Structural instability of IncP-1 plasmids in Pseudomonas aeruginosa PAT involves interaction with plasmid pVS1

The structural instability exhibited by IncP-1 plasmids in Pseudomonas aeruginosa strain PAT was shown to be Rec+ dependent and involved interaction with the resident plasmid pVS1. Structural instability resulted from deletion of plasmid deoxyribonucleic acid at a frequency of ca. 10(-2)/cell per generation. Deletants could be stabilized by transduction into P. aeruginosa strain PAO, but in strain PAT deletants had only a transient existence, as continued deletion led eventually to the loss of the entire plasmid. The patterns of markers lost in PAT were used to demonstrate a marker order for R68 similar to that published elsewhere for RP4 (Barth and Grinter, J. Mol. Biol. 113:455-474, 1977), except that only one Tra region was found. R68 also exhibited Rec+-dependent structural instability in PAO(pVS1) derivatives but, unlike the case in PAT, instability was not accompanied by chromosome mobilization. We isolated deletants of pVS1 which were unable to promote structural instability.     

Reconsideration of the previously classified incompatibility groups of plasmids,IncP-1 and IncP-11

This study presents the reassessment of earlier published data with reference to the article published in Environmental Microbiology entitled ‘IncP-type plasmids carrying genes for antibiotic resistance or aromatic compound degradation are prevalent in sequenced Aromatoleum and Thauera strains’ by Lo et al. This correspondence clarifies misperceptions of plasmids classified under incompatibility (Inc) groups IncP-1 and IncP-11.     

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.     

Impact of conjugal transfer on the stability of IncP-1 plasmid pKJK5 in bacterial populations

The intrinsic stability of IncP-1 plasmid pKJK5 was assessed in both an Escherichia coli and a Kluyvera sp. population maintained in bacterial mats and in liquid nutrient broth without selective pressure. A fluorescence tagging/flow cytometry approach was used to detect and quantify plasmid loss from populations harboring either conjugation-proficient or -deficient pKJK5 derivatives. The results show that the plasmid's ability to conjugate plays an important role in its stable maintenance in populations of both species. This effect was most pronounced in dense bacterial populations and to a far lesser extent during growth in liquid broth. Furthermore, conjugation-proficient plasmids were able to spread infectiously in the bacterial mats initiated with various ratios of plasmid-harboring cells, resulting in a nearly exclusively plasmid-harboring population.     

Utilization of IncP-1 plasmids as vectors for transposon mutagenesis in myxobacteria

No free plasmid has ever been found in the myxobacterium Myxococcus xanthus, but IncP-1 plasmids are able to integrate into the chromosome of this bacterium. The frequency of integration depends greatly upon the structure of the IncP-1 plasmid used. This property has been used to devise new delivery systems for transposon mutagenesis in this species. Plasmids with low integration efficiencies have proved to be efficient donors of Tn5, while plasmids with very high frequencies of integration could be used directly to generate mutations. These vectors have also proved efficient for Tn5 transfer into other species of myxobacteria, which have not so far been susceptible to genetic analysis.     

Multimerization of high copy number plasmids causes instability: CoIE1 encodes a determinant essential for plasmid monomerization and stability

Although the natural multicopy plasmid CoIE1 is maintained stably under most growth conditions, plasmid cloning vectors related to it are relatively unstable, being lost at frequencies of 10(-2)-10(-5) per cell per generation. Evidence suggests that CoIE1 and related plasmids are partitioned randomly at cell division and that plasmid stability is correlated inversely with plasmid multimerization; factors or conditions that reduce multimerization increase stability. Cells containing plasmid multimers segregate plasmid-free cells because the multimers are maintained at lower copy numbers than monomers, as predicted by origin-counting models for copy number control. CoIE1 is stable because it encodes a determinant, cer, that is necessary for recA-, recF-, and recE-independent recombination events that efficiently convert any multimers to monomers. We have localized monomerizing and stability determinants of CoIE1 to within a 0.38 kb region that, when cloned into plasmid vectors, greatly increases their stability.     

Increased abundance of IncP-1beta plasmids and mercury resistance genes in mercury-polluted river sediments: first discovery of IncP-1beta plasmids with a complex mer transposon as the sole accessory element

Although it is generally assumed that mobile genetic elements facilitate the adaptation of microbial communities to environmental stresses, environmental data supporting this assumption are rare. In this study, river sediment samples taken from two mercury-polluted (A and B) and two nonpolluted or less-polluted (C and D) areas of the river Nura (Kazakhstan) were analyzed by PCR for the presence and abundance of mercury resistance genes and of broad-host-range plasmids. PCR-based detection revealed that mercury pollution corresponded to an increased abundance of mercury resistance genes and of IncP-1beta replicon-specific sequences detected in total community DNA. The isolation of IncP-1beta plasmids from contaminated sediments was attempted in order to determine whether they carry mercury resistance genes and thus contribute to an adaptation of bacterial populations to Hg pollution. We failed to detect IncP-1beta plasmids in the genomic DNA of the cultured Hg-resistant bacterial isolates. However, without selection for mercury resistance, three different IncP-1beta plasmids (pTP6, pTP7, and pTP8) were captured directly from contaminated sediment slurry in Cupriavidus necator JMP228 based on their ability to mobilize the IncQ plasmid pIE723. These plasmids hybridized with the merRTDeltaP probe and conferred Hg resistance to their host. A broad host range and high stability under conditions of nonselective growth were observed for pTP6 and pTP7. The full sequence of plasmid pTP6 was determined and revealed a backbone almost identical to that of the IncP-1beta plasmids R751 and pB8. However, this is the first example of an IncP-1beta plasmid which had acquired only a mercury resistance transposon but no antibiotic resistance or biodegradation genes. This transposon carries a rather complex set of mer genes and is inserted between Tra1 and Tra2.     

Direct selection for curing and deletion of Rhizobium plasmids using transposons carrying the Bacillus subtilis sacB gene

We have constructed derivatives of the transposon Tn5 carrying the mob site (oriT) of plasmid RP4, and an nptI-sacB-sacR cassette [Ried and Collmer, Gene 57 (1987) 239-246]. The mob site, in conjunction with the antibiotic-resistance markers carried on the transposons, allows identification of transposon inserts in cryptic plasmids by mobilisation to other strains. The sacB-sacR genes allow direct selection for the loss or curing of plasmids, because only strains which no longer contain an active sacB gene are able to grow on media containing sucrose. We have tested these transposons in four strains of Rhizobium leguminosarum and two strains of Rhizobium meliloti, and have been able to demonstrate curing of several large cryptic plasmids, and generation of large deletions in many other plasmids. This method has enabled us to show that the R. leguminosarum plasmids pRL12JI and pR1eVF39f carry auxotrophic markers, and that the plasmid pR1eVF39c carries genes which affect colony morphology.     

Roles of long and short replication initiation proteins in the fate of IncP-1 plasmids

Broad-host-range IncP-1 plasmids generally encode two replication initiation proteins, TrfA1 and TrfA2. TrfA2 is produced from an internal translational start site within trfA1. While TrfA1 was previously shown to be essential for replication in Pseudomonas aeruginosa, its role in other bacteria within its broad host range has not been established. To address the role of TrfA1 and TrfA2 in other hosts, efficiency of transformation, plasmid copy number (PCN), and plasmid stability were first compared between a mini-IncP-1β plasmid and its trfA1 frameshift variant in four phylogenetically distant hosts: Escherichia coli, Pseudomonas putida, Sphingobium japonicum, and Cupriavidus necator. TrfA2 was sufficient for replication in these hosts, but the presence of TrfA1 enhanced transformation efficiency and PCN. However, TrfA1 did not contribute to, and even negatively affected, long-term plasmid persistence. When trfA genes were cloned under a constitutive promoter in the chromosomes of the four hosts, strains expressing either both TrfA1 and TrfA2 or TrfA1 alone, again, generally elicited a higher PCN of an IncP1-β replicon than strains expressing TrfA2 alone. When a single species of TrfA was produced at different concentrations in E. coli cells, TrfA1 maintained a 3- to 4-fold higher PCN than TrfA2 at the same TrfA concentrations, indicating that replication mediated by TrfA1 is more efficient than that by TrfA2. These results suggest that the broad-host-range properties of IncP-1 plasmids are essentially conferred by TrfA2 and the intact replication origin alone but that TrfA1 is nonetheless important to efficiently establish plasmid replication upon transfer into a broad range of hosts.     

IncP-1 R plasmids decrease the serum resistance and the virulence of Pseudomonas aeruginosa

Pseudomonas aeruginosa strain PAO1 was compared to PAO1 strains containing an IncP-1 R plasmid (RP1, R68, or R68.45) in an experimental mouse burn infection model. All R plasmids tested caused a 10- to 400-fold increase in mean lethal dose (LD50). The decrease in virulence produced by plasmids R68 and R68.45 was significantly greater than the decrease caused by the closely related plasmid RP1. All plasmids also led to an increased sensitivity of strain PAO1 to human serum bactericidal activity. Virulence and serum resistance of strain PAO1 were restored by curing of the entire plasmid R68.45 but not by deletions in the plasmid's transfer gene regions.     

Partial-pKD1 plasmids provide enhanced structural stability for heterologous protein production in Kluyveromyces lactis

The stability of pKD1-based vectors in the yeast Kluyveromyces lactis was investigated during short- and long-term culture. The vectors carried an expression/secretion cassette consisting of the Saccharomyces cerevisiaeSUC2 gene under the control of the S. cerevisiaeα-factor promoter and leader. The first set of vectors contained the entire pKD1 sequence linearized at either the unique EcoRI or the unique SphI site of the pKD1 plasmid. During long-term sequential batch culture in selective medium with either vector, invertase activity rapidly dropped while the plasmid-bearing population increased from 60% to 100%. This apparently contradictory behavior was due to structural instability. The enzyme restriction patterns of recovered plasmid DNA retained the pKD1 band while the band containing the SUC2 cassette had decreased substantially in size. To overcome this structural instability, a vector carrying the pKD1 replication origin and the cis-acting stability locus (lacking the inverted repeats) was employed in a pKD1⁺ (but otherwise isogenic) strain. With this plasmid, invertase activity remained constant (for at least 70 generations). While the new vector was significantly more stable, initial invertase activity was substantially lower than that for the vectors containing the full pKD1 sequence. Southern hybridization confirmed that this decrease was primarily due to reduced copy number. The results indicate that full-pKD1 vectors may be preferred for batch culture, while partial-pKD1 vectors are more suitable for long-term (e.g. fed-batch or continuous) culture. Received: 24 June 1997 / Received revision: 14 November 1997 / Accepted: 29 November 1997     

A plasmid cloning vector for the direct selection of strains carrying recombinant plasmids

A plasmid cloning vector with ampicillin-resistance and streptomycin-sensitivity markers is suitable for the direct selection of strains carrying recombinant plasmids. The selection for plasmid transformants utilizes their ampicillin resistance whereas selection for recombinant plasmids is based on the inactivation of the rpsL gene contained on the plasmid. When streptomycin-resistant Escherichia coli strains are used as recipients in transformation, transformants carrying the parental plasmid are phenotypically sensitive to streptomycin while those carrying hybrid plasmids are resistant to streptomycin.     

Genetic analysis of insertion mutations of the promiscuous IncP-1 plasmid R18 mapping near oriT which affect its host range

Transposon Tn7 insertion mutations of the promiscuous IncP-1 plasmid R18 which affect its conjugational transmissibility from Pseudomonas aeruginosa to Escherichia coli C, a strain of E. coli K12, Salmonella typhimurium and P. maltophilia have been mapped physically. They map to coordinate 53.5 kb in the Tral region of the plasmid. An 800-bp fragment mapping between R18 coordinates 52.85 and 53.65 kb, which complemented the host range defect of the mutants when tested with E. coli C as recipient, has been identified. However, complementation occurred only when the 800-bp cloned fragment was provided in the E. coli C recipient but not when situated in the P. aeruginosa donor. It is concluded that a trans-acting gene product of R18 is required, in the transcipient, for conjugative DNA metabolism during, or immediately following, the conjugational transfer of this plasmid between certain donor and recipient hosts.     

Behavior of the IncP-7 carbazole-degradative plasmid pCAR1 in artificial environmental samples

In artificial environmental samples, the behavior of the IncP-7 conjugative plasmid pCAR1, which is involved in the catabolism of carbazole, was monitored. Sterile soil and water samples supplemented with carbazole were prepared. After inoculation with Pseudomonas putida harboring pCAR1, seven species of the genus Pseudomonas, and three other bacterial species, were monitored for carbazole degradation, bacterial survival, and conjugative transfer of pCAR1. In artificial soils, more than 90% of the carbazole was degraded in samples with high water content, suggesting that the water content is a key factor in carbazole degradation in artificial soils. In three of the artificial environmental water samples, more than 95% of the carbazole was degraded. Transconjugants were detected in some artificial water samples, but not in the artificial soil samples, suggesting that pCAR1 is preferably transferred in aqueous environments. Composition analysis of the artificial water samples and examination of conjugative transfer indicated that the presence of the divalent cations Ca(2+) and Mg(2+) promoted the plasmid transfer. The presence of carbazole also increases in incidence of transconjugants, probably by enhancing their growth. In contrast, humic acids in the liquid layer of artificial soil samples appeared to prevent conjugative transfer.     

Sequence of plasmid pBS228 and reconstruction of the IncP-1alpha phylogeny

The antibiotic resistance plasmid pBS228 has been completely sequenced, and revealed to be descended from a plasmid virtually identical to the Birmingham IncP-1alpha plasmid RK2/RP4/RP1. However, it has three additional transposon insertions, one of which is responsible for the extra antibiotic resistances conferred. Loss of kanamycin resistance, which is characteristic of most IncP-1alpha plasmids, is the result of this insertion. A second transposon causes inactivation of the mating pair formation apparatus, rendering the plasmid non-self-transmissible. Comparison with the published data for other IncP-1alpha plasmids gives insight into the recent evolutionary history of this group as well as the acquisition and transmission of one of the first ampicillin resistance transposons discovered.     

Structural instability and stabilization of IncP-1 plasmids integrated into the chromosome of Myxococcus xanthus

IncP-1 plasmids are self transmissible to Myxococcus xanthus and maintained integrated into the host chromosome where they are liable to structural instability: deletions can span through the integrated plasmid; the frequency of these events depends on the recipient strain and on the localization of the insertion on the chromosome, but not on the structure of the plasmid.It is possible to isolate stabilized insertion, even from one of the most unstable strains, by growing immobilized cells in carrageenan beads in continuous nonselective culture. The remaining resistant cells are stabilized. Both the structural instability and the possible stabilization of the insertion can be useful when IncP-1 plasmids are used as cloning vectors in Myxococcus xanthus.     

DNA recognition by the KorA proteins of IncP-1 plasmids RK2 and R751

The KorA repressor proteins of IncP-1 plasmids belong to a growing family of plasmid-encoded repressors that regulate partitioning genes, and in the IncP-1 plasmids coordinate these with expression of replication and transfer genes as well. Both KorA(RK2) (IncP-1 alpha) and KorA(R751) (IncP-1 beta) recognise the 5'-GTTTAGCTAAAC-3' palindrome. Reporter gene assays showed that KorA proteins from these two main subgroups of IncP-1 plasmids show specificity for their own promoter/operators and this preference was confirmed with in vitro binding studies using gel mobility shift assays on one representative promoter. Class I (high affinity) operators for KorA(RK2) are flanked by an A-A-A/T sequence in the upstream half; the T base was shown to greatly influence strong repression. A C-A-G triplet was present in the same region in the R751 O(A) sequences and the G base was accordingly found to be important for strong KorA(R751) repression. An obvious difference between the two KorA proteins is a histidine to serine change at the C-proximal end of the putative recognition helix of the HTH motif (aa 56). An IncP-1 alpha KorAH56S mutant protein had higher affinity for all operators but had improved more on R751 operators than on RK2 operators. This indicates that KorA of RK2 is not maximised for DNA binding activity and that the aa difference at position 56 may play a role in differentiation between alpha and beta KorA operators.     

New molecular variants of epsilon and beta IncP-1 plasmids are present in estuarine waters

In this work the presence of broad-host-plasmids in an estuary in Portugal has been investigated. Pseudomonas putida KT2442 was used as model recipient bacteria in biparental matings with tetracycline and mercury to select for resistance phenotypes. As a result, 7 transconjugants were shown to carry broad-host-plasmids from the IncP-1 group, as seen by PCR amplification of the trfA gene. Sequence analysis confirmed the isolation of 4 plasmids from β-1 subgroup and 3 assigned to the recently described ε subgroup. To our knowledge this is the first report concerning the detection and isolation of IncP-1β and ε plasmids in estuarine waters. Moreover it is shown that, even though the retrieved plasmids are phylogenetically close to previously characterized plasmids, such as pB10 and pKJK5, respectively, they constitute new molecular variants.