scholarly journals Gross Chromosomal Rearrangements in Kluyveromyces marxianus Revealed by Illumina and Oxford Nanopore Sequencing

2020 ◽  
Vol 21 (19) ◽  
pp. 7112
Author(s):  
Lin Ding ◽  
Harrison D. Macdonald ◽  
Hamilton O Smith ◽  
Clyde A. Hutchison III ◽  
Chuck Merryman ◽  
...  
Keyword(s):  
Kluyveromyces Marxianus ◽  
Chromosomal Rearrangements ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Illumina Miseq ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Random Integration ◽  
Gross Chromosomal Rearrangements ◽  
Oxford Nanopore

Kluyveromyces marxianus (K. marxianus) is an increasingly popular industrially relevant yeast. It is known to possess a highly efficient non-homologous end joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus otherwise wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the gross chromosomal rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in eukaryotes, which could potentially provide insights for cancer research.

scholarly journals Gross Chromosomal Rearrangements in Kluyveromyces Marxianus Revealed by Illumina and Oxford Nanopore Sequencing

2020 ◽  
Author(s):  
Lin Ding ◽  
Harrison D. Macdonald ◽  
Hamilton O Smith ◽  
Clyde A. Hutchison III ◽  
Chuck Merryman ◽  
...  
Keyword(s):  
Kluyveromyces Marxianus ◽  
Chromosomal Rearrangements ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Illumina Miseq ◽  
Sequencing Analysis ◽  
Nanopore Sequencing ◽  
Random Integration ◽  
Gross Chromosomal Rearrangements ◽  
Oxford Nanopore

Kluyveromyces marxianus (K. marxianus) is a newly emerging industrially relevant yeast. It is known to possess a highly efficient Non-Homologous End Joining (NHEJ) pathway that promotes random integration of non-homologous DNA fragments into its genome. The nature of the integration events was traditionally analyzed by Southern blot hybridization. However, the precise DNA sequence at the insertion sites were not fully explored. We transformed a PCR product of the Saccharomyces cerevisiae URA3 gene (ScURA3) into an uracil auxotroph K. marxianus wildtype strain and picked 24 stable Ura+ transformants for sequencing analysis. We took advantage of rapid advances in DNA sequencing technologies and developed a method using a combination of Illumina MiSeq and Oxford Nanopore sequencing. This approach enables us to uncover the Gross Chromosomal Rearrangements (GCRs) that are associated with the ScURA3 random integration. Moreover, it will shine a light on understanding DNA repair mechanisms in Eukaryotes, which could potentially provide insights for cancer research.

scholarly journals High-Temperature Ethanol Fermentation and Transformation with Linear DNA in the Thermotolerant Yeast Kluyveromyces marxianus DMKU3-1042

2008 ◽  
Vol 74 (24) ◽  
pp. 7514-7521 ◽  
Author(s):  
Sanom Nonklang ◽  
Babiker M. A. Abdel-Banat ◽  
Kamonchai Cha-aim ◽  
Nareerat Moonjai ◽  
Hisashi Hoshida ◽  
...  
Keyword(s):  
High Temperature ◽  
Ethanol Production ◽  
Kluyveromyces Marxianus ◽  
Blot Hybridization ◽  
Molecular Genetic ◽  
Cost Effective ◽  
Southern Blot Hybridization ◽  
Heterologous Proteins ◽  
Linear Dna ◽  
Ethanol Producer

ABSTRACT We demonstrate herein the ability of Kluyveromyces marxianus to be an efficient ethanol producer and host for expressing heterologous proteins as an alternative to Saccharomyces cerevisiae. Growth and ethanol production by strains of K. marxianus and S. cerevisiae were compared under the same conditions. K. marxianus DMKU3-1042 was found to be the most suitable strain for high-temperature growth and ethanol production at 45°C. This strain, but not S. cerevisiae, utilized cellobiose, xylose, xylitol, arabinose, glycerol, and lactose. To develop a K. marxianus DMKU3-1042 derivative strain suitable for genetic engineering, a uracil auxotroph was isolated and transformed with a linear DNA of the S. cerevisiae ScURA3 gene. Surprisingly, Ura+ transformants were easily obtained. By Southern blot hybridization, the linear ScURA3 DNA was found to have inserted randomly into the K. marxianus genome. Sequencing of one Lys− transformant confirmed the disruption of the KmLYS1 gene by the ScURA3 insertion. A PCR-amplified linear DNA lacking K. marxianus sequences but containing an Aspergillus α-amylase gene under the control of the ScTDH3 promoter together with an ScURA3 marker was subsequently used to transform K. marxianus DMKU3-1042 in order to obtain transformants expressing Aspergillus α-amylase. Our results demonstrate that K. marxianus DMKU3-1042 can be an alternative cost-effective bioethanol producer and a host for transformation with linear DNA by use of S. cerevisiae-based molecular genetic tools.

scholarly journals Evaluation of Oxford Nanopore MinIONTM Sequencing for 16S rRNA Microbiome Characterization

2017 ◽  
Author(s):  
Xiao Ma ◽  
Elyse Stachler ◽  
Kyle Bibby
Keyword(s):  
16S Rrna ◽  
Pure Culture ◽  
Community Sample ◽  
Operational Taxonomic Unit ◽  
Illumina Miseq ◽  
Rrna Gene ◽  
Ribosomal Database Project ◽  
Nanopore Sequencing ◽  
Sequencing Platform ◽  
Oxford Nanopore

AbstractIn this manuscript we evaluate the potential for microbiome characterization by sequencing of near-full length 16S rRNA gene region fragments using the Oxford Nanopore MinION (hereafter ‘Nanopore’) sequencing platform. We analyzed pure-culture E. coli and P. fluorescens, as well as a low-diversity mixed community sample from hydraulic fracturing produced water. Both closed and open reference operational taxonomic unit (OTU) picking failed, necessitating the direct use of sequences without OTU picking. The Ribosomal Database Project classifier against the Green Genes database was found to be the optimal annotation approach, with average pure-culture annotation accuracies of 93.8% and 82.0% at the phyla and genus levels, respectively. Comparative analysis of an environmental sample using Nanopore and Illumina MiSeq sequencing identified high taxonomic similarity when using a weighted metric (Bray-Curtis), and significantly reduced similarity when using an unweighted metric (Jaccard). These results highlight the great potential of Nanopore sequencing to analyze broad microbial community trends, and the challenge of applying Nanopore sequencing to discern rare taxa in mixed microbial communities. Finally, we observed that between-run carryover following washes on the same flowcell accounted for >10% of sequence reads, necessitating future development to either prevent carryover or filter sequences of interest (e.g. barcoding).

scholarly journals Genetic characterization of multidrug resistance in Shigella spp. from Japan

2006 ◽  
Vol 55 (12) ◽  
pp. 1685-1691 ◽  
Author(s):  
Ashraf M. Ahmed ◽  
Kimi Furuta ◽  
Kei Shimomura ◽  
Yoshio Kasama ◽  
Tadashi Shimamoto
Keyword(s):  
Shigella Flexneri ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Shigella Sonnei ◽  
Sequencing Analysis ◽  
Class 1 Integron ◽  
Gene Cassettes ◽  
Class 1 ◽  
Shigella Spp

This study characterized the genetic basis of antimicrobial resistance of a number of Shigella spp. isolated from humans from 2000 to 2004 in Hiroshima prefecture, Japan. A total of 26 isolates of Shigella spp. were included in this study. Antimicrobial susceptibility tests revealed high levels of resistance, especially to ampicillin, streptomycin, trimethoprim, tetracycline, nalidixic acid and ciprofloxacin. PCR and DNA sequencing were used for screening and characterization of antibiotic-resistance determinants. PCR sequencing analysis revealed the presence of only one type of class 1 integron in one isolate of Shigella sonnei. This class 1 integron was 1904 bp and contained two gene cassettes: a probable esterase/lipase (estX) and aadA1, which confers resistance to streptomycin and spectinomycin. Two types of class 2 integron were identified in this study. One was the classic type (2158 bp) and carried the three conserved resistance gene cassettes of the class 2 integron, dfrA1, sat1 and aadA1, which confer resistance to trimethoprim, streptothricin and streptomycin/spectinomycin, respectively. This type was detected in both Shigella sonnei (14 isolates) and Shigella flexneri (five isolates). The other type was shorter (1313 bp) and carried only two gene cassettes, dfrA1 and sat1. This integron was detected in a single isolate of Shigella sonnei. PFGE patterns showed limited diversity within clusters of the same species. Furthermore, an extended-spectrum β-lactamase gene, bla OXA-30, which confers resistance to ampicillin, was characterized in all isolates of Shigella flexneri except the oldest strain, which was isolated in 2000. Southern blot hybridization and conjugation experiments showed that bla OXA-30 was located in the chromosome.

scholarly journals Identification of an IS6110 insertion site in plcD, the unique phospholipase C gene of Mycobacterium bovis

2006 ◽  
Vol 55 (4) ◽  
pp. 451-457 ◽  
Author(s):  
Cristina Viana-Niero ◽  
Cesar Alejandro Rosales Rodriguez ◽  
Fabiana Bigi ◽  
Marcos Santos Zanini ◽  
José Soares Ferreira-Neto ◽  
...  
Keyword(s):  
Phospholipase C ◽  
Mycobacterium Bovis ◽  
Blot Hybridization ◽  
Insertion Site ◽  
Direct Repeat ◽  
Southern Blot Hybridization ◽  
Single Copy ◽  
Genomic Variation ◽  
Organ Distribution ◽  
Sequencing Analysis

The IS6110 repetitive element is present in multiple copies in most Mycobacterium tuberculosis complex bacteria, except for Mycobacterium bovis strains, which usually contain a single copy of IS6110 located on a 1·9 kb PvuII fragment of the direct repeat region. IS6110 transposition can disrupt coding regions and is a major force of genomic variation. In a previous work it was demonstrated that phospholipase C genes are preferential loci for IS6110 transposition in M. tuberculosis clinical strains. Bacterial phospholipase C enzymes participate in pathogenic mechanisms used by different organisms, and have been implicated in intracellular survival, cytolysis and cell-to-cell spread. Four phospholipase C genes (plcA, plcB, plcC and plcD) were detected in the genomes of M. tuberculosis, Mycobacterium africanum, Mycobacterium microti and ‘Mycobacterium canettii’. M. bovis and the vaccine strain M. bovis Bacillus Calmette–Guérin contain only the plcD gene. In the present work, the existence of IS6110 insertions within plcD, the unique phospholipase C gene of M. bovis, has been investigated by PCR, Southern blot hybridization and sequencing analysis. In 18 (7·3 %) of 245 isolates analysed, the plcD gene was interrupted by the insertion of one copy of IS6110, which in all cases was transposed in the same orientation and at the same position, 1 972 894, relative to the genome of M. bovis AF2122/97. These 18 isolates were distributed in 6 different spoligotype patterns and contained 4 to 8 IS6110 copies. In contrast, strains showing an intact plcD gene contained one (87 %), two (9·4 %) or three (2·4 %) IS6110 copies, and only a single isolate (1·2 %) had four IS6110 copies. The implications of plcD gene disruption in M. bovis have not been fully investigated, but no differences in the organ distribution of the disease were detected when animals infected with strains from the same spoligotype patterns bearing plcD : : IS6110 and intact plcD were compared.

scholarly journals Physical Analysis of Tn10- and IS10-Promoted Transpositions and Rearrangements

Genetics ◽  
1987 ◽  
Vol 116 (3) ◽  
pp. 359-369
Author(s):  
Michael M Shen ◽  
Elisabeth A Raleigh ◽  
Nancy Kleckner
Keyword(s):  
Escherichia Coli ◽  
Southern Blot ◽  
Chromosomal Rearrangements ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Physical Analysis ◽  
Present Evidence ◽  
Target Sites ◽  
Minimal Media

ABSTRACT We have investigated by Southern blot hybridization the rate of IS10 transposition and other Tn10/IS10-promoted rearrangements in Escherichia coli and Salmonella strains bearing single chromosomal insertions of Tn10 or a related Tn10 derivative. We present evidence for three primary conclusions. First, the rate of IS10 transposition is approximately 10-4 per cell per bacterial generation when overnight cultures are grown and plated on minimal media and is at least ten times more frequent than any other Tn10/IS10-promoted DNA alteration. Second, all of the chromosomal rearrangements observed can be accounted for by two previously characterized Tn10-promoted rearrangements: deletion/inversions and deletions. Together these rearrangements occur at about 10% the rate of IS10 transposition. Third, the data suggest that intramolecular Tn10-promoted rearrangements preferentially use nearby target sites, while the target sites for IS10 transposition events are scattered randomly around the chromosome.

scholarly journals Emergence of mcr-1-Harboring Salmonella enterica Serovar Sinstorf Type ST155 Isolated From Patients With Diarrhea in Jiangsu, China

2021 ◽  
Vol 12 ◽  
Author(s):  
Guoye Liu ◽  
Huimin Qian ◽  
Jingwen Lv ◽  
Benshun Tian ◽  
Changjun Bao ◽  
...  
Keyword(s):  
Southern Blot ◽  
Gel Electrophoresis ◽  
Blot Hybridization ◽  
Southern Blot Hybridization ◽  
Pulsed Field Gel Electrophoresis ◽  
Sequencing Analysis ◽  
Colistin Resistance ◽  
Pulsed Field ◽  
Extended Spectrum Beta Lactamase ◽  
Pcr Method

Background: This study analyzed the antimicrobial resistance phenotypes and mechanisms of quinolone, cephalosporins, and colistin resistance in nontyphoidal Salmonella from patients with diarrhea in Jiangsu, China.Methods: A total of 741 nontyphoidal Salmonella isolates were collected from hospitals in major cities of Jiangsu Province, China between 2016 and 2017. Their susceptibility to commonly used antibiotics was evaluated by broth micro-dilution and sequencing analysis of resistance genes screened by a PCR method. For mcr-1 positive isolates, genetic relationship study was carried out by pulsed-field gel electrophoresis and multiloci sequence typing analysis. The transferability of these plasmids was measured with conjugation experiments and the genetic locations of mcr-1 were analyzed by pulsed-field gel electrophoresis profiles of S1-digested genomic DNA and subsequent Southern blot hybridization.Results: Among 741 nontyphoidal Salmonella isolates, the most common serotypes identified were S. Typhimurium (n=257, 34.7%) and S. Enteritidis (n=127, 17.1%), and the isolates showed 21.7, 20.6, and 5.0% resistance to cephalosporins, ciprofloxacin, and colistin, respectively. Among the 335 nalidixic acid-resistant Salmonella, 213 (63.6%) and 45 (13.4%) had at least one mutation in gyrA and parC. Among the plasmid-borne resistance, qnrS1 (85; 41.9%) and aac(6')-Ib-cr4 (75; 36.9%) were the most common quinolone resistance (PMQR) genes, while blaCTX-M-14 (n=35) and blaCTX-M-55 (n=46) were found to be dominant extended-spectrum beta-lactamase (ESBL) genes in nontyphoidal Salmonella. In addition, eight mcr-1-harboring strains were detected since 2016 and they were predominate in children under the age of 7years. Conjugation assays showed the donor Salmonella strain has functional and transferable colistin resistance and Southern blot hybridization revealed that mcr-1 was located in a high molecular weight plasmid.Conclusion: In nontyphoidal Salmonella, there is a rapidly increasing trend of colistin resistance and this is the first report of patients harboring mcr-1-positive Salmonella with a new ST type ST155 and new serotype S. Sinstorf. These findings demonstrate the necessity for cautious use and the continuous monitoring of colistin in clinical applications.

scholarly journals Expression of the mef(E) Gene Encoding the Macrolide Efflux Pump Protein Increases in Streptococcus pneumoniae with Increasing Resistance to Macrolides

2005 ◽  
Vol 49 (11) ◽  
pp. 4635-4640 ◽  
Author(s):  
Aleksandra K. Wierzbowski ◽  
Dave Boyd ◽  
Michael Mulvey ◽  
Daryl J. Hoban ◽  
George G. Zhanel
Keyword(s):  
Streptococcus Pneumoniae ◽  
Efflux Pump ◽  
Blot Hybridization ◽  
Gene Copy Number ◽  
Southern Blot Hybridization ◽  
Macrolide Resistance ◽  
Start Codon ◽  
Gene Copy ◽  
Sequencing Analysis ◽  
E Gene

ABSTRACT Active macrolide efflux is a major mechanism of macrolide resistance in Streptococcus pneumoniae in many parts of the world, especially North America. In Canada, this active macrolide efflux in S. pneumoniae is predominantly due to acquisition of the mef(E) gene. In the present study, we assessed the mef(E) gene sequence as well as mef(E) expression in variety of low- and high-level macrolide-resistant, clindamycin-susceptible (M-phenotype) S. pneumoniae isolates (erythromycin MICs, 1 to 32 μg/ml; clindamycin MICs, ≤0.25 μg/ml). Southern blot hybridization with mef(E) probe and EcoRI digestion and relative real-time reverse transcription-PCR were performed to study the mef(E) gene copy number and expression. Induction of mef(E) expression was analyzed by Etest susceptibility testing pre- and postincubation with subinhibitory concentrations of erythromycin, clarithromycin, azithromycin, telithromycin, and clindamycin. The macrolide efflux gene, mef(E), was shown to be a single-copy gene in all 23 clinical S. pneumoniae isolates tested, and expression post-macrolide induction increased 4-, 6-, 20-, and 200-fold in isolates with increasing macrolide resistance (erythromycin MICs 2, 4, 8, and 32 μg/ml, respectively). Sequencing analysis of the macrolide efflux genetic assembly (mega) revealed that mef(E) had a 16-bp deletion 153 bp upstream of the putative start codon in all 23 isolates. A 119-bp intergenic region between mef(E) and mel was sequenced, and a 99-bp deletion was found in 11 of the 23 M-phenotype S. pneumoniae isolates compared to the published mega sequence. However, the mef(E) gene was fully conserved among both high- and low-level macrolide-resistant isolates. In conclusion, increased expression of mef(E) is associated with higher levels of macrolide resistance in macrolide-resistant S. pneumoniae.

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.

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