Analysis of the Mobilization Functions of the Vancomycin Resistance Transposon Tn 1549 , a Member of a New Family of Conjugative Elements

Abstract

Conjugative transfer from Clostridium symbiosum to enterococci of Tn1549, which confers VanB-type vancomycin resistance, has been reported. This indicates the presence of a transfer origin (oriT) in the element. Transcription analysis of Tn1549 indicated that orf29, orf28, orfz, and orf27 were cotranscribed. A pACYC184 derivative containing 250 bp intergenic to orf29-orf30 of Tn1549 was mobilized in Escherichia coli recA::RP4::⌬nic provided that orf28 and orf29 were delivered simultaneously. These open reading frame (ORF) genes were able to promote mobilization in trans, but a cis-acting preference was observed. On the basis of a mobilization assay, a minimal 28-bp oriT was delimited, although the frequency of transfer was significantly reduced compared to that of a 130-bp oriT fragment. The minimal oriT contained an inverted repeat and a core, which was homologous to the cleavage sequence found in certain Gram-positive rolling-circle replicating (RCR) plasmids. While Orf29 was a mobilization accessory component similar to MobC proteins, Orf28 was identified as a relaxase belonging to a new phyletic cluster of the MOB p superfamily. The nick site was identified within oriT by an oligonucleotide cleavage assay. Closely related oriTs linked to mobilization genes were detected in data banks; they were found in various integrative and conjugative elements (ICEs) originating mainly from anaerobes. These results support the notion that Tn1549 is a member of a MOB p clade. Interestingly, the Tn1549-derived constructs were mobilized by RP4 in E. coli, suggesting that a relaxosome resulting from DNA cleavage by Orf28 interacted with the coupling protein TraG. This demonstrates the capacity of Tn1549 to be mobilized by a heterologous transfer system.Tn1549 is a 34-kb transposon which confers vancomycin resistance in Enterococcus spp. (22). This entirely sequenced element, which harbors the vanB operon, is transferred passively among enterococci by conjugative plasmids or by the exchange of large chromosomal fragments (9,17,22). Tn1549 has also been reported in Gram-positive anaerobes (18, 46), and transfer from Clostridium symbiosum to Enterococcus has been demonstrated (28). This element is likely to be a conjugative transposon consisting of three functional modules involved in transposition, transfer, and antibiotic resistance. The transposition module encodes an excisionase and an integrase which cooperatively direct excision of the element. Excision is followed by circularization, resulting in a heteroduplex at the joined ends according to the tyrosine recombinase model of the Tn916 prototype. Analysis of Enterococcus transconjugants indicated that acquisition of VanB-type resistance was due to transposition of Tn1549 (28). However, it remains to be established if Tn1549 is a conjugative or a mobilizable transposon, since a single retransfer event has been obtained but in the presence of Tn916 (28). In both instances, whether active or passive transfer is being considered, presence of a transfer origin (oriT) is requi...

Green Fluorescent Protein-Labeled Monitoring Tool To Quantify Conjugative Plasmid Transfer between Gram-Positive and Gram-Negative Bacteria

“…The mean mobilization rate from three independent experiments was 1.42 · 10 Ϫ5 Ϯ 3.11 · 10 Ϫ6 transconjugants per recipient cell. Mobilization rates were approximately three times lower than pIP501 transfer rates in experiments under the same conditions, suggesting a possible cis-acting preference of the Orf1 relaxase in agreement with observations made for the TraA relaxase of plasmid pRetCF2d and relaxase Orf28 of the conjugative transposon Tn1549 (32,40). …”

Comparison of direct cultivation on a selective solid medium, polymerase chain reaction from an enrichment broth, and the BD GeneOhm™ VanR Assay for identification of vancomycin-resistant enterococci in screening specimens

“…Since genotypes vanE to vanG (and most probably the previously identified vanL and vanM, too; [Boyd et al, 2008] and [Xu et al, 2010]) still remain rare among clinical enterococcal isolates encountered in hospitals worldwide (Werner et al, 2008), this does not complicate substantially the performance and predictive value of the present assay in detecting and identifying VRE colonizers. Quite contrarily, the prevalence of vanB, vanD, vanE, and vanG genes among nonenterococcal, intestinal bacteria may thwart the good performance of a putative assay capable of detecting all acquired van genotypes ( [Domingo et al, 2005], [Domingo et al, 2007], [Graham et al, 2008], [Launay et al, 2006] and [Tsvetkova et al, 2010]). Performance of the assay in identifying a set of 20 reference and test strains (vanA/vanB positive and van negative) performed at 3 laboratories showed 100% sensitivity and specificity.…”

Current Trends of Emergence and Spread of Vancomycin-Resistant Enterococci

“…In strains of Clostridium symbiosum an entire vanB2 type Tn1549 cluster was identified which was transferable in vitro and in vivo in the digestive tract of mice highlighting the important role that commensal, intestinal, non-enterococcal hosts may play for acquiring, preserving and distributing (vancomycin) resistance genes to nosocomial pathogens (Launay et al, 2006). The corresponding conjugative transposon Tn1549 encodes all necessary functions for a successful transfer of the element across species and genus barriers also demonstrating its potential to transfer vanB type vancomycin resistance from Enterococcus to other important nosocomial pathogens like Staphylococcus spp, C. difficile and others (Tsvetkova et al, 2010). A large number of the C. difficile genome of the multi-drug resistant, clinical strain 630 consisted of mobile, genetic elements (11%) including a tet(M)-encoding self-conjugative transposon Tn5397 (Sebaihia et al, 2006).…”

“…The conjugative vanB transposon Tn1549 or its backbone is widely prevalent among vanB type enterococci and related Gram-positive bacteria such as Clostridium spp. (see chapter 5; (Launay et al, 2006;Tsvetkova et al, 2010)). Conjugative transposons have been known for a long time in Enterococcus and Bacteroides and were lately also identified in Gram-negatives.…”