Genetic Organization of Transfer-Related Determinants on the Sex Factor F and Related Plasmids

Ippen‐Ihler, Karin; Skurray, Ronald A.

doi:10.1007/978-1-4757-9357-4_2

Cited by 35 publications

(37 citation statements)

References 163 publications

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“…Nucleotide sequencing of tra region DNA had revealed a number of additional tra region loci (8,19), and during the course of these studies, F and pOX38 derivatives that carry kan insertion mutations in many of these genes also became available. Table 2 also summarizes the results of JEL92 and JEL93 Western blot assays with inner membranes prepared from these strains.…”

Section: Resultsmentioning

confidence: 99%

The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F pilin

et al. 1993

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The Escherichia coli F plasmid gene required for amino-terminal acetylation of F-pilin subunits was identified. Using Western blots (immunoblots), we assayed the reaction of monoclonal antibodies with F-pilin polypeptides in inner membrane preparations from various F mutant strains. It was known that JEL92 recognizes an internal pilin epitope and JEL93 recognizes the acetylated amino-terminal sequence (L.S. Frost, J.S. Lee, D.G. Scraba, and W. Paranchych, J. Bacteriol. 168:192-198, 1986). As expected, neither antibody reacted with inner membranes from F- cells or Flac derivatives that do not synthesize pilin. Mutations that affected the individual activities of F tra genes traA, -B, -C, -D, -E, -F, -G, -H, -I, -J, -K, -L, -M, -N, -P, -R, -U, -V and -W or trb genes trbA, -B, -C, -D, -E, -G, -H, and -I did not prevent JEL92 or JEL93 recognition of membrane pilin. However, Hfr deletion mutants that lacked the most-distal transfer region genes did not express pilin that reacted with JEL93. Nevertheless, all strains that retained traA and traQ did express JEL92-reactive pilin polypeptides. Analysis of strains expressing cloned tra segments showed that traA and traQ suffice for synthesis of JEL92-reactive pilin, but synthesis of JEL93-reactive pilin is additionally dependent on traX. We concluded that the traX product is required for acetylation of F pilin. Interestingly, our data also showed that TraA+ TraQ+ cells synthesize two forms of pilin which migrate at approximately 7 and 8 kDa. In TraX+ cells, both become acetylated and react with JEL93. Preparations of wild-type F-pilus filaments contain both types of subunits.

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Section: Resultsmentioning

confidence: 99%

The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F pilin

et al. 1993

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“…The transferred DNA is then circularized and a complementary strand is synthesized in the recipient cells (Wilkins and Lanka 1993). The genes coding for the conjugal transfer are primarily located in the plasmids (Heinemann and Ankenbauer 1993;Ippen-Ihler and Skurray 1993). Each incompatibility group of plasmids differs in its conjugal transfer system.…”

Section: Discussionmentioning

confidence: 99%

Characterization of plasmids that encode streptomycin‐resistance in bacterial epiphytes of apple

Huang

Burr

1999

Journal of Applied Microbiology

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T .-C. H UA NG A ND T. J . B UR R . 1999. Streptomycin resistance in strains of Pseudomonas syringae pv. papulans, Pantoea agglomerans and a yellow-pigmented, nonfluorescent Pseudomonas sp. (Py), isolated from apple orchards in New York and Washington states, is predominantly associated with strA-strB genes carried on conjugal plasmids (R plasmids). None of 128 resistant Erwinia amylovora strains from the eastern and western USA hybridized with a strA-strB probe, SMP3. Resistant Py strains transfered R plasmids to Ps. syringae pv. papulans and to Py in vitro at frequencies of 10 −1 -10 −2 per recipient cell whereas Ps. syringae pv. papulans transferred its plasmids at frequencies of 10 −2 to below detectable levels. Transfer of R plasmids to P. agglomerans was not detected and resistant P. agglomerans did not transfer their R plasmids to any recipients. R plasmids were found to be highly diverse as measured by DNA fingerprint analysis. Transfer-deficient transposon mutants of R plasmid pCPP519 were generated, and 3·9 kb EcoRI and 3·0 kb SmaI fragments that hybridized with a Tn5 probe were cloned and sequenced. The deduced amino acid sequences of the 3·9 kb fragment were similar to proteins involved in replication, nicking at oriT, and piliation in other bacteria.

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“…TraG is highly conserved in all known conjugation systems, and all TraG homologs have hydropathy profiles that suggest an integral membrane topology. The homologous protein of F (TraD) binds single-stranded DNA in vitro and is required for efficient DNA replacement synthesis in donor cells (33). TrbB and TrbD, like their IncP counterparts, have nucleotide binding motifs (36) and may provide energy for the export of DNA or the export of other Trb proteins.…”

Section: Discussionmentioning

confidence: 99%

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes

et al. 1996

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We have determined the DNA sequences of two unlinked regions of octopine-type Ti plasmids that contain genes required for conjugal transfer. Both regions previously were shown to contain sequences that hybridize with tra genes of the nopaline-type Ti plasmid pTiC58. One gene cluster (designated tra) contains a functional oriT site and is probably required for conjugal DNA processing, while the other gene cluster (designated trb) probably directs the synthesis of a conjugal pilus and mating pore. Most predicted Tra and Trb proteins show relatively strong sequence similarity (30 to 50% identity) to the Tra and Trb proteins of the broad-host-range IncP plasmid RP4 and show significantly weaker sequence similarity to Vir proteins found elsewhere on the Ti plasmid. An exception is found in the Ti plasmid TraA protein, which is predicted to be a bifunctional nickase-helicase that has no counterpart in IncP plasmids or among Vir proteins but has homologs in at least six other self-transmissible and mobilizable plasmids. We conclude that this Ti plasmid tra system evolved by acquiring genes from two or three different sources. A similar analysis of the Ti plasmid vir region indicates that it also evolved by appropriating genes from at least two conjugal transfer systems. The widely studied plasmid pTiA6NC previously was found to be nonconjugal and to have a 12.65-kb deletion of DNA relative to other octopine-type Ti plasmids. We show that this deletion removes the promoter-distal gene of the trb region and probably accounts for the inability of this plasmid to conjugate.It is widely appreciated that Agrobacterium strains are able to transfer segments of oncogenic DNA from their large Ti plasmids to the nuclei of infected plant cells, resulting in the formation of crown gall tumors. This conjugation-like process is mediated by the products of approximately 25 Ti plasmidencoded vir genes (59). It may be less widely appreciated that the vir region contains only one of two DNA transfer systems found on Ti plasmids, since most Ti plasmids are able to undergo conjugal transfer between bacterial hosts (16). It was postulated some years ago that a single conjugation system might mediate both kinds of DNA transfer, but more recently it was shown that these processes are genetically distinct. Although two reports indicate that these two DNA transfer systems may interact (26, 51), other studies indicate that tra gene mutations do not affect T-DNA transfer to plants and, conversely, vir gene mutations do not abolish interbacterial conjugation (4,8,30). This suggests that, at least under most conditions, these transfer systems are functionally quite distinct. However, before the present study was initiated, it still remained possible that these two systems were the products of an evolutionarily recent gene duplication. We provide data that argue against such an origin and suggest a more complicated evolutionary lineage for tra and vir functions.Several laboratories are studying how Agrobacterium tumefaciens perceives and responds to...

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Genetic Organization of Transfer-Related Determinants on the Sex Factor F and Related Plasmids

Cited by 35 publications

References 163 publications

The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F pilin

The Escherichia coli K-12 F plasmid gene traX is required for acetylation of F pilin

Characterization of plasmids that encode streptomycin‐resistance in bacterial epiphytes of apple

The conjugal transfer system of Agrobacterium tumefaciens octopine-type Ti plasmids is closely related to the transfer system of an IncP plasmid and distantly related to Ti plasmid vir genes

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