Transposition genes of the Bacteroides mobilizable transposon Tn4555: role of a novel targeting gene

Conjugative transposons have been identified in several bacterial species, most notably the Gram-positive Enterococci and the Gram-negative Bacteroides. In Bacteroides species, these elements encode a complete conjugative machinery, which mediates their own intercellular transfer, and they can mobilize in trans co-resident elements. One such mobilizable element is the antibiotic resistance transposon, Tn4555, which was previously found to integrate into a specific genome target site via a site-specific recombination mechanism. In this work, we demonstrate that three Tn4555 genes were involved in integration of the element. These were int encoding a lambda-type integrase, which was absolutely required for integration of the transposon, and two accessory genes, which increased the frequency of integration. Interestingly, one of these accessory gene products, TnpA, directed the insertion of Tn4555 into the genome target site; in the absence of tnpA, the insertion pattern was essentially random. This is the first example of a site-specific recombinase that uses a specific targeting protein.     

The Bacteroides mobilizable transposon Tn4555 integrates by a site-specific recombination mechanism similar to that of the gram-positive bacterial element Tn916.

The Bacteroides mobilizable transposon Tn4555 is a 12.2-kb molecule that encodes resistance to cefoxitin. Conjugal transposition is hypothesized to occur via a circular intermediate and is stimulated by coresident tetracycline resistance elements and low levels of tetracycline. In this work, the ends of the transposon were identified and found to consist of 12-bp imperfect inverted repeats, with an extra base at one end. In the circular form, the ends were separated by a 6-bp "coupling sequence" which was associated with either the left or the right transposon terminus when the transposon was inserted into the chromosome. Tn4555 does not duplicate its target site upon insertion. Using a conjugation-based transposition assay, we showed that the coupling sequence originated from 6 bases of genomic DNA flanking either side of the transposon prior to excision. Tn4555 preferentially transposed into a 589-bp genomic locus containing a 207-bp direct repeat. Integration occurred before or after the repeated sequence, with one integration site between the two repeats. These observations are consistent with a transposition model based on site-specific recombination. In the bacteriophage lambda model for site-specific recombination, the bacteriophage recombines with the Escherichia coli chromosome via a 7-bp "crossover" region. We propose that the coupling sequence of Tn4555 is analogous in function to the crossover region of lambda but that unlike the situation in lambda, recombination occurs between regions of nonhomologous DNA. This ability to recombine into divergent target sites is also a feature of the gram-positive bacterial transposon Tn916.     

Analysis of the zinc finger domain of TnpA, a DNA targeting protein encoded by mobilizable transposon Tn4555

The mobilizable transposon Tn4555, found in Bacteroides spp., is an important antibiotic resistance element encoding a broad spectrum beta-lactamase. Tn4555 is mobilized by conjugative transposons such as CTn341 which can transfer the transposon to a wide range of bacterial species where it integrates into preferred sites on the host chromosome. Selection of the preferred target sites is mediated by a DNA-binding protein TnpA which has a prominent zinc finger motif at the N-terminus of the protein. In this report the zinc finger motif was disrupted by site directed mutagenesis in which two cysteine residues were changed to serine residues. Elemental analysis indicated that the wild-type protein but not the mutated protein was able to coordinate zinc at a molar ration of 1/1. DNA binding electrophoretic mobility shift assays showed that the ability to bind the target site DNA was not significantly affected by the mutation but there was about a 50% decrease in the ability to bind single stranded DNA. Consistent with these results, electrophoretic mobility shift assays incorporating zinc chelators did not have a significant on affect the binding of DNA target. In vivo, the zinc finger mutation completely prevented transposition/integration as measured in a conjugation assay. This was in contrast to results in which a TnpA knockout was still able to insert into host genomes but there was no preferred target site selection. The phenotype of the zinc finger mutation was not effectively rescued by providing wild-type TnpA in trans. Taken together these results indicated that the zinc finger is not required for DNA binding activity of TnpA but that it does have an important role in transposition and it may mediate protein/protein interactions with integrase or other Tn4555 proteins to facilitate insertion into the preferred sites.     

Transposition of Tn4551 in Bacteroides fragilis: identification and properties of a new transposon from Bacteroides spp.

Tn4551, a clindamycin resistance (Ccr) transposon from the R plasmid pBI136, was cloned onto an Escherichia coli-Bacteroides shuttle vector which could replicate normally in E. coli but was maintained unstably in Bacteroides fragilis. To aid in cloning and to ensure maintenance of Tn4551 in E. coli, a kanamycin resistance determinant (Kmr) was inserted in the transposon. The transposon-bearing shuttle vector pFD197 was transformed into B. fragilis 638, and putative insertions of Tn4551::Kmr were identified by screening for resistance to clindamycin and plasmid content. Southern hybridization analyses were used to verify integration of the transposon in the B. fragilis chromosome, and the frequency of insertion was estimated at 7.8 X 10(-5) events per generation. In 57% of the isolates tested a second integration event also occurred. This second insertion apparently involved just a single copy of the 1.2-kilobase repeat sequence which flanks the transposon. In addition, Tn4551::Kmr appeared to function as a transposon in E. coli. Evidence for this was obtained by the isolation of transposon insertions into the bacteriophage P1 genome. Finally, the transposon vector, pFD197, could be mobilized to other B. fragilis strains in which transposition was detected. Mobilization from the strain 638 background was via a conjugation like process, but occurred in the absence of known conjugative elements or other detectable plasmids. This result suggested the presence of a host-encoded transfer system in this B. fragilis strain.     

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.     

Identification of transposition proteins encoded by the bacterial transposon Tn7.

The bacterial transposon, Tn7, encodes an elaborate array of transposition genes, tnsABCDE. We report here the direct identification of the TnsA, TnsB, TnsC and TnsD polypeptides by immunoblotting. Our results demonstrate that the complexity of the protein information devoted to Tn7 transposition is considerable: the aggregate molecular size of the five Tns polypeptides is about 300 kDa. We also report the sequence of the tnsA gene and of the 5' ends of tnsB and tnsD. This analysis reveals that all five tns genes are oriented in the same direction within Tn7.     

Tn4399, a conjugal mobilizing transposon of Bacteroides fragilis.

Conjugal transposons play an important role in the dissemination of antibiotic resistance determinants in the streptococci and have been postulated to exist in Bacteroides fragilis. To investigate the presence of conjugal transposons in B. fragilis, we employed a Tra- derivative of the transfer factor pBFTM10 contained in the chimeric plasmid pGAT400 delta BglII. We attempted to restore transferability to this plasmid from a series of transconjugants generated by crossing B. fragilis TMP230 containing the TET transfer factor with B. fragilis TM4000, a standard recipient. Transconjugant TM4.2321 transferred pGAT400 delta BglII to Escherichia coli HB101 at almost the same frequency as did the Tra+ parental plasmid, pGAT400. Analysis of the transferred plasmids revealed the presence of 9.6 kilobases of additional DNA in every case but at different positions in independent isolates. The presence of this DNA, designated Tn4399, allowed the pGAT400 delta BglII derivatives to retransfer from the TM4000 background to B. fragilis or E. coli recipients. DNA hybridization studies demonstrated the presence of one copy of Tn4399 in TMP230 and three copies at new sites in TM4.2321. Tn4399 is a new B. fragilis transposon with unique transfer properties that may play a role in the dissemination of drug resistance genes. It differs from previously described conjugal transposons by its ability to mobilize nonconjugal plasmids in cis.     

Bacteroides fragilis transfer factor Tn5520: the smallest bacterial mobilizable transposon containing single integrase and mobilization genes that function in Escherichia coli

Many bacterial genera, including Bacteroides spp., harbor mobilizable transposons, a class of transfer factors that carry genes for conjugal DNA transfer and, in some cases, antibiotic resistance. Mobilizable transposons are capable of inserting into and mobilizing other, nontransferable plasmids and are implicated in the dissemination of antibiotic resistance. This paper presents the isolation and characterization of Tn5520, a new mobilizable transposon from Bacteroides fragilis LV23. At 4,692 bp, it is the smallest mobilizable transposon reported from any bacterial genus. Tn5520 was captured from B. fragilis LV23 by using the transfer-deficient shuttle vector pGAT400DeltaBglII. The termini of Tn5520 contain a 22-bp imperfect inverted repeat, and transposition does not result in a target site repeat. Tn5520 also demonstrates insertion site sequence preferences characterized by A-T-rich nucleotide sequences. Tn5520 has been sequenced in its entirety, and two large open reading frames whose predicted protein products exhibit strong sequence similarity to recombinase-integrase enzymes and mobilization proteins, respectively, have been identified. The transfer, mobilization, and transposition properties of Tn5520 have been studied, revealing that Tn5520 mobilizes plasmids in both B. fragilis and Escherichia coli at high frequency and also transposes in E. coli.     

Tn4400, a compound transposon isolated from Bacteroides fragilis, functions in Escherichia coli.

Transfer factor pBFTM10, isolated from the obligate anaerobic bacterium Bacteroides fragilis, carries a clindamycin resistance determinant which we have suggested is part of a transposable element. DNA homologous to this determinant is found in many Clnr Bacteroides isolates, either in the chromosome or on plasmids. We have now established that Ccr resides on a transposon, Tn4400. In addition to the Ccr determinant that functions under anaerobic conditions in B. fragilis, Tn4400 also carries a determinant for tetracycline resistance (Tcr) which only functions in Escherichia coli under aerobic conditions. The presence of Tn4400 on pBFTM10 does not confer tetracycline resistance on B. fragilis cells containing it. DNA from pBFTM10 was cloned in E. coli, with pDG5 as the cloning vector, to form pGAT500. Using a mobilization assay involving pGAT500 and an F factor derivative, pOX38, we determined that a 5.6-kilobase region of pBFTM10 DNA was capable of mediating replicon fusion and transposition. Most of the mobilization products resulted from inverse transposition reactions, while some were the result of true cointegrate formation. Analysis of the cointegrate molecules showed that three were formed by the action of one of the ends of Tn4400 (IS4400), and one was formed by the action of the whole element (Tn4400). The cointegrate molecule carrying intact copies of Tn4400 at the junction of the two plasmids could resolve to yield an unaltered donor plasmid (pGAT500) and a conjugal plasmid containing a copy of Tn4400 or a copy of one insertion sequence element (pOX38::Tn4400 or pOX38::IS4400). Thus, Tn4400 is a compound transposon containing active insertion sequence elements as directly repeated sequences at its ends.     

Bacteroides fragilis mobilizable transposon Tn5520 requires a 71 base pair origin of transfer sequence and a single mobilization protein for relaxosome formation during conjugation

Tn5520 is the smallest known bacterial mobilizable transposon and was isolated from an antibiotic resistant Bacteroides fragilis clinical isolate. When a conjugation apparatus is provided in trans, Tn5520 is mobilized (transferred) efficiently within, and from, both Bacteroides spp. and Escherichia coli. Only two genes are present on Tn5520; one encodes an integrase, and the other a multifunctional mobilization (Mob) protein BmpH. BmpH is essential for Tn5520 mobility. The focus of this study was to identify the Tn5520 origin of conjugative transfer (oriT) and to study BmpH-oriT binding. We delimited the functional Tn5520 oriT to a 71 bp sequence upstream of the bmpH gene. A plasmid vector harbouring this minimal 71 bp oriT was mobilized at the same frequency as that of intact Tn5520. The minimal oriT contains one 17 bp inverted repeat (IR) sequence. We constructed and tested multiple IR mutants and showed that the IR was essential in its entirety for mobilization. A nick site sequence (5'-GCTAC-3') was also identified within the minimal oriT; this sequence resembled nick sites found in plasmids of Gram positive origin. We further showed that mutation of a highly conserved GC dinucleotide in the nick site sequence completely abolished mobilization. We also purified BmpH and showed that it specifically bound a Tn5520 oriT fragment in electrophoretic mobility shift assays. We also identified non-nick site sequences within the minimal oriT that were essential for mobilization. We hypothesize that transposon-based single Mob protein systems may contribute to efficient gene dissemination from Bacteroides spp., because fewer DNA processing proteins are required for relaxosome formation.     

Tn4451 from Clostridium perfringens is a mobilizable transposon that encodes the functional Mob protein, TnpZ

The 6.3 kb Clostridium perfringens transposon Tn 4451 encodes a 50 kDa protein, TnpZ, which has amino acid sequence similarity to a group of plasmid mobilization and recombination proteins that comprise the Mob/Pre family. Members of this family interact with an upstream palindromic sequence called an RS_A site, and an RS_A-like sequence has been identified upstream of the tnpZ gene. In Escherichia coli , in the presence of a chromosomally integrated derivative of the broad-host-range IncP plasmid, RP4, TnpZ was able to promote plasmid mobilization in cis and was able to function in trans to enable the mobilization of a co-resident plasmid carrying an RS_A site. It was also able to mediate the conjugative transfer of plasmids from E. coli to C. perfringens . Site-directed mutagenesis of two bases within the RS_A site resulted in a significant reduction in mobilization frequency, demonstrating that the RS_A site is required for efficient plasmid mobilization. TnpZ is the only Mob/Pre protein known to be associated with a transposable genetic element, and Tn 4451 is the first mobilizable but non-self-transmissible transposon to be identified from a Gram-positive bacterium.     

Kinetic and structural analysis of a cleaved donor intermediate and a strand transfer intermediate in Tn10 transposition.

Tn10 transposes by a nonreplicative "cut and paste" mechanism. We describe here two protein-DNA complexes that are reaction intermediates in the Tn10 transposition process: a cleaved donor complex whose DNA component consists of transposon sequences cleanly excised from flanking donor DNA, and a strand transfer complex whose DNA component contains transposon termini specifically joined to a target site. The kinetic behavior of the first species suggests that it is an early intermediate in the transposition reaction. These two Tn10 complexes are closely analogous to complexes identified in the pathway for replicative "cointegrate" formation by bacteriophage Mu and thus represent intermediates that may be common to both nonreplicative and replicative transposition. These and other results suggest that the Tn10 and Mu reactions are fundamentally very similar despite their very different biological outcomes. The critical difference between the two reactions is the fate of the DNA strand that is not joined to target DNA.     

Tn5099, a xylE promoter probe transposon for Streptomyces spp.

Tn5099, a promoter probe transposon for Streptomyces spp., was constructed by inserting a promoterless xylE gene and a hygromycin resistance gene into IS493. Tn5099 transposed into different sites in the Streptomyces griseofuscus genome, and the xylE reporter gene was expressed in some of the transposition mutants. Strains containing Tn5099 insertions that gave regulated expression of the xylE gene were identified.     

Activation of a cryptic streptomycin-resistance gene in the Bacteroides erm transposon, Tn4551

Bacteroides compound transposons encoding erm resistance are highly homologous but previous studies have shown some divergence of Tn4551. Results presented here describe a novel Tn4551 streptomycin-resistance gene, aadS, that was phenotypically silent in wild-type Bacteroides. However, aadS expression could be activated by a trans-acting chromosomal mutation. The aadS-encoded peptide displayed significant homology to Gram-positive streptomycin-dependent adenyltransferases, and enzymatic analysis confirmed the production of this activity. Examination of the nucleotide sequence showed that 200 bp upstream of aadS, the DNA base composition changed abruptly from 31% G+C to 48% G+C. These two regions were demarcated by a DNA sequence with homology to the recombination hot spots reported for Tn21 and the Bacteroides ermFU gene and to sequences at the ends of the chromosomal Bacteroides conjugal element, XBU4422.     

Analysis of chromosomal insertion sites for Bacteroides Tn4555 and the role of TnpA

Tn4555, a mobilizable transposon carrying cefoxitin resistance, is directed to a preferred target site in the Bacteroides fragilis chromosome by a transposon-encoded targeting protein TnpA. In an effort to characterize target site selection for Tn4555, the existence of preferred target sites in other species of Bacteroides and in Escherichia coli was examined. For these analyses a Tn4555 mini element, pFD660, was transferred from E. coli donors to Bacteroides thetaiotaomicron or Bacteroides ovatus recipients and the resulting sites of insertion analyzed. A similar construct, pFD794 was used to determine insertion sites in E. coli, and preferred sites were found in all bacteria tested. Also the ability of TnpA to bind to various targets was examined in mobility shift assays. Although TnpA bound to all tested sequences, it displayed higher affinity for the target sites. The binding characteristics of TnpA and the lack of significant base sequence homology between targets suggested that secondary structure of the sites was important for TnpA binding. Circular permutation tests supported the idea that TnpA targets bent DNA.     

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.     

NBU1, a mobilizable site-specific integrated element from Bacteroides spp., can integrate nonspecifically in Escherichia coli.

NBU1 is an integrated Bacteroides element that can he mobilized from Bacteroides donors to Bacteroides recipients. Previous studies have shown that a plasmid carrying the internal mobilization region of NBU1 could be transferred by conjugation from Bacteroides thetaiotaomicron to Escherichia coli. In this report, we show that NBU1 can integrate in E. coli. Whereas integration of NBU1 in B. thetaiotaomicron is site specific, integration of NBU1 in E. coli was relatively random, and the insertion frequency of NBU1 into the E. coli chromosome was 100 to 1,000 times lower than the frequency of integration in B. thetaiotaomicron. The frequency of NBU1 integration in E. coli could be increased about 10- to 70-fold, to a value close to that seen with B. thetaiotaomicron, if the primary integration site from B. thetaiotaomicron, BT1-1, was provided on a plasmid in the E. coli recipient or the NBU1 integrase gene, intN1, was provided on a high-copy-number plasmid to increase the amount of integrase available in the recipient. When the primary integration site was available in the recipient, NBU1 integrated site specifically in E. coli. Our results show that NBUs have a very broad host range and are capable of moving from Bacteroides spp. to distantly related species such as E. coli. Moreover, sequence analysis of NBU1 integration sites provided by integration events in E. coli has helped to identify some regions of the NBU1 attachment site that may play a role in the integration process.     

Tn7 transposition in vitro proceeds through an excised transposon intermediate generated by staggered breaks in DNA.

We have developed a cell-free system in which the bacterial transposon Tn7 inserts at high frequency into its preferred target site in the Escherichia coli chromosome, attTn7; Tn7 transposition in vitro requires ATP and Tn7-encoded proteins. Tn7 transposes via a cut and paste mechanism in which the element is excised from the donor DNA by staggered double-strand breaks and then inserted into attTn7 by the joining of 3' transposon ends to 5' target ends. Neither recombination intermediates nor products are observed in the absence of any protein component or DNA substrate. Thus, we suggest that Tn7 transposition occurs in a nucleoprotein complex containing several proteins and the substrate DNAs and that recognition of attTn7 within this complex provokes strand cleavages at the Tn7 ends.     

Tn5381, a conjugative transposon identifiable as a circular form in Enterococcus faecalis.

We have identified two 19-kb conjugative transposons (Tn5381 and Tn5383) in separate strains of multiply resistant Enterococcus faecalis. These transposons confer resistance to tetracycline and minocycline via a tetM gene, are capable of both chromosomal and plasmid integration in a Rec- environment, and transfer between strains in the absence of detectable plasmid DNA at frequencies ranging from < 1 x 10(-9) to 2 x 10(-5) per donor CFU, depending on the donor strain and the growth conditions. Hybridization studies indicate that these transposons are closely related to Tn916. We have identified bands of ca. 19 kb on agarose gel separations of alkaline lysis preparations from E. faecalis strains containing chromosomal copies of Tn5381, which we have confirmed to be a circularized form of this transposon. This phenomenon has previously been observed only when Tn916 has been cloned in Escherichia coli. Overnight growth of donor strains in the presence of subinhibitory concentrations of tetracycline results in an approximately 10-fold increase in transfer frequency of Tn5381 into enterococcal recipients and an increase in the amount of the circular form of Tn5381 as detectable by hybridization. These results suggest that Tn5381 is a Tn916-related conjugative transposon for which the appearance of a circular form and the conjugative-transfer frequency are regulated by a mechanism(s) affected by the presence of tetracycline in the growth medium.     

The Transfer Origin for Bacteroides Mobilizable Transposon Tn4555 Is Related to a Plasmid Family from Gram-Positive Bacteria

Conjugal transfer of Bacteroides mobilizable transposon Tn4555 was examined with an Escherichia coli-based assay system. It was shown that mobilization required the cis-acting oriT_Tn region and that the Tn4555 mobA_Tn gene and RK231 must be present in trans. With alkaline agarose gel electrophoresis and filter blot hybridizations, it was shown that at oriT_Tn there was a site- and strand-specific cleavage event that was dependent on mobA_Tn. The 5′ end of this cleavage site was mapped by primer extension, and the nucleotide sequence surrounding the site had homology to a family of oriT nick sites found in mobilizable plasmids of gram-positive bacteria. Removal of the nick site by deletion of 18 bp surrounding the site resulted in a significant loss of transfer activity.