Modified mariner Transposons for Random Inducible-Expression Insertions and Transcriptional Reporter Fusion Insertions in Bacillus subtilis

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-␤-Dthiogalactopyranoside (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.

Defects in the Flagellar Motor Increase Synthesis of Poly-γ-Glutamate in Bacillus subtilis

“…The sequence CCAACCTGT marks the end of the Mariner transposon insertion sequence. The next two bases are TA, according to the mechanism of mariner transposition, and mark the beginning of the chromosomal DNA specific to the insertion site (adapted from Pozsgai et al [114]). …”

The Activity of V, an Extracytoplasmic Function Factor of Bacillus subtilis, Is Controlled by Regulated Proteolysis of the Anti- Factor RsiV

“…Each pool was serially diluted, plated for single colonies on LB plus kanamycin, and grown overnight. The resulting colonies were replicate plated to LB plus X-Gal (100 g/ml), IPTG (1 mM), and lysozyme (20 g/ml) and incubated at 37°C for 6 h. Cells which were unable to induce the expression of P sigV -lacZ were isolated, and the location of the mutation determined by sequencing as previously described (44).…”

“…We performed a transposon screen to identify mutations which altered V activation in response to lysozyme. We utilized the transposon TnHyJump, a TnYLB transposon with an outwardfacing constitutive promoter, which allowed us to identify both null mutations and insertions which resulted in the overexpression of a gene downstream of the insertion site (44). A strain containing two copies of a P sigV -lacZ reporter was mutagenized with TnHyJump, and more than 20,000 colonies were screened for either increased or decreased P sigV -lacZ expression in the presence of lysozyme.…”

Synthesis, Release, and Recapture of Compatible Solute Proline by Osmotically Stressed Bacillus subtilis Cells

“…In the first of these bioassays, release of proline was manifested and visualized by the growth of a lac ϩ proline auxotrophic (proC46:: Tn5) E. coli strain as a blue halo forming around B. subtilis cells spotted onto a lawn of the indicator cells in the presence of the LacZ indicator dye X-Gal; B. subtilis does not possess a strong ␤-galactosidase activity (43). As assessed by this bioassay, the ⌬(opuE::tet)1 mutant BLOB9 released proline, whereas neither the opuE wild-type strain JH642 nor strain BLOB26, the abovedescribed derivative of BLOB9 carrying an extra copy of the intact opuE gene, was surrounded by a blue halo, indicative of proline excretion (Fig.…”