Analysis of F transfer genes involved in F-pilus biogenesis in Escherichia coli K12 : the traQ region

Abstract

Genes and gene products in the transfer region of the Escherichia coli plasmid F were investigated. A detailed restriction map of F DNA in the traB-traG region was constructed, and a series of plasmids expressing SmaI, HincII, and PstI fragments from this region were obtained. The tra activities and products of these clones were identified. The traQ gene, known to express an activity for processing of F traA product to F-pilin was of particular interest. Functional analyses showed that traQ was contained in a 1.2 kilobase (kb) HincII-SmaI fragment. The DNA sequence of a 1.5 kb PstI-SmaI fragment with included this region was obtained. DNA from an F traFl3 amber mutant contained a C -> T transition in base 58 of the sequenced region, indicating that a 127 amino acid open reading frame in the promoter proximal portion of the sequenced segment specified the carboxy-terminal portion of TraF protein. In vitro deletion mutants were constructed to affect the three additional open reading frames specified by the sequence. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of S³⁵-methionine labelled proteins synthesized in maxi-cells containing the deletion mutants showed that expression of a polypeptide migrating at an apparent molecular weight (M[subscript a]) of 12,500 was associated with traQ activity. This polypeptide derived from a segment of the DNA sequence which encoded a 94 amino acid (10,867 dalton) protein, defining traQ. Three additional transfer genes, trbA, artA, and trbB were also designated by these analyses. The DNA sequence shows that the trbA gene product (M[subscript a] 14,200 on SDS-PAGE) is a 115 amino acid (12,947 dalton) polypeptide and that the artA gene specifies a 104 amino acid (12,133 dalton) protein. The trbB gene is located in the region immediately downstream from traQ and encodes an M[subscript a] 18,400 protein detected by SDS-PAGE. Interestingly, the artA gene is transcribed in the opposite direction from traQ, and has been found to have its own promoter. The functional role of the trbA, trbB, and artA products, and other products found to stem from the traC-F interval will require further investigation.