Transposition of the IS21-related element IS1415 in Rhodococcus erythropolis

Abstract

Three copies of the IS21-related transposable element IS1415 were identified in Rhodococcus erythropolis NI86/21. Adjacent to one of the IS1415 copies, a 47-bp sequence nearly identical to the conserved 5 end of integrons was found. Accurate transposition of IS1415 carrying a chloramphenicol resistance gene (Tn5561) was demonstrated following delivery from a suicide vector to R. erythropolis SQ1.The genus Rhodococcus is characterized by a remarkable ability to degrade or convert a broad range of organic compounds (reviewed in reference 31). Rhodococcus rhodochrous has already found application in the industrial production of acrylamide (32). A steadily increasing number of reports reveal the potential of these nocardioform actinomycetes for environmental biotechnology. However, progress in this area is still hampered by a lack of appropriate genetic tools, such as (broad-host) vectors and transpositional mutagenic systems (10). In particular, the existence of transposable elements in Rhodococcus species is poorly documented. Two recent reports describe the identification of IS elements of the IS256 family by sequence analysis of DNA regions containing biodegradative genes (5, 18), but transposition has not yet been demonstrated. In this report, we describe the identification of the Rhodococcus transposable element IS1415, representing the first member of the IS21 family isolated from an actinomycete. We further show that a transposon constructed from IS1415 is capable of faithful transposition following delivery from a suicide vector to Rhodococcus cells.IS1415: a member of the IS21 family. The thcRBCD gene cluster, required for biodegradation of thiocarbamate herbicides (21) and s-triazine herbicides (20), is located in the region downstream of the cobalamin (vitamin B 12 ) biosynthetic operon cobLMK of Rhodococcus erythropolis NI86/21 (4). Further sequence analysis of the original LambdaGEM-12 clone (FAJ2028) (21) indicated that the N-terminal part of a putative transposase gene was present on this SacI fragment. With the 900-bp SalI-SacI fragment (Fig. 1A) as a probe, the overlapping clone FAJ2031 was isolated from the EMBL3 library of the R. erythropolis NI86/21 genome (30). A hybridizing 6.8-kb BamHI subfragment was cloned in pUC18 (pFAJ2546) and provided about 3.7 kb of additional DNA sequence. Further DNA sequencing of both strands on overlapping fragments subcloned in pUC18 was carried out with an automated sequencer (ALF; Pharmacia Biotech). Potential coding regions (ORF1-ORF4Ј, IstA, and IstB) were identified with the GCWIND program (28). No obvious open reading frames (ORFs) were detectable between ORF1 and IstA, consistent with the lower GϩC content of this region (58.8%) compared to those of the other regions of the fragment (64.7%). Database searches revealed no known homologs for ORF1, ORF2, or ORF3. The hydropathy pattern of the partial ORF4 amino acid sequence showed several potential transmembrane helices (data not shown), consistent with a low but extended sequence similarity (21.4% identity i...

Biodegradation of the xenobiotic organic disulphide 4,4′-dithiodibutyric acid by Rhodococcus erythropolis strain MI2 and comparison with the microbial utilization of 3,3′-dithiodipropionic acid and 3,3′-thiodipropionic acid

“…After identification and characterization of the genes involved, the construction of metabolically engineered strains suitable for production of 4MB-containing PTEs should become possible. The system of transposon mutagenesis (Nagy et al, 1997) used in this study, which was the only available and applicable method at the time, is not suitable for the genotypic characterization of transposon-induced mutants, as also described elsewhere (Sallam et al, 2006). However, a promising system for the construction of transposon mutant libraries of strains of R. erythropolis has been developed just recently (Sallam et al, 2007).…”

“…For transposon mutagenesis, the suicide vectors pFAJ2571 and pFAJ2527 (Nagy et al, 1997) were transferred into the chloramphenicol-susceptible isolate R. erythropolis MI2 by electroporation. Transposon-induced mutants were selected on MSM agar plates containing 50 mg chloramphenicol ml 21 (MSM Cm ) and 0.2 % (w/v) sodium gluconate (master plates).…”

“…Therefore, this isolate was subjected to transposon mutagenesis employing the suicide vector plasmids pFAJ2571 and pFAJ2572 (Nagy et al, 1997), conferring chloramphenicol resistance to the recipient. This mutagenesis aimed at the generation of mutants with defective growth on DTDB to investigate the DTDB catabolism.…”

Advances in the Development of Genetic Tools for the Genus Rhodococcus

“…To date, several insertion sequences (IS) have been identified in the Rhodococcus species [43][44][45][46][47][48][49][50] . However, of all these IS, only a few have been shown to be transpositionally active.…”

Application of real-time PCR array to the multiple detection of antibiotic resistant genes in glacier ice samples

“…Horizontal gene transfer is a key phenomenon for antibiotic resistance genes to persist in a new environment (Van Elsas and Bailey, 2002). Indeed, the antibiotic resistance genes detected here have been reported to reside on the mobile genetic elements (Agerso et al, 2006;Bauernfeind et al, 1996;Bradford et al, 1997;Chopra and Roberts, 2001;Desomer et al, 1992;Furushita et al, 2003;Matsuoka et al, 1997;Nagy et al, 1997;Van Overbeek et al, 2002;Wohlleben et al, 1989).…”