Terminal nucleotide sequences of Tn551, a transposon specifying erythromycin resistance in Staphylococcus aureus: homology with Tn3

The erythromycin resistance determinant of Staphylococcus aureus plasmid pI258 resides on a 5.3 kb transposon, Tn551. We have determined DNA sequences surrounding the junctions between the transposon and the flanking DNA in the wild-type plasmid, in an insertion into a second plasmid, and in two transposon-related deletions. The ends of the transposon consist of an inverted repeat of 40 base pairs flanked by a direct repeat of 5, thus placing the transposon in the same class as Tn3, IS2, Tn501, gamma delta, and bacteriophage Mu. Interestingly, we find that the terminal sequences of the 40 base pairs inverted repeat are very similar to the ends of Tn3, a transposon which one would not have expected to show any relation to Tn551. This result suggests common ancestry for Tn3 and Tn551. The inverted repeat sequence of Tn551 also contains (with one additional inserted base) the internal heptanucleotide sequence which has been found to be common to most of the transposable elements that generate 5-base pair direct repeat sequences.     

Isolation of transposon Tn551 insertions near chromosomal markers of interest in Staphylococcus aureus.

A procedure was developed to isolate insertions of transposon Tn551 near other markers of interest on the chromosome of Staphylococcus aureus NCTC 8325. When an inoculum of strain 8325-4 carrying a thermosensitive mutant of plasmid pI258 (on which Tn551 resides) was inoculated into brain heart infusion agar plus erythromycin and grown to saturation at 43 degrees C, the transforming DNA extracted from this population of cells contained a random collection of different chromosomal insertions of Tn551; this DNA is referred to as pooled Tn551 DNA. When erythromycin-sensitive recipient strains containing chromosomal markers of interest were transformed with pooled Tn551 DNA, and the resulting Emr transformants were screened for coinheritance of the donor allele of the marker of interest, insertions of Tn551 were isolated near several markers, including fus-149, tet-3490, mec-4916, pig-131, ilv-129, pur-140, and uraA141. Many of the insertions were within the linkage groups that contained these markers, and several insertions occupied different positions between the linkage groups in heretofore undefined regions of the circular chromosomal map of S. aureus. These insertions of transposon Tn551 extend the known limits of the existing linkage groups, provide linkage data and map locations for markers not previously mapped, and provide a means to map markers which cannot be directly selected.     

Insertional inactivation of staphylococcal methicillin resistance by Tn551.

Transposon Tn551 was translocated into the chromosome of a methicillin-resistant (mec) strain of Staphylococcus aureus by heat inactivation of a thermo-sensitive plasmid carrying Tn551 and selection for erythromycin-resistant (Emr) survivors. Two independent chromosomal insertions of Tn551 were obtained which reduced the level of the methicillin resistance by a factor of 50 to 100, making the strains phenotypically methicillin sensitive (Mecs). Each of the Tn551 insertions was on the largest fragment produced by EcoRI digestion of the chromosomal DNA of these strains. The integration sites lie about 1 kilobase apart. These Mecs strains reverted to Mecr at frequencies of 2.4 X 10(-8) and 3.6 X 10(-5), respectively. The majority of Mecr revertants still were Emr; only a few lost the Emr phenotype concomitantly with reversion to the Mecr phenotype. Hybridization data with labeled Tn551 showed complex rearrangements and deletions in the region of the insertion. These two Tn551 insertions do not lie on the same linkage group, II, as the mec determinant. The phenotypic expression of methicillin resistance, therefore, is also dependent upon a chromosomal genetic marker not physically linked to the mec determinant.     

Distribution of Tn551 insertion sites responsible for auxotrophy on the Staphylococcus aureus chromosome

A method was devised to efficiently select isolates of Staphylococcus aureus 8325 in which Tn551, a transposon originating on the pI258 plasmid responsible for erythromycin resistance (Emr), had translocated to the host chromosome. This method consisted of selecting for Emr at 43 degrees C with a strain in which the pI258 plasmid was unable to replicate at 43 degrees C because of a temperature-sensitive plasmid mutation. By selecting isolates that were Emr at 43 degrees C and auxotrophic for nutrients not required by the parent strain. Tn551-induced auxotrophic mutants were readily isolated. The incidence of auxotrophic classes was not random; 80% of the isolates in one experiment were Trp-, whereas only a single example of each of some of the other classes was isolated. Among the Trp- mutants, the distribution of trp genes affected and the frequency of precise excision of Tn551 from individual sites varied. When analyzed by transformation, the Tn551-induced ala, his, ilv, lys, rib, thrA, thrB, and trp mutations were shown to occupy sites previously defined by nitrosoguanidine-induced mutations. Tn551-induced mutagenesis provided three previously unrecognized classes of auxotrophs (tyr, met, and thrC), and the Tn551 integration sites resulting in these mutations have been identified. In addition, a chromosomal region (uraB) was identified by Tn551 mutagenesis that is distinct from uraA (previously defined by chemical mutagenesis). Some Tn551-induced mutations (most notably pur) could not be linked to the known linkage groups of the chromosome by transformation. With the exception of two pur mutations, all of the Tn551-induced auxotrophic mutational sites cotransformed at unity with Tn551 and, in cases in which they were selected, prototrophic transformants were always Ems. Thus, the Tn551 and auxotrophic sites are identical.     

Transformation of a conditionally peptidoglycan-deficient mutant of Staphylococcus aureus with plasmid DNA.

A simple and reliable method for polyethylene glycol-induced plasmid transformation of a temperature-sensitive peptidoglycan-deficient mutant of Staphylococcus aureus is described. The procedure uses strains carrying the tofA372 mutation grown under conditions that yield osmotically fragile cells capable of efficient wall regeneration. The peptidoglycan-deficient cells were transformed with plasmids pE194 and pI258 at frequencies comparable with those obtained with protoplasts prepared with lysostaphin treatment. A readily portable tofA372 mutation was constructed by isolating an insertion of the erythromycin resistance transposon Tn551 adjacent to tofA372. tofA372 was shown by protoplast fusion and transformation analyses to be in the gene order hly-421-omega [Chr::Tn551]1059-tofA372-uraB232-omega [Chr::Tn916]1101-thrB106 on the chromosome of S. aureus NCTC 8325.     

Insertion sites and the terminal nucleotide sequences of the Tn4 transposon.

The nucleotide sequences at the ends of the Tn4 transposon (mercury spectinomycin and sulfonamide resistance) have been determined. They are inverted repeated sequences of 38 nucleotides with three mismatched base pairs. These sequences are strongly homologous with the terminal sequences of Tn501 (mercury resistance) but less so with those of Tn3 (ampicillin resistance). The Tn4 transposon generates pentanucleotide members (Tn3, Tn1000, Tn501, Tn551, IS2) with the exception of Tn1721 and bacteriophage Mu. Among the three Tn4 insertion sites examined here, two of them occurred near a nonanucleotide sequence in perfect homology with part of the terminal inverted-repeat sequence of Tn4 and the third insertion occurred near a sequence of partial homology to one end of Tn4. All three insertions were in the same orientation such that IRb is proximal to its homologous sequence on the recipient DNA.     

Inter- and intrageneric transfer of Tn916 between Streptococcus faecalis and Clostridium tetani

We report that the streptococcal resistance transposon, Tn916, is conjugally transferred to Clostridium tetani (Utrecht) in intergenic matings. Streptococcus faecalis CG180, harboring a 41-kb plasmid (pAM180) containing Tn916 (15 kb), transferred the transposon-associated tetracycline resistance (Tcr) to C. tetani in filter matings at a frequency of about 10(-4)/donor. An erythromycin resistance marker carried by pAM180 was not transferred, indicating lack of plasmid conjugation or stable inheritance of plasmid sequences. DNA extracted from C. tetani transconjugants was probed with radiolabeled Tn916 using Southern blot analysis and these results indicated that the transposon integrated at multiple host genomic sites. Tn916-carrying C. tetani strains were able to transfer Tcr to suitable recipient strains of C. tetani as well as to S. faecalis recipients. These results indicate that this transposon is able to be disseminated and expressed in obligately anaerobic gram-positive bacteria. Moreover, this system opens avenues for the implementation of transposon mutagenesis in this important pathogenic species.     

Genetic analysis of Staphylococcus aureus with Tn4001.

Tn4001, a 4.5-kilobase composite transposon with IS256 ends that confers resistance to gentamicin (Gmr), tobramycin, and kanamycin in Staphylococcus aureus, can transpose to diverse chromosomal sites in S. aureus. Chromosomal insertions of Tn4001 were isolated either after UV irradiation of transducing lysates carrying pII147::Tn4001 or by selection for thermoresistant Gmr isolates with strains containing thermosensitive derivatives of plasmids pI258 and pII147 carrying Tn4001. Frequent integration of the entire delivery plasmid occurred under these selective conditions in recombination-proficient hosts. When selection for thermoresistant Gmr isolates was done with these plasmids in recombination-deficient hosts, 99% or more of the Gmr isolates resulted from transposition of Tn4001 in the absence of plasmid integration. Efficient isolation of Tn4001 insertions near markers of interest and the isolation of insertional auxotrophs were achieved. Reversion frequencies of insertional auxotrophs were between 10(-6) and 10(-7) (higher than those observed with Tn551 and Tn917). About 50% of the prototrophic revertants were Gms, and these are attributed to precise excision of Tn4001. The Gmr prototrophic revertants were due to intergenic suppression.     

The role of transposons in replication: Tn5 suppresses ts-mutation for plasmid RP1 replication in Escherichia coli cells

Effect of restoration by transposon Tn5 of genetic damage in RP1 plasmid replication (named transposon suppression) was described. Hybrid plasmid, a derivative of RP1 and RP4, having ts mutation for replication--tsr12 and deletion in the aphA gene controlling kanamycin resistance, was constructed. Five of derivatives of this plasmid containing transposon Tn5 were made, and the strains containing both the Tn5 integrated into the chromosome and intact hybrid plasmid or the parental plasmid with the replication ts mutation, were constructed. It was shown that transposon Tn5 comprised within the hybrid plasmid or in the chromosome promotes maintenance of these replication defective plasmids in the bacterial culture at a non-permissive temperature and thus suppresses plasmid mutation tsr12. It was determined that the extent of suppression of plasmid replication ts mutation depends on the localization of transposon Tn5.     

Suppression of autolysis and cell wall turnover in heterogeneous Tn551 mutants of a methicillin-resistant Staphylococcus aureus strain.

Isogenic Tn551 mutants of a highly and uniformly methicillin-resistant strain of Staphylococcus aureus were tested for their rates of autolysis and cell wall degradation in buffer and for cell wall turnover during growth. The normal (relatively fast) autolysis and turnover rates of the parent strain were retained in a Tn551 mutant in which the insert was located within the mec gene and which produced undetectable levels of penicillin-binding protein 2A. On the other hand, autolysis and cell wall turnover rates were greatly reduced in auxiliary mutants, i.e., mutants in which the transposon caused conversion of the high-level and uniform resistance of the parent strain to a variety of distinct heterogeneous expression types and greatly decreased resistance levels. All of these mutants contained an intact mec gene and produced normal amounts of penicillin-binding protein 2A, and one of the mutations was located in the femA region of the staphylococcal chromosome (B. Berger-Bachi, L. Barberis-Maino, A. Strassle, and F. H. Kayser, Mol. Gen. Genet. 219:263-269, 1989). Autolysis rates were related to the degree of residual methicillin resistance and to the sites of Tn551 insertion. Fast cell wall turnover may help expression of high-level methicillin resistance by providing a mechanism for the excision of abnormal (and potentially lethal) structural elements of the cell wall synthesized by the bacteria in the presence of methicillin.     

Evolution of complex resistance transposons from an ancestral mercury transposon.

The molecular interrelationship of a transposon family which confers multiple antibiotic resistance and is assumed to have been generated from an ancestral mercury transposon was analyzed. Initially, the transposons Tn2613 (7.2 kilobases), encoding mercury resistance, and Tn2608 (13.5 kilobases), encoding mercury, streptomycin, and sulfonamide resistances, were isolated and their structures were analyzed. Next, the following transposons were compared with respect to their genetic and physical maps: Tn2613 and Tn501, encoding mercury resistance; Tn2608 and Tn21, encoding mercury, streptomycin, and sulfonamide resistance; Tn2607 and Tn4, encoding streptomycin, sulfonamide, and ampicillin resistance; and Tn2603, encoding mercury, streptomycin, sulfonamide, and ampicillin resistance. The results suggest that the transposons encoding multiple resistance were evolved from an ancestral mercury transposon.     

Analysis of a Clostridium difficile PCR ribotype 078 100 kilobase island reveals the presence of a novel transposon, Tn6164

ABSTRACT: BACKGROUND: Clostridium difficile is the main cause of antibiotic associated diarrhea. In the past decade, the number of C. difficile patients has increased dramatically, coinciding with the emergence of two PCR ribotypes, 027 and 078. PCR ribotype 078 is also frequently found during C. difficile outbreaks in pigfarms. Previously, the genome of the PCR ribotype 078 strain M120, a human isolate, was described to contain a unique insert of 100 kilobases. RESULTS: Analysis of this insert revealed over 90 open reading frames, encoding proteins originating from transposons, phages and plasmids. The insert was shown to be a transposon (Tn6164), as evidenced by the presence of an excised and circularised molecule, containing the ligated 5'and 3'ends of the insert. Transfer of the element could not be shown through filter-mating experiments. Whole genome sequencing of PCR ribotype 078 strain 31618, isolated from a diarrheic piglet, showed that Tn6164 was not present in this strain. To test the prevalence of Tn6164, a collection of 231 Clostridium difficile PCR ribotype 078 isolates from human (n = 173) and porcine (n = 58) origin was tested for the presence of this element by PCR. The transposon was present in 9 human, tetracycline resistant isolates, originating from various countries in Europe, and none of the pig strains. Nine other strains, also tetracycline resistant human isolates, contained half of the transposon, suggesting multiple insertion steps yielding the full Tn6164. Other PCR ribotypes (n = 66) were all negative for the presence of the transposon. Multi locus variable tandem repeat analysis revealed genetic relatedness among transposon containing isolates. Although the element contained several potential antibiotic resistance genes, it did not yield a readily distinguishable phenotype. CONCLUSIONS: Tn6164 is a newly described transposon, occurring sporadically in C. difficile PCR ribotype 078 strains. Although no transfer of the element could be shown, we hypothesize that the element could serve as a reservoir of antibiotic resistance genes for other bacteria. Further research is needed to investigate the transfer capabilities of the element and to substantiate the possible role of Tn6164 as a source of antibiotic resistance genes for other gut pathogens.     

Plasmid-borne cadmium resistance genes in Listeria monocytogenes are present on Tn5422, a novel transposon closely related to Tn917.

The complete (6,449-bp) nucleotide sequence of the first-described natural transposon of Listeria monocytogenes, designated Tn5422, was determined. Tn5422 is a transposon of the Tn3 family delineated by imperfect inverted repeats (IRs) of 40 bp. It contains two genes which confer cadmium resistance (M. Lebrun, A. Audurier, and P. Cossart, J. Bacteriol. 176:3040-3048, 1994) and two open reading frames that encode a transposase (TnpA) and a resolvase (TnpR) of 971 and 184 amino acids, respectively. The cadmium resistance genes and the transposition genes are transcribed in opposite directions and are separated by a putative recombination site (res). The structural elements presumed to be involved in transposition of Tn5422 (IRs, transposase, resolvase, and res) are very similar to those of Tn917, suggesting a common origin. The transposition genes were not induced by cadmium. Analysis of sequences surrounding Tn5422 in nine different plasmids of L. monocytogenes indicated that Tn5422 is a functional transposon, capable of intramolecular replicative transposition, generating deletions. This transposition process is probably the reason for the size diversity of the L. monocytogenes plasmids. Restriction analysis and Southern hybridization revealed the presence of Tn5422 in all the plasmid-mediated cadmium-resistant L. monocytogenes strains tested but not in strains encoding cadmium resistance on the chromosome.     

Presence of chromosomal elements resembling the composite structure Tn3701 in streptococci.

Tn3701, carried by Streptococcus pyogenes A454, is the first chromosomal composite element to be described; it contains in its central region Tn3703, a transposon similar to Tn916. A comparison by DNA-DNA hybridization of Tn3701 with omega(cat-tet) and Tn3951, carried by Streptococcus pneumoniae BM6001 and by Streptococcus agalactiae B109, respectively, revealed that the two latter structures are also Tn3701-like composite elements. The DNAs of 27 other antibiotic-resistant group A, B, C, and G streptococci and of S. pneumoniae BM4200 showed sequence homologies to Tn3701 (14 strains, including BM4200), to the regions of Tn3701 outside of Tn3703 (5 strains), and to Tn916 alone (8 strains). The DNAs of five strains did not detectably hybridize with any probe. The tetM gene was identified in most chromosomal genetic elements coding for tetracycline-minocycline resistance. Since Tn3701-like elements are widely disseminated among antibiotic-resistant streptococci (47% of the 34 strains studied), we propose that Tn3701 be considered the prototype of chromosomal composite elements.     

Tn917 transposition in Lactobacillus plantarum using the highly temperature-sensitive plasmid pTV1Ts as a vector

pTV1Ts, a temperature-sensitive plasmid coding for chloramphenicol (Cm) resistance and carrying the macrolide-lincosamide-steptogramin B (MLS) resistance transposon Tn917, was introduced into strains of Lactobacillus plantarum by electroporation. After two passages in broth medium selecting for MLS resistance at 40 degrees C and subsequent plating on solid medium, two strains, L. plantarum NC4Ts1 and L. plantarum NC7Ts5, lost chloramphenicol resistance but retained MLS resistance, indicative of Tn917 transposition into host DNA. Analysis of DNA from MLSrCms isolates from both strains revealed Tn917 insertions into resident plasmids. Restriction analysis of plasmid DNA from four MLSrCms isolates from NC7Ts5 indicated four different insertion sites.     

Isolation and characterization of a Tn551-autolysis mutant of Staphylococcus aureus.

A Lyt- mutant with reduced autolytic activity was isolated after Tn551 mutagenesis of the methicillin-susceptible Staphylococcus aureus laboratory strain RN450. The Lyt- phenotype could be transferred back into the parent and into a variety of other S. aureus strains by transduction of the transposon marker. Southern analysis has located the Tn551 insert to a 3.2-kb HindIII DNA fragment on the SmaI B fragment of the staphylococcal chromosome. The Lyt- phenotype included reduced rates of cell wall turnover and autolysis induced by detergent or methicillin treatment; however, the rate of methicillin-induced killing was not affected. Peptidoglycans prepared from the parental and mutant cells showed identical muropeptide compositions, as resolved by a high-resolution high-pressure liquid chromatography technique. On the other hand, LiCl extracts of the mutant cells contained reduced amounts of total protein and lower specific cell wall-degrading activity compared with those of extracts of parental cells. The profile of bacteriolytic enzymes as detected by sodium dodecyl sulfate-polyacrylamide gel electrophoresis showed multiple band differences between mutant and parental cells; a major lytic band with properties characteristic of the staphylococcal endo-beta-N-acetylglucosaminidase was completely absent from the Lyt- cells. The Lyt- phenotype transduced into a series of methicillin-resistant strains of both homogeneous and heterogeneous phenotypes caused only a modest decrease in the level of methicillin resistance, as determined by population analysis.     

Molecular genetic analysis of the Tn5041 transposition system

A study was made of the transposition of the mercury resistance transposon Tn5041 which, together with the closely related toluene degradation transposon Tn4651, forms a separate group in the Tn3 family. Transposition of Tn5041 was host-dependent: the element transposed in its original host Pseudomonas sp. KHP41 but not in P. aeruginosa PAO-R and Escherichia coli K12. Transposition of Tn5041 in these strains proved to be complemented by the transposase gene (tnpA) of Tn4651. The gene region determining the host dependence of Tn5041 transposition was localized with the use of a series of hybrid (Tn5041 x Tn4651) tnpA genes. Its location in the 5'-terminal one-third of the transposase gene is consistent with the data that this region is involved in the formation of the transposition complex in transposons of the Tn3 family. As in other transposons of this family, transposition of Tn5041 occurred via cointegrate formation, suggesting its replicative mechanism. However, neither of the putative resolution proteins encoded by Tn5041 resolved the cointegrates formed during transposition or an artificial cointegrate in E. coli K12. Similar data were obtained with the mercury resistance transposons isolated from environmental Pseudomonas strains and closely related to Tn5041 (Tn5041 subgroup).     

A chromosomally located transposon in Pseudomonas aeruginosa

A new transposon, Tn2521, coding for carbenicillin, streptomycin, spectinomycin, and sulfanilamide resistance, has been identified in Pseudomonas aeruginosa. The transposon occurs naturally in the chromosome of clinical strains of P. aeruginosa isolated in geographically separated hospitals. This has been demonstrated by its transductional linkage to the pur-136 marker and also by Southern hybridization. Tn2521 is 6.8 kilobases, can transpose from the chromosome to both IncP-1 and IncP-2 plasmid genomes, and has a pattern of restriction endonuclease sites unlike that of any previously described transposon. The carbenicillin resistance carried by Tn2521 is due to the PSE-4 type of beta-lactamase.