Structural-functional organization of R-plasmid pBS222 with a broad range of bacterial hosts

The broad host-range plasmid pBS222 is compatible with broad host-range plasmids of all known incompatibility groups and codes for tetracycline resistance. pBS222 is efficiently mobilized by Inc P-1 plasmid RP4 and is also capable of conjugal transfer with low efficiency to different gramnegative microorganisms. The size of the plasmid (17.2 Kb) has been determined and its physical map has been constructed. The plasmid harbours the unique sites for restriction endonucleases BglII, HindIII, HpaI, KpnI, SmaI and XbaI cleawage. The plasmid derivatives pBS352-pBS355 have been obtained that carry kan- and cam-determinants in addition to tet-gene. Plasmid pBS355 has been used to clone EcoRI-fragments of phage lambda DNA. The plasmid pBS222 regions essential for replication and maintenance have been localized by DNA hybridization analysis of its mini-derivatives pBS356 and 357. pBS222 is a convenient model for investigations of the plasmid replication and maintenance mechanisms in different bacterial hosts as well as for the construction of broad host-range vectors.     

Genetic characteristics and physical organization of the R-plasmid pBS52 with a broad range of bacterial hosts

The genetic and physical data on Pseudomonas aeruginosa plasmid pBS52 coding for the resistances to ampicillin, streptomycin and sulfonamids have been obtained. This conjugative plasmid is transferable to a broad range of gram-negative bacterial hosts and compatible with the broad host-range plasmids from all known incompatibility groups. The plasmid size has been determined (38 Kb) and a physical map has been constructed using restriction endonucleases EcoRI, EcoRV, BamHI, BglII, PstI, PvuII, SalI, SlaI. The presence of a fragment, approximately 200 bp in size, which contains the sites for many of widely used restriction endonucleases is a characteristic feature of the plasmid pBS52.     

A new plasmid pBS1001 with broad range of hosts

A new broad host-range plasmid capable of conjugative transfer has been isolated and characterized. The plasmid has the high frequency of conjugation transfer, is capable of conjugative transfer mobilization of nonconjugative plasmids, carries no known phenotypic markers. The plasmid demonstrates the specific interaction with the plasmids of P incompatibility group. The comparatively small size of the plasmid permits one to use it efficiently for comparative study of organization of the broad host range plasmids.     

Plasmid Transfer by Electroporation and Conjugation in Tetragenococcus and Pediococcus Genera and Evidence of Plasmid-Linked Metabolic Traits

Various strains of Pediococcus genus were successfully transformed by electroporation using the broad host-range plasmid pSA3 and the lactococcal Rep22 based-replicon pUCB304. Failure to transform Tetragenococcus strains by electroporation have led to use conjugation as an alternative plasmid DNA transfer mechanism. Intergeneric matings conducted with the broad host-range conjugative plasmid pVA797 from Lactococcus lactis subsp. lactis SL2/797A to Tetragenococcus halophilus and Pediococcus pentosaceus strains have shown that Tetragenococcus strains are not impervious to plasmid DNA transfer. Plasmid and metabolic profiles of wild and mutant strains of Pediococcus pentosaceus NCDO990 have shown that many metabolic traits including lactose utilization are plasmid linked.     

Unidirectional replication of the P-group plasmid RK2.

The mode of replication of the broad host-range plasmid RK2 has been determined from examination of molecular replicative forms cleaved with the restriction endonucleases EcoRI and Hind III. Replication is unidirectional, and proceeds from a unique origin. The location of the origin and other evidence suggests that genes involved in plasmid maintenance are not tightly clustered.     

Studies of colicin E1 plasmid functions by analysis of deletions and TnA insertions of the plasmid.

The further identification of regions of the colicin E1 plasmid that affect plasmid functions has been achieved by studying deletions and TnA insertions of the plasmid. Colicin production, colicin immunity, relaxation of plasmid deoxyribonucleic acid, and plasmid incompatibility functions have been examined. A strong correlation has been observed between the ability of colicin E1 plasmid deoxyribonucleic acid to be relaxed and the ability of that plasmid to be transferred by conjugation.     

Region-specific insertion of transposons in combination with selection for high plasmid transferability and stability accounts for the structural similarity of IncP-1 plasmids

The overall architecture of IncP-1 plasmids is very conserved in that the accessory genes are typically located in one or two specific regions: between oriV and trfA and between the tra and trb operons. Various hypotheses have been formulated to explain this, but none have been tested experimentally. We investigated whether this structural similarity is due to region-specific transposition alone or also is reliant on selection for plasmids with insertions limited to these two regions. We first examined the transposition of Tn21Km into IncP-1beta plasmid pBP136 and found that most Tn21Km insertions (67%) were located around oriV. A similar experiment using the oriV region of IncP-1beta plasmid pUO1 confirmed these results. We then tested the transferability, stability, and fitness cost of different pBP136 derivatives to determine if impairment of these key plasmid characters explained the conserved plasmid architecture. Most of the pBP136 derivatives with insertions in transfer genes were no longer transferable. The plasmids with insertions in the oriV-trfA and tra-trb regions were more stable than other plasmid variants, and one of these also showed a significantly lower fitness cost. In addition, our detailed sequence analysis of IncP-1 plasmids showed that Tn402/5053-like transposons are situated predominantly between the tra and trb operons and close to the putative resolution site for the ParA resolvase, a potential hot spot for those transposons. Our study presents the first empirical evidence that region-specific insertion of transposons in combination with selection for transferable and stable plasmids explains the structural similarity of IncP-1 plasmids.     

Tn5 mutagenesis of Rhizobium trifolii host-specific nodulation genes result in mutants with altered host-range ability

Summary A 14 kb DNA fragment from the Sym plasmid of the Rhizobium trifolii strain ANU843, known to carry common nodulation nod and host specific nodulation hsn genes, was extensively mutagenised with transposon Tn5. A correlation between the site of Tn5 insertion and the induced nodulation defect led to the identification of three specific regions (designated I, II, III) which affected nodulation ability. Twenty-three Tn5 insertions into region I (ca. 3.5 kb) affected normal root hair curling ability and abolished infection thread formation. The resulting mutants were unable to nodulate all tested plant species. Tn5 insertions in regions II and III resulted in mutants which showed an exaggerated root hair curling (Hac⁺⁺) response on clover plants. Ten region II mutants which occurred over a 1.1 kb area showed a greatly reduced nodulation ability on clovers and produced aborted, truncated infection threads. Tn5 insertions into region III (ca. 1.5 kb) altered the outcome of crucial early plant recognition and infection steps by R. trifolii. Seven region III mutants displayed host-range properties which differed from the original parent strain. Region III mutants were able to induce marked root hair distortions, infection threads, and nodules on Pisum sativum including the recalcitrant Afghanistan variety. In addition region III mutants showed a poor nodulation ability on Trifolium repens even though the ability to induce infection threads was retained on this host. The altered host-range properties of region III mutants could only be revealed by mutation and the mutant phenotype was shown to be recessive.     

In vivo packaging of cosmids in transposon-mediated mutagenesis.

A technique was developed that permits the analysis of large regions of DNA by transposition mutagenesis. Large fragments of the pTiA6NC plasmid were cloned into the broad host range cosmid pHK17 and subjected to transposition mutagenesis by Tn3. Cosmids containing Tn3 insertions were selected by in vivo packaging by lambda cI857 and transduction to a new host. The insertions were localized by DNA restriction endonuclease analysis and transferred to the Ti-plasmid by marker exchange.     

Nucleotide sequence of the trfA gene of broad host-range plasmid RK2

The nucleotide sequence of a 1622 base-pair segment of the broad host-range IncP plasmid RK2 (identical to RP1, RP4, R18 and R68) was determined. This region includes the trfA gene, encoding a trans-acting product essential for vegetative plasmid replication. The nucleotide sequence, together with the results described in the accompanying paper by Shingler & Thomas, indicates that the trfA gene encodes two polypeptide products (of 382 and 285 amino acids) by utilizing different translational start points within a single open reading frame. The region common to both trfA polypeptides includes a sequence with homology to a number of proteins that bind to double-stranded DNA. The trfA gene is preceded by another open reading frame, encoding a polypeptide of 116 amino acids of unknown function. Both cistrons are transcribed from a promoter outside the region of sequence reported here; however, much higher levels of the short polypeptide than of either of the trfA gene products are observed. Possible mechanisms for the control of the relative levels of the products of this operon are discussed, together with features of the trfA gene that may be important for its function in the diverse gram-negative bacterial species in which RK2 can be maintained.     

Identification of Rhizobium plasmid sequences involved in recognition of Psophocarpus, Vigna, and other legumes

Symbiotic DNA sequences involved in nodulation by Rhizobium must include genes responsible for recognizing homologous hosts. We sought these genes by mobilizing the symbiotic plasmid of a broad host-range Rhizobium MPIK3030 (= NGR234) that can nodulate Glycine max, Psophocarpus tetragonolobus, Vigna unguiculata, etc., into two Nod- Rhizobium mutants as well as into Agrobacterium tumefaciens. Subsequently, cosmid clones of pMPIK3030a were mobilized into Nod+ Rhizobium that cannot nodulate the chosen hosts. Nodule development was monitored by examining the ultrastructure of nodules formed by the transconjugants. pMPIK3030a could complement Nod- and Nif- deletions in R. leguminosarum and R. meliloti as well as enable A. tumefaciens to nodulate. Three non-overlapping sets of cosmids were found that conferred upon a slow-growing Rhizobium species, as well as on R. loti and R. meliloti, the ability to nodulate Psophocarpus and Vigna, thus pointing to the existence of three sets of host-specificity genes. Recipients harboring these hsn regions had truly broadened host-range since they could nodulate both their original hosts as well as MPIK3030 hosts.     

Analysis of copy number control elements in the region of the vegetative replication origin of the broad host range plasmid RK2.

Broad host-range plasmid RK2 is able to replicate in a controlled manner in most Gram negative bacterial species. To analyze the elements of its control mechanism, we have measured the copy number in Escherichia coli of mini-RK2 replicons isogenic except for defined deletions in regions adjacent to the vegetative replication origin, oriVRK2, which have previously been implicated in copy number control because of their expression of plasmid incompatibility. The results indicate that while the previously defined 700-bp HaeII oriVRK2 fragment carries one copy control element (copA), a second (copB) lies at least partly outside this fragment towards the tetracycline resistance genes of RK2. Deletions affecting both these regions give a mini replicon with a copy number of 35-40 compared with 4-7 for parental RK2. Further incompatibility experiments indicate that targets for both incA (copA) and incB (copB) lie within the 700-bp HaeII oriVRK2 fragment.     

Monitoring the conjugal transfer of plasmid RP4 in activated sludge and in situ identification of the transconjugants

A GFPmut3b-tagged derivative of broad host-range plasmid RP4 was used to monitor the conjugative transfer of the plasmid from a Pseudomonas putida donor strain to indigenous bacteria in activated sludge. Transfer frequencies were determined to be in the range of 4 x 10(-6) to 1 x 10(-5) transconjugants per recipient. In situ hybridisation with fluorescently labeled, rRNA-targeted oligonucleotides was used to phylogenetically affiliate the bacteria that had received the plasmid.     

Transcription of the Agrobacterium Ti plasmid in the bacterium and in Crown Gall tumors

Regions of the Agrobacterium tumefaciens tumor-inducing (Ti) plasmid which are transcribed in the bacterium or in two tobacco Crown Gall tumors were localized. Complementary DNA (cDNA) probes made to bacterial or tumor RNA were hybridized to blots of the Ti-plasmid or cloned “T”-DNA restriction endonuclease fragments digested with various restriction endonucleases. Extensive regions of the Ti plasmid are transcribed in the bacterium grown in minimal or rich medium. An additional region of the plasmid, which has previously been defined genetically as coding for proteins responsible for octopine utilization and conjugative T-plasmid transfer, is transcribed when the bacteria are induced with octopine. This region is transcribed constitutively in a mutant which is constitutive for octopine utilization. Another additional region of the plasmid is transcribed when the bacteria are induced with agropine. All sections of the “T” DNA are weakly transcribed in the bacterium. In contrast to this, specific regions of the “T” DNA are transcribed into both polyadenylated and nonpolyadenylated RNA in the tumors. The selectivity with which regions are transcribed in the tumor may indicate that the “T” DNA has “evolved” for best use in a eucaryotic cell.     

Tn1000-mediated insertion mutagenesis of the histidine utilization (hut) gene cluster from Klebsiella aerogenes: genetic analysis of hut and unusual target specificity of Tn1000.

The histidine utilization (hut) genes from Klebsiella aerogenes were cloned in both orientations into the HindIII site of plasmid pBR325, and the two resulting plasmids, pCB120 and pCB121, were subjected to mutagenesis with Tn1000. The insertion sites of Tn1000 into pCB121 were evenly distributed throughout the plasmid, but the insertion sites into pCB120 were not. There was a large excess of Tn1000 insertions in the "plus" or gamma delta orientation in a small, ca. 3.5-kilobase region of the plasmid. Genetic analysis of the Tn1000 insertions in pCB120 and pCB121 showed that the hutUH genes form an operon transcribed from hutU and that the hutC gene (encoding the hut-specific repressor) is independently transcribed from its own promoter. The hutIG cluster appears not to form an operon. Curiously, insertions in hutI gave two different phenotypes in complementation tests against hutG504, suggesting either that hutI contains two functionally distinct domains or that there may be another undefined locus within the hut cluster. The set of Tn1000 insertions allowed an assignment of the gene boundaries within the hut cluster, and minicell analysis of the polypeptides expressed from plasmids carrying insertions in the hut genes showed that the hutI, hutG, hutU, and hutH genes encode polypeptides of 43, 33, 57, and 54 kilodaltons, respectively.     

Nucleotide sequence and functional properties of DNA encoding incompatibility in the broad host-range plasmid R1162

Summary A 370 base pair (bp) fragment of R1162 DNA encoding the incompatibility determinant has been cloned and sequenced. The DNA is located between 6.1 and 6.5 on the R1162 map, near the origin of replication. The sequence contains three perfectly conserved 20 bp direct repeats, with 11 bp of this sequence repeated a fourth time. The direct repeat unit shows some homology with that of another, unrelated broad host-range plasmid, RK2. The cloned DNA has two other properties: it lowers the copy number of R1162 when cloned into this plasmid, and it is required in cis for replication of R1162 satellite plasmids.     

DNA synthesis is initiated at two positions within the origin of replication of plasmid R1162.

DNA synthesis of broad host-range plasmid R1162 is initiated from two positions, flanking a large (40 bp stem, 40 bp loop) inverted repeat. Each start-point is located within a highly conserved, but oppositely oriented, 10 base-pair sequence. Synthesis from the two positions converges within the intervening inverted repeat. An analysis of deletions suggests that both start positions must be present for synthesis. A model describing possible early events in replication of plasmid R1162 is presented.