Genetic characteristics of bla NDM-1-positive plasmid in Citrobacter freundii isolate separated from a clinical infectious patient

2013 ◽  
Vol 62 (9) ◽  
pp. 1332-1337 ◽  
Author(s):  
Xiao-Xing Du ◽  
Jian-Feng Wang ◽  
Ying Fu ◽  
Feng Zhao ◽  
Yan Chen ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Variable Region ◽  
Southern Blot Hybridization ◽  
Potential Threat ◽  
High Sequence Similarity ◽  
S1 Nuclease ◽  
Genetic Characteristics ◽  
Clinical Patient

This study reports an infectious case involving an NDM-1-producing Citrobacter freundii and further explored the potential threat of the bla NDM-1 gene by analysing the characteristics of the NDM-1-encoding plasmid sequence. A bla NDM-1-positive C. freundii with high resistance to carbapenems was separated from a clinical patient suffering from a urinary tract infection. S1 nuclease-based plasmid analysis followed by Southern blot hybridization, a conjugation experiment and electrotransformation confirmed that the bla NDM-1 gene was located on a plasmid. High-throughput sequencing of the bla NDM-1-postive plasmid (pCFNDM-CN) showed that it was a 54 kb IncX-type plasmid and contained a backbone region and a variable region with two β-lactamase genes (bla NDM-1 and bla SHV-12). The NDM-1 composite transposon in the variable region was surrounded by IS26 and IS5-truncated ISAba125, and shared a high sequence similarity to the bla NDM-1 surrounding structure in Acinetobacter spp. Our research suggested that the NDM-1 composite transposon might play an essential role in mobilization of the bla NDM-1 gene from Acinetobacter spp. to Enterobacteriaceae.

scholarly journals Cloning, Sequencing, and Expression of the Leukotoxin Gene from Fusobacterium necrophorum†

2001 ◽  
Vol 69 (9) ◽  
pp. 5447-5455 ◽  
Author(s):  
Sanjeev Kumar Narayanan ◽  
T. G. Nagaraja ◽  
M. M. Chengappa ◽  
George C. Stewart
Keyword(s):  
Molecular Weight ◽  
Western Blot ◽  
Sequence Similarity ◽  
Blot Hybridization ◽  
Flow Cytometric Analysis ◽  
Southern Blot Hybridization ◽  
Fusobacterium Necrophorum ◽  
Inverse Pcr ◽  
Western Blot Assay ◽  
Polyclonal Antisera

ABSTRACT Fusobacterium necrophorum is a gram-negative, rod-shaped, anaerobic bacterium that is a primary or secondary etiological agent in a variety of necrotic purulent infections in animals and humans. Included are diseases of cattle such as liver abscesses and foot rot, which have economically important consequences for the cattle industry. The major virulence factor of this bacterium is leukotoxin, a secreted protein of high molecular weight active against leukocytes from ruminants. The screening of a genomic DNA library with polyclonal antisera raised against native affinity-purified leukotoxin and further extension of the sequence using inverse PCR led to the cloning of the entire leukotoxin gene. The leukotoxin gene open reading frame (ORF; lktA) consists of 9,726 bp and encodes a protein of 3,241 amino acids with an overall molecular weight of 335,956. The leukotoxin does not have sequence similarity with any other bacterial leukotoxin. Five truncated overlapping polypeptides covering the wholelktA ORF were used to immunize rabbits. In Western blot assays, polyclonal antisera raised against all five truncated polypeptides recognized affinity-purified leukotoxin fromF. necrophorum culture supernatant in a Western blot assay. Antisera directed against two of the five polypeptides had neutralizing activity against the toxin. The entire leukotoxin ORF was expressed in Escherichia coli. Flow-cytometric analysis showed that the recombinant leukotoxin was active against bovine polymorphonuclear leukocytes and was inhibited with antiserum raised against the F. necrophorum leukotoxin. Southern blot hybridization analysis revealed different patterns of lktAhybridizing bands between isolates of the two subspecies ofF. necrophorum.

scholarly journals An Oligoribonuclease Gene inStreptomyces griseus

2000 ◽  
Vol 182 (16) ◽  
pp. 4647-4653 ◽  
Author(s):  
Yasuo Ohnishi ◽  
Yoko Nishiyama ◽  
Rie Sato ◽  
Shogo Kameyama ◽  
Sueharu Horinouchi
Keyword(s):  
Sequence Similarity ◽  
Streptomyces Griseus ◽  
Aerial Mycelium ◽  
Morphological Development ◽  
Developmentally Regulated ◽  
High Sequence Similarity ◽  
S1 Nuclease ◽  
Reading Frame ◽  
Reverse Transcription Pcr ◽  
Nuclease Mapping

ABSTRACT In Streptomyces griseus, A-factor (2-isocapryloyl-3R-hydroxymethyl-γ-butyrolactone) serves as a microbial hormone that switches on many genes required for streptomycin production and morphological development. An open reading frame (Orf1) showing high sequence similarity to oligoribonucleases of various origins is present just downstream of adpA, one of the A-factor-dependent genes. Orf1 was named OrnA (oligoribonuclease A) because it showed 3′-to-5′ exo-oligoribonuclease activity, releasing [32P]CMP from ApCpC[32P]pC used as a substrate. Reverse transcription-PCR and S1 nuclease mapping analyses revealed that ornA was transcribed from two promoters; one was a developmentally regulated, A-factor-dependent promoter in front of adpA, and the other was a constitutive promoter in front of the ornA coding sequence. Transcription ofornA was thus additively enhanced at the initiation stage for secondary metabolism and aerial mycelium formation.ornA-disrupted strains grew slowly and scarcely formed aerial mycelium. ornA homologues were distributed in a wide variety of Streptomyces species, including S. coelicolor A3(2), as determined by Southern hybridization analysis. Disruption of the ornA homologue in S. coelicolor A3(2) also caused phenotypes similar to those of theS. griseus ΔornA strains. The OrnA oligoribonucleases inStreptomyces species are therefore not essential but play an important role in vegetative growth and in the initiation of differentiation.

scholarly journals Class 1 integron element in Thai Acinetobacter baumannii reveals a linkage to the European clone I

2012 ◽  
Vol 1 (1) ◽  
pp. 24-28
Author(s):  
B Thapa ◽  
C Tribuddharat ◽  
S Srifuengfung ◽  
C Dhiraputra
Keyword(s):  
Acinetobacter Baumannii ◽  
Blot Hybridization ◽  
Variable Region ◽  
Southern Blot Hybridization ◽  
Multidrug Resistant ◽  
Dot Blot ◽  
Class 1 Integron ◽  
Integrase Gene ◽  
Class 1 ◽  
Resistance Island

BACKGROUND: Class 1 integron element is innate to most of the multidrug resistant Acinetobacter baumannii and its spread is common among international clones worldwide. The aim of this study was to document the presence of blaVEB-1 harboring class 1 integron element and its gene cassettes in Thai A. baumannii in relation to A. baumannii European clone I, AYE strain. MATERIALS AND METHODS: Thirty seven carbapenem resistant A. baumannii isolates identified in routine microbiology laboratory of Siriraj Hospital, Bangkok were studied. The dot blot hybridization was performed to detect class 1 integron element integrase gene. PCR was used to amplify blaVEB-1, arr2, cmlA, blaOXA-10 resistance cassettes, and variable region of class 1 integron element. blaVEB-1 gene was localized by southern blot hybridization. RESULTS: The prevalence of class 1 integron element was 86.48% in the isolates studied. The blaVEB-1 was present in 7 isolates however the location of blaVEB-1 gene was different in different isolate. Four isolates (Ab03-168, Ab04-28, Ab08-20, and Ab08-22) harbored calss 1 integron element variable region sized 5.5 kb as described in strain AYE. However, blaVEB-1 was only amplified from Ab03-168. The cassette organization in this isolate was 5’CS-aadB-blaVEB-1-arr2-cmlA-blaOXA- 10-aadA1-3’CS. The class 1 integron element similar to the element identified in genomic resistance island, AbaRI of European clone I, AYE was identified in Thai A. baumannii. CONCLUSIONS: blaVEB-1 harboring class 1 integron element with minor cassette variation was identified in Thai A. baumanni isolate which might suggest the spread of this resistant cassette or the spread of the European clone I in Thailand. Monitoring of the global spread of multi-resistant A. baumannii is mandatory to control the spread of resistant genes and this multi-resistant pathogen. DOI: http://dx.doi.org/10.3126/ijim.v1i1.6715Int J Infect Microbiol 2012;1(1):24-28

scholarly journals Characterization of RNA content in individual phase-separated coacervate microdroplets

2021 ◽  
Author(s):  
Damian Wollny ◽  
Benjamin Vernot ◽  
Jie Wang ◽  
Maria Hondele ◽  
Anthony Hyman ◽  
...  
Keyword(s):  
High Throughput Sequencing ◽  
Rna World ◽  
Sequence Similarity ◽  
Central Component ◽  
Sequence Motifs ◽  
High Sequence Similarity ◽  
Specific Sequence ◽  
Rna Content ◽  
Wide Range

AbstractLiquid-liquid phase separation or condensation is a form of macromolecular compartmentalization. Condensates formed by complex coacervation were hypothesized to have played a crucial part during the origin-of-life. In living cells, condensation organizes biomolecules into a wide range of membraneless compartments. Although RNA is a key component of condensation in cells and the central component of the RNA world hypothesis, little is known about what determines RNA accumulation in condensates and how single condensates differ in their RNA composition. Therefore, we developed an approach to read the RNA content from single condensates using high-throughput sequencing. We find that RNAs which are enriched for specific sequence motifs efficiently accumulate in condensates. These motifs show high sequence similarity to short interspersed elements (SINEs). We observed similar results for protein-derived condensates, demonstrating applicability across different in vitro reconstituted membraneless organelles. Thus, our results provide a new inroad to explore the RNA content of phase-separated droplets at single condensate resolution.

scholarly journals Genetic Diversity, Symbiotic Evolution, and Proposed Infection Process of Bradyrhizobium Strains Isolated from Root Nodules of Aeschynomene americana L. in Thailand

2012 ◽  
Vol 78 (17) ◽  
pp. 6236-6250 ◽  
Author(s):  
Rujirek Noisangiam ◽  
Kamonluck Teamtisong ◽  
Panlada Tittabutr ◽  
Nantakorn Boonkerd ◽  
Uchiumi Toshiki ◽  
...  
Keyword(s):  
Sequence Similarity ◽  
Root Nodules ◽  
Blot Hybridization ◽  
Pcr Amplification ◽  
Southern Blot Hybridization ◽  
Housekeeping Genes ◽  
Infection Process ◽  
Rrna Gene ◽  
Content Type ◽  
Group 3

ABSTRACTThe diversity of bacteria nodulatingAeschynomene americanaL. in Thailand was determined from phenotypic characteristics and multilocus sequence analysis of the 16S rRNA gene and 3 housekeeping genes (dnaK,recA, andglnB). The isolated strains were nonphotosynthetic bacteria and were assigned to the genusBradyrhizobium, in whichB. yuanmingensewas the dominant species. Some of the other species, includingB. japonicum,B. liaoningense, andB. canariense, were minor species. These isolated strains were divided into 2 groups—nod-containing and divergentnod-containing strains—based on Southern blot hybridization and PCR amplification ofnodABCgenes. The divergentnodgenes could not be PCR amplified and failed to hybridizenodgene probes designed fromB. japonicumUSDA110, but hybridized to probes from other bradyrhizobial strains under low-stringency conditions. The grouping based on sequence similarity ofnodgenes was well correlated with the grouping based on that ofnifHgene, in which thenod-containing and divergentnod-containing strains were obviously distinguished. The divergentnod-containing strains and photosynthetic bradyrhizobia shared closenifHsequence similarity and an ability to fix nitrogen in the free-living state. Surprisingly, the strains isolated fromA. americanacould nodulateAeschynomeneplants that belong to different cross-inoculation (CI) groups, includingA. afrasperaandA. indica. This is the first discovery of bradyrhizobia (nonphotosynthetic andnod-containing strain) originating from CI group 1 nodulating roots ofA. indica(CI group 3). An infection process used to establish symbiosis onAeschynomenedifferent from the classical one is proposed.

scholarly journals Dissemination ofblaOXA-23in Acinetobacter spp. in China: Main Roles of Conjugative Plasmid pAZJ221 and Transposon Tn2009

2015 ◽  
Vol 59 (4) ◽  
pp. 1998-2005 ◽  
Author(s):  
Li-Lin Liu ◽  
Shu-Juan Ji ◽  
Zhi Ruan ◽  
Ying Fu ◽  
Yi-Qi Fu ◽  
...  
Keyword(s):  
Blot Hybridization ◽  
Carbapenem Resistance ◽  
Southern Blot Hybridization ◽  
Conjugative Plasmid ◽  
S1 Nuclease ◽  
Content Type ◽  
Carbapenem Resistant ◽  
Copy Numbers ◽  
Ca 50 ◽  
Genomic Locations

ABSTRACTProduction of the OXA-23 carbapenemase is the most common reason for the increasing carbapenem resistance inAcinetobacterspp. This study was conducted to reveal the genetic basis ofblaOXA-23dissemination inAcinetobacterspp. in China. A total of 63 carbapenem-resistant OXA-23-producingAcinetobactersp. isolates, representing different backgrounds, were selected from 28 hospitals in 18 provinces for this study. Generally, two patterns of plasmids carryingblaOXA-23were detected according to S1-nuclease pulsed-field gel electrophoresis and Southern blot hybridization. A ca. 78-kb plasmid, designated pAZJ221, was found in 23Acinetobacter baumanniiand threeAcinetobacter nosocomialisisolates, while a novel ca. 50-kb plasmid was carried by only two otherA. baumanniiisolates. Three of these isolates had an additional copy ofblaOXA-23on the chromosome. Transformation of the two plasmids succeeded, but only pAZJ221 was conjugative. Plasmid pAZJ221 was sequenced completely and found to carry no previously known resistance genes exceptblaOXA-23. TheblaOXA-23gene of the remaining 35 isolates was chromosome borne. TheblaOXA-23genetic environments were correlated with Tn2009in 57 isolates, Tn2008in 5 isolates, and Tn2006in 1 isolate. The MIC values for the carbapenems with these isolates were not significantly associated with the genomic locations or the copy numbers ofblaOXA-23. Overall, these observations suggest that the plasmid pAZJ221 and Tn2009have effectively contributed to the wide dissemination ofblaOXA-23inAcinetobacterspp. in China and that horizontal gene transfer may play an important role in dissemination of theblaOXA-23gene.

Analysis of genetic polymorphisms affecting the four phospholipase C (plc) genes in Mycobacterium tuberculosis complex clinical isolates

Microbiology ◽  
2004 ◽  
Vol 150 (4) ◽  
pp. 967-978 ◽  
Author(s):  
C. Viana-Niero ◽  
P. E. de Haas ◽  
D. van Soolingen ◽  
S. C. Leão
Keyword(s):  
Mycobacterium Tuberculosis ◽  
Phospholipase C ◽  
Genetic Polymorphisms ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Clinical Isolates ◽  
Significant Similarity ◽  
Genomic Deletions ◽  
Genes Encoding ◽  
Mycobacterium Africanum

The Mycobacterium tuberculosis genome contains four highly related genes which present significant similarity to Pseudomonas aeruginosa genes encoding phospholipase C enzymes. Three of these genes, plcA, plcB and plcC, are organized in tandem (locus plcABC). The fourth gene, plcD, is located in a different region. This study investigates variations in plcABC and plcD genes in clinical isolates of M. tuberculosis, Mycobacterium africanum and ‘Mycobacterium canettii’. Genetic polymorphisms were examined by PCR, Southern blot hybridization, sequence analysis and RT-PCR. Seven M. tuberculosis isolates contain insertions of IS6110 elements within plcA, plcC or plcD. In 19 of 25 M. tuberculosis isolates examined, genomic deletions were identified, resulting in loss of parts of genes or complete genes from the plcABC and/or plcD loci. Partial plcD deletion was observed in one M. africanum isolate. In each case, deletions were associated with the presence of a copy of the IS6110 element and in all occurrences IS6110 was transposed in the same orientation. A mechanism of deletion resulting from homologous recombination of two copies of IS6110 was recognized in a group of genetically related M. tuberculosis isolates. Five M. tuberculosis isolates presented major polymorphisms in the plcABC and plcD regions, along with loss of expression competence that affected all four plc genes. Phospholipase C is a well-known bacterial virulence factor. The precise role of phospholipase C in the pathogenicity of M. tuberculosis is unknown, but considering the potential importance that the plc genes may have in the virulence of the tubercle bacillus, the study of isolates cultured from patients with active tuberculosis bearing genetic variations affecting these genes may provide insights into the significance of phospholipase C enzymes for tuberculosis pathogenicity.

scholarly journals Efficient CRISPR/Cas9 mediated Pooled-sgRNAs assembly accelerates targeting multiple genes related to male sterility in cotton

Plant Methods ◽  
2021 ◽  
Vol 17 (1) ◽  
Author(s):  
Mohamed Ramadan ◽  
Muna Alariqi ◽  
Yizan Ma ◽  
Yanlong Li ◽  
Zhenping Liu ◽  
...  
Keyword(s):  
Genetic Transformation ◽  
Sequence Similarity ◽  
High Specificity ◽  
Transformation Method ◽  
High Sequence Similarity ◽  
Single Experiment ◽  
Next Generation Sequencing Technology ◽  
Cotton Transformation ◽  
Assembly Method ◽  
Multiple Genes

Abstract Background Upland cotton (Gossypium hirsutum), harboring a complex allotetraploid genome, consists of A and D sub-genomes. Every gene has multiple copies with high sequence similarity that makes genetic, genomic and functional analyses extremely challenging. The recent accessibility of CRISPR/Cas9 tool provides the ability to modify targeted locus efficiently in various complicated plant genomes. However, current cotton transformation method targeting one gene requires a complicated, long and laborious regeneration process. Hence, optimizing strategy that targeting multiple genes is of great value in cotton functional genomics and genetic engineering. Results To target multiple genes in a single experiment, 112 plant development-related genes were knocked out via optimized CRISPR/Cas9 system. We optimized the key steps of pooled sgRNAs assembly method by which 116 sgRNAs pooled together into 4 groups (each group consisted of 29 sgRNAs). Each group of sgRNAs was compiled in one PCR reaction which subsequently went through one round of vector construction, transformation, sgRNAs identification and also one round of genetic transformation. Through the genetic transformation mediated Agrobacterium, we successfully generated more than 800 plants. For mutants identification, Next Generation Sequencing technology has been used and results showed that all generated plants were positive and all targeted genes were covered. Interestingly, among all the transgenic plants, 85% harbored a single sgRNA insertion, 9% two insertions, 3% three different sgRNAs insertions, 2.5% mutated sgRNAs. These plants with different targeted sgRNAs exhibited numerous combinations of phenotypes in plant flowering tissues. Conclusion All targeted genes were successfully edited with high specificity. Our pooled sgRNAs assembly offers a simple, fast and efficient method/strategy to target multiple genes in one time and surely accelerated the study of genes function in cotton.

scholarly journals Evolution of Chi motifs in Proteobacteria

2021 ◽  
Author(s):  
Angélique Buton ◽  
Louis-Marie Bobay
Keyword(s):  
Sequence Similarity ◽  
Bacterial Species ◽  
Double Strand Breaks ◽  
Dna Double Strand Breaks ◽  
Large Dataset ◽  
High Sequence Similarity ◽  
Strand Breaks ◽  
E Coli ◽  
Dna Motif ◽  
And Staphylococcus Aureus

Abstract Homologous recombination is a key pathway found in nearly all bacterial taxa. The recombination complex allows bacteria to repair DNA double strand breaks but also promotes adaption through the exchange of DNA between cells. In Proteobacteria, this process is mediated by the RecBCD complex, which relies on the recognition of a DNA motif named Chi to initiate recombination. The Chi motif has been characterized in Escherichia coli and analogous sequences have been found in several other species from diverse families, suggesting that this mode of action is widespread across bacteria. However, the sequences of Chi-like motifs are known for only five bacterial species: E. coli, Haemophilus influenzae, Bacillus subtilis, Lactococcus lactis and Staphylococcus aureus. In this study we detected putative Chi motifs in a large dataset of Proteobacteria and we identified four additional motifs sharing high sequence similarity and similar properties to the Chi motif of E. coli in 85 species of Proteobacteria. Most Chi motifs were detected in Enterobacteriaceae and this motif appears well conserved in this family. However, we did not detect Chi motifs for the majority of Proteobacteria, suggesting that different motifs are used in these species. Altogether these results substantially expand our knowledge on the evolution of Chi motifs and on the recombination process in bacteria.

Meiotic Recombination Between Paralogous RBCSB Genes on Sister Chromatids of Arabidopsis thaliana

Genetics ◽  
2004 ◽  
Vol 166 (2) ◽  
pp. 947-957 ◽  
Author(s):  
John G Jelesko ◽  
Kristy Carter ◽  
Whitney Thompson ◽  
Yuki Kinoshita ◽  
Wilhelm Gruissem
Keyword(s):  
Gene Cluster ◽  
Dna Sequences ◽  
Meiotic Recombination ◽  
Sequence Similarity ◽  
Specific Gene ◽  
High Sequence Similarity ◽  
Paralogous Genes ◽  
Chimeric Genes ◽  
Unequal Recombination ◽  
Sister Chromatids

Abstract Paralogous genes organized as a gene cluster can rapidly evolve by recombination between misaligned paralogs during meiosis, leading to duplications, deletions, and novel chimeric genes. To model unequal recombination within a specific gene cluster, we utilized a synthetic RBCSB gene cluster to isolate recombinant chimeric genes resulting from meiotic recombination between paralogous genes on sister chromatids. Several F1 populations hemizygous for the synthRBCSB1 gene cluster gave rise to Luc+ F2 plants at frequencies ranging from 1 to 3 × 10-6. A nonuniform distribution of recombination resolution sites resulted in the biased formation of recombinant RBCS3B/1B::LUC genes with nonchimeric exons. The positioning of approximately half of the mapped resolution sites was effectively modeled by the fractional length of identical DNA sequences. In contrast, the other mapped resolution sites fit an alternative model in which recombination resolution was stimulated by an abrupt transition from a region of relatively high sequence similarity to a region of low sequence similarity. Thus, unequal recombination between paralogous RBCSB genes on sister chromatids created an allelic series of novel chimeric genes that effectively resulted in the diversification rather than the homogenization of the synthRBCSB1 gene cluster.

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