Sequence analysis of the family of penicillinase-producing plasmids of Neisseria gonorrhoeae

The exact nature of the sequence differences between the medically important family of gonococcal penicillinase-producing plasmids has been ascertained. The entire DNA sequence of the Asia-type plasmid, pJD4, demonstrated that it is 7426 bp and contains two direct repeats (DR30) that are implicated in the formation of deletion variant plasmids, such as the Africa-type plasmid. We have identified putative DnaA and IHF binding sites, various open reading frames that are thought to specify functional proteins, and some important DNA sequences involved with conjugative transfer of gonococcal beta-lactamase plasmids. The deletion in the Africa-type plasmid is 1827 bp and one of the DR30 repeats is also missing. The deletion in the Rio-type plasmid and several Toronto-type plasmids was determined to be 2273 bp and the sequence spanning the deletion was identical irrespective of geographic or temporal origin. The &Ncirc;imes-type plasmid is an Africa-type plasmid and also contains an IS5 insertion sequence. Since IS5 has not been identified in gonococcal isolates, we suggest that this sequence may have been inserted after the original gonococcal plasmid was transformed into Escherichia coli. The New Zealand plasmid is an Asia-type plasmid that contains an endogenous tandem duplication of 1883 bp and the direct DR2 is implicated in this duplication. The nature of the defined truncation of Tn2 present in the various plasmids is also discussed.     

Sequence and distribution of IS866, a novel T region-associated insertion sequence from Agrobacterium tumefaciens

We have identified a new insertion sequence, IS866, located in the auxin synthesis gene TA iaaH of Tm4, a wide host range biotype III octopine/cucumopine type Agrobacterium tumefaciens strain with two T regions on its tumor-inducing (Ti) plasmid, TA, and TB. IS866 is 2716 bp long, has inverted repeats of 27 bp with three mismatches, and generates 8-bp direct repeats upon integration. In addition to IS866, pTiTm4 carries two copies of a related element, IS867, associated with TA and TB, respectively. A systematic study of 92 virulent Agrobacterium strains has shown that among the three biotypes all octopine/cucumopine and vitopine biotype III isolates contain IS866-like elements. The various octopine/cucumopine Ti plasmids always carry IS866 and IS867 at the same position as in pTiTm4. The chromosomes of the bacteria which contain these Ti plasmids also carry IS866 and IS867 copies but in varying numbers and locations.     

Characterization of insertion sequence IS946, an Iso-ISS1 element, isolated from the conjugative lactococcal plasmid pTR2030.

The self-transmissible plasmid pTR2030 mobilized nonconjugative heterologous cloning vectors pGK12 (Cmr Emr) and pSA3 (Emr) at frequencies of 10(-5) to 10(-6) per input donor. Transconjugants harbored a 51- or 58-kilobase (kb) plasmid not found in the parental strains that cotransferred at high frequency with Cmr Emr and pTR2030-encoded phage resistance (Hsp+) in second-round matings (10(-1) per input donor). Restriction endonuclease mapping and DNA-DNA hybridization identified the 51- to 58-kb plasmids as pTR2030::vector cointegrates. Examination of four cointegrates indicated that pGK12 and pSA3 had inserted within two locations on pTR2030. Resolution of the cointegrates generated vector derivatives containing a 0.8-kb insert of pTR2030 DNA. Restriction analyses of several resolution plasmids indicated that the 0.8-kb element had inserted into various positions within pGK12 and pSA3 and in certain cases had inactivated the Cmr or Emr marker of pGK12. A conjugative mobilization assay demonstrated that the 0.8-kb element, designated IS946, mediated transpositional recombination. Nucleotide sequence determination identified IS946 as an 808-base-pair (bp) insertion sequence sharing ca. 96% homology with lactococcal insertion sequence ISS1. IS946 differed by 27 and 31 bp from ISS1S and ISS1T, respectively, and in 2 of 226 amino acids in the deduced sequence of the putative transposase. IS946 has perfect 18-bp terminal inverted repeats, identical to ISS1, and similarly generated 8-bp direct repeats of the target site upon insertion.     

The pCoo plasmid of enterotoxigenic Escherichia coli is a mosaic cointegrate

CS1 is the prototype of a class of pili of enterotoxigenic Escherichia coli (ETEC) associated with diarrheal disease in humans. The genes encoding this pilus are carried on a large plasmid, pCoo. We report the sequence of the complete 98,396-bp plasmid. Like many other virulence plasmids, pCoo is a mosaic consisting of regions derived from multiple sources. Complete and fragmented insertion sequences (IS) make up 24% of the total DNA and are scattered throughout the plasmid. The pCoo DNA between these IS elements has a wide range of G+C content (35 to 57%), suggesting that these regions have different ancestries. We find that the pCoo plasmid is a cointegrate of two functional replicons, related to R64 and R100, which are joined at a 1,953-bp direct repeat of IS100. Recombination between these repeats in the cointegrate generates the two smaller replicons which coexist with the cointegrate in the culture. Both of the smaller replicons have plasmid stability genes as well as genes that may be important in pathogenesis. Examination by PCR of 17 other unrelated CS1 ETEC strains with a variety of serotypes demonstrated that all contained at least parts of both replicons of pCoo and that strains of the O6 genotype appear to contain a cointegrate very similar to pCoo. The results suggest that this family of CS1-encoding plasmids is evolving rapidly.     

4',4' adenyltransferase activity on conjugative plasmids isolated from Staphylococcus aureus is encoded on an integrated copy of pUB110.

In staphylococci, linked resistance to the aminoglycosides kanamycin, neomycin, paromomycin, and tobramycin (KmNmPmTmr) is generally mediated by an aadD determinant which encodes production of an adenyltransferase aminoglycoside modifying enzyme, AAD(4',4'). The aadD resistance determinant is located on small multicopy plasmids such as pUB110, and has also been found on large multiresistance plasmids and on the chromosome in some strains. Examination of two conjugative plasmids from strains of Staphylococcus aureus isolated in North America indicated that the aadD determinant on these plasmids is located on an integrated copy of pUB110. The integrated pUB110 is flanked by direct repeats of the staphylococcal insertion sequence IS257. Analysis of the conjugative plasmid pSK41 showed an 8-bp duplication of the pUB110 sequence immediately adjacent to flanking IS257 elements, suggesting that integration of pUB110 was mediated by IS257.     

Recombination in recA cells between direct repeats of insertion element IS1.

The IS1 sequences that flank the Tn9 chloramphenicol acetyltransferase gene as direct repeats recombine after transformation into an Escherichia coli recA strain. The recombination requires the lambda pL promoter on the plasmid. A plasmid that contains mutant IS1 elements does not recombine. These results indicate that this recombination requires an IS1-specific gene product. The recombinational activity of IS1 may resolve transient cointegrates formed during the transposition of IS1. I discuss a possible role for the lambda pL promoter.     

recA-independent recombination between repeated IS50 elements is not caused by an IS50-encoded function.

Certain pBR322-related plasmids containing direct repeats of the insertion element IS50 appear to be unstable in recA Escherichia coli because smaller recombinant derivatives accumulate rapidly in plasmid DNA populations. We show here that (i) this instability is plasmid specific, but not IS50 specific; (ii) it is due to a detrimental effect exerted by these plasmids on bacterial growth; and (iii) the growth impairment is alleviated in cells harboring the smaller recombinant plasmids. Although a recent report had concluded that accumulation of recombinants reflected an IS50-specific recombination function, when correction is made for the relative growth rates of cells containing the parental and recombinant plasmids the evidence for such a recombination function disappears.     

Direct repeats flanking the Bacteroides transposon Tn4351 are insertion sequence elements.

The clindamycin-erythromycin resistance (Ccr Emr) region of the Bacteroides transposon Tn4351 is flanked by direct repeats. This study showed that the direct repeats are insertion sequence (IS) elements. Although both IS elements can mediate transfer of the chloramphenicol (Cmr) marker on pBR328 by cointegrate formation with the conjugal IncW plasmid R388, IS4351R-mediated transfer of Cmr occurred at a consistently lower frequency than did the transfer mediated by IS4351L. Analysis of plasmids from the resultant transconjugants revealed IS-mediated activities such as deletions, tandem duplication of IS4351L, and excision of IS4351R.     

The multiple antibiotic resistance IncP-1 plasmid pKJK5 isolated from a soil environment is phylogenetically divergent from members of the previously established alpha, beta and delta sub-groups

The 54,383bp plasmid pKJK5 was recovered from a soil environment by exogenous plasmid isolation and conveys resistance towards tetracycline and trimethoprim. Sequencing and annotation revealed a high level of structural similarity of the backbone genes to other IncP-1 plasmids containing a Tra1 and Tra2 region, a central control module and a replication initiation module. A considerable degree of divergence was associated with the backbone genes of pKJK5 as compared to homologous genes in the alpha, beta and delta subgroups, which indicates that pKJK5 may belong to a novel subgroup of IncP-1 plasmids, which may also accommodate the partially sequenced non-subgroup classified plasmid pEMT3. Individual backbone genes in pKJK5 have a GC-content, which is consistently lower (average 6.3%) than the homologous genes from the archetype IncP-1beta plasmid R751 indicating homogenous amelioration of IncP-1 plasmid backbone genes. Two discrete accessory elements of 2145bp (load 1) and 11678bp (load 2) respectively are situated between the Tra1 and Tra2 regions of pKJK5, both bounded by inverted repeats and direct flanking repeats indicative of transposon-mediated insertion. Load 1 consists of an insertion sequence ISPa17 and load 2 is a Tn402-derivative containing a class 1 integron, IS1326 and a fragment identical to a region of plasmid pTB11 harboring a tetracycline resistance determinant and part of an IncP-1alphaoriV region.     

Transfer of "artificial transposons" constructed on the basis of insertion element IS1

Terminal inverted repeats of the insertion element IS1 were synthesized chemically and plasmids containing these sequences flanking kanamycin-resistance gene in different combinations were constructed. Further incorporation of a whole-sized copy of the IS1 into such plasmids caused in some cases the autonomous transfer of Km-resistance from plasmid to bacteriophage lambda DNA. The transposition of the Km-resistance gene was only observed in those cases when the gene was enclosed between IS1 copy and one of the terminal repeats. The data obtained are discussed with regard to the evolution of bacterial transposons.     

A novel type of transposon generated by insertion element Is102 present in a psc101 derivative

We describe a novel type of transposon in the tetracycline resistance plasmid pYM103, a derivative of pSC101 carrying a single copy of an insertion element IS102. The new transposons we found were identified as DNA segments, approximately 6 kb (Tn1021) and 10 kb (Tn1022) in length, able to mediate the cointegration of pYM1O3 with plasmid Col E1. The resulting cointegrate contains either of these pYM1O3 segments duplicated in a direct orientation at the junctions of the parent plasmids. A direct duplication of a 9 bp sequence at the target site in Col E1 is found at the junctions for cointegration. Both transposons have IS1O2 at one end and also contain different lengths of the pYM103 DNA adjacent to IS102, including the tetracycline resistance gene. Each transposon contains terminal inverted repeats of a short nucleotide sequence. These results and the fact that IS102 can itself mediate plasmid cointegration, giving rise to a duplication of a 9 bp target sequence, indicate that IS102 is responsible for generation of Tn1021 and Tn1022. They are quite different from the common IS-associated transposons, which are always flanked by two copies of an IS element, and may be similar to transposons such as those of the Tn3 family and phage Mu.     

Mobilization of the pACYC184 plasmid by hybrid pAS8-121 delta plasmids in Escherichia coli cells

The plasmid pACYC184 is shown to be mobilized for conjugal transfer in Escherichia coli cells by the deleted (Tn7-TcR) derivatives of the hybrid conjugative plasmid pAS8-121 (RP4-Co1E1). Both the mobilized and mobilizing plasmids are autonomously inherited by the recipient cells when the mobilizing plasmid carries single copy of IS8 (the plasmid pAS8-121 delta 16). Cointegrates pAS8-121 delta 16D:: ::pACYC184 are found in the recipient cells with pACYC184 being inserted between two repeats of IS8 if the derivate plasmid pAS8-121 delta 16D having the duplication of IS8 is used to mobilize pACYC184 for conjugal transfer. The insertion of pACYC184 between IS8 repeats in the plasmid pAS8-121 delta 16D eliminates the plasmid ability to be inserted with high frequency into the chromosome of the phototrophic bacterium R. sphaeroides 2R. The cointegrate pAS8-121 delta 16D:: pACYC184 is stable but can be resolved during the transformation deriving the plasmid pACYC184:: IS8. The latter may be used as a probe for isolation and analysis of IS8 DNA sequences and for constructing the vectors on the basis of pACYC184.     

Analysis of DNA repeats in bacterial plasmids reveals the potential for recurrent instability events

Structural instability has been frequently observed in natural plasmids and vectors used for protein expression or DNA vaccine development. However, there is a lack of information concerning hotspot mapping, namely, DNA repeats or sequences identical to the host genome. This led us to evaluate the abundance and distribution of direct, inverted, and tandem repeats with high recombination potential in 36 natural plasmids from ten bacterial genera, as well as in several widely used bacterial and mammalian expression vectors. In natural plasmids, we observed an overrepresentation of close direct repeats in comparison to inverted ones and a preferential location of repeats with high recombination potential in intergenic regions, suggesting a highly plastic and dynamic behavior. In plasmid vectors, we found a high density of repeats within eukaryotic promoters and non-coding sequences. As a result of this in silico analysis, we detected a spontaneous recombination between two 21-bp direct repeats present in the human cytomegalovirus early enhancer/promoter (huCMV EEP) of the pCIneo plasmid. This finding is of particular importance, as the huCMV EEP is one of the most frequently used regulatory elements in plasmid vectors. Because pDNA integration into host gDNA can have adverse consequences in terms of plasmid processing and host safety, we also mapped several regions with high probability to mediate integration into the Escherichia coli or human genomes. Like repeated regions, some of these were located in non-coding regions of the plasmids, thus being preferential targets to be removed.     

Intramolecular recombination during plasmid transformation of Bacillus subtilis competent cells.

We have constructed plasmids carrying direct internal repeats 260-2000 bp long. Monomers of such plasmids transformed Bacillus subtilis competent cells. The efficiency of transformation varied with the square of the length of repeats. The transformed clones harbored either the entire transforming plasmid and the plasmid arising by recombination between the repeats, or only the latter plasmid. Internally-repeated plasmids linearized by in vitro cleavage with restriction endonuclease could transform, yielding clones which exclusively harbored a plasmid resulting from recombination between the repeats. When the transforming plasmid carried repeats which differed slightly, conversion of one repeat into the other could occur. The following model of plasmid transformation accounts for these data: (1) plasmid DNA is cleaved and rendered linear in contact with competent cells; (2) a linear, at least partially double-stranded plasmid molecule is introduced or formed by repair within the cell; (3) a circular viable plasmid is produced by recombination between repeats carried on this molecule; (4) alternatively, a viable plasmid is produced by repairing the cut within one of the repeats by DNA synthesis which uses the other repeat as a template.     

Plasmid maintenance of derivatives of oriP of Epstein-Barr virus.

oriP is the origin of plasmid replication of Epstein-Barr virus. Replication from oriP requires both the cis-acting elements (the family of repeats and the dyad symmetry element) and the viral origin-binding protein, EBNA-1. The ability of plasmids containing oriP to be maintained stably in EBNA-1-positive cells reflects the efficiency both of their replication and of their segregation each cell cycle. The efficiency of plasmid maintenance was determined for plasmids containing derivatives of oriP with one copy of the dyad symmetry element and two copies of the family of repeats by measuring the rate at which they were lost from cells in the absence of selection. These measurements demonstrated that plasmids with derivatives of oriP with two copies of the family of repeats in one orientation are maintained only slightly less efficiently than is wild-type oriP. To determine whether plasmid maintenance could be affected by reinitiation at the dyad symmetry element (T. A. Gahn and C. L. Schildkraut, Cell 58:527-535, 1989), plasmids containing derivatives of oriP with two copies of the dyad symmetry element and one copy of the family of repeats were compared with plasmids containing wild-type oriP in EBNA-1-positive cells. These measurements showed that plasmids containing a derivative of oriP with two copies of the dyad symmetry element are maintained as efficiently as is wild-type oriP and are not amplified relative to wild-type oriP. These observations indicate that the trans-acting factors that regulate DNA to replicate once per S phase are insensitive to multiple cis-acting regulatory sites within a replicon.     

IS-elements and their role in genetic recombination]

The data concerning the biological functions and properties of short specific polynucleotide sequences (so called insertion sequences--IS) are reviewed. IS elements integrated in a genome can lead to strongly polar mutations in Escherichia coli, its bacteriophages and plasmids, while some IS (IS2) being integrated in inverted orientation turn on the gene activity. Several copies of the IS elements are present in the E. coli chromosome. A characteristic feature of IS is their ability to recA-independent migration along the bacterial chromosome. Possible mechanisms of IS integration are discussed. IS elements play the key role in the majority of recA-independent recombinational events: F-prime and partially Hfr-formation, plasmid recombination and dissociation, some cases of deletion formation etc. IS elements participate in recombination in the form of direct or inverted repeats. Direct repeats probably determine the processes of dissociation of the complete multicomponent R-factors and other plasmids. Inverted repeats (some of them are palindromes) are responsible for the migration of several drug-resistance determinants called transposons. Possible mechanisms of IS-dependent and probably IS-controlled recombination are discussed.     

Genomic comparison of archaeal conjugative plasmids from Sulfolobus

All of the known self-transmissable plasmids of the Archaea have been found in the genus Sulfolobus. To gain more insight into archaeal conjugative processes, four newly isolated self-transmissable plasmids, pKEF9, pHVE14, pARN3 and pARN4, were sequenced and subjected to a comparative sequence analysis with two earlier sequenced plasmids, pNOB8 and pING1. The analyses revealed three conserved and functionally distinct sections in the genomes. Section A is considered to encode the main components of the conjugative apparatus, where two genes show low but significant sequence similarity to sections of genes encoding bacterial conjugative proteins. A putative origin of replication is located in section B, which is highly conserved in sequence and contains several perfect and imperfect direct and inverted repeats. Further downstream, in section C, an operon encoding six to nine smaller proteins is implicated in the initiation and regulation of replication. Each plasmid carries an integrase gene of the type that does not partition on integration, and there is strong evidence for their integration into host chromosomes, where they may facilitate intercellular exchange of chromosomal genes. Two plasmids contain hexameric short regularly spaced repeats (SRSR), which have been implicated in plasmid maintenance, and each plasmid carries multiple recombination motifs, concentrated in the variable regions, which likely provide sites for genomic rearrangements.     

Theta-type DNA replication stimulates homologous recombination in the Bacillus subtilis chromosome

To test the effects of theta-type replication on homologous DNA recombination, we integrated in the chromosome of Bacillus subtilis a structure comprising a conditional replication region and direct repeats of ∼ 4 kb. The replicon was derived from a broad-host-range plasmid, pAMβ1, which replicates by a unidirectional theta mechanism and is thermosensitive. The direct repeats were derived from plasmid pBR322 and flanked the chloramphenicol-resistance gene of plasmid pC194. Recombination between the repeats could therefore lead to a loss of the resistance gene or the appearance of additional repeats. The integrated replicon was active at the permissive temperature, and ∼ 25% of the integrated plasmids could be isolated as Y-shaped molecules after restriction, having a branch at the replication origin. Replicon activity stimulated recombination four- to fivefold, as estimated from the proportion of chloramphenicol-sensitive cells at the restrictive and permissive temperature, and also led to the appearance of additional direct repeats. We conclude that theta-type replication stimulates homologous recombination and suggest that many or even most recombination events between long homologous sequences present in a bacterial genome may be the consequence of DNA replication.