Division of Microbiology, Central Drug Research Institute, Lucknow 226001, India1
Author for correspondence: Ranjana Srivastava. Tel: +91 522 220908. Fax: +91 522 223405. e-mail: root{at}cscdri.ren.nic.in
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ABSTRACT |
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Keywords: insertion sequence, Mycobacterium fortuitum, reporter gene, molecular trap
Abbreviations: IS, insertion sequence; Kan, kanamycin; ts, temperature sensitive
The EMBL accession number for the sequence reported in this paper is Y18875 MF018875
a Present address: Department of Radiation Medicine, Biochemistry & Molecular Biology, Georgetown University Medical Centre, Washington DC, USA.
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INTRODUCTION |
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Most ISs in mycobacteria have been identified as IS-like elements found within repetitive DNA (McFadden et al., 1987 ; Thierry et al., 1990
), by hybridization to drug-resistance markers (Martin et al., 1990
) or through transposon traps (Cirillo et al., 1991
; Guilhot et al., 1992
). The second approach led to the isolation of transposon Tn610 from Mycobacterium fortuitum (Martin et al., 1990
). Transposon trapping appears to be the most attractive alternative as it allows isolation of a true IS element through transposition into a reporter gene whose inactivation can be easily monitored. We have described in this paper an IS trap system that uses a temperature sensitive (ts) mycobacteriumEscherichia coli shuttle vector, pCD4, to trap ISs in the lacZ reporter system during replication in the mycobacterial host. Two clones with disrupted lacZ genes were isolated as white colonies on X-Gal plates in both mycobacterial as well as E. coli hosts. By using this vector, a new element, IS219, was isolated from M. fortuitum.
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METHODS |
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Genetic methods.
For electroporation, mycobacterial cells were grown to an OD650 of 1·0 in LB broth containing glycerol and Tween 80. The cells were electroporated as described by Connell (1994) . Competent E. coli cells for transformation were prepared by the standard calcium chloride/rubidium chloride method (Sambrook et al., 1989
).
Plasmid isolation was done by the alkaline-SDS lysis procedure (Sambrook et al., 1989 ). Plasmid DNA from mycobacterial cells was isolated by the alkaline-SDS lysis method with slight modifications. Cells were harvested at early stationary phase and inactivated at 80 °C for 1 h prior to isolation of plasmid DNA. Sequential incubation in lysozyme (1 mg ml-1; 1 h at 37 °C), SDS/NaOH solution (10 min at 45 °C) and high salt/acetate mix (1618 h) were done. Plasmid DNA was precipitated with 2-propanol.
For Southern blot hybridization, chromosomal DNA from mycobacterial strains was isolated as described by Connell (1994) . The DNA was digested with restriction enzymes and electrophoresed through 0·8% agarose in TAE (Sambrook et al., 1989
). The fragments were transferred to a nitrocellulose membrane overnight by capillary transfer. The DNA was cross-linked to the membrane by using a UV Stratalinker (Stratagene). The DNA probe was labelled with digoxigenin; labelling and detection were done as described by Boehringer Mannheim. Restriction enzymes, biochemicals, T4 DNA ligase etc. were purchased from Promega, New England Biolabs and Sigma.
Cloning and subcloning of the IS for sequencing purposes was done using standard methodologies. DNA sequences were determined from double-stranded plasmid DNA on an automated DNA sequencer (ABI PRISM model 377; Applied Biosystems). Universal forward and reverse M13 and internal primers were used to sequence fragments cloned in pUC vectors. The sequences were analysed using the DNASIS package (Pharmacia). The BLASTX program (Altschul et al., 1997 ) was used for homology searches within the GenBank/EMBL databases.
Construction of pCD4.
M. smegmatis mc2155[pMV261::lacZ] was exposed to 66 µg NTG ml-1 for 45 min to give 5% survival (Holland & Ratledge, 1971 ). The cells were washed and plated on NA plates containing Tween 80 and Kan at a density of 300400 c.f.u. per plate and incubated at 30 °C until the colonies were visible (3 d). The plates were then shifted to 40 °C, which prevented clones carrying plasmids with a ts mutation to grow in size. After confirmation of their ts nature in mycobacteria, the plasmids were transformed into E. coli. The stability of lacZ was checked with respect to the parent pMV261::lacZ plasmid by looking at the segregation of blue colonies into white and blue colonies in the presence of X-Gal after 72 h growth at 30 °C. One of the ts plasmids was named pCD4 and used for further studies.
Molecular trapping of ISs from M. fortuitum.
The ts plasmid pCD4 was electroporated into M. fortuitum and blue colonies were selected on MB agar plates containing Kan and X-Gal at 30 °C. One such blue clone was grown in liquid medium at 30 °C in the presence of Kan for 72 h. Dilutions of the culture were plated to obtain well isolated colonies. Some white clones obtained were analysed for insertion in the lacZ expression cassette by examination of the BamHI restriction profile. Further localization of the insert was performed using a combination of restriction enzymes.
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RESULTS AND DISCUSSION |
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Isolation of ISs from M. fortuitum
M. fortuitum carrying pCD4 was grown in NB containing Kan and Tween for 72 h at 30 °C and plated on MB agar containing Kan and X-Gal. Both blue and white colonies appeared after 45 d. White colonies appeared at a frequency of 5x10-5. The plasmids from the white mutants thus obtained were analysed and their BamHI restriction profile inspected, using pCD4 as the control. BamHI cleaves pCD4 into two fragments of 3·078 (lacZ) and 4·5 kb (rest of the plasmid) (Fig. 2, lane 20). Plasmids from white colonies digested with BamHI yielded fragments of different sizes. Upon analysis they revealed either deletions within the lacZ structural gene or Phsp60lacZ region, deleting one BamHI site (Fig. 2
, lanes 3, 4, 6, 8, 12, 13 and 19). In seven cases, the restriction profile remained the same (Fig. 2
, lanes 2, 9, 10 and 1518). When this gel was hybridized with M. fortuitum genomic DNA, only the upper bands in lanes 5 and 14 showed a signal. In these two cases, the 3·078 kb fragment remained the same but the 4·5 kb fragment was larger in size (Fig. 2
, lanes 5 and 14) due to acquisition of DNA sequence from M. fortuitum as confirmed by Southern hybridization. These two plasmids were selected and named pCD48 and pCD49.
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Molecular analysis of pCD48 and pCD49
The BamHI profiles of pCD48 and pCD49 indicated that the ISs were not located in the lacZ gene. Their location in the Phsp60 region was confirmed by digestion with BamHI, BglII, BamHI/BglII, HpaI and HpaI/XbaI (Fig. 1) and subsequent hybridization with genomic DNA from M. fortuitum (Fig. 3
) and a Phsp60 fragment (not shown) as probes. The fragments in pCD48 and pCD49 representing junctions of the IS element and Phsp60 hybridized with both M. fortuitum genomic DNA and Phsp60 while true internal fragments hybridized only with M. fortuitum.
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pCD49.
In pCD49, the 1·4 and 0·76 kb fragments from the BamHI/BglII digest represented Phsp60IS junctions (Fig. 1). The two fragments were subcloned into the BamHI site of pUC13 and sequenced using commercially available forward and reverse primers of pUC plasmids (New England Biolabs) and internal primers. The Phsp60IS junctions were confirmed by nucleotide sequence homology to the Phsp60 sequence. The sequencing of junction fragments displayed a duplication of the 6 bp CTTAGC present in the Phsp60 fragment of pCD4. Hence it corresponds to the duplication of the target sequence during the transposition event. The nucleotide sequence of the full IS element was obtained and the IS element was named IS219.
Characterization of IS219
IS219 is 1653 bp long with 8 bp imperfect inverted repeats at its termini. Analysis of the nucleotide sequence revealed a G+C content of 63·7 mol%, which is typical of mycobacteria. Two major ORFs were identified, ORFA (bases 125913) of 272 aa having a potential RBS 9 nt upstream of the start codon (GTG), and ORFB (bases 7561656) of 300 aa with ATG as the start codon. A potential RBS is present 10 nt upstream of the start codon. Two direct repeats and two inverted repeats were found within IS219. Using the BLASTX program (Altschul et al., 1997 ), the putative mycobacterial protein encoded by ORFB showed 27% identity and 40% positive matches over 158 aa to a putative transposase from Acetobacter pasteurianus (Takemura et al., 1991
) (Fig. 4
) but no significant homology to any other known IS element was found. This suggests that the element discovered by us through the lacZ trap system is a unique one not reported so far.
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M. fortuitum is associated with a wide variety of infections involving lungs, skin, bone, central nervous system, prosthetic heart valves and also in disseminated disease (Silcox et al., 1981 ; Woods & Washington, 1987
; Wallace et al., 1991
). It will be interesting to investigate the occurrence of IS219 in different M. fortuitum strains to establish its use in diagnostic and epidemiological studies.
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ACKNOWLEDGEMENTS |
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REFERENCES |
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Received 11 August 1999;
revised 9 November 1999;
accepted 7 February 2000.
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