Suppression of tumorigenicity by the IncW R plasmid pSa in Agrobacterium tumefaciens

Summary The effect of the IncW R plasmid, pSa, on tumorigenicity and on the expression and maintenance of the Ti plasmid in tumorigenic strains of A. tumefaciens was determined. Plasmid pSa could be transferred into and stably maintained by both octopine-and nopaline-utilizing A. tumefaciens strains. The R plasmid had no effect on Ti plasmid maintenance or on Ti plasmid functions, such as octopine utilization or conjugal bacterial transfer. However, A. tumefaciens strains harboring both the R plasmid and the Ti plasmid in most instances failed to induce tumors on a number of plant species. This effect on tumorigenicity is specific to pSa. When pSa is cured from the A. tumefaciens transconjugants or when their Ti plasmids are genetically transferred to an appropriate recipient, the resultant strains lacking the R plasmid regain tumorigenicity. Restriction endonuclease analysis of plasmid DNA isolated from transconjugants harboring pSa showed no difference in Ti plasmid cleavage patterns when compared to plasmid DNA isolated from the tumorigenic parent strain. These results indicate that pSa does not induce detectable permanent genetic alteration of the Ti plasmid. Rather, it appears that the R plasmid suppresses some Ti plasmid function(s) necessary for tumorigenicity.     

Genetic map of the crown gall suppressive IncW plasmid pSa

Summary A genetic map of the W incompatibility group plasmid pSa has been prepared through the construction of deletion derivatives of pSa and the cloning of various fragments of pSa in pBR322. Phenotypic analysis of these derivatives has identified the location of genes encoding resistance to chloramphenicol, sulfonamides, spectinomycin, streptomycin, kanamycin, gentamycin, and tobramycin. Information sufficient for the replication of the plasmid in both Escherichia coli and Agrobacterium tumefaciens is contained within a 4 kilobase pair region. Two regions have been identified as involved in the transfer of the plasmid; one of these regions is also involved in the inhibition of oncogenesis by pSa when it is present in an oncogenic strain of A. tumefaciens. Certain of the deletion derivatives of pSa are potential vectors for the cloning and analysis of A. tumefaciens Ti plasmid DNA.     

Convenience of plasmid R6K higher-copy-number deletion derivative for cloning of larger DNA fragments

Vector properties of plasmid pNH602, a higher-copy-number deletion mutant of plasmid R6K, were tested by cloning the 6.5 Mg/molBam HI pSa fragment carrying determinants of resistance to four antibiotics in the uniqueBam HI site of pNH602. The resultingin vitro constructed recombinant plasmid pNH606 was found to be stable, conjugative, multicopy (20 copies of pNH606 perE. coli chromosome were estimated) and to ensure the increased expression of different genes responsible for the antibiotic resistance. The pSa fragment inserted in theBam HI site of plasmid pNH602 (located in Tn2660) was proved to be transposable to other replicons. Recombinant plasmid pNH606 was analyzed using restriction enzymesBam HI andEco RI and its physical and genetic map was constructed.     

A plasmid which can be transferred between Escherichia coli and Pasteurella haemolytica by electroporation and conjugation

Three broad-host-range plasmids (pRK290, pSa4 and pKT230) and one native Pasteurella haemolytica plasmid (pPH33) were used in transformation experiments with P. haemolytica strains T179 (serotype A1), Y216 (serotype A2) and its capsular-deficient variant Y216/NS1. No transformants were detected with either heat-shock or freeze-thaw techniques. However, by electroporation, all P. haemolytica strains were transformed by pPH33 but not by pRK290 or pSa4. The highest frequency obtained was 91 x 10(4) transformants per microgram of pPH33 DNA with P. haemolytica strain Y216/NS1. Although pPH33 itself was non-transmissible by conjugation, it could be mobilized from Escherichia coli, using the transfer function of the IncP plasmid pRK2013, into P. haemolytica at a frequency of 0.3-2.2 x 10(-3) per recipient cell.     

Effect of Plasmid pSa and of Auxin on Attachment of Agrobacterium tumefaciens to Carrot Cells

When the plasmid pSa is introduced into Agrobacterium tumefaciens, its presence results in the suppression of bacterial virulence. A. tumefaciens(pSa) cells are virulent on Bryophyllum diagremontiana only when inoculated with auxin. A. tumefaciens(pSa) cells also bind to plant cells only in the presence of auxin. The effect of auxin is on the bacteria rather than on the plant cells, since the bacteria require auxin to bind to heat-killed carrot cells. Bacteria containing pSa and grown in the absence of auxin showed a lag in binding to carrot cells in auxin-containing medium. This lag was not seen during the binding of wild-type strains. Tetracycline inhibited the binding of A. tumefaciens(pSa) in auxin-containing medium, suggesting that bacterial protein synthesis is required for the auxin effect. No difference was seen in the size or ability to inhibit bacterial binding of lipopolysaccharides from bacteria containing or lacking pSa and grown with or without auxin. A. tumefaciens(pSa) cells grown in the absence of auxin lacked surface polypeptide(s) found in bacteria grown in the presence of auxin and in the wild-type bacteria, which do not contain pSa. Thus, the presence of certain polypeptides appears to be associated with the ability of the bacteria to bind to plant cells.     

The region essential for efficient autonomous replication of pSa in Escherichia coli

Summary Comparative analyses were made between plasmid pSa17, a deletion derivative of pSa that is capable of replicating efficiently in Escherichia coli and plasmid pSa3, a derivative that is defective for replication. By comparing the restriction maps of these two derivatives, the regions essential for replication and for stable maintenance of the plasmid were determined. A 2.5 kb DNA segment bearing the origin of DNA replication of pSa17 was sequenced. A 36 kDa RepA protein was encoded in the region essential for replication. Downstream of the RepA coding region was a characteristic sequence including six 17 bp direct repeats, the possible binding sites of RepA protein, followed by AT-rich and GC-rich sequences. Furthermore, an 8 bp incomplete copy of the 17 bp repeat was found in the promoter region of the repA gene. Based on the hypothesis that RepA protein binds to this partial sequence as well as to intact 17 bp sequences, an autoregulatory system for the synthesis of RepA protein may be operative. Another open reading frame (ORF) was found in the region required for the stability of the plasmid. The putative protein encoded in this ORF showed significant homology to several site-specific recombination proteins. A possible role of this putative protein in stable maintenance of the plasmid is discussed.     

Natural transformation of Pseudomonas stutzeri by plasmids that contain cloned fragments of chromosomal deoxyribonucleic acid

Natural transformation of plasmids by Pseudomonas stutzeri was found to depend on their bearing inserts of chromosomal DNA. A set of plasmids derived from the nonconjugative broad host range plasmid pSa151 was constructed by integrating various chromosomal DNA fragments into the single EcoR1 site of pSa151. Selection for the kanamycin resistance determined by pSa151 demonstrated that the derivative plasmids were taken into the cells by natural transformation and stably maintained; each could be reisolated unchanged. Thirty-two different derivative plasmids, 14 to 31 kbase pairs in size, all transformed. The frequency of transformation increased with the size of the chromosomal insert over a twenty fold range. These results suggest that the mechanism of transformation of plasmids by the Gram-negative P. stutzeri is similar to those proposed to operate in Gram-positive bacteria.Dedicated to Prof. Dr. H.-G. Schlegel on the occasion of his 60 th birthday     

A comparison of the origin of replication of pSa with R6K

Summary The plasmid pOri3 is a derivative of the origin of replication of pSa. Replication is defective as a result of a truncated repA gene, the product of which is required for plasmid replication. The defective replication is complemented by the presence of the intact repA gene of pSa, or by the presence of the plasmid R6K. The basis of this complementation has been examined by comparing the nucleotide sequence of the origin of pSa with that of R6K. A 13 base pair sequence present twice in the origin of pSa has homology with a 13 base pair sequence that is present fourteen times in the origin of R6K. These sequences may be the binding sites for the initiator proteins of these two plasmids. The location of these binding sites relative to the genes for the initiator proteins suggests that an autoregulatory control mechanism for the synthesis of the initiator proteins may also play a role in the control of plasmid copy number.     

Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains.

Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.     

Mobilization of Thiobacillus ferrooxidans plasmids among Escherichia coli strains

Nonconjugative Thiobacillus ferrooxidans plasmids were mobilized at high frequencies among Escherichia coli strains by the IncP plasmid RP4 and at low frequencies by the IncN plasmid R46, but not by the IncW plasmid pSa. The mobilization region of a nonconjugative T. ferrooxidans plasmid was located on a 5.3-kilobase T. ferrooxidans DNA fragment.     

Plasmid shuttle vector with two insertionally inactivable markers for coryneform bacteria

A new shuttle vector pCEM500 replicating inEscherichia coli and inBrevibacterium flavum was constructed. It carries two antibiotic resistance determinants (Km^r/Gm^r from plasmid pSa of Gram-negative bacteria and Sm^r/Sp^r from plasmid pCG4 ofCorynebacterium glutamicum) which are efficiently expressed in both hosts and can be inactivated by insertion of DNA fragments into the unique restriction endonuclease sites located within them. This vector was found to be stably maintained inB. flavum and can be used for transfer of the cloned genes into this amino-acid-producing coryneform bacterium.     

Conjugal transfer of plasmid and chromosomal markers between strains of Thiobacillus versutus

In experiments on conjugation in Thiobacillus versutus with the use of pTAV1-less strains as recipients, we have proved that the derivative of the wild-type T. versutus cryptic plasmid pTAV1 (107 kb) marked with Tn1721 (Tc^r) transposon demonstrates Tra^- phenotype but can be mobilized for transfer by pSa Tra⁺ broad-host-range helper plasmid at a low frequency. The possibility of chromosomal gene exchange between different auxotrophic and drug-resistant T. versutus mutants has been confirmed. The previously assumed participation of plasmid pTAV1 in the above process must be excluded because conjugal transfer of chromosomal markers can be observed even when two pTAV1-free strains are mated. Formation of some classes of transconjugants can be reasonably explained only when two-directional chromosomal DNA transfer (retrotransfer) is considered. At this stage of our studies we can not propose any hypothesis on the mechanism of chromosomal gene transfer. The possible role of the megaplasmids discovered in T. versutus in chromosome mobilization needs to be elucidated.     

Modification of T-DNA of nopaline Ti plasmid by intermediary vector and utilization of agrocin 84 sensitivity as simple criterion of conjugation transfer of modified Ti plasmid

The chloramphenicol resistance gene from pSa was introduced into T-DNA of pTi T37 of Agrobacterium tumefaciens by cointegration with intermediary plasmid based on pBR322. The resulting intermediary vector was mobilized to A. tumefaciens T37 by conjugative plasmid pRK2. The RK2 plasmid also forms contegrates with pTi due to the Tn3 transposon which was used for the mobilization of modified pTi into plasmid-less A. tumefaciens strain. Transconjugants were selected on the basis of their antibiotic resistance markers and tested for agrocin sensitivity as proof of Ti plasmid transfer. Agrocin sensitivity of tranconjugants together with chloramphenicol resistance was shown to be a sufficient and simple criterion of transfer of modified Ti plasmids. Agrobacterium strains with modified Ti plasmids showed decreased virulence in consequence of the presence of additional borderline sequence inside their T-DNA.     

Stabilization of a miniderivative of the broad-host-range IncW plasmid pSa by insertion of plasmid R1parB region

The plasmid vector pEM100 (13.5 kb) constructed from pGV1106, a miniderivative of the broad-host-range IncW pSa plasmid, and the pAM330 plasmid ofBrevibacterium lactofermentum is not stably maintained inEscherichia coli host cells under nonselective growth conditions. By insertion of a 0.9 kb DNA fragment containing theparB locus (responsible for the maintenance of plasmid R1 inE. coli cells) to plasmid pEM100, plasmid pEM110 was prepared which is maintained in a population ofE. coli cells growing without a selection pressure very stably. Translated by Č. Novotny     

Restriction mapping of plasmid pSa1, isolated from Streptomyces albus G strain

A novel plasmid designated pSa1 has been isolated from Streptomyces albus G strain producing SalGI restriction endonuclease. Molecular weight of the plasmid is 3.4 +/- 0.2 mD. The action of 12 restriction endonucleases on the plasmid DNA was studied. Restriction map of pSa1 DNA was established for SmaI, HindII, XbaI and KpnI endonucleases.     

Electroporation and conjugal plasmid transfer to members of the genus Aquaspirillum

Electroporation methods and conjugal matings were used to transfer several plasmid vectors to Aquaspirillum dispar and Aquaspirillum itersonii. The incompatibility P class plasmid RP4 was conjugally transferred from Escherichia coli HB101 to these spirilla, and the transconjugants subsequently donated the molecule to plasmid-free E. coli and A. dispar strains via conjugal matings. High-voltage electrotransformation was used to transfer plasmids pUCD2, pSa151 and RP4 to A. dispar and A. itersonii, at efficiencies as high as 3×10⁴ transformants per μg plasmid DNA. RP4 DNA isolated from spirillum hosts, but not RP4 from E. coli cells was successfully transferred to A. dispar and A. itersonii by electrotransformation, suggesting that modification and/or restriction activity may be present in these Aquaspirillum species.     

The effect of Sa and MiniSa plasmids on the induction of plant crown galls and hairy roots byAgrobacterium tumefaciens andAgrobacterium rhizogenes

The Inc-W group plasmid Sa or its derivative MiniSa were introduced into two strains ofAgrobacterium tumefaciens with Ti plasmids, one strain ofA. tumefaciens with the Ri plasmid and oneA. rhizogenes strain with the Ri plasmid. The effect was similar in allAgrobacterium strains. The pSa suppressed fully the virulence ofAgrobacterium strains (i.e. their ability to induce tumor growths - crown galls or hairj⁷ roots) inKalanchoe plants and carrot root slices. The MiniSa plasmid caused only a slight decrease of the frequency and size of tumor growths induced. The mechanism of suppression of virulence by the Sa plasmid inAgrobacterium tumefaciens andAgrobacterium rhizogenes seems to be similar.     

Detailed restriction enzyme map of crown gall-suppressive IncW plasmid pSa, showing ends of deletion causing chloramphenicol sensitivity.

Detailed restriction enzyme maps of the antibiotic resistance plasmid pSa and of a chloramphenicol-sensitive spontaneous deletion mutant of pSa were constructed. The smaller plasmid contained one set of three restriction enzyme sites that appeared at both ends of the deleted region in the intact plasmid. The frequency of loss of chloramphenicol resistance was 1% after eight logs of growth and was dependent on the recA gene function of Escherichia coli. A previously published map lacks an 11-kilobase-pair SstII fragment present on this map.     

Isolation and chromosomal localization of new MITE-like sequences from <Emphasis Type="Italic">Secale</Emphasis>

A species-specific DNA sequence (marker) that can detect the presence of Secale cereale chromatin in common wheat background was developed by using wheat microsatellite primer Xgwm614. One rye-specific DNA amplified fragment of 416bp (pSa614₄₁₆) was obtained from Secale africanum and a wheat — S. africanum amphiploid. The primer Xgwm614 also gave rise to specific bands in all Chinese Spring-Imperial rye addition lines 1R to 7R. Sequence analysis revealed that pSa614₄₁₆ was strongly homologous to a miniature inverted transposable element (MITE) stowaway-like element. Results of fluorescence in situ hybridization showed that the signal of pSa614₄₁₆ was distributed along all S. cereale. cv Jingzhou chromosomes, but the signal strengths were unbalanced on the seven rye genome chromosomes. This repetitive element may be useful as a molecular marker for the introgression of rye germplasm into the wheat genome.