Transposon mutagenesis of the Pseudomonas aeruginosa PAO chromosome and the isolation of high frequency of recombination donors

Abstract The transposons Tn 5 and Tn 2521 have been loaded on to a temperature-sensitive, replication-defective mutant of the promiscuous IncP-1 plasmid R68 and used for transposon mutagenesis of the Pseudomonas aeruginosa PAO chromosome. Tn 5 was found to be particularly useful for the generation of new auxotrophic mutations and Tn 2521 for the generation of a range of high frequency of recombination donors by plasmid integration into the chromosome. The new origins and directions of chromosome transfer mediated by this conditional plasmid replication system will greatly facilitate improved genetic mapping of PAO.     

Role of IncP-1 plasmid primase in conjugation between Pseudomonas species

Abstract A Tn7 insertion in the DNA primase gene of the promiscuous IncP-1 plasmid R18 specifically reduced plasmid conjugational transfer from Pseudomanas aeruginosa donors to Pseudomonas stutzeri recipients. The cloned primase gene was found to efficiently complement the mutation in both the donor and in the recipient suggesting that the primase is required for priming single-stranded plasmid DNA in the donor prior to its transit to the recipient where it is converted to the double- stranded form.     

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.     

Recalibration of the Pseudomonas aeruginosa Strain Pao Chromosome Map in Time Units Using High-Frequency-of-Recombination Donors

High-frequency-of-recombination donors of P. aeruginosa strain PAO were generated using a temperature-sensitive, replication mutant of the IncP-1 plasmid R68, loaded with the transposon Tn2521. Fourteen donors so isolated mobilized the chromosome in a polarized manner from a number of different transfer origins. The donors were used to construct a time of entry map of the entire chromosome and this was achieved by determining the time of entry of 32 randomly dispersed markers in crosses using nalidixic acid to interrupt chromosome transfer. Analysis of the time of entry data enabled the recalibration of the chromosome map to 75 min.     

The 79,370-bp conjugative plasmid pB4 consists of an IncP-1beta backbone loaded with a chromate resistance transposon,the strA-strB streptomycin resistance gene pair,the oxacillinase gene bla(NPS-1), and a tripartite antibiotic efflux system of the resistance-nodulation-division family

Plasmid pB4 is a conjugative antibiotic resistance plasmid, originally isolated from a microbial community growing in activated sludge, by means of an exogenous isolation method with Pseudomonas sp. B13 as recipient. We have determined the complete nucleotide sequence of pB4. The plasmid is 79,370 bp long and contains at least 81 complete coding regions. A suite of coding regions predicted to be involved in plasmid replication, plasmid maintenance, and conjugative transfer revealed significant similarity to the IncP-1beta backbone of R751. Four resistance gene regions comprising mobile genetic elements are inserted in the IncP-1beta backbone of pB4. The modular 'gene load' of pB4 includes (1) the novel transposon Tn 5719 containing genes characteristic of chromate resistance determinants, (2) the transposon Tn 5393c carrying the widespread streptomycin resistance gene pair strA-strB, (3) the beta-lactam antibiotic resistance gene bla(NPS-1) flanked by highly conserved sequences characteristic of integrons, and (4) a tripartite antibiotic resistance determinant comprising an efflux protein of the resistance-nodulation-division (RND) family, a periplasmic membrane fusion protein (MFP), and an outer membrane factor (OMF). The components of the RND-MFP-OMF efflux system showed the highest similarity to the products of the mexCD-oprJ determinant from the Pseudomonas aeruginosa chromosome. Functional analysis of the cloned resistance region from pB4 in Pseudomonas sp. B13 indicated that the RND-MFP-OMF efflux system conferred high-level resistance to erythromycin and roxithromycin resistance on the host strain. This is the first example of an RND-MFP-OMF-type antibiotic resistance determinant to be found in a plasmid genome. The global genetic organization of pB4 implies that its gene load might be disseminated between bacteria in different habitats by the combined action of the conjugation apparatus and the mobility of its component elements.     

Plasmid pB8 is closely related to the prototype IncP-1beta plasmid R751 but transfers poorly to Escherichia coli and carries a new transposon encoding a small multidrug resistance efflux protein

The IncP-1beta plasmid pB8, which confers resistance to amoxicillin, spectinomycin, streptomycin, and sulfonamides, was previously isolated from a sewage treatment plant. It was found to possess abnormal conjugative transfer properties, i.e., transfer to Escherichia coli by conjugation or electroporation could not be detected. We showed in this study that plasmid pB8 is transferable to E. coli by conjugation, but only at low frequencies and under specific experimental conditions, a phenomenon that is very unusual for IncP-1 plasmids. Determination of the complete 57,198bp pB8 nucleotide sequence revealed that the backbone of the plasmid consists of a complete set of IncP-1beta-specific genes for replication initiation, conjugative plasmid transfer, stable inheritance, and plasmid control with an organisation identical to that of the prototype IncP-1beta plasmid R751. All of the minor differences in the pB8 backbone sequence compared to that of R751 were also found in other IncP-1beta plasmids known to transfer to and replicate in E. coli. Plasmids pB8 and R751 can be distinguished with respect to their accessory genetic elements. First, the pB8 region downstream of the replication initiation gene trfA contains two transposable elements one of which is similar to Tn5501. The latter transposon encodes a putative post-segregational-killing system and the small multidrug resistance (SMR) protein QacF, mediating quaternary ammonium compound resistance. The accessory genes in this region are not responsible for the poor plasmid transfer to E. coli since a pB8 deletion derivative devoid of all genes in that region showed the same conjugative transfer properties as pB8. A Tn5090/Tn402 derivative carrying a class 1 integron is located between the conjugative transfer modules. The Tn5090/Tn402 integration-sites are exactly identical on pB8 and R751 but in contrast to R751 the pB8 element carries the resistance gene cassettes oxa-2 for amoxicillin resistance and aadA4 for streptomycin/spectinomycin resistance, the integron-specific conserved segment consisting of the genes qacEDelta1, sul1, and orf5, and a truncated tni transposition module (tniAB). Although future work will have to determine the molecular basis for the poor transfer of pB8 to E. coli, our findings demonstrate that the host-range of typical IncP-1 plasmids may be less broad than expected.     

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.     

A chromosomally located transposon in Pseudomonas aeruginosa

A new transposon, Tn2521, coding for carbenicillin, streptomycin, spectinomycin, and sulfanilamide resistance, has been identified in Pseudomonas aeruginosa. The transposon occurs naturally in the chromosome of clinical strains of P. aeruginosa isolated in geographically separated hospitals. This has been demonstrated by its transductional linkage to the pur-136 marker and also by Southern hybridization. Tn2521 is 6.8 kilobases, can transpose from the chromosome to both IncP-1 and IncP-2 plasmid genomes, and has a pattern of restriction endonuclease sites unlike that of any previously described transposon. The carbenicillin resistance carried by Tn2521 is due to the PSE-4 type of beta-lactamase.     

Conjugative transfer of plasmid RP1 to soil isolates of Pseudomonas fluorescens is facilitated by certain large RP1 deletions

Abstract The broad-host-range IncP plasmid RP1 could not be transferred by conjugation from Escherichia coli to Pseudomonas fluorescens strain CHA0. However, this conjugative transfer was possible with RP1 derivatives which had large deletions extending from the primase gene towards the Tra-2 region, thus lacking the kanamycin resistance gene and IS 21 . Such RP1 deletion derivatives permitted IncP cosmid mobilization to P. fluorescens CHA0 and could be used as vectors for transposon mutagenesis with a newly constructed Tn 5 derivative (carrying kanamycin and mercury resistance determinants) in strain CHA0 and another P. fluorescens soil isolate, strain S9.     

Molecular genetic analysis of bacterial plasmid promiscuity

Abstract The molecular genetic basis of the promiscuity of the wide host range conjugative IncP-1α plasmids has been investigated by transposon mutagenesis and by the construction of minireplicons. The former has identified the origin of plasmid vegetative replication, the replication genes needed for initiation of plasmid replication, the DNA primase gene and a gene encoding a polypeptide of 52 kDa and mapping near the origin of plasmid transfer as all contributing to promiscuity. Minireplicon constructions confirm this conclusion but in addition establish that the origins of replication, transfer and other genomic regions produce complex interactions with respect to host range. DNA sequence analysis within the origin of replication show that the first direct repeat of the cluster of five repeats and sequences immediately 5' to it appear to be required in some ( Escherichia coli ) but not in other ( Pseudomonas aeruginosa ) hosts for plasmid replication.     

Molecular genetic analysis of bacterial plasmid promiscuity

The molecular genetic basis of the promiscuity of the wide host range conjugative IncP-1 alpha plasmids has been investigated by transposon mutagenesis and by the construction of minireplicons. The former has identified the origin of plasmid vegetative replication, the replication genes needed for initiation of plasmid replication, the DNA primase gene and a gene encoding a polypeptide of 52 kDa and mapping near the origin of plasmid transfer as all contributing to promiscuity. Minireplicon constructions confirm this conclusion but in addition establish that the origins of replication, transfer and other genomic regions produce complex interactions with respect to host range. DNA sequence analysis within the origin of replication show that the first direct repeat of the cluster of five repeats and sequences immediately 5' to it appear to be required in some (Escherichia coli) but not in other (Pseudomonas aeruginosa) hosts for plasmid replication.     

Insertions of the Transposon Tn1 into the PSEUDOMONAS AERUGINOSA Chromosome

The transposon Tn1 has been translocated to the chromosome of Pseudomonas aeruginosa from plasmid R18, following hydroxylamine mutagenesis of the plasmid. Twelve insertions were mapped to six distinct sites distal to 55 min of the origin of chromosome transfer by the plasmid FP2. These map locations were confirmed by host chromosome mobilization tests mediated by plasmids R18 or R91-5, due to Tn1 homology between plasmid and host chromosome. All the Tn1 chromosomal inserts were retransposable to other plasmids (Sa, R931 and R38). The behavior of Tn1 in P. aeruginosa was very similar to its behavior in Escherichia coli with respect to regional specificity, orientation of insertion and in serving as regions of homology for host chromosome mobilization by plasmids. This last property has permitted the demonstration that Tn1 on R18 and R91-5 is in opposite orientation with respect to the origin of transfer (oriT) of the two plasmids.     

Translocation of ampicillin transposon Tn1 in Escherichia coli cells during sexual reproduction

The efficiency of Tn1 transposition has been shown to increase considerably in course of bacterial conjugation. Usually, the frequency of Tn1 transposition from plasmid pSA2001, a derivative of RP4, into the chromosome never exceeded 0.1% per cell. Percentage of His+ transconjugants, marked by transposon Tn1 during conjugation between Hfr donor, carrying plasmid pSA2001, and auxotrophic recipient, was about 30%. Transposon Tn1 transfer into the recipient cells does not depend on the recA+ gene function in donor cells or on conjugative transfer of plasmid pSA2001. The transfer requires the recA+ gene function in recipients as well as the Hfr function in donor cells. Southern's blot-hybridization revealed the insertion of transposon Tn1 into the different sites of the chromosome of His+ transconjugants. The transposon inserted during conjugation retains the ability to potential further translocation into new sites on the chromosomal DNA.     

Tn3702, a conjugative transposon in Enterococcus faecalis

Enterococcus faecalis strain D434 was found to carry on its chromosome a determinant encoding tetracycline-minocycline resistance (Tcr-Mnr) and to harbor both an R plasmid and a cryptic conjugative plasmid, pIP1141. The determinant coding for Tcr-Mnr was located on a conjugative transposon, designated Tn3702. The transposition of Tn3702 on to both pIP1141 and the hemolysin plasmid pIP964 yielded different derivatives each of which contained an 18.5-kilobase insert. The structure of Tn3702 is similar to that of the conjugative transposon Tn916.     

Vibrio cholerae hybrid sex factor that contains ampicillin transposon Tn1.

The ampicillin resistance transposon Tn1 was translocated from the R plasmid RP4 to the Vibrio cholerae conjugative plasmid, P. The hybrid sex factor P::Tn1 was highly transmissible and expressed the biological activities of the P factor. In addition, P::Tn1 facilitated transfer of RP4 to V. cholerae recipients. Physical studies of P::Tn1 indicated that the Tn1 transposon was added to the otherwise unaltered P plasmid.     

Transposon-facilitated recombination in Vibrio cholerae

Summary Improved Vibrio cholerae donors were constructed by introducing the ampicillin transposon, Tn1, into both the conjugative plasmid, P, and the bacterial chromosome to provide portable regions of homology. The resulting Tfr (Transposon-facilitated recombination) donors transferred genes at high frequency from origins specified by the chromosomally inserted Tn1 copies. Tn1 was transposed into the chromosome from a deleted P::Tn1 vector, which was eliminated from the cells by superinfection with a thermosensitive P::Tn9 (chloramphenicol) mutant plasmid. After eliminating the thermosensitive plasmid, the chromosomally resistant isolates were converted into donors with a P::Tn1 conjugative plasmid. Tfr donors were also obtained by isolating Tn1 insertion mutations in a gene for thymine biosynthesis. Chromosomal sites of Tn1 relative to bacterial genes were determined by measuring gene transfer frequencies and genetic linkage. In one case, linkage of the amp gene to the chromosomal genes that defined its location was demonstrated. Chromosomal transfer by Tfr donors was reversed by isolating P:Tn1 plasmids that contained Tn1 inserted in the opposite orientation.     

Mutagenesis and chromosome mobilization in Hyphomicrobium facilis B-522.

Spontaneously derived antibiotic-resistant mutants of Hyphomicrobium facilis B-522, a restricted facultative methylotroph, occurred at a high frequency on agar plates with low antibiotic concentrations. Mutants specifically defective in methanol oxidation have been obtained using an allyl alcohol direct selection technique. By chemical mutagenesis with N-methyl-N'-nitro-N-nitrosoguanidine in the presence of chloramphenicol several stable auxotrophic mutants could be isolated: three leucine auxotrophs, two threonine auxotrophs, and two leucine-methionine double auxotrophic mutants. Optimal conditions for transposon mutagenesis have been developed by comparing several transposon delivery vectors. With the suicide plasmid pRK2013 as a vector, the tetracycline resistance conferring transposon Tn5-132 was introduced into the genome of H. facilis B-522. The following insertion mutants have been obtained: leu-3::Tn5-132, ilv-1::Tn5-132, and pur-1::Tn5-132. Broad host range IncP-1 plasmids could be successfully transferred by interspecific matings. Chromosome mobilization was demonstrated with the conjugative IncP-1 plasmids RP1, R68.45, pMO60, and H. facilis 2189 (leu-2, met-1, mox-1, nfs-1, str-12) as recipient strain. Transconjugants occurred at frequencies ranging from 10(-6) to 10(-8) for each marker.     

Role of sog polypeptides specified by plasmid ColIb-P9 and their transfer between conjugating bacteria.

The sog gene of the conjugative plasmid ColIb-P9 specifies two sequence-related polypeptides with the N-terminal third of the larger product having DNA primase activity. To resolve the function of the C-terminal portion of the polypeptides, we constructed a ColIb mutant containing a Tn5 insertion in the 3' region of sog. The mutation truncated sog gene products without inactivating DNA primase and rendered the plasmid defective in conjugation. Tests for the presence of conjugative pili, for complementation by a sog+ recombinant, and for mobilization of small origin of transfer (oriT) recombinant plasmids indicated that the mutant ColIb allows conjugative aggregation of cells but it is defective in DNA transfer at some stage subsequent to its initiation at oriT. Physical evidence is given that normal sog polypeptides are among a group of proteins transferred selectively from the donor to the recipient cell by a conjugation-specific process. No transfer of the mutant sog proteins was detected. It is proposed that the C-terminal region of sog polypeptides facilitates transfer of single-stranded ColIb DNA between conjugating cells following initiation of transfer at the oriT site, and that in this role the proteins are transmitted to the recipient cell.     

Transposon insertion mutagenesis of Pseudomonas aeruginosa with a Tn5 derivative: application to physical mapping of the arc gene cluster

For insertional mutagenesis of Pseudomonas aeruginosa, a derivative of the kanamycin-resistance (KmR) transposon Tn5 was constructed (Tn5-751) that carried the trimethoprim-resistance (TpR) determinant from plasmid R751 as an additional marker. Double selection for KmR and TpR avoided the isolation of spontaneous aminoglycoside-resistant mutants which occur at high frequencies in P. aeruginosa. As a delivery system for the recombinant transposon, plasmid pME305, a derivative of the broad-host-range plasma RP1, proved effective; pME305 is temperature-sensitive at 43 degrees C for maintenance in Escherichia coli and P. aeruginosa and deleted for IS21 and the KmR and primase genes. In matings with an E. coli donor carrying pME9(= pME305::Tn5-751), transposon insertion mutants of P. aeruginosa PAO were recovered at approx. 5 X 10(-7)/donor at 43 degrees C. Among Tn5-751 insertional mutants 0.9% were auxotrophs. A thr::Tn5-751 mutation near the recA-like locus rec-102 is useful for the construction of recombination-deficient strains. Several arc::Tn5-751 mutants could be isolated that were defective in anaerobic utilization of arginine as an energy source. From three of these mutants the arc gene region was cloned into an E. coli vector plasmid. Since Tn5-751 has a single EcoRI site between the TpR and KmR genes, EcoRI-generated fragments carrying either resistance determinant plus adjacent chromosomal DNA could be selected separately in E. coli. Thus, a restriction map of the arc region was constructed and verified by hybridization experiments. The arc genes were tightly clustered, confirming earlier genetic evidence.     

Tn2301, a transposon construct carrying the entire transfer region of the F plasmid.

The largest R . BamHI fragment of the plasmid F, which carries the entire F conjugation system, has been cloned into the single R . BamHI site of the ampicillin (Ap) resistance transposon TN1. pDS1106 (ColE1 mob::Tn1) was the vector plasmid, and the resultant conjugative plasmid, pED830, was characterized both genetically and by restriction enzyme analysis. The transposon construct, denoted Tn2301, was transposable at frequencies similar to Tn1 to small nonconjugative plasmids or to the Escherichia coli host chromosome. In the former case, Apr conjugative plasmids were obtained, whereas in the latter case, Hfr strains resulted. Representative Hfr strains were characterized by quantitative and interrupted mating experiments. Extension of this technique for Hfvr formation should aid chromosome mapping both in E. coli and in other bacterial genera.