Insertion mutations in the promiscuous IncP-1 plasmid R18 which affect its host range between Pseudomonas species

Fifty-one host range mutants of the promiscuous plasmid R18 were isolated by Tn7 insertion mutagenesis by using Pseudomonas aeruginosa as the permissive, and P. stutzeri as the nonpermissive, host. Endonuclease cleavage mapping of 40/51 mutants showed that 37 mutations mapped to kilobase coordinates 40.3-43.8 in the two overlapping genes encoding plasmid DNA primase. Thus by this procedure it has been possible readily to isolate a large number of primase mutants. The majority of these mutations mapped to the overlapping DNA whereas a few also mapped to the nonoverlap region encoding the larger 118-kDa polypeptide. Among these mutants were four which had long deletions within the overlapping segment and extending to varying lengths anticlockwise of it. The genetic defect in these mutants has been correlated with greatly reduced in vitro primase enzyme activity. The primase mutations drastically affected the mutant's ability to mobilize a nonconjugative, wide-host-range IncP-4(Q) plasmid from P. aeruginosa to P. stutzeri although mobilization within P. aeruginosa was affected to a lesser degree. Other insertion mutations were mapped to the regions of plasmid origin of transfer (oriT) and origin of replication (oriV), but their physical location was different to previously identified similar mutations obtained using Escherichia coli as the nonpermissive host. Their physically distinct locations were correlated with differences in their transmissibility from P. aeruginosa into enteric bacterial species and into other Pseudomonas species.     

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.     

DNA sequence analysis of host range mutants of the promiscuous IncP-1 plasmids R18 and R68 with Tn7 insertions in oriV

Transposon Tn7 insertions in the origin of vegetative replication (oriV) result in host range mutants of the promiscuous IncP-1 plasmids R18 and R68 which affect plasmid replication in Escherichia coli but not in Pseudomonas aeruginosa. The sites of these insertions have been analyzed by DNA sequence analysis. In two mutants, the insertions generated direct duplications of 5′GTATT3′ at the target site which included the first base at the 5′ end of the fourth 17-bp direct repeat in oriV. In a third mutant the duplication of 5′GACAC3′ also involved the same direct repeat also at the 5′ end but contiguous with the previous duplication. DNA sequence analysis of another Tn7-induced host range mutant of R18, characterized by reduced conjugational transmissibility into P. stutzeri while retaining normal transmissibility within P. aeruginosa, showed that the insertion generated a 474-bp deletion which brought the insertion 20 bp 5′ to the 17-bp direct repeat between oriV and the oxytetracycline hydrochloride-resistant gene. The analysis of the DNA sequence data at the site of the Tn7 insertions shows that particular segments of the DNA sequence in oriV are differentially required for the replication of these plasmids in different bacterial hosts and thus of importance to the promiscuity of these plasmids.     

Plasmid donor affects host range of promiscuous IncP-1beta plasmid pB10 in an activated-sludge microbial community

Horizontal transfer of multiresistance plasmids in the environment contributes to the growing problem of drug-resistant pathogens. Even though the plasmid host cell is the primary environment in which the plasmid functions, possible effects of the plasmid donor on the range of bacteria to which plasmids spread in microbial communities have not been investigated. In this study we show that the host range of a broad-host-range plasmid within an activated-sludge microbial community was influenced by the donor strain and that various mating conditions and isolation strategies increased the diversity of transconjugants detected. To detect transconjugants, the plasmid pB10 was marked with lacp-rfp, while rfp expression was repressed in the donors by chromosomal lacI(q). The phylogeny of 306 transconjugants obtained was determined by analysis of partial 16S rRNA gene sequences. The transconjugants belonged to 15 genera of the alpha- and gamma-Proteobacteria. The phylogenetic diversity of transconjugants obtained in separate matings with donors Pseudomonas putida SM1443, Ralstonia eutropha JMP228, and Sinorhizobium meliloti RM1021 was significantly different. For example, the transconjugants obtained after matings in sludge with S. meliloti RM1021 included eight genera that were not represented among the transconjugants obtained with the other two donors. Our results indicate that the spectrum of hosts to which a promiscuous plasmid transfers in a microbial community can be strongly influenced by the donor from which it transfers.     

Genetic organization of the broad-host-range IncP-1 plasmid R751.

We have identified regions encoding conjugal transfer, plasmid maintenance, and trimethoprim resistance on the IncP-1 plasmid R751 by complementation tests with cloned deoxyribonucleic acid fragments and self-replicating derivatives constructed in vitro. The genes for replication and transfer show a scattered organization similar to that previously determined for RK2, another IncP-1 plasmid. Derivatives of RK2 are able to complement R751 derivatives defective in these functions. Restriction enzyme cleavage sites in R751 deoxyribonucleic acid are clustered in regions of the plasmid physical map. Neither region is required for plasmid maintenance or transfer, although one determines resistance to trimethoprim. A similar clustering of cleavage sites is seen with RK2, which nevertheless has a very different restriction map.     

Broad-host-range IncP-4 plasmid R1162: effects of deletions and insertions on plasmid maintenance and host range

R1162 is an 8.7-kilobase (kb) broad-host-range replicon encoding resistance to streptomycin and sulfa drugs. In vitro deletion of 1.8-kb DNA between coordinates 3.0 and 5.3 kb did not affect plasmid maintenance, but a Tn1 insertion at coordinate 6.3 kb led to a recessive defect in plasmid maintenance. The only cis-acting region necessary for plasmid replication appears to lie between the Tn1 insertion at coordinate 6.3 kb and a second Tn1 insertion at coordinate 6.5 kb. All R1162 sequences between position 6.5 kb and the EcoRI site at coordinate 8.7/0 kb were dispensible for replication in Escherichia coli and Pseudomonas putida. Plasmids carrying insertions in a variety of restriction sites in an R1162::Tn1 derivative were unstable in P. putida but stable in E. coli. Tn5 insertions in R1162 showed a hot spot at coordinate 7.5 kb. A Tn5 insertion at coordinate 8.2 kb appeared to mark the 3' end of the streptomycin phosphotransferase coding sequence. All R1162::Tn5 derivatives showed specific instability in Pseudomonas strains but not in E. coli. The instability could be relieved by internal deletions of Tn5 sequences. In the haloaromatic-degrading Pseudomonas sp. strain B13, introduction of an unstable R1162::Tn5 plasmid led to loss of ability to utilize m-chlorobenzoate as a growth substrate. Our results showed that alteration of plasmid sequence organization in nonessential regions can result in restriction of plasmid host range.     

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.     

质粒RSF1010寄主范围特性的遗传学分析

采用Tn5插入诱变、限制性核酸内切酶作图以及DNA转化等方法,对广泛寄主范围型质粒SF 1010的衍生体－pKT 2 40进行研究。证实质粒的寄主围决定于它在遗传背景不同的寄主中复制并保存自身的能力,而repA,rcpB和repC基因为该质拉复制所必需。     

Analysis of nonpolar insertion mutations in the trfA gene of IncP plasmid RK2 which affect its broad-host-range property

Replication of broad-host-range plasmid RK2 requires the protein product(s) of the plasmid-encoded trfA gene to initiate replication at oriV, the vegetative replication origin. The trfA gene contains two translational starts which direct translation of two polypeptides, of 382 and 285 amino acids, which differ by the 97 amino acids at their N-terminus. Nonpolar insertions which abolish expression of the larger TrfA polypeptide but otherwise retain the trfA gene's normal expression signals severely reduce plasmid replication efficiency in Pseudomonas aeruginosa and to a lesser extent in Pseudomonas putida, but have very little effect in Escherichia coli. This indicates that the organization of the trfA gene, producing two polypeptides products, plays an important part in the broad-host-range of plasmid RK2 by providing a degree of flexibility in the way the plasmid's replication system interacts with host biochemistry.     

Application of pM3, a broad host range IncP-9 plasmid, for genetic analysis in Enterobacteriaceae

The possibility of using a transposon-carrying variant of broad host range plasmid pM3 (IncP-9) as a universal vector for transposon mutagenesis and as a chromosome-mobilizing factor was demonstrated in bacteria of the family Enterobacteriaceae.     

Genetic analysis of the parB+ locus of plasmid R1

Summary Megaplasmid DNA from mutants has been analysed physically for deletions and insertions in order to identify the location of hydrogenase (hox) genes in Alcaligenes eutrophus. Four classes of mutants have been examined: mutants defective in genes coding for soluble NAD-dependent hydrogenase (hoxS), mutants impaired in the membrane-bound hydrogenase (hoxP), mutants altered in the regulation of hox gene expression (hoxC) and mutants with lesions in the carbon dioxide fixing enzyme system (cfx). A comparison of the restriction patterns with EcoRI, BamHI and HindIII, complementation studies with cloned DNA and DNA - DNA hybridization experiments showed that genes coding for hox and cfx are clustered on a 100-kb region of the 450-kb plasmid pHG1.     

Cultivation-independent examination of horizontal transfer and host range of an IncP-1 plasmid among gram-positive and gram-negative bacteria indigenous to the barley rhizosphere

The host range and transfer frequency of an IncP-1 plasmid (pKJK10) among indigenous bacteria in the barley rhizosphere was investigated. A new flow cytometry-based cultivation-independent method for enumeration and sorting of transconjugants for subsequent 16S rRNA gene classification was used. Indigenous transconjugant rhizosphere bacteria were collected by fluorescence-activated cell sorting and identified by cloning and sequencing of 16S rRNA genes from the sorted cells. The host range of the pKJK10 plasmid was exceptionally broad, as it included not only bacteria belonging to the alpha, beta, and gamma subclasses of the Proteobacteria, but also Arthrobacter sp., a gram-positive member of the Actinobacteria. The transfer frequency (transconjugants per donor) from the Pseudomonas putida donor to the indigenous bacteria was 7.03 x 10(-2) +/- 3.84 x 10(-2). This is the first direct documentation of conjugal transfer between gram-negative donor and gram-positive recipient bacteria in situ.     

Nucleotide sequence and functions of the oriT operon in IncI1 plasmid R64.

Two transfer genes of IncI1 plasmid R64, tentatively designated nikA and nikB, were cloned and sequenced. They are located adjacent to the origin of transfer (oriT) and appear to be organized into an operon, which we call the oriT operon. On the basis of the DNA sequence, nikA and nikB were concluded to encode proteins with 110 and 899 amino acid residues, respectively. Complementation analysis indicated that these two genes are indispensable for the transfer of R64 but are not required for the mobilization of ColE1. By the maxicell procedure, the product of nikA was found to be a 15-kDa protein. On treating a cleared lysate prepared from cells harboring a plasmid containing oriT, nikA, and nikB with sodium dodecyl sulfate or proteinase K, superhelical plasmid DNA in the cleared lysate was converted to an open circular form (relaxation). Relaxation of plasmid DNA was found to require the oriT sequence in cis and the nikA and nikB sequences in trans. It would thus follow that the products of nikA and nikB genes form a relaxation complex with plasmid DNA at the oriT site.     

Cloning and nucleotide sequence of the oriT region of the IncI1 plasmid R64.

The nucleotide sequence at the oriT region of the IncI1 plasmid R64 was determined. A recombinant plasmid carrying a 141-base-pair R64 sequence was mobilized with a normal frequency, while a plasmid carrying only 44 base pairs of this R64 sequence was mobilized with a frequency 1/10 that of the original plasmid. The oriT region of the R64 plasmid contains two inverted-repeat sequences.     

Determination of the nick site at oriT of IncI1 plasmid R64: global similarity of oriT structures of IncI1 and IncP plasmids.

The nick site at the origin of transfer, oriT, of IncI1 plasmid R64 was determined. A site-specific and strand-specific cleavage of the phosphodiester bond was introduced during relaxation of the oriT plasmid DNA. Cleavage occurred between 2'-deoxyguanosine and thymidine residues, within the 44-bp oriT core sequence. The nick site was located 8 bp from the 17-bp repeat. A protein appeared to be associated with the cleaved DNA strand at the oriT site following relaxation. This protein was observed to bind to the 5' end of the cleaved strand, since the 5'-phosphate of the cleaved strand was resistant to the phosphate exchange reaction by polynucleotide kinase. In contrast, the 3' end of the cleaved strand appeared free, since it was susceptible to primer extension by DNA polymerase I. The global similarity of the oriT structures of IncI1 and IncP plasmids is discussed.     

Genetic heteroduplex analysis of the R6K plasmid]

The results of genetic studies of R6K Tra1- and R6Kdelta[Sm1] mutants of R6K plasmid and those of heteroduplex analysis of DNAs have shown that DNA of this drug-resistant factor contains three loops flanked by the inverted repeats. The latter are designated as IR1, IR2 and IR3 and are of 50, 100 and 120 nucleotides in size respectively. IR1 is inserted into the loop flanked by IR2. Loops with these two repeats are located in major EcoR1 fragment, IR3 having been found in minor EcoRI fragment of the plasmid. The evidence obtained from the analysis of heteroduplex R6K/RSF2124 has shown that the loop with IR1 is corresponding to transposon Tn3. The extent of the deletion deltaSm1 indicates that IR2 may be a part of a transposon bearing the resistance to streptomycin. By comparing present data with those obtaine from the analysis of the RSF1040 factor of DNA replication initiation sites (Grosa et al., 1976), it has been suggested that the loop with IR3 represents a transposon with replicative functions (TnRep). The deletion of the mutant plasmid R6Kdelta[Sm1] (7.2 . 10(6) daltons in size) which affected one of the EcoRI sites not only confers the sensitivity to streptomycin but enhances also the efficiency of conjugational transfer and results in the loss of the R6K ability to bring about integrative suppression and to inhibit the fertility of the plasmids from IncP and IncN groups. The deletion mutant proved to have lost the property of incompatibility with the initial plasmid R6K and with itself.