Identification and nucleotide sequence analysis of a transfer-related region in the streptococcal conjugative transposon Tn5252

Abstract

To obtain a functional map of TnS252, a 47.5-kb streptococcal conjugative transposon, a series of defined deletion and insertion mutations were introduced within the transposon. Interruptions at several regions were found to affect the conjugal transposition functions of the element in filter-mating experiments. The nucleotide sequence of the left terminus of Tn5252 showed two open reading frames, ORFR and ORF2, adjoining the at site. The organization of this region and the structure of the predicted integrase encoded by ORFi were found to be similar to those of other site-specific recombination systems.Even though Streptococcus pneumoniae can receive and stably maintain a variety of plasmids from other streptococci, endogenous plasmids are rare in this species. However, since the 1970s multiple antibiotic resistance has begun to appear in this species and a novel class of mobile elements conveniently termed "conjugative transposons" has been shown to be the chief carriers of the multiple antibiotic resistance (5,9,10,14,17,24). The chromosomally integrated conjugative transposons are able to transfer themselves to other cells by a process requiring cell-to-cell contact (5,10,12,23). Two classes of streptococcal conjugative transposons have been recognized (2, 16). Even though both types are capable of conjugal transposition, no structural similarity has been observed (2, 15). Also, the class of elements, typified by the well-characterized Tn916 and Tn1545, integrate at several sites in the recipient's genome (2,7,8), whereas the larger conjugative transposons such as Tn5252 and Tn3701 preferred to integrate at a unique spot (2, 28). We have been studying the biology of Tn5253, a 65.5-kb conjugative transposon carrying resistance to tetracycline and chloramphenicol (2,(27)(28)(29). In a previous article (2), we showed that TnS253 is a composite element of two conjugative transposons (Fig. 1). The smaller element, now termed TnS251 (18 kb), was observed to be structurally and functionally similar to the Tn916 class of elements (2) and did not show homology to any other region in the parental element, TnS253 (2). The sequences beyond TnS251 within TnS253, now called Tn5252, were themselves capable of conjugative transposition (2). While considerable information regarding the Tn916 (TnS251) class of elements has been obtained (6), very little is known of the Tn5252 class of elements (6). To understand the molecular details of this novel class of elements, we had focused our efforts on studying TnS252. In this article, we report our studies relating to the genetic organization and nucleotide sequence of the left end of this transposon.Strategy for creating insertion and deletion mutations within Tn5252. The availability of Escherichia coli recombinant plasmids carrying DNA fragments derived from TnS252 facilitated generation of deletion and insertion mutations within the passenger segments. The basic strategy employed in this 744-6790. study, illustrated in Fig. 2, was as follows. DNA restriction fragments rangi...

Composite Structure of Streptococcus pneumoniae Containing the Erythromycin Efflux Resistance Gene mef (I) and the Chloramphenicol Resistance Gene catQ

“…Such extensive genome rearrangements are consistent with the well-established recombination-mediated genetic plasticity that is a distinctive feature of S. pneumoniae (9). The apparent nonmobility of the 5216IQ complex is consistent with the loss of conjugation-related ORFs of both Tn916 (10) and Tn5252 (19). On the other hand, owing to the greater similarity to TIGR4 than to R6, a lack of zones of homology with the pneumococcal chromosome could account for the unsuccessful transformation experiments, considering that the standard Rx1 recipient has the same derivation (from Avery's historical D39) as R6.…”

Naturally Occurring Lactococcal Plasmid pAH90 Links Bacteriophage Resistance and Mobility Functions to a Food-Grade Selectable Marker

“…The replication regions of pAH33 and pAH82 exhibited 73% identity and are similar to the replication determinants of several theta-replicating lactococcal plasmids, as first identified for plasmid pCI305 (25). In pAH82, a repB gene (orf13) is capable of encoding a protein (386 amino acids) with high identity to RepB proteins of the lactococcal plasmids pND608 (98%; accession number AAA67955), pIL2614 (98% [58]), pWV02 (79% [32]), and pIL103 (79% [58]). The region upstream of repB has features conserved in pCI305-type theta replicons and is also proposed to contain the origin of replication (ori).…”

“…cremoris 712 (61) and pRS01 sex factors (41), both of which are associated with group II introns. MobD also showed homology to several gram-positive intron-free mobilization proteins, including 29% identity with the Tn5252-encoded relaxase enzyme in Streptococcus pneumoniae (32) and 22% identity to the RLX protein of Staphylococcus aureus (5). Mobilization and relaxase proteins are known to play an essential role in conjugation and act by introducing a single-stranded nick at the origin of transfer (oriT).…”

Tn 5253 Family Integrative and Conjugative Elements Carrying mef (I) and catQ Determinants in Streptococcus pneumoniae and Streptococcus pyogenes

“…As shown by early studies (10,33), the cat pC194 -containing DNA region is flanked by direct repeats, the recombination of which may lead to spontaneous curing of the region. Very recently, the issue was thoroughly investigated by Iannelli et al (11), who described an ⍀cat(pC194) (7,627 bp), flanked by two 1,169-bp direct repeats.…”