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Entry exclusion is a process whereby plasmid transfer between donor and recipient cells harboring identical or closely related conjugative plasmids is inhibited. Exclusion proteins in the recipient cells are responsible for entry exclusion. Although IncI1 Plasmid R64 and IncIγ plasmid R621a exhibit similar genome structure in replication, transfer, and leading regions, they belong to different incompatibility and exclusion groups. The amino acid sequences of TraY and ExcA proteins are significantly different between R64 and R621a. In the present study, TraY proteins of R64 and R621a were exchanged. Transfer of R64 derivative carrying R621a TraY was inhibited by recipient R621a ExcA but not R64 ExcA and transfer of R621a derivative carrying R64 TraY was inhibited by recipient R64 ExcA but not R621a ExcA. This indicates that R64 and R621a TraY proteins in the donor cells are the targets of cognate ExcA proteins in the recipient proteins. Since two segments, an internal and a C-terminal segment, were found to vary between R64 and R621a TraY proteins, various chimera TraY proteins were constructed. Conjugation experiments suggested that the R64 internal variable segment recognizes R64 ExcA protein and the R621a C-terminal variable segment recognizes R621a ExcA protein., (Copyright © 2012 Elsevier Inc. All rights reserved.)

We present the complete genome sequence of the tetracycline resistance plasmid R621a isolated from Salmonella typhimurium, which belongs to the incompatibility group Iγ. In the 93,185bp circular double-stranded R621a genome, 96 complete ORFs are predicted. In addition, one and six different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of four regions: replication, leading, transfer, and miscellaneous regions. The R621a genome is similar to those of IncI1 plasmids such as R64 and ColIb-P9 and particularly to those of pEK204 and pEC_Bactec. Three major differences including inc, parAB, and excA regions were noted between R621a and prototype IncI1 plasmids. Seven nucleotide replacements and one nucleotide deletion in the putative Inc RNA sequence are found between R621a and IncI1 plasmids irrespective of close similarity in the other parts of the rep system. The sequences of R621a parAB and excA genes are significantly different from those of R64 and ColIb-P9, while those of R621a parAB and excA genes exhibit close similarity to those of pEK204 and pEC_Bactec, respectively. The R621a genome is suggested to be formed by acquiring parAB and excA genes from pEK204 and pEC_Bactec genomes, respectively, and then novel inc function by the mutations. The insertions in the R621a, pEK204, and pEC_Bactec genomes are flanked by direct repeats, suggesting that insertions accompanied by long target duplications have also played an important role in the evolution of IncI plasmids., (Copyright © 2011 Elsevier Inc. All rights reserved.)

A streptomycin and tetracycline resistance plasmid R64 isolated from Salmonella enterica serovar Typhimurium belongs to the incompatibility group I1 (IncI1). The DNA sequence of the R64 conjugative transfer region was described previously (Komano et al., 2000). Here, we report the complete genome sequence of R64. In the circular double-stranded R64 genome with 120,826bp, 126 complete ORFs are predicted. In addition, 2 and 6 different kinds of proteins are produced by translational reinitiation and shufflon multiple inversions, respectively. The genome consists of five major regions: replication, drug resistance, stability, transfer leading, and conjugative transfer regions in clockwise order. The nucleotide sequence essential for autonomous replication of R64 is completely identical to that of IncI1 colicinogenic plasmid ColIb-P9, an indication that these two plasmids share the same mechanisms for replication and copy number control. Tetracycline and streptomycin resistance genes are encoded in transposons Tn10 and Tn6082, respectively. These transposons and two insertion elements, IS2 and IS1133, were inserted stepwise into the arsenic-resistant gene, arsA1, present in the drug resistance region. The stability and transfer leading regions contain various important genes such as parAB, resD, ardA, psiAB, or ssb for plasmid maintenance, recombination and transfer reactions. When the genome of R64 was compared with those of other plasmids, varying levels of similarity were observed. It is suggested that genetic recombinations including the site-specific rfsF-ResD system have played an important role in diversity of genomes related to R64. It was found that R64 exhibits highly organized genome structure., (Copyright 2010 Elsevier Inc. All rights reserved.)

The predicted amino acid sequences of the pilL and pilN genes, required for the thin pilus formation of IncI1 plasmids R64 and ColIb-P9, contain N-terminal lipoprotein signal peptide motifs. The pilL and pilN products were labeled with [(3)H]palmitic acid as 38- and 57-kDa proteins, respectively, indicating that they are lipoproteins. Both PilL and PilN were localized to the outer membrane., (Copyright 2000 Academic Press.)

The sog gene of the IncI1 plasmid R64 was sequenced and characterized. The sog gene was shown to express two acidic proteins, SogL and SogS, with 1255 and 844 amino acid residues, respectively. The SogS protein was expressed by translational reinitiation within the SogL reading frame. Analysis of dnaG-suppression activity using the Escherichia coli dnaG strain indicated that the domain for this activity was located within the N-terminal one-third segment of the SogL protein. A Deltasog mutation was constructed by replacing most of the sog coding sequence with a DNA fragment encoding a tetracycline resistance gene. Introduction of the Deltasog mutation into an R64 derivative resulted in approximately a 50-fold reduction in transfer frequency. It was observed that only a limited portion of the SogL or SogS protein corresponding to an internal 0.94-kb EcoRV-SnaBI segment of the sog gene was required for the conjugal transfer of R64.

We have cloned and sequenced the exc gene determining surface exclusion of IncI1 plasmid R64. R64 exc gene is highly homologous to the R144 exc gene. The R64 exc gene may encode two proteins with 220 and 147 amino acid residues by inframe reinitiation of translation, as the R144 exc gene does, while the larger protein with 220 amino acids was shown to be essential for surface exclusion function. R64 derivatives carrying deletion or insertion mutations in the exc gene transferred at a frequency similar to that of wild-type R64, indicating that the exc gene is not essential for the transfer of R64.

The broad host range plasmid RSF1010 requires for its replication in Escherichia coli three plasmid-encoded proteins and specific nucleotide sequences ssiA, ssiB, and iterons in the oriVRSF1010. In Pseudomonas aeruginosa, a recombinant mini-RSF1010 plasmid lacking ssiB lost its replication ability, but a miniplasmid lacking ssiA or carrying a primosome assembly site in place of ssiA could replicate. Moreover, ssiA, as a sole ssi signal, in the orientation that ssiB had originally taken was sufficient for replication of the miniplasmid. These results indicated that only one RSF1010-specific ssi signal in the orientation that ssiB takes in wild-type oriVRSF1010 was essential for replication of RSF1010. Replication of the miniplasmids was dependent on the three plasmid-encoded proteins, RepA, B', and C, as in E. coli.

Taking advantage of the plaque morphology method, we identified a single-strand initiation (ssi) signal in plasmid pSM32, a mini-Co1Ib plasmid. This ssi signal was situated in the 350-nt HaeIII segment of the 1.8-kb S7 fragment, and located nearly 400 nt downstream of the origin of DNA replication. Introduction of the ssi signal into a mutant of filamentous phage M13 lacking oric resulted in restoration of phage growth and RFI DNA synthesis. Interestingly, DNA homology studies showed that the nucleotide sequence of the ssi signal was extremely homologous with that of the "G4-type" ssi signal in plasmid R100.

A physical map of the 75.1-kb IncI2 plasmid R721 was constructed by using 15 restriction enzymes, and the regions of several genetic determinants including the origins of replication and of conjugal DNA transfer were located on the physical map. It was found that R721 bears a DNA region which undergoes DNA rearrangement similar to the shufflon of R64.

The R64 shufflon is a novel type of DNA rearrangement in which four DNA segments invert independently or in groups. The related plasmid ColIb carries a variant shufflon. The present sequence analysis shows that the ColIb shufflon consists of three DNA segments that are highly homologous to the A, B, and C segments of the R64 shufflon. The 329-bp D segment of R64 is not present in the ColIb shufflon. As in the case of R64, the ColIb shufflon may act as a biological switch to select one of the six open reading frames in which the N-terminal region is constant while the C-terminal region is variable.

Taking advantage of the plaque morphology method, we detected two single-stranded initiation (ssi) signals in the plasmid pSY343; one was in the 170-nucleotide (nt) EcoRV-ThaI segment (170P), and the other was in the 93-nt DraI-FnuDII segment (93F), which were designated as ssiA and ssiB, respectively. We cloned the two ssi signals in the filamentous phage vectors M13 delta lac184 and flR199. A conserved 7-nt consensus sequence involved in the n' recognition site for priming DNA initiation on single-stranded (ss) DNA templates (A. Van der Ende, R. Teerstra, H. Van der Avoort, and P.J. Weisbeek, 1983, Nucleic Acids Res. 11, 4957-4975) was found, three copies in 170P and one in 93F. These two ssi signals contain possible stem and loop structures. The 170P overlapped partly with the origin (ori) region of pSY343 and the 93F was away from the ori region. Growth of chimera phages such as M13 delta lac184/ssiA and M13 delta lac184/ssiB was 38- and 71-fold greater, respectively, than that of M13 delta lac184, 8 h after phage infection. The conversion efficiency in vivo of ss to replicative form (RF) DNA of these chimera phages carrying ssiA and ssiB was 1.9- and 2.2-fold greater, respectively, than that of M13 delta lac184, 50 min after infection.