Regulation of the “tetCD” genes of transposon Tn101

J. Mol. Biol. (1997) 270, 14±25

Regulation of the ``tetCD'' Genes of Transposon Tn10 Cynthia M. Pepe, Chise Suzuki, Cynthia Laurie and Robert W. Simons* Department of Microbiology and Molecular Genetics, the Molecular Biology Institute and the Jonsson Comprehensive Cancer Center, University of California, Los Angeles, CA USA

In addition to the genes involved in tetracycline resistance, the loop region of the composite transposon Tn10 contains two other known genes, tetC and tetD, whose functions are unclear. Using primarily a genetic approach, we examined tetCD gene expression and regulation. The tetC gene product, TetC, is a diffusible repressor of both tetC and tetD transcription. Despite an earlier claim by others, we do not detect induction of either tetC or tetD by tetracycline (Tc) or several of its analogs. Although the 50 ends of the tetC and tetD messages overlap due to transcription from convergent promoters, we ®nd no evidence for anti-sense RNA control. The operator for the TetC repressor has been localized. We also demonstrate that transcription from the tetD promoter probably terminates within IS10-Right and does not apparently interfere with Tn10 or IS10-Right transposition or its regulation. # 1997 Academic Press Limited

*Corresponding author

Keywords: tetracycline resistance; transposition; transcription; gene regulation

Introduction Tn10 is a well-characterized composite transposon (for reviews, see Kleckner, 1989, 1990) consisting of a 6.5 kb unique loop region ¯anked by nearly identical inverted repeats of insertion sequence IS10 (Figures 1 to 3). Four genes, tetA, tetR, tetC, and tetD, are located in the right half of the loop (Jorgensen & Reznikoff, 1979; Wray et al., 1981; Hillen & Schollmeier, 1983; Postle et al., 1984; Braus et al., 1984; Schollmeier & Hillen, 1984). IS10Right (IS10-R) is structurally and functionally intact and codes for the IS10 transposase (tnp), whose expression is rate-limiting for Tn10 and IS10 transposition (Halling et al., 1982; Morisato et al., 1983). IS10-Left (IS10-L) is structurally intact but does not encode a functional transposase (Foster et al., 1981; Halling et al., 1982). The transposase acts at the outside ends of the IS10-R and IS10-L to promote Tn10 transposition, or at the outside and Present address: C. M. Pepe, U.S. Department of Commerce National Marine Fisheries Service, Northwest Fisheries Science Center, 2725 Montlake Blvd. E. Seattle, WA 98112, USA; C. Suzuki, National Food Research Institute, 2-1-2 Kannondai, Tsukuba-shi Ibaraki 305, Japan. Abbreviations used: Tc, tetracycline; IPTG, isopropylb-D-galactopyranoside; ADTc, 2-acetyl-2decarboxyamide Tc; Cm, chloramphenicol; Km, kamamycin; R, Right; L, Left; CITc, chlortetracycline; AHTc, anhydrotetracycline. 0022±2836/97/260014±12 $25.00/0/mb971094

inside ends of the IS10 elements themselves, to promote their independent transposition (Kleckner, 1989). Expression of the IS10-R tnp gene is carefully controlled at a number of levels (Kleckner, 1989, 1990). There are two well-characterized promoters in IS10-R (Figure 1; Simons et al., 1983; Case et al., 1988). pIN speci®es the tnp message, RNA-IN. pOUT speci®es a 69 nt anti-sense RNA, RNA-OUT, which binds speci®cally to RNA-IN and blocks translation of the tnp gene, presumably to prevent accumulation of Tn10 or IS10 elements to detrimental levels (Simons & Kleckner, 1983; Ma & Simons, 1990; J. Matsunaga & R.W.S., unpublished observations). Mutations in pOUT that decrease RNA-OUT expression are suppressed by heterologous upstream promoters, which produce longer transcripts (generally termed RNA-OUT0 ) that are rapidly processed by ribonuclease E to RNAOUT* (C.M.P., C.S. & R.W.S., unpublished results), which differs from RNA-OUT by two additional 50 nucleotides (Case et al., 1988). Case et al. (1988) proposed that a naturally occurring promoter within Tn10, possibly the tetD promoter, might produce RNA-OUT*, although this possibility was not speci®cally examined. Interestingly, transcription across the outside end (OE) of IS10-R inhibits transposition, presumably by interfering with transposase binding (Davis et al., 1985). Whether transcription from the tetD (or another) promoter # 1997 Academic Press Limited

Tn10 Gene Expression

15

Figure 1. The structure of Tn10. The right half of transposon Tn10 is shown to scale and numbered from the outside end (OE) of IS10-Right (IS10-R), which encodes the transposase gene (tnp) (Foster et al., 1981; Halling et al., 1982). The tetC, tetD, tetA, and tetR genes located in the Tn10 loop region are also indicated. Straight arrows show the direction of open reading frames. IS10-R and tetD overlap slightly. Bent arrows indicate the principal promoters. pIN and pOUT specify the IS10-R tnp mRNA and anti-sense RNA, respectively. pC and pD specify the tetC and tetD mRNAs, respectively. The ®lled box between tetC and tetA represents a bidirectional transcription terminator (ter) (Schollmeier et al., 1985). IE, inside end of IS10-R.

similarly affects inside end (IE) activity is not known. The tetA and tetR genes are involved in Tn10 Tc resistance (TcR; Jorgensen & Reznikoff, 1979) and are transcribed from divergent, overlapping promoters, pA and pR (Figure 1; Bertrand et al., 1983; Wray et al., 1981). The tetA gene product affords TcR (McMurry et al., 1980; Levy, 1992). tetR encodes a Tc-inducible repressor that negatively regulates transcription of both tetA and tetR (Beck et al., 1982; Wray et al., 1981; Jorgensen & Reznikoff, 1979; Hillen et al., 1983, 1984; Bertrand et al., 1983). The function of the tetCD genes is unclear and there is con¯icting evidence that they play a role in TcR. Deletion analysis suggested that tetCD might play some role in high level Tc resistance (Jorgensen & Reznikoff, 1979; Coleman & Foster,

1981), but Braus et al. (1984) were unable to con®rm this observation. The tetC and tetD genes are transcribed in opposite directions from the convergent pC and pD promoters (Figures 1 and 2; Schollmeier & Hillen, 1984). Palindromic sequences near pC and pD may serve as operators (Figure 2; Schollmeier & Hillen, 1984). Braus et al. (1984) demonstrated tetCD expression and suggested that the tetD gene product is membrane-associated and induced by the Tc analog, 2-acetyl-2-decarboxyamide tetracycline (ADTc), which also induces the tetAR system but is not an antibiotic (Inoue et al., 1977). We reasoned that transcription from pD could, in principle, alter IS10-R activity by perturbing inside end (IE) function, interfering with transcription from the opposing tnp promoter, pIN, or increasing anti-sense control by producing RNA-

Figure 2. Sequence of the tetC/tetD control region. Base-pairs 1675 to 1974 of Tn10 (numbered from the outside end of IS10-R) are shown along with relevant restriction sites, the conserved ÿ10 and ÿ35 regions (boxed) of the pC and pD promoters, the 50 ends of the tetC and tetD transcripts (bent arrows at base-pairs 1802 and 1815, respectively), the putative start codons for tetC and tetD (boxed ATGs at base-pairs 1830 and 1742, respectively), two dyad symmetries centered at base-pairs 1806 and 1964 (broken arrows), and the sequences of primers used for analysis of the tetC and tetD mRNAs (boxed).

16

Tn10 Gene Expression

Figure 3. Tn10 plasmids. Shown is a set of related multicopy plasmids bearing different portions (shaded boxes) of Tn10. pCP47, pCP57, pCP51, pCP34 and pCP58 were constructed by inserting fragments into pBR333 (Foster et al., 1981). pCP52 is pCP57 with a ®lled-in BglII site. pCP38 was constructed by inserting the DraI-ClaI fragment into the ®lled-in BamHI site of pRS475. pCP49, pCP48 and pCP50 are pCP38 with ®lled-in BglII or HindIII sites (*), or a deletion between those sites (broken line), respectively. Repression of tetC and tetD fusion expression by each plasmid is summarized as ‡ or ÿ and was taken from Table 1 or similar experiments.

OUT*. To address these questions we ®rst needed to analyze tetCD expression and regulation . Here, we show that TetC represses transcription from both pC and pD, and that transcription from pD probably proceeds only partway into IS10-R. We ®nd that pD transcription has no apparent effect on RNA-OUT or transposase expression, nor any effect on inside end activity under the conditions analyzed.

The major 50 ends of the tetC and tetD transcripts map, respectively, to base-pairs 1802 and 1815 of Tn10 (numbered from the outside end of IS10-R; see Figures 1 and 2), and these termini are the same in both the tetC‡ (repressed) and tetCÿ (derepressed) cases. They differ

Experimental Results Disruption of the tetC gene increases tetC and tetD transcription We used primer extension analysis to identify the tetC and tetD mRNAs expressed in vivo from multicopy plasmids containing intact or mutated tetC and tetD genes (Figure 3). When both genes are intact (pCP47; Figure 3), the tetD transcript is easily detected (Figure 4, lane 1). The tetC transcript is more dif®cult to detect (Figure 4, lane 5), and neither transcript is detected in vivo with plasmids lacking these genes (data not shown). With pCP58, in which tetC is partially deleted (Figure 3), both the tetC and tetD transcript levels increase substantially (Figure 4, lanes 3 and 7). On the other hand, with pCP57, in which tetD is partially deleted, no increase in either transcript is seen (Figure 4, lanes 2 and 6). Finally, with pCP34 (Figure 3), where both tetC and tetD are partially deleted, transcript levels are the same (Figure 4, lanes 4 and 8) as seen with the partial deletion of tetC alone. Thus, the tetC gene product apparently represses transcription from pC and pD, whereas tetD plays no obvious role in this regard.

Figure 4. Primer extension analysis of the tetC and tetD mRNAs. Total cellular RNA was extracted from DR459 transformed with an isogenic set of multicopy plasmids containing mutations in the tetC and/or tetD genes, and analyzed by primer extension essentially as described (Pepe et al., 1994). The tetD and tetC primers shown in Figure 2 were used to produce the left and right panels, respectively. Plasmids were pCP47 (lanes 1 and 5), pCP57 (2 and 6), pCP58 (3 and 7) and pCP34 (4 and 8). Lanes 3 and 4 were loaded with 0.1 mg total cellular RNA, 7 and 8 with 1 mg, and all others with 10 mg. Since the two panels were derived from separate experiments, relative band intensities are comparable within, but not between, panels. Sequence ladders were generated from pCP34 DNA with the same primers and labeled to correspond to the respective mRNA sequences.

17

Tn10 Gene Expression

Figure 5. tetC and tetD fusion to the lacZ gene. Transcriptional (lacZ‡) or translational (`lacZ) fusions were constructed on plasmids by inserting the indicated elements (refer to Figure 1) into pRS1274 (elements 1, 3 and 9), pRS415 (elements 5, 6, 7 and 10), pRS591 (elements 2 and 4), or pRS414 (element 8). Element 6 was derived from element 5 by ®lling in the HindIII site (*). Elements 3 and 4 extend from NheI to the ClaI site in tetA. Element 10 extends from EcoRI to the BglII site downstream of tetR. Fusions were crossed to lRS45 to yield the listed phages, which were integrated at the latt site of DR459 and b-galactosidase activities determined, all as described (Simons et al., 1987). The values shown are the averages of at least three independent determinations (standard errors were <5% of the mean values). Monomers of b-galactosidase per cell were estimated according to Miller (1992). Bent arrows depict the pC, pD, pA and pR promoters (refer to Figure 1). Bg, BglII; Nh, NheI; Mb, MboII; Ec, EcoRI. slightly from the termini identi®ed earlier by S1 analysis (Schollmeier & Hillen, 1984), which correspond to basepairs 1800 and 1817, respectively (see Figure 2), and we do not detect the minor tetD transcript reported in that earlier study (corresponding to base-pair 1781 in Figure 2). These differences are minor and probably re¯ect differences between the primer extension and S1 analyses. The putative ÿ10 and ÿ35 promoter sequences lying upstream of the 50 ends are indicated in Figure 2 and are the same as those proposed earlier (Schollmeier & Hillen, 1984). The magnitude of derepression is 100fold for both tetC and tetD. tetC disruption also increases tetC-lacZ and tetDlacZ fusion expression To further analyze tetCD expression, we constructed a series of single-copy genetic fusions between tetC or tetD and the lacZ reporter gene (Figure 5). As expected, expression from an operon (transcriptional) fusion between the tetD promoter and an intact lacZ reporter gene lacking its own promoter (pD-lacZ‡) is 20-fold lower in the presence of the upstream tetC gene in its native context (lRS430), than when that tetC gene is partially deleted (lRS417). The same is true for isogenic tetD0 -0 lacZ protein (translational) fusions in which the tetD gene is fused inframe to a truncated 0 lacZ reporter gene (Figure 5; cf. lRS427 and lRS416). Similarly, expression from a pClacZ‡ operon fusion, whose fusion junction is immediately downstream of the intact tetC gene (lRS469), is

about eightfold lower than an isogenic fusion in which the tetC gene is disrupted (lRS471). These results are consistent with the primer extension studies described above, although the magnitude of derepression is less, possibly due to gene dosage effects (multicopy in the RNA studies; single copy in the fusions). tetC encodes a trans-acting repressor We also measured expression from single-copy tetC and tetD transcriptional and translational fusions lacking an intact tetC gene (Figure 5; lRS412, 413, 416 and 417) in the presence of multicopy plasmids carrying various portions of Tn10 (see Figure 3), to determine if tetC represses in trans, and whether any other Tn10 element is involved. These results are shown in Table 1 and summarized in Figure 3. Whenever the multicopy plasmid contains an intact tetC gene (pNK81, pNK82, pCP47, and pCP57), expression from the tetC and tetD operon and protein fusions is repressed 5 and 20-fold, respectively. No repression is seen with plasmids in which tetC is lacking (pNK83) or disrupted [pCP34 and pCP58 (Table 1 and Figure 3); pCP51 and pCP52 (Figure 3)]. No Tn10 element other than tetC appears to in¯uence tetC or tetD expression (cf. pNK81 and pCP57). Moreover, since we observe equivalent repression of isogenic protein and operon fusions, control appears to occur at the transcriptional level. When the multicopy plasmid contains a placUV5-tetC fusion (pCP38) in which the tetC gene (lacking pC) is

18

Tn10 Gene Expression Table 1. Repression of tetC and tetD fusion expression by tetC in trans Fusion expression (units b-galactosidase) fromb

Multicopy plasmida No pNK81 pNK82 pNK83 pCP47 pCP57 pCP58 pCP34 pCP38 pCP38 pCP48 pCP48

Genotype

IPTG

pD-lacZ‡ (lRS417)

tetD0 -0 lacZ (lRS416)

pC-lacZ‡ (lRS413)

tetC0 -0 lacZ (lRS412)

Tn10 Tn10 (Rt. 12) Tn10 (Lt. 12) tetC‡ tetD‡ tetC‡ tetDÿ tetCÿ tetD‡ tetCÿ tetDÿ placUV5-tetC‡ placUV5-tetC‡ placUV5-tetCÿ placUV5-tetCÿ

na na na na na na na ÿ ‡ ÿ ‡

365 265 6825 355 335 5880 6220 200 40 5750 4745

45 50 1180 50 45 1430 1200 60 35 4620 4980

50 40 265 54 50 265 235 10 9 370 345

8 6 50 8 7 40 35 3 1 185 140

a

Multicopy plasmids (ApR) are as described for Figure 3. DR459 was lysogenized with the indicated phage (Figure 5) and transformed with plasmids listed (Figure 3). For the placUV5 containing plasmids (pCP38, pCP48), the lysogens also contained the F0 lacIQ lacZ::Tn5 episome from NK7386 (Davis et al., 1985). b-Galactosidase activities were determined as described (Simons et al., 1987) and the values shown are the averages of at least three independent determinations (standard deviations were <15% of mean values). na, not applicable. b

transcribed from the inducible lacUV5 promoter and operator, repression of the single-copy tetC and tetD fusions to lacZ is induced by IPTG (about tenfold for operon fusions and two- to threefold for protein fusions). No repression is seen when the tetC gene in this plasmid is disrupted (pCP48). The level of repression achieved with the fully induced placUV5-tetC construct is about tenfold greater than repression by pC-tetC (cf. pCP38 (‡IPTG) with pCP57), consistent with the observation that placUV5 is stronger than pC. In the absence of IPTG there is 10 to 30-fold repression by placUV5-tetC (cf. pCP38(ÿIPTG) with pCP34). This apparent failure to fully repress placUV5 in the absence of IPTG may result from interference by the opposing pD promoter, which is present in the placUV5-tetC construct (under these experimental conditions there is suf®cient lac repressor to fully repress a multicopy operator; Simons et al., 1987). In any case, these observations, together with the genetic and transcriptional studies described above, show that tetC encodes a diffusible repressor of tetC and tetD transcription. tetC and tetD expression levels The level of b-galactosidase expression from the fusion vectors used here is a good indicator of relative transcriptional and translational activity (Simons et al., 1987). Accordingly, we estimate that the fully derepressed pD promoter is 20-fold stronger than the fully derepressed pC promoter (cf. lRS417 and lRS413, Figure 5). In addition, the well-characterized placUV5 promoter is about fourfold weaker than pD and about sixfold stronger than pC (cf. lRS74 with lRS417 and lRS413, Figure 5). The tetD gene is translated about 50% more ef®ciently than tetC (cf. the ratios of lRS416 to lRS417 and lRS412 to lrid="f5">RS413, Figure 5), and both genes are translated less ef®ciently than lacZ (cf. lRS416 with lRS417, and lRS412 with lRS413, Figure 5). Assuming similar stabilities of the tetD and tetD0 -0 lacZ gene products, we estimate that there are 200 TetD monomers/cell when tetD is repressed and 6000/cell when fully derepressed (see the legend to Figure 5). Similarly, we estimate that there are 200 TetC monomers/cell when tetC is fully derepressed.

Tetracycline does not induce tetC or tetD fusion expression The tetC and tetD genes were named on the basis of experiments suggesting that the tetD gene product was inducible by ADTc (Braus et al., 1984), despite the fact that these genes play no obvious role in Tc resistance (Coleman & Foster, 1981; Braus et al., 1984). We re-examined this question with the fusions described above. In all of these experiments, both protein (not shown) and operon fusions to tetC and tetD were examined. As a control, we also examined a [tetR‡]-pA-lacZ‡ operon fusion (lRS535; Figure 5), which is inducible by Tc and its analogs (see below). ADTc had no effect on tetC and tetD fusion expression, nor on the [tetR‡]-pA-lacZ‡ fusion (not shown), suggesting that the stock of ADTc made available to us was not active. When we tested two other widely used Tc analogs, autoclaved chlortetracycline (ClTc; Bochner et al., 1980) and anhydrotetracycline (AHTc; Moyed & Bertrand, 1983), both induced the [tetR‡]-pA-lacZ‡ fusion, but neither induced any of a variety of tetC or tetD fusions (Table 2 and data not shown). Finally, we examined the effects of Tc itself, in cells rendered TcR by the presence of an intact Tn10 element (Table 2, lines 6 to 10), a miniTn10 element (an abbreviated Tn10 element containing only the tetAR genes; data not shown), or pBR322 (data not shown). Although induction of the [tetR‡]-pA-lacZ‡ fusion is clearly seen with both Tc and ClTc under these conditions, no induction is seen with any tetC or tetD fusion (Table 2, lines 6 to 10). In these experiments there was considerably less repression of pD-lacZ‡ expression by the unlinked Tn10 element (which presumably encodes a normal tetC gene), than when the tetC gene is located directly upstream of this fusion (cf. lines 1 and 6 to lines 1 and 2: Table 2). Whether this re¯ects preferential cis-action of the TetC repressor is not known. We also examined a number of other antibiotics including ampicillin, chloramphenicol, gentamicin, nalidixic acid, novobiocin and penicillin, and found that none induced the [tetC‡]-tetD0 -0 lacZ fusion (lRS427) at sublethal concentrations (data not shown).

19

Tn10 Gene Expression Table 2. Effect of tetracycline on tetC, tetD and tetA fusion expression Fusion expression (units b-gal) withb No 1 2 3 4 5 6 7 8 9 10

Prophageb lRS417 lRS430 lRS469 lRS471 lRS535 lRS417 lRS430 lRS469 lRS471 lRS535

Operon fusion ‡

pD-lacZ [tetC‡]-pD-lacZ‡ pC-[tetC‡]-lacZ‡ pC-[tetCÿ]-lacZ‡ [tetR‡]-pA-lacZ‡ pD-lacZ‡ [tetC‡]-pD-lacZ‡ pC-[tetC‡]-lacZ‡ pC-[tetCÿ]-lacZ‡ [tetR‡]-pA-lacZ‡

TcR/S

±

Tc

ClTc

S S S S S R R R R R

7405 295 30 125 115 2290 190 15 44 95

± ± ± ± ± 970 85 5 21 4475

6260 250 25 115 3065 1380 130 10 24 3355

a

Phage (see Figure 5) were integrated at the latt sites of DR459 (TcS; S) or RS6359 (TcR; R). b-Galactosidase assays were done as for Table 1. When present, Tc and autoclaved ClTc were added at 15 and 50 mg/ml, respectively. b

Tetracycline does not induce the synthesis of tetC or tetD transcription Although Tc does not induce the tetC and tetD fusions described above, it remained possible that induction might occur with fully intact tetC and tetD genes in the context (or presence) of Tn10. To address this possibility, we examined the effects of Tc on the steady-state levels of tetC and tetD mRNAs expressed either from a single-copy, intact Tn10 element (data not shown) or from a multicopy tetC‡D0 plasmid (pCP57) transformed into a strain containing a singlecopy mini-Tn10 (TcR) element (which lacks tetCD; Table 3). Surprisingly, we occasionally observed that the steady-state levels of both transcripts increased slightly when these cells are grown in the presence of Tc, and that this apparent ``induction'' was also seen with a plasmid (pCP51) in which the tetC and tetD transcripts are already derepressed by a tetC mutation (data not shown). This effect was inconsistent, ranged from two to sevenfold, and was only observed with Tc concentrations above 10 mg/ml (data not shown). Given these observations and our failure to detect induction of the tetD and tetC fusions, we sought an alternative explanation for the apparent ``induction'' of the mRNAs. It is known that Tc and chloramphenicol Table 3. Effects of tetracycline and chloramphenicol on the tetD mRNA Cells grown in presence of:a ± Tc Cm

Relative tetD mRNA levelb

tetD mRNA t12 (minutes)c

1.0 3.6 3.1

2.5 8.0 10.0

a RS6510 (TcR CmS) was transformed with pCP57 (tetD0 tetC‡ tetAÿ), grown to mid-log in LB medium Tc (15 mg/ml), or to mid-log in LB and then for 30 minutes with Cm (200 mg/ml), aliquots removed at zero, 0.5, one, two, three, and four minutes after the addition of rifampicin (200 mg/ml), and total cellular RNA extracted and analyzed by primer extension with the tetD primer as described for Figure 4. b Relative intensities (quantitated by densitometry) of autoradiographic bands corresponding to the tetD mRNA 50 end at zero minute. c Half-lives (t12) calculated from a plot of tetD 50 end band intensity versus time in each case, as described (Pepe et al., 1994).

(Cm) stabilize mRNAs, by a process thought to involve the stalling of elongating ribosomes (Flache et al., 1992). We reasoned that if Tc ``induced'' the tetC and tetD transcripts by such means, there would be a corresponding effect on transcript stability. Therefore, we measured both the steady-state levels and half-lives of the tetD mRNA in the presence and absence of Tc or Cm. Table 3 shows that, in a typical experiment, both Tc and Cm cause comparable increases in both steady state-level and half-life (three to fourfold), demonstrating that the apparent ``induction'' occurs at the level of transcript stability, not transcription initiation. Therefore, we conclude that Tc does not induce transcription of the tetC or tetD genes, even in the context of a normal Tn10 element. It is worth noting that, since these Tc ``induction'' experiments were performed with cells expressing the TetA protein, which reduces the intracellular Tc concentration (McMurry et al., 1980), we may not have achieved Tc levels suf®cient to stall ribosomes. However, this argument is moot since the effects seen are at the level of transcript stability and they occur when tetC and tetD expression is constitutive (i.e. in the absence of TetC). Moreover, they are modest and also occur with Cm. The TetC binding site is located in the tetCD promoter region It is reasonable to assume that the TetC binding site(s) is located in the region of the tetC and tetD promoters. To examine this likelihood, we measured the ability of multicopy plasmids containing fragments from this region to titrate repression of a single-copy [tetC‡]-tetD0 0 lacZ fusion (lRS427). Multicopy plasmids containing intact promoters (pCP72 and pCP75; Figure 6) titrate repression 10 to 16-fold, whereas plasmids lacking this region (pCP73 and pCP74) titrate repression less than twofold. These results suggest that the TetC binding site(s) lies in the DNA between the A¯II and BglII sites at positions 1760 and 1942, respectively. These results are also consistent with the presence of a site in the tetC and/or tetD mRNAs. However, since both protein and operon fusions respond equivalently to tetC inhibition (Table 1), we do not favor this possibility. As indicated earlier, the tetC and tetD transcripts overlap for 14 nucleotides, raising the possibility of antisense RNA control of tetC and/or tetD expression. However, we see no effect of the multicopy levels of tetC and tetD transcripts expressed from pCP72 or pCP75 on the

20

Tn10 Gene Expression

Figure 6. Titration of fusion expression with multicopy plasmids containing the tetCD control region. Various portions of Tn10 (shaded bars) carried on multicopy plasmids were examined for their effects on the expression of a single-copy [tetC‡]-tetD0 -0 lacZ protein fusion (lRS427; Figure 5) in DR459. The indicated fragments were inserted into the polylinker of pCP73, a pUC19 (Yanisch-Perron et al., 1985) derivative in which rrnB T1 transcriptional terminators (®lled boxes) ¯ank the polylinker site to prevent read-through transcription from adjacent sequences. b-Galactosidase activities were determined as for Figure 5. expression of derepressed tetC or tetD fusions (data not shown). pD transcription probably terminates within IS10-R When the tetC gene is disrupted, transcription is readily detected across the junction between tetD and

IS10-R (data not shown). To determine the extent to which such pD-speci®ed transcription proceeds into the IS10-R element, we carried out a series of related experiments. In the ®rst (Figure 7A), we used multicopy fusions to compare the relative levels of pD-speci®ed transcription at two fusion junctions in Tn10: within the tetD gene itself (pCP37 and pCP195) or just downstream

Figure 7. Analysis of transcription termination in IS10 and tetD. A related set of lacZ fusions were constructed on multicopy plasmids and their levels of expression measured in DR459 as described for Figure 5 (standard errors were <15% of the mean values). A, Operon fusions were constructed by inserting portions of Tn10 (indicated by shaded bars) in front of the lacZ‡ gene of pRS415 (for pCS015 and pCS023) or pRS1274 (for pCP37 and pCP195). pCP195 and pCS023 were constructed by ®lling in the HindIII sites of pCP37 and pCS015, respectively. pCS015 and pCS023 contain the mci11 pOUT-down mutation (Case et al., 1988). B, Composite operon fusions were constructed by inserting portions of Tn10 (shaded bars) between the placUV5 promoter (open arrow) and lacZ‡ gene of pRS475. Values are relative to pRS475 activity (14,000 units).

21

Tn10 Gene Expression

Figure 8. Suppression of the pOUT-mci11 mutation by transcription from the upstream pD promoter. Multicopy plasmids containing various portions of Tn10 (shaded bars; refer to Figure 1) were examined for their effects in RS6224, which contains a single-copy tnp0 -0 lacZ protein fusion whose expression is inhibited by the IS10-R anti-sense RNA, RNA-OUT (Simons et al., 1983; Case et al., 1988, 1989). The mci11 point mutation abolishes pOUT activity (broken arrow; Case et al., 1988). H3* indicates a tetC gene disrupted by ®lling in the HindIII site. Broken lines indicate deleted sequences. b-Galactosidase activities were determined as for Figure 5 (standard errors were <6% of the mean values). Anti-sense control is summarized as ‡ (1 unit),  (7 units) or ÿ (10 to 15 units). of the pOUT promoter in IS10-R (pCS015 and pCS023). As expected, when pD transcription is derepressed, expression within tetD increases 20-fold (cf. pCP37 and pCP195). In contrast, derepression increases transcription at the pOUT region of IS10-R no more than twofold when the pOUT promoter is abolished by the mci11 pOUT-down point mutation (cf. pCS015 and pCS023), and an insigni®cant amount when pOUT is wild-type (cf. values in parentheses). In the second experiment (Figure 7B), we inserted various IS10-R and/or tetD sequences between the placUV5 promoter and the lacZ‡ gene on a multicopy plasmid. These results show that signi®cant transcription termination probably occurs between the NheI site in tetD and the NcoI site in IS10-R. Presumably, this termination would also act on pD transcription. In the third experiment (Figure 8), we measured the ability of pD-speci®ed transcription to suppress the mci11 pOUT-down mutation, thereby restoring antisense RNA control of a single-copy tnp0 -0 lacZ fusion. Such inhibition has been shown elsewhere to be a sensitive and speci®c measure of functional RNA-OUT levels (Simons & Kleckner, 1983; Case et al., 1988, 1989). The results show that pD transcription does, indeed, suppress mci11, but only when most of the DNA between pOUT and tetD is deleted (cf. lines 3 and 4 with lines 5 to 8). Together, these results suggest that little or no pDspeci®ed transcription proceeds into IS10-R, certainly not as far as the pOUT region. Most likely, termination stops at more than one site. When we analyzed these sequences we found no putative rho-independent terminator-like structures, suggesting that termination occurs by a rho-dependent mechanism. This notion is supported by the observation that transcription from the fusions shown in Figure 7 increases four to sixfold in a rho201 mutant (data not shown). We note, however, that other explanations, including effects on transcript stability, are not ruled out.

pD transcription does not alter IS10 or Tn10 transposition We conducted a series of experiments to assess the possible effects of pD-speci®ed transcription on the rates of IS10 and Tn10 transposition. In the ®rst experiment (Figure 9A), we examined the effect of pD-speci®ed transcription on the ability of a multicopy IS10-R element to inhibit the transposition of an intact, single-copy Tn10 element by anti-sense control. The results show that wildtype IS10-R (pRS459) inhibits about threefold (cf. with pRS460, where the mciR5 mutation abolishes anti-sense control), and that this inhibition is not perturbed by derepression of pD (cf. pRS459 and pCS001), consistent with the observations shown in Figure 8. It should be noted that, in this experiment, transposition of the single-copy Tn10 element is not signi®cantly affected by transposase expressed from the multicopy IS10-R element, since transposase acts preferentially in cis (i.e. on the element from which it is expressed; Morisato et al., 1983). In the second experiment, we simultaneously measured the effects of pD derepression on self-driven transposition of a multicopy Tn10 element as well as the ability of that multicopy element to complement a function-defective single-copy Tn10 element in trans (here, the relatively weak trans-activity of the transposase can be detected). These results (Figure 9B) show that pD derepression decreases self-driven transposition (TcR) no more than twofold, with little or no effect on complementation of the single-copy element (KmR). In the third experiment, we examined the effect of pD transcription on transposition of the IS10-R element itself. To do this we used a marked IS10-R element (Roberts et al., 1985) in which the inner end of IS10-R is duplicated so as to permit insertion of a kanamycin resistance (KmR) gene between an intact tnp gene and an intact inside end. Figure 9C shows that the rate of marked IS10-R transposition is unaffected by pD transcription.

22

Tn10 Gene Expression

Figure 9. Effects of pD transcription on Tn10 and IS10 transposition. Transposition of single and/or multicopy Tn10 or IS10 elements to F-pOX38 was measured in a ``mating-out'' assay (Morisato et al., 1983). The recipient strain was NK6441 in all experiments. The donor strains either lack a Tn10 element in their chromosome (CP317), contain a single-copy intact Tn10 element (CP315) conferring TcR (Foster et al., 1981), or contain a single-copy mini-Tn10 element (RS6146) conferring KmR (Way et al., 1984). Donor strains also contain various multicopy plasmids, depending on the following experimental designs. A, Anti-sense control of single-copy Tn10 transposition. pRS459 (see Figure 8) expresses the IS10-R anti-sense RNA, RNA-OUT, which acts in trans to inhibit tnp expression from a single-copy, wild-type Tn10 element (TcR). The mciR5 point mutation in the isogenic pRS460 plasmid abolishes anti-sense control (Case et al., 1988). pCS001 differs from pRS459 by a mutation in tetC (*; ®lled-in HindIII site), which only partially derepresses pD in this cell, since the tetC gene in the chromosomal Tn10 element remains intact. B, Complementation of the tnp-defective mini-Tn10 element. pNK214, which contains an intact IS10-R element and a truncated ``left'' end, provides tnp function for its own transposition (TcR) as well as that of the tnp-defective mini-Tn10 element (KmR). pCP59 differs from pNK214 in that pD is fully derepressed by a mutation in tetC (*). C, IS10-R transposition. The ``marked'' IS10 element (KmR) catalyzes its own transposition. In pNK1166, a portion of IS10-R and the tetD gene (from StuI to XhoII; hatched) was duplicated so that the KmR gene (kan) could be inserted between an intact tnp gene and the IS10-R inside end (Roberts et al., 1985). pCP205 differs from pNK1166 in that pD is fully derepressed by a mutation (*) in tetC. D, Complementation by the marked IS10-R element. pCP208 and pCP207 are KmS versions of pNK1166 and pCP205, respectively (by virtue of a ®lled-in HindIII site in the kan gene), thereby enabling measurement of complemented transposition by the KmR mini-Tn10 element.

In the fourth experiment, we examined the effect of pD transcription on the ability of this same IS10-R element (rendered kanamycin-sensitive; KmS) to complement a single-copy, function-defective KmR Tn10 element. Once again, derepression had little or no effect on the level of complementing function produced. In general, the marked IS10-R element complements transposition of the single-copy KmR Tn10 element about fourfold less well than a multicopy Tn10 element (cf. ``kan hops'' in D and B). This difference might be due to expression of the KmR gene from the marked IS10-R element, whose transcription is directed opposite that of the tnp gene (Roberts et al., 1985). Finally, in these and closely related experiments, we found that Tc had no effect on anti-sense control, complementation, or self-driven transposition of Tn10 or IS10-R (data not shown). Together, these results show that pD transcription across the inside end of IS10-R does not detectably perturb the level of tnp expression or its regulation by RNA-OUT, nor does it affect the activities of the inside or outside ends of IS10-R.

Discussion TetC represses tetC and tetD transcription The results presented here show that the tetC and tetD genes are negatively regulated at the transcriptional level by the tetC gene product, TetC. The genetic arrangement of tetCD super®cially resembles that of tetAR. Both sets of genes are transcribed from divergent promoters, and one of the genes in each pair encodes a repressor that regulates transcription of itself and its partner. The tetCD genes may also have a complex promoter/ operator arrangement analogous to that of tetAR. However, while there are two palindromes that may serve as TetC operators (Figure 2), their sequences are different and only the palindrome near the start sites of both pC and pD is within the region that titrates TetC repression, suggesting a single TetC binding site. Unlike tetAR, Tc does not

23

Tn10 Gene Expression

induce tetCD transcription. The apparent transcript induction we did observe can be attributed to stabilization of these mRNAs by Tc. Flache et al. (1992) reported similar effects of Tc on a tetA-lacZ translational fusion and on the ompA mRNA. Braus et al. (1984) claimed to identify the tetC and tetD gene products in minicells. In addition, they argued that ADTc induced the tetD gene product, as well as the tetA and tetR gene products, from a plasmid in which tetC is partially deleted (thus, the tetD gene would have been transcribed constitutively). When we repeated these minicell experiments as closely as possible, we were unable to detect the TetC protein, although we did detect low levels of the TetD protein if the tetC gene was partially deleted (C.M.P. & R.W.S., unpublished results). We did not observe induction of TetC or TetD proteins by AHTc or autoclaved ClTc, whether or not the tetC gene was intact. tetD function is unknown Based on these and earlier observations by others (Coleman & Foster, 1981; Braus et al., 1984), we conclude that the tetCD genes do not play an obvious role in Tc resistance. TetC shows no signi®cant similarity to other sequences in the protein databases. Interestingly, TetD shows signi®cant similarity to a number of known or putative transcriptional regulators comprising the XylS/AraC family (Gallegos et al., 1993). In particular, TetD is 57% identical to the Escherichia coli Rob protein, a member of a smaller group of proteins related to SoxS and MarA, which are involved in the cell's response to oxidative and other stress, including multiple antibiotic resistance (for a review, see Hidalgo & Demple, 1996). Although Rob and MarA are able to activate transcription of some of the genes in the SoxS regulon (Hidalgo & Demple, 1996), preliminary experiments reveal no obvious effects of TetC or TetD in this regard (E. Simons & R.W.S., unpublished observations). We point out, however, that since the tetD stop codon lies within IS10-R (Schollmeier & Hillen, 1984), Tn10 formation from its original components (presumably two IS10 elements and the genes they now ¯ank) may have truncated the original tetD gene, rendering it inactive. pD transcription does not interfere with IS10 or Tn10 activity Although pD transcription most likely proceeds at least partway into the adjacent IS10-R element, we found that inside end function was unaffected. This observation is in contrast to that of Davis et al. (1985), who found that plac transcription across either the inside or outside ends of Tn10 decreased transposition activity. There are several possible explanations for this difference. First, the experiments reported here were performed in the natural IS10-R/tetD context, as opposed to the arti®cial context in which the lac

promoter directs transcription into IS10-R. Second, the current experiments were performed with multicopy elements, whereas Davis et al. (1985) used single-copy elements, raising the possibility that transcription across the inner end does not affect IS10-R transposition when the element is in multicopy, but does so in singlecopy. Third, it is formally possible that the tetD gene product functions to ``desensitize'' the inner terminus to the effects of transcription. We also found that pD transcription does not affect the expression or function of the anti-sense RNA, RNA-OUT, nor expression of the tnp gene. We believe this may be due, in part, to termination of pD transcription at several rho-dependent and/ or rho-independent sites within IS10-R that have not been mapped precisely. This may be a general mechanism IS10 employs to maintain correct levels of transposase and anti-sense RNA expression when the inside end of the element is positioned adjacent to an active promoter.

Experimental Procedures Media, enzymes and chemicals Media, growth conditions and transformation procedures were as described (Miller, 1992; Simons et al., 1987; Bochner et al., 1980). When used, supplements were added at the following concentrations: ampicillin, 150 mg/ml; Km, 35 mg/ml; streptomycin (Sm), 150 mg/ml; Tc, 15 mg/ml; ClTc, 50 mg/ml; AHTc, 10 mg/ml; ADTc, 5 mg/ml; Xgal, 40 mg/ml. Media and chemicals were purchased from Difco, Sigma, 5-Prime to 3-Prime, ICN or IBI. ADTc was a gift from P®zer, Inc. Enzymes were purchased from New England Biolabs, US Biochemicals or Promega Biotech. Oligonucleotides were obtained from the UCLA DNA Synthesizing Facility. [g-32P]dATP was purchased from New England Nuclear/Dupont. Kodak XAR-5 ®lm and Amersham Hyper®lm-MP were used for autoradiography. Strains DR459 (
lacX74 galOP308 rpsL
(tonB-trpA)905 trpR) and NK6641 (
lac proXIII recA56 thy su StrR lR; Foster et al., 1981) were obtained from N. Kleckner. RS6359 (DR459 srl::Tn10), RS6510 (DR459 zzz::mini-Tn10 (TcR)), RS6146 (F-pOX38:: mini-lac/arg
lac proXIII nalR rifR recA56 ara su Tn10 del16 del17 (KmR TetS)), CP315 (F-pOX38::mini-lac/
lacX74 galOP308 rpsL
(tonB-trpA)905 trpR srl::Tn10 recA56) and CP317 (F-pOX38::mini-tet/arg
lac proXIII nalR rif R recA56 ara su ) were constructed by routine methods. F-pOX38 is a deletion derivative of plasmid F that no longer carries the insertion sequences present on wild-type F (Guyer et al., 1980). The pOX38 plasmids used here are described by Way et al. (1984). pNK81, pNK82, pNK83 and pNK214 have been described (Foster et al., 1981; Way et al., 1984) and

24 these or isogenic point mutant plasmids were the source of Tn10 fragments contained in the plasmids shown in Figures 3 and 5 to 9. pRS414, pRS415, pRS591, pRS1274 (same as pRS528), pRS475, lRS74 and lRS45 are lacZ fusions or vectors and have been described (Simons et al., 1987). Other plasmids and phages were constructed by routine methods (Simons et al., 1987; Miller, 1992; Sambrook et al., 1989) and are described in Figures 3 and 5 to 9. Complete details of all strain constructions are available upon request. Transposition assays All mating-out transposition assays were performed as described (Morisato et al., 1983) with the following modi®cations. The donor strains were either RS6146, CP315 or CP317. The frequency of exconjugants was determined in the case of RS6146 by direct selection of Sm Lac‡ colonies, for CP315 by direct selection of Trp‡ Lac‡ colonies, and for CP317 by direct selection of SmR TcR colonies. For RS6146, transposition exconjugants were selected by plating a total of 1 ml of the mating mixture on LB-Sm-Km media or 0.4 ml on LB-Sm-Tc; for CP315, 1 ml on MinA containing glucose, proline, arginine, thymine, and Tc; for CP317, 0.5 ml on LB-Sm-Km.

Acknowledgements We thank Rick Wolf for unpublished information and useful discussion, Kevin Bertrand for plasmids, the UCLA RIBS computing center for assistance with computational analysis, and all members of the laboratory for continuous support and discussion. C.M.P. was supported in part by an NIH Predoctoral training grant in cellular and molecular biology (T32-GM1075) and an Ursula Mandel Award. This research was supported by grants from the National Institutes of Health (GM 35322) and the American Cancer Society (JFRA-00130).

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Edited by K. Yamamoto (Received 9 May 1996; received in revised form 4 February 1997; accepted 4 April 1997)