Summary auto-generated
This research article describes the development of genetic tools for studying gene regulation in root nodule bacteria, particularly Sinorhizobium meliloti. The researchers constructed several cassettes and minitransposons containing antibiotic resistance markers (kanamycin or gentamicin) combined with a promoterless gusA reporter gene encoding β-glucuronidase. The key innovation was modifying the gusA gene with stop codons in all three reading frames to ensure proper translational fusion detection. These constructs were designed for insertional mutagenesis and transcriptional signal localization studies. Four main minitransposon variants were created (mTn5-Nm, mTn5-Gm, mTn5-GNm, and mTn5-GGm) with reduced restriction sites to facilitate cloning. A broad-host-range promoter probe vector (pFUS1) was also developed using an IncP1 plasmid backbone. The constructs were successfully tested in S. meliloti, revealing acid-inducible gene fusions. The researchers demonstrated that these tools can identify genes regulated by specific environmental signals, such as pH-responsive phrR promoter activity. The minitransposons exhibited transposition rates of 1×10⁻⁵ and maintained antibiotic resistance at high concentrations, making them valuable for mutagenesis studies in Gram-negative bacteria.
Key findings
- Novel cassettes and minitransposons were constructed with reduced restriction sites, combining promoterless gusA reporter genes with kanamycin or gentamicin resistance markers for insertional mutagenesis and transcriptional analysis in root nodule bacteria.
- A broad-host-range promoter probe vector (pFUS1) was developed based on IncP1 plasmid backbone to enable monitoring of transcriptional activity across different bacterial genetic backgrounds.
- The minitransposons demonstrated efficient transposition rates of 1×10⁻⁵ in S. meliloti and Bradyrhizobium species, with antibiotic resistance maintained at high concentrations up to 1000 μg/mL for kanamycin.
- Acid-inducible promoter fusions were successfully identified and characterized, particularly the phrR gene which showed fivefold induction at pH 5.7 compared to pH 7.0 in S. meliloti WSM419.
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Abstract
Cassettes have been developed that contain an antibiotic resistance marker with and without a promoterless gusA reporter gene. The nptII (encoding kanamycin resistance) or aacCI (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)-nptII-T(trpA)) and CAS-Gm (P(tac)/P(aacCI)-aacCI-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)-nptII-T(trpA)) or CAS-GGm (gusA- P(tac)/P(aacCI)-aacCI-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)-nptII-T(trpA)) and mTn5-Gm (containing P(tac)/P(aacCI)-aacCI-T(trpA)) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-P(tac)-nptII-T(trpA)) and mTn5-GGm (containing gusA-P(tac)/P(aacCI)-aacCI-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.