Analysis of secondary, integration sites for IS117 in Streptomyces lividans and their role in the generation of chromosomal deletions

IS117, the 2.6 kb mini-circle of Streptomyces coelicolor A3(2), is a transposable element previously shown to be integrated into two distant sites in the chromosome. When introduced into S. lividans, IS117 integrates into one preferred chromosomal site, but when this site was artificially deleted, IS117 integrated into many secondary sites. Nucleotide sequence analysis of several secondary integration sites revealed varying degrees of similarity with the preferred site, but no consensus sequence. Nevertheless, sites more similar to the preferred site tended to be occupied more often than those that are less similar. Insertion of IS117 into secondary sites in the chromosome of S. lividans sometimes mediated chromosomal rearrangements. It was shown that some strains containing IS117 integrated into secondary sites had suffered deletions of chromosomal DNA. Deletions were adjacent to the inserted element and were at least several kilobases long. The proposed model implicates homologous recombination between IS117 copies integrated into two different secondary sites in the same chromosome as a cause of the deletions.     

Transposition of IS 117, the 2.5 kb Streptomyces coelicolor A3(2) 'minicircle': roles of open reading frames and origin of tandem insertions

IS 117 is a 2527 bp transposable element from Streptomyces coelicolor A3(2) with a circular transposition intermediate. Disruption of 0RF1 of IS 117, presumed to encode a transposase, abolished transposition. Deletion or mutation of 0RF2 and 0RF3, which overlap each other on opposite strands of IS 117, caused a c. 20-fold reduction in integration frequency of the circular form of IS 117 into the Streptomyces lividans chromosome or into the preferred chromosomal target site cloned on a plasmid in transformation experiments. In contrast, inactivation of ORF2/3 did not significantly influence transposition of IS 117 derivatives from an already integrated state in the chromosome to the preferred target site cloned on a plasmid. 0RF2 mutants apparently excised readily from the S. lividans chromosome, whereas excision of integrated wild-type IS 117 derivatives to yield the unoccupied site was not detected; presumably, therefore, the circular transposition intermediate normally arises replicatively. Attempts to promote integration of a plasmid carrying the attachment site of IS 117 by providing the ORF1 product in trans were unsuccessful. Most transformation of S. lividans with circular IS 117 derivatives yielded tandem chromosomal insertions, which arose by co-transformation rather than dimerization of a monomeric insert. Typically, two to three transforming elements gave a transformed strain, suggesting a local concentration of transposase as a limit on integration.     

Excision of pIJ408 from the chromosome of Streptomyces glaucescens and its transfer into Streptomyces lividans

Summary Streptomyces glaucescens GLA000 contains the integrated 15 kb DNA element pIJ408 which, during mating of the parent strain with S. lividans, can be transferred into recipient cells. In S. lividans cells, pIJ408 was found in an autonomously replicating form and in a chromosomally integrated state. In the majority of the S. lividans transconjugants studied, a deletion derivative pIJ408. 1 (12.4 kb) occurred. The deletion form was found in some strains only as a free plasmid, in others it was also chromosomally integrated. The integration region of pIJ408 was subcloned and precisely mapped by hybridization, restriction and sequencing analyses. The DNA junction fragments of the integrated plasmid in S. glaucescens, as well as the DNA fragment containing the attachment site of the S. lividans chromosome, were also cloned, submitted to detailed restriction analysis and sequenced. The attachment site of pIJ408 (attP) and the junctions of its integrated form with the chromosomal DNA in S. glaucescens (attL and attR) contain an identical 43 bp sequence. The chromosomal attachment site in S. lividans (attB) differs from the S. glaucescens att sequence by a single base substitution. The similarities between attachment sites of SLP1, pMEA100, pSAM2 and pIJ408 are discussed.     

Plasmid formation in Streptomyces: excision and integration of the SLP1 replicon at a specific chromosomal site

Summary We present data showing that the SLP1 plasmids found in Streptomyces lividans after mating with S. coelicolor strain A3(2) orginate as deletion mutants of a 17 kb segment of the S. coelicolor chromosome. Excision of the entire 17 kb segment yields a transiently existing plasmid containing a site for integration into the chromosome of recipient SLP1^- S. lividans strains at a unique locus that corresponds to the original chromosomal location of SLP1 in S. coelicolor. The deletion mutants of SLP1 lack the attachment site and/or other regions required for its integration, and thus persist in the recipient as autonomously replicating plasmids. Plasmids that contain the complete 17 kb sequence of the chromosomally integrated SLP1 segment were constructed in vitro by circularization of restriction endonuclease-generated fragements of chromosomal DNA carrying a tandemly-duplicated integrant of SLP1. Transformation of an SLP1^- S. lividans strain with such plasmids results in chromosomal integration of the SLP1 sequence at the same site at which it is integrated in S. lividans cells that acquire the sequence by mating with S. coelicolor. A model for the site-specific excision and integration of SLP1 is presented.     

IS6110-Mediated Deletions of Wild-Type Chromosomes of Mycobacterium tuberculosis

The ipl locus is a site for the preferential insertion of IS6110 and has been identified as an insertion sequence, IS1547, in its own right. Various deletions around the ipl locus of clinical isolates of Mycobacterium tuberculosis were identified, and these deletions ranged in length from several hundred base pairs up to several kilobase pairs. The most obvious feature shared by these deletions was the presence of an IS6110 copy at the deletion sites, which suggested two possible mechanisms for their occurrence, IS6110 transposition and homologous recombination. To clarify the mechanism, an investigation was conducted; the results suggest that although deletion transpositionally mediated by IS6110 was a possibility, homologous recombination was a more likely one. The implications of such chromosomal rearrangements for the evolution of M. tuberculosis, for IS6110-mediated mutagenesis, and for the development of genetic tools are discussed. The deletion of genomic DNA in isolates of M. tuberculosis has previously been noted at only a few sites. This study examined the deletional loss of genetic material at a new site and suggests that such losses may occur elsewhere too and may be more prevalent than was previously thought. Distinct from the study of laboratory-induced mutations, the detailed analysis of clinical isolates, in combination with knowledge of their evolutionary relationships to each other, gives us the opportunity to study mutational diversity in isolates that have survived in the human host and therefore offers a different perspective on the importance of particular genetic markers in pathogenesis.     

Site-specific integration of plasmid pSAM2 in Streptomyces lividans and S. ambofaciens

Summary Streptomyces ambofaciens strain ATCC23877 contains the 11.1 kb plasmid pSAM2 stably integrated into its chromosome. This plasmidic sequence is able to loop out and to be transferred at high frequency to S. lividans where it is found simultaneously as both free and integrated plasmid. When a UV derivative of strain ATCC23877 (strain ATCC15154) is used, the resident copy of pSAM2 can be transferred to S. lividans, but only the integrated form is found in this strain. In both cases, the integration occurs at a unique chromosomal region through the same plasmidic integration site as that in strain ATCC23877. The resident copy of strain ATCC15154 can also be transferred at low frequency to S. ambofaciens DSM40697 (devoid of any pSAM2 sequence). In this case, as several copies of pSAM2 are integrated, the integration pattern is complicated. Integration of a complete pSAM2 sequence in this strain occurs in a region that hybridizes with the integration zones of S. lividans and of S. ambofaciens strain ATCC23877. Comparison of the cloned integration zone of S. lividans before and after the integration event showed that the restriction pattern of the resident pSAM2 in strain ATCC15154 is similar to that of the free form of pSAM2 found naturally in another UV derivative of strain ATCC23877 (strain JI3212).     

Internal size variations in Tn1546-like elements due to the presence of IS1216V

In this study, internal size variations in the VanA gene cluster Tn1546, encoding resistance to glycopeptides, is described. Studies of previously uncharacterized size variations of an internal region, encoding the vanX and vanY genes of Tn1546, revealed that these variations were due to the presence of the IS sequence, IS1216V. This IS sequence has previously been found integrated in Tn1546. Integration of the IS1216V element created both deletions and a duplication in a non-essential region of Tn1546. In several isolates, the entire vanY gene was deleted, proving that this gene is non-essential for vancomycin resistance.     

ISLpl1 Is a Functional IS30-Related Insertion Element in Lactobacillus plantarum That Is Also Found in Other Lactic Acid Bacteria

We describe the first functional insertion sequence (IS) element in Lactobacillus plantarum. ISLpl1, an IS30-related element, was found on the pLp3 plasmid in strain FB335. By selection of spontaneous mutants able to grow in the presence of uracil, it was demonstrated that the IS had transposed into the uracil phosphoribosyltransferase-encoding gene upp on the FB335 chromosome. The plasmid-carried IS element was also sequenced, and a second potential IS element was found: ISLpl2, an IS150-related element adjacent to ISLpl1. When Southern hybridization was used, the copy number and genome (plasmid versus chromosome) distribution data revealed different numbers and patterns of ISLpl1-related sequences in different L. plantarum strains as well as in Pediococcus strains. The ISLpl1 pattern changed over many generations of the strain L. plantarum NCIMB 1406. This finding strongly supports our hypothesis that ISLpl1 is a mobile element in L. plantarum. Database analysis revealed five quasi-identical ISLpl1 elements in Lactobacillus, Pediococcus, and Oenococcus strains. Three of these elements may be cryptic IS, since point mutations or 1-nucleotide deletions were found in their transposase-encoding genes. In some cases, ISLpl1 was linked to genes involved in cold shock adaptation, bacteriocin production, sugar utilization, or antibiotic resistance. ISLpl1 is transferred among lactic acid bacteria (LAB) and may play a role in LAB genome plasticity and adaptation to their environment.     

Transposition of IS1181 in the genomes of Staphylococcus and Listeria

The recombinant plasmid pIP1713 was constructed to analyse the transpositional activity of the insertion sequence IS1181 in Staphylococcus aureus RN4220, Staphylococcus carnosus TM300 and Listeria monocytogenes EGD. This 11.3-kb plasmid contains two genetically different elements: (i) a pE194ts-derived replicon, the ermC gene of which confers resistance to erythromycin in Gram-positive bacteria of several species, and (ii) a copy of IS1181, cloned from S. aureus BM3121, in which the tetracycline resistance gene, tet(T), has been inserted between the transposase-encoded gene and the downstream inverted repeat. When introduced by electroporation into the three bacterial hosts, pIP1713 delivered IS1181Ωtet(T) to various chromosomal sites. Cointegrate structures between pIP1713 and the host chromosome were occasionally detected. Transposition was associated with 8-bp repeats at the insertion sites. IS1181Ωtet(T) could be used for random mutagenesis in Gram-positive bacteria.     

Sequence organization and insertion specificity of the novel chimeric ISHp609 transposable element of Helicobacter pylori

Here we describe ISHp609 of Helicobacter pylori, a new member of the IS605 mobile element family that is novel and contains two genes whose functions are unknown, jhp960 and jhp961, in addition to homologs of two other H. pylori insertion sequence (IS) element genes, orfA, which encodes a putative serine recombinase-transposase, and orfB, whose homologs in other species are also often annotated as genes that encode transposases. The complete four-gene element was found in 10 to 40% of strains obtained from Africa, India, Europe, and the Americas but in only 1% of East Asian strains. Sequence comparison of 10 representative ISHp609 elements revealed higher levels of DNA sequence matches (99%) than those seen in normal chromosomal genes (88 to 98%) or in other IS elements (95 to 97% for IS605, IS606, and IS607) from the same H. pylori populations. Sequence analysis suggested that ISHp609 can insert at many genomic sites with its left end preferentially next to TAT, with no target specificity for its right end, and without duplicating or deleting target sequences. A deleted form of ISHp609, containing just jhp960 and jhp961 and 37 bp of orfA, found in reference strain J99, was at the same chromosomal site in 15 to 40% of the strains from many geographic regions but again in only 1% of the East Asian strains. The abundance and sequence homogeneity of ISHp609 and of this nonmobile remnant suggested a recent bottleneck and then rapid spread in H. pylori populations, possibly selected by the contributions of the elements to bacterial fitness.     

Isolation and genetic structure of IS112, an insertion sequence responsible for the inactivation of the SalI restriction-modification system of Streptomyces albus G.

Summary IS112 is a transposable element identified in Streptomyces albus G by its frequent mutagenic insertion into the genes for the SalI restriction-modification system. IS112 is present in several copies in the genome of S. albus G. Homologous sequences were detected in other Streptomyces strains. Sequence analysis revealed that IS112 has a length of 883 by with a GC content of 67.4%. The copy that was isolated contained imperfect inverted repeats (16/20 match) at its ends and was flanked by a 2 by duplication at the target site, which was located within the gene (salIR) for the Sall endonuclease. A long open reading frame (ORF) encoding a putative polypeptide of 256-253 amino acids spans almost the entire sequence. Significant homology was detected between this polypeptide and that corresponding to ORFB of IS493, an insertion sequence recently isolated from Streptomyces lividans 66.

---

     

A 2.6 kb DNA sequence of Streptomyces coelicolor A3(2) which functions as a transposable element

Summary Streptomyces coelicolor A3(2) contains CCC DNA molecules, 2.6 kb in size, with an average copy number of less than one per ten chromosomes. Southern hybridisation revealed, in addition, two linear, integrated copies (A and B) of this mini-circle sequence per chromosome. The two integrated copies have similar (if not identical) ends and are present in the same locations in various S. coelicolor A3(2) derivatives. The mini-circle sequence is absent from S. lividans 66 and S. violaceolatus ISP5438 and from several Streptomyces species less closely related to S. coelicolor A3(2). None of a variety of Streptomyces plasmids tested contained homology to the mini-circle sequence. When a 1.8 kb fragment of the mini-circle lacking the ends of the integrated copies was inserted into KC515 (a derivative of the temperate phage C31 which is unable to lysogenise host strains by the natural route because the phage attachment site has been deleted) the resulting phage lysogenised S. coelicolor A3(2) (integrating into the genome of this host by homologous recombination with resident minicircle sequences) but not S. lividans or a variety of other C31 hosts. In contrast, a KC515 derivative (KC591) carrying the entire 2.6 kb mini-circle sequence linearised at its single BclI site (and therefore containing the integration site of the free mini-circle) lysogenised not only S. coelicolor A3(2) but also S. lividans 66 and most other strains normally lysogenised by C31. The KC591 lysogens of the eight Streptomyces species tested contained a linear, integrated prophage with termini apparently identical to those of the linear mini-cricle copies of S. coelicolor. In S. lividans, KC591 integrated preferentially at a site apparently homologous to the site occupied by mini-circle sequence A in S. coelicolor A3(2) strains, but integration into secondary sites also occurred.     

Three insertion sequences from the cyanobacterium Synechocystis PCC6803 support the occurrence of horizontal DNA transfer among bacteria

Three insertion sequences were characterized from the widely-used cyanobacterium Synechocystis PCC6803. They all harbored a putative transposase sequence flanked by two imperfect inverted repeats, seemed to have duplicated their target insertion site and occurred as multiple copies in the host genome. They exhibited no obvious homology with any other cyanobacterial ISs and were termed IS5S (871 bp), IS4S (1299 bp) and ISS1987 (949 bp) because they were, respectively, homologous to IS5- and IS4-bacterial elements, and to several members of the IS630-Tc1-mariner superfamily of IS elements occurring in a wide range of hosts. This suggests that these IS-elements were spread through horizontal transfer between evolutionary distant organisms. Three IS5S-copies were isolated as a rescue insertion into a replicating plasmid (IS5Sa), or subsequently cloned from a Synechocystis DNA-library probed with IS5Sa (IS5Sb and IS5Sc), and appeared to be almost identical. In the vicinity of IS5Sb, we found the ISS1987 element inserted into the IS4S element. This indicates that the ISS1987 element has been, and could still be, mobile since its transposase sequence is not interrupted with stop codons or translational frameshifts, unlike that which is found in most members of the IS630-Tc1-mariner superfamily of transposable elements.     

Genetic organization and transposition properties of IS511

IS511 is an endogenous insertion sequence (IS) of the bacterium Caulobactercrescentus strain CB15 and it is the first Caulobacter IS to be characterized at the molecular level. We determined the 1266-bp nucleotide sequence of IS511 and investigated its genetic organization, relationship to other ISs, and transposition properties. IS511 belongs to a distinct branch of the IS3 family that includes ISRI, IS476, and IS1222, based on nucleotide sequence similarity. The nucleotide sequence of IS511 encodes open reading frames (orfs) designated here as orfA and orfB, and their relative organization and amino acid sequences of the predicted protein products are very similar to those of orfAs and orfBs of other IS3 family members. Nuclease S1 protection assays identified an IS511 RNA, and its 5′ end maps approximately 16 nucleotides upstream of orfA and about six nucleotides downstream of a sequence that is similar to the consensus sequence of C. crescentus housekeeping promoters. Evidence is presented that IS511 is capable of precise excision from the chromosome, and transposition from the chromosome to a plasmid. Transpositional insertions of IS511 occurred within sequences with a relatively high G?+?C content, and they were usually, but not always, flanked by a 4-bp direct repeat that matches a sequence at the site of insertion. We also determined the nucleotide sequence flanking the four endogenous IS511 elements that reside in the chromosome of C. crescentus. Our findings demonstrate that IS511 is a transposable IS that belongs to a branch of the IS3 family.     

Secondary chromosomal attachment site and tandem integration of the mobilizable Salmonella genomic island 1

Background

The Salmonella genomic island 1 is an integrative mobilizable element (IME) originally identified in epidemic multidrug-resistant Salmonella enterica serovar Typhimurium (S. Typhimurium) DT104. SGI1 contains a complex integron, which confers various multidrug resistance phenotypes due to its genetic plasticity. Previous studies have shown that SGI1 integrates site-specifically into the S. enterica, Escherichia coli, or Proteus mirabilis chromosome at the 3′ end of thdF gene (attB site).

Methodology/Principal Findings

Here, we report the transfer of SGI1 to a ΔthdF mutant of S. Typhimurium LT2. In the absence of thdF, the frequency of transconjugant formation was reduced by around thirty times of magnitude. Through DNA sequencing SGI1 was shown to integrate specifically into a secondary attachment site (2^nd attB), which is located in the intergenic region between the chromosomal sodB and purR genes. At this 2^nd attB site, we found that a significant fraction of SGI1 transconjugants (43% of wild type and 100% of ΔthdF mutant) contained tandem SGI1 arrays. Moreover, in wild type S. Typhimurium LT2 transconjugants, SGI1 integrated into both attachment sites, i.e., thdF and sodB-purR. The formation of SGI1 tandem arrays occurred in both specific attB sites. There was heterogeneity in the size of the SGI1 tandem arrays detected in single transconjugant colonies. Some arrays consisted as far as six SGI1s arranged in tandem. These tandem arrays were shown to persist during serial passages with or without antibiotic selection pressure.

Conclusions/Significance

The ability of integration into two distinct chromosomal sites and tandem array formation of SGI1 could contribute to its spread and persistence. The existence of a secondary attachment site in the Salmonella chromosome has potential implications for the mobility of SGI1, which may integrate in other attachment sites of other bacterial pathogens that do not possess the 1^st or 2^nd specific SGI1 attB sites of Salmonella.     

Characterization of the genes and attachment sites for site-specific integration of plasmid pSE101 in Saccharopolyspora erythraea and Streptomyces lividans

The 11.3 kb plasmid pSE101 integrates into the chromosome of Saccharopolyspora erythraea at a specific attB site and into the chromosome of Streptomyces lividans at many sites. Multisite integration in S. lividans was also observed when a 1.9 kb segment of pSE101 containing attP and adjacent plasmid sequence was used to transform a pSE101^– S. lividans host. Nucleotide sequencing of this segment revealed the presence of a complete open reading frame (ORF) designated int, encoding a putative polypeptide of 448 amino acids that shows similarities to site-specific recombinases of the integrase family. Sequencing of the 1.3 kb segment upstream of int revealed the presence of three additional ORFs: the one most distal to int encodes a putative 76 amino acid basic polypeptide analogous to the Xis proteins of a number of bacteriophages. Nucleotide sequencing of attP, and the attB, attL and attR sites from Sac. erythraea revealed a 46 by sequence common to all sites with no duplications of chromosomal sequences in the integrated state. A putative structural gene for a tRNA^Thr was found to overlap the 46 by common sequence at attB. Sequencing of four pSE101 integration sites (attB) and corresponding attL and attR sites in S. lividans showed that the 46 by sequence was present at each attR site, whereas only the first three bases, CTT, were retained at each attL and attB site. A feature common to the four attB sites and to attB is a highly conserved 21 by segment with inverted repeats flanking the CTT sequence. This indicates that crossover at each attB site in S. lividans employed attP and a site within a 5 by sequence in attB and suggests that the secondary structure of the 21 by sequence is important for site-specific integration at attB or attB.     

Intergeneric Conjugation in Streptomyces peucetius and Streptomyces sp. Strain C5: Chromosomal Integration and Expression of Recombinant Plasmids Carrying the chiC Gene

Intergeneric conjugal transfer of plasmid DNA from Escherichia coli to Streptomyces circumvents problems such as host-controlled restriction and instability of foreign DNA during the transformation of Streptomyces protoplasts. The anthracycline antibiotic-producing strains Streptomyces peucetius and Streptomyces sp. strain C5 were transformed using E. coli ET12567(pUZ8002) as a conjugal donor. When this donor species, carrying pSET152, was mated with Streptomyces strains, the resident plasmid was mobilized to the recipient and the transferred DNA was also integrated into the recipient chromosome. Analysis of the exconjugants showed stable integration of the plasmid at a single chromosomal site (attB) of the Streptomyces genome. The DNA sequence of the chromosomal integration site was determined and shown to be conserved. However, the core sequence, where the crossover presumably occurred in C5 and S. peucetius, is TTC. These results also showed that the C31 integrative recombination is active and the phage attP site is functional in S. peucetius as well as in C5. The efficiency and specificity of C31-mediated site-specific integration of the plasmid in the presence of a 3.7-kb homologous DNA sequence indicates that integrative recombination is preferred under these conditions. The integration of plasmid DNA did not affect antibiotic biosynthesis or biosynthesis of essential amino acids. Integration of a single copy of a mutant chiC into the wild-type S. peucetius chromosome led to the production of 30-fold more chitinase.     

Physical and genetic analysis of IS110, a transposable element of Streptomyces coelicolor A3(2)

Summary On at least three independent occasions a 1.6 kb segment of Streptomyces coelicolor DNA was detected in apparently the same location in an attP-deleted derivative of the temperature phage C31 that carried a selectable viomycin resistance gene. This sequence (termed IS110) allowed integration of the phage (giving viomycin-resistant transductants) at homologous sequences (detected by Southern hybridisation) at several locations in the S. coelicolor genome. The inserted prophages facilitated genetic mapping of two IS110 copies in the chromosomal linkage map. A third copy did not exhibit simple segregation with chromosomal markers, and there appeared to be a frequent DNA rearrangement close to this copy. Some variation in the number of copies of IS110 and their location has taken place in the pedigree of S. coelicolor derivatives. IS110 did not hybridise to any known S. coelicolor plasmid, nor to any of several other IS-like elements previously described in other Streptomyces plasmids or phages. It hybridised strongly to DNA from only a small minority of other Streptomyces species and was absent from S. lividans, a close relative of S. coelicolor.     

The insertion sequence IS21 of R68.45 and the molecular basis for mobilization of the bacterial chromosome

The IncP-1 plasmid mutant R68.45, which is able to mobilize the chromosomes of many Gram-negative bacteria, was shown to carry a 2.10-kb insertion sequence designated IS21. This sequence transposed to the small multicopy plasmid pED815 at a high frequency (2 × 10^?3) and in two pED815::IS21 derivatives inactivated the tetracycline-resistance and replication functions, respectively. We propose that the chromosome-mobilizing ability of R68.45 is due to the formation of an R68.45-chromosome cointegrate during transposition of IS21. This would account for its high efficiency and the absence of a fixed chromosomal origin of transfer in Pseudomonas aeruginosa PAO, and its ability to function in a variety of bacterial hosts. R68.45 is formed from R68 by duplication of a 2.1-kb DNA segment including a distinctive cluster of seven restriction endonuclease sites. The two copies of the duplicated segment are probably contiguous and so might have arisen by a transition type of mechanism. IS21 is similar in length to the duplicated segment and includes the same set of seven cleavage sites located at similar distances from the two termini. However, the single copy of the duplicated segment in R68 transposed at an undetectably low frequency (<6 × 10^?8); either the duplicated segment and IS21, although overlapping, are not identical, or they are identical but the transposition system is nonfunctional in R68. Our further investigations of R68.45 and of several independently isolated chromosome-mobilizing derivatives of R68 demonstrated that these were indistinguishable from each other and that they did not include any P. aeruginosa PAO DNA. Furthermore, we searched without success for sequences corresponding to IS21, and to the Escherichia coli K-12 insertion sequences IS1, IS2, and IS3, on the chromosomes of P. aeruginosa PAO and PAT and P. putida PPN, and on several Pseudomonas plasmids. The contribution of homology to low-frequency chromosomal mobilization by these plasmids is discussed.