Identification and characterization of the products from the traJ and traY genes of plasmid R100

Abstract

The nucleotide sequence of part of the tra region of R100 including traJ and traY was determined, and the products of several tra genes were identified. The nucleotide sequence of traJ, encoding a protein of 223 amino acids, showed poor homology with the corresponding segments of other plasmids related to R100, but the deduced amino acid sequences showed low but significant homology. The first four amino acids at the N-terminal region of the TraJ protein were not essential for positive regulation of expression of traY, the first gene of the traYZ operon. The nucleotide sequence of traY shows that this gene may use TTG as the initiation codon and that it encodes a protein of 75 amino acids. Analysis of the traY gene product, which was obtained as the fusion protein with ,l-galactosidase, showed that the N-terminal region of the product has an amino acid sequence identical to that deduced from the assigned frame but lacks formylmethionine. traY of plasmid F, which encodes a larger protein than the TraY protein of R100, is thought to use ATG as an initiation codon. However, a TTG initiation codon was found in the preceding region of the previously assigned traY coding frame of F. Interestingly, when translation of traY of F was initiated from TTG, the amino acid sequence homologous to the TraY protein of R100 appeared in tandem in the TraY protein of F. This may suggest that traY of F has undergone duplication of a gene like the traY gene of R100.Resistance (R) plasmids such as R100 and Rl, belonging to the FII incompatibility group (4), confer conjugal transfer ability, a process in which DNA is transmitted from one bacterial host to another; this requires cell-to-cell contact and synthesis of sex pili by donor bacteria (for reviews, see references 21, 47, and 48). These plasmids share homology with the fertility factor, F, in the tra region, which is responsible for DNA transfer (41) and which is about 33 kilobase pairs in length. The tra region of F contains at least 26 genes which are organized into three main operons, traM, traJ, and traYZ (2, 19; for a review, see reference 21). Transcription of the tra YZ operon in F and Rl has been shown to be positively regulated at promoter pyz by the product of traJ (8,12,13,16,32). Expression of traJ itself is repressed by the fertility inhibition complex, FinOP, which is composed of the products of two genes, finO and finP, encoded by the R plasmids and F (11,16,32). The initial event in DNA transfer upon expression of the tra YZ operon is strand-and site-specific nicking at the origin of transfer, oriT, by the plasmid-specified endonuclease, which is thought to be a complex of the products of tra Y and traZ (5).In this paper, we report the nucleotide sequence of the promoter-proximal region of tra of R100, including traJ and traY, and the identification of gene products of this region. The plasmids used were R100 (33), pUC19 (49), and pJG200 (17). The pSI plasmids used, except pSI200, are listed in Fig. 1. They were constructed as described below.Construction of pSI ...

Swapping single-stranded DNA sequence specificities of relaxases from conjugative plasmids F and R100

“…The relaxosome may serve to generate the single-stranded DNA (ssDNA) substrate that the relaxase targets. Relaxases cleave ssDNA in a Mg 2ϩ -dependent transesterification reaction that proceeds through a stable phosphotyrosyl intermediate (9)(10)(11)(12)(13)(14)(15). At the end of conjugative transfer, relaxases are thought to ligate the plasmid ends together (9,(16)(17)(18)(19).…”

Large Scale Purification and Characterization of TraI Endonuclease Encoded by Sex Factor Plasmid R100

“…Plasmids, pUC119 (Vieira and Messing, 1987), pSI87-XE1 (Inamoto et al, 1988), and pYY35-1 (Inamoto et al, 1991), were derivatives of pUC18 or pUC19 (Yanisch-Perron et al, 1985) with a DNA segment of plasmid R100. Plasmid pHF11 was constructed by inserting a 236-bp BamHI fragment containing oriT of R100 into pUC18.…”

“…The TraI protein encoded by plasmid F or R100 has been purified by monitoring its ATPase activity or by its molecular size, respectively. The TraI proteins purified introduce the strand-and site-specific nick, such that it is covalently linked with the 5Ј end of the nick (Inamoto et al, 1991(Inamoto et al, , 1994Reygers et al, 1991;Matson and Morton, 1991). The TraI proteins have been known to be DNA helicase (AbdelMonem and Hoffman-Berling, 1976;Inamoto et al, 1994), which is supposed to unwind the duplex DNA from the nick introduced in the plasmid to provide the single-stranded DNA (Abdel-Monem et al, 1983), which is known to be transferred to the recipient cell (Ohki and Tomizawa, 1968;Rupp and Ihler, 1968).…”

The finM promoter and the traM promoter are the principal promoters of the traM gene of the antibiotic resistance plasmid R100

“…VAR23 and VAR26 are R100-1 mutants carrying deletions in the traY gene. In our laboratory co-ordinates of the traY gene (based on the data of Inamoto et al, 1988), the traY ORF extends from base number 2667 to 2894(UAG). VAR23 is deleted from base 2688 to 2752.…”