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.     

Regions of broad-host-range plasmid RK2 involved in replication and stable maintenance in nine species of gram-negative bacteria.

The replication and maintenance properties of the broad-host-range plasmid RK2 and its derivatives were examined in nine gram-negative bacterial species. Two regions of RK2, the origin of replication (oriV) and a segment that encodes for a replication protein (trfA delta kilD, designated trfA*), are sufficient for replication in all nine species tested. However, stable maintenance of this minimal replicon (less than 0.3% loss per generation under nonselection conditions) is observed only in Escherichia coli, Pseudomonas aeruginosa, Pseudomonas putida, and Azotobacter vinelandii. Maintenance of this minimal replicon is unstable in Rhizobium meliloti, Agrobacterium tumefaciens, Caulobacter crescentus, Acinetobacter calcoaceticus, and Rhodopseudomonas sphaeroides. A maintenance function has been localized to a 3.1-kilobase (kb) region of RK2 encoding three previously described functions: korA (trfB korB1 korD), incP1-(II), and korB. The 3.1-kb maintenance region can increase or decrease the stability of maintenance of RK2 derivatives dependent on the host species and the presence or absence of the RK2 origin of conjugal transfer (oriT). In the case of A. calcoaceticus, stable maintenance requires an RK2 segment that includes the promoter and the kilD (kilB1) functions of the trfA operon in addition to the 3.1-kb maintenance region. The broad-host-range maintenance requirements of plasmid RK2, therefore, are encoded by multiple functions, and the requirement for one or more of these functions varies among gram-negative bacterial species.     

Positioning and the specific sequence of each 13-mer motif are critical for activity of the plasmid RK2 replication origin

The minimal replication origin of the broad-host-range plasmid RK2, oriV, contains five iterons which are binding sites for the plasmid-encoded replication initiation protein TrfA, four DnaA boxes, which bind the host DnaA protein, and an AT-rich region containing four 13-mer sequences. In this study, 26 mutants with altered sequence and/or spacing of 13-mer motifs have been constructed and analysed for replication activity in vivo and in vitro. The data show that the replacement of oriV 13-mers by similar but not identical 13-mer sequences from Escherichia coli oriC inactivates the origin. In addition, interchanging the positions of the oriV 13-mers results in greatly reduced activity. Mutants with T/A substitutions are also inactive. Furthermore, introduction of single-nucleotide substitutions demonstrates very restricted sequence requirements depending on the 13-mer position. Only two of the mutants are host specific, functional in Pseudomonas aeruginosa but not in E. coli. Our experiments demonstrate considerable complexity in the plasmid AT-rich region architecture required for functionality. It is evident that low internal stability of this region is not the only feature contributing to origin activity. Our studies suggest a requirement for sequence-specific protein interactions within the 13-mers during assembly of replication complexes at the plasmid origin.     

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.     

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.     

Interactions of DnaA proteins from distantly related bacteria with the replication origin of the broad host range plasmid RK2

Replication initiation of the broad host range plasmid RK2 requires binding of the host-encoded DnaA protein to specific sequences (DnaA boxes) at its replication origin (oriV). In contrast to a chromosomal replication origin, which functionally interacts only with the native DnaA protein of the organism, the ability of RK2 to replicate in a wide range of Gram-negative bacterial hosts requires the interaction of oriV with many different DnaA proteins. In this study we compared the interactions of oriV with five different DnaA proteins. DNase I footprint, gel mobility shift, and surface plasmon resonance analyses showed that the DnaA proteins from Escherichia coli, Pseudomonas putida, and Pseudomonas aeruginosa bind to the DnaA boxes at oriV and are capable of inducing open complex formation, the first step in the replication initiation process. However, DnaA proteins from two Gram-positive bacteria, Bacillus subtilis and Streptomyces lividans, while capable of specifically interacting with the DnaA box sequences at oriV, do not bind stably and fail to induce open complex formation. These results suggest that the inability of the DnaA protein of a host bacterium to form a stable and functional complex with the DnaA boxes at oriV is a limiting step for plasmid host range.     

Gene transfer in magnetic bacteria: transposon mutagenesis and cloning of genomic DNA fragments required for magnetosome synthesis.

Broad-host-range IncP and IncQ plasmids have been transferred to the aerobic magnetic bacterium Aquaspirillum sp. strain AMB-1. Conjugal matings with Escherichia coli S17-1 allowed high-frequency transfer of the RK2 derivative pRK415 (4.5 x 10(-3) transconjugant per recipient cell) and the RSF1010 derivative pKT230 (3.0 x 10(-3) transconjugant per recipient). These plasmids successfully formed autonomous replicons in transconjugants and could be isolated and transformed back into E. coli, illustrating their potential as shuttle vectors. A mobilizable plasmid containing transposon Tn5 was transferred to Aquaspirillum sp. strain AMB-1 and also to the obligately microaerophilic magnetic bacterium Aquaspirillum magnetotacticum MS-1. Five nonmagnetic kanamycin-resistant mutants of Aquaspirillum sp. strain AMB-1 in which Tn5 was shown to be integrated into the chromosome were obtained. Different genomic fragments containing the mutagenized regions were cloned into E. coli. Two genomic fragments were restriction mapped, and the site of Tn5 insertion was determined. They were shown to be identical, although derived from independent transposon insertions. One of these clones was found to hybridize strongly to regions of the A. magnetotacticum MS-1 chromosome. This is the first report of gene transfer in a magnetic bacterium.     

The kil-kor regulon of broad-host-range plasmid RK2: nucleotide sequence, polypeptide product, and expression of regulatory gene korC.

Broad-host-range plasmid RK2 encodes several kil operons (kilA, kilB, kilC, kilE) whose expression is potentially lethal to Escherichia coli host cells. The kil operons and the RK2 replication initiator gene (trfA) are coregulated by various combinations of kor genes (korA, korB, korC, korE). This regulatory network is called the kil-kor regulon. Presented here are studies on the structure, product, and expression of korC. Genetic mapping revealed the precise location of korC in a region near transposon Tn1. We determined the nucleotide sequence of this region and identified the korC structural gene by analysis of korC mutants. Sequence analysis predicts the korC product to be a polypeptide of 85 amino acids with a molecular mass of 9,150 daltons. The KorC polypeptide was identified in vivo by expressing wild-type and mutant korC alleles from a bacteriophage T7 RNA polymerase-dependent promoter. The predicted structure of KorC polypeptide has a net positive charge and a helix-turn-helix region similar to those of known DNA-binding proteins. These properties are consistent with the repressorlike function of KorC protein, and we discuss the evidence that KorA and KorC proteins act as corepressors in the control of the kilC and kilE operons. Finally, we show that korC is expressed from the bla promoters within the upstream transposon Tn1, suggesting that insertion of Tn1 interrupted a plasmid operon that may have originally included korC and kilC.     

A broad host range replicon with different requirements for replication initiation in three bacterial species.

Plasmid RK2 is unusual in its ability to replicate stably in a wide range of Gram-negative bacteria. The replication origin (oriV) and a plasmid-encoded initiation protein (TrfA; expressed as 33 and 44 kDa forms) are essential for RK2 replication. To examine initiation events in bacteria unrelated to Escherichia coli, the genes encoding the replicative helicase, DnaB, of Pseudomonas putida and Pseudomonas aeruginosa were isolated and used to construct protein expression vectors. The purified proteins were tested for activity along with E.coli DnaB at RK2 oriV. Each helicase could be recruited and activated at the RK2 origin in the presence of the host-specific DnaA protein and the TrfA protein. Escherichia coli or P.putida DnaB was active with either TrfA-33 or TrfA-44, while P.aeruginosa DnaB required TrfA-44 for activation. Moreover, unlike the E.coli DnaB helicase, both Pseudomonas helicases could be delivered and activated at oriV in the absence of an ATPase accessory protein. Thus, a DnaC-like accessory ATPase is not universally required for loading the essential replicative helicase at a replication origin.     

Conjugal transfer of bacterial chromosomes mediated by the RK2 plasmid transfer origin cloned into transposon Tn5.

We report here a novel system for the conjugal transfer of bacterial chromosomes which utilizes the transfer origin (oriT) of plasmid RK2 cloned into transposon Tn5. Tn5 with oriT was inserted by transposition into the chromosomes of Escherichia coli and Rhizobium meliloti. The oriT sequence then served as the origin of high-frequency chromosome transfer when a helper RK2 plasmid was present in the same cell. The broad host range features of RK2 make this system of oriented chromosome mobilization applicable to most gram-negative bacteria.     

Regeneration of insertionally inactivated streptococcal DNA fragments after excision of transposon Tn916 in Escherichia coli: strategy for targeting and cloning of genes from gram-positive bacteria

The conjugative transposon Tn916 (15 kilobases), originally identified in Streptococcus faecalis DS16, has been cloned as an intact element on the pBR322-derived vector pGL101 in Escherichia coli. The EcoRI F' (EcoRI F::Tn916) fragment of pAM211 (pAD1::Tn916) was cloned into the single EcoRI site of pGL101 to form the chimera, pAM120, by selecting for the expression of Tn916-encoded tetracycline resistance (Tcr). Interestingly, in the absence of continued selection for Tcr, Tn916 excised from pAM120 at high frequency. This excision event resulted in a plasmid species consisting of the pGL101 vector and a 2.7-kilobase restriction fragment comigrating with the EcoRI F fragment of pAD1 during agarose gel electrophoresis. Filter blot hybridization experiments showed the 2.7-kilobase fragment generated as a result of Tn916 excision to be homologous with the EcoRI F fragment of pAD1. Analogous results were obtained with another chimera, pAM170, generated by ligating the EcoRI D' (EcoRI D::Tn916) fragment of pAM210 (pAD1::Tn916) to EcoRI-digested pGL101. Comparison of the AluI and RsaI cleavage patterns of the EcoRI F fragment isolated after Tn916 excision with those from an EcoRI F fragment derived from pAD1 failed to detect any difference in the two fragments: data in support of a precise Tn916 excision event in E. coli. Subcloning experiments showed that an intact transposon was required for Tn916 excision and located the Tcr determinant near the single HindIII site on Tn916. Although excision occurred with high frequency in E. coli, Tn916 insertion into the E. coli chromosome was a much rarer event. Tcr transformants were not obtained when pAM120 DNA was used to transform a polA1 strain, E. coli C2368.     

Identification of Yersinia enterocolitica genes affecting survival in an animal host using signature-tagged transposon mutagenesis

Pathogenic Yersinia species are associated with both localized and systemic infections in mammalian hosts. In this study, signature-tagged transposon mutagenesis was used to identify Yersinia enterocolitica genes required for survival in a mouse model of infection. Approximately 2000 transposon insertion mutants were screened for attenuation. This led to the identification of 55 mutants defective for survival in the animal host, as judged by their ability to compete with the wild-type strain in mixed infections. A total of 28 mutants had transposon insertions in the virulence plasmid, validating the screen. Two of the plasmid mutants with severe virulence defects had insertions in an uncharacterized region. Several of the chromosomal insertions were in a gene cluster involved in O-antigen biosynthesis. Other chromosomal insertions identified genes not previously demonstrated as being required for in vivo survival of Y. enterocolitica. These include genes involved in the synthesis of outer membrane components, stress response and nutrient acquisition. One severely attenuated mutant had an insertion in a homologue of the pspC gene (phage shock protein C) of Escherichia coli. The phage shock protein operon has no known biochemical or physiological function in E. coli, but is apparently essential for the survival of Y. enterocolitica during infection.     

Intramolecular transposition and inversion in plasmid R6K

Selection was made in Escherichia coli K-12 recA hosts carrying plasmid R6K for ampicillin hyperresistance. Twenty-two selected strains were found to carry mutant plasmids, which, from electron microscopy and restriction enzyme analysis, were concluded to arise by a duplication of transposon Tn2660, which confers ampicillin resistance, in all cases the duplicate transposon being in an inverted orientation with respect to the resident Tn2660. A mutant of R6K, pSJC301, which was temperature sensitive for ampicillin resistance was produced by in vitro hydroxylamine treatment of R6K deoxyribonucleic acid. A plasmid hybrid, pSJC102, was constructed by cloning the EcoRI R6K fragment carrying the wild-type beta-lactamase gene into the EcoRI site of ColE1. pSJC301 and pSJC102 were transformed into the same recA host strain to form a stable biplasmid strain. Ampicillin-hyperresistant mutants were selected from this strain and screened for plasmids with a duplication of transposon Tn2660, which occurred with equal frequency in either pSJC301 or pSJC102; of 12 characterized, all were inverse repeats of the resident transposon. All six Tn2660 inserts into pSJC301 determined temperature-sensitive ampicillin resistance, and all six inserts into pSJC102 determined wild-type ampicillin resistance, from which it was inferred that transposition of a duplicate Tn2660 occurs predominantly as an intramolecular event, at least in the multicopy R6K plasmid. In all 28 insertion mutants of R6K, there was an inversion of the deoxyribonucleic acid between the two transposons, whereas in only one of six insertion mutants of pSJC102, inversion had occurred. These results are discussed in terms of current models of transposition.     

Novel system for recognizing and eliminating foreign DNA in Pseudomonas putida.

Derivatives of pQSR49 (R1162::Tn1) containing cloned fragments of Escherichia coli chromosomal DNA are stable in E. coli but unstable in Pseudomonas putida. Similar derivatives containing P. putida chromosomal DNA are stable in both species. Instability is a consequence of plasmid loss during growth and is not due to death or inhibition of growth of plasmid-containing cells. Average copy numbers per cell of the unstable hybrid plasmids are similar to that of pQSR49, indicating that instability is not the result of reduced replication of plasmid DNA. A plasmid unrelated to pQSR49, RK2, becomes unstable in the presence of the unstable hybrids but only when it also contains foreign DNA. The data suggest an inducible mechanism in P. putida active in the elimination of plasmid-borne foreign DNA from the cell.     

Insertional mutagenesis and recovery of interrupted genes of Streptococcus mutans by using transposon Tn917: preliminary characterization of mutants displaying acid sensitivity and nutritional requirements.

New vectors were constructed for efficient transposon Tn917-mediated mutagenesis of poorly transformable strains of Streptococcus mutans(pTV1-OK) and subsequent recovery of interrupted genes in Escherichia coli (pT21delta2TetM). In this report, we demonstrate the utility of Tn917 mutagenesis of a poorly transformable strain of S. mutans (JH1005) by showing (i) the conditional replication of pTV1-OK, a repA(Ts) derivative of the broad-host-range plasmid pWVO1 harboring Tn9l7, in JH1005 at the permissive temperature (30 degrees C) versus that at the nonpermissive temperature (45 degrees C); (ii) transposition frequencies similar to those reported for Bacillus subtilis (10(-5) to 10(-4)) with efficient plasmid curing in 90 to 97% of the erythromycin-resistant survivors following a temperature shift to 42 to 45 degrees C; and (iii) the apparent randomness of Tn917 insertion as determined by Southern hybridization analysis and the ability to isolate nutritional mutants, mutants in acid tolerance, and mutants in bacteriocin production, at frequencies ranging from 0.1 to 0.7%. Recovery of transposon-interrupted genes was achieved by two methods: (i) marker rescue in E. coli with the recovery vector pTV21delta2TetM, a tetracycline-resistant and ampicillin-sensitive Tn9l7-pBR322 hybrid, and (ii) "shotgun" cloning of genomic libraries of Tn917 mutants into pUC19. Sequence analyses revealed insertions at five different genetic loci in sequences displaying homologies to Clostridium spp.fhs (66% identity), E. coli dfp (43% identity), and B. subtilis ylxM-ffh (58% identity), icd (citC [69% identity]), and argD (61% identity). Insertions in icd and argD caused nutritional requirements; the one in ylxM-ffh caused acid sensitivity, while those in fhs and dfp caused both acid sensitivity and nutritional requirements. This paper describes the construction of pTV1-OK and demonstrates that it can be efficiently employed to deliver Tn917 into S. mutans for genetic analyses with some degree of randomness and that insertions in the chromosome can be easily recovered for subsequent characterization. This represents the first published report of successful Tn9l7 mutagenesis in the genus Streptococcus.