role for point mutations and deletions in transposon ... - Europe PMC

The EMBO Journal Vol.1 No.6

pp.755-759, 1982

Phenotypic reversion of an ISI-mediated deletion mutation: a combined role for point mutations and deletions in transposon evolution

Shigeru lida*, Roberto Marcoli1, and Thomas A. Bickle Department of Microbiology, Biozentrum, University of Basel, Klingelbergstrasse 70, CH-4056 Basel, Switzerland Communicated by T.A. Bickle Received on 24 June 1982

We have physically characterised a deletion mutant of the R plasmid R100 which has lost all of the antibiotic resistances, including chloramphenicol resistance (Cmr), coded by its IS1flanked r-determinant. The deletion was mediated by one of the flanking IS1 elements and terminates within the carboxyl terminus of the Cmr gene. DNA sequence analysis showed that the mutated gene would produce a protein 20 amino acids longer than the wild-type due to fusion with an open reading frame in the IS element. Surprisingly for a deletion mutation, rare, spontaneous Cmr revertants could be recovered. Two of the four revertants studied had frame shifts due to the insertion of a single AT base pair at the same position; the revertants would code for a protein five amino acids shorter than the wild-type. The other two revertants had acquired duplications of the 34-bp inverted terminal repeat sequences of the IS1 element and would direct the synthesis of a protein six amino acids longer than the wild-type. The reverted Cmr markers were still capable of transposition. These observations suggest a role for point mutations and small DNA rearrangements in the formation of new gene organisations produced by mobile genetic elements. Key words: IS-flanked transposons/ISI/Cmr gene/spontaneous mutations/evolution

Introduction In recent years it has become clear that many transposons are flanked by IS elements and that it is the IS elements that provide the transposition functions (Calos and Miller, 1980; Starlinger, 1980; Kleckner, 1981; Iida et al., 1982). Th-ese IS flanked transposons are thought to have evolved by the sequential acquisition of IS elements on either side of the gene or genes destined to become part of the transposon (Iida et al., 1980, 1982). On the other hand, many IS elements have a more or less marked target specificity (Kleckner, 1981; Iida et al., 1982). ISi, for example, integrated preferentially into DNA sequences that have some homology with its termini and that lie within A + T-rich DNA segments while some other IS elements have more stringent specificities (Galas et al., 1980; Klaer et al., 1980; Meyer et al., 1980; Saedler et al., 1980; Engler and Van Bree, 1981; Halling and Kleckner, 1981). These target specificities might be important for the evolution and structure of new transposons. An IS element could integrate into the control region in front of a gene or into the structural gene itself. If such a mutated gene acquired another copy of the IS element in its vicinity, it might become transposable but without conferring a phenotype. Occasion-

ally, point mutations or small DNA rearrangements may restore the function of the mutated gene converting the cryptic transposon to a normal one. Alternatively, a gene within an IS flanked transposon may be mutated due to the activities of its IS elements, particularly IS-mediated deletion (Reif and Saedler, 1975; lida and Arber, 1980), converting an active transposon into one that is phenotypically silent. Again, further spontaneous mutation may restore the phenotype. Here, we describe an example of the latter process observed in an ISI flanked chloramphenicol resistance (Cmr) transposon derived from TN2670, the r-determinant of the R-plasmid R100 (Hu et al., 1975; Arber et al., 1978; lida and Arber, 1980; lida et al., 1981a). Earlier, we had isolated a variety of ISI-mediated deletion derivatives of this transposon that still carried the cat gene for Cmr (Arber et al., 1978; lida and Arber, 1980). In the course of these studies we found that the cat gene product also confers resistance to fusidic acid (Far) (Marcoli et al., 1980; Volker et al., 1982). We were, therefore, intrigued by a report that another deletion derivative of TN2670 on a R100 derivative, called pEDR104, provided resistance to Cm but not to Fa (Dempsey and Willetts, 1976). We analysed strains containing this plasmid and have cleared up the contradiction. These strains are sensitive to chloramphenicol itself but are resistant to the chloramphenicol succinate (Cmsuc) used in the earlier study (Dempsey and Willets, 1976). Despite the fact that the Cms and Fae phenotype of strains carrying pEDR104 is clearly due to deletion of the carboxyl terminus of the cat gene, spontaneous revertants that have regained resistance to both Cm and Fa can be isolated. The properties and structure of both the mutant and the pseudo-revertants are presented.

'Present address: ISREC, Chemin des Boveresses, CH-1066 Epalinges s/Lausanne, Switzerland. *To whom reprint requests should be sent.

Results The physical structure of pEDR104 The plasmid pEDR104 was isolated as a temperatureresistant revertant of a strain harbouring RIOO::XcI857susS7b5l5b519 (Dempsey and Willetts, 1976). In the parent strain, the X prophage was integrated between the genes for resistance to mercury (Hgr) and sulphonamide (Sur) of the r-determinant, or Tn2670. The revertant was still resistant to tetracycline (Tc) and Cmsuc but was no longer resistant to streptomycin (Sm), sulphonamide, mercury, or fusidic acid. From these results, Dempsey and Willets (1976) concluded that pEDR104 had a deletion in the r-determinant that removed the X prophage, the genes for Far, Smr, Sur, and Hgr but left the cat gene for Cmr intact. We have analysed the plasmid pEDR104 by restriction cleavage and Southern hybridisation (Figure 1) and have constructed the physical map shown in Figure 2. As predicted by Dempsey and Willetts (1976), most of the r-determinant has been deleted. The deletion was mediated by ISIb and pEDR104 still carries two copies of ISI. The length of the DNA between them is 0.06 kb less than that of the smallest Cmr transposon that we know of, Tn981 (or TnCm2O4) which contains 921 bp of DNA between its IS1 elements (Arber et al., 1978; Marcoli et al., 1980; Reif, 1980; Figure 2).

© IRL Press Limited, Oxford, England. 0261-4189/82/0106-0755$2.00/0.

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S. Uda, R. Marcoli and T.A. Bickle

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Fig. 1. Restriction cleavage and hybridisation analysis of pEDR104 DNA. The electrophoresis was on 0.8%7o agarose gels. A. EcoRI fragments of R100-1; B, EcoRI fragments of pEDR104 DNA; C, BglIl fragments of R10-l DNA; D, BgllI fragments of pEDR104 DNA. The arrows indicate fragments containing sequences of IS1 detected by hybridisation with 32Plabelled Xrl4::ISI. ISla lies in the longest EcoRI and the shorter BgllI fragment of R100-l (lida, 1980). Note that the Cm segment between the two IS elements of pEDR104 contains a single EcoRI site and no BglII site. All of the fragments from the RTF segment of RIOO are present in pEDR104 whereas most of those from the r-determinants are absent (lida, 1980). E, PstI fragments of PIA::Tn981; F, PstI fragments of pEDR104; G, PMtI fragments of pSHI161, a pBR322 derivative containing the PstI Cmr fragment from pEDR104-1; H, hybridisation of the PstI fragments of pEDR104 with Xrl4::ISl; I and J, hybridisation of PMtI digests of PIA::Tn981 and pEDR104, respectively, with 32P-labelled pBR325 DNA. PIA::Tn981 is a PlCm204 derivative carrying two copies of IS1 (Arber et al., 1978; Reif, 1980). The arrows labelled y and z point to the Cmr segments generated by PstI digestion. They are 1689 (Tn981) and 1627 (Tn2670A104) bp long (Ohtsubo and Ohtsubo, 1978; Marcoli et al., 1980; this study). The arrow x indicates the position of the 1870-bp PstI Cmr fragment from Tn9 (Alton and Vapnek, 1979).

Antibiotic resistance properties ofpEDR104 Although the strain Escherichia coli K12 DB10 (pEDR104) is resistant to Cmsuc, it is sensitive to Cm at the normally used concentration of 25 itg/ml as well as to Fa. However, this strain is slightly resistant to Cm and can form colonies on plates containing 2.5 jg/ml or less of Cm (Table I). Rare revertants for resistance to Cm at 25 ,tg/ml have been isolated and these revertants are also resistant to Fa. The enhanced resistance to Cm is due to a mutation of the plasmid rather than the host chromosome because passage of the plasmid into E. coli K12 AB1 157 and then back into E. coli K12 DB10 led again to CmrFar cells. The copy number of the revertants pEDR104-1, -19, and -26 does not seem to be higher than that of the parent plasmid, as judged from the intensity of the plasmid bands in cleared lysates following agarose gel electrophoresis and the yield of DNA after CsCl-ethidium bromide centrifugation. Tandem duplications of the cat gene in ISI-flanked transposons has been observed repeatedly in E. coli K12 and this is accompanied by an increased resistance to Cm and Fa (Hashimoto and Round, 1975; Chandler et al., 1979; Meyer and Iida, 1979). Furthermore, cloning of the Cmr segment of pEDR104 into the high copy number plasmid pBR322 leads to increased resistance to both Cm and Fa (Table I). However, comparison of the restriction cleavage patterns of plasmids carrying the reversions to higher Cmr with the parent pEDR104 indicated these plasmids had no such tandem gene amplification (data not shown). 756

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Fig. 2. The structure of pEDR104 compared to RIOO. The physical map of RIOO and the location of the drug resistance genes in the r-determinant have been described earlier (Arber et al., 1978; lida, 1980). Tra indicates the region coding for conjugative transfer genes and Rep the region necessary for autonomous replication including the origin of DNA replication. The RTF part flanked by two ISi elements contains the Tcr transposon TnlO, Tra genes, and Rep region. The arrow marked X on the Tn2670 map shows the integration site of the X genome in the RIOO::X cointegrate which is the precursor of pEDR104 (Dempsey and Willetts, 1976; Dempsey et al., 1978). In pEDR104, an ISlb-mediated deletion has removed most of the r-determinant together with the integrated X DNA and produced Tn267OA104. ISI elements are drawn as boxes and the vertical line within the boxes marks the position of the single Pstl site and indicates the orientation of the element (Ohtsubo and Ohtsubo, 1978). The horizontal arrow under the Cm/Fa shows the position and orientation of the cat gene for Cmr (Alton and Vapnek, 1979; Marcoli et al., 1980). The bottom part of the figure shows the strategy used for sequencing the junction between ISlb and the cat gene in pEDR104 and its revertants to Cm'. The restriction sites used were the HaeIII site in the cat gene at position 704 (Marcoli et al., 1980) and the AluI and Tthl 11I at positions 702 and 712, respectively, in the ISI (Ohtsubo and Ohtsubo, 1978).

DNA sequence analysis of the cat gene carried on pEDR104 and its revertants We have cloned the cat gene from pEDR104 and from four independently isolated revertants to high Cmr into the plasmid pBR322 either in vitro or in vivo (see Materials and methods) and determined the DNA sequence around the carboxyl terminus of these cat genes. The strategy used for the DNA sequencing is shown in Figure 2 and the results are summarised in Figure 3. The DNA sequence confirms that pEDR104 has an ISibmediated deletion into the carboxyl terminus of the cat gene. The deletion removes 21 bp from the end of the gene and leaves it in phase with an open reading frame in the S11 element. The protein that this gene would produce is 20 amino acids longer than the wild-type protein and the last 27 amino acids would be garbled. These drastic alterations to the carboxyl terminus of the cat gene product are clearly responsible for the reduced resistance to Fa and Cm. All four of the spontaneous revertants have small DNA rearrangements that result in the production of a protein lacking much of the extraneous carboxyl terminal tail of the mutant. Two of them (pEDR104-1 and -26) are identical and are insertions of a single ATf base pair that result in the production of a protein five amino acids shorter than the wild-type. The DNA sequence of the regulatory region preceding the cat

Post-transposiffonal modulation

Table 1. Drug resistance phenotypes of strains harbouring pEDR104 and its related plasmids Efficiency of colony formation on LA plates containing drug Cm Cmsuc 2.5 Ag/ml 5 ,g/ml 10 1tg/ml 50 1g/ml

Strain

DBIO DBlO (pEDR104) DBI0 (pEDR104-1) DB1O(pEDR104-7) DBIO (R100-1) DBIO (pBR322:Cm-104) DBIO (pBR322:Cm-104-1) DBIO (pBR325) AB1157 AB1 157 (pEDR104) AB1 157 (pEDR104-1) AB1157 (pEDR104-7) AB1157 (R100-1)