scholarly journals Construction of Nontoxigenic Mutants of Nonproteolytic Clostridium botulinum NCTC 11219 by Insertional Mutagenesis and Gene Replacement

2016 ◽  
Vol 82 (10) ◽  
pp. 3100-3108 ◽  
Author(s):  
Charlien Clauwers ◽  
Kristof Vanoirbeek ◽  
Laurence Delbrassinne ◽  
Chris W. Michiels
Keyword(s):  
Validation Studies ◽  
Clostridium Botulinum ◽  
Food Challenge ◽  
Basic Research ◽  
Gene Replacement ◽  
Safe Alternative ◽  
Process Validation ◽  
Content Type ◽  
Replacement Method ◽  
New Gene

ABSTRACTGroup II nonproteolyticClostridium botulinum(gIICb) strains are an important concern for the safety of minimally processed ready-to-eat foods, because they can grow and produce botulinum neurotoxin during refrigerated storage. The principles of control of gIICb by conventional food processing and preservation methods have been well investigated and translated into guidelines for the food industry; in contrast, the effectiveness of emerging processing and preservation techniques has been poorly documented. The reason is that experimental studies withC. botulinumare cumbersome because of biosafety and biosecurity concerns. In the present work, we report the construction of two nontoxigenic derivatives of the type E gIICb strain NCTC 11219. In the first strain, the botulinum toxin gene (bont/E) was insertionally inactivated with a retargeted intron using the ClosTron system. In the second strain,bont/Ewas exchanged for an erythromycin resistance gene using a new gene replacement strategy that makes use ofpyrEas a bidirectional selection marker. Growth under optimal and stressed conditions, sporulation efficiency, and spore heat resistance of the mutants were unaltered, except for small differences in spore heat resistance at 70°C and in growth at 2.3% NaCl. The mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with gIICb. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in gIICb and other clostridia.IMPORTANCEThe nontoxigenic mutants described in this work provide a safe alternative for basic research as well as for food challenge and process validation studies with psychrotrophicClostridium botulinum. In addition, this work expands the clostridial genetic toolbox with a new gene replacement method that can be applied to replace any gene in clostridia.

scholarly journals The T788G Mutation in thecyp51CGene Confers Voriconazole Resistance in Aspergillus flavus Causing Aspergillosis

2012 ◽  
Vol 56 (5) ◽  
pp. 2598-2603 ◽  
Author(s):  
Wei Liu ◽  
Yi Sun ◽  
Wei Chen ◽  
Weixia Liu ◽  
Zhe Wan ◽  
...  
Keyword(s):  
Aspergillus Flavus ◽  
Resistant Strain ◽  
Antifungal Susceptibility ◽  
Gene Replacement ◽  
Content Type ◽  
Replacement Method ◽  
First Choice ◽  
The One ◽  
A Site ◽  
First Time

ABSTRACTWith voriconazole (VRC) being approved as the first choice in treating invasive aspergillosis (IA) and its increasing use in treatment, a VRC-resistant strain ofAspergillus flavus, the second leading cause of IA afterAspergillus fumigatus, has emerged. The VRC-resistant strain ofA. flavuswas isolated for the first time from the surgical lung specimen of an IA patient with no response to VRC therapy. In order to ascertain the mechanism of VRC resistance, the azole target enzyme genes in this strain ofA. flavuswere cloned and sequenced, and 4 mutations generating amino acid residue substitutions were found in thecyp51Cgene. To further determine the role of this mutated gene for VRC resistance inA. flavus, anAgrobacterium tumefaciens-mediated gene replacement approach was applied. Consequently, the mutatedcyp51Cgene from thisA. flavusstrain was proven to confer the VRC resistance. Finally, to discern the one out of the four mutations in thecyp51Cgene that is responsible for contributing to VRC resistance, a site-directed gene mutagenesis procedure combined with a gene replacement method was performed. As a result, the T788G missense mutation in thecyp51Cgene was identified as responsible for VRC resistance inA. flavus. These findings indicated that the detection of this mutation inA. flavuscould serve as an indicator for physicians to avoid the use of VRC during IA treatment. Further comprehensive surveillance for antifungal susceptibility, as well as intensive study on the mechanism of azole resistance inA. flavuscausing IA, would be required to fully understand this mechanism.

scholarly journals Closed Genome Sequence of Clostridium botulinum Strain CFSAN064329 (62A)

2018 ◽  
Vol 6 (26) ◽  
Author(s):  
Travis G. Wentz ◽  
Kuan Yao ◽  
Kristin M. Schill ◽  
N. Rukma Reddy ◽  
Guy E. Skinner ◽  
...  
Keyword(s):  
Genome Sequence ◽  
Clostridium Botulinum ◽  
Botulinum Neurotoxin ◽  
Food Challenge ◽  
Strictly Anaerobic ◽  
Botulinum Neurotoxin A ◽  
Gram Positive ◽  
Content Type

Clostridium botulinum is a strictly anaerobic, Gram-positive, spore-forming bacterium that produces botulinum neurotoxin, a potent and deadly proteinaceous exotoxin. Clostridium botulinum strain CFSAN064329 (62A) produces an A1 serotype/subtype botulinum neurotoxin and is frequently utilized in food challenge and detection studies.

scholarly journals Targeted Disruption of the α-Amylase Gene in the Hyperthermophilic Archaeon Sulfolobus solfataricus

2003 ◽  
Vol 185 (2) ◽  
pp. 482-488 ◽  
Author(s):  
Penny Worthington ◽  
Viet Hoang ◽  
Francisco Perez-Pomares ◽  
Paul Blum
Keyword(s):  
Genetic Manipulation ◽  
Amylase Activity ◽  
Sulfolobus Solfataricus ◽  
Protein A ◽  
Gene Replacement ◽  
Coding Sequence ◽  
Replacement Method ◽  
Tandem Mass Spectroscopy ◽  
New Gene

ABSTRACT Sulfolobus solfataricus secretes an acid-resistant α-amylase (amyA) during growth on starch as the sole carbon and energy source. Synthesis of this activity is subject to catabolite repression. To better understand α-amylase function and regulation, the structural gene was identified and disrupted and the resulting mutant was characterized. Internal α-amylase peptide sequences obtained by tandem mass spectroscopy were used to identify the amyA coding sequence. Anti-α-amylase antibodies raised against the purified protein immunoprecipitated secreted α-amylase activity and verified the enzymatic identity of the sequenced protein. A new gene replacement method was used to disrupt the amyA coding sequence by insertion of a modified allele of the S. solfataricus lacS gene. PCR and DNA sequence analysis were used to characterize the altered amyA locus in the recombinant strain. The amyA::lacS mutant lost the ability to grow on starch, glycogen, or pullulan as sole carbon and energy sources. During growth on a non-catabolite-repressing carbon source with added starch, the mutant produced no detectable secreted amylase activity as determined by enzyme assay, plate assay, or Western blot analysis. These results clarify the biological role of the α-amylase and provide additional methods for the directed genetic manipulation of the S. solfataricus genome.

scholarly journals Mutation of RNA Polymerase β Subunit (rpoB) Promotes hVISA-to-VISA Phenotypic Conversion of Strain Mu3

2011 ◽  
Vol 55 (9) ◽  
pp. 4188-4195 ◽  
Author(s):  
Miki Matsuo ◽  
Tomomi Hishinuma ◽  
Yuki Katayama ◽  
Longzhu Cui ◽  
Maria Kapi ◽  
...  
Keyword(s):  
Rna Polymerase ◽  
Response Regulator ◽  
Single Copy ◽  
Gene Replacement ◽  
Vancomycin Resistance ◽  
Β Subunit ◽  
Gene Encoding ◽  
Content Type ◽  
Replacement Method ◽  
Two Component

ABSTRACTThe clinical vancomycin-intermediateStaphylococcus aureus(VISA) strain Mu50 carries two mutations in thevraSRandgraRStwo-component regulatory systems (TCRSs), namely,vraS(I5N) andgraR(N197S) (hereinafter designatedgraR*). The clinical heterogeneously vancomycin-intermediateS. aureus(hVISA) strain Mu3 shares with Mu50 the mutation invraSthat encodes the VraS two-component histidine kinase. Previously, we showed that introduction of the plasmid pgraR*, carrying the mutated two-component response regulatorgraR*, converted the hVISA strain Mu3 into VISA (vancomycin MIC = 4 mg/liter). Subsequently, however, we found that the introduction of a single copy ofgraR* into the Mu3 chromosome by a gene replacement method did not confer on Mu3 the VISA phenotype. The gene-replaced strain Mu3graR* thus obtained remained hVISA (MIC ≤ 2 mg/liter), although a small increase in vancomycin MIC was observed compared to that of the parent strain Mu3. Reevaluation of the Mu3 and Mu50 genomes revealed the presence of another mutation responsible for the expression of the VISA phenotype in Mu50. Here, we demonstrate that in addition to the two regulator mutations, a third mutation found in the Mu50rpoBgene, encoding the RNA polymerase β subunit, was required for Mu3 to achieve the level of vancomycin resistance of Mu50. The selection of strain Mu3graR* with rifampin gave rise torpoBmutants with various levels of increased vancomycin resistance. Furthermore, 3 (33%) of 10 independently isolated VISA strains established from the heterogeneous subpopulations of Mu3graR* were found to possessrpoBmutations with or without an accompanying rifampin-resistance phenotype. The data indicate that a sizable proportion of the resistant hVISA cell subpopulations is composed of spontaneousrpoBmutants with various degrees of increased vancomycin resistance.

scholarly journals Whole-Genome Single-Nucleotide-Polymorphism Analysis for Discrimination of Clostridium botulinum Group I Strains

2014 ◽  
Vol 80 (7) ◽  
pp. 2125-2132 ◽  
Author(s):  
Narjol Gonzalez-Escalona ◽  
Ruth Timme ◽  
Brian H. Raphael ◽  
Donald Zink ◽  
Shashi K. Sharma
Keyword(s):  
Single Nucleotide Polymorphism ◽  
Clostridium Botulinum ◽  
Toxin Gene ◽  
Polymorphism Analysis ◽  
Snp Analysis ◽  
Whole Genome ◽  
Nucleotide Polymorphism ◽  
Single Nucleotide ◽  
Content Type ◽  
Group I

ABSTRACTClostridium botulinumis a genetically diverse Gram-positive bacterium producing extremely potent neurotoxins (botulinum neurotoxins A through G [BoNT/A-G]). The complete genome sequences of three strains harboring only the BoNT/A1 nucleotide sequence are publicly available. Although these strains contain a toxin cluster (HA+OrfX−) associated with hemagglutinin genes, little is known about the genomes of subtype A1 strains (termed HA−OrfX+) that lack hemagglutinin genes in the toxin gene cluster. We sequenced the genomes of three BoNT/A1-producingC. botulinumstrains: two strains with the HA+OrfX−cluster (69A and 32A) and one strain with the HA−OrfX+cluster (CDC297). Whole-genome phylogenic single-nucleotide-polymorphism (SNP) analysis of these strains along with other publicly availableC. botulinumgroup I strains revealed five distinct lineages. Strains 69A and 32A clustered with theC. botulinumtype A1 Hall group, and strain CDC297 clustered with theC. botulinumtype Ba4 strain 657. This study reports the use of whole-genome SNP sequence analysis for discrimination ofC. botulinumgroup I strains and demonstrates the utility of this analysis in quickly differentiatingC. botulinumstrains harboring identical toxin gene subtypes. This analysis further supports previous work showing that strains CDC297 and 657 likely evolved from a common ancestor and independently acquired separate BoNT/A1 toxin gene clusters at distinct genomic locations.

scholarly journals Identification of the ATPase Subunit of the Primary Maltose Transporter in the Hyperthermophilic Anaerobe Thermotoga maritima

2017 ◽  
Vol 83 (18) ◽  
Author(s):  
Raghuveer Singh ◽  
Derrick White ◽  
Paul Blum
Keyword(s):  
Thermotoga Maritima ◽  
Mutant Cell ◽  
Sugar Metabolism ◽  
Genetic System ◽  
Gene Replacement ◽  
Atpase Subunit ◽  
Content Type ◽  
Fermentative Hydrogen Production ◽  
Atpase Subunits ◽  
Fermentative Hydrogen

ABSTRACT Thermotoga maritima is a hyperthermophilic anaerobic bacterium that produces molecular hydrogen (H2) by fermentation. It catabolizes a broad range of carbohydrates through the action of diverse ABC transporters. However, in T. maritima and related species, highly similar genes with ambiguous annotation obscure a precise understanding of genome function. In T. maritima, three putative malK genes, all annotated as ATPase subunits, exhibited high identity to each other. To distinguish between these genes, malK disruption mutants were constructed by gene replacement, and the resulting mutant cell lines were characterized. Only a disruption of malK3 produced a defect in maltose catabolism. To verify that the mutant phenotype arose specifically from malK3 inactivation, the malK3 mutation was repaired by recombination, and maltose catabolism was restored. This study demonstrates the importance of a maltose ABC-type transporter and its relationship to sugar metabolism in T. maritima. IMPORTANCE The application and further development of a genetic system was used here to investigate gene paralogs in the hyperthermophile Thermotoga maritima. The occurrence of three ABC transporter ATPase subunits all annotated as malK was evaluated using a combination of genetic and bioinformatic approaches. The results clarify the role of only one malK gene in maltose catabolism in a nonmodel organism noted for fermentative hydrogen production.

scholarly journals Genome Sequence of Clostridium botulinum Strain Adk2012 Associated with a Foodborne Botulinum Case in Tottori, Japan, in 2012

2017 ◽  
Vol 5 (34) ◽  
Author(s):  
Hiroshi Asakura ◽  
Shiori Yamamoto ◽  
Yoshika Momose ◽  
Haru Kato ◽  
Masaaki Iwaki ◽  
...  
Keyword(s):  
Genome Size ◽  
Genome Sequence ◽  
Clostridium Botulinum ◽  
Draft Genome ◽  
Draft Genome Sequence ◽  
Content Type ◽  
Foodborne Botulism

ABSTRACT We report here a draft genome sequence of Clostridium botulinum Adk2012 responsible for a foodborne botulism case that occurred in Tottori, Japan, in 2012. Its genome size was 2,904,173 bp, with 46 rRNAs and 54 tRNAs, at a coverage of 14.5×.

scholarly journals Alternative Sigma Factor SigK Has a Role in Stress Tolerance of Group I Clostridium botulinum Strain ATCC 3502

2013 ◽  
Vol 79 (12) ◽  
pp. 3867-3869 ◽  
Author(s):  
Elias Dahlsten ◽  
David Kirk ◽  
Miia Lindström ◽  
Hannu Korkeala
Keyword(s):  
Stress Tolerance ◽  
Sigma Factor ◽  
Clostridium Botulinum ◽  
Alternative Sigma Factor ◽  
Strain Atcc ◽  
Content Type ◽  
Osmotic Stress Tolerance ◽  
Group I ◽  
Temperature Downshift

ABSTRACTThe role of the alternative sigma factor SigK in cold and osmotic stress tolerance ofClostridium botulinumATCC 3502 was demonstrated by induction ofsigKafter temperature downshift and exposure to hyperosmotic conditions and by impaired growth of thesigKmutants under the respective conditions.

scholarly journals Plasmid-Borne Type E Neurotoxin Gene Clusters in Clostridium botulinum Strains

2013 ◽  
Vol 79 (12) ◽  
pp. 3856-3859 ◽  
Author(s):  
Zhen Zhang ◽  
Hannamari Hintsa ◽  
Ying Chen ◽  
Hannu Korkeala ◽  
Miia Lindström
Keyword(s):  
Gene Cluster ◽  
Gel Electrophoresis ◽  
Clostridium Botulinum ◽  
Southern Hybridization ◽  
Gene Clusters ◽  
Pulsed Field Gel Electrophoresis ◽  
Content Type ◽  
Pulsed Field ◽  
Large Plasmids

ABSTRACTA collection of 36Clostridium botulinumtype E strains was examined by pulsed-field gel electrophoresis (PFGE) and Southern hybridization with probes targeted tobotEandorfX1in the neurotoxin gene cluster. Three strains were found to contain neurotoxin subtype E1 gene clusters in large plasmids of about 146 kb in size.

Knowledge mapping of microfinance performance research: a bibliometric analysis

2021 ◽  
Vol 48 (3) ◽  
pp. 399-418
Author(s):  
Shabiha Akter ◽  
Md Hamid Uddin ◽  
Ahmad Hakimi Tajuddin
Keyword(s):  
Financial Performance ◽  
Bibliometric Analysis ◽  
Social Performance ◽  
Credit Scoring ◽  
Basic Research ◽  
Future Research ◽  
Knowledge Mapping ◽  
Microfinance Institutions ◽  
Content Type ◽  
Bibliometric Review

PurposePerformance assessment of microfinance institutions (MFIs) has long been a question of considerable research interest. The dual goals – financial performance and social performance of MFIs widely studied yet remain unsolved in the existing literature. To assess the knowledge structure of research in this area and to aid future research, we review the literature with bibliometric analysis.Design/methodology/approachOur study has used bibliographic data of 1,252 scientific documents indexed in the Scopus database from 1995 to 2020 (June 05). We have used the “bibliometrix” package in R language to analyze the data and illustrate the findings.FindingsWe find that there has been an increasing trend in publications, especially from 2006 onwards. Various bibliometric indicators allow us to follow the progression of knowledge along with identifying the most contributing and impactful authors, publication sources, institutions and countries. We illustrate the major research themes and identify that “poverty alleviations”, “group lending” and “credit scoring” are the major emerging and specialized themes besides the basic research evolved around “microfinance” or “microcredit”. Our further analysis of thematic evolution over different time frames reveals that “financial performance” aspect is getting more attention in recent times in evaluating the performance of MFIs.Originality/valueThe insights of knowledge accumulated from our bibliometric review and thematic analysis provide researchers with an efficient comprehension of the advancement of the research on microfinance performance and offer avenues for future scientific endeavors.

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