Transformation of Mycoplasma pulmonis: demonstration of homologous recombination, introduction of cloned genes, and preliminary description of an integrating shuttle system

The transposons Tn916 and Tn4001 and a series of integrating plasmids derived from their antibiotic resistance genes were used to examine polyethylene glycol-mediated transformation of Mycoplasma pulmonis. Under optimal conditions, Tn916 and Tn4001 could be introduced into M. pulmonis at frequencies of 1 x 10(-6) and 5 x 10(-5) per CFU, respectively. Integrating plasmids were constructed with the cloned antibiotic resistance determinants of Tn916 and Tn4001, a pMB1-derived plasmid replicon, and mycoplasmal chromosomal DNA and were used to examine recombinational events after transformation into M. pulmonis. Under optimal conditions, chromosomal integrations could be recovered at a frequency of 1 x 10(-4) to 1 x 10(-6) per CFU, depending on the size and nature of the chromosomal insert and the parental plasmid. Integrated plasmids were stable in the absence of selection and could be rescued in Escherichia coli along with adjacent mycoplasma DNA. These studies provide the first direct evidence of a recombination system in the Mollicutes and describe the first E. coli-M. pulmonis shuttle vectors.     

Random insertion of the gentamicin resistance transposon Tn4001 in Mycoplasma pulmonis

The staphylococcal transposon Tn4001 was introduced into Mycoplasma pulmonis using an Escherichia coli-derived vector by polyethylene glycol-mediated transformation. Using a reaction mixture containing 10 micrograms plasmid DNA, 10 micrograms yeast tRNA, and 34-35% polyethylene glycol per 1 x 10(8) cells, Tn4001 could be introduced into M. pulmonis at a frequency of 5 x 10(-5) per colony forming unit. DNA-DNA hybridization studies illustrated that Tn4001 could occupy a diversity of insertion sites in the M. pulmonis chromosome. These data indicated that Tn4001 is a potentially useful tool for the introduction of mutations and for genetic studies in M. pulmonis.     

Genetic exchange of transposon and integrative plasmid markers in Mycoplasma pulmonis.

Matings of genetically marked derivatives of Mycoplasma pulmonis resulted in the exchange of chromosomal DNA and the appearance of doubly marked transconjugants. Transposons Tn916 and Tn4001, and a series of integrative plasmids derived from their cloned antibiotic resistance genes, were used to construct antibiotic-resistant mycoplasmal derivatives to examine this phenomenon at the molecular level. Genetic exchange occurred on agar surfaces at frequencies ranging from 3.3 X 10(-4) to 6.4 X 10(-8) transconjugants per CFU. Examination of chromosomal DNA from transconjugants by hybridization revealed that the transposons or integrated plasmids were in the same chromosomal locations as in the parental strains, indicating that exchange involved the transfer of chromosomal DNA and homologous recombination. Transfer was not affected by DNase, polyethylene glycol, EDTA, or calcium chloride but was affected by treatment of either parent with trypsin. Mixing of mating strains before plating had no effect on mating frequencies, but mating did occur in liquid media. The ability to exchange chromosomal markers was limited to selected strains of M. pulmonis; mating did not occur with Acholeplasma laidlawii or M. gallisepticum. Heat and UV inactivation studies revealed that nonviable cells could act as donors in matings. The evidence presented supports a conjugationlike mechanism involving specific trypsin-sensitive membrane components.     

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.     

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.     

Construction of mini-Tn4001tet and its use in Mycoplasma gallisepticum

The Mollicutes are a group of cell-wall-less bacteria and are important plant and animal pathogens. Progress toward analyzing their pathogenic mechanisms has been hampered by the few available genetic tools. Of the two transposons shown to function in mycoplasmas, only Tn4001 is readily amenable to modification and development. One disadvantage of using Tn4001 in mycoplasmas has been independent insertion of the insertion sequence, IS256, probably as a result of inadequate control of the transposase expression in mycoplasmas. In this study, we describe the construction of a mini-Tn4001 containing the tetM antibiotic resistance gene from Tn916. The transposase gene was placed outside the inverted repeats to lower the frequency of independent transposition events. Transposition of mini-Tn4001tet in Mycoplasma gallisepticum occurred at a frequency of 1-8 x 10(-6), a frequency similar to that of the parent transposon. Insertions of mini-Tn4001tet were random and only single insertions were observed. Several unique restriction sites between the inverted repeat sequences provide for further development of mini-Tn4001.     

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.     

Construction and use of derivatives of transposon Tn4001 that function in Mycoplasma pulmonis and Mycoplasma arthritidis

Previous attempts to introduce transposon Tn4001 into Mycoplasma pulmonis and Mycoplasma arthritidis have not been successful, possibly due to functional failure of the transposon's gentamicin resistance determinant. Tn4001C and Tn4001T were constructed, respectively, by insertion of a chloramphenicol acetyltransferase gene and the tetM tetracycline resistance determinant into Tn4001. Both Tn4001C and Tn4001T transposed in M. pulmonis, and Tn4001T transposed in M. arthritidis. The incorporation of a Tn4001T derivative that contained lacZ into either Mycoplasma species resulted in transformants with readily detectable LacZ activity. Tn4001T may be of general utility for use as a mycoplasma cloning vehicle because tetM functions in all species of Mycoplasma examined thus far.     

Physical map of Mycoplasma gallisepticum.

Physical chromosomal maps of two Mycoplasma gallisepticum strains, R and ATCC 19610, were constructed by using field inversion gel electrophoresis. To assist in the ordering of chromosomal fragments and the construction of the chromosomal maps, the gram-positive transposon Tn4001 was modified to serve as a mobile restriction site. The total sizes of the M. gallisepticum R and ATCC 19610 genomes were estimated to be 1,037 and 998 kb, respectively. The restriction enzyme locations for EagI and SmaI were determined along with several transposon insertion sites. The two strain maps were similar except for three small deletions and one additional EagI site in strain ATCC 19610.     

Identification of Tn4451 and Tn4452, chloramphenicol resistance transposons from Clostridium perfringens.

The recombinant plasmids pJIR45 and pJIR97 contain the chloramphenicol resistance determinants derived from the Clostridium perfringens R plasmids pIP401 and pJIR27, respectively. Escherichia coli cultures which harbored these recombinant plasmids rapidly became chloramphenicol sensitive when grown in the absence of chloramphenicol. The loss of resistance was associated with the loss of 6.2-kilobase (kb) segments from both plasmids. Detailed restriction analysis of E. coli- and C. perfringens-derived deletion plasmids indicated that deletion of these segments was essentially precise. Transposition of the 6.2-kb segments was demonstrated by cloning the determinants into a temperature-sensitive plasmid, curing the recombinant plasmids, and selecting chloramphenicol-resistant, plasmid-free clones. Southern hybridization analysis of chromosomal DNA isolated from these recA E. coli clones indicated that the 6.2-kb segments had transposed to different sites on the chromosome. Heteroduplex analysis and restriction mapping indicated that the transposons, Tn4451 (pIP401) and Tn4452 (pJIR27), were closely related and did not contain large inverted or directly repeated sequences. These transposons represent the first transposable elements from the clostridia to be identified and characterized.     

Characterization of conjugative transposon Tn5251 of Streptococcus pneumoniae

Abstract Tn5251 belongs to the Tn916-Tn1545 family of conjugative transposons (CT) and was found integrated into CT Tn5252 , to form the composite element Tn5253 of Streptococcus pneumoniae . We show that Tn5251 is identical in structure and size to Tn916 . DNA sequence analysis of a 4,419-bp segment containing the tet(M) gene showed that only 73 nucleotides out of 4,419 were different in the the two CT. Essentially all differences (66 / 73) were clustered in a 688-bp segment of tet(M) , which was 90% identical to Tn916 and 100% identical to the tet(M) genes of Tn1545 from S. pneumoniae and pOZ101 from Neisseria gonorrhoeae . DNA sequence analysis of the Tn5251/Tn5252 junction fragments allowed us (i) to determine Tn5251 termini, (ii) to define the 6-bp coupling sequences flanking the CT, and (iii) to infer the structure of the integration site ( attB ) of Tn5251 into Tn5252 . Conjugal transfer of Tn5251 independent from Tn5253 could not be detected, even if we could show excision and formation of Tn5251 circular intermediates at a level of 5.4 copies per 10⁶ chromosomes.     

Transposons Tn916 and Tn925 can transfer from Enterococcus faecalis to Leuconostoc oenos

Abstract The streptococcal transposons Tn916 and Tn925 were transferred to several strains of Leuconostoc (Ln.) oenos using the filter mating method. The insertion of both transposons into the chromosome occurred at different sites. Transconjugants of Ln. oenos carrying Tn916 could serve as donors in mating experiments with Lactococcus lactis LM2301. Further analysis of L. lactis LM2301 transconjugants showed that the insertion of the transposon Tn916 into the chromosome was site-specific. These studies establish a basis for the initiation of genetic studies in this Leuconostoc species since there are no efficient conjugal or transformation systems previously described for this microorganism.     

Tn5053 family transposons are res site hunters sensing plasmidal res sites occupied by cognate resolvases

DNA sequence database search revealed that most of Tn5053/Tn402 family transposons inserted into natural plasmids were located in putative res regions upstream of genes encoding various resolvase-like proteins. Some of these resolvase genes belonged to Tn3 family transposons and were closely related to the tnpR genes of Tn1721 and a recently detected Tn5044. Using recombinant plasmids containing fragments of Tn1721 or Tn5044 as targets in transposition experiments, we have demonstrated that Tn5053 displays striking insertional preference for the res regions of these transposons: more than 70% of Tn5053 insertion events occur in clusters inside the target res regions, while most remaining insertion events occur no further than 200 base pairs away from both sides of the res regions. We demonstrate that Tn5053 insertions (both into and outside a res region of the target plasmid) require the presence of a functional cognate resolvase gene either in cis or in trans. To our knowledge, this is the first case when a site-specific recombination system outside a transposon has been shown to be involved in transposition.     

Construction and analysis of a modified Tn4001 conferring chloramphenicol resistance in Mycoplasma pneumoniae

The Staphylococcus aureus transposon Tn4001 and derivatives thereof have been transformed successfully in several mycoplasma species. In order to expand the versatility of Tn4001 for other genetic manipulations and for use in mycoplasma species resistant to gentamicin (Gm), chloramphenicol acetyltransferase (Cat) from S. aureus was evaluated as a selectable marker. The cat gene was cloned in both orientations into a modified Tn4001 and transformed into Mycoplasma pneumoniae, conferring resistance to Cm and Gm. Replacement of the gene for GmR in Tn4001 with cat likewise conferred CmR when transformed into M. pneumoniae. The minimum inhibitory concentration to Cm in transformants with cat derivatives of Tn4001 was 300-500 microg/ml, and Cat enzyme activity was demonstrated by using a fluorescent substrate.     

Tn1721 derivatives for transposon mutagenesis, restriction mapping and nucleotide sequence analysis

New derivatives of the tetracycline-resistance transposon Tn1721 that carry resistances to chloramphenicol, tetracycline, kanamycin and streptomycin are described. These elements are provided on various plasmid vehicles and as chromosomal insertions to extend the range of targets for Tn mutagenesis. Single EcoRI sites at the ends of these transposons proved most useful for physical mapping, for the generation of new EcoRI sites in cloning experiments, for end-labelling and for sequencing of DNA adjacent to an insertion.     

Characterization of Tn2411 and Tn2410, two transposons derived from R-plasmid R1767 and related to Tn2603 and Tn21

Two transposable elements, Tn2410 and Tn2411, were isolated from Salmonella typhimurium R-factor R1767. They have sizes of 18.5 and 18.0 kilobases, respectively. Tn2411 mediates resistance to streptomycin, sulfonamides, and mercury. In Tn2410, the streptomycin resistance gene was replaced by a gene coding for the production of the beta-lactamase OXA-2, which is responsible for ampicillin resistance. Physical and functional maps of both transposons were compared with those of Tn21, Tn4, and Tn2603. From these data it appeared that Tn21 could be an ancestral transposon from which Tn2411, Tn2410, Tn2603, and Tn4 were evolved by the addition or deletion of small DNA segments.     

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.     

Definition of three resolvase binding sites at the res loci of Tn21 and Tn1721.

The dual functions of resolvase, site-specific recombination and the regulation of its own expression from tnpR, both require the interaction of this protein with the DNA sequence at res, but the specificity of this interaction differs between groups of Tn3-like elements. In this study, DNA fragments that contained res from Tn21 or Tn1721 were subjected to either cleavage by DNase I or methylation by dimethyl sulphate in the presence of the purified resolvase from Tn21 or Tn1721. These experiments showed that each resolvase bound to the same three sites (I, II and III) within res from Tn1721 and to an equivalent series of three sites on Tn21: the differences in the amino acid sequences of the two proteins did not affect their interaction with either DNA. The DNA sequences at each site had some similarities and, in conjunction with data from the related transposon Tn501, a consensus was established. However, the three sites are functionally distinct: site I (tnpR-distal) spans the recombination cross-over point and sites II and III (tnpR-proximal) overlap the promoter of tnpR. The binding sites on these transposons were compared with those in the gamma delta/Tn3 system: the similarities between the two groups of transposons revealed some general features of resolvase-DNA interactions while the differences in fine structure elucidated the specificity of each resolvase.     

Isolation and characterization of transposon Tn4001-generated, cytadherence-deficient transformants of Mycoplasma pneumoniae and Mycoplasma genitalium

Abstract Cytadherence and subsequent parasitism of host cells by the human pathogens, Mycoplasma pneumoniae and Mycoplasma genitalium , are mediated by adhesins and adherence-related accessory proteins. In this report we demonstrate the use of transposon Tn 4001 to generate Tn-induced transformants displaying cytadherence-deficient characteristics. Mycoplasma pneumoniae Tn-generated transformant, designated 8R, lacked the high-molecular weight adherence-accessory proteins HMW1/4 and was deficient in hemadsorption and cytadherence capabilities. In transformant 8R, Tn 4001 was not localized in or near the hmw 1 gene or in the upstream adhesin (p30/hmw3) locus, suggesting an alternate site associated with the regulation of hmw 1 gene expression. Sequence analysis identified the transposon insertion site at the crl locus previously reported, although the protein characteristics of transformant 8R differed from the earlier described transformants. The M. genitalium Tn-transformant, designated G26, was also defective in hemadsorption and cytadherence. However, transformant G26 synthesized adhesins P140 and P32 suggesting that Tn 4001 transposed into a new gene or site previously unlinked to cytadherence, namely ORF MG032. This study demonstrates the utility of Tn 4001 mutagenesis for both M. pneumoniae and M. genitalium which, in the latter case, has special relevance in light of the recent complete characterization of its continuous total genomic sequence.     

DNA sequences of and complementation by the tnpR genes of Tn21, Tn501 and Tn1721

DNA sequences that encode the tnpR genes and internal resolution (res) sites of transposons Tn21 and Tn501, and the res site and the start of the tnpR gene of Tn1721 have been determined. There is considerable homology between all three sequences. The homology between Tn21 and Tn501 extends further than that between Tn1721 and Tn501 (or Tn21), but in the homologous regions, Tn1721 is 93% homologous with Tn501, while Tn21 is only 72-73% homologous. The tnpR genes of Tn21 and Tn501 encode proteins of 186 amino acids which show homology with the tnpR gene product of Tn3 and with other enzymes that carry out site-specific recombination. However, in all three transposons, and in contrast to Tn3, the tnpR gene is transcribed towards tnpA gene, and the res site is upstream of both. The res site of Tn3 shows no obvious homology with the res regions of these three transposons. Just upstream of the tnpR gene and within the region that displays common homology between the three elements, there is a 50 bp deletion in Tn21, compared to the other two elements. A TnpR- derivative of Tn21 was complemented by Tn21, Tn501 and Tn1721, but not by Tn3.