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

Abstract

* Office of theCassettes 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 (PtaC) and the trpA terminator ( T , ) and then cloned between Not1 sites to construct the CAS-Nm (Ptac-npt//-TtpA) and CAS-Gm ( Pt , CP, , , c/ -aacC/ -T, )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 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 IncPl plasmid. This construct will enable transcriptional activity to be monitored in different genetic backgrounds. Each cassette was cloned as a Not1 fragment into the Not1 site of a PUT derivative to construct four minitransposons. The mTn5-Nm (containing Ptac-npt//-TtwA) and mTn5-Gm (containing P, CP, , , , -aacC/ -TtwA) minitransposons have been constructed specifically for insertional inactivation studies. The minitransposons mTn5-GNm (containing gusA-P,,-npt / / -T, ) and mTn5-GGm (containing gusA-Pt,~P,,,-dacC/-TtpA) can be used for transcription signal localization or insertional inactivation. The TAC-31 R 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 Gramnegative bacteria.CAS-G Nm (gUSA-Pt,,-npt//-TtpA) or CAS-GGm (gUSA-Pt,CP,,,c/-da~CI-TtpA)

The plant signal salicylic acid shuts down expression of the vir regulon and activates quormone-quenching genes in Agrobacterium

“…A348 contains the same genome components as C58 except for the Ti plasmid, which originated from strain A6. The virB1 promoter was amplified and cloned into the pFUS1 vector to generate a virB1::gusA transcriptional gene fusion (36). Plasmid pWT160 (37) was used to measure virG expression.…”

Disruption of sitA Compromises Sinorhizobium meliloti for Manganese Uptake Required for Protection against Oxidative Stress

“…Transductions with M12 were performed as described previously (15). Random mini-Tn5-GusNm (mTn5) mutagenesis and triparental matings were performed as described previously (13,37). The protocols of Sambrook and Russell (39) were used for routine manipulations of plasmid and chromosomal DNAs.…”

Identification and analysis of a siderophore biosynthetic gene cluster from Agrobacterium tumefaciens C58

“…Amplification primers were designed containing PstI restriction enzyme recognition sites at the ends, and they were digested with PstI and cloned into pJQ200mp18 (Quandt & Hynes, 1993) digested with PstI, generating pMTA1 and pMTA2 respectively. pCRS487 (Reeve et al, 1999) was digested with NotI, and the gusA-nptII cartridge from this plasmid was cloned directly into the NotI site in pMTA1 or was blunt-ended with Klenow enzyme and cloned into the blunted XhoI site in pMTA2. pMTA5 was generated by digesting pHP45V (Prentki & Krisch, 1984) with SmaI and cloning the spectinomycin resistance cassette into blunted NotI-digested pMTA1.…”

“…Plasmid pCRS538 (Reeve et al, 1999) was used to mutagenize strain MTA100. The plasmid was delivered by conjugation from E. coli S17-1, and exconjugants were selected using gentamicin and kanamycin.…”