Sequence rearrangements in the plasmid ColV,I-K94

ColV,I-K94 is a conjugative (F-like) plasmid specifying colicins V and I. Numerous rearrangements were observed in ColV,I-K94 plasmids from three culture collections and in derivatives which specified larger inhibition zones and increased titers of colicin V. Several of the rearrangements in ColV,I-K94 derivatives specifying higher titers of colicin V involved a 1.3 ± 0.08-kb inverted repeat sequence which was separated by a 3.3 ± 0.2-kb sequence. This was rearranged in one derivative, pKH46, such that the 3.3-kb sequence became flanked by a 2.5 ± 0.15-kb inverted repeat which was subsequently altered to form a 2.1 ± 0.1-kb inverted repeat. In two other derivatives, the 3.3-kb sequence was deleted together with all of the flanking inverted repeat (in pKH39-2) or part of each repeat (in pKH39-1). A 0.5 ± 0.1-kb sequence adjacent to one of the 1.3-kb repeats was inverted or substituted in both of these plasmids. A novel 12.9 ± 0.7-kb inverted duplication separated by a 7.3 ± 0.4-kb sequence was present in the ColV,I-K94 derivative, pKH41, and insertion of the γδ element was detected in another derivative, pKH46-1, after this nonconjugative plasmid had been mobilized by the F plasmid. The genes for colicin V and for immunity to colicin V were mapped to a 3.6-kb sequence. The copy numbers of both pKH39-2 and pKH46-1::γδ were greater than those of pKH38 from which they were derived.     

Characterization of the stability functions and inverted repeat structure X3 of the IncFI plasmid ColV2-K94

A region of the IncFI plasmid ColV2-K94 which showed homology to the sop partitioning genes of F was cloned and characterized in an attempt to study the stability functions of this element. The sop region contained the incD incompatibility determinant common to many IncFI plasmids, but could not confer on ColV2-K94 miniplasmids the same stable inheritance found in the intact ColV2-K94; thus, other functions appear to be required for efficient plasmid maintenance. Adjacent to the area of sop homology was the X3 region, which was found to contain three inverted IS1-like sequences. The X3 region of ColV2-K94 was similar in organization to the aerobactin iron uptake region of ColV3-K30, but ColV2-K94 lacked the ability to synthesize either the aerobactin siderophore or its outer membrane receptor.     

The plasmid cloning vector pBR325 contains a 482 base-pair-long inverted duplication

The plasmid pBR325 is a cloning vector constructed in vitro by addition of the chloramphenicol resistance (Cmr) gene of an IS1-flanked transposon to pBR322 (Bolivar, 1978). It is a 5 995 bp plasmid carrying no sequence originating from IS1. DNA-sequence data suggest that its Cmr segment was derived from a Cm transposon longer than Tn9. The plasmid pBR325 carries between the Cmr and Tcr genes a 482 bp sequence which duplicates, in the opposite orientation, a section pf pBR322 located at the end of the tcr gene. The same structure was found in pBR328, a deletion derivative of pBR325 (Soberon et al., 1980). The possible implications of this inverted duplication on cloning experiments are discussed.     

Contributions of traT and iss genes to the serum resistance phenotype of plasmid ColV2-K94

Abstract A genetic determinant for serum resistance, designated iss , has been found previously on the colicinogenic plasmid ColV2-K94. In this work we have identified a second serum resistance gene, traT , on ColV2-K94. The serum resistance mediated by derivatives of ColV2-K94 was due to presence of one or both of the iss and traT genes. Plasmid pWS12 (TraT⁺ Iss⁺) contained the kanamycin (Km) resistance transposon Tn 903 inserted near the origin of replication of ColV2-K94, and plasmids pWS15 (TraT⁺), pWS16 (TraT⁺) and pWS18 (TraT⁺ Iss⁺) were deletion derivatives of pWS12 constructed in vitro and in vivo. pWS12 and pWS18 conferred a 20-fold increase in relative resistance to 20% guinea pig serum when introduced into the serum-susceptible, genetically defined recA strain of Escherichia coli K-12, AB2463. Plasmids pWS15 and pWS16, from which iss had been deleted, still conferred 5-fold increases in relative resistance on AB2463. The level of resistance conferred on this strain by the antibiotic resistance plasmid R100–1 (which expresses the traT serum resistance gene) was comparable to that of plasmids pWS15 and pWS16. The 25-kDa traT gene product was identified by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) analysis of the outer membrane proteins of strain AB2463 carrying ColV2-K94. This protein cross-reacted immunologically with the traT protein expressed by F or R100–1. Our results indicated that both traT and iss are capable of mediating serum resistance in ColV2-K94.     

Genetic structure and stability of a copy number mutant of IncFI group plasmid ColV-K94 in Escherichia coli K-12

Summary Mutants pWS10, pWS11, and pWS12 were derived from an, IncFI group plasmid ColV-K94 by the insertion of a transposon Tn903 (Km^r). These plasmids were all approximately 130 kb in length. The plasmid pWS12 resembled the wild type ColV-K94 in transmissibility, incompatibility and stable maintenance. Cells harboring pWS11 were poor conjugal donors but resistant to the same level of kanamycin as pWS12 containing hosts. In contrast, pWS10 conferred a higher resistance to kanamycin and exhibited reduced incompatibility properties in comparison with pWS12. The higher drug resistance associated with pWS10 appeared to be a consequence of an increase in its copy number and the generation of miniplasmids of varying sizes.Electron microscope analysis of the copy mutant pWS10 revealed that Tn903 was located at a site adjacent to a region 32.6F to 35.3F. The latter region appears to be the primary replicon of ColV-K94 and is homologous with the secondary replicon of F. The insertional mutagenesis with Tn903 brought about an extensive DNA rearrangement including the duplication and translocation of the stems of two inverted repeat structures. The DNA alterations of pWS10 were distinguishable through comparison of its EcoRI digestion patterns with those of pWS11 and pWS12.     

Structure and mode of transposition of Tn2555 carrying sucrose utilization genes

The sucrose transposon Tn2555 from Escherichia coli, which has an unstable structure, was studied in more detail. Sequence analysis of one of the transposon variants, designated Tn2555.3, revealed the presence of two direct IS26 copies on its flanks, and a third inverted IS26 copy inside the transposon. The sucrose utilization genes of Tn2555.3 were found to be identical to those of the previously described pUR400 plasmid. It was demonstrated that Tn2555.3 translocation from pBR325 to RP4 occurs via a cointegrate formation, mediated by one of the three IS26 copies, followed by its resolution due to RecA-dependent recombination between two direct IS26 copies flanking the donor replicon.     

Amplification of drug resistance genes flanked by inversely repeated IS1 elements: involvement of IS1-promoted DNA rearrangements before amplification.

Tn2653 contains one copy of the tet gene and two copies of the cat gene derived from plasmid pBR325 and is flanked by inverted repeats of IS1. Transposed onto the P1-15 prophage, it confers a chloramphenicol resistance phenotype to the Escherichia coli host. Because the prophage is perpetuated as a plasmid at about one copy per host chromosome, the host cell is still tetracycline sensitive even though P1-15 is carrying one copy of the tet gene. We isolated P1-15::Tn2653 mutants conferring a tetracycline resistance phenotype, in which the whole transposon and variable flanking P1-15 DNA segments were amplified. Amplification was most probably preceded by IS1-mediated DNA rearrangements which led to long direct repeats containing Tn2653 sequences and P1-15 DNA. Subsequent recombination events between these direct repeats led to amplification of a segment containing the tetracycline resistance gene in tandem arrays.     

Second replicon in ColV2-K94 mediates the stable coexistence of two incompatible plasmids.

The colicin-producing plasmid pWS12, a Tn903 derivative of ColV2-K94, was found to be incompatible with the IncFI plasmids KLF1 and R386. It was compatible with the IncFII plasmids R538 and R100. Three overlapping mini-ColV derivatives, pWS15, pWS16 and pWS17, were obtained by restriction digestion of pWS12. Unlike pWS12, pWS16 exhibited incompatibility with both IncFI and IncFII plasmids, whereas the pWS15 and pWS17 plasmids expressed IncFII incompatibility but not the IncFI incompatibility of their parental ColV plasmid. We show that, although pWS12 has an IncFII replicon, Rep1, it does not normally express IncFII incompatibility because a second replicon, Rep2 (homologous to the secondary replicon of F), functions during the stable coexistence of the plasmid with IncFII plasmids. When Rep2 is deleted (as in the mini-ColV plasmids) or made nonfunctional (as in a PolA mutant strain), ColV then behaves as an IncFII plasmid.     

Characteristics of a colicin plasmid in Escherichia coli strain B177 isolated from a septicemic calf

A colicin plasmid in Escherichia coli strain B177 isolated from a septicemic calf was characterized. The colicin type was identified as ColV by using reference ColV producers. The colicin plasmid was labeled with transposon Tn903 and subjected to conjugation. The transconjugants examined suggest that the colicin plasmid confers serum resistance. There was no difference in siderophore utilization ability between the transconjugants and host strain SF800. Bioassay for siderophore suggests that the colicin plasmid specifies the production of iron-chelating compounds available for the host strain.     

Tn903 induces inverted duplications in the chromosome of bacteriophage lambda

Specialized transducing strains of bacteriophage lambda have been isolated that carry the transposable kanamycin resistance element, Tn903. Tn903 carries an inverted duplication of 1130 base-pairs flanking the kanamycin resistance gene. Often, when λ::Tn903 particles are infected into bacterial cells, the lambda chromosome is rearranged into a defective lambda plasmid which replicates with the bacterial cell. The formation of the defective plasmids (called Tn903λdv) is most likely induced by the Tn903 insertion itself. This follows from the fact that the novel DNA sequence found in these plasmids, with respect to the ancestral λTn903 chromosome, is always adjacent to the Tn903 element. Physical chromosomal mapping of these plasmids shows that they contain large inverted duplications of lambda sequences situated about the Tn903 insertion. The formation of the Tn903λdv plasmids from the ancestral λTn903 is not dependent on the recombination functions provided through the phage red gene or the host recA gene.     

Insertion element IS102 resides in plasmid pSC101.

In vivo recombination was found to occur between plasmid pHS1, a temperature-sensitive replication mutant of pSC101 carrying tetracycline resistance, and plasmid ColE1 after selection for tetracycline resistance at the restrictive temperature, 42 degrees C. Extensive analysis of the physical structures of three of these recombinant plasmids, using restriction endonucleases and the electron microscope heteroduplex method, revealed that the plasmid pHS1 was integrated into different sites on ColE1. The recombinant plasmids contained a duplication of a unique 1-kilobase (kb) sequence of pHS1 in a direct orientation at the junctions between the two parental plasmid sequences. This was confirmed by comparing the nucleotide sequence of the recombinants and their parental plasmids. Nucleotide sequence analysis further revealed that nine nucleotides at the site of recombination of ColE1 were duplicated at the junction of each of the 1-kb sequences. The formation of recombinants was independent of RecA function. Based on our previous finding that a plasmid containing a deoxyribonucleic acid insertion (IS) element can recombine with a second plasmid to generate a duplication of the IS element, we conclude that the 1-kb sequence is an insertion sequence, which we named IS102. For convenience, we have also denoted the IS102 sequence as eta theta to assign the orientation of the sequence. Eighteen nucleotides at one end (eta end) were found to be repeated in an inverted orientation at the other end (theta end) of IS102. The nucleotide sequence of the eta end of the sequence was found to be identical to the sequence at the ends of the transposon Tn903, which is responsible for transposition of the kanamycin resistance gene.     

Simple assay for quantitation of Tn5 inversion events in Escherichia coli and use of the assay in determination of plasmid copy number.

In this report, we describe a simple method for measuring the frequency of sequence inversion in the transposable element Tn5 as a result of recombination across its duplicated IS50 elements. The structure of Tn5 was manipulated so that the neomycin phosphotransferase gene of the transposon would be expressed only if a sequence inversion event occurred. This highly sensitive assay also served as the basis for a novel means of estimating plasmid copy number.     

Comparison of the CopB systems of plasmids R1 and Co1V2-K94: A single base alteration in CopB gene is responsible for the increased copy number of the low copy number plasmid CoIV2-K94

Summary We have isolated a deletion mutation and a point mutation in the copB gene of the replication region Repl of the IncFI plasmid Co1V2-K94. Subsequently, this copB gene with and without point mutation was cloned and sequenced, and the point mutation was mapped in the coding region of copB with a change of one amino acid from arginine to serine. Furthermore, this copB mutant had an approximately 10-fold increase in copy number. The CopB-phenotype of Co1V2-K94 could be complemented in trans by the copB gene of coresident IncFII plasmids such as R1 and R538, but not R100, suggesting that ColV2-K94 and R1 or R538 contain the same copB allele.     

IS1-mediated tandem duplication of plasmid pBR322. Dependence on recA and on DNA polymerase I

Transposable-element-mediated fusion of the conjugal plasmid pOX38::Tn9 with pBR322 results in the appearance of cointegrates composed of a single copy of each plasmid, and cointegrates which carry a single copy of pOX38 but multiple tandem copies of pBR322. These plasmids are separated by directly repeated copies of the transposable element. We demonstrate here that such multimers can be generated from monomeric cointegrates, probably by unequal crossing over between the flanking Tn9(IS1) elements. Their appearance is thus not necessarily associated with the original transposition (fusion) event. Our study demonstrates that the process of duplication is strongly dependent on the homologous recombination system of Escherichia coli, since it is undetectable by our methods in recA- strains. It is also strongly dependent on the presence of a functional DNA polymerase I in the cell. The major pathway(s) for this duplication thus appears to rely on both the homologous recombination system and the replication of the duplicated segment.     

Identification of citrate utilization transposon Tn3411 from a naturally occurring citrate utilization plasmid.

We have isolated a new transposon, Tn3411, encoding citrate-utilizing ability, from a naturally occurring citrate utilization (Cit) plasmid, pOH3001. Citrate transposon Tn3411 was transposed from pOH3001 to lambda b519 b515 cI857 S7 (abbreviated lambda bb) phage, and further from the resulting lambda bb:Tn3411 to a vector plasmid, pBR322, in recA-deficient strains. The Cit+ plasmids (pOH2 and pOH3) constructed by the integration of Tn3411 into pBR322 were examined by restriction endonuclease and heteroduplex analysis. The results obtained were as follows: (i) Tn3411 was 7.4 kilobases long and flanked by small inverted repeats, and it contained one more pair of inverted repeats at the opposite orientation in the internal region, thus making alternate repeats; and (ii) the Cit+ structure gene was located on the fragment (5.5 kilobases) between two SalI cleavage sites on Tn3411.     

Transposition of a tetracycline-resistance determinant (Tn1523) and cointegration events mediated by the pIP231 plasmid in Escherichia coli

A 10.8-kb transposable DNA sequence conferring resistance to tetracycline resides on the IncY Escherichia coli plasmid pIP231. This sequence, designated Tn1523, was shown to insert into different sites of the replicons of the IncY prophage P1Cm c1.100 and the IncI1 plasmid pIP112. This process is not dependent on the host recombination system recA. Genetic results indicate that Tn1523 transposition involves the formation of a cointegrate intermediate, either between pIP231 and P1Cm c1.100, or between pIP231 and pIP112. These intermediates were found to be resolved into donor and recipient plasmids, each harboring a copy of the Tn1523 transposon. A stable structure formed by fusion of the pIP231 plasmid with the pIP112 plasmid was also observed. This event occurs in the absence of the bacterial recA gene product and seems to involve a site-specific reciprocal recombination between "IS-like" elements.     

A physical and genetic map of the IncFI plasmid ColV2-K94

A restriction enzyme map of the IncFI plasmid ColV2-K94 was generated using EcoRI, BamHI, HindIII, and XhoI; the genetic features of this element were then mapped from previous heteroduplex studies.     

Promoter activity associated with the left inverted terminal repeat of the killer plasmid k1 from yeast.

The killer plasmid k1 of Kluyveromyces lactis has terminal inverted repeats of 202 base pairs (bp). The left terminal repeat is contiguous to the transcribed open reading frame, ORF1, which is supposed to code for a DNA polymerase. A 266-bp fragment (called Pk1) containing most of the terminal repeat sequence was isolated and examined for promoter activity. Pk1 was fused, in either original or inversed orientation, with a promoter-less lacZ gene of E coli and a promoter-less G418 resistance gene of Tn903. These fusions were introduced into a pKD1-derived circular vector, and transformed into a lactose-negative (lac4), and a G418-sensitive K lactis host. Lac+ and G418-resistant transformants were obtained with either orientation of Pk1. The promoter activity of Pk1 fragment was independent of the presence or absence of killer plasmids. It is not known whether Pk1 can also function bidirectionally on the natural k1 plasmid. The possible functions of Pk1 for killer plasmid gene expression and plasmid replication are discussed.