scholarly journals Construction of a Derivative of Agrobacterium tumefaciens C58 That Does Not Mutate to Tetracycline Resistance

2001 ◽  
Vol 14 (1) ◽  
pp. 98-103 ◽  
Author(s):  
Zhao-Qing Luo ◽  
Thomas E. Clemente ◽  
Stephen K. Farrand
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Host Range ◽  
High Frequency ◽  
Tetracycline Resistance ◽  
Physiological Traits ◽  
Selection Marker ◽  
Detectable Effect ◽  
Broad Host Range ◽  
Binary Vectors ◽  
Agrobacterium Tumefaciens C58

Agrobacterium tumefaciens C58 mutates to tetracycline resistance at high frequency, complicating the use of many broad-host-range cloning and binary vectors that code for resistance to this antibiotic as the selection marker. Such mutations are associated with a resistant gene unit, tetC58, that is present in the genome of this strain. By deleting the tetC58 locus, we constructed NTL4, a derivative of C58 that no longer mutates to tetracycline resistance. The deletion had no detectable effect on genetic or physiological traits of NTL4 or on the ability of this strain to transform plants.

Mutagenesis and chromosome mobilization in Hyphomicrobium facilis B-522

1992 ◽  
Vol 38 (11) ◽  
pp. 1167-1174 ◽  
Author(s):  
Christian G. Gliesche ◽  
Peter Hirsch
Keyword(s):  
High Frequency ◽  
Tetracycline Resistance ◽  
Transposon Mutagenesis ◽  
Chemical Mutagenesis ◽  
Direct Selection ◽  
Broad Host Range ◽  
Antibiotic Resistant ◽  
Auxotrophic Mutants ◽  
Restricted Facultative Methylotroph ◽  
Insertion Mutants

Spontaneously derived antibiotic-resistant mutants of Hyphomicrobium facilis B-522, a restricted facultative methylotroph, occurred at a high frequency on agar plates with low antibiotic concentrations. Mutants specifically defective in methanol oxidation have been obtained using an allyl alcohol direct selection technique. By chemical mutagenesis with N-methyl-N′ -nitro-N-nitrosoguanidine in the presence of chloramphenicol several stable auxotrophic mutants could be isolated: three leucine auxotrophs, two threonine auxotrophs, and two leucine–methionine double auxotrophic mutants. Optimal conditions for transposon mutagenesis have been developed by comparing several transposon delivery vectors. With the suicide plasmid pRK2013 as a vector, the tetracycline resistance conferring transposon Tn5-132 was introduced into the genome of H. facilis B-522. The following insertion mutants have been obtained: leu-3::Tn5-132, ilv-1::Tn5-132, and pur-1::Tn5-132. Broad host range IncP-1 plasmids could be successfully transferred by interspecific matings. Chromosome mobilization was demonstrated with the conjugative IncP-1 plasmids RP1, R68.45, pMO60, and H. facilis 2189 (leu-2, met-1, mox-1, nfs-1, str-12) as recipient strain. Transconjugants occurred at frequencies ranging from 10−6 to 10−8 for each marker. Key words: methylotrophs, Hyphomicrobium, N-methyl-N′-nitro-N-nitrosoguanidine mutagenesis, transposon mutagenesis, promiscuous plasmids, chromosome mobilization.

scholarly journals Cloning and Characterization of a Tetracycline Resistance Determinant Present in Agrobacterium tumefaciens C58

1999 ◽  
Vol 181 (2) ◽  
pp. 618-626 ◽  
Author(s):  
Zhao-Qing Luo ◽  
Stephen K. Farrand
Keyword(s):  
Agrobacterium Tumefaciens ◽  
Promoter Region ◽  
Tetracycline Resistance ◽  
Homologous Region ◽  
Polycyclic Compounds ◽  
Repressor Proteins ◽  
Repressive Effect ◽  
Agrobacterium Tumefaciens C58 ◽  
Spontaneous Mutants

ABSTRACT Agrobacterium tumefaciens C58 and its derivatives give rise to spontaneous mutants resistant to tetracycline at a high frequency. We observed that a mutation affecting a tRNA processing function significantly affected the emergence of such mutants, suggesting that C58 contained a positively acting gene conferring resistance to tetracycline. A cosmid clone conferring resistance to tetracycline in Escherichia coli andAgrobacterium was isolated from a genomic bank of one such mutant. Subcloning, transposon mutagenesis, and DNA sequence analysis revealed that this DNA fragment contained two divergently transcribed genes, tetA and tetR, encoding products that were very similar to proteins of the Tet(A) class of tetracycline resistance systems. In the clone from this mutant, tetR was disrupted by an IS426. The homologous region from wild-type NT1 contained an intact tetR gene and did not confer resistance to tetracycline. Hybridization analysis showed that of 22 members of the genus Agrobacterium surveyed, only strains C58 and T37 contained the tet determinant. Moreover, only these two strains mutated to resistance to this antibiotic. Unlike other Tet(A) systems, neither tetracycline nor a series of its derivatives induced the expression of this tet gene unit. Other polycyclic compounds, including many of plant origin, also did not induce this tet gene system. The divergent promoter region of this tet system contained a single inverted repeat element identical to one such operator repeat in the promoter region of the tet determinant from the IncP1α R plasmid RP4. TetR repressor proteins from the Agrobacterium tetsystem and from RP4 interacted with the heterologous operators. While the repressive effect of the TetR protein from strain C58 (TetRC58) on the tetA gene from strain RP4 (tetA RP4) was not relieved by tetracycline, repression of tetA C58 by TetRRP4was lifted by this antibiotic.

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