Transposon-facilitated chromosome mobilization in Agrobacterium tumefaciens

Abstract

We improved chromosomal gene transfer in Agrobacterium tumefaciens strain 15955 by constructing donors containing homologous transposons on both the sex factor plasmid and chromosome. First, we constructed plasmid pDP35, a kanamycin-sensitive derivative of R68.45. We then constructed derivatives of pDP35 that contained insertions of the kanamycin resistance transposon Tn5. By restriction endonuclease analysis, we identified two plasmids, pDP37 and pDP38, in which Tn5 was inserted in the same region of the plasmid but in opposite orientations. We also constructed isolates of A. tumefaciens containing an insertion of Tn5 in the chromosome. We transferred pDP37 or pDP38 into these chromosomal Tn5 strains and tested their ability to mobilize chromosomal markers to a series of auxotrophic recipients. Mobilization was observed at frequencies ranging from 10(-4) to 10(-7) recombinants per input donor for most markers tested. Both the plasmid and the chromosomal Tn5 elements were found to be required for mobilization at these higher frequencies. Donors were shown to transfer chromosomal markers in a polarized fashion. Recombinants coinherited unselected markers at frequencies of from 100 to 0.3 percent. The improved transfer frequencies and the observed polarity in chromosome transfer suggest that with this method we can genetically characterize A. tumefaciens chromosomal functions.

Studies on Inc-P plasmids inErwinia carotovorasubsp.carotovora

“…Frequency of transfer of R-factor (pMO850) from P. aeruginosa to E. carotovora strains was similar to R18-1 transfer reported earlier, but was at least 3 orders of magnitude higher than RP1 and R91 [5]. Unhke pMO850, the transfer of pDP37, another derivative of R68.45 [33], was not detected in E. caroto•ora (data not shown). Plasmid pJB4JI, a hybrid plasmid of RP4 and Mu containing Tn5, was transferred from E. coil to E. carotovora at a frequency of 5 x 10 -6 per donor cell, confirming results reported by Zink et al [35].…”

“…The conjugative gene transfer ability of many plasmids is enhanced if they contain a sequence of DNA homologous to a DNA sequence present on the bacterial chromosome [15]. This has been demonstrated by insertion of transposons Tnl, Tn501 and Tn5 to both chromosome and plasmid DNA [21,31,33]. In an attempt to determine if RP4 and R751 can mobilize chromosomal markers in E. carotovora when a homologous DNA sequence is present on both the plasrnid and the bacterial chromosome, the following experiments were per- Table 4 Conjugal transfer frequencies of pLAFR1/pRK2013 binary plasmid system for E. carotovora Mating was performed as described in MATERIALS AND METHODS.…”

Characterization of transposon Tn5-facilitated donor strains and development of a chromosomal linkage map for Agrobacterium tumefaciens

“…In strain 15955, however, this plasmid showed inefficient mobilizing activity (13; D. L. Pischl and S. K. Farrand, unpublished data). We therefore investigated whether a transposon-facilitated mobilizing system would demonstrate more efficient gene transfer activity (26). We constructed donor strains that contain a copy of TnS in both the sex factor plasmid (a derivative of R68.45) and in the chromosome (26).…”

“…We therefore investigated whether a transposon-facilitated mobilizing system would demonstrate more efficient gene transfer activity (26). We constructed donor strains that contain a copy of TnS in both the sex factor plasmid (a derivative of R68.45) and in the chromosome (26). In addition to an improved transfer frequency, such donor strains have acquired the ability to transmit the chromosome in a polarized fashion.…”

“…Strains and plasmids are listed in Table 1. The construction of derivatives of strain SA101 harboring chromosomal insertions of TnS has been described previously (26). These isolates were assigned sequential strain numbers beginning with SA5000.…”