Transfer of transposable drug-resistance elements Tn5, Tn7, and Tn76 to Azotobacter beijerinckii: use of plasmid RP4::Tn76 as a suicide vector

Transposable elements Tn5, Tn7, and Tn76 were transferred to Azotobacter beijerinckii. Evidence was obtained for the transposition of Tn5 but cells of the majority of presumptive transposition isolates had abnormal morphologies and rapidly lost viability when subcultured. Data are presented that indicate that plasmid RP4::Tn76 behaves as a suicide vector upon transfer to this host, allowing the isolation of A. beijerinckii::Tn76 isolates at a high frequency. Nitrogen-fixing mutants and leucine and adenine auxotrophs were isolated from cultures in which the transposition of Tn76 occurred.     

Tn7 insertion mutations affecting the host range of the promiscuous IncP-1 plasmid R18

The genetic basis of plasmid host range has been investigated by Tn7 insertion mutagenesis of the promiscuous plasmid R18 in Pseudomonas aeruginosa. Six mutants have been isolated on the basis of greatly reduced transferability into Escherichia coli C while retaining normal transferability within P. aeruginosa. Their physical mapping shows that two of them map at coordinate 11.72 ± 0.14 kb, in the region of the origin of plasmid replication (oriV) and one at 18.0 ± 0.3 kb, in the trans-acting gene essential for initiation of replication at oriV (trfA). Three map at 48.4 ± 0.5 kb in the region of the origin of plasmid transfer (oriT) and the site at which a single-strand nick is introduced in the plasmid DNA-protein relaxation complex (rlx). Consistent with the postulated defective replication of the oriV and trfA mutants was their inability to transform E. coli C or K12 while being able to transform P. aeruginosa. As expected the oriT/rlx mutants transformed both hosts as effectively as R18. Furthermore the trfA mutant was readily curable by mitomycin C in a DNA polymerase I-proficient P. aeruginosa and spontaneously lost from a polymerase-deficient mutant of P. aeruginosa suggesting a role of this polymerase in the replication of R18. Extensive transfer tests from P. aeruginosa into a range of enteric bacteria, other Pseudomonas species and into other Gram-negative bacteria indicated a complex host range pattern for these mutants. It appears that both plasmid replication and conjugation genes are responsible for host range in addition to the involvement of host gene products.     

Complementation analysis in Pseudomonas aeruginosa of the transfer genes of the wide host range R plasmid R18

A method of transductional complementation was developed in Pseudomonas aeruginosa to identify the cistrons involved in the conjugal transfer of the wide host range R plasmid R18. This used the P. aeruginosa bacteriophage E79tv-2 and has led to the identification of eight tra cistrons encoded by this plasmid. Plasmids mutant in six cistrons, traA, traB, traC, traD, traE, and traG were resistant to donor-specific phage (Dps^?) while traF and traH mutant plasmids retained phage sensitivity. Some traB mutants were unable to inhibit the replication of phage G101 (Phi(G101)^?) while some were also deficient in entry exclusion (Eex^?). Two traB mutants which were also Eex^? were suppressible by an amber suppressor. Three tra mutants selected directly as being Phi(G101)^? were found to be also Dps^?Eex^? and mutant in traB. These data suggest a relationship between traB, Eex, and Phi(G101). In order to facilitate future genetic comparison of the tra genes of R18 and other wide host range plasmids and the role of the host in conjugation, R18 DNA was compared with that of RP4, by restriction enzyme fragment patterns and found to be identical.     

Cloning and molecular analysis of the finO region from the antibiotic-resistance plasmid R6-5

The cloning of the finO region from the antibiotic-resistance plasmid R6-5 is reported. On the basis of DNA deletion analysis and Tn5 transposon insertional mutagenesis of finO+ chimeric plasmids, finO has been located within the coordinates 94.0-94.85 on the R6-5 map. A 32,000-Da polypeptide (32K), which is encoded within 92.75-94.25R6-5, has been identified and shown not to be associated with the FinO phenotype.     

Regions on the F plasmid which affect plasmid maintenance and the ability to segregate into Escherichia coli minicells

Certain derivative mini-F plasmids were found to segregate into Escherichia coli minicells, in contrast to the intact mini-F plasmid which does not. Segregation was not related to the presence or absence of the normal origin of vegetative replication, but appeared to be affected by regions of F which encode replication, incompatibility, copy number control, and partitioning functions. Segregation of mini-F plasmids into minicells was not random; the plasmid concentration in minicells did not correlate with the plasmid concentration in cells. Genes, or gene products, of F from the region spanning the sequences 44.1–49.3F appeared to affect the ability of mini-F plasmids to segregate into minicells. Segregation of mini-F plasmids into minicells was not directly related to stable plasmid inheritance. These results argue for the sequestration of mini-F plasmids in host cells.     

A technique for mapping transfer RNA genes by electron microscopy of hybrids of ferritin-labeled transfer RNA and DNA: the phi-80hpsu+3-system

A method for mapping transfer RNA genes on single strands of DNA is described. tRNA is covalently coupled to the electron-opaque label, ferritin. The ferritinlabeled tRNA, Fer-tRNA, is hybridized to a single strand of DNA, or to a single- strand region of a DNA in a heteroduplex. The sites where the Fer-RNA binds to the complementary sequence on the DNA are then mapped by electron microscopy. Several alternative coupling procedures are described (see Fig. 1). In HzI a — COCH₂Br group is attached to ferritin by acylation. 3'-Oxidized tRNA is joined to HSRCONHNH₂ by hydrazone formation. Ferritin is then coupled to tRNA by reaction of the CBr and SH bonds. In the BI procedure a lysine amino group of ferritin is coupled by Schiff base formation with 3'-oxidized RNA. The conjugate is stabilized by borohydride reduction. In the BII procedure, a —COCH₂Br group is attached to ferritin. (H₂NCH₂CH₂S—)₂ is coupled to oxidized tRNA by Schiff base formation and borohydride reduction. An SH group is exposed by reduction. This HS-tRNA is coupled to a —COCH₂Br group attached to ferritin. All the procedures work but BII is recommended. Methods for purifying the Fer-tRNA and the Fer-tRNA-DNA hybrid are described. For the transducing phages, φ80hpsu^+,?_III and φ80hpsu^?_III, the DNA molecules each carry a piece of bacterial DNA of length 0·066±0·007 λ unit (3100 nucleotide pairs; we find the length of λ is 8·99 φX174 units) replacing a piece of phage DNA of φ80h of length 0·045±0·005 λ unit. The left junction of this bacterial DNA with phage DNA (referred to as P-B′) is at or close to the att site. The two tandem tyrosine genes of φ80hpsu^+,?_III and the single tRNA gene of φ80hpsu^?_III have been mapped at a position 1100 nucleotides to the right of the left (P·B′) junction of phage DNA and bacterial DNA, by hybridizing Escherichia coli Fer-tRNA to φ80hpsu_III/φ80h heteroduplexes. The separation of the two ferritin labels in φ80hpsu^+,?_III hybrids gives 140±20 nucleotides as the size of a single tyrosine tRNA gene.     

A comparison of virulence determinants in an octopine Ti plasmid, a nopaline Ti plasmid, and an Ri plasmid by complementation analysis of Agrobacterium tumefaciens mutants

Transposon-insertion mutants with vir^? Ti plasmids were characterized and then used in complementation experiments. One of the mutants (LBA 1517) had a mutation in a newly discovered vir locus called virF. The virF mutation led to a strongly diminished virulence on tomato and tobacco, but not on certain other plant species. Also a mutant (LBA 1505) was isolated with a mutation somewhere in the bacterial genome but outside the octopine Ti plasmid that caused a restriction in host range for tumor induction. Introduction of a nopaline Ti plasmid or an Ri plasmid into LBA 1505 did not restore normal virulence, showing that the vir gene affected in LBA 1505 determines a factor which is essential for normal tumor induction both by different types of Ti plasmids and by the Ri plasmid. The introduction of R primes containing part or all of the octopine Ti plasmid virulence region led to a restoration of virulence in strains with a vir^? nopaline Ti plasmid. Also the transfer of an Ri plasmid to a large number of different vir^? octopine or nopaline Ti plasmid mutants rendered these strains virulent. These results indicate that the octopine Ti plasmid, the nopaline Ti plasmid, and the Ri plasmid each have a similar virulence system which can mediate the transfer of T-DNA to plant cells from different types of Ti or Ri plasmids. In complementation experiments between vir^? octopine Ti plasmid mutations and vir^? nopaline Ti plasmid mutations it was found that equivalent functions are determined by the areas of DNA homology in the virulence regions of these two types of Ti plasmids. The previously defined octopine Ti plasmid virC locus appeared to consist of two different loci. One of these loci was found to be in a region of the octopine Ti plasmid which does not share DNA homology with the nopaline Ti plasmid, and was therefore called virO (octopine Ti plasmid specific). For the other locus the name virC was retained. Whereas mutations in the virC locus were avirulent on all plant species tested, mutations in virO were avirulent on tomato and pea, but virulent on sunflower and Nicotiana rustica. VirO^? mutants produced rooty tumors on Kalanchoë tubiflora.     

Study of the incompatibility and replication of the 70-kb virulence plasmid of Yersinia

The 70-kb virulence plasmid, vir, from four Yersinia enterocolitica and one Y. pseudotuberculosis strains are incompatible with IncFI plasmids FLac and R386 while they are compatible with plasmids representing nine other incompatibility groups. Hybridization experiments carried out on one of these virulence plasmids showed that it contains the F incompatibility determinant D, incD. This determinant was cloned onto pACYC184 and the recombinant clone expressed incompatibility with FLac. We conclude that the incompatibility observed between F or R386 and the 70-kb virulence plasmid of Y. enterocolitica and Y. pseudotuberculosis is mediated by incD. Replication genes (rep) from the same plasmid were cloned independently in Escherichia coli. Rep and incD map on two different BamHI fragments. Surprisingly, the replicon isolated is not sensitive to inc D incompatibility. Apart from incD, vir and F share extremely little homology. In particular, there is no evidence for the presence of an F-like transfer operon on vir.     

F plasmid incompatibility and copy number genes: Their map locations and interactions

The genes coding for vegetative F plasmid replication, replication control, and incompatibility are known to map between the kilobase coordinates 40.3 and 49.3 (abbreviated 40.3–49.3F). We have subdivided this region of the F genome by a combination of in vivo and in vitro genetic techniques and have constructed F:pSC101 hybrid plasmids which contain the F DNA sequences having the approximate coordinates 41–43, 43–46, and 46–49F. We find that hybrids with regions 43–46 and 46–49F are incompatible with an F′lac⁺ plasmid while the hybrid with the region 41–43F is compatible. We have also constructed similar F:pSC101 hybrid plasmids with the regions 43–46 and 46–49F derived from mini-F plasmid copy number mutants. We find that hybrids made from three independent F copy number mutants show a loss of the incompatibility function associated with the 43–46F region and retention of the incompatibility function associated with 46–49F region. Moreover, spontaneous revertants, selected for regain of the 43–46F incompatibility function, have also regained normal control over their copy numbers. We also find that copy number mutations map in the 43–46F region. From our results we conclude (i) that F contains at least two inc⁺ loci, designated incA⁺ (46–49F) and incB⁺ (43–46F), and (ii) that gene(s) regulating F copy number may be related to the incB⁺ gene(s).     

A host-dependent hybrid plasmid suitable as a suicidal carrier for transposable elements

Plasmid pAS8Tc^s rep-1::Tn7 (abbreviated pAS8Rep-1), a derivative of the RP4-ColE1 hybrid plasmid pAS8 displaying ColE1-dependent replication/maintenance, was found capable of the introduction of transposon Tn7 into the genome of phytopathogenic Pseudomonas. The plasmid is potentially useful as a general purpose suicidal Tn carrier for bacteria that do not support stable replication/maintenance of ColE1 but are within the conjugational host range of RP4.     

Prophage lambda at unusual chromosomal locations: III. The components of the secondary attachment sites

The integration of phage λ occurs by a reciprocal genetic exchange, promoted by the product of phage int gene, at specific sites on the phage and bacterial genomes (att's). Lysogenic bacteria thus contain two att's which bracket the inserted prophage. Genetically, the phage, bacterial and prophage att's differ from each other, indicating that each site has specific elements which segregate during recombination.In hosts that lack the bacterial att, phage integration occurs at about 0.5% the normal frequency. It results from Int-promoted recombination between the phage att and any one of many secondary sites in the bacterial genome. To analyze these sites, we measured Int-promoted recombination at the secondary prophage att's. We found that they differed from the normal prophage att's and from the phage att. The secondary sites, therefore, do not appear to carry any of the specific elements of the phage or bacterial att's.The transducing phage isolated from secondary site lysogens integrate at two loci. In the absence of helper, they insert via homology with the bacterial DNA. Co-infection with helper results in their integration at the normal bacterial att.     

A convective mass transfer model for determining intestinal wall permeabilities: laminar flow in a circular tube

A convective mass transfer model as analyzed and developed for use in determining intestinal wall permeabilities from external perfusion experiments. Analysis of the model indicates that the ratio of the exit to inlet concentration $\text{C}{}_{\mathrm{m}}\text{C}{}_0$ is a function of only two dimensionless independent variables, the wall permeability, $\text{P}{}_{\mathrm{w}}{}^1$ and Graetz number, Gz = πDL/2Q. The Graetz number contains the independent variables of interest, length, diflusivity, and flow rate. The radius of the intestine is included implicitly in the flow rate. Since $\text{C}{}_{\mathrm{m}}\text{C}{}_0$ and Gz are the experimental quantities, and the solution to the model system contains P_ω¹ implicitly, a convenient approximate method is developed which allows a direct calculation of P_ω¹. This method is in error by 10–20% in the worst cases. The approach is illustrated by application to the determination of the wall permeabilities for two non polar compounds.     

Conditional lethal mutants of the kilB determinant of broad host range plasmid RK2

We have examined the relationship of kilB to the other known determinants which map in the 14'-22' region of RK2. These are trfA, which encodes a diffusible replication function, and tra3, which specifies a function required for plasmid transmissibility. We found that, in addition to kilB, both tra3 and trfA functions are expressed by the cloned 14'-22' region of RK2. Four temperature-sensitive mutants of kilB were isolated by in vitro mutagenesis of the cloned segment. At 42 degrees C these mutant plasmids can be maintained in Escherichia coli cells which lack a korB+ helper plasmid. At 30 degrees C the helper plasmid is required. Our analysis of these mutants revealed that kilB function is distinct from those of trfA and tra3. One mutant plasmid was temperature-sensitive for maintenance of an RK2 ori plasmid, but this phenotype was shown to be independent of the KilB(ts) phenotype. Thus, kilB appears to be a separate new locus in this portion of the RK2 genome. In addition, these mutants allowed us to test for the existence of an essential replication determinant (trfB) in the 50.4'-56.4' region of RK2. Our results demonstrate that this region is non-essential for replication from the RK2 ori in E. coli. We propose an alternative hypothesis to explain the role of the RK2 trfB region for plasmid maintenance in E. coli.     

The multicomponent nature of teratoma cell adhesion factor

The multicomponent nature of teratoma cell adhesion factor has been demonstrated. Fractionation of crude ascites fluid on a DEAE cellulose ion exchange column shows that two or more components are involved in teratoma adhesion factor (TAF) activity. Glycoproteins (or proteoglycans) in fractionated ascites fluid were localized in polyacrylamide gels. The possible role of these sugar-containing molecules in teratoma cell adhesion and current hypotheses on the mechanism of carbohydrate involvement in intercellular adhesion are discussed.     

Effect of complexation of flavin radical with tryptophan on electron transfer rates: a model for flavin-protein interactions

The rates of oxidation of lumiflavin radical by ferricyanide, indole radical and oxygen are decreased by factors of four to ten as a result of complexation with tryptophan. Tyrosine, methionine and glycine were found not to measurably alter the flavin radical reactivity. Similar results were obtained using flavinyl peptides in which tryptophan or methionine were covalently linked to the flavin. These observations suggest that one of the consequences of the interaction between the flavin and a tryptophan side chain in the coenzyme binding site of the flavodoxins is to deactivate the semiquinone form of the enzyme towards oxidizing agents, thereby increasing its stability.     

Stabilization of the cloning vector pACYC184 by insertion of F plasmid leading region sequences

The leading region of the F plasmid is, by definition, the first part of the plasmid DNA to be transferred to the recipient cell during conjugation. Restriction fragments of the leading region, when cloned into the plasmid vector pACYC184, extended the maintenance of the normally unstable p15A-derived vector replicon in rec+ Escherichia coli K-12 cells. Mutations in the host's general recombination systems were found to influence the maintenance of these hybrid plasmids.     

Specific pattern of instability of Escherichia coli HisG gene cloned in Bacillus subtilis via the Staphylococcus aureus plasmid pCS194

The plasmid pCS194, generated in vivo by recombination of two Staphylococcus aureus plasmids, pC194 and pS194, coding, respectively, for chloramphenicol (Cm) and streptomycin (Sm) resistance, can be replicated also in Bacillus subtilis in the presence of either of the two antibiotics. In their absence, no segregation of the individual components is observed, but the whole plasmid is lost at a rate of about 10% per generation. The unique EcoRI site of pCS194 is located in the Sm^R determinant. EcoRI-cleaved pCS194 has been joined to an EcoRI-linearized Escherichia coli replicon, the in vitro recombinant pHisG plasmid, composed of the vector pBR313 plus a BglII-segment of E. coli chromosomal DNA, containing a functional hisG gene. The ligation mixture has been used to transform either E. coli or B. subtilis. Following E. coli transformation and selection for Ap^R and Cm^R (the latter is expressed in E. coli by the pC194 determinant), two his⁺ clones were picked at random and the plasmids extracted. These appear identical and contain the original segments. Conversely, after transformation of B. subtilis and selection for Cm^R, only his^? clones have been obtained. From them, deleted plasmids have been extracted. They have lost part or, more frequently, all of the E. coli DNA insert. In the latter case also most of the bracketing pS194 sequence has been lost, and the resulting plasmids are almost identical to pC194, the Cm^R parent of pCS194. When the intact recombinant plasmids, isolated from his⁺ Ap^R Cm^RE. coli clones, have been used to transform B. subtilis cells for Cm^R, again deleted plasmids almost identical to pC194 have been obtained. The events causing these rearrangements occur after in vitro ligation, during either transformation or early propagation of the plasmids, and are probably caused by a translocatable element present in pCS194. A detailed physical map of pC194, carrying the restriction sites for HindIII, HaeIII, HpaII, MboII, AluI, HhaI, and BglI, is presented.