-
Nature Biotechnology Jan 2024Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an...
Conventional genome engineering with CRISPR-Cas9 creates double-strand breaks (DSBs) that lead to undesirable byproducts and reduce product purity. Here we report an approach for programmable integration of large DNA sequences in human cells that avoids the generation of DSBs by using Type I-F CRISPR-associated transposases (CASTs). We optimized DNA targeting by the QCascade complex through protein design and developed potent transcriptional activators by exploiting the multi-valent recruitment of the AAA+ ATPase TnsC to genomic sites targeted by QCascade. After initial detection of plasmid-based integration, we screened 15 additional CAST systems from a wide range of bacterial hosts, identified a homolog from Pseudoalteromonas that exhibits improved activity and further increased integration efficiencies. Finally, we discovered that bacterial ClpX enhances genomic integration by multiple orders of magnitude, likely by promoting active disassembly of the post-integration CAST complex, akin to its known role in Mu transposition. Our work highlights the ability to reconstitute complex, multi-component machineries in human cells and establishes a strong foundation to exploit CRISPR-associated transposases for eukaryotic genome engineering.
Topics: Humans; CRISPR-Cas Systems; Transposases; Plasmids; DNA; Genome; Gene Editing
PubMed: 36991112
DOI: 10.1038/s41587-023-01748-1 -
Biochemical Society Transactions Aug 2023SMC and SMC-like complexes promote chromosome folding and genome maintenance in all domains of life. Recently, they were also recognized as factors in cellular immunity...
SMC and SMC-like complexes promote chromosome folding and genome maintenance in all domains of life. Recently, they were also recognized as factors in cellular immunity against foreign DNA. In bacteria and archaea, Wadjet and Lamassu are anti-plasmid/phage defence systems, while Smc5/6 and Rad50 complexes play a role in anti-viral immunity in humans. This raises an intriguing paradox - how can the same, or closely related, complexes on one hand secure the integrity and maintenance of chromosomal DNA, while on the other recognize and restrict extrachromosomal DNA? In this minireview, we will briefly describe the latest understanding of each of these complexes in immunity including speculations on how principles of SMC(-like) function may explain how the systems recognize linear or circular forms of invading DNA.
Topics: Humans; Cell Cycle Proteins; Chromosomes; DNA; Plasmids
PubMed: 37584323
DOI: 10.1042/BST20221395 -
Microbial Cell Factories Jan 2024Antibiotic-based plasmid selection and maintenance is a core tool in molecular biology; however, while convenient, this strategy has numerous drawbacks for biological...
BACKGROUND
Antibiotic-based plasmid selection and maintenance is a core tool in molecular biology; however, while convenient, this strategy has numerous drawbacks for biological manufacturing. Overuse of antibiotics and antibiotic resistance genes (ARG) contributes to the development of antimicrobial resistance, which is a growing threat to modern medicine. Antibiotics themselves are costly and therefore often omitted in fermentations, leading to plasmid loss and a corresponding loss in product yield. Furthermore, constitutive expression of a plasmid-encoded antibiotic resistance gene imposes a significant metabolic burden on the cells. For many fermentation products (e.g., in nutrition and medicine), the use of antibiotic resistance genes is subject to strict regulations and should be avoided. We present a method for plasmid selection and maintenance with stringent selection pressure that is independent of antibiotics and ARG. Furthermore, it can be used without any restrictions regarding culture medium and temperature.
RESULTS
The developed method involves modification of a bacterial strain such that an essential gene is expressed genomically under the control of an inducible promoter. A copy of the same essential gene with the endogenous promoter is supplied on a plasmid for selection. In the absence of the inducer for the genomic copy of the essential gene, cells rely on expression of the plasmid-encoded gene copy, leading to tight selection for plasmid maintenance. Induction of the genomic copy of the essential gene enables the engineered strain to be propagated in the absence of a plasmid. Here, we describe the genetic setup and demonstrate long-term, tight selection for plasmid maintenance with a variety of different plasmids and E. coli strains.
CONCLUSIONS
This method facilitates plasmid-based fermentations by eliminating the need for antibiotic selection and improving plasmid maintenance.
Topics: Anti-Bacterial Agents; Fermentation; Escherichia coli; Plasmids; Promoter Regions, Genetic
PubMed: 38212806
DOI: 10.1186/s12934-023-02291-z -
Nucleic Acids Research Sep 2023Bacterial conjugation was first described by Lederberg and Tatum in the 1940s following the discovery of the F plasmid. During conjugation a plasmid is transferred... (Review)
Review
Bacterial conjugation was first described by Lederberg and Tatum in the 1940s following the discovery of the F plasmid. During conjugation a plasmid is transferred unidirectionally from one bacterium (the donor) to another (the recipient), in a contact-dependent manner. Conjugation has been regarded as a promiscuous mechanism of DNA transfer, with host range determined by the recipient downstream of plasmid transfer. However, recent data have shown that F-like plasmids, akin to tailed Caudovirales bacteriophages, can pick their host bacteria prior to transfer by expressing one of at least four structurally distinct isoforms of the outer membrane protein TraN, which has evolved to function as a highly sensitive sensor on the donor cell surface. The TraN sensor appears to pick bacterial hosts by binding compatible outer membrane proteins in the recipient. The TraN variants can be divided into specialist and generalist sensors, conferring narrow and broad plasmid host range, respectively. In this review we discuss recent advances in our understanding of the function of the TraN sensor at the donor-recipient interface, used by F-like plasmids to select bacterial hosts within polymicrobial communities prior to DNA transfer.
Topics: Bacteria; Bacterial Proteins; Conjugation, Genetic; DNA, Bacterial; F Factor; Membrane Proteins; Plasmids
PubMed: 37592747
DOI: 10.1093/nar/gkad678 -
MBio Aug 2023Conjugative plasmids play a vital role in bacterial evolution and promote the spread of antibiotic resistance. They usually cause fitness costs that diminish the growth...
Conjugative plasmids play a vital role in bacterial evolution and promote the spread of antibiotic resistance. They usually cause fitness costs that diminish the growth rates of the host bacteria. Compensatory mutations are known as an effective evolutionary solution to reduce the fitness cost and improve plasmid persistence. However, whether the plasmid transmission by conjugation is sufficient to improve plasmid persistence is debated since it is an inherently costly process. Here, we experimentally evolved an unstable and costly plasmid pHNSHP24 under laboratory conditions and assessed the effects of plasmid cost and transmission on the plasmid maintenance by the plasmid population dynamics model and a plasmid invasion experiment designed to measure the plasmid's ability to invade a plasmid-free bacterial population. The persistence of pHNSHP24 improved after 36 days evolution due to the plasmid-borne mutation A51G in the 5'UTR of gene . This mutation largely increased the infectious transmission of the evolved plasmid, presumably by impairing the inhibitory effect of FinP on the expression of . We showed that increased conjugation rate of the evolved plasmid could compensate for the plasmid loss. Furthermore, we determined that the evolved high transmissibility had little effect on the -deficient ancestral plasmid, implying that high conjugation transfer is vital for maintaining the -bearing plasmid. Altogether, our findings emphasized that, besides compensatory evolution that reduces fitness costs, the evolution of infectious transmission can improve the persistence of antibiotic-resistant plasmids, indicating that inhibition of the conjugation process could be useful to combat the spread of antibiotic-resistant plasmids. IMPORTANCE Conjugative plasmids play a key role in the spread of antibiotic resistance, and they are well-adapted to the host bacteria. However, the evolutionary adaptation of plasmid-bacteria associations is not well understood. In this study, we experimentally evolved an unstable colistin resistance () plasmid under laboratory conditions and found that increased conjugation rate was crucial for the persistence of this plasmid. Interestingly, the evolved conjugation was caused by a single-base mutation, which could rescue the unstable plasmid from extinction in bacterial populations. Our findings imply that inhibition of the conjugation process could be necessary for combating the persistence of antibiotic-resistance plasmids.
Topics: Plasmids; Drug Resistance, Microbial; Bacteria; Mutation; Anti-Bacterial Agents
PubMed: 37314200
DOI: 10.1128/mbio.00442-23 -
ACS Synthetic Biology Oct 2023Although cell-free protein expression has been widely used for the synthesis of single proteins, cell-free synthetic biology has rapidly expanded to new, more complex...
Although cell-free protein expression has been widely used for the synthesis of single proteins, cell-free synthetic biology has rapidly expanded to new, more complex applications. One such application is the prototyping or implementation of complex genetic networks involving the expression of multiple proteins at precise ratios, often from different plasmids. However, expression of multiple proteins from multiple plasmids may inadvertently result in unexpected, off-target changes to the levels of the proteins being expressed, a phenomenon termed plasmid crosstalk. Here, we show that the effects of plasmid crosstalk─even at the qualitative level of increases vs decreases in protein expression─depend on the concentration of plasmids in the reaction and the type of transcriptional machinery involved in the expression. This crosstalk can have a significant impact on genetic circuitry function and even interpretation of simple experimental results and thus should be taken into consideration during the development of cell-free applications.
Topics: Plasmids; Protein Processing, Post-Translational; Gene Regulatory Networks; Cell Physiological Phenomena; Cell-Free System
PubMed: 37756020
DOI: 10.1021/acssynbio.3c00412 -
Infection, Genetics and Evolution :... Aug 2024Our study highlights the escalating issue of beta-lactam resistance in nosocomial pathogens, driven by the broad spectrum of antibiotic-degrading enzymes and plasmid...
Our study highlights the escalating issue of beta-lactam resistance in nosocomial pathogens, driven by the broad spectrum of antibiotic-degrading enzymes and plasmid exchange. We catalogued known beta-lactamases across 230 bacterial genera, identified 2349 potential beta-lactamases across over 673 genera, and anticipate discovering many new types, underscoring the need for targeted gene analysis in combating resistance. This study also elucidates the complex relationship between the diversity and frequency of beta-lactamase genes across bacterial genera, highlighting the need for genus-specific approaches in combating antibiotic resistance and emphasizing these genes' significant global distribution and host-specific prevalence. We report many transcriptional regulators, transposases and other factors in the genomes of 20 different bacterial isolates, some of which are consistent with the ability of these species to adapt to different environments. Although we could not determine precisely which factors regulate the presence of beta-lactamases in specific bacteria, we found that the proportion of regulatory genes, the size of the genome, and other factors are not decisive. Further studies are needed to elucidate key aspects of this process.
Topics: beta-Lactamases; Bacteria; Genome, Bacterial; Phylogeny; beta-Lactam Resistance; Anti-Bacterial Agents; Humans; Plasmids
PubMed: 38810790
DOI: 10.1016/j.meegid.2024.105610 -
Nature Microbiology Jan 2024Gut environments harbour dense microbial ecosystems in which plasmids are widely distributed. Plasmids facilitate the exchange of genetic material among microorganisms...
Gut environments harbour dense microbial ecosystems in which plasmids are widely distributed. Plasmids facilitate the exchange of genetic material among microorganisms while enabling the transfer of a diverse array of accessory functions. However, their precise impact on microbial community composition and function remains largely unexplored. Here we identify a prevalent bacterial toxin and a plasmid-encoded resistance mechanism that mediates the interaction between Lactobacilli and Enterococci. This plasmid is widespread across ecosystems, including the rumen and human gut microbiota. Biochemical characterization of the plasmid revealed a defence mechanism against reuterin, a toxin produced by various gut microbes, such as Limosilactobacillus reuteri. Using a targeted metabolomic approach, we find reuterin to be prevalent across rumen ecosystems with impacts on microbial community structure. Enterococcus strains carrying the protective plasmid were isolated and their interactions with L. reuteri, the toxin producer, were studied in vitro. Interestingly, we found that by conferring resistance against reuterin, the plasmid mediates metabolic exchange between the defending and the attacking microbial species, resulting in a beneficial relationship or mutualism. Hence, we reveal here an ecological role for a plasmid-coded defence system in mediating a beneficial interaction.
Topics: Humans; Animals; Symbiosis; Ecosystem; Plasmids; Propane; Limosilactobacillus reuteri; Enterococcus
PubMed: 38151647
DOI: 10.1038/s41564-023-01521-9 -
Nature Communications Mar 2024Bacterial evolution is affected by mobile genetic elements like phages and conjugative plasmids, offering new adaptive traits while incurring fitness costs. Their...
Bacterial evolution is affected by mobile genetic elements like phages and conjugative plasmids, offering new adaptive traits while incurring fitness costs. Their infection is affected by the bacterial capsule. Yet, its importance has been difficult to quantify because of the high diversity of confounding mechanisms in bacterial genomes such as anti-viral systems and surface receptor modifications. Swapping capsule loci between Klebsiella pneumoniae strains allowed us to quantify their impact on plasmid and phage infection independently of genetic background. Capsule swaps systematically invert phage susceptibility, revealing serotypes as key determinants of phage infection. Capsule types also influence conjugation efficiency in both donor and recipient cells, a mechanism shaped by capsule volume and conjugative pilus structure. Comparative genomics confirmed that more permissive serotypes in the lab correspond to the strains acquiring more conjugative plasmids in nature. The least capsule-sensitive pili (F-like) are the most frequent in the species' plasmids, and are the only ones associated with both antibiotic resistance and virulence factors, driving the convergence between virulence and antibiotics resistance in the population. These results show how traits of cellular envelopes define slow and fast lanes of infection by mobile genetic elements, with implications for population dynamics and horizontal gene transfer.
Topics: Phenotype; Plasmids; Serogroup; Genome, Bacterial; Bacteriophages
PubMed: 38448399
DOI: 10.1038/s41467-024-46147-5 -
Nature Microbiology Jul 2023Conjugative plasmids are self-transmissible mobile genetic elements that transfer DNA between host cells via type IV secretion systems (T4SS). While T4SS-mediated...
Conjugative plasmids are self-transmissible mobile genetic elements that transfer DNA between host cells via type IV secretion systems (T4SS). While T4SS-mediated conjugation has been well-studied in bacteria, information is sparse in Archaea and known representatives exist only in the Sulfolobales order of Crenarchaeota. Here we present the first self-transmissible plasmid identified in a Euryarchaeon, Thermococcus sp. 33-3. The 103 kbp plasmid, pT33-3, is seen in CRISPR spacers throughout the Thermococcales order. We demonstrate that pT33-3 is a bona fide conjugative plasmid that requires cell-to-cell contact and is dependent on canonical, plasmid-encoded T4SS-like genes. Under laboratory conditions, pT33-3 transfers to various Thermococcales and transconjugants propagate at 100 °C. Using pT33-3, we developed a genetic toolkit that allows modification of phylogenetically diverse Archaeal genomes. We demonstrate pT33-3-mediated plasmid mobilization and subsequent targeted genome modification in previously untransformable Thermococcales species, and extend this process to interphylum transfer to a Crenarchaeon.
Topics: Archaea; Plasmids; DNA; Bacteria; Genome, Archaeal
PubMed: 37277532
DOI: 10.1038/s41564-023-01387-x