SOS-induction of the RP4 plasmid tet-determinant

Induction of the tetracycline-resistance genes function by the inducer of the DNA-repair and mutability SOS-system, UV-light, has been tested. Activity of the tet-genes residing on the plasmid RP4 in Escherichia coli cells has been shown to be inducible by the low doses of tetracycline as well as by the mutagenic doses of UV-light. The induction was quantitatively registered by measuring the activity of beta-galactosidase of bacteriophage Mud1 (Ap, Lac) inserted into the tet-genes of the plasmid RP4. The bacteriophage integration inactivates the tet-genes function of the constructed plasmid fusing the lac-operon to a promoter of inactivated genes. Precise excision of bacteriophage restores the activity of the tet-genes proving together with the plasmid DNA-restriction analysis the fusion of tet-promoter with Iac-operon. The tet-genes of RP4 are concluded to be a part of the SOS-regulon, a set of genes inducible by the conditions harming the bacterial cell. Preliminary data on the mutagenic activity of tetracycline obtained in the bacterial test-system of mutagens are discussed.     

pMH2, a small plasmid bearing the nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette

A small plasmid containing the entire nif gene cluster of Enterobacter agglomerans 333 as an excisable cassette has been constructed, using pACYC177 as a vector. Two cosmid clones taken from a gene library of E. agglomerans plasmid pEA3 were used as a source of nif genes. A SmaI fragment of peaMS2-2, containing the H,D,K,Y,E,N,X,U,S,V,W,Z,M,L,A and B genes and an ApaI fragment of peaMS2-16 containing nifA,B,Q,F and J were selected to construct pMH2. The resulting plasmid of 33 kb carries the complete nif gene cluster as a nif cassette on a single XbaI fragment. The nif construct pMH2 in Escherichia coli strains has significant nitrogenase activity compared to wild-type E. agglomerans 333. The nif gene cluster construct was found to be very stable.     

Selection, analysis and mapping of mutations in the gene for resistance to kanamycin in plasmid RP4

We have tested possibilities of Escherichia coli strains dependent on drugs streptomycin and paromomycin for selection of spontaneous mutations in the RP4 kan gene specifying resistance to aminoglycosids--kanamycin, neomycin and paromycin. A set of kan gene mutations were obtained, classified ad mapped.     

Physical and genetic map of the major nif gene cluster from Azotobacter vinelandii.

Determination of a 28,793-base-pair DNA sequence of a region from the Azotobacter vinelandii genome that includes and flanks the nitrogenase structural gene region was completed. This information was used to revise the previously proposed organization of the major nif cluster. The major nif cluster from A. vinelandii encodes 15 nif-specific genes whose products bear significant structural identity to the corresponding nif-specific gene products from Klebsiella pneumoniae. These genes include nifH, nifD, nifK, nifT, nifY, nifE, nifN, nifX, nifU, nifS, nifV, nifW, nifZ, nifM, and nifF. Although there are significant spatial differences, the identified A. vinelandii nif-specific genes have the same sequential arrangement as the corresponding nif-specific genes from K. pneumoniae. Twelve other potential genes whose expression could be subject to nif-specific regulation were also found interspersed among the identified nif-specific genes. These potential genes do not encode products that are structurally related to the identified nif-specific gene products. Eleven potential nif-specific promoters were identified within the major nif cluster, and nine of these are preceded by an appropriate upstream activator sequence. A + T-rich regions were identified between 8 of the 11 proposed nif promoter sequences and their upstream activator sequences. Site-directed deletion-and-insertion mutagenesis was used to establish a genetic map of the major nif cluster.     

Detection of plasmid RP4 transfer in soil and rhizosphere,and the occurrence of homology to RP4 in soil bacteria

Plasmid RP4 transfer between introduced pseudomonads was studied in non-rhizosphere and rhizosphere soil. The addition of nutrients to the non-rhizosphere soil stimulated plasmid transfers between introduced donor and recipient cells, and no transfer was detected in nonamended soil. Transfer was also detected in soil in a model rhizosphere, but not in corresponding non-rhizosphere soil. Colony hybridization with whole plasmid RP4 DNA as a probe was employed to detect transfers to indigenous organisms in soil. Although transfers to introduced recipient cells were easily detected in parallel controls, no indigenous organisms were identified that had received RP4. Background levels of soil organisms with the RP4 resistance pattern were considerable, and about 10% of these populations contained DNA sequences with homology to RP4. However, no plasmids could be detected in any of 20 isolates, nor was resistance transfer to aPseudomonas fluorescens recipient detected in filter matings.     

Localization and physical mapping of a plasmid-borne 23-kb nif gene cluster from Enterobacter agglomerans showing homology to the entire nif gene cluster of Klebsiella pneumoniae M5a1

A physical and genetical map of the plasmid pEA3 indigenous to Enterobacter agglomerans is presented. pEA3 is a 111-kb large plasmid containing a 23-kb large cluster of nif genes which shows extensive homology (Southern hybridization and heteroduplex analysis) to the entire nif gene cluster of Klebsiella pneumoniae (Kp) M5a1. All the nif genes on pEA3 are organized in the same manner as in K. pneumoniae, except nifJ, which is located on the left end of pEA3 nif gene cluster (near nifQB). A BamHI restriction map of pEA3 and a detailed restriction map of the 23-kb nif region on pEA3 is also presented. The nif genes of pEA3 showed a low level of acetylene reduction in Escherichia coli, demonstrating that these genes are functional and contain the whole genetic information required to fix nitrogen. The origin of vegetative replication (OriV) of pEA3 was localized about 5.5 kb from the right end of the nif gene cluster. In addition to pEA3, large plasmids from four other strains of E. agglomerans showed homology to all the Kp nif genes tested, indicating that in diazotrophic strains of E. agglomerans nif genes are usually located on plasmids. In contrast, in most of the free-living, nitrogen-fixing bacteria the nif genes are on chromosome.     

Transfer of plasmid RP4 in the spermosphere and rhizosphere of barley seedling

Transfer of plasmid RP4 to indigenous bacteria in bulk soil could only be detected in soil with nutrient amendment. Lack of physiological active donor and recipient cells was apparently one of the limiting factors in un-amended bulk soil. Plasmid transfer was detected both in the spermosphere and rhizosphere of barley seedlings. Transfer occured from seed coated donor bacteria (i) to introduced recipient bacteria and (ii) to indigenous bacteria present in soil. Plasmid transfer was also detected from donor bacteria introduced to the soil to seed coated recipient bacteria. Transfer efficiencies in the rhizosphere were significantly below the transfer efficiencies obtained in the spermosphere. The transfer efficiencies detected in the barley spermosphere were among the highest reported from any natural environment.     

Transfer of prophage Mu into methylotrophic bacteria in the plasmid RP4

Bacteriophage Mu genome has been transferred into the cells of Pseudomonas methanolica and Methylobacterium sp. SKF240, that are naturally resistant to the bacteriophage, as a fragment of a hybrid plasmid RP4::Mu cts62. Temperature induction of the bacteriophage results in host cell lysis. Plasmid RP::Mu cis62 is maintained in methylotrophic cells presenting a cointegrative structure.The genetic and electrophoretic, analyses of the DNA isolated from transconjugant cells have confirmed the conclusion. Bacteriophage Mu propagation has been shown to be restricted in methylotrophic cells.     

Chromosomal transfer promoted by the promiscuous plasmid RP4.

We have studied the properties of the recombinant plasmid RP4λatt. This plasmid possesses the EcoRI-generated fragment of phage λ containing the genes att-int-xis (srIλ2–3) inserted into the single EcoRI site of the promiscuous plasmid RP4. The insertion of this λ fragment has no detectable effect on normal plasmid functions. However, it confers the ability to promote low-frequency polarized chromosomal transfer by int-promoted integration into the host λ attachment site attλ. We have succeeded in isolating an Hfr derivative which has the plasmid stably integrated at attλ. The Hfr derivative is unusual in having both an integrated and an autonomous RP4λatt stably coexisting in the same cell.     

Effect of plasmid F'Col+trp+ on the transfer of plasmid RP4]

Interaction of conjugative plasmids F'colV colB trp and PR4 in Escherichia coli host was studied during the transfer of the plasmids from cell to cell. The plasmid F'colV colB trp is found to stimulate the transfer of RP4 from the diplasmid strain. This seems to be due to stabilization of the conjugating pairs which require normal pili coded by the plasmid F'colV colB trp.     

Two mechanisms necessary for the stable inheritance of plasmid RP4

Plasmid RP4 is stably maintained in strains of Escherichia coli and other Gram-negative bacteria. Inactivation of the plasmid primase gene (pri) or removal of the PstIC fragment gave RP4 derivatives that are slightly unstably maintained in E. coli. Removal of the Tn 1 multimer resolution system (res and tnpR) did not lead to any detectable plasmid loss. Removal of all three of these regions, however, gave rise to pNJ5000 which is lost at high frequency. We have dissected the mechanisms causing this phenomenon. In contrast to RP4, pNJ5000 accumulates significantly as plasmid multimers in a Rec+ host; in a recA host, multimers are not seen and the plasmid is stably maintained. Multimers therefore appear to form by recA-mediated homologous recombination and cause plasmid instability, perhaps by interfering with partition. We demonstrate a mechanism provided by the PstIC fragment which acts on multimers analogously to the Tn1/3 resolution system on plasmid cointegrates, being effective only when cloned in cis. The loss of pri, on the other hand, can be complemented in trans. Our results are consistent with the view that primase prevents multimers forming (rather than resolving them once formed), perhaps by binding specifically to single-stranded regions of the plasmid and preventing homologous pairing.     

Sym plasmid genes of Rhizobium trifolii expressed in Lignobacter and Pseudomonas strains.

A 14-kilobase (kb) fragment of Rhizobium trifolii Sym plasmid containing nodulation (nod) genes or the pSym plasmid of R. trifolii cointegrated with a broad-host-range vector R68.45 (pPN1) were transferred to Lignobacter strain K17 and Pseudomonas aeruginosa strain PAO5 by conjugation. Lignobacter transconjugants carrying Sym plasmid pPN1 formed nodules on white, red, and subterranean clover plants. Lignobacter transconjugants containing a 14-kb fragment of nod genes cloned into a multicopy plasmid nodulated only white and subterranean clover plants, whereas transconjugants carrying the same fragment cloned into a low-copy plasmid vector nodulated only white clover plants. All nodules formed by Lignobacter transconjugants showed bacterial release from the infection threads into the host cytoplasm. Pseudomonas transconjugants with plasmid pPN1 formed nodule-like structures on white clover plants. These structures were not invaded by bacteria; however, a few bacteria were found within the intercellular spaces of the outermost cells of the structures. Pseudomonas transconjugants carrying the 14-kb fragment of R. trifolii nod genes did not form nodules on tested clover plants. All clover plants inoculated with either Pseudomonas or Lignobacter transconjugants containing a 14-kb fragment of nod genes (but not entire Sym plasmid) showed the "thick-and-short-root" response when compared to the control plants inoculated with the R. trifolii wild-type strain.     

Characterization of in vitro constructed IS30-flanked transposons

In order to facilitate functional studies on the mobile genetic element IS30, a resident of the Escherichia coli chromosome, transposon structures with two copies of IS30 flanking the chloramphenicol-resistance gene cat were constructed in vitro. Transposons containing IS30 as direct repeats (Tn2700 and Tn2702) transpose from multicopy plasmids into the genome of phage P1-15, thus giving rise to special transduction for cat with frequencies between 10(-5) and 10(-8)/plaque-forming unit. In contrast, transposon structures with IS30 in inverted repeat (Tn2701 and Tn2703) showed no detectable (less than 10(-9] transposition activity in vivo. By restriction analysis, two insertion sites of Tn2700 and Tn2702 on the phage P1-15 genome were indistinguishable from those observed earlier with a single copy of the IS30 element. These two insertion sites were used several times independently by Tn2700 and Tn2702. This confirms the non-random target selection by the element and it indicates that transposition of Tn2700 and Tn2702 follows the same rules as that of IS30.     

Use of RP4-prime plasmids constructed in vitro to promote a polarized transfer of the chromosome in Escherichia coli and Rhizobium meliloti.

Summary RP4-prime plasmids containing chromosomal fragments of either Escherichia coli or Rhizobium meliloti were constructed in vitro. When introduced into E. coli or R. meliloti respectively, they promoted a polarized transfer of the chromosome as demonstrated either by the gradient of transfer of various markers or by the study of the genetic constitution of recombinants. In E. coli, mobilization was shown to be dependent upon the presence of a functional rec A system. Inheritance of markers was due to their integration into the chromosome of the recipient as shown by the need for a functional rec A system in the recipient E. coli or by mobilization of recessive markers in R. meliloti. The system described could be applied to genetic mapping in any Gram negative bacteria.     

Properties of plasmids constructed by the in vitro insertion of DNA fromRhizobium leguminosarum orProteus mirabilis into RP4

Summary Plasmids have been constructed by insertion of DNA fromRhizobium leguminosarum orProteus mirabilis into RP4 (an R factor of group P). Such recombinant plasmids retain the wide host range of the parental plasmid, being as efficiently transmissible as the unmodified RP4 and are stably maintained in rapidly growing cultures.The recombinant plasmids, even though each contained a DNA sequence absolutely identical with that of the host strain, are no more efficient at mobilizing the transfer of chromosomal genetic information from that host strain than was unmodified RP4. We therefore conclude that an unknown factor must be essential in the process of chromosome mobilization and rate limiting for that process.     

Characterization of the virA gene of the agropine-type plasmid pRiA4 of Agrobacterium rhizogenes

We sequenced a 4.2-kb DNA region encompassing the vir A locus of the hairy-root-inducing plasmid pRiA4, and compared its sequence with the published vir A region sequences of four tumor-inducing plasmids. An open reading frame capable of coding for 829 amino acids was identified for vir A. Deletion mutants of vir A constructed by fusing to lacZ, but not the wild-type game itself, were efficiently expressed in Escherichia coli when they were put downstream front the lac promoter. These fused gene products became soluble or insoluble depending on the length of their lacZ moieties.