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

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 beta-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.     

Nucleotide sequences of five IncF plasmid finP alleles.

The nucleotide sequences of five finP alleles from various IncF plasmids (finP types I to V) as well as of three finP mutations were determined and compared. The finP gene specificity could be attributed to a variable, six-to-seven-nucleotide loop located between inverted repeats, and the sequence data were consistent with the product of finP being an RNA molecule rather than a protein. The finP mutations interrupted a proposed finP promoter or destabilized a predicted stem-and-loop structure in the finP RNA molecule.     

Origin of transfer of IncF plasmids and nucleotide sequences of the type II oriT, traM, and traY alleles from ColB4-K98 and the type IV traY allele from R100-1.

The complete nucleotide sequences of the ColB4-K98 (ColB4) plasmid transfer genes oriT, traM, and traY as well as the traY gene of R100-1 are presented and compared with the corresponding regions from the conjugative plasmids F, R1, and R100. The sequence encoding the oriT nick sites and surrounding inverted repeats identified in F was conserved in ColB4. The adenine-thymine-rich sequence following these nick sites was conserved in R1 and ColB4 but differed in F and R100, indicating that this region may serve as the recognition site for the traY protein. A series of direct repeats unique to the ColB4 plasmid was found in the region of dyad symmetry following this AT-rich region. This area also encodes 21-base-pair direct repeats which are homologous to those in F and R100. The traM gene product may bind in this region. Overlapping and following these repeats is the promoter(s) for the traM protein. The traM protein from ColB4 is similar to the equivalent products from F, R1, and R100. The traY protein from ColB4 is highly homologous to the R1 traY gene product, while the predicted R100-1 traY product differs at several positions. These differences presumably define the different alleles of traM and traY previously identified for IncF plasmids by genetic criteria. The translational start codons of the ColB4 and R100-1 traY genes are GUG and UUG, respectively, two examples of rare initiator codon usage.     

Specific binding of the TraY protein to oriT and the promoter region for the traY gene of plasmid R100

The traY gene product of plasmid R100 was purified as a hybrid protein, TraY-collagen-beta-galactosidase. The hybrid protein as well as the TraY' protein, which was obtained by collagenolysis of the hybrid protein, specifically binds to an AT-rich 36-base pair sequence (here called sbyA) within the region including the origin of transfer, oriT. The oriT region consists of highly conserved and nonconserved regions among R100-related plasmids, and sbyA was located within the nonconserved region immediately adjacent to the conserved region. This supports the idea that the TraY protein has a role as a component of endonuclease in recognizing its own oriT sequence. Unexpectedly, however, the hybrid protein and the TraY' protein were also found to bind to two different AT-rich sequences (each 24 base pairs in length) in the promoter region preceding the traY gene (here called sbyB and sbyC). This suggests that the TraY protein may have another role in regulating the expression of its own gene. The "TAA(A/T)T" sequence motif observed in these binding sites might constitute a core sequence recognized by the TraY protein. Mg2+ is not required for the specific binding of the TraY protein.     

Expression of F plasmid traT: independence of traY----Z promoter and traJ control

We have shown, using an F-derived Tra+ cosmid in conjunction with the infected-cell translational system and a time-course study, that one of the surface exclusion genes, traT, can be expressed independently of the promoter of the traY----Z operon, PYZ, and in the absence of a normal quantity of traJ gene product. Studies with deleted derivatives of the cosmid pRS2405 confirmed this independence and also indicated that expression of traD can be independent of PYZ. We propose that the expression of traT by these deleted plasmids is directed from a traJ-independent promoter, PT, located adjacent to traT.     

Nucleotide sequence and organization of genes flanking the transfer origin of promiscuous plasmid RP4.

The nucleotide sequence of the relaxase operon and the leader operon which are part of the Tra1 region of the promiscuous plasmid RP4 was determined. These two polycistronic operons are transcribed divergently from an intergenic region of about 360 bp containing the transfer origin and six close-packed genes. A seventh gene completely overlaps another one in a different reading frame. Conjugative DNA transfer proceeds unidirectionally from oriT with the leader operon heading the DNA to be transferred. The traI gene of the relaxase operon includes within its 3' terminal region a promoter controlling the 7.2-kb polycistronic primase operon. Comparative sequence analysis of the closely related IncP plasmid R751 revealed a similarity of 74% at the nucleotide sequence level, indicating that RP4 and R751 have evolved from a common ancestor. The gene organization of relaxase- and leader operons is conserved among the two IncP plasmids. The transfer origins and the genes traJ and traK exhibit greater sequence divergence than the other genes of the corresponding operons. This is conceivable, because traJ and traK are specificity determinants, the products of which can only recognize homologous oriT sequences. Surprisingly, the organization of the IncP relaxase operons resembles that of the virD operon of Agrobacterium tumefaciens plasmid pTiA6 that mediates DNA transfer to plant cells by a process analogous to bacterial conjugation. Furthermore, the IncP TraG proteins and the product of the virD4 gene share extended amino acid sequence similarity, suggesting a functional relationship.     

Gene 19 of plasmid R1 is required for both efficient conjugative DNA transfer and bacteriophage R17 infection.

F-like plasmids require a number of genes for conjugation, including tra operon genes and genes traM and traJ, which lie outside the tra operon. We now establish that a gene in the "leading region," gene 19, provides an important function during conjugation and RNA phage infection. Mutational inactivation of gene 19 on plasmid R1-16 by introduction of two nonpolar stop codons results in a 10-fold decrease in the conjugation frequency. Furthermore, infection studies with the male-specific bacteriophage R17 revealed that the phage is not able to form clear plaques in Escherichia coli cells carrying an R1-16 plasmid with the defective copy of gene 19. The total number of cells infected by phage R17 is reduced by a factor of 10. Both the conjugation- and infection-attenuated phenotypes caused by the defective gene 19 can be complemented in trans by introducing gene 19 alleles encoding the wild-type protein. Restoration of the normal phenotypes is also possible by introduction of the pilT gene encoded by the unrelated IncI plasmid R64. Our functional studies and similarities of protein 19 to proteins encoded by other DNA transfer systems, as well as the presence of a conserved motif in all of these proteins (indicative for a putative muramidase activity) suggest that protein 19 of plasmid R1 facilitates the passage of DNA during conjugation and entry of RNA during phage infection.     

A stable core region of the tra operon mRNA of plasmid R1-19.

The degradation of the polycistronic tra-mRNA of the resistance plasmid R1-19 leads to the accumulation of a well defined series of stable mRNA species. The majority of the most stable mRNAs contains the message for the traA gene only. The differently sized stable mRNAs possess a common 3'terminus within the traL gene but vary at their 5' ends. The 3'terminus probably results from protection against exoribonucleases by a secondary structural feature. We propose that the 5' ends are generated by endoribonucleolytic cleavage. The stability of this part of the tra-mRNA exceeds 30 minutes and probably increases the rate of expression of the traA gene product propilin, the precursor of the sex pilus subunit. The expression of propilin and its processing into a protein of the molecular weight of mature pilin is demonstrated with the isolated gene. The sequence of the so far unknown genes traL and traE of R1-19 is presented.