Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria

Citation
Wg. Reeve et al., Constructs for insertional mutagenesis, transcriptional signal localization and gene regulation studies in root nodule and other bacteria, MICROBIO-UK, 145, 1999, pp. 1307-1316
Citations number
28
Categorie Soggetti
Microbiology
Journal title
MICROBIOLOGY-UK
ISSN journal
13500872 → ACNP
Volume
145
Year of publication
1999
Part
6
Pages
1307 - 1316
Database
ISI
SICI code
1350-0872(199906)145:<1307:CFIMTS>2.0.ZU;2-1
Abstract
Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kan amycin resistance) or aacCI (encoding gentamicin resistance) genes were equ ipped with the tac promoter (P-tac) and the trpA terminator (T-trpA) and th en cloned between NotI sites to construct the GAS-Nm (P-tac-nptII-T-trpA) a nd CAS-Cm (P-tac/P-aaccCI-aacCI-T-trpA) cassettes. The markers were also cl oned downstream to a modified promoterless Escherichia coli gusA gene (cont aining TGA step codons in all three reading frames prior to its RES and sta rt codon) to construct the GAS-CNm (gusA-P-tac-nptII-T-trpA) or CAS-CGm (gu sA- P-tac/P-aacCI-aacCI-T-trpA) cassettes. Cassettes containing the promote rless gusA create type I fusions with a target DNA sequence to detect trans criptional activity. The promoterless gusA gene has also been cloned into a broad-host-range IncP1 plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette w as cloned as a NotI fragment into the NotI site of a pUT derivative to cons truct four minitransposons. The mTn5-Nm (containing P-tac-nptII-T-trpA) and mTn5-Cm (containing P-tac/P-aacCI-T-trpA) minitransposons have been constr ucted specifically for insertional inactivation studies. The minitransposon s mTn5-GNm (containing gusA-P-tac-nptII-T-trpA) and mTn5-GCm (containing gu sA-P-tac/CPaacCI-T-trpA) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used t o sequence DNA flanking both sides of GAS-Nm, GAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sid es of CAS-GNm, CAS-GCm, mTn5-GNm and mTn5-GGm. The specific application of these constructs to generate acid- or nodule-inducible fusions is presented . The new constructs provide useful tools for insertional mutagenesis, tran scriptional signal localization and gene regulation studies in the root nod ule bacteria and possibly other Gram-negative bacteria.