Gene replacement method for determining conditions in which Bacillus subtilis genes are essential or dispensable for cell viability

2004 ◽  
Vol 64 (3) ◽  
pp. 382-386 ◽  
Author(s):  
H. Yakhnin ◽  
P. Babitzke
Keyword(s):  
Bacillus Subtilis ◽  
Cell Viability ◽  
Gene Replacement ◽  
Replacement Method

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 Erratum: BRaf signaling principles unveiled by large-scale human mutation analysis with a rapid lentivirus-based gene replacement method

2017 ◽  
Vol 31 (8) ◽  
pp. 846-846
Author(s):  
Chae-Seok Lim ◽  
Xi Kang ◽  
Vincent Mirabella ◽  
Huaye Zhang ◽  
Qian Bu ◽  
...  
Keyword(s):  
Mutation Analysis ◽  
Large Scale ◽  
Gene Replacement ◽  
Replacement Method ◽  
Human Mutation

scholarly journals Functional analysis of subunits III and IV of Bacillus subtilis aa3-600 quinol oxidase by in vitro mutagenesis and gene replacement

1995 ◽  
Vol 1232 (1-2) ◽  
pp. 67-74 ◽  
Author(s):  
Gaetano Villani ◽  
Maria Tattoli ◽  
Nazzareno Capitanio ◽  
Philippe Glaser ◽  
Sergio Papa ◽  
...  
Keyword(s):  
Functional Analysis ◽  
Bacillus Subtilis ◽  
Gene Replacement ◽  
In Vitro Mutagenesis ◽  
Quinol Oxidase

scholarly journals Effects of Bacillus Subtilis-Fermented White Sword Bean Extract on Adipogenesis and Lipolysis of 3T3-L1 Adipocytes

Foods ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1423
Author(s):  
Yujeong Choi ◽  
Da-Som Kim ◽  
Min-Chul Lee ◽  
Seulgi Park ◽  
Joo-Won Lee ◽  
...  
Keyword(s):  
Bacillus Subtilis ◽  
Cell Viability ◽  
Dietary Supplement ◽  
Free Glycerol ◽  
Glycerol Content ◽  
Treatment Groups ◽  
Protein Levels ◽  
Triglyceride Accumulation ◽  
Before And After

To investigate the adipogenesis and lipolysis effects of the Bacillus subtilis-fermented white sword bean extract (FWSBE) on 3T3-L1 adipocytes, we treated 3T3-L1 preadipocytes before and after differentiation with FWSBE and measured triglyceride, free glycerol, mRNA, and protein levels. First, FWSBE reduced the cell viability of 3T3-L1 pre-adipocytes under 1000 µg/mL conditions. Triglyceride accumulation in 3T3-L1 pre-adipocytes was suppressed, and free glycerol content in mature 3T3-L1 adipocytes was increased in the FWSBE treatment groups, indicating that FWSBE has anti-obesity effects. Further, FWSBE suppressed adipogenesis in 3T3-L1 pre-adipocytes by lowering the protein levels of C/EBPα, PPARγ, and FAS and increasing the level of pACC and pAMPK. Additionally, FWSBE promoted lipolysis in mature 3T3-L1 adipocytes by increasing the transcription levels of Ppara, Acox, and Lcad and the protein levels of pHSL and ATGL. Thus, we suggest that FWSBE can be a potential dietary supplement because of its anti-obesity properties.

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 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 Genetic Requirements for Potassium Ion-Dependent Colony Spreading in Bacillus subtilis

2005 ◽  
Vol 187 (24) ◽  
pp. 8462-8469 ◽  
Author(s):  
Rebecca F. Kinsinger ◽  
Daniel B. Kearns ◽  
Marina Hale ◽  
Ray Fall
Keyword(s):  
Bacillus Subtilis ◽  
Ph Control ◽  
Growth Conditions ◽  
Gene Replacement ◽  
Potassium Ion ◽  
Nucleotide Biosynthesis ◽  
Surface Colonization ◽  
Transposon Insertion ◽  
Colony Spreading ◽  
Sliding Motility

ABSTRACT Undomesticated strains of Bacillus subtilis exhibit extensive colony spreading on certain soft agarose media: first the formation of dendritic clusters of cells, followed by spreading (pellicle-like) growth to cover the entire surface. These phases of colonization are dependent on the level of potassium ion (K+) but independent of flagella, as verified with a mutant with a hag gene replacement; this latter finding highlights the importance of sliding motility in colony spreading. Exploring the K+ requirement, directed mutagenesis of the higher-affinity K+ transporter KtrAB, but not the lower-affinity transporter KtrCD, was found to inhibit surface colonization unless sufficient KCl was added. To identify other genes involved in K+-dependent colony spreading, transposon insertion mutants in wild-type strain 3610 were screened. Disruption of genes for pyrimidine (pyrB) or purine (purD, purF, purH, purL, purM) biosynthetic pathways abolished the K+-dependent spreading phase. Consistent with a requirement for functional nucleic acid biosynthesis, disruption of purine synthesis with the folic acid antagonist sulfamethoxazole also inhibited spreading. Other transposon insertions disrupted acetoin biosynthesis (the alsS gene), acidifying the growth medium, glutamine synthetase (the glnA gene), and two surfactin biosynthetic genes (srfAA, srfAB). This work identified four classes of surface colonization mutants with defective (i) potassium transport, (ii) surfactin formation, (iii) growth rate or yield, or (iv) pH control. Overall, the ability of B. subtilis to colonize surfaces by spreading is highly dependent on balanced nucleotide biosynthesis and nutrient assimilation, which require sufficient K+ ions, as well as growth conditions that promote sliding motility.

scholarly journals Cytochemical studies of cell viability and gene expression in Bacillus subtilis macrofibres

Microbiology ◽  
1997 ◽  
Vol 143 (12) ◽  
pp. 3713-3721 ◽  
Author(s):  
N. H. Mendelson ◽  
B. Salhi ◽  
K. E. Sundahl
Keyword(s):  
Gene Expression ◽  
Bacillus Subtilis ◽  
Cell Viability
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