Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless reporter gene. The (encoding kanamycin resistance) or (encoding gentamicin resistance) genes were equipped with the promoter (P) and the terminator (T) and then cloned between I sites to construct the CAS-Nm (P--T) and CAS-Gm (P/P --T) cassettes. The markers were also cloned downstream to a modified promoterless gene (containing TGA stop codons in all three reading frames prior to its RBS and start codon) to construct the CAS-GNm (-P--T) or CAS-GGm ( P/P--T) cassettes. Cassettes containing the promoterless create type I fusions with a target DNA sequence to detect transcriptional activity. The promoterless 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 I fragment into the I site of a pUT derivative to construct four minitransposons. The mTn5-Nm (containing P--T) and mTn5-Gm (containing -P--T) and mTn5-GGm (containing -P/P--T ) 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.

Keyword(s): gusA , induction , phr genes , reporter and Rhizobium

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