SHORT COMMUNICATION Transposition-Induced Structural Instability of Escherichia coli–Mycobacteria Shuttle Vectors Mamta Chawla and Sujoy Kr. Das Gupta 1 Department of Microbiology, Bose Institute (Centenary Building), P1/12 C.I.T. Scheme VIIM, Calcutta 700054, India Received September 4, 1998; revised October 19, 1998 Escherichia coli–mycobacteria shuttle vectors, derived from pAL5000 (a mycobacterial plas- mid) and pUC19, were frequently found to undergo structural alterations due to transposition of IS1096, a Mycobacterium smegmatis transposable element, at a cluster of sites located within a small region of 60 bp, immediately upstream of a kanamycin resistance gene present in these vectors. The structural alterations led to deletion of large regions of the vector which, in several cases, were found to extend into the ORF2 (RepB) coding sequences of the pAL5000 replication region without affecting its replication capability. This suggests that the entire ORF2 coding sequences of the pAL5000 replication region may not be essential for replication of pAL5000- derived vectors. The deletion derivatives, which contain the minimal sequences required for replication and selection in mycobacteria, were found to be structurally stable and therefore these could be potentially used as stable vector systems for the transformation of mycobacteria. © 1999 Academic Press In recent years significant effort has been made to understand the molecular biology of Mycobacterium tuberculosis with the objective of developing newer methods for the prevention and cure of Tuberculosis (Hatfull, 1993). The availability of Escherichia coli–mycobacteria shuttle vectors based on the M. fortuitum plas- mid pAL5000 (Rauzier et al., 1988) has been very useful in this context (Snapper et al., 1990, Das Gupta et al., 1993, 1998; Hatfull, 1993). Although these vectors have been used often, the mechanisms of their replication and stability are not known in detail, which limits their us- age. While studying the replication of E. coli– mycobacteria shuttle vectors in M. smegmatis, we came across a unique phenomenon of trans- position of IS1096, an M. smegmatis transposon (Cirillo et al., 1991), at a cluster of closely linked sites in the vectors, resulting in large- scale deletions. The information provided in this study, will give a better insight into the aspect of vector instability in mycobacteria and therefore help in designing improved mycobac- terial vectors. The shuttle vectors were constructed using standard techniques (Sambrook et al., 1989) as shown in Fig. 1. Fragments of different sizes derived from the replication region of the plas- mid pAL5000 were cloned at the AccI/HincII sites of the vector pBC1, a modified pUC19 vector carrying a kanamycin resistance marker (kan) in addition to that of ampicillin (amp). The plasmid pMC1 harbors the EcoRV–HpaI fragment of pAL5000, which has been used widely as a replication cassette for mycobacte- rial vectors (Snapper et al., 1990). In pMC 2, 3, and 6 smaller fragments derived from within this region have been cloned. The fragment in pMC6 is the smallest replication-competent fragment derived from pAL5000 (Stolt and Stoker, 1996a). Essentially this region consists of the tandemly arranged ORFs 1 and 2 (Fig. 1) which code for the putative replication proteins 1 To whom correspondence should be addressed. Fax: 91-33-334-3886. E-mail: sujoy@boseinst.ernet.in. 135 0147-619X/99 $30.00 Copyright © 1999 by Academic Press All rights of reproduction in any form reserved. Plasmid 41, 135–140 (1999) Article ID plas.1998.1384, available online at http://www.idealibrary.com on