Modified mariner Transposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis
TLDR
This work modifications an existing mariner transposon, TnYLB, such that it can easily be genetically manipulated and introduced into Bacillus subtilis, and generates a series of three new mariner derivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes.Abstract:
Transposons are mobile genetic elements bounded by insertion sequences that are recognized by a specific mobilizing transposase enzyme. The transposase may mobilize not only the insertion sequences but also intervening DNA. mariner is a particularly efficient transposon for the random chromosomal integration of genes and insertional mutagenesis. Here, we modify an existing mariner transposon, TnYLB, such that it can easily be genetically manipulated and introduced into Bacillus subtilis. We generate a series of three new mariner derivatives that mobilize spectinomycin, chloramphenicol, and kanamycin antibiotic resistance cassettes. Furthermore, we generate a series of transposons with a strong, outward-oriented, optionally isopropyl-β-d-thiogalactopyranoside (IPTG)-inducible promoter for the random overexpression of neighboring genes and a series of transposons with a promoterless lacZ gene for the random generation of transcriptional reporter fusions. We note that the modification of the base transposon is not restricted to B. subtilis and should be applicable to any mariner-compatible host organism, provided that in vitro mutagenesis or an in vivo species-specific delivery vector is employed.read more
Citations
More filters
Journal ArticleDOI
Identification of Poly-N-acetylglucosamine as a Major Polysaccharide Component of the Bacillus subtilis Biofilm Matrix
Damien Roux,Colette Cywes-Bentley,Yi-Fan Zhang,Yi-Fan Zhang,Stéphanie Pons,Melissa A. Konkol,Daniel B. Kearns,Dustin J. Little,P. Lynne Howell,David Skurnik,Gerald B. Pier +10 more
TL;DR: Genes in the epsHIJK locus needed for biofilm formation encode proteins synthesizing the conserved bacterial polysaccharide poly-N-acetylglucosamine (PNAG).
Journal ArticleDOI
Synthesis, release, and recapture of compatible solute proline by osmotically stressed Bacillus subtilis cells.
Tamara Hoffmann,Carsten von Blohn,Agnieszka Stanek,Susanne Moses,Helena Barzantny,Erhard Bremer +5 more
TL;DR: The data reported here show that the OpuE transporter not only possesses the previously reported role for the scavenging of exogenously provided proline as an osmoprotectant but also functions as a physiologically highly important recapturing device for proline that is synthesized de novo and subsequently released by salt-stressed B. subtilis cells.
Journal ArticleDOI
Low Concentrations of Vitamin C Reduce the Synthesis of Extracellular Polymers and Destabilize Bacterial Biofilms.
Santosh Pandit,Vaishnavi Ravikumar,Alyaa M. Abdel-Haleem,Abderahmane Derouiche,V. R. S. S. Mokkapati,Carina Sihlbom,Katsuhiko Mineta,Takashi Gojobori,Xin Gao,Fredrik Westerlund,Ivan Mijakovic +10 more
TL;DR: The quantitative proteomics analysis shows that non-lethal concentrations of vitamin C inhibit bacterial quorum sensing and other regulatory mechanisms underpinning biofilm development, and that the EPS biosynthesis in reduced, and especially the polysaccharide component of the matrix is depleted.
Journal ArticleDOI
Defects in the Flagellar Motor Increase Synthesis of Poly-γ-Glutamate in Bacillus subtilis
TL;DR: It is shown that B. subtilis requires only the MotA/MotB stator during swarming motility and that the residues required for stator force generation are highly conserved from the Proteobacteria to the Firmicutes.
Journal ArticleDOI
Dissecting and engineering metabolic and regulatory networks of thermophilic bacteria for biofuel production
TL;DR: Recent advances in dissecting and engineering the metabolic and regulatory networks of thermophilic bacteria for improving the traits of key interest in biofuel industry: cellulose degradation, pentose-hexose co-utilization, and tolerance of thermal, osmotic, and solvent stresses are reviewed.
References
More filters
BookDOI
Mobile DNA III
Nancy L. Craig,Michael Chandler,Martin Gellert,Alan M. Lambowitz,Phoebe A. Rice,Suzanne Sandmeyer +5 more
TL;DR: This new edition of the bestselling series on movable genetic elements highlights the many exciting advances in the field over the last decade, including conservative site-specific recombination, programmed rearrangements, DNA-only transposons, and LTR, and non-LTR retrotransposons.
Journal ArticleDOI
Fruiting body formation by Bacillus subtilis
Steven S. Branda,José Eduardo González-Pastor,Sigal Ben-Yehuda,Richard Losick,Roberto Kolter +4 more
TL;DR: Fruiting body formation depended on regulatory genes required early in sporulation and on genes evidently needed for exopolysaccharide and surfactin production, an indication that multicellularity has been lost during domestication of B. subtilis.
Journal ArticleDOI
Lactose genes fused to exogenous promoters in one step using a Mu-lac bacteriophage: in vivo probe for transcriptional control sequences
TL;DR: The lactose structural genes, without the lactose promoter, have been incorporated into the bacteriophage Mu genome to form a Mu-lac specialized transducing phage as discussed by the authors.
Journal ArticleDOI
Antibiotic-resistance cassettes for Bacillus subtilis
TL;DR: The genes encoding resistance to four different antibiotics were cloned in the polylinker of various Escherichia coli plasmid vectors to create tagged chromosomal disruptions after recombination into Bs and selection in the presence of the appropriate antibiotic.
Journal ArticleDOI
Nucleotide sequence and functional map of pE194, a plasmid that specifies inducible resistance to macrolide, lincosamide, and streptogramin type B antibodies.
TL;DR: By means of site-specific cleavage with restriction endonucleases and cloning resultant fragments, determinants of the two major biological functions of p E194, i.e., inducible MLS resistance and replication, could be localized and assigned to specific sequences in the plasmid.