Cloning of the pif region of the F sex factor and identification of a pif protein product

This paper reports a detailed investigation of the pif region of the F factor responsible for inhibition of development of T7 and related "female-specific" phages. We have mapped a series of pif::Tn5 insertions to a region between 39.6 and 42.8 kilobases on the physical map of F. All pif::Tn5 insertions plated T7 at full efficiency; most were clustered in a 1.8-kilobase interval on both sides of the EcoRI site located at F coordinate 40.3 kilobases. A 5.2-kilobase Pst-I fragment with F coordinates 38.9 to 44.1 has been cloned into a pSC101 vector to create the Pif+ plasmid pGS103. A series of Pif- deletion mutants and nonsense mutants were isolated from pGS103. Using minicells carrying pGS103 or its derivatives, we have identified a 70,000-dalton pif protein.     

Plasmid cointegrates of Flac and lambda prophage.

Fifteen cointegrates of the plasmid Flac and prophage lambda that had suffered no detectable change in plasmid phenotype were isolated and characterized. The locations of the prophage insertions were determined by genetic analysis of deletion mutants obtained from each cointegrate as survivors of growth at 42 degrees C. In 11 cointegrates, the prophage was inserted between traI and lac, although probably in more than one location; in 3 others, it was on one side or the other of lac; and in 1 it was between lac and pif. Deletions covering all or part of the transfer region, as well as of lac and of pif, were obtained in the course of this analysis. Deletion mutants that had lost all known transfer genes were also oriT, but they retained the capacity to recircularize after transfer. Attempts were made to isolate lambda transducing phages for nearby plasmid genes from the cointegrates, and lambdaptraGD, lambdaptraD, lambdaptraI, and lambdadtraDI phages were obtained.     

Regulation of the F-factor pif operon: pifO, a site required in cis for autoregulation, titrates the pifC product in trans.

F factor pifC, pifA, and pifB gene expression is subject to negative regulation by the product of the pifC locus (J.F. Miller and M. H. Malamy, J. Bacteriol. 156:338-347, 1983). In this paper, we describe the properties of a new regulatory site in the pif region, pifO, which is required in cis for autoregulation of pif gene expression. Spontaneous pifO mutations were isolated that allow expression of a pifC-lacZ protein fusion in the presence of pifC product in trans. Recombination of these pifO mutations onto F'lac results in increased pifA and pifB activity. Thus, a single regulatory element, pifO, regulates pifC, pifA, and pifB expression in cis. The presence of multiple copies of a fragment from the pif region carrying wild-type pifO sequences (F coordinates 42.9 to 42.43 kilobases) in trans to F'lac results in an increase in pifA and pifB activity as measured by inhibition of T7 plating. When the pifO mutations are recombined onto a plasmid carrying pifO, the resulting recombinants are greatly decreased in the ability to increase F'lac pif expression. These results suggest that increased F'lac pifA and pifB expression caused by pifO sequences in trans is a consequence of titration of pifC product and derepression of the pif operon.     

F factor inhibition of conjugal transfer of broad-host-range plasmid RP4: requirement for the protein product of pif operon regulatory gene pifC.

By the use of deletions, point mutations, and gene fusions, we show that the protein product of the F factor pifC gene is responsible for F factor inhibition of plasmid RP4 conjugal transfer. Deletion analysis of pif sequences carried by pSC101-F chimeric plasmids demonstrated that removal of all or part of the pifC coding sequence greatly decreased or abolished the ability of these plasmids to inhibit RP4 transfer. Amber mutations in the pifC gene eliminated inhibition in an Su- host strain but not in and Su+ (supF) host. Plasmids carrying nonpolar pifC mutations did not decrease the efficiency of RP4 transfer when present in trans. Whereas pifC+ plasmids inhibited RP4 transfer, the presence of RP4 in the same cell as F' lac increased F'lac Pif activity approximately 1,000-fold. This effect most likely resulted from the binding of the pifC product to RP4 DNA and concomitant derepression of the F factor pif operon. PifC inhibited trans mobilization of pMS204, a nonconjugative plasmid carrying the RP4 oriT locus, by the RP1 derivative pUB307. pMS204 had no trans effect on pif operon expression, whereas pUB307 increased F'lac Pif expression, as did RP4. Our results suggest that the pifC product inhibits expression of one or more RP4 genes, the products of which are required for conjugal transfer of RP4 and are required in trans for mobilization of nonconjugal RP4 oriT containing plasmids.     

Identification of the pifC gene and its role in negative control of F factor pif gene expression

The pif region of the F factor includes two genes, pifA and pifB, that lead to abortive T7 infection. We have identified a new gene in this region, pifC, by constructing an in vitro fusion of pif DNA at 41.6 kilobases on the F factor physical map to the lacZ gene. A PifC-LacZ fusion protein of 149,000 daltons has been identified by immunoprecipitation and polyacrylamide gel electrophoresis. This allows us to assign the N terminus of pifC to 42.5 kilobases on the F map. Using fusions of pifC, pifA, and pifB to lacZ, we have studied the regulation of pif gene expression and have shown that the product of pifC negatively controls its own expression and that of pifA and pifB.     

Physical mapping of genes on the F plasmid of Escherichia coli responsible for inhibition of growth of female-specific bacteriophages.

By correlating the resistance or sensitivity to female-specific phages of strains carrying F plasmids with deletions for part of the region 32.6 to 42.9 F, cloned F fragments, and other plasmids, it was shown that the pif loci are located near and clockwise to a point on F with coordinate 38.3 F.     

Transfer functions of the Streptococcus faecalis plasmid pAD1: organization of plasmid DNA encoding response to sex pheromone.

The conjugative plasmid pAD1 (59.6 kilobases) of Streptococcus faecalis shows a 10,000-fold increase in transfer frequency following induction by the sex pheromone cAD1. Mutagenesis of the plasmid with transposon Tn917 was undertaken to determine the region(s) of pAD1 required for the mating response. The relevant genetic material was found to be distributed over a 31.2-kilobase contiguous region of the plasmid. Although insertions in two previously identified regions (traA and traB) exhibited increased transfer frequencies, insertions in five new regions (D, E, F, G, and H) decreased the ability of pAD1 to transfer. Insertions in region H allowed the cells to form visible mating aggregates, but the plasmid transfer frequency was decreased to levels below detection during a 1-h broth mating. Mutants with mutations in region G were able to form aggregates; however, insertions in regions D, E, and F prevented aggregate formation. Insertions in region C decreased the sensitivity of the cell to exogenous cAD1 and exhibited increased activity of the pheromone inhibitor iAD1. Surface protein profiles produced by a number of these mutants were examined, and in some cases were found to be different from those of the wild type. A map showing the various regions is presented, and related aspects of the regulation of the pAD1 mating response are discussed.     

A rapid, small scale method for characterization of plasmid insertions in the Dictyostelium genome.

A rapid, simple method for characterization of plasmid insertions in the Dictyostelium discoideum genome was developed. It is based on the capability of linear plasmid multimers in the insertions to recircularize efficiently in Escherichia coli cells. This recombinational recircularization of plasmid multimers provides a highly sensitive and reliable tool for determining whether individual Dictyostelium transformants resulted from restriction enzyme-mediated integration (REMI) or from recombinational integration of plasmid (RIP). The method also reveals any rearrangements in RIP insertions and provides an estimate of the vector copy number in any particular transformant.     

In vivo Himar1-based transposon mutagenesis of Francisella tularensis

Francisella tularensis is the intracellular pathogen that causes human tularemia. It is recognized as a potential agent of bioterrorism due to its low infectious dose and multiple routes of entry. We report the development of a Himar1-based random mutagenesis system for F. tularensis (HimarFT). In vivo mutagenesis of F. tularensis live vaccine strain (LVS) with HimarFT occurs at high efficiency. Approximately 12 to 15% of cells transformed with the delivery plasmid result in transposon insertion into the genome. Results from Southern blot analysis of 33 random isolates suggest that single insertions occurred, accompanied by the loss of the plasmid vehicle in most cases. Nucleotide sequence analysis of rescued genomic DNA with HimarFT indicates that the orientation of integration was unbiased and that insertions occurred in open reading frames and intergenic and repetitive regions of the chromosome. To determine the utility of the system, transposon mutagenesis was performed, followed by a screen for growth on Chamberlain's chemically defined medium (CDM) to isolate auxotrophic mutants. Several mutants were isolated that grew on complex but not on the CDM. We genetically complemented two of the mutants for growth on CDM with a newly constructed plasmid containing a nourseothricin resistance marker. In addition, uracil or aromatic amino acid supplementation of CDM supported growth of isolates with insertions in pyrD, carA, or aroE1 supporting the functional assignment of genes within each biosynthetic pathway. A mutant containing an insertion in aroE1 demonstrated delayed replication in macrophages and was restored to the parental growth phenotype when provided with the appropriate plasmid in trans. Our results suggest that a comprehensive library of mutants can be generated in F. tularensis LVS, providing an additional genetic tool to identify virulence determinants required for survival within the host.     

Virulence-associated genetic regions comprising 31 kilobases of the 230-kilobase plasmid in Shigella flexneri 2a.

By random transposon Tn5 insertions, we previously identified six virulence-associated SalI fragments, B, D, F, G, H, and P, in the 230-kilobase plasmid pMYSH6000 of Shigella flexneri 2a. In this study, we analyzed the sites of 134 independent Tn5 insertions on four contiguous SalI fragments, B, P, H, and D, of pMYSH6000 and identified five virulence-associated regions; four were associated with inducing a positive Sereny test (Ser), invasion into epithelial cells (Inv), binding to Congo red (Pcr), and inhibition of bacterial growth (Igr), and one was associated with the Ser and Inv but not with the Pcr or Igr phenotypes. Hybridization studies revealed that these virulence-associated DNA regions were highly conserved among 15 other virulence plasmids of four species of Shigella and enteroinvasive Escherichia coli. These data indicate that at least seven separate genetic determinants on the virulence plasmid are required for full expression of the virulence phenotype of shigellae.     

Characterization of conjugal transfer functions of Agrobacterium tumefaciens Ti plasmid pTiC58.

Physical characterization of 13 transposon Tn5 insertions within the agrocinopine-independent, transfer-constitutive Ti plasmid pTiC58Trac identified three separate loci essential for conjugation of this nopaline/agrocinopine A + B-type Ti plasmid. Complementation analysis with relevant subcloned DNAs indicated that the three physically separated blocks of conjugal genes constitute distinct complementation groups. Two independent Tn5 insertions within the wild-type, agrocinopine-dependent, repressed pTiC58 plasmid resulted in constitutive expression of conjugal transfer. These two insertions were physically indistinguishable and could not be complemented in trans. However, the Trac phenotype resulted when the Tn5-mutated fragment cointegrated into the wild-type Ti plasmid. While the spontaneous Trac mutant Ti plasmids were also derepressed for agrocinopine catabolism, those generated by Tn5 insertions remained inducible, indicating that this apparent cis-acting site is different from that affected in the spontaneous mutants. No chromosomal Tn5 insertion mutations were obtained that affected conjugal transfer. An octopine-type Ti plasmid, resident in different Agrobacterium tumefaciens chvB mutants, transferred at normal frequencies, demonstrating that this virulence locus affecting plant cell binding is not required for Ti plasmid conjugation. None of our conjugal mutants limited tumor development on Kalanchoe diagremontiana. Known lesions in pTiC58 vir loci had no effect on conjugal transfer of this Ti plasmid. These results show that pTiC58 Ti plasmid conjugal transfer occurs by functions independent of those required for transfer of DNA to plant cells.     

The tra region of the nopaline-type Ti plasmid is a chimera with elements related to the transfer systems of RSF1010, RP4, and F.

The Ti plasmids of Agrobacterium tumefaciens encode two transfer systems. One mediates the translocation of the T-DNA from the bacterium to a plant cell, while the other is responsible for the conjugal transfer of the entire Ti plasmid from one bacterium to another. The determinants responsible for conjugal transfer map to two regions, tra and trb, of the nopaline-type Ti plasmid pTiC58. By using transposon mutagenesis with Tn3HoHo1, we localized the tra determinants to an 8.5-kb region that also contains the oriT region. Fusions to lacZ formed by transposon insertions indicated that this region is expressed as two divergently transcribed units. We determined the complete nucleotide sequence of an 8,755-bp region of the Ti plasmid encompassing the transposon insertions defining tra. The region contains six identifiable genes organized as two units divergently transcribable from a 258-bp inter-genic region that contains the oriT site. One unit encodes traA, traF, and traB, while the second encodes traC, traD, and traG. Reporter insertions located downstream of both sets of genes did not affect conjugation but were expressed, suggesting that the two units encode additional genes that are not involved in transfer under the conditions tested. Proteins of the predicted sizes were expressible from traA, traC, traD, and traG. The products of several Ti plasmid tra genes are related to those of other conjugation systems. The 127-kDa protein expressed from traA contains domains related to MobA of RSF1O1O and to the helicase domain of TraI of plasmid F. The translation product of traF is related to TraF of RP4, and that of traG is related to TraG of RP4 and to VirD4 of the Ti plasmid T-DNA transfer system. Genetic analysis indicated that at least traG and traF are essential for conjugal transfer, while sequence analysis predicts that traA also encodes an essential function. traB, while not essential, is required for maximum frequency of transfer. Patterns of sequence relatedness indicate that the oriT and the predicted cognate site-specific endonuclease encoded by traA share lineage with those of the transfer systems of RSF1010 and plasmid F, while genes of the Ti plasmid encoding other essential tra functions share common ancestry with genes of the RP4 conjugation system.     

Construction and characterization of derivatives carrying insertion mutations in F plasmid transfer region genes, trbA, artA, traQ, and trbB.

We devised a method for construction of insertion mutations in F plasmid tra region genes as a means of investigating the functions associated with previously uncharacterized loci. First, we constructed mutations in vitro, by insertion of a kanamycin resistance gene into a unique restriction site within a tra region fragment carried by a small, chimeric plasmid. Second, we crossed the insertion mutations, in vivo, onto a plasmid containing the complete F tra region sequence (either F lac, or pOX38, a Tra+ F plasmid derivative). Using this method, we obtained F lac mutant derivatives carrying KmR gene insertions in traQ, and a set of pOX38 mutant derivatives carrying a KmR gene insertion in trbA, artA, traQ, or trbB. Analysis of these derivatives showed that insertion of a kan gene at the NsiI site of traQ resulted in transfer deficiency, F-pilus-specific-phage resistance and an absence of detectable F-pilin subunit synthesis. Since the traQ mutants regained a wild-type phenotype when complemented with a traQ+ plasmid clone, we concluded that traQ expression is essential to transfer and F-pilus synthesis. However, pOX38 derivatives carrying kan gene inserts in genes trbA, artA, or trbB retained F-pilus-specific phage sensitivity and transferred at normal levels. Thus, these three gene products may not be essential for F-transfer from Escherichia coli K-12 under standard mating conditions.     

Rhizobium phaseoli symbiotic mutants with transposon Tn5 insertions.

Rhizobium phaseoli CFN42 DNA was mutated by random insertion of Tn5 from suicide plasmid pJB4JI to obtain independently arising strains that were defective in symbiosis with Phaseolus vulgaris but grew normally outside the plant. When these mutants were incubated with the plant, one did not initiate visible nodule tissue (Nod-), seven led to slow nodule development (Ndv), and two led to superficially normal early nodule development but lacked symbiotic nitrogenase activity (Sna-). The Nod- mutant lacked the large transmissible indigenous plasmid pCFN42d that has homology to Klebsiella pneumoniae nitrogenase (nif) genes. The other mutants had normal plasmid content. In the two Sna- mutants and one Ndv mutant, Tn5 had inserted into plasmid pCFN42d outside the region of nif homology. The insertions of the other Ndv mutants were apparently in the chromosome. They were not in plasmids detected on agarose gels, and, in contrast to insertions on indigenous plasmids, they were transmitted in crosses to wild-type strain CFN42 at the same frequency as auxotrophic markers and with the same enhancement of transmission by conjugation plasmid R68.45. In these Ndv mutants the Tn5 insertions were the same as or very closely linked to mutations causing the Ndv phenotype. However, in two mutants with Tn5 insertions on plasmid pCFN42d, an additional mutation on the same plasmid, rather than Tn5, was responsible for the Sna- or Ndv phenotype. When plasmid pJB4JI was transferred to two other R. phaseoli strains, analysis of symbiotic mutants was complicated by Tn5-containing deleted forms of pJB4JI that were stably maintained.     

Heterozygosity and instability of amplified chromosomal insertions in the radioresistant bacterium Deinococcus radiodurans.

Natural transformation, duplication insertion, and plasmid transformation in Deinococcus radiodurans, a bacterium that contains 4 to 10 chromosomes per cell, were studied. Duplication insertions were often heterozygous, with some chromosomes containing highly amplified insertions and others containing no insertions. Large amplified regions were apparently deleted by intrachromosomal recombination, generating as by-products extrachromosomal circles consisting of multiple tandem repeats of the amplified sequence. The circles were of heterogenous integer sizes, containing as many as 10 or more amplification units. Two strains that are defective in natural transformation and sensitive to DNA-damaging agents were further characterized. Both strains were defective in duplication insertion. While on strain was normal for plasmid transformation, the other was totally defective in this regard, suggesting that plasmid transfer in D. radiodurans may require recombinational functions.     

Agrobacterium tumefaciens mutants affected in crown gall tumorigenesis and octopine catabolism.

Mutants of Agrobacterium tumefaciens which affect virulence or the ability to catabolize octopine were isolated after Tn5-induced mutagenesis. Of 8,900 colonies tested, 7 mutants with Tn5 insertions in a specific region of other Ti plasmid unable to catabolize octopine were isolated. Thirty-seven mutants affected in tumorigenesis resulted from insertions in the Ti plasmid and the Agrobacterium chromosome. Of these mutations, 12 were chromosomal and 25 mapped on the plasmid. Twenty-three mapped within a 20-megadalton region, which is distinct from the Ti plasmid sequences found stably integrated into the plant cell genome T-deoxyribonucleic acid). Included in these were mutants that were either a virulent or produced tumors with unusual morphologies. Three mutants contained insertions in the T-deoxyribonucleic acid. These three mutants incited tumors which synthesized octopine but had an altered morphology due to either extensive proliferation of shoots or roots from the tumor callus. Three additional mutants not caused by Tn5 contained mutations in the Ti plasmid.     

ColIb plasmid genes that inhibit the replication of T5 and T7 bacteriophage

The colicin Ib (ColIb) plasmid genes that inhibit the replication of the T5-like and T7 bacteriophage have been cloned on an approximately 7200-bp ClaI fragment and their sites relative to each other and to the colicin immunity (imm) gene have been mapped. The inhibition of wild-type T7 by the clone is shown to be caused by the same gene or genes (pic) that cause the inhibition of T7 kinase-negative mutants and is a different gene than the one that causes inhibition of T5 (ibf or abi). The pic gene does not hybridize to the pif genes of the F plasmid that also cause the replication of T7 to be inhibited. The abi gene and the pic gene map very closely together but are under the control of different promoters. The abi gene has a maximum size of 900 bp and lies approximately 3000 bp away from the immunity gene, distal to the colicin gene. A site which maps in or near the gene binds very tightly to Escherichia coli RNA polymerase. The pic gene or genes lie between the abi gene and the imm gene and are contiguous with abi. Promoters for pic have been mapped and hypotheses to explain the inhibition of T7 by a cloned gene but not the whole ColIb plasmid are presented.