# | Rank | Similarity | Title + Abs. | Year | PMID |
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 |
| 288 | 0 | 1.0000 | A new series of mycobacterial expression vectors for the development of live recombinant vaccines. Recombinant BCG (bacillus Calmette-Guérin) is a promising candidate as a live vaccine delivery system. Thus far, however, only autoreplicative plasmids carrying the heterologous genes to be expressed in BCG, together with antibiotic-resistance genes, have been successfully used. This could potentially lead to the spreading of antibiotic resistance among other bacteria, and might therefore be unsafe for the environment. In this study, we present a series of three Escherichia coli-Mycobacteria shuttle vectors which enable expression and secretion of antigens without the use of antibiotic-resistance markers. All these plasmids confer mercury resistance to the host bacteria as the only selectable marker and contain a unique restriction site to allow for single-step in-frame cloning of open reading frames downstream from the Mycobacterium tuberculosis 85A antigen promoter and export signal. The system was used to express the free beta-subunit of human chorionic gonadotropin (hCG beta), a potential target of an immunotherapeutic vaccine. | 1996 | 8918246 |
| 9297 | 1 | 0.9996 | Xer recombination for the automatic deletion of selectable marker genes from plasmids in enteric bacteria. Antibiotic resistance genes are widely used to select bacteria transformed with plasmids and to prevent plasmid loss from cultures, yet antibiotics represent contaminants in the biopharmaceutical manufacturing process, and retaining antibiotic resistance genes in vaccines and biological therapies is discouraged by regulatory agencies. To overcome these limitations, we have developed X-mark™, a novel technology that leverages Xer recombination to generate selectable marker gene-free plasmids for downstream therapeutic applications. Using this technique, X-mark plasmids with antibiotic resistance genes flanked by XerC/D target sites are generated in Escherichia coli cytosol aminopeptidase (E. coli pepA) mutants, which are deficient in Xer recombination on plasmids, and subsequently transformed into enteric bacteria with a functional Xer system. This results in rapid deletion of the resistance gene at high resolution (100%) and stable replication of resolved plasmids for more than 40 generations in the absence of antibiotic selective pressure. This technology is effective in both Escherichia coli and Salmonella enterica bacteria due to the high degree of homology between accessory sequences, including strains that have been developed as oral vaccines for clinical use. X-mark effectively eliminates any regulatory and safety concerns around antibiotic resistance carryover in biopharmaceutical products, such as vaccines and therapeutic proteins. Graphical Abstract. | 2022 | 35601876 |
| 6313 | 2 | 0.9996 | A Novel Nonantibiotic, lgt-Based Selection System for Stable Maintenance of Expression Vectors in Escherichia coli and Vibrio cholerae. Antibiotic selection for the maintenance of expression plasmids is discouraged in the production of recombinant proteins for pharmaceutical or other human uses due to the risks of antibiotic residue contamination of the final products and the release of DNA encoding antibiotic resistance into the environment. We describe the construction of expression plasmids that are instead maintained by complementation of the lgt gene encoding a (pro)lipoprotein glyceryl transferase essential for the biosynthesis of bacterial lipoprotein. Mutations in lgt are lethal in Escherichia coli and other Gram-negative organisms. The lgt gene was deleted from E. coli and complemented by the Vibrio cholerae-derived gene provided in trans on a temperature-sensitive plasmid, allowing cells to grow at 30°C but not at 37°C. A temperature-insensitive expression vector carrying the V. cholerae-derived lgt gene was constructed, whereby transformants were selected by growth at 39°C. The vector was successfully used to express two recombinant proteins, one soluble and one forming insoluble inclusion bodies. Reciprocal construction was done by deleting the lgt gene from V. cholerae and complementing the lesion with the corresponding gene from E. coli The resulting strain was used to produce the secreted recombinant cholera toxin B subunit (CTB) protein, a component of licensed as well as newly developed oral cholera vaccines. Overall, the lgt system described here confers extreme stability on expression plasmids, and this strategy can be easily transferred to other Gram-negative species using the E. coli-derived lgt gene for complementation.IMPORTANCE Many recombinant proteins are produced in bacteria from genes carried on autonomously replicating DNA elements called plasmids. These plasmids are usually inherently unstable and rapidly lost. This can be prevented by using genes encoding antibiotic resistance. Plasmids are thus maintained by allowing only plasmid-containing cells to survive when the bacteria are grown in medium supplemented with antibiotics. In the described antibiotic-free system for the production of recombinant proteins, an essential gene is deleted from the bacterial chromosome and instead provided on a plasmid. The loss of the plasmid becomes lethal for the bacteria. Such plasmids can be used for the expression of recombinant proteins. This broadly applicable system removes the need for antibiotics in recombinant protein production, thereby contributing to reducing the spread of genes encoding antibiotic resistance, reducing the release of antibiotics into the environment, and freeing the final products (often used in pharmaceuticals) from contamination with potentially harmful antibiotic residues. | 2018 | 29222103 |
| 388 | 3 | 0.9996 | Improved bacterial hosts for regulated expression of genes from lambda pL plasmid vectors. The construction and use of a set of Escherichia coli strains with defective lambda prophages that facilitate expression of genes cloned in lambda pL-plasmid vectors is described. These bacteria allow high and regulated expression of such genes, whereas a kanamycin-resistance marker (KmR) on the prophage allows easy identification and genetic transfer from strain to strain. Optimal conditions for examining gene expression with the pL-vector systems using these strains are discussed. | 1993 | 8406046 |
| 9403 | 4 | 0.9995 | Molecular genetics and pathogenesis of Clostridium perfringens. Clostridium perfringens is the causative agent of a number of human diseases, such as gas gangrene and food poisoning, and many diseases of animals. Recently significant advances have been made in the development of C. perfringens genetics. Studies on bacteriocin plasmids and conjugative R plasmids have led to the cloning and analysis of many C. perfringens genes and the construction of shuttle plasmids. The relationship of antibiotic resistance genes to similar genes from other bacteria has been elucidated. A detailed physical map of the C. perfringens chromosome has been prepared, and numerous genes have been located on that map. Reproducible transformation methods for the introduction of plasmids into C. perfringens have been developed, and several genes coding for the production of extracellular toxins and enzymes have been cloned. Now that it is possible to freely move genetic information back and forth between C. perfringens and Escherichia coli, it will be possible to apply modern molecular methods to studies on the pathogenesis of C. perfringens infections. | 1991 | 1779929 |
| 9283 | 5 | 0.9995 | Vibrio cholerae: Measuring Natural Transformation Frequency. Many bacteria can become naturally competent to take up extracellular DNA across their outer and inner membranes by a dedicated competence apparatus. Whereas some studies show that the DNA delivered to the cytoplasm may be used for genome repair or for nutrition, it can also be recombined onto the chromosome by homologous recombination: a process called natural transformation. Along with conjugation and transduction, natural transformation represents a mechanism for horizontal transfer of genetic material, e.g., antibiotic resistance genes, which can confer new beneficial characteristics onto the recipient bacteria. Described here are protocols for quantifying the frequency of transformation for the human pathogen Vibrio cholerae, one of several Vibrio species recently shown to be capable of natural transformation. | 2014 | 25367272 |
| 6314 | 6 | 0.9995 | Identification of genes involved in the resistance of mycobacteria to killing by macrophages. The survival of M. leprae and M. tuberculosis in the human host is dependent upon their ability to produce gene products that counteract the bactericidal activities of macrophages. To identify such mycobacterial genes and gene products, recombinant DNA libraries of mycobacterial DNA in E. coli were passed through macrophages to enrich for clones carrying genes that endow the normally susceptible E. coli bacteria with an enhanced ability to survive within macrophages. Following three cycles of enrichment, 15 independent clones were isolated. Three recombinants were characterized in detail, and each confers significantly enhanced survival on E. coli cells carrying them. Two of the cloned genetic elements also confer enhanced survival onto M. smegmatis cells. Further characterization of these genes and gene products should provide insights into the survival of mycobacteria within macrophages and may identify new approaches of targets for combatting these important pathogens. | 1994 | 8080180 |
| 6312 | 7 | 0.9995 | D-serine deaminase is a stringent selective marker in genetic crosses. The presence of the locus for D-serine deaminase (dsd) renders bacteria resistant to growth inhibition by D-serine and enables them to grow with D-serine as the sole nitrogen source. The two properties permit stringent selection in genetic crosses and make the D-serine deaminase gene an excellent marker, especially in the construction of strains for which the use of antibiotic resistance genes as selective markers is not allowed. | 1995 | 7814336 |
| 9402 | 8 | 0.9995 | Phage resistance in lactic acid bacteria. The interactions between lactic acid bacteria and their phages are commercially significant. Current research has focused on the elucidation of the mechanisms and genetics of phage resistance. Phage resistance genes have been linked to plasmid DNA for Streptococcus lactis and Streptococcus cremoris, and preliminary studies suggest the operation of mechanisms such as the prevention of phage adsorption, restriction/modification, and abortive infection. Some phage resistance plasmids can be conjugally transferred, providing a means of dissemination among phage-sensitive strains for the construction of phage-resistant starter cultures. | 1988 | 3139060 |
| 261 | 9 | 0.9995 | Suicide vectors for antibiotic marker exchange and rapid generation of multiple knockout mutants by allelic exchange in Gram-negative bacteria. Allelic exchange is frequently used in bacteria to generate knockout mutants in genes of interest, to carry out phenotypic analysis and learn about their function. Frequently, understanding of gene function in complex processes such as pathogenesis requires the generation of multiple mutant strains. In Pseudomonads and other non-Enterobacteriaceae, this is a time-consuming and laborious process based on the use of suicide vectors and allelic exchange of the appropriate mutant version of each gene, disrupted by a different antibiotic marker. This often implies the generation of a series of mutants for each gene, each disrupted by a different antibiotic marker, in order to obtain all possible double or multiple mutant combinations. In this work, we have modified this method by developing a set of 3 plasmid derivatives from the previously described suicide vector for allelic exchange, pKAS32, to make antibiotic marker exchange easier and thus accelerate the entire process. Briefly, the construction of each single gene knockout mutant is carried out by allelic exchange of the chromosomal gene with a mutant allele disrupted by the insertion of a kanamycin resistance cassette. When a double mutant strain is required, antibiotic marker exchange is performed in either one of the single mutants, using any of the three plasmid derivatives that carry the kanamycin resistance gene disrupted by either a chloramphenicol, gentamycin, or streptomycin resistance cassette. The single mutant strain, carrying now an antibiotic resistance marker other than kanamycin, can be used to introduce a second mutation using the original plasmid constructs, to generate a double mutant. The process can be repeated sequentially to generate multiple mutants. We have validated this method by generating strains carrying different combinations of mutations in genes encoding different transcriptional regulators of the Hrp type III secretion system in Pseudomonas syringae. We have also tested the genetic organisation and stability of the resulting mutant strains during growth in laboratory conditions as well as in planta. | 2006 | 16750581 |
| 284 | 10 | 0.9995 | Expression of a transposable antibiotic resistance element in Saccharomyces. Some eukaryotic genes can be expressed in bacteria but there are few examples of the expression of prokaryotic genes in eukaryotes. Antibiotic G418 is a 2-deoxystreptamine antibiotic that is structurally related to gentamicin but has inhibitory activity against a much wider variety of pro- and eukaryotic organisms. In bacteria, resistance to G418 can be determined by several plasmid-encoded modifiying enzymes and, in view of the broad spectrum of activity of G418, we considered that this antibiotic might be useful as a selective agent for the introduction of these antibiotic resistance genes into a eukaryotic organism such as Saccharomyces cerevisiae. Additional impetus for these experiments came from the knowledge that certain of the G418-resistance determinants in bacteria are carried on transposable elements; a study of the properties of these elements in eukaryotes would be intriguing. | 1980 | 6253817 |
| 9197 | 11 | 0.9995 | Temperature-sensitive bacterial pathogens generated by the substitution of essential genes from cold-loving bacteria: potential use as live vaccines. Temperature-sensitive (TS) viruses have been used for decades as vaccines capable of limited replication in their hosts. Although attenuated bacteria, such as the Bacille Calmette-Guérin anti-tuberculosis vaccine, have been used for almost a century, it is only recently that there has been progress in using TS bacterial strains as live vaccines. Decades of work on essential bacterial genes and the recent explosion in the number of available bacterial genomic sequences set the groundwork for the identification of essential genes from diverse bacteria. This knowledge has allowed for the substitution of essential genes from cold-loving bacteria into the chromosomes of pathogenic bacteria. Many of these gene substitutions generated TS pathogenic bacterial strains, and some were demonstrated to provide protective immunity in mice. This work opens the possibility of engineering many pathogenic bacteria to create TS strains that can be used as vaccines. | 2011 | 21229224 |
| 6307 | 12 | 0.9995 | High-density transposon libraries utilising outward-oriented promoters identify mechanisms of action and resistance to antimicrobials. The use of bacterial transposon mutant libraries in phenotypic screens is a well-established technique for determining which genes are essential or advantageous for growth in conditions of interest. Standard, inactivating, transposon libraries cannot give direct information about genes whose over-expression gives a selective advantage. We report the development of a system wherein outward-oriented promoters are included in mini-transposons, generation of transposon mutant libraries in Escherichia coli and Pseudomonas aeruginosa and their use to probe genes important for growth under selection with the antimicrobial fosfomycin, and a recently-developed leucyl-tRNA synthase inhibitor. In addition to the identification of known mechanisms of action and resistance, we identify the carbon-phosphorous lyase complex as a potential resistance liability for fosfomycin in E. coli and P. aeruginosa. The use of this technology can facilitate the development of novel mechanism-of-action antimicrobials that are urgently required to combat the increasing threat worldwide from antimicrobial-resistant pathogenic bacteria. | 2020 | 33186989 |
| 9298 | 13 | 0.9995 | Delivering "Chromatic Bacteria" Fluorescent Protein Tags to Proteobacteria Using Conjugation. Recently, we published a large and versatile set of plasmids, the chromatic bacteria toolbox, to deliver eight different fluorescent protein genes and four combinations of antibiotic resistance genes to Gram-negative bacteria. Fluorescent tags are important tools for single-cell microbiology, synthetic community studies, biofilm, and host-microbe interaction studies. Using conjugation helper strain E. coli S17-1 as a donor, we show how plasmid conjugation can be used to deliver broad host range plasmids, Tn5 transposons delivery plasmids, and Tn7 transposon delivery plasmids into species belonging to the Proteobacteria. To that end, donor and recipient bacteria are grown under standard growth conditions before they are mixed and incubated under non-selective conditions. Then, transconjugants or exconjugant recipients are selected on selective media. Mutant colonies are screened using a combination of tools to ensure that the desired plasmids or transposons are present and that the colonies are not containing any surviving donors. Through conjugation, a wide range of Gram-negative bacteria can be modified without prior, often time-consuming, establishment of competent cell and electroporation procedures that need to be adjusted for every individual strain. The here presented protocol is not exclusive for the delivery of Chromatic bacteria plasmids and transposons, but can also be used to deliver other mobilizable plasmids to bacterial recipients. | 2019 | 33654996 |
| 3824 | 14 | 0.9995 | Screening for novel antibiotic resistance genes. Knowledge of novel antibiotic resistance genes aids in the understanding of how antibiotics function and how bacteria fight them. This knowledge also allows future generations of an antibiotic or antibiotic group to be altered to allow the greatest efficacy. The method described here is very simple in theory. The bacterial strains are screened for antibiotic resistance. Cultures of the strain are grown, and DNA is extracted. A partial digest of the extraction is cloned into Escherichia coli, and the transformants are plated on selective media. Any colony that grows will possess the antibiotic resistance gene and can be further examined. In actual practice, however, this technique can be complicated. The detailed protocol will need to be optimized for each bacterial strain, vector, and cell line chosen. | 2010 | 20830570 |
| 8225 | 15 | 0.9995 | Basic peptide-morpholino oligomer conjugate that is very effective in killing bacteria by gene-specific and nonspecific modes. Basic peptides covalently linked to nucleic acids, or chemically modified nucleic acids, enable the insertion of such a conjugate into bacteria grown in liquid medium and mammalian cells in tissue culture. A unique peptide, derived from human T cells, has been employed in a chemical synthesis to make a conjugate with a morpholino oligonucleotide. This new conjugate is at least 10- to 100-fold more effective than previous peptides used in altering the phenotype of host bacteria if the external guide sequence methodology is employed in these experiments. Bacteria with target genes expressing chloramphenicol resistance, penicillin resistance, or gyrase A function can effectively be reduced in their expression and the host cells killed. Several bacteria are susceptible to this treatment, which has a broad range of potency. The loss in viability of bacteria is not due only to complementarity with a target RNA and the action of RNase P, but also to a non-gene-specific tight binding of the complexed nontargeted RNA to the basic polypeptide-morpholino oligonucleotide. | 2011 | 21949365 |
| 9707 | 16 | 0.9995 | Towards safer vectors for the field release of recombinant bacteria. The prospect of the deliberate environmental release of genetically manipulated microorganisms has given rise to a great deal of polemic. Amongst the rational scientific concerns are those concerned with the fate of the released bacteria, the fate of the recombinant genes that they carry, the selective pressures acting upon them in different environmental situations and the long term effects on the environment and human health. All recombinant DNA is carried by vectors (plasmids, transposons or bacteriophage or remnants of these). Thus the way in which recombinant constructions are made may itself lead to potential biosafety concerns, irrespective of the host bacterium and the recombinant DNA fragment of primary interest. The purpose of the present review is to assess progress in improved vector design aimed at eliminating risks due to the way recombinant vectors are constructed. Improved vector constructions include the avoidance of the use, or removal, of antibiotic resistance genes, the use of defective transposons rather than plasmids in order to reduce horizontal transfer and the development of conditionally lethal suicide systems. More recently, new site-specific recombination systems have permitted transposon vectors to be manipulated following strain construction, but before environmental release, so that virtually all recombinant DNA not directly involved in the release experiment is eliminated. Such bacteria are thus pseudo-wild type in that they contain no heterologous DNA other than the genes of interest. | 2002 | 15612252 |
| 262 | 17 | 0.9994 | Genome scanning in Haemophilus influenzae for identification of essential genes. We have developed a method for identifying essential genes by using an in vitro transposition system, with a small (975 bp) insertional element containing an antibiotic resistance cassette, and mapping these inserts relative to the deduced open reading frames of Haemophilus influenzae by PCR and Southern analysis. Putative essential genes are identified by two methods: mutation exclusion or zero time analysis. Mutation exclusion consists of growing an insertional library and identifying open reading frames that do not contain insertional elements: in a growing population of bacteria, insertions in essential genes are excluded. Zero time analysis consists of monitoring the fate of individual insertions after transformation in a growing culture: the loss of inserts in essential genes is observed over time. Both methods of analysis permit the identification of genes required for bacterial survival. Details of the mutant library construction and the mapping strategy, examples of mutant exclusion, and zero time analysis are presented. | 1999 | 10438768 |
| 9357 | 18 | 0.9994 | The bifunctional enzymes of antibiotic resistance. The evolutionary union of two genes--each encoding proteins of complementary enzymatic activity--into a single gene so as to allow the coordinated expression of these activities as a fusion polypeptide, is an increasingly recognized biological occurrence. The result of this genetic union is the bifunctional enzyme. This fusion of separate catalytic activities into a single protein, whose gene is regulated by a single promoter, is seen especially where the coordinated expression of the separate activities is highly desirable. Increasingly, a circumstance driving the evolution of the bifunctional enzyme in bacteria is the resistance response of bacteria to antibiotic chemotherapy. We summarize the knowledge on bifunctional antibiotic-resistance enzymes, as possible harbingers of clinically significant resistance mechanisms of the future. | 2009 | 19615931 |
| 4437 | 19 | 0.9994 | The activity of glycopeptide antibiotics against resistant bacteria correlates with their ability to induce the resistance system. Glycopeptide antibiotics containing a hydrophobic substituent display the best activity against vancomycin-resistant enterococci, and they have been assumed to be poor inducers of the resistance system. Using a panel of 26 glycopeptide derivatives and the model resistance system in Streptomyces coelicolor, we confirmed this hypothesis at the level of transcription. Identification of the structural glycopeptide features associated with inducing the expression of resistance genes has important implications in the search for more effective antibiotic structures. | 2014 | 25092694 |