trfA Gene Research Articles

Analysis of the trfA region of broad host-range plasmid RK2 by transposon mutagenesis and identification of polypeptide products

Broad host-range plasmid RK2 is a member of the Escherichia coli incompatibility group P. Unlike most other groups of plasmids, members of the P group are capable of efficient transfer between and maintenance in most gram-negative bacterial species. It is of interest whether this broad host-range results from differences between the mechanism of replication of broad and narrow host-range plasmids. The regions of RK2 required for replication in E. coli have previously been defined as an origin of vegetative replication, oriV RK2, and a gene, trfA, specifying a positively required trans-acting product. In this study Tn1723 transposon insertions have been used to map the trfA gene and determine its functional gene product. The Tn1723 insertions define the outer limits of the gene, a promoter region, a “leader” region not essential for trfA activity and a coding region. Three polypeptides of 13 × 10 3, 43 × 10 3 and 32 × 10 3 molecular weight are produced from this region and the production of a 32 × 10 3M r polypeptide is shown to be correlated with trfA activity in E. coli. Analysis of polypeptides produced from transposon insertion derivatives in which all but 35 base-pairs of inserted DNA is deleted, along with the effect of these insertions on trfA activity, suggest that the 43 × 10 3 and 32 × 10 3M r polypeptide coding sequences overlap in the same reading frame and that all three polypeptides (13 × 10 3, 32 × 10 3 and 43 × 10 3M r) may be translated from the same initial transcript.

Essential genes of plasmid RK2 in Escherichia coli: trfB region controls a kil gene near trfA.

Plasmid RK2 encodes several kil determinants whose lethal action on Escherichia coli host cells is prevented by RK2 kor genes. Here we show that the mini-RK2 plasmid, pRK248, specifies a kilB component (kilB1) in the region of the replication gene trfA. kilB1 is different from trfA and is completely encoded within the pRK248 HaeII A fragment. Transformation of E. coli cells with hybrid plasmids containing the cloned kilB1 determinant is very inefficient and results in the selection of variant kil- plasmids, many of which show genetic and physical evidence of deletions. If another pRK248 gene (korB1) is present in the cells, kilB1+ plasmids can be established at high efficiency and without any detectable changes. KorB1 is encoded by the trfB region of pRK248 because recombinant plasmids with this region are able to control kilB1 in trans. These results substantiate our earlier explanation for the structure of pRK248 and for the perplexing requirement of the trfB region in this plasmid.