The osa gene of pSa encodes a 21.1-kilodalton protein that suppresses Agrobacterium tumefaciens oncogenicity

Abstract

The incompatibility group W plasmid pSa suppresses Agrobacterium tumefaciens oncogenicity (J. Loper and C. Kado, J. Bacteriol. 139:591-596, 1979). The oncogenic suppressive activity was localized to a 3.1-kb region of pSa by TnS mutagenesis and deletion analysis. Within this fragment, a 1.1-kb subclone bearing oncogenic suppressive activity was subjected to further characterization. Nucleotide sequencing of the 1.1-kb fragment revealed a 570-bp open reading frame (ORF1) that has a coding capacity for a protein of 21.1 kDa. Sequencing of flanking regions revealed a second ORF (ORF2) located 3 bp upstream of ORF1, with a coding capacity for a protein of 22.8 kDa. Gene fusions of these ORFs to a T74i10 expression system in Escherichia coli resulted in the synthesis of polypeptides of the predicted sizes. An E. coli promoter consensus sequence was not found in the expected positions in the region preceding ORF1. However, several sequences with similarity to the consensus -10 sequence of the A. tumefaciens vir gene promoters were found upstream of ORF1. Potential translational start signals are upstream of ORF1 and ORF2. These sequences showed no significant similarity at the nucleotide or amino acid levels with those in available data bases. However, the C-terminal portion of the ORF1 protein is rich in hydrophobic residues. Perhaps oncogenicity suppression is effected by an association of this protein with the Agrobacterium membrane such that T-DNA transfer is blocked.The 39-kb IncW plasmid pSa, originally isolated from Shigella flexneri (49), confers resistance to streptomycin, chloramphenicol, kanamycin, spectinomycin, and sulfonamides (22) and is capable of self-mobilization and replication in a wide range of gram-negative bacteria (45). An interesting trait of pSa is its ability to completely abolish the oncogenic activity of Agrobacterium tumefaciens (32). The plasmid also prevents hairy-root-forming ability of the related species A. rhizogenes (47). As long as pSa is present in the bacterium, oncogenic suppression by pSa is absolute. The presence of pSa does not cause instability of the oncogenic Ti plasmid, and oncogenicity is regained when the strain is cured of pSa (15). Also, pSa does not block conjugative transfer of the Ti plasmid, and when the Ti plasmid is transferred from a pSa-bearing strain to a recipient strain, oncogenicity is conferred on the recipient (15).To identify the region of pSa that confers oncogenic suppressive (OS) activity Zaitlin (54) tested several subclones of pSa and located the OS activity to a 7.6-kb fragment cloned in pUCD111. The number of loci and location of these loci which conferred OS activity were not defined, however. Furthermore, the function of the products of the OS locus remained unknown. We have shown, however, that OS activity is unrelated to bacterial auxin production (24), which was proposed by Chernin et al. to be involved in tumorigenesis (8). Also, induction of the virulence genes of the Ti plasmid and T-DNA border cleavage do occur in the presence of pSa (9...

Osa Protein Constitutes a Strong Oncogenic Suppression System That Can Block vir -Dependent Transfer of IncQ Plasmids between Agrobacterium Cells and the Establishment of IncQ Plasmids in Plant Cells

“…The RSF1010 derivatives pJB31 and pJW323 were shown previously to suppress tumorigenesis by the octopine-type strain A348 on N. tabacum (tobacco) and K. daigremontiana (4,33), whereas pSa inhibited transformation of numerous plant species by both strain C58 (7,9,11,23) and strain A208 (19), which contain a nopalinetype Ti plasmid, and by strains A348 (19) and 1D1 (11), which contain an octopine-type Ti plasmid. Oncogenic suppression by pSa was more effective on A. tumefaciens C58 than suppression of strain 1D1 was (11).…”

“…The osa (oncogenic suppressive activity) gene of pSa is sufficient to inhibit plant transformation (7,9). This phenomenon, called oncogenic suppression, resembles fertility inhibition of conjugative plasmid transfer by plasmids of different incompatibility groups (16,24,27).…”

Inhibition of Agrobacterium tumefaciens oncogenicity by the osa gene of pSa

“…osa is part of an operon. Previous sequence analyses of the 3.1-kb DNA fragment revealed the presence of four orfs, one of which was osa (6,7). No obvious promoters or potential binding sites for a positive regulator (e.g., inverted repeats) were found in the regions where promoters are usually found with respect to the translational start site of each ort.…”

“…orf-3 is followed by the 570-bp osa gene. Although previous studies have indicated that osa plays a major part in oncogenic inhibition (6), the contribution of each upstream gene, such as nuc, to this activity has remained unclear.…”

“…The osa gene is located between the region containing conjugative transfer genes (trw genes) and the region containing the repA and recA genes and the origin of plasmid DNA replication (orn) (6,35). There are three orfs upstream of osa in the 3.1-kb pSa segment conferring oncogenic suppression, the most distal being orf-1 (630 bp), which encodes a 23.2-kDa polypeptide, followed by the 525-bp nuc gene, which encodes a 19.7-kDa DNase (7), and orf-3 (603 bp), which encodes a 22.8-kDa polypeptide of unknown function.…”