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

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.