalysis site analysis were characterized. Our study provides the methodology for discovery of novel integrases and increases the capacity of weapon pool for genetic engineering in bacteria.

Immortalization Reversibility in the Context of Cell Therapy Biosafety.

Immortalization (genetically induced prevention of replicative senescence) is a promising approach to obtain cellular material for cell therapy or for bio-artificial organs aimed at overcoming the problem of donor material shortage. Immortalization is reversed before cells are used in vivo to allow cell differentiation into the mature phenotype and avoid tumorigenic effects of unlimited cell proliferation. However, there is no certainty that the process of de-immortalization is 100% effective and that it does not cause unwanted changes in the cell. In this review, we discuss various approaches to reversible immortalization, emphasizing their advantages and disadvantages in terms of biosafety. We describe the most promising approaches in improving the biosafety of reversibly immortalized cells: CRISPR/Cas9-mediated immortogene insertion, tamoxifen-mediated self-recombination, tools for selection of successfully immortalized cells, using a decellularized extracellular matrix, and ensuring post-transplant safety with the use of suicide genes. The last process may be used as an add-on for previously existing reversible immortalized cell lines.

Strategies for site-specific recombination with high efficiency and precise spatiotemporal resolution.

Site-specific recombinases (SSRs) are invaluable genome engineering tools that have enormously boosted our understanding of gene functions and cell lineage relationships in developmental biology, stem cell biology, regenerative medicine, and multiple diseases. However, the ever-increasing complexity of biomedical research requires the development of novel site-specific genetic recombination technologies that can manipulate genomic DNA with high efficiency and fine spatiotemporal control. Here, we review the latest innovative strategies of the commonly used Cre-loxP recombination system and its combinatorial strategies with other site-specific recombinase systems. We also highlight recent progress with a focus on the new generation of chemical- and light-inducible genetic systems and discuss the merits and limitations of each new and established system. Finally, we provide the future perspectives of combining various recombination systems or improving well-established site-specific genetic tools to achieve more efficient and precise spatiotemporal genetic manipulation.

Shortening the biologics clinical timeline with a novel method for generating stable, high-producing cell pools and clones.

Reducing drug development timelines is an industry-wide goal to bring medicines to patients in need more quickly. This was exemplified in the coronavirus disease 2019 pandemic where reducing development timelines had a direct impact on the number of lives lost to the disease. The use of drug substances produced using cell pools, as opposed to clones, has the potential to shorten development timelines. Toward this goal, we have developed a novel technology, GPEx® Lightning, that allows for rapid, reproducible, targeted recombination of transgenes into more than 200 Dock sites in the Chinese hamster ovary cell line genome. This allows for rapid production of high-expressing stable cell pools and clones that reach titers of 4-12 g/l in generic fed-batch production. These pools and clones are highly stable in both titer and glycosylation, showing strong similarities in glycosylation profiles.

The TLCΦ satellite phage harbors a Xer recombination activation factor.

The circular chromosomes of bacteria can be concatenated into dimers by homologous recombination. Dimers are solved by the addition of a cross-over at a specific chromosomal site, dif, by 2 related tyrosine recombinases, XerC and XerD. Each enzyme catalyzes the exchange of a specific pair of strands. Some plasmids exploit the Xer machinery for concatemer resolution. Other mobile elements exploit it to integrate into the genome of their host. Chromosome dimer resolution is initiated by XerD. The reaction is under the control of a cell-division protein, FtsK, which activates XerD by a direct contact. Most mobile elements exploit FtsK-independent Xer recombination reactions initiated by XerC. The only notable exception is the toxin-linked cryptic satellite phage of Vibrio cholerae, TLCΦ, which integrates into and excises from the dif site of the primary chromosome of its host by a reaction initiated by XerD. However, the reaction remains independent of FtsK. Here, we show that TLCΦ carries a Xer recombination activation factor, XafT. We demonstrate in vitro that XafT activates XerD catalysis. Correspondingly, we found that XafT specifically interacts with XerD. We further show that integrative mobile elements exploiting Xer (IMEXs) encoding a XafT-like protein are widespread in gamma- and beta-proteobacteria, including human, animal, and plant pathogens.

[Genetic Engineering in Mycobacteria].

Genetic tools for targeted modification of the mycobacterial genome contribute to the understanding of the physiology and virulence mechanisms of mycobacteria. Human and animal pathogens, such as the Mycobacterium tuberculosis complex, which causes tuberculosis, and M. leprae, which causes leprosy, are of particular importance. Genetic research opens up novel opportunities to identify and validate new targets for antibacterial drugs and to develop improved vaccines. Although mycobacteria are difficult to work with due to their slow growth rate and a limited possibility to transfer genetic information, significant progress has been made in developing genetic engineering methods for mycobacteria. The review considers the main approaches to changing the mycobacterial genome in a targeted manner, including homologous and site-specific recombination and use of the CRISPR/Cas system.

Mobilization of pdif modules in Acinetobacter: A novel mechanism for antibiotic resistance gene shuffling?

XerCD-dif site-specific recombination is a well characterized system, found in most bacteria and archaea. Its role is resolution of chromosomal dimers that arise from homologous recombination. Xer-mediated recombination is also used by several plasmids for multimer resolution to enhance stability and by some phage for integration into the chromosome. In the past decade, it has been hypothesized that an alternate and novel function exists for this system in the dissemination of genetic elements, notably antibiotic resistance genes, in Acinetobacter species. Currently the mechanism underlying this apparent genetic mobility is unknown. Multidrug resistant Acinetobacter baumannii is an increasingly problematic pathogen that can cause recurring infections. Sequencing of numerous plasmids from clinical isolates of A. baumannii revealed the presence of possible mobile modules: genes were found flanked by pairs of Xer recombination sites, called plasmid-dif (pdif) sites. These modules have been identified in multiple otherwise unrelated plasmids and in different genetic contexts suggesting they are mobile elements. In most cases, the pairs of sites flanking a gene (or genes) are in inverted repeat, but there can be multiple modules per plasmid providing pairs of recombination sites that can be used for inversion or fusion/deletion reactions; as many as 16 pdif sites have been seen in a single plasmid. Similar modules including genes for surviving environmental toxins have also been found in strains of Acinetobacter Iwoffi isolated from permafrost cores; this suggests that these mobile modules are an ancient adaptation and not a novel response to antibiotic pressure. These modules bear all the hallmarks of mobile genetic elements, yet, their movement has never been directly observed to date. This review gives an overview of the current state of this novel research field.

Potential Mobilization of mcr-10 by an Integrative Mobile Element via Site-Specific Recombination in Cronobacter sakazakii.

mcr-10 is a newly identified plasmid-borne colistin resistance gene, but its mobilization mechanism remains unclear. In this study, mcr-10 was found on an IncFIB plasmid carrying virulence genes mrkABCDFJ, iucABCD/iutA, and eitCBAD in a Cronobacter sakazakii isolate. By comparison with closely related plasmids, two recombination sites were identified flanking the genetic element containing mcr-10 and an integrase-encoding gene, suggesting that site-specific recombination mediated by an integrase of an integrative mobile element is a potential mechanism for mobilizing mcr-10.

In vitro characterization of the site-specific recombination system based on genus Habenivirus ϕRSM small serine integrase.

The genus Habenivirus which includes Ralstonia virus ϕRSM encodes a site-specific integrase of a small serine recombinase belonging to the resolvase/invertase family. Here we describe the integrative/excisive recombination reactions mediated by ϕRSM integrase using in vitro assays. The products of attP/attB recombination, i.e. attL and attR, were exactly identical to those found in the prophage ϕRSM in R. solanacearum strains. The minimum size of attB required for integration was determined to be 37 bp, containing a 13 bp core and flanking sequences of 4 bp on the left and 20 bp on the right. ϕRSM integrative recombination proceeds efficiently in vitro in the absence of additional proteins or high-energy cofactors. Excision of a functional phage genome from a prophage fragment was demonstrated in vitro, demonstrating two-way activity of ϕRSM1 integrase. This is the first example of a small serine recombinase from the resolvase/invertase group that functions in integrative and excisive recombination for filamentous phages. This serine integrase could be used as a tool for several genome engineering applications.

DNA-Topology Simplification by Topoisomerases.

The topological properties of DNA molecules, supercoiling, knotting, and catenation, are intimately connected with essential biological processes, such as gene expression, replication, recombination, and chromosome segregation. Non-trivial DNA topologies present challenges to the molecular machines that process and maintain genomic information, for example, by creating unwanted DNA entanglements. At the same time, topological distortion can facilitate DNA-sequence recognition through localized duplex unwinding and longer-range loop-mediated interactions between the DNA sequences. Topoisomerases are a special class of essential enzymes that homeostatically manage DNA topology through the passage of DNA strands. The activities of these enzymes are generally investigated using circular DNA as a model system, in which case it is possible to directly assay the formation and relaxation of DNA supercoils and the formation/resolution of knots and catenanes. Some topoisomerases use ATP as an energy cofactor, whereas others act in an ATP-independent manner. The free energy of ATP hydrolysis can be used to drive negative and positive supercoiling or to specifically relax DNA topologies to levels below those that are expected at thermodynamic equilibrium. The latter activity, which is known as topology simplification, is thus far exclusively associated with type-II topoisomerases and it can be understood through insight into the detailed non-equilibrium behavior of type-II enzymes. We use a non-equilibrium topological-network approach, which stands in contrast to the equilibrium models that are conventionally used in the DNA-topology field, to gain insights into the rates that govern individual transitions between topological states. We anticipate that our quantitative approach will stimulate experimental work and the theoretical/computational modeling of topoisomerases and similar enzyme systems.

Novel conditional plasmids regulated by chemical switches provide versatile tools for genetic engineering in Escherichia coli.

Engineering bacterial genomes or foreign DNA cloned as bacterial artificial chromosomes (BACs) relies on usage of helper plasmids, which deliver the desired tools transiently into the bacteria to be modified. After the anticipated action is completed the helper plasmids need to be cured. To make this efficient, plasmids are used that are maintained by conditional amplicons or carry a counter-selection marker. Here, we describe new conditional plasmids that can be maintained or cured by using chemical induction or repression. Our method is based on the dependency of plasmids carrying ori6Kγ origin of replication on the presence of protein Π. Ori6Kγ based plasmids are tightly regulated conditional constructs, but they require usually special E. coli strains to operate. To avoid this, we placed the Π protein expression under the control of a co-expressed conditional repressor. Regulating the maintenance of plasmids with administration or removal of chemicals is fully compatible with any other conditional amplicons applied to date. Here, we describe methods for inducing sites specific recombination of BACs as an example. However, the same strategy might be used to construct appropriate helper plasmids for any other transient components of genome editing methodologies such as λred recombinases or CRISPR/Cas components.

Recombination of the Phase-Variable spnIII Locus Is Independent of All Known Pneumococcal Site-Specific Recombinases.

Streptococcus pneumoniae is one of the world's leading bacterial pathogens, causing pneumonia, septicemia, and meningitis. In recent years, it has been shown that genetic rearrangements in a type I restriction-modification system (SpnIII) can impact colony morphology and gene expression. By generating a large panel of mutant strains, we have confirmed a previously reported result that the CreX (also known as IvrR and PsrA) recombinase found within the locus is not essential for hsdS inversions. In addition, mutants of homologous recombination pathways also undergo hsdS inversions. In this work, we have shown that these genetic rearrangements, which result in different patterns of genome methylation, occur across a wide variety of serotypes and sequence types, including two strains (a 19F and a 6B strain) naturally lacking CreX. Our gene expression analysis, by transcriptome sequencing (RNAseq), confirms that the level of creX expression is impacted by these genomic rearrangements. In addition, we have shown that the frequency of hsdS recombination is temperature dependent. Most importantly, we have demonstrated that the other known pneumococcal site-specific recombinases XerD, XerS, and SPD_0921 are not involved in spnIII recombination, suggesting that a currently unknown mechanism is responsible for the recombination of these phase-variable type I systems.IMPORTANCEStreptococcus pneumoniae is a leading cause of pneumonia, septicemia, and meningitis. The discovery that genetic rearrangements in a type I restriction-modification locus can impact gene regulation and colony morphology led to a new understanding of how this pathogen switches from harmless colonizer to invasive pathogen. These rearrangements, which alter the DNA specificity of the type I restriction-modification enzyme, occur across many different pneumococcal serotypes and sequence types and in the absence of all known pneumococcal site-specific recombinases. This finding suggests that this is a truly global mechanism of pneumococcal gene regulation and the need for further investigation of mechanisms of site-specific recombination.

Characterization of the Chromosome Dimer Resolution Site in Caulobacter crescentus.

Chromosome dimers occur in bacterial cells as a result of the recombinational repair of DNA. In most bacteria, chromosome dimers are resolved by XerCD site-specific recombination at the dif (deletion-induced filamentation) site located in the terminus region of the chromosome. Caulobacter crescentus, a Gram-negative oligotrophic bacterium, also possesses Xer recombinases, called CcXerC and CcXerD, which have been shown to interact with the Escherichia colidif site in vitro Previous studies on Caulobacter have suggested the presence of a dif site (referred to in this paper as dif1CC ), but no in vitro data have shown any association with this site and the CcXer proteins. Using recursive hidden Markov modeling, another group has proposed a second dif site, which we call dif2CC , which shows more similarity to the dif consensus sequence. Here, by using a combination of in vitro experiments, we compare the affinities and the cleavage abilities of CcXerCD recombinases for both dif sites. Our results show that dif2CC displays a higher affinity for CcXerC and CcXerD and is bound cooperatively by these proteins, which is not the case for the original dif1CC site. Furthermore, dif2CC nicked substrates are more efficiently cleaved by CcXerCD, and deletion of the site results in about 5 to 10% of cells showing an altered cellular morphology.IMPORTANCE Bacteria utilize site-specific recombination for a variety of purposes, including the control of gene expression, acquisition of genetic elements, and the resolution of dimeric chromosomes. Failure to resolve dimeric chromosomes can lead to cell division defects in a percentage of the cell population. The work presented here shows the existence of a chromosomal resolution system in C. crescentus Defects in this resolution system result in the formation of chains of cells. Further understanding of how these cells remain linked together will help in the understanding of how chromosome segregation and cell division are linked in C. crescentus.

Molecular Mechanisms of hsdS Inversions in the cod Locus of Streptococcus pneumoniae.

Streptococcus pneumoniae (pneumococcus), a major human pathogen, is well known for its adaptation to various host environments. Multiple DNA inversions in the three DNA methyltransferase hsdS genes (hsdSA, hsdSB, and hsdSC) of the colony opacity determinant (cod) locus generate extensive epigenetic and phenotypic diversity. However, it is unclear whether all three hsdS genes are functional and how the inversions mechanistically occur. In this work, our transcriptional analysis revealed active expression of hsdSA but not hsdSB and hsdSC, indicating that hsdSB and hsdSC do not produce functional proteins and instead act as sources for altering the sequence of hsdSA by DNA inversions. Consistent with our previous finding that the hsdS inversions are mediated by three pairs of inverted repeats (IR1, IR2, and IR3), this study showed that the 15-bp IR1 and its upstream sequence are strictly required for the inversion between hsdSA and hsdSB Furthermore, a single tyrosine recombinase PsrA catalyzes the inversions mediated by IR1, IR2, and IR3, based on the dramatic loss of these inversions in the psrA mutant. Surprisingly, PsrA-independent inversions were also detected in the hsdS sequences flanked by the IR2 (298 bp) and IR3 (85 bp) long inverted repeats, which appear to occur spontaneously in the absence of site-specific or RecA-mediated recombination. Because the HsdS subunit is responsible for the sequence specificity of type I restriction modification DNA methyltransferase, these results have revealed that S. pneumoniae varies the methylation patterns of the genome DNA (epigenetic status) by employing multiple mechanisms of DNA inversion in the cod locus.IMPORTANCEStreptococcus pneumoniae is a major pathogen of human infections with the capacity for adaptation to host environments, but the molecular mechanisms behind this phenomenon remain unclear. Previous studies reveal that pneumococcus extends epigenetic and phenotypic diversity by DNA inversions in three methyltransferase hsdS genes of the cod locus. This work revealed that only the hsdS gene that is in the same orientation as hsdM is actively transcribed, but the other two are silent, serving as DNA sources for inversions. While most of the hsdS inversions are catalyzed by PsrA recombinase, the sequences bound by long inverted repeats also undergo inversions via an unknown mechanism. Our results revealed that S. pneumoniae switches the methylation patterns of the genome (epigenetics) by employing multiple mechanisms of DNA inversion.