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 nptll (encoding kanamycin resistance) or aacCl (encoding gentamicin resistance) genes were equipped with the tac promoter (P tac ) and the trpA terminator (T trpA ) and then cloned between NotI sites to construct the CAS-Nm (P tac -nptll-T trpA ) and CAS-Gm (P tac /P aacCl -aacCl-T trpA ) cassettes. The markers were also cloned downstream to a modified promoterless Escherichia coli gusA gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (gusA-P tac -nptll-T trpA ) or CAS-GGm (gusA- P tac /P aacCl -aacCl-T trpA ) cassettes. Cassettes containing the promoterless gusA create type I fusions with a target DNA sequence to detect transcriptional 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 was cloned as a NotI fragment into the NotI site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing P tac -nptll-T trpA ) and mTn5-Gm (containing gusA-P tac -nptll-T trpA ) and mTn5-GGm (containing gusA-P tac /P aacCl -aacCl-T trpA ) can be used for transcription signal localization or insertional inactivation. The TAC-31R and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-Nm, CAS-Gm, mTn5-Nm and mTn5-Gm. The WIL3 and TAC-105F primers can be used to sequence DNA flanking both sides of CAS-GNm, CAS-GGm, 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, transcriptional signal localization and gene regulation studies in the root nodule bacteria and possibly other Gram-negative bacteria.