The Bacteroides mobilizable insertion element, NBU1, integrates into the 3' end of a Leu-tRNA gene and has an integrase that is a member of the lambda integrase family.

NBU1 is a 10.3-kbp integrated Bacteroides element that can be induced to excise from the chromosome and can be mobilized to a recipient by trans-acting functions provided by certain Bacteroides conjugative transposons. The NBU1 transfer intermediate is a covalently closed circle, which is presumed to be the form that integrates into the recipient genome. We report here that a 2.4-kbp segment of NBU1 was all that was required for site-specific integration into the chromosome of Bacteroides thetaiotaomicron 5482. This 2.4-kbp region included the joined ends of the NBU1 circular form (attN1) and a single open reading frame, intN1, which encoded the integrase. Previously, we had found that NBU1 integrates preferentially into a single site in B. thetaiotaomicron 5482. We have now shown that the NBU1 target site is located at the 3' end of a Leu-tRNA gene. The NBU1 integrase gene, intN1, was sequenced. The predicted protein had little overall amino acid sequence similarity to any proteins in the databases but had limited carboxy-terminal similarity to the integrases of lambdoid phages and to the integrases of the gram-positive conjugative transposons Tn916 and Tn1545. We also report that the intN1 gene is expressed constitutively.     

Characterization of a Bacteroides mobilizable transposon, NBU2, which carries a functional lincomycin resistance gene

The mobilizable Bacteroides element NBU2 (11 kbp) was found originally in two Bacteroides clinical isolates, Bacteroides fragilis ERL and B. thetaiotaomicron DOT. At first, NBU2 appeared to be very similar to another mobilizable Bacteroides element, NBU1, in a 2.5-kbp internal region, but further examination of the full DNA sequence of NBU2 now reveals that the region of near identity between NBU1 and NBU2 is limited to this small region and that, outside this region, there is little sequence similarity between the two elements. The integrase gene of NBU2, intN2, was located at one end of the element. This gene was necessary and sufficient for the integration of NBU2. The integrase of NBU2 has the conserved amino acids (R-H-R-Y) in the C-terminal end that are found in members of the lambda family of site-specific integrases. This was also the only region in which the NBU1 and NBU2 integrases shared any similarity (28% amino acid sequence identity and 49% sequence similarity). Integration of NBU2 was site specific in Bacteroides species. Integration occurred in two primary sites in B. thetaiotaomicron. Both of these sites were located in the 3' end of a serine-tRNA gene NBU2 also integrated in Escherichia coli, but integration was much less site specific than in B. thetaiotaomicron. Analysis of the sequence of NBU2 revealed two potential antibiotic resistance genes. The amino acid sequences of the putative proteins encoded by these genes had similarity to resistances found in gram-positive bacteria. Only one of these genes was expressed in B. thetaiotaomicron, the homolog of linA, a lincomycin resistance gene from Staphylococcus aureus. To determine how widespread elements related to NBU1 and NBU2 are in Bacteroides species, we screened 291 Bacteroides strains. Elements with some sequence similarity to NBU2 and NBU1 were widespread in Bacteroides strains, and the presence of linA(N) in Bacteroides strains was highly correlated with the presence of NBU2, suggesting that NBU2 has been responsible for the spread of this gene among Bacteroides strains. Our results suggest that the NBU-related elements form a large and heterogeneous family, whose members have similar integration mechanisms but have different target sites and differ in whether they carry resistance genes.     

Excision, transfer, and integration of NBU1, a mobilizable site-selective insertion element.

The Bacteroides species harbor a family of conjugative transposons called tetracycline resistance elements (Tcr elements) that transfer themselves from the chromosome of a donor to the chromosome of a recipient, mobilize coresident plasmids, and also mediate the excision and circularization of members of a family of 10- to 12-kbp insertion elements which share a small region of DNA homology and are called NBUs (for nonreplicating Bacteroides units). The NBUs are sometimes cotransferred with Tcr elements, and it was postulated previously that the excised circular forms of the NBUs were plasmidlike forms and were transferred like plasmids and then integrated into the recipient chromosome. We used chimeric plasmids containing one of the NBUs, NBU1, and a Bacteroides-Escherichia coli shuttle vector to show that this hypothesis is probably correct. NBU1 contained a region that allowed mobilization by both the Tcr elements and IncP plasmids, and we used these conjugal elements to allow us to estimate the frequencies of excision, mobilization, and integration of NBU1 in Bacteroides hosts to be approximately 10(-2), 10(-5) to 10(-4), and 10(-2), respectively. Although functions on the Tcr elements were required for the excision-circularization and mobilization of NBU1, no Tcr element functions were required for integration into the recipient chromosome. Analysis of the DNA sequences at the integration region of the circular form of NBU1, the primary insertion site in the Bacteroides thetaiotaomicron 5482 chromosome, and the resultant NBU1-chromosome junctions showed that NBU1 appeared to integrate into the primary insertion site by recombining within an identical 14-bp sequence present on both NBU1 and the target, thus leaving a copy of the 14-bp sequence at both junctions. The apparent integration mechanism and the target selection of NBU1 were different from those of both XBU4422, the only member of the conjugal Tcr elements for which these sequences are known, and Tn4399, a mobilizable Bacteroides transposon. The NBUs appear to be a distinct type of mobilizable insertion element.     

NBU1 integrase: evidence for an altered recombination mechanism

NBU1 is a 10.3 kbp Bacteroides mobilizable transposon. A previous study had identified a 2.7 kbp segment of the excised circular intermediate that was sufficient to mediate integration of the element after transfer. This segment contained an integrase gene, intN1, and a region spanning the ends of the circular form within which integration occurred (attN1). The integrase protein, IntN1, appeared to be a member of the tyrosine recombinase family because it contains the canonical C-terminal RKHRHY [RK(H/K)R(H/W)Y] motif that characterizes members of that family. In this study, we describe an Escherichia coli-based integration assay system that has allowed us to characterize attN1 in detail. We first localized attN1 to a 250 bp region. We then used site-directed mutations to identify directly repeated sequences within attN1 that were required for site-specific integration. The locus of NBU1 site-specific integration in the Bacteroides thetaiotaomicron chromosome, attBT1-1, contains a 14 bp sequence that is identical to a 14 bp sequence that spans the joined ends of the NBU1 attN1 site (common core sequences). The effects of mutations in the common core were different from the expected results if NBU1 integration was similar to lambda integration. In particular single base changes near one end of the common core region, which introduced heterology, actually increased the frequency of integration. By contrast, compensating changes that restored homology in the common core region reduced the integration frequency. The recombination mechanism also differs from the one used by conjugative transposons that have coupling sequences between the sites of strand cleavage and exchange. These results indicate that although NBU1 integrase is considered to be a member of the tyrosine recombinase family, it catalyses an integrative recombination reaction that occurs by a different crossover mechanism.     

Multiple gene products and sequences required for excision of the mobilizable integrated Bacteroides element NBU1

NBU1 is an integrated 10.3-kbp Bacteroides element, which can excise and transfer to Bacteroides or Escherichia coli recipients, where it integrates into the recipient genome. NBU1 relies on large, >60-kbp, conjugative transposons for factors that trigger excision and for mobilization of the circular form to recipients. Previously, we showed that a single integrase gene, intN1, was necessary and sufficient for integration of NBU1 into its target site on the Bacteroides or E. coli genome. We now show that an unexpectedly large region of NBU1 is required for excision. This region includes, in addition to intN1, four open reading frames plus a large region downstream of the fourth gene, prmN1. This downstream sequence was designated XRS, for "excision-required sequence." XRS contains the oriT of the circular form of NBU1 and about two-thirds of the adjacent mobilization gene, mobN1. This is the first time an oriT, which is involved in conjugal transfer of the circular form, has been implicated in excision. Disruption of the gene immediately downstream of intN1, orf2, completely abolished excision. The next open reading frame, orf2x, was too small to be disrupted, so we still do not know whether it plays a role in the excision reaction. Deletions were made in each of two open reading frames downstream of orf2x, orf3 and prmN1. Both of these deletions abolished excision, indicating that these genes are also essential for excision. Attempts to complement various mutations in the excision region led us to realize that a portion of the excision region carrying prmN1 and part of the XRS (XRS(HIII)) inhibited excision when provided in trans on a multicopy plasmid (8 to 10 copies per cell). However, a fragment carrying prmN1, XRS, and the entire mobilization gene, mobN1, did not have this effect. The smaller fragment may be interfering with excision by attracting proteins made by the intact NBU1 and thus removing them from the excision complex. Our results show clearly that excision is a complex process that involves several proteins and a cis-acting region (XRS) which includes the oriT. We suggest that this complex excision machinery may be necessary to allow NBU1 to coordinate nicking at the ends during excision and nicking at the oriT during conjugal transfer, to prevent premature nicking at the oriT before NBU1 has excised and circularized.     

Evidence that the clindamycin-erythromycin resistance gene of Bacteroides plasmid pBF4 is on a transposable element.

We constructed a shuttle vector, pE5-2, which can replicate in both Bacteroides spp. and Escherichia coli. pE5-2 contains a cryptic Bacteroides plasmid (pB8-51), a 3.8-kilobase (kb) EcoRI-D fragment from the 41-kb Bacteroides fragilis plasmid pBF4, and RSF1010, an IncQ E. coli plasmid. pE5-2 was mobilized by R751, an IncP E. coli plasmid, between E. coli strains with a frequency of 5 X 10(-2) to 3.8 X 10(-1) transconjugants per recipient. R751 also mobilized pE5-2 from E. coli donors to Bacteroides uniformis 0061RT and Bacteroides thetaiotaomicron 5482 with a frequency of 0.9 X 10(-6) to 2.5 X 10(-6). The Bacteroides transconjugants contained only pE5-2 and were resistant to clindamycin and erythromycin. Thus, the gene for clindamycin and erythromycin resistance must be located within the Eco RI-D fragment of BF4. A second recombinant plasmid, pSS-2, which contained 33 kb of pBF4 (including the EcoRI-D fragment and contiguous regions) could also be mobilized by R751 between E. coli strains. In some transconjugants, a 5.5-kb (+/- 0.3 kb) segment of the pBF4 portion of pSS2 was inserted into one of several sites on R751. In some other transconjugants this same 5.5-kb segment was integrated into the E. coli chromosome. This segment could transfer a second time onto R751. Transfer was RecA independent. The transferred segment included the entire EcoRI-D fragment, and thus the clindamycin-erythromycin resistance determinant, from pBF4.     

Construction and characterization of a Bacteroides thetaiotaomicron recA mutant: transfer of Bacteroides integrated conjugative elements is RecA independent.

We report the construction and analysis of a Bacteroides thetaiotaomicron recA disruption mutant and an investigation of whether RecA is required for excision and integration of Bacteroides mobile DNA elements. The recA mutant was deficient in homologous recombination and was more sensitive than the wild-type strain to DNA-damaging agents. The recA mutant was also more sensitive to oxygen than the wild type, indicating that repair of DNA contributes to the aerotolerance of B. thetaiotaomicron. Many Bacteroides clinical isolates carry self-transmissible chromosomal elements known as conjugative transposons. These conjugative transposons can also excise and mobilize in trans a family of unlinked integrated elements called nonreplicating Bacteroides units (NBUs). The results of a previous study had raised the possibility that RecA plays a role in excision of Bacteroides conjugative transposons, but this hypothesis could not be tested in Bacteroides spp. because no RecA-deficient Bacteroides strain was available. We report here that the excision and integration of the Bacteroides conjugative transposons, as well as NBU1 and Tn4351, were unaffected by the absence of RecA activity.     

Use of targeted insertional mutagenesis to determine whether chondroitin lyase II is essential for chondroitin sulfate utilization by Bacteroides thetaiotaomicron.

Bacteroides thetaiotaomicron produces two inducible chondroitin lyases (I and II) when it is grown on chondroitin sulfate. Both enzymes have very similar biochemical properties. To determine whether both enzymes are required for growth on chondroitin sulfate, we constructed a Bacteroides suicide vector, pE3-1, and used it to create an insertional mutation that interrupts the chondroitin lyase II gene of Bacteroides thetaiotaomicron. pE3-1 contains a 4.4-kilobase cryptic B. eggerthii plasmid (pB8-51), the Escherichia coli cloning vector pBR328, and the EcoRI D fragment from the conjugative B. fragilis plasmid pBF4. A 0.8-kilobase fragment from the center of the B. thetaiotaomicron chondroitin lyase II gene was inserted in pE3-1 to create pEG817. Although, pEG817 is stably maintained in E. coli and can be mobilized into B. thetaiotaomicron by the IncP plasmid R751, pEG817 is not maintained as a plasmid in Bacteroides spp. When pEG817 was mobilized into B. thetaiotaomicron, with selection for a drug marker on pEG817, transconjugants were obtained which had pEG817 inserted into the chondroitin lyase II gene. Western blot analysis was used to confirm that intact chondroitin lyase II is not produced in the mutant. The mutant was able to utilize chondroitin sulfate as a sole source of carbon, although no active chondroitin lyase II was produced. Thus chondroitin lyase I alone appears to be sufficient for growth on chondroitin sulfate. The mutant also had some minor changes in its outer membrane protein profile. However, there was no evidence that any of the major chondroitin sulfate-associated polypeptides in the outer membrane were affected by the insertion in the chondroitin lyase II gene.     

Factors required in vitro for excision of the Bacteroides conjugative transposon, CTnDOT

Four genes have been found to be essential for excision of the Bacteroides conjugative transposon CTnDOT in vivo: intDOT, orf2c, orf2d, and exc. The intDOT gene encodes an integrase that is essential for integration and excision. The function of the other genes is still uncertain. Previously, we developed an in vitro system for the integration reaction. We have now developed an in vitro system for excision. In this system, the left and right junctions of CTnDOT, attL, and attR, are provided on separate plasmids. The excision reaction produced a cointegrate which contained the attDOT (the joined ends of CTnDOT) and attB (the chromosomal target site). Cointegrate formation was observed after electroporation of Escherichia coli with the assay mixture and was also detected directly in the assay mixture by Southern hybridization. The highest reaction frequencies (10(-3)) were obtained with a mixture that contained purified IntDOT and a cell extract from Bacteroides thetaiotaomicron 4001, which contained the excision region of CTnDOT carried on a plasmid. An unexpected finding was that the addition of purified Exc, which is essential for excision in vivo, was not required for excision in vitro, nor did it increase the frequency of cointegrate formation.     

Tetracycline-dependent appearance of plasmidlike forms in Bacteroides uniformis 0061 mediated by conjugal Bacteroides tetracycline resistance elements.

Some human colonic Bacteroides strains carry conjugal tetracycline resistance (Tcr) elements, which are thought to be chromosomal. We have found that some of these Tcr elements can mediate the appearance of plasmidlike forms in Bacteroides uniformis 0061. When B. uniformis 0061, containing a conjugal Tcr element designated Tcr ERL, was grown in medium containing tetracycline (1 microgram/ml), two circular DNA forms were found in the alkaline plasmid preparations: NBU1 (10.3 +/- 0.5 kilobases) and NBU2 (11.5 +/- 0.5 kilobases). Restriction analysis of NBU1 and NBU2 showed that they were not identical, although Southern blot analysis indicated that they did contain some region(s) of homology. Results of Southern blot analysis also demonstrated that both NBU1 and NBU2 were normally integrated in the chromosome of B. uniformis or in some undetected large plasmid. Although we were unable to determine the exact structure and location of the integrated forms of NBU1 and NBU2 in B. uniformis, they appear to be in close proximity to each other. Neither NBU1 or NBU2 could be detected as a plasmidlike form in cells exposed to UV light, thymidine starvation, mitomycin C, or autoclaved chlortetracycline (50 micrograms/ml). Four conjugal Tcr elements other than the Tcr ERL element were able to mediate the appearance of NBU1 alone, and two Tcr elements did not mediate the excision of either NBU1 or NBU2. Three strains from different Bacteroides species contained some DNA sequences which had homology to NBU1 and NBU2.     

A cryptic 65-kilobase-pair transposonlike element isolated from Bacteroides uniformis has homology with Bacteroides conjugal tetracycline resistance elements.

A 65-kilobase-pair element, XBU4422, which has some transposonlike characteristics but carries no known antibiotic resistance genes, has been isolated from Bacteroides uniformis 0061. XBU4422 was trapped on Bacteroides-Escherichia coli shuttle vectors during experiments in which one of the conjugal Bacteroides tetracycline resistance (Tcr) elements was being used to mobilize the shuttle vectors to Bacteroides recipients. Results of Southern hybridization experiments showed that XBU4422 is normally integrated in the B. uniformis 0061 chromosome and is found only in some strains. Insertion of XBU4422 in the shuttle vectors was site specific and orientation specific. Nonmobilizable vectors that had acquired XBU4422 became transmissible and could be transferred to Bacteroides or E. coli recipients. In B. uniformis transconjugants, the XBU4422 insertion in the vectors was usually intact, but XBU4422 was always lost in matings with E. coli, Bacteroides thetaiotaomicron, or B. ovatus. The loss of XBU4422 did not visibly alter the vector; in the case of E. coli, the loss of the insertion appeared to be RecA dependent. Although XBU4422 carried no antibiotic resistances, it shared regions of homology with six conjugal Bacteroides Tcr elements; this homology was strongest with the ends of XBU4422. Using a strain of B. thetaiotaomicron that contains no XBU4422-hybridizing sequences, we showed that the ends of XBU4422 were probably reacting with the ends of the Tcr elements. These results provide the first direct evidence that the Tcr elements, like XBU4422, are integrated in the chromosome and that insertion of the least some Tcr elements, such as TcrEmr DOT, is relatively site specific.     

The Salmonella genomic island 1 is an integrative mobilizable element

Salmonella genomic island 1 (SGI1) is a genomic island containing an antibiotic resistance gene cluster identified in several Salmonella enterica serovars. The SGI1 antibiotic resistance gene cluster, which is a complex class 1 integron, confers the common multidrug resistance phenotype of epidemic S. enterica Typhimurium DT104. The SGI1 occurrence in S. enterica serovars Typhimurium, Agona, Paratyphi B, Albany, Meleagridis and Newport indicates the horizontal transfer potential of SGI1. Here, we report that SGI1 could be conjugally transferred from S. enterica donor strains to non-SGI1 S. enterica and Escherichia coli recipient strains where it integrated into the recipient chromosome in a site-specific manner. First, an extrachromosomal circular form of SGI1 was identified by PCR which forms through a specific recombination of the left and right ends of the integrated SGI1. Chromosomal excision of SGI1 was found to require SGI1-encoded integrase which presents similarities to the lambdoid integrase family. Second, the conjugal transfer of SGI1 required the presence of a helper plasmid. The conjugative IncC plasmid R55 could thus mobilize in trans SGI1 which was transferred from the donor to the recipient strains. By this way, the conjugal transfer of SGI1 occurred at a frequency of 10(-5)-10(-6) transconjugants per donor. No transconjugants could be obtained for the SGI1 donor lacking the int integrase gene. Third, chromosomal integration of SGI1 occurred via a site-specific recombination between a 18 bp sequence found in the circular form of SGI1 and a similar 18 bp sequence at the 3' end of thdF gene in the S. enterica and E. coli chromosome. SGI1 appeared to be transmissible only in the presence of additional conjugative functions provided in trans. SGI1 can thus be classified within the group of integrative mobilizable elements (IMEs).     

Conjugal transfer of a shuttle vector from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4.

Prevotella ruminicola (formerly Bacteroides ruminicola) is an anaerobic, gram-negative, polysaccharide-degrading bacterium which is found in the rumina of cattle. Since P. ruminicola is thought to make an important contribution to digestion of plant material in rumina, the ability to alter this strain genetically might help improve the efficiency of rumen fermentation. However, previously there has been no way to introduce foreign DNA into P. ruminicola strains. In this study we transferred a shuttle vector, pRDB5, from the colonic species Bacteroides uniformis to P. ruminicola B(1)4. The transfer frequency was 10(-6) to 10(-7) per recipient. pRDB5 contains sequences from pBR328, a cryptic colonic Bacteroides plasmid pB8-51, and a colonic Bacteroides tetracycline resistance (Tcr) gene. pRDB5 was mobilized out of B. uniformis by a self-transmissible Bacteroides chromosomal element designated Tcr Emr 12256. pRDB5 replicated in Escherichia coli as well as in Bacteroides spp. and was also mobilized from E. coli to B. uniformis by using IncP plasmid R751. However, direct transfer from E. coli to P. ruminicola B(1)4 was not detected. Thus, to introduce cloned DNA into P. ruminicola B(1)4, it was necessary first to mobilize the plasmid from E. coli to B. uniformis and then to mobilize the plasmid from B. uniformis to P. ruminicola B(1)4.     

Conjugal transfer of a shuttle vector from the human colonic anaerobe Bacteroides uniformis to the ruminal anaerobe Prevotella (Bacteroides) ruminicola B(1)4.

Prevotella ruminicola (formerly Bacteroides ruminicola) is an anaerobic, gram-negative, polysaccharide-degrading bacterium which is found in the rumina of cattle. Since P. ruminicola is thought to make an important contribution to digestion of plant material in rumina, the ability to alter this strain genetically might help improve the efficiency of rumen fermentation. However, previously there has been no way to introduce foreign DNA into P. ruminicola strains. In this study we transferred a shuttle vector, pRDB5, from the colonic species Bacteroides uniformis to P. ruminicola B(1)4. The transfer frequency was 10(-6) to 10(-7) per recipient. pRDB5 contains sequences from pBR328, a cryptic colonic Bacteroides plasmid pB8-51, and a colonic Bacteroides tetracycline resistance (Tcr) gene. pRDB5 was mobilized out of B. uniformis by a self-transmissible Bacteroides chromosomal element designated Tcr Emr 12256. pRDB5 replicated in Escherichia coli as well as in Bacteroides spp. and was also mobilized from E. coli to B. uniformis by using IncP plasmid R751. However, direct transfer from E. coli to P. ruminicola B(1)4 was not detected. Thus, to introduce cloned DNA into P. ruminicola B(1)4, it was necessary first to mobilize the plasmid from E. coli to B. uniformis and then to mobilize the plasmid from B. uniformis to P. ruminicola B(1)4.     

Evidence that the Bacteroides thetaiotaomicron chondroitin lyase II gene is adjacent to the chondro-4-sulfatase gene and may be part of the same operon.

The chondroitin lyase II gene from Bacteroides thetaiotaomicron has previously been cloned in Escherichia coli on a 7.8-kilobase (kb) fragment (pA818). In E. coli, the chondroitin lyase II gene appeared to be expressed from a promoter that was about 0.5 kb from the beginning of the gene. However, when a subcloned 5-kb fragment from pA818 which contained the chondroitin lyase II gene and the promoter from which the gene is expressed in E. coli was introduced into B. thetaiotaomicron on a multicopy plasmid (pEG800), the chondroitin lyase specific activity of B. thetaiotaomicron was not altered. Further evidence that the promoter that is recognized in E. coli may not be the promoter from which the chondroitin lyase II gene is transcribed in B. thetaiotaomicron was obtained by making an insertion in the B. thetaiotaomicron chromosome at a point which is 1 kb upstream from the chondroitin lyase II gene. This insertion stopped synthesis of the chondroitin lyase II gene product, as would be predicted if the gene was part of an operon and was transcribed in B. thetaiotaomicron from a promoter that was at least 1 kb upstream from the chondroitin lyase II gene. A region of pA818 which was adjacent to the chondroitin lyase II gene and which included the region used to make the insertional mutation was found to code for chondro-4-sulfatase, an enzyme that breaks down one of the products of the chondroitin lyase reaction. The upstream insertion mutant of B. thetaiotaomicron which stopped synthesis of chondroitin lyase II had no detectable chondro-4-sulfatase activity. This mutant was still able to grow on chondroitin sulfate, although the rate of growth was slower than that of the wild type.     

Development of an in vitro integration assay for the Bacteroides conjugative transposon CTnDOT

Integrated self-transmissible elements called conjugative transposons (CTns) are responsible for the transfer of antibiotic resistance genes in many different species of bacteria. One of the best characterized of these newly recognized elements is the Bacteroides CTn, CTnDOT. CTnDOT is thought to have a circular transfer intermediate that transfers to and integrates into the genome of the recipient cell. Previous investigations of the mechanism of CTnDOT integration have been hindered by the lack of an in vitro system for checking this model of integration and determining whether the CTnDOT integrase alone was sufficient to catalyze the integration reaction or whether host factors might be involved. We report here the development of an in vitro system in which a plasmid containing the joined ends of CTnDOT integrates into a plasmid carrying a CTnDOT target site. To develop this in vitro system, a His-tagged version of the integrase gene of CTnDOT was cloned and shown to be active in vivo. The protein produced by this construct was partially purified and then added to a reaction mixture that contained the joined ends of the circular form of CTnDOT and a plasmid carrying one of the CTnDOT target sites. Integration was demonstrated by using a fairly simple mixture of components, but integration was stimulated by a Bacteroides extract or by purified Escherichia coli integration host factor. The results of this study demonstrate both that the circular form of CTnDOT is the form that integrates into the target site and that host factors are involved in the integration process.     

The bacteroides NBU1 integrase performs a homology-independent strand exchange to form a holliday junction intermediate

The Bacteroides mobilizable transposon NBU1 uses an integrase (IntN1) that is a tyrosine recombinase for its integration and excision from the host chromosome. Previously we showed that IntN1 makes 7-bp staggered cuts within the NBU1 att sites, and certain mismatches within the crossover region of the attN1 site (G(-2)C attN1) or the chromosomal target site (C(-3)G attBT1-1) enhanced the in vivo integration efficiency. Here we describe an in vitro integration system for NBU1. We used nicked substrates and a Holliday junction trapping peptide to show that NBU1 integration proceeds via formation of a Holliday junction intermediate that is formed by exchange of bottom strands. Some mismatches next to the first strand exchange site (in reactions with C(-3)G attBT1-1 or G(-2)C attN1 with their wild-type partner site) not only allowed formation of the Holliday junction intermediate but also increased the rate of recombinant formation. The second strand exchange appears to be homology-dependent. IntN1 is the only tyrosine recombinase known to catalyze a reaction that is more efficient in the presence of mismatches and where the first strand exchange is homology-independent. The possible mechanisms by which the mismatches stimulate recombination are discussed.     

Regions in Bacteroides plasmids pBFTM10 and pB8-51 that allow Escherichia coli-Bacteroides shuttle vectors to be mobilized by IncP plasmids and by a conjugative Bacteroides tetracycline resistance element.

Bacteroides-Escherichia coli shuttle vectors containing a nonmobilizable pBR322 derivative and either pBFTM10 (pDP1, pCG30) or pB8-51 (pEG920) were mobilized by IncP plasmid R751 or pRK231 (an ampicillin-sensitive derivative of RK2) between E. coli strains and from E. coli to Bacteroides recipients. IncI alpha R64 drd-ll transferred these vectors 1,000 times less efficiently than did the IncP plasmids. pDP1, pCG30, and pEG920 could be mobilized from B. uniformis donors to both E. coli and Bacteroides recipients by a conjugative Bacteroides Tcr (Tcr ERL) element which was originally found in a clinical Bacteroides fragilis strain (B. fragilis ERL). However, the shuttle vector pE5-2, which contains pB8-51 cloned in a restriction site that prevents its mobilization by IncP or IncI alpha plasmids, also was not mobilized at detectable frequencies from Bacteroides donors by the Tcr ERL element. The mobilization frequencies of pCG30, pDP1, and pEG920 by the Tcr ERL element in B. uniformis donors to E. coli recipients was about the same as those to isogenic B. uniformis recipients. Transfer of the shuttle vectors from B. uniformis donors to E. coli occurred at the same frequencies when the matings were done aerobically or anaerobically. Growth of the B. uniformis donors in tetracycline (1 microgram/ml) prior to conjugation increased the mobilization frequencies of the vectors to both E. coli and Bacteroides recipients 50 to 100 times.     

Characterization of the integrase of NBU1, a Bacteroides mobilizable transposon

NBU1 is a Bacteroides mobilizable transposon (MTn) that is integrated within the host chromosome and requires CTnDOT functions for its excision and transfer into a new host. The NBU1 integrase IntN1 has been classified as a tyrosine recombinase based on the presence of conserved residues. We created alanine mutants of the residues R291, K314, H393, R396, H419 and the conserved substitution Y429F and tested them for integration efficiency. The results suggest that these residues in IntN1 are important for integration, and Y429 could be the catalytic nucleophile. We employed suicide substrates and partially purified IntN1 to determine the positions of IntN1 cleavage within the 14 bp common core region that is identical in both NBU1 att sites. We show that IntN1 makes 7 bp staggered cuts on the top and bottom strands. From previous mutational analysis of the att sites, we show that two specific mutations near the site of bottom strand cleavage within this 7 bp region increased integration, and mutations of the two bases near top strand cleavage site had no effect on integration. These results indicate that IntN1 lacks the strict requirement for homology between the recombining sites seen with other tyrosine recombinases. We also show that phosphorothioate substitutions at the cleavage site and 1 bp downstream inhibited cleavage by IntN1. This differs from other studied tyrosine recombinases where inhibition occurs by substitutions at the cleavage site only.     

The Shiga-toxin VT2-encoding bacteriophage varphi297 integrates at a distinct position in the Escherichia coli genome

The plaque-forming VT2-encoding lambdoid bacteriophage varphi297 was isolated from a Belgian clinical Escherichia coli O157:H7 isolate. PCR walking, starting from the int gene of phage varphi297, demonstrated that the varphi297 prophage integrated in the yecE gene of a lysogenic E. coli K12 strain. This integration site, in E. coli K12 and in the original clinical O157:H7 isolate, was confirmed by PCR using primers flanking this site. The excisionase protein of phage varphi297 is identical to the excisionase of VT1-encoding phage VT1-Sakai, while the integrases, which are 82% identical, show significant sequence divergence in the central and C-terminal region. This can explain the different integration sites of both prophages. The activity of the integrase was proven by its ability to mediate the integration of a suicide plasmid, carrying the attachment site of varphi297, at the appropriate position in the E. coli chromosome.