# | Rank | Similarity | Title + Abs. | Year | PMID |
|---|---|---|---|---|---|
| 0 | 1 | 2 | 3 | 4 | 5 |
| 376 | 0 | 0.9886 | Construction of a reporter plasmid for screening in vivo promoter activity in Francisella tularensis. Francisella tularensis is a facultative intracellular bacterium that survives and multiplies inside macrophages. Here we constructed a new promoter probe plasmid denoted pKK214 by introduction of a promoter-less chloramphenicol acetyltransferase (cat) gene into the shuttle vector pKK202. A promoter library was created in F. tularensis strain LVS by cloning random chromosomal DNA fragments into pKK214. Approximately 15% of the recombinant bacteria showed chloramphenicol resistance in vitro. The promoter library was also used to infect macrophages in the presence of chloramphenicol and after two cycles of infection the library contained essentially only chloramphenicol resistance clones which shows that pKK214 can be used to monitor F. tularensis genes that are expressed during infection. | 2001 | 11728719 |
| 3766 | 1 | 0.9885 | Identification of Genome-Wide Mutations in Ciprofloxacin-Resistant F. tularensis LVS Using Whole Genome Tiling Arrays and Next Generation Sequencing. Francisella tularensis is classified as a Class A bioterrorism agent by the U.S. government due to its high virulence and the ease with which it can be spread as an aerosol. It is a facultative intracellular pathogen and the causative agent of tularemia. Ciprofloxacin (Cipro) is a broad spectrum antibiotic effective against Gram-positive and Gram-negative bacteria. Increased Cipro resistance in pathogenic microbes is of serious concern when considering options for medical treatment of bacterial infections. Identification of genes and loci that are associated with Ciprofloxacin resistance will help advance the understanding of resistance mechanisms and may, in the future, provide better treatment options for patients. It may also provide information for development of assays that can rapidly identify Cipro-resistant isolates of this pathogen. In this study, we selected a large number of F. tularensis live vaccine strain (LVS) isolates that survived in progressively higher Ciprofloxacin concentrations, screened the isolates using a whole genome F. tularensis LVS tiling microarray and Illumina sequencing, and identified both known and novel mutations associated with resistance. Genes containing mutations encode DNA gyrase subunit A, a hypothetical protein, an asparagine synthase, a sugar transamine/perosamine synthetase and others. Structural modeling performed on these proteins provides insights into the potential function of these proteins and how they might contribute to Cipro resistance mechanisms. | 2016 | 27668749 |
| 382 | 2 | 0.9883 | Visualizing pneumococcal infections in the lungs of live mice using bioluminescent Streptococcus pneumoniae transformed with a novel gram-positive lux transposon. Animal studies with Streptococcus pneumoniae have provided valuable models for drug development. In order to monitor long-term pneumococcal infections noninvasively in living mice, a novel gram-positive lux transposon cassette, Tn4001 luxABCDE Km(r), that allows random integration of lux genes onto the bacterial chromosome was constructed. The cassette was designed so that the luxABCDE and kanamycin resistance genes were linked to form a single promoterless operon. Bioluminescence and kanamycin resistance only occur in a bacterial cell if this operon has transposed downstream of a promoter on the bacterium's chromosome. S. pneumoniae D39 was transformed with plasmid pAUL-A Tn4001 luxABCDE Km(r), and a number of highly bioluminescent colonies were recovered. Genomic DNA from the brightest D39 strain was used to transform a number of clinical S. pneumoniae isolates, and several of these strains were tested in animal models, including a pneumococcal lung infection model. Strong bioluminescent signals were seen in the lungs of the animals containing these pneumococci, allowing the course and antibiotic treatment of the infections to be readily monitored in real time in the living animals. Recovery of the bacteria from the animals showed that the bioluminescent signal corresponded to the number of CFU and that the lux construct was highly stable even after several days in vivo. We believe that this lux transposon will greatly expand the ability to evaluate drug efficacy against gram-positive bacteria in living animals using bioluminescence. | 2001 | 11292758 |
| 9737 | 3 | 0.9882 | Zinc Finger Nuclease: A New Approach to Overcome Beta-Lactam Antibiotic Resistance. BACKGROUND: The evolution of antibiotic-resistant bacteria (ARB) and antibiotic-resistance genes (ARGs) has been accelerated recently by the indiscriminate application of antibiotics. Antibiotic resistance has challenged the success of medical interventions and therefore is considered a hazardous threat to human health. OBJECTIVES: The present study aimed to describe the use of zinc finger nuclease (ZFN) technology to target and disrupt a plasmid-encoded β-lactamase, which prevents horizontal gene transfer-mediated evolution of ARBs. MATERIALS AND METHODS: An engineered ZFN was designed to target a specific sequence in the ampicillin resistance gene (amp(R)) of the pTZ57R plasmid. The Escherichia coli bacteria already contained the pZFN kanamycin-resistant (kana(R)) plasmid as the case or the pP15A, kana(R) empty vector as the control, were transformed with the pTZ57R; the ability of the designed ZFN to disrupt the β-lactamase gene was evaluated with the subsequent disturbed ability of the bacteria to grow on ampicillin (amp) and ampicillin-kanamycin (amp-kana)-containing media. The effect of mild hypothermia on the ZFN gene targeting efficiency was also evaluated. RESULTS: The growth of bacteria in the case group on the amp and amp-kana-containing media was significantly lower compared with the control group at 37°C (P < 0.001). Despite being more efficient in hypothermic conditions at 30°C (P < 0.001), there were no significant associations between the incubation temperature and the ZFN gene targeting efficiency. CONCLUSIONS: Our findings revealed that the ZFN technology could be employed to overcome ampicillin resistance by the targeted disruption of the ampicillin resistance gene, which leads to inactivation of β-lactam synthesis. Therefore, ZFN technology could be engaged to decrease the antibiotic resistance issue with the construction of a ZFN archive against different ARGs. To tackle the resistance issue at the environmental level, recombinant phages expressing ZFNs against different ARGs could be constructed and released into both hospital and urban wastewater systems. | 2016 | 27099691 |
| 9066 | 4 | 0.9881 | VRprofile: gene-cluster-detection-based profiling of virulence and antibiotic resistance traits encoded within genome sequences of pathogenic bacteria. VRprofile is a Web server that facilitates rapid investigation of virulence and antibiotic resistance genes, as well as extends these trait transfer-related genetic contexts, in newly sequenced pathogenic bacterial genomes. The used backend database MobilomeDB was firstly built on sets of known gene cluster loci of bacterial type III/IV/VI/VII secretion systems and mobile genetic elements, including integrative and conjugative elements, prophages, class I integrons, IS elements and pathogenicity/antibiotic resistance islands. VRprofile is thus able to co-localize the homologs of these conserved gene clusters using HMMer or BLASTp searches. With the integration of the homologous gene cluster search module with a sequence composition module, VRprofile has exhibited better performance for island-like region predictions than the other widely used methods. In addition, VRprofile also provides an integrated Web interface for aligning and visualizing identified gene clusters with MobilomeDB-archived gene clusters, or a variety set of bacterial genomes. VRprofile might contribute to meet the increasing demands of re-annotations of bacterial variable regions, and aid in the real-time definitions of disease-relevant gene clusters in pathogenic bacteria of interest. VRprofile is freely available at http://bioinfo-mml.sjtu.edu.cn/VRprofile. | 2018 | 28077405 |
| 258 | 5 | 0.9881 | An efficient system for the generation of marked genetic mutants in members of the genus Burkholderia. To elucidate the function of a gene in bacteria it is vital that targeted gene inactivation (allelic replacement) can be achieved. Allelic replacement is often carried out by disruption of the gene of interest by insertion of an antibiotic-resistance marker followed by subsequent transfer of the mutant allele to the genome of the host organism in place of the wild-type gene. However, due to their intrinsic resistance to many antibiotics only selected antibiotic-resistance markers can be used in members of the genus Burkholderia, including the Burkholderia cepacia complex (Bcc). Here we describe the construction of improved antibiotic-resistance cassettes that specify resistance to kanamycin, chloramphenicol or trimethoprim effectively in the Bcc and related species. These were then used in combination with and/or to construct a series enhanced suicide vectors, pSHAFT2, pSHAFT3 and pSHAFT-GFP to facilitate effective allelic replacement in the Bcc. Validation of these improved suicide vectors was demonstrated by the genetic inactivation of selected genes in the Bcc species Burkholderia cenocepacia and B. lata, and in the non-Bcc species, B. thailandensis. | 2017 | 27825973 |
| 349 | 6 | 0.9879 | Mini-Tn5 transposon derivatives for insertion mutagenesis, promoter probing, and chromosomal insertion of cloned DNA in gram-negative eubacteria. A collection of Tn5-derived minitransposons has been constructed that simplifies substantially the generation of insertion mutants, in vivo fusions with reporter genes, and the introduction of foreign DNA fragments into the chromosome of a variety of gram-negative bacteria, including the enteric bacteria and typical soil bacteria like Pseudomonas species. The minitransposons consist of genes specifying resistance to kanamycin, chloramphenicol, streptomycin-spectinomycin, and tetracycline as selection markers and a unique NotI cloning site flanked by 19-base-pair terminal repeat sequences of Tn5. Further derivatives also contain lacZ, phoA, luxAB, or xylE genes devoid of their native promoters located next to the terminal repeats in an orientation that affords the generation of gene-operon fusions. The transposons are located on a R6K-based suicide delivery plasmid that provides the IS50R transposase tnp gene in cis but external to the mobile element and whose conjugal transfer to recipients is mediated by RP4 mobilization functions in the donor. | 1990 | 2172217 |
| 378 | 7 | 0.9878 | Construction and use of a self-cloning promoter probe vector for gram-negative bacteria. Transposon Tn5 has been used extensively for the genetic analysis of Gram- bacteria. We describe here the construction and use of a Tn5 derivative which contains the ColE1 origin of DNA replication, thereby allowing the cloning of DNA adjacent to the Tn without the need for construction of genomic libraries. The Tn is derived from Tn5-B21 [Simon et al., Gene 80 (1989) 161-169] and contains a promoter-probe lacZ gene and genes encoding resistance to tetracycline and beta-lactams. It is housed within a mobilisable suicide plasmid which can be transferred to a wide range of Gram- bacteria. The Tn was tested using pyoverdine siderophore-synthesis genes (pvd) from Pseudomonas aeruginosa. The simple cloning procedure allowed 15.9 kb of pvd-associated DNA to be cloned; in addition, the lacZ reporter gene allowed the transcription of pvd genes to be studied. The bacteria were resistant to carbenicillin only if the Tn (and hence the beta-lactamase-encoding gene) was downstream from an active promoter. | 1993 | 8386128 |
| 9228 | 8 | 0.9878 | In Situ Cas12a-Based Allele-Specific PCR for Imaging Single-Nucleotide Variations in Foodborne Pathogenic Bacteria. In situ profiling of single-nucleotide variations (SNVs) can elucidate drug-resistant genotypes with single-cell resolution. The capacity to directly "see" genetic information is crucial for investigating the relationship between mutated genes and phenotypes. Fluorescence in situ hybridization serves as a canonical tool for genetic imaging; however, it cannot detect subtle sequence alteration including SNVs. Herein, we develop an in situ Cas12a-based amplification refractory mutation system-PCR (ARMS-PCR) method that allows the visualization of SNVs related to quinolone resistance inside cells. The capacity of discriminating SNVs is enhanced by incorporating optimized mismatched bases in the allele-specific primers, thus allowing to specifically amplify quinolone-resistant related genes. After in situ ARMS-PCR, we employed a modified Cas12a/CRISPR RNA to tag the amplicon, thereby enabling specific binding of fluorophore-labeled DNA probes. The method allows to precisely quantify quinolone-resistant Salmonella enterica in the bacterial mixture. Utilizing this method, we investigated the survival competition capacity of quinolone-resistant and quinolone-sensitive bacteria toward antimicrobial peptides and indicated the enrichment of quinolone-resistant bacteria under colistin sulfate stress. The in situ Cas12a-based ARMS-PCR method holds the potential for profiling cellular phenotypes and gene regulation with single-nucleotide resolution at the single-cell level. | 2024 | 38277772 |
| 8458 | 9 | 0.9878 | A PCR-based approach for isolating pathogen resistance genes from potato with potential for wide application in plants. Plant genes for pathogen resistance can be used to engineer disease resistant crops. Oligonucleotides were designed from sequence motifs conserved between resistance genes of tobacco and Arabidopsis thaliana and used as PCR primers in potato DNA. Amplification products were obtained that were homologous to known resistance genes and linked without recombination with the nematode resistance locus Gro1 and the Phytophthora infestans resistance locus R7 of potato. Map positions of PCR-derived potato gene fragments were also correlated with resistance loci of the related tomato and tobacco genomes. Our results indicate that plant resistance genes that are effective against nematodes, fungi, viruses and bacteria may be isolated based on common sequence motifs and PCR methodology. | 1996 | 8944022 |
| 5121 | 10 | 0.9877 | Rapid Nanopore Whole-Genome Sequencing for Anthrax Emergency Preparedness. Human anthrax cases necessitate rapid response. We completed Bacillus anthracis nanopore whole-genome sequencing in our high-containment laboratory from a human anthrax isolate hours after receipt. The de novo assembled genome showed no evidence of known antimicrobial resistance genes or introduced plasmid(s). Same-day genomic characterization enhances public health emergency response. | 2020 | 31961318 |
| 351 | 11 | 0.9877 | Rapid and efficient cloning of proviral flanking fragments by kanamycin resistance gene complementation. We have developed a technique for the rapid cloning of unknown flanking regions of transgenic DNA. We complemented a truncated kanamycin resistance gene of a bacterial plasmid with a neomycin resistance gene fragment from a gene transfer vector. Optimized transformation conditions allowed us to directly select for kanamycin-resistant bacteria. We cloned numerous proviral flanking fragments from growth factor-independent cell mutants that were obtained after infection with a replication incompetent retroviral vector and identified integrations into the cyclin D2 and several unknown genomic sequences. We anticipate that our method could be adapted to various vector systems that are used to tag and identify genes and to map genomes. | 1999 | 9863001 |
| 354 | 12 | 0.9877 | New cloning vectors to facilitate quick allelic exchange in gram-negative bacteria. New cloning vectors have been developed with features to enhance quick allelic exchange in gram-negative bacteria. The conditionally replicative R6K and transfer origins facilitate conjugation and chromosomal integration into a variety of bacterial species, whereas the sacB gene provides counterselection for allelic exchange. The vectors have incorporated the lacZ alpha fragment with an enhanced multicloning site for easy blue/white screening and priming sites identified for efficient in vivo assembly or other DNA assembly cloning techniques. Different antibiotic resistance markers allow versatility for use with different bacteria, and transformation into an Escherichia coli strain capable of conjugation enables a quick method for allelic exchange. As a proof of principle, the authors used these vectors to inactivate genes in Vibrio cholerae and Salmonella typhimurium. | 2021 | 33492170 |
| 375 | 13 | 0.9877 | A mariner transposon vector adapted for mutagenesis in oral streptococci. This article describes the construction and characterization of a mariner-based transposon vector designed for use in oral streptococci, but with a potential use in other Gram-positive bacteria. The new transposon vector, termed pMN100, contains the temperature-sensitive origin of replication repATs-pWV01, a selectable kanamycin resistance gene, a Himar1 transposase gene regulated by a xylose-inducible promoter, and an erythromycin resistance gene flanked by himar inverted repeats. The pMN100 plasmid was transformed into Streptococcus mutans UA159 and transposon mutagenesis was performed via a protocol established to perform high numbers of separate transpositions despite a low frequency of transposition. The distribution of transposon inserts in 30 randomly picked mutants suggested that mariner transposon mutagenesis is unbiased in S. mutans. A generated transposon mutant library containing 5000 mutants was used in a screen to identify genes involved in the production of sucrose-dependent extracellular matrix components. Mutants with transposon inserts in genes encoding glycosyltransferases and the competence-related secretory locus were predominantly found in this screen. | 2014 | 24753509 |
| 5464 | 14 | 0.9877 | Genomic and resistome analysis of Alcaligenes faecalis strain PGB1 by Nanopore MinION and Illumina Technologies. BACKGROUND: Drug-resistant bacteria are important carriers of antibiotic-resistant genes (ARGs). This fact is crucial for the development of precise clinical drug treatment strategies. Long-read sequencing platforms such as the Oxford Nanopore sequencer can improve genome assembly efficiency particularly when they are combined with short-read sequencing data. RESULTS: Alcaligenes faecalis PGB1 was isolated and identified with resistance to penicillin and three other antibiotics. After being sequenced by Nanopore MinION and Illumina sequencer, its entire genome was hybrid-assembled. One chromosome and one plasmid was assembled and annotated with 4,433 genes (including 91 RNA genes). Function annotation and comparison between strains were performed. A phylogenetic analysis revealed that it was closest to A. faecalis ZD02. Resistome related sequences was explored, including ARGs, Insert sequence, phage. Two plasmid aminoglycoside genes were determined to be acquired ARGs. The main ARG category was antibiotic efflux resistance and β-lactamase (EC 3.5.2.6) of PGB1 was assigned to Class A, Subclass A1b, and Cluster LSBL3. CONCLUSIONS: The present study identified the newly isolated bacterium A. faecalis PGB1 and systematically annotated its genome sequence and ARGs. | 2022 | 35443609 |
| 8453 | 15 | 0.9876 | In silico analysis of gene content in tomato genomic regions mapped to the Ty-2 resistance gene. Tomato yellow leaf curl virus is one of the main diseases affecting tomato production worldwide. Previous studies have shown that Ty-2 is an important resistance gene located between molecular markers C2_At2g28250 (82.3 cM) and T0302 (89.0 cM), and exhibits strong resistance to tomato yellow leaf curl virus in Asia. In this study, Ty-2 candidate genes were subjected to bioinformatic analysis for the sequenced tomato genome. We identified 69 genes between molecular markers C2_At2g28250 and T0302, 22 of which were disease-related resistant genes, including nucleotide binding site-leucine-rich repeat disease resistance genes, protease genes (protein kinase, kinase receptor, and protein isomerase), cytochromes, and transcription factors. Expressed sequence tag analysis revealed that 77.3% (17/22) of candidate disease-resistance genes were expressed, involving 143 expressed sequence tags. Based on full-length cDNA sequence analysis, 7 candidate genes were found, 4 of which were involved in tomato responses to pathogens. Microarray expression analysis also showed that most candidate genes were involved in the tomato responses to multiple pathogens, including fungi, viruses, and bacteria. RNA-seq expression analysis revealed that all candidate genes participated in tomato growth and development. | 2015 | 26214476 |
| 377 | 16 | 0.9876 | Construction of improved plasmid vectors for promoter characterization in Pseudomonas aeruginosa and other gram-negative bacteria. We report the construction of two broad host range promoter-probe plasmid vectors for rapid analysis of promoters in Gram-negative bacteria. The new vectors, pME4507 and pME4510, carry carbenicillin and gentamycin resistance genes, respectively, and are small sized (4 kb) with a flexible multiple cloning site to facilitate directional cloning of putative promoter elements. The vectors allow rapid plate-based screening for promoter activities, using beta-galactosidase as the reporter enzyme. In the absence of an inserted promoter fragment, they display very low background activity, making them a useful tool for analysis of low expression level promoters. | 1998 | 9851050 |
| 450 | 17 | 0.9876 | One-step inactivation of chromosomal genes in Escherichia coli K-12 using PCR products. We have developed a simple and highly efficient method to disrupt chromosomal genes in Escherichia coli in which PCR primers provide the homology to the targeted gene(s). In this procedure, recombination requires the phage lambda Red recombinase, which is synthesized under the control of an inducible promoter on an easily curable, low copy number plasmid. To demonstrate the utility of this approach, we generated PCR products by using primers with 36- to 50-nt extensions that are homologous to regions adjacent to the gene to be inactivated and template plasmids carrying antibiotic resistance genes that are flanked by FRT (FLP recognition target) sites. By using the respective PCR products, we made 13 different disruptions of chromosomal genes. Mutants of the arcB, cyaA, lacZYA, ompR-envZ, phnR, pstB, pstCA, pstS, pstSCAB-phoU, recA, and torSTRCAD genes or operons were isolated as antibiotic-resistant colonies after the introduction into bacteria carrying a Red expression plasmid of synthetic (PCR-generated) DNA. The resistance genes were then eliminated by using a helper plasmid encoding the FLP recombinase which is also easily curable. This procedure should be widely useful, especially in genome analysis of E. coli and other bacteria because the procedure can be done in wild-type cells. | 2000 | 10829079 |
| 4778 | 18 | 0.9875 | DNA extraction of microbial DNA directly from infected tissue: an optimized protocol for use in nanopore sequencing. Identification of bacteria causing tissue infections can be comprehensive and, in the cases of non- or slow-growing bacteria, near impossible with conventional methods. Performing shotgun metagenomic sequencing on bacterial DNA extracted directly from the infected tissue may improve time to diagnosis and targeted treatment considerably. However, infected tissue consists mainly of human DNA (hDNA) which hampers bacterial identification. In this proof of concept study, we present a modified version of the Ultra-Deep Microbiome Prep kit for DNA extraction procedure, removing additional human DNA. Tissue biopsies from 3 patients with orthopedic implant-related infections containing varying degrees of Staphylococcus aureus were included. Subsequent DNA shotgun metagenomic sequencing using Oxford Nanopore Technologies' (ONT) MinION platform and ONTs EPI2ME WIMP and ARMA bioinformatic workflows for microbe and antibiotic resistance genes identification, respectively. The modified DNA extraction protocol led to an additional ~10-fold reduction of human DNA while preserving S. aureus DNA. Including the DNA sequencing and bioinformatics analyses, the presented protocol has the potential of identifying the infection-causing pathogen in infected tissue within 7 hours after biopsy. However, due to low number of S. aureus reads, positive identification of antibiotic resistance genes was not possible. | 2020 | 32076089 |
| 5131 | 19 | 0.9875 | Conjugative Transfer of the pVA1-Type Plasmid Carrying the pirAB(vp) Genes Results in the Formation of New AHPND-Causing Vibrio. Acute hepatopancreatic necrosis disease (AHPND) has caused sharp declines in aquaculture industries of whiteleg shrimp Penaeus vannamei in Asia and the Americas since 2010. Vibrio parahaemolyticus, V. campbellii, V. owensii, and V. punensis have been proved to cause AHPND. However, the mechanisms underlying the burgeoning number of Vibrio species that cause AHPND is not known. All of AHPND-causing Vibrio bacteria (V(AHPND)) harbor a highly homologous plasmid (designated as pVA1-type) carrying pirAB(vp) toxin genes. In this study, we demonstrate conclusively that the pVA1-type plasmid can be transferred from V(AHPND) to non-pathogenic bacteria. We constructed a pVPGX1-Cm(r) plasmid (a pVA1-type plasmid) by adding a chloramphenicol resistance gene as a marker in a donor AHPND-causing V. parahaemolyticus 20130629002S01 (Vp2S01). Horizontal transfer of this plasmid was successfully performed from the AHPND-Vp2S01 to a non-pathogenic strain of V. campbellii at the transfer efficiency of 2.6×10(-8) transconjugant/recipient, and DNase I treatment did not eliminate the transfer. The recipient V. campbellii acquired the pVA1-type plasmid and was shown to produce pirAB(vp) RNA and proteins. Challenge studies using the transconjugant caused 100% mortality in exposed groups of P. vannamei. The challenged shrimp, infected with the transconjugant bacteria, showed typical gross signs and histological lesions of AHPND. These results demonstrated the conjugative transfer of an AHPND pVA1-type plasmid. It provides timely information for explaining the increased species of AHPND-causing Vibrio bacteria and will be useful in the development of management strategies leading to the prevention and control of AHPND. | 2019 | 31231618 |