Complete nucleotide sequence of insertion element IS4351 from Bacteroides fragilis

Abstract

The nucleotide sequence and genetic analyses of one of the directly repeated sequences flanking the macrolide-lincosamide-streptogramin B drug resistance determinant, ermF, from the Bacteroides fragilis R plasmid, pBF4, suggested that this region is an insertion sequence (IS) (3,9,14,17,18,40).Within the obligately anaerobic Bacteroides spp., three independently isolated R plasmids, pBF4 (44), pBFTM1O (41), and pBI136 (36), have been described which confer resistance to macrolide, lincosamide, and streptogramin B (MLS)-type antibiotics. In each of these plasmids, the MLS resistance genes are flanked by directly repeated (DR) DNA sequences of approximately 1.1 kb (12,31,35). The MLS resistance determinant and the flanking DR sequences on pBF4 (33, 34) and pBFTM10 (28) have been demonstrated to undergo transposition in both Escherichia coli and Bacteroides spp. and have been designated transposons Tn4351 and Tn4400, respectively. A similar observation has been noted with the analogous region of pBI136 (C. J. Smith, submitted for publication).During the course of studying the genetic basis of MLS resistance in the Bacteroides fragilis plasmid pBF4, we determined that this resistance gene, ermF, is transcriptionally dependent on an adjacent DR sequence (27). In the present study, we report the nucleotide sequence of one member of the DR segments of pBF4. Computer analyses of this nucleotide sequence revealed structural similarities of this element to procaryotic IS elements, although no significant homology was seen at the nucleotide level. We also examined the biological activity of this element in E. coli and present evidence that it can undergo recA-independent transposition. We propose to designate this element IS4351. MATERIALS AND METHODSBacterial strains, plasmids, and culture conditions. Bacterial strains and plasmids are shown in Table 1. B. fragilis V479-1 or V598, V600, and V689 were maintained on supplemented brain heart infusion broth (22) containing 5 ,ug of clindamycin per ml or 10 jig of tetracycline per ml as appropriate. E. coli JM101 was maintained on YT medium (23). Other E. coli strains were maintained on LB medium (23) with antibiotics as follows: V831, 10 ,ug of tetracycline per ml; V1317, 50 jig of ampicillin per ml; V1337, 300 ,ug of erythromycin per ml; and V1348, 50 ,ug of ampicillin per ml, 50 jig of chloramphenicol per ml, and 10 jig of tetracycline per ml.Genetic transformation of E. coli. E. coli strains JM101 and HB101 were transformed with bacteriophage and plasmid DNA, respectively, as previously described (27). Plasmid DNA and M13

Conjugal transfer of antibiotic resistance factors in Bacteroides fragilis: the btgA and btgB genes of plasmid pBFTM10 are required for its transfer from Bacteroides fragilis and for its mobilization by IncP beta plasmid R751 in Escherichia coli

“…In the major category, of which TGD555, TGD1, TGD4, and TGD5 are examples, the Tn1000 insertion probably lies outside of the transfer region, since these plasmids could be transferred to both recipients at frequencies close to those of the parental plasmids. In contrast, coordinates of pBFTM10 starting at the second EcoRl site located in the left direct repeat of Tn4400 (19). pGAT550 differs from pGAT400 by the insertion point of pDG5 at the EcoRI site at approximately 12.2 kb of pGAT400.…”

Cloning and sequencing of IS1086, an Alcaligenes eutrophus insertion element related to IS30 and IS4351

“…Two sequences displaying significant levels of homology with IS1086 were found. Both are IS elements: IS30 isolated from E. coli (5,6) and IS4351 isolated from Bacteroides fragilis (25). Figure 3 shows an alignment of the three sequences.…”

Bacteroides: the Good, the Bad, and the Nitty-Gritty

“…This is the case for the IS-borne promoters that can be found upstream of the cfiA or ccrA gene (189), macrolide resistance genes (205), and metronidazole resistance nim genes (259,278).…”