Replication of an origin-containing derivative of plasmid RK2 dependent on a plasmid function provided in trans

Abstract

pRK212.2, a derivative of the broad host range plasmid RK2, contains two EcoRI cleavage fragments, A and B, neither of which can replicate by itself in Escherichia coli. Fragment A (41.7 kilobases), but not fragment B (14.4 kilobases), can be cloned by insertion into the unrelated plasmids mini-F and ColEl. Fragment B contains the origin of replication and the ampicillin-resistance determinant of RK2. Transformation of E. coli cells containing the mini-F-fragment A hybrid plasmid with fragment B DNA results in the recircularization and replication of fragment B as a nonmobilizable plasmid (pRK2O67) with the copy number and incompatibility properties of RK2. Fragment B cannot be cloned in the absence of fragment A because the latter fragment suppresses a function, specified by fragment B, that results in loss of host cell viability. A small segment (2.4 kilobases) of fragment B that contains the RK2 origin of replication but no longer affects host cell growth in the absence of fragment A had been cloned previously by insertion into a ColEl plasmid. This hybrid plasmid, designated pRK256, will replicate in E. coli polA mutants only when a fragment A-bearing helper plasmid is present. These results demonstrate that the potentially lethal function specified by fragment B of RK2 is not necessary for replication and that at least one trans-acting function is directly involved in RK2 replication.Bacterial plasmids of the P-I incompatibility group (Inc P-1) are capable of replication in a wide range of Gram-negative bacteria (1-4). We have been examining the replication properties of RK2, an Inc P-1 antibiotic-resistance plasmid (5), in an effort to determine the molecular basis of its broad host range. RK2 [56.4 kilobases (kb)] is self-transmissible and codes for resistance to kanamycin (Km), ampicillin (Ap), and tetracycline (Tc) (5, 6). In Escherichia coli the molecule replicates unidirectionally from a fixed origin located approximately 12 kb from the single EcoRI cleavage site (7). From studies of RK2 molecules manipulated in tntro with restriction endonucleases, it appears that the genes essential for replication and maintenance in E. coli are distributed over a 24-kb portion of the plasmid that includes the origin of replication (ref. 8; unpublished data). This finding is in contrast to the considerably smaller replication regions of plasmids of other incompatibility groups that appear to have a more limited host range than does RK2.To locate and characterize individual genes required for replication, the RK2 essential region has been further subdivided by the construction of hybrid plasmids carrying RK2 genes on unrelated replicons. In this paper, we report that at least one RK2 function can act in trans to permit the replication and maintenance of a recircularized fragment of RK2 that contains the RK2 origin of replication. This origin-containing region is a defective replicon but can be maintained in the cell as a separate covalently closed circular (CCC) molecule in the presence of the cloned gene(s...

Transformation of Azospirillum brasilense Cd with an ACC Deaminase Gene from Enterobacter cloacae UW4 Fused to the Tet r Gene Promoter Improves Its Fitness and Plant Growth Promoting Ability

“…Plasmid DNA was transferred from E. coli DH5a to A. brasilense Cd by triparental mating using pRK2013 [14] in E. coli HB101 as the helper plasmid. The construct pRKTACC (the ACC deaminase gene under the control of the Tet r promoter cloned in pRK415) was transferred from E. coli DH5a to A. brasilense Cd as described previously [25].…”

The Mechanism of Killing by the Proline-Rich Peptide Bac7(1–35) against Clinical Strains of Pseudomonas aeruginosa Differs from That against Other Gram-Negative Bacteria

“…The resulting plasmid, psbmA1, was mobilized from E. coli into P. aeruginosa PAO1 via triparental mating with the helper strain of E. coli (pRK2013) (44). Transconjugants were then selected on LB agar in the presence of 300 g/ml tetracycline, 100 g/ml ampicillin, and 25 g/ml nalidixic acid.…”

Identification, characterization of levoglucosan kinase, and cloning and expression of levoglucosan kinase cDNA from Aspergillus niger CBX-209 in Escherichia coli

“…The minimum number of transformants needed for the cloning of a specific gene is calculated according to the following equation: N ¼ lnð1 À P Þ= lnð1 À f Þ [39], in which P refers to the probability that a random cDNA fragment appears in a cDNA bank; f is the size ratio of a insert fragment to the whole genome; and N is the total number of transformants. Since the genome of A. niger is about 10,000 kb, in a cDNA bank with an average fragment size of approx.…”