D-serine deaminase is a stringent selective marker in genetic crosses. - Related Documents




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631201.0000D-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.19957814336
26010.9998Improved antibiotic resistance gene cassette for marker exchange mutagenesis in Ralstonia solanacearum and Burkholderia species. Marker exchange mutagenesis is a fundamental approach to understanding gene function at a molecular level in bacteria. New plasmids carrying a kanamycin resistance gene or a trimethoprim resistance gene were constructed to provide antibiotic resistance cassettes for marker exchange mutagenesis in Ralstonia solanacearum and many antibiotic-resistant Burkholderia spp. Insertion sequences present in the flanking sequences of the antibiotic resistance cassette were removed to prevent aberrant gene replacement and polar mutation during mutagenesis in wild-type bacteria. Plasmids provided in this study would be convenient for use in gene cassettes for gene replacement in other Gram-negative bacteria.201121538255
926820.9998The expression of integron arrays is shaped by the translation rate of cassettes. Integrons are key elements in the rise and spread of multidrug resistance in Gram-negative bacteria. These genetic platforms capture cassettes containing promoterless genes and stockpile them in arrays of variable length. In the current integron model, expression of cassettes is granted by the P(c) promoter in the platform and is assumed to decrease as a function of its distance. Here we explored this model using a large collection of 136 antibiotic resistance cassettes and show the effect of distance is in fact negligible. Instead, cassettes have a strong impact in the expression of downstream genes because their translation rate affects the stability of the whole polycistronic mRNA molecule. Hence, cassettes with reduced translation rates decrease the expression and resistance phenotype of cassettes downstream. Our data puts forward an integron model in which expression is contingent on the translation of cassettes upstream, rather than on the distance to the P(c).202439455579
631430.9998Identification 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.19948080180
26140.9998Suicide 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.200616750581
631150.9998Development of an antibiotic marker-free platform for heterologous protein production in Streptomyces. BACKGROUND: The industrial use of enzymes produced by microorganisms is continuously growing due to the need for sustainable solutions. Nevertheless, many of the plasmids used for recombinant production of proteins in bacteria are based on the use of antibiotic resistance genes as selection markers. The safety concerns and legal requirements surrounding the increased use of antibiotic resistance genes have made the development of new antibiotic-free approaches essential. RESULTS: In this work, a system completely free of antibiotic resistance genes and useful for the production of high yields of proteins in Streptomyces is described. This system is based on the separation of the two components of the yefM/yoeBsl (antitoxin/toxin) operon; the toxin (yoeBsl) gene, responsible for host death, is integrated into the genome and the antitoxin gene (yefMsl), which inactivates the toxin, is located in the expression plasmid. To develop this system, the toxin gene was integrated into the genome of a strain lacking the complete operon, and the antibiotic resistance gene integrated along with the toxin was eliminated by Cre recombinase to generate a final host strain free of any antibiotic resistance marker. In the same way, the antibiotic resistance gene from the final expression plasmid was removed by Dre recombinase. The usefulness of this system was analysed by checking the production of two hydrolases from different Streptomyces. Production of both proteins, with potential industrial use, was high and stable over time after strain storage and after serial subcultures. These results support the robustness and stability of the positive selection system developed. CONCLUSIONS: The total absence of antibiotic resistance genes makes this system a powerful tool for using Streptomyces as a host to produce proteins at the industrial level. This work is the first Streptomyces antibiotic marker-free system to be described. Graphical abstract Antibiotic marker-free platform for protein expression in Streptomyces. The antitoxin gene present in the expression plasmid counteracts the effect of the toxin gene in the genome. In absence of the expression plasmid, the toxin causes cell death ensuring that only plasmid-containing cells persist.201728950904
932860.9998Man-made cell-like compartments for molecular evolution. Cellular compartmentalization is vital for the evolution of all living organisms. Cells keep together the genes, the RNAs and proteins that they encode, and the products of their activities, thus linking genotype to phenotype. We have reproduced this linkage in the test tube by transcribing and translating single genes in the aqueous compartments of water-in-oil emulsions. These compartments, with volumes close to those of bacteria, can be recruited to select genes encoding catalysts. A protein or RNA with a desired catalytic activity converts a substrate attached to the gene that encodes it to product. In other compartments, substrates attached to genes that do not encode catalysts remain unmodified. Subsequently, genes encoding catalysts are selectively enriched by virtue of their linkage to the product. We demonstrate the linkage of genotype to phenotype in man-made compartments using a model system. A selection for target-specific DNA methylation was based on the resistance of the product (methylated DNA) to restriction digestion. Genes encoding HaeIII methyltransferase were selected from a 10(7)-fold excess of genes encoding another enzyme.19989661199
930470.9998Variation of the flagellin gene locus of Campylobacter jejuni by recombination and horizontal gene transfer. The capacity of Campylobacter jejuni to generate genetic diversity was determined for its flagellar region. Recombination within a genome, as well as recombination after the uptake of exogenous DNA, could be demonstrated. The subunit of the flagellar filament of C. jejuni is encoded by two tandem genes, flaA and flaB, which are highly similar and therefore subject to recombination. A spontaneous recombination within this locus was demonstrated in a bacterial clone containing an antibiotic-resistance gene inserted in flaA. A recombinant was isolated in which the antibiotic-resistance gene had been repositioned into flaB, indicating that genetic information can be exchanged between the two flagellin genes of C. jejuni. The occurrence of recombinational events after the uptake of exogenous DNA by naturally competent bacteria was demonstrated with two mutants containing different antibiotic-resistance markers in their flagellin genes. Double-resistant transformants were formed when purified chromosomal donor DNA was added to a recipient strain, when the two bacterial cultures were mixed under conditions that induce natural competence, or when the two strains were cocultured. Both mechanisms of recombination may be used by the pathogenic organism to escape the immunological responses of the host or otherwise adapt to the environment.19957894725
631380.9998A 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.201829222103
630590.9997Antimicrobial genes from Allium sativum and Pinellia ternata revealed by a Bacillus subtilis expression system. Antimicrobial genes are found in all classes of life. To efficiently isolate these genes, we used Bacillus subtilis and Escherichia coli as target indicator bacteria and transformed them with cDNA libraries. Among thousands of expressed proteins, candidate proteins played antimicrobial roles from the inside of the indicator bacteria (internal effect), contributing to the sensitivity (much more sensitivity than the external effect from antimicrobial proteins working from outside of the cells) and the high throughput ability of screening. We found that B. subtilis is more efficient and reliable than E. coli. Using the B. subtilis expression system, we identified 19 novel, broad-spectrum antimicrobial genes. Proteins expressed by these genes were extracted and tested, exhibiting strong external antibacterial, antifungal and nematicidal activities. Furthermore, these newly isolated proteins could control plant diseases. Application of these proteins secreted by engineered B. subtilis in soil could inhibit the growth of pathogenic bacteria. These proteins are thermally stable and suitable for clinical medicine, as they exhibited no haemolytic activity. Based on our findings, we speculated that plant, animal and human pathogenic bacteria, fungi or even cancer cells might be taken as the indicator target cells for screening specific resistance genes.201830266995
3824100.9997Screening 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.201020830570
262110.9997Genome 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.199910438768
388120.9997Improved 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.19938406046
6335130.9997Gene Amplification Uncovers Large Previously Unrecognized Cryptic Antibiotic Resistance Potential in E. coli. The activation of unrecognized antibiotic resistance genes in the bacterial cell can give rise to antibiotic resistance without the need for major mutations or horizontal gene transfer. We hypothesize that bacteria harbor an extensive array of diverse cryptic genes that can be activated in response to antibiotics via adaptive resistance. To test this hypothesis, we developed a plasmid assay to randomly manipulate gene copy numbers in Escherichia coli cells and identify genes that conferred resistance when amplified. We then tested for cryptic resistance to 18 antibiotics and identified genes conferring resistance. E. coli could become resistant to 50% of the antibiotics tested, including chloramphenicol, d-cycloserine, polymyxin B, and 6 beta-lactam antibiotics, following this manipulation. Known antibiotic resistance genes comprised 13% of the total identified genes, where 87% were unclassified (cryptic) antibiotic resistance genes. These unclassified genes encoded cell membrane proteins, stress response/DNA repair proteins, transporters, and miscellaneous or hypothetical proteins. Stress response/DNA repair genes have a broad antibiotic resistance potential, as this gene class, in aggregate, conferred cryptic resistance to nearly all resistance-positive antibiotics. We found that antibiotics that are hydrophilic, those that are amphipathic, and those that inhibit the cytoplasmic membrane or cell wall biosynthesis were more likely to induce cryptic resistance in E. coli. This study reveals a diversity of cryptic genes that confer an antibiotic resistance phenotype when present in high copy number. Thus, our assay can identify potential novel resistance genes while also describing which antibiotics are prone to induce cryptic antibiotic resistance in E. coli. IMPORTANCE Predicting where new antibiotic resistance genes will rise is a challenge and is especially important when new antibiotics are developed. Adaptive resistance allows sensitive bacterial cells to become transiently resistant to antibiotics. This provides an opportune time for cells to develop more efficient resistance mechanisms, such as tolerance and permanent resistance to higher antibiotic concentrations. The biochemical diversity harbored within bacterial genomes may lead to the presence of genes that could confer resistance when timely activated. Therefore, it is crucial to understand adaptive resistance to identify potential resistance genes and prolong antibiotics. Here, we investigate cryptic resistance, an adaptive resistance mechanism, and identify unknown (cryptic) antibiotic resistance genes that confer resistance when amplified in a laboratory strain of E. coli. We also pinpoint antibiotic characteristics that are likely to induce cryptic resistance. This study may help detect novel antibiotic resistance genes and provide the foundation to help develop more effective antibiotics.202134756069
8387140.9997Construction and Analysis of Two Genome-Scale Deletion Libraries for Bacillus subtilis. A systems-level understanding of Gram-positive bacteria is important from both an environmental and health perspective and is most easily obtained when high-quality, validated genomic resources are available. To this end, we constructed two ordered, barcoded, erythromycin-resistance- and kanamycin-resistance-marked single-gene deletion libraries of the Gram-positive model organism, Bacillus subtilis. The libraries comprise 3,968 and 3,970 genes, respectively, and overlap in all but four genes. Using these libraries, we update the set of essential genes known for this organism, provide a comprehensive compendium of B. subtilis auxotrophic genes, and identify genes required for utilizing specific carbon and nitrogen sources, as well as those required for growth at low temperature. We report the identification of enzymes catalyzing several missing steps in amino acid biosynthesis. Finally, we describe a suite of high-throughput phenotyping methodologies and apply them to provide a genome-wide analysis of competence and sporulation. Altogether, we provide versatile resources for studying gene function and pathway and network architecture in Gram-positive bacteria.201728189581
6310150.9997Use of the lambda Red recombinase system to produce recombinant prophages carrying antibiotic resistance genes. BACKGROUND: The Red recombinase system of bacteriophage lambda has been used to inactivate chromosomal genes in E. coli K-12 through homologous recombination using linear PCR products. The aim of this study was to induce mutations in the genome of some temperate Shiga toxin encoding bacteriophages. When phage genes are in the prophage state, they behave like chromosomal genes. This enables marker genes, such as antibiotic resistance genes, to be incorporated into the stx gene. Once the phages' lytic cycle is activated, recombinant Shiga toxin converting phages are produced. These phages can transfer the marker genes to the bacteria that they infect and convert. As the Red system's effectiveness decreased when used for our purposes, we had to introduce significant variations to the original method. These modifications included: confirming the stability of the target stx gene increasing the number of cells to be transformed and using a three-step PCR method to produce the amplimer containing the antibiotic resistance gene. RESULTS: Seven phages carrying two different antibiotic resistance genes were derived from phages that are directly involved in the pathogenesis of Shiga toxin-producing strains, using this modified protocol. CONCLUSION: This approach facilitates exploration of the transduction processes and is a valuable tool for studying phage-mediated horizontal gene transfer.200616984631
9311160.9997Various plasmid strategies limit the effect of bacterial restriction-modification systems against conjugation. In bacteria, genes conferring antibiotic resistance are mostly carried on conjugative plasmids, mobile genetic elements that spread horizontally between bacterial hosts. Bacteria carry defence systems that defend them against genetic parasites, but how effective these are against plasmid conjugation is poorly understood. Here, we study to what extent restriction-modification (RM) systems-by far the most prevalent bacterial defence systems-act as a barrier against plasmids. Using 10 different RM systems and 13 natural plasmids conferring antibiotic resistance in Escherichia coli, we uncovered variation in defence efficiency ranging from none to 105-fold protection. Further analysis revealed genetic features of plasmids that explain the observed variation in defence levels. First, the number of RM recognition sites present on the plasmids generally correlates with defence levels, with higher numbers of sites being associated with stronger defence. Second, some plasmids encode methylases that protect against restriction activity. Finally, we show that a high number of plasmids in our collection encode anti-restriction genes that provide protection against several types of RM systems. Overall, our results show that it is common for plasmids to encode anti-RM strategies, and that, as a consequence, RM systems form only a weak barrier for plasmid transfer by conjugation.202439413206
9408170.9997Genomic evidence for antibiotic resistance genes of actinomycetes as origins of antibiotic resistance genes in pathogenic bacteria simply because actinomycetes are more ancestral than pathogenic bacteria. Although in silico analysis have suggested that the antibiotic resistance genes in actinomycetes appear to be the origins of some antibiotic resistance genes, we have shown that recent horizontal transfer of antibiotic resistance genes from actinomycetes to other medically important bacteria have not taken place. Although it has been speculated in Benveniste and Davies' attractive hypothesis that antibiotic resistance genes of actinomycetes are origins of antibiotic resistance genes in pathogenic bacteria because the actinomycetes require mechanisms such as metabolic enzymes (encoded by the antibiotic resistance genes) to degrade the antibiotics they produce or to transport the antibiotics outside the bacterial cells, this hypothesis has never been proven. Both the phylogenetic tree constructed using 16S rRNA gene sequences and that constructed using concatenated amino acid sequences of 15 housekeeping genes extracted from 90 bacterial genomes showed that the actinomycetes is more ancestral to most other bacteria, including the pathogenic Gram-negative bacteria, Gram-positive bacteria, and Chlamydia species. Furthermore, the tetracycline resistance gene of Bifidobacterium longum is more ancestral to those of other pathogenic bacteria and the actinomycetes, which is in line with the ancestral position of B. longum. These suggest that the evolution of antibiotic resistance genes of antibiotic-producing bacteria in general parallels the evolution of the corresponding bacteria. The ancestral position of the antibiotic resistance genes in actinomycetes is probably unrelated to the fact that they produce antibiotics, but simply because actinomycetes are more ancestral than pathogenic bacteria.200616824692
6334180.9997Epigenetic inheritance based evolution of antibiotic resistance in bacteria. BACKGROUND: The evolution of antibiotic resistance in bacteria is a topic of major medical importance. Evolution is the result of natural selection acting on variant phenotypes. Both the rigid base sequence of DNA and the more plastic expression patterns of the genes present define phenotype. RESULTS: We investigated the evolution of resistant E. coli when exposed to low concentrations of antibiotic. We show that within an isogenic population there are heritable variations in gene expression patterns, providing phenotypic diversity for antibiotic selection to act on. We studied resistance to three different antibiotics, ampicillin, tetracycline and nalidixic acid, which act by inhibiting cell wall synthesis, protein synthesis and DNA synthesis, respectively. In each case survival rates were too high to be accounted for by spontaneous DNA mutation. In addition, resistance levels could be ramped higher by successive exposures to increasing antibiotic concentrations. Furthermore, reversion rates to antibiotic sensitivity were extremely high, generally over 50%, consistent with an epigenetic inheritance mode of resistance. The gene expression patterns of the antibiotic resistant E. coli were characterized with microarrays. Candidate genes, whose altered expression might confer survival, were tested by driving constitutive overexpression and determining antibiotic resistance. Three categories of resistance genes were identified. The endogenous beta-lactamase gene represented a cryptic gene, normally inactive, but when by chance expressed capable of providing potent ampicillin resistance. The glutamate decarboxylase gene, in contrast, is normally expressed, but when overexpressed has the incidental capacity to give an increase in ampicillin resistance. And the DAM methylase gene is capable of regulating the expression of other genes, including multidrug efflux pumps. CONCLUSION: In this report we describe the evolution of antibiotic resistance in bacteria mediated by the epigenetic inheritance of variant gene expression patterns. This provides proof in principle that epigenetic inheritance, as well as DNA mutation, can drive evolution.200818282299
9276190.9997In Vitro Assessment of the Fitness of Resistant M. tuberculosis Bacteria by Competition Assay. Bacteria become resistant by a number of different mechanisms, and these include mutation in chromosomal genes (1), acquisition of plasmids (2), insertion of bacteriophage, transposon or insertion sequence DNA (3-5), or gene mosaicism (6). There is a dogma that bacteria that become resistant pay a significant physiological price and that if antimicrobial prescribing is controlled it will result in the eradication of resistant organisms. There are only very few studies that investigate the physiology of resistance acquisition and these do show that a physiological price is paid for this change (7, 8). Once an organism acquires resistance through mutation, acquisition of resistance genes via plasmids, transposons and bacteriophages the initial physiological defect is compensated by the antibiotic selective pressure, which balances the physiological deficit imposed by the resistant mutation or additional DNA (8, 9).200121374423