Gain-Of-Function Mutations in Tnsc, an ATP-Dependent Transposition Protein That Activates the Bacterial Transposon Tn7

Gain-of-Function Mutations in TnsC, an ATP-Dependent Transposition Protein That Activates the Bacterial Transposon Tn7

Anne E. Stellwagen and Nancy L. Craig Department of Molecular Biology and Genetics, Howard Hughes Medical Institute, Johns Hopkins University School of Medicine, Baltimore, Maryland 21205-2185 Manuscript received September 18, 1996 Accepted for publication December 2, 1996

ABSTRACT The bacterial transposon Tn 7encodes five genes whose protein products inare different used combina- tions to direct transposition to different types of target sites. TnsABC+D directs transpositiona specific to site in the Escherichia coli chromosome called attTn7, whereas TnsABC+E directs transposition to non- attTn7 sites. These transposition reactions can also recognize and avoid “immune” targets that already contain a copy of Tn 7. TnsD and TnsE are required to activate TnsABC as well as to selecta target site; no transposition occurs with wild-type TnsABC alone. Here, we describe the isolation of TnsC gainaf- function mutants that activate the TnsA+B transposase in the absenceof TnsD or TnsE. Some of these TnsC mutants enable the TnsABC machinery to execute transposition without sacrificing its ability to discriminate between different typesof targets. OtherTnsC mutants appear to constitutively activatethe TnsABC machinery so that it bypasses target signals.We also present experiments that suggest that target selection occurs earlyin the Tn7 transposition pathway in vivo: favorableattTn7targets appearto promote the excision of Tn 7 from the chromosome, whereas immune targets do not allow transposon excision to occur. This work supports the view that TnsC plays a central role in the evaluation and utilization of ” target DNAs.

RANSPOSONS are DNA elements that can move The bacterial transposon Tn7 is distinctive in that T from one position to another in the genome of it uses several element-encoded accessory proteins to their host organism. The selection of a new insertion evaluate potential targetDNAs for positive and negative site is usually not arandom process; instead, many features, and to select a target site (for a recent review, transposons show characteristic preferences for certain see CRAIG 1996). Tn 7encodesfive genes whose protein types of target sites. In some cases, target selection re- products mediate its transposition (ROGERSet al. 1986; flects the DNA sequence preferencesof the transposase WADDELLand CRAIG1988). Two of these proteins, TnsA itself. The bacterial transposon TnlO, for example,pref- and TnsB, collaborate to execute the catalytic steps of erentially inserts into “hotspots” with a common se- Tn 7 transposition (MAY and CRAIG1996; SARNOVSKYet quence motif in vivo (HALLING and KLECKNER 1982), al. 1996); thus TnsA+B constitutes the Tn7transposase. and TnlO transposase binds to hotspot sequences in The activity of this transposase is modulated by the re- vitro (D. HANIFORD,personal communication). In other maining proteins, TnsC, TnsD, and TnsE, and also by cases, the transposase is directed to particular targets the nature of the target DNA. TnsABC+D directs trans- through interactions with cellular proteins or transpo- position at high frequency to a specific site in the E. son-encoded factors. The transcription factors TFIIIB coli chromosome called attTn7 (BARTHet al. 1976; LICH- and TFIIIC, for example, recruitthe yeast retrotranspo- TENSTEIN and BRENNER1982; ROGERSet al. 1986; WAD- son Ty3 to insert into the promoters of genes tran- DELL and CRAIG 1988; KUBOand CRAIG 1990).An alter- scribed by RNA polymerase I11 (KIRCHNER et al. 1995), native combination of proteins, TnsABC+E, directs whereas bacteriophage Mu insertions are targeted to transposition at lower frequency to many non-attTn7 DNA molecules bound by the phage-encodedaccessory sites in the chromosome (ROGERSet al. 1986; WADDELL factor MuB (CRAIGIEand MIZUUCHI1987; SURETTEet and CRAIG1988; KUBOand CRAIG1990) and, preferen- al. 1987; ADZUMA and MIZUUCHI 1988). Understanding tially,to conjugable plasmids (WOLKOWet al. 1996). how these factors modulate transposase activity to im- Thus, attTn7 and conjugable plasmids contain positive pose target site preferences will lend insight into the signals that recruit thetransposon to these target DNAs. spread of transposons and viruses and may suggest ways The Tn7 transposition machinery can also recognize to manipulate those target preferences. and avoid targets that are unfavorable for insertion. Tn 7 transposition occurs only once into a given target Corresponding authur: Nancy L. Craig, Howard Hughes Medical Insti- molecule; repeated transposition events into the same tute, Department of Molecular Biology and Genetics, Johns Hopkins University School of Medicine, 725 N. Wolfe St., Room 615 PCTB, target are specifically inhibited (HAUERand SHAPIRO Baltimore, MD 21205-2185. E-mail: [email protected] 1984; ARCISZEWSKA et al. 1989).Therefore, a pre-ex-

Genetics 145: 573-585 (March, 1997) 574 A. E. andStellwagen N. L. Craig isting copy of Tn 7 in a potential target DNA generates The proposal that TnsC regulates the connection be- a negative signal which renders that target “immune” tween the transposase and the target site predicts that to further insertion. The negative target signal affects TnsC mutants could be isolated which would constitu- both TnsD- and TnsE-activated transposition reactions tively activate Tn7 transposition. In this paper, we re- and is dominant to any positive signals present on a port theidentification of gain-of-function TnsC mutants potential target molecule (ARCISZEWSKA et al. 1989). that can activate the TnsA+B transposase in the ab- Several other transposons, such as Mu and members of sence of TnsD or TnsE. We have characterized the abil- the Tn? family, also displaythis form of negative target ity of these mutants to promote insertions into various regulation (LEE et al. 1983; REYES et al. 1987; ADZUMA targets and to respond to regulatory signals on those and MIZUUCHI1988). targets. Interestingly, one class of TnsC mutants acti- Target selection could be an early or late event in vates transposition in a way that is still sensitiveto target the course of a transposition reaction. For example, a signals, whereas a second class of TnsC mutants acti- transposon could constitutively excise from its donor vates transposition in a way that appearsto bypass target position, and theexcised transposon could then be cap- signals. As had been observed in vitro, the critical com- tured at different frequencies by different types of tar- munication between the transposon and the target get molecules. TnlO appears to follow this course of DNA appears to be an early event in the Tn 7 reaction events in vitro, excising from its donor position before pathway in vivo, preceding the double-strand breaks at any interactions with target DNA occur (SAKAIet al. the transposon ends that initiate transposition. 1995; KLECKNER et al. 1996). Alternatively, the process of transposon excision could itself be dependent on the MATERIALS AND METHODS identification of a favorable target site. Tn 7 transposition shows an early dependence on target DNA signals Media, chemicals, and enzymes: LB broth and agar were in vitro: neither transposition intermediates nor inser- prepared as described by MILLER(1972). Trimethoprim selection products areseen in theabsence of an attTn7target tion was on Isosensitest agar (Oxoid). Lac phenotypes were evaluated on MacConkey lactose agar (Difco).Antibiotic con- (BAINTONet al. 1991, 1993). Thus, the nature of the centrations used were 100 pg/ml carbenicillin (Cb), 30 ,ug/ target DNA appears to regulate the initiation of Tn7 ml chloramphenicol (Cm), 7.5 pg/ml gentamicin (Gn), 50 transposition in vitro. yg/ml kanamycin (Km), 10 ,ug/ml nalidixic acid (Nal), 20 How are positive and negative target signals commu- pg/ml tetracycline (Tet) and 100 pg/ml trimethoprim (Tp). nicated to the Tn7 transposase? Dissection of the Hydroxylamine was purchased from Sigma. DNA modifying enzymes were purchased from commercial sources and used TnsABC+D reaction in vitro has implicated TnsC as a as recommended by the manufacturer. pivotal connector between the transposase and the tar- Bacterial strains, phage and plasmids: BR293 is E. coli F- get DNA. TnsC is an ATP-dependent, nonspecific DNA A(lacpro)thi TpsL A(gal - hG) + lucZ pL CI’~~~pRS7 bindingprotein (GAMASand CRAIG1992). However, (EL.ESPURUand YARMOLINSKY1979; YARMOLINSKY and STEVENS TnsC can be specifically recruited to attTn7 targets 1983). BR293 is identical to NK8027 (HANIFORD et al. 1989), and was generously provided by NANCYKL.EC:KNER. NLC51 is through interactionswith TnsD, an attTn7-specific DNA E. coli F- araDI39 A(argF-lac) U169 qsLl50 relAl j’bB5301 binding protein (BAINTONet al. 1993). TheTnsC-TnsD- deoCl ptsF.25 rbsRValR recA56 (MCKOWNet al. 1987).CW51 is attTn7 complex has been visualized by DNA protection E. coli F- ara arg Alac+roXIII recA56 NalRRip (WADDELLand and mobility-shiftassays, and the preassembled com- CRAIG 1988). hKKl is lambda780 hisG9424::Tn10 dell6 dell 7 : : attTn7:: miniTn 7-KmR (MCKONW et al. 1988). Tns plex can attract insertions (BAINTONet al. 1993). Direct transpositionproteins were provided bypCW15 (tnsABC), interactions between TnsC and the TnsA+B transpc- pCW23 (tnsD), pCW30 (tnsE), or pCW4 (tnsABCDE) (WAD- sase have alsobeen observed (A. STELLWAGENand N. L. DELL and CRAIG1988). Target plasmids were derivatives of CRAIG, unpublished results). Therefore,TnsC may serve POX-G, a conjugable derivative of the F plasmid that carries as a “connector” or “matchmaker” between the trans- GnK (JOHNSON and REZNIKOFF 1984). POX-attTn7carries a (-342 to +165) attTn7 sequence (ARCISZEWSKA et al. 1989). posase and the TnsD attTn7 target complex (BAIN- + The immune plasmid POX-attTn7 EP-1: : miniTn 7-CmR was TON et al. 1993; SANCAR and HEARST1993). This connec- made by transposingminiTn7-CmR (MGKOWN ~t al. 1988) tion is not constitutive, but instead appears to be onto POX-attTn7 using TnsABC+E to direct the insertion regulated by the ATP state of TnsC. Only the ATP- into a non-attTn7position. Constructionof the immune target bound form of TnsC is competent to interact with target plasmidPOX-G : : miniTn 7-dhfr is describedbelow. The transposon donorplasmid for the papillation assay was pox- DNAs and activate the TnsA+B transposase; the ADP- G:: miniTn 7lac, containing promoterless lacZY between the bound form of TnsC has neither of these activities and transposon ends (HUGHES1993). The high copy transposon cannot participate in Tn 7 transposition (GAM and donor for mating-out assays was pEMA, containing miniTn7- CRAIG 1992;BAINTON et al. 1993). TnsC hydrolyzes ATP KmR (BAINTONet al. 1993). at a modest rate (A. STELLWAGENand N. L. CRAIG, un- Manipulation and characterization of DNA Phage and plasmid isolation, transformation, and standard cloning tech- published results) and therefore can switch from an niqueswere performed as described by SAMBROOKet al. active to an inactive state. The modulation of TnsC’s (1989). Conjugation andP1 transduction were performedas ATP state may be a central mechanism for regulating described by MILLER(1972). DNA sequencing was done on Tn 7 transposition. an automated AB1 sequencer. Two plasmids were constructed Tn 7 Gain-of-Function Mutants 575 in this work: (1) POX-G:: miniTn 7-dhfr. MiniTn 7-dhfi-wascon- structed by replacing the Km' cassette in pLA1 (~CISZEWSKA et al. 1989) with a dhfr cassette from pSD511 (DEVINEand BOEKE1994), which had been amplified by PCR to add flank- ing Sua sites. The PCR fragment was ligated into the TA vector (Invitrogen), the dhfr cassette was then removed by S+lI digestion and inserted into the SalI site of pLA1,replacing the Km' gene. The resulting plasmid was transformed into NLC51 + pCW4 + POX-G, and grown for several days to allow transposition to occur. POX-G plasmids which had received a miniTn 7-dhfr insertion were identified b mating into CW51 and selecting for Tp'. (2) TnsA(BCA"2'''). The lnrABC plasmid pCWl5 was briefly treated with BamHI, and molecules that had lost the 0.9-kb fragment from the 5' end of tnscwere identified by gel electrophoresis, using low melting tempera- ture agarose (Seaplaque, FMC Corp., Marine Colloids Div.). Gel slices containing these molecules were isolated and the DNA was religated to generate tnsA(BC?"2y3). SOS induction assays: SOS induction was evaluated in FIGURE1.-Papillation phenotypes of the TnsC gain-of- BR293 strains. Tn 7 and miniTn 7-Km' elements were intro- function mutants. Cells were patched on MacConkey lactose duced byP1 transduction; target plasmids were introduced plates and photographed after three days' incubation at 30". by conjugation. lacZ expression served as a reporter for the TnsA+B was present in each strain; the TnsC species present SOS state of the cells, and was evaluated by patching cells on is indicated below each patch. MacConkey lactoseplates at 30" for 18-36 hr. P-galactosidase activity was quantitated in cultures that were grown at 30", using the assay described by MILLER(1972). and miniTn7-Km' exconjugants were selected on KmNal Mutagenesis of hnsC: The tnsABC plasmid pCW15 was ex- plates. Transposition frequencies are expressed as the num- posed to 1 M hydroxylamine hydrochloride in 0.45 M NaOH ber of Tp'- or Km'exconjugants/totaI number of exconju- (final pH -7.0) at 37" for 20 hr (ROSE et al. 1990). The DNA gants. was recovered by multiple ethanol precipitations, and PouII- SphI fragments containing mutagenized tnrC were subcloned into untreated pCW15, replacing the wild-typetnsC. These RESULTS plasmids were then introduced into CW51 + pOX-G::mini- Tn 7Zucby electroporation, and transformants were selected Isolation of TnsC gain-of-functionmutants To focus on MacConkey lactose plates containing Cm. The plates were on the contributions of TnsC to the evaluation and incubated at 30" for 3-4 days and screened for the emergence utilization of target DNAs, we sought to isolate gain-of- of Lac' papillae, indicating transposition of miniTn 7lac. function TnsC mutants that could activate the TnsA+B A hop transposition assay: Tn 7transposition was evaluated transposase in the absence of TnsD or TnsE. Since over- in NLC51 strains into which tnsfunctions were introduced by expression of wild-type TnsC does not relieve the re- transformation, and POX-G was introduced by conjugation (for Figure 5). The protocol of MCKOM'N et al. (1988) was quirementfor TnsD or TnsE (WADDELLand CRAIG followed: Cells were grown in LB and 0.2% maltose at 37" to 1988), these gain-of-function mutations are predicted an ODm of 0.4-0.6 and then concentrated to 1.6 X lo9cells/ to affect the biochemical properties of TnsC, rather ml by centrifugation and resuspension in 10 mM MgSO,; 0.1 than its expression or stability. ml cells werecombined with 0.1 mlAKKl containing miniTn 7- A visual assay for Tn 7transposition has been devel- Km ' Km' at a multiplicity of infection of 0.1 phage per cell. The infection proceeded for 15min at 37" and was terminated by oped to facilitate the identification of transposition mu- the addition of 10 mM sodium citrate in 0.8 ml LB. Cells were tants (HUGHES1993; HUISMANand KLECKNER 1987). allowed to recover with aeration for 60 min at 37" and then This assay uses a miniTn71ac element that carries pro- spread on plates containing Km and citrate. Transposition moterless lady genes between the cisacting sequences frequency is expressed as the number of Km' colonies/pfu at the transposon ends. The miniTn71ac element is lo- of AKKl. Mating-out transposition assay: Tn 7transposition was eval- cated in a transcriptionally silent position on a donor uated in the derivatives of BR293 used to monitorSOS induc- plasmid; cells containing this plasmid are phenotypi- tion (Table 3) or in NLC51 strains into which tns functions cally Lac-. When Tns functions are provided in trans, were introduced by transformation, and p0X-G or POX- miniTn 71ac can transpose to new sites in the E. coli G::miniTn 7-dhfr were introduced by conjugation. MiniTn 7- chromosome. Some of those transposition events place Km ' Km' was present in the NLC51 strains either in the chromo- theelement downstream from active promoters, re- somal attTn7 site (Figure 4 and Table 1) or the high copy plasmid pEMA (Table 2). The protocol was adapted from sulting in increased ZucZ expression. This is observed WADDELLand CRAIG(1988): The donor strains described on MacConkey lactose color indicator plates as the above and the recipient strain cMr51 were grown at 37" to an emergence of red (Lac+)papillae in an otherwise white ODbooof 0.4-0.6 with gentle aeration. Donors and recipients (Lac-) colony. Therefore, the number of papillae re- were mixed at a ratio of 1:5, and growth was continued for flects the amountof transposition that occurred during another hour. Mating was disrupted by vigorous vortexing, and the cells were diluted and plated. The total number of the growth of that colony. exconjugants was determined by selection on GnNal plates. Cells containing miniTn 71ac and' various Tns func- Tn 7-containing exconjugants were selected on TpNal plates, tions were patched on color indicator plates (Figure 1). Virtually no Lac+ papillae were seen in cells containing ( only TnsABC"', whereas cells containing TnsABC"' + E produced many Lac+ papillae. Southern blotting has demonstrated that TnsABC"' + E papillae result from translocations of miniTn71ac to a variety of chromosomal locations rather than from intramolecular rearrangements of the donor plasmid (HUGHES1993). It should be noted that most TnsABC" + D events are silent in this assay, because there is no appropriately oriented promoter adjacent to attTn7which would fire into miniTn 7Zac insertions in that target site (HUGHES 1993; R. DEBOYand N. L. CRAIG,unpublished results). We used this visual assay to screen forTnsC mutants I S401F S4U1 YMUZ that had acquired the ability to activate Tn7 transposi- A282T tion in the absence of TnsD or TnsE. Randomly mutagenized tnsCwas clonedinto a plasmid containing Class I1 TnsC mutants tnsAB these tns genes were introduced into cells containing miniTn71ac and evaluated for their ability to FIGUREP.-hino acid changes in the TnsC mutants. The promote papillation. Six gain-of-functionTnsC mutants TnsC protein sequence is cartooned, with the residues altered in the Class I mutants indicated above the protein and the were identified, from -3000 transformants screened Class I1 mutants below the protein. Hatched boxes represent (Figure 1). Transposition activated by these TnsC mu- Walker A and Walker B motifs. tants still required the TnsA+B transposase and intact transposon ends, and comparable levelsof wild-type TnsC mutantspromote intermolecular transposi- and mutant TnsC proteins were observed by Western tion: The papillation assayis a powerful screen for blotting (data not shown). The papillation phenotypes transposition activity, but it does notnecessarily report of the TnsC mutants varied considerably, suggesting only intermolecular transposition events. Internal rear- thatdifferent mutants were activating different rangements of the miniTn71uc donor plasmid, which amounts of miniTn 71uc transposition. Several TnsC mu- fortuitously place the miniTn 7hc elementdownstream tants promoted more transposition than TnsABC"' + from a promoter, would also produce Lac+ papillae. E, with TnsABCS~OIYA~OZ achieving the highest level of Therefore, we investigated whether the TnsC mutants transposition. Indeed, cells containing TnsCS4n1YA4n2 facilitate the TnsA+B transposase to do intramolecular produced so many papillae that, afterseveral days' incu- recombination or whether the mutants promote inter- bation, the entirepatch of cellsappeared red. At earlier molecular transposition. time points, however, individual papillae could be seen The A hop assay measures the translocation of a mini- in the T~SABC""~~~~~patches as well (data not shown). Tn 7-KmR element from a replication- and integration- The amino acid changes responsible for the mutant defective A phage to the bacterial chromosome during phenotypes were determined by DNA subcloning and a transientinfection. The miniTn 7-KmRelement carries sequencing. tnsCencodes a protein of 555 amino acids, a kanamycin resistance cassette with a constitutive pro- with Walker A and B motifs in the amino-terminal half moter. Therefore, theh hop assay reports thetotal num- of the protein (FLOES et ul. 1990). Walker A and B ber of transposition events occurring into the chromo- motifs have been implicated by structural and muta- some, unlike the papillation assay, which only scores tional analyses to be directly involvedin nucleotide those events which occur downstream from active pro- binding and/or hydrolysis in a variety of ATPases and moters. TnsABC"' had no detectable transposition activ- GTPases (WALKERet al. 1982; see SARASTEet al. 1990 ity in the A hop assay, whereas TnsABCW+ E generated for review). The tnsC mutations primarily result in sin- 2.2 X io" KmRcoIonies/pfu, a transposition frequency gle amino acid substitutions whose locations are scat- 220-fold above the threshold of sensitivity (Figure 3). tered across the TnsC protein sequence (Figure 2). As Transposition promoted by TnsABC"' + D occurred at will be described below, the TnsC mutants segregate a much greater frequency, generating 1.8 X lO-4 KmR into two phenotypic classes, which are indicated above colonies/pfu. All of the TnsC mutants could promote and below the cartoon. Transposition reactions acti- the translocation of miniTn7-KmR. TnsABCAZ2"' and vated by the Class I mutants are sensitive to immune TnsABC%01YA402had striking activities, promoting eight- targets and the target selection factors TnsD and TnsE, and 50-fold more transposition than TnsABC"' + E. whereas transposition reactions activated by the Class Other TnsC mutants promoted more modest levels of I1 mutants arecompromised in their responses to these transposition. Although all of the TnsABC""""' reac- signals. Interestingly, the residues affected in two of the tions show a gain-of-function over TnsABC"', none of mutants (TnsCA22.'>\'and ~~~cE233K) lie in or very close the mutant reactions reach the level of transposition to TnsC's Walker B motif. seen with TnsABC"' + TnsD. Tn 7 Gain-of-F1unction Mutants 577

10 -3 -fn d - C 3 m e- .-a, lo? - C 10" .z0 - a, 8 -m U c E 2 10-4 y 10-s . L x m 0 C tn a, .- 10-6 a,CJ, g 10-5 - a, C .-0 U .-L E Lo Y 0 10.7 - :: 6 10-6 F C I- al m 10 4 a, cu - 0 K 9 .- 10-7 -7 .-0 Lo N WP 0 zs4s"ocu ++ !? x r2Er2zz C 2 zzzk5k W MMMMMMM 3% I- cubOa,OO + g;gcgcg ce r2a12zz r MMMMMMM M FIGURE3.-TnsC mutants promote transposition to the cggcggg g chromosome. Frequencies of transposition of miniTn 7-KmR FIGURE4.-TnsC mutants promote transposition to conju- from a A phage to the chromosome were measured by the A hop assay. TnsA+B was present in each strain; the TnsC spe- gable plasmids. Frequencies of transposition of miniTn 7-KmR cies present is indicated below each column. from the chromosome to the conjugable target plasmid POX- G were measured by the mating-out assay. TnsA+R was present in each strain;the TnsC species present is indicated below The mating-out assay was used to explore the ability each column. of the TnsC mutants to promote translocations into a different type of target molecule. This assay measures (data not shown).This suggests that the TnsC mutants the frequency of transposition of miniTn7-KmR from primarily activate inter- ratherthan intramolecular the chromosome to POX-G, a conjugable derivative of transposition events. the E. coli F factor. The TnsABC"' + E machinery prefer- Responseto the target selectors TnsD and TnsE: entially selects conjugable plasmids as targets for trans- TnsD and TnsE are required to activate the TnsABC"' position, whereas the TnsABC"' + D machinery does machinery and to direct transposition into particular not recognize POX-G unless it contains attTn7 se- target DNAs (ROGERSet al. 1986; WADDELLand CRAIG quences (ROGERSet al. 1986; WADDELLand CRAIG 1988; 1988; KUBO and CRAIG 1990; WOLKOWet al. 1996). The WOLKOWet al. 1996). The TnsC mutants could promote TnsABC""'""' machineries, by definition, do not require transposition to POX-G (Figure 4), with the frequencies the inputs of TnsD or TnsE. However, we investigated of transposition ranging over two orders of magnitude, whether TnsD or TnsE could influence the frequencies from the modestly active TnsABCr233Kto the strongly or distribution of transposition events promoted by the active TnsABCS4n'YA4"2. However, none of the mutant TnsC mutants. reactions promoted more transposition to POX-G than The A hop assay was used to evaluate the effects of TnsABC"' + E. This is in contrast to results from the TnsD and an available attTn7site on transposition pro- papillation and A hop assays, in which several of the moted by the TnsC mutants (Figure 5). All of the mu- TnsC mutant reactions promoted more transposition tant reactions were responsive to TnsD + attTn7, but than TnsABC"' + E. Since the A hop assay reports the those responses varied widely. Reactions activated by total amount of transposition in a cell, rather than just TnsABCk225"and T~SABC~~"~were strongly stimulated transposition to POX-G, we hypothesize that the major- by TnsD + attTn7, promoting 500- and 5000-fold more ity of the transposition events activated by the TnsC transposition, respectively, in the presence of TnsD + mutants are directedto the chromosome. The distribu- attTn7 than with TnsABCA2'" or T~sABC"*"~alone. tion of TnsABCln'''a"t transposition events between the The remaining mutant reactions were less profoundly chromosome and POX-G is addressed below. influenced by TnsD: T~SABC~~"~reactions showed a Thus, we have demonstrated that thegain-of-function moderate (50-fold) stimulation, whereas reactions acti- TnsC mutants can promote intermolecular transposi- vated byTnsCE2"', TnsCS4"yA402and TnsCA2'" were tion, using two independent assays. We havealso looked somewhat inhibited in the presence of TnsD. directly for intramolecular rearrangementsof the mini- We also examined the effects of TnsEand the conjuga- Tn 7lacdonor plasmid in the papillation strains; no Lac+ ble plasmid p0X-G on the transposition events activated rearrangements were seen out of 24 papillae examined by the TnsC mutants. Transposition frequencies were 578 A. E. Stellwagen and N. L. Craig

TABLE 1 Sensitivity of TnsC mutant reactions to transposition immunity

Fold inhihition of insertion into TnsC immune targetimmune TnsC C"' + E 83 (11) Class I mutants TnsCMT!.5V 104 (6) TnsCE'7:3" 50 (6) Class I1 mutants TnsC"'"" 3.6 (6) T~~G\~HST 0.6 (6) ~ns~.4011'A4fl!! 2.0 (6) Tns&%f)1F 0.9 (6)

FIGURE!%--Effects of the target selectionfactors TnsD and The frequencies of miniTn7-Km' transposition from the TnsE. Frequencies of transpositionof miniTn 7-Km' from a A chromosome into the nonimmune targetPOX-G and the im- phage to the chromosome and/or POX-G were measured in mune target POX-G::miniTn 7-dqrwere determined for each strainscontaining TnsA+B and the TnsC mutants, either TnsC species, and the fold reduction of insertions into the alone or in combination with TnsD or TnsE. The preferred immunetarget is indicated.TnsA+B was present in each target for TnsD reactions,attTn7, was present in the chromo- strain. The number of trials is given in parentheses; the vari- somes of these strains. The preferred target for TnsE reac- abilitv of a given experiment was less than twofold. tions, POX-G, was introduced by conjugation into strains containing the TnsC mutants or the TnsC mutants + TnsE. The T~~cF~R~KT~~C\~X!?T ~~~cS40IYA402 and T~~c%~(I~F-is distribution of miniTn7-Km' insertions between the chromo- I , some andPOX-G was determined by mating the POX-G plas- not responsive to the positive effects of TnsD or TnsE mids from the Km' products of a A hop assay into the Kms or both. By these criteria, TnsC"401F is proposed to strain CW51, and testing whether Km resistance was plasmid be a member of Class 11: although TnsC"'o'F-activated linked. No transposition was detected in strains containing reactions are somewhat stimulated by TnsD, the distri- TnsABC" alone or TnsABC"""" + TnsD. bution of insertions in TnsCS"O'F-activatedreactions is not affected by TnsE. The grouping of the TnsC mu- measured by the A hop assay (Figure 5), and thedistribu- tants into these two classes is supported by the differen- tion of insertions between the chromosome and p0X-G tial responses of the Tn"BC'"l''s''' reactions to immune was also determined (data notshown). In the absence of targets, as described below. TnsE, the vast majority ofthe TnsABC"'"'""' transposition Recognition of negativetarget signals: TheTn7 event5were targeted to the chromosome rather than transposition machinery evaluates potential target p0X-G. However, in the presence of TnsE, preferential DNAs not only for positive signals, such as nllTn7, but insertion into p0X-G was observed with some of the also for negative signals, such as the presence of a pre- TnsC mutants. Tn~C';.~'~~-activatedreactions were most existing copy of Tn 7. While TnsD and TnsE are clearly clearly affected by TnsE: 20-fold more transposition was involved in the recognition of positive target signals, seen with T~sABC"?':~"+ TnsE than withTnsABC';.2"K the mechanism by which negative target signals are rec- alone, and themajority ofthe T~sABC"~"~~+ TnsE trans- ognized is unknown. Is this information also communi- position events weretargeted to p0X-G. The distribution cated by TnsD or TnsE, or is TnsC more directly in- of insertions in TnsC"'""-activated reactions was also af- volved? We examined whether transposition activated fected by TnsE: targeting to p0X-G was detected in the by the TnsC mutants was responsive to negative target majority of the TnsABCN2?"' + TnsE reactions examined, regulation by comparing frequencies of insertion into although the frequency of transposition wa5 unchanged. immune and nonimmunePOX-G targets in the mating- By contrast, transposition activated by the remaining out assay (Table 1). Consistent with previous observa- TnsC mutants was predominantly targeted to the chro- tions (ARCISZEWSKA~t nl. 1989),TnsABC"' + E transposi- mosome despite the presence of TnsE, and the frequen- tion showed a strong immune response: insertions into cies of those reactions were unchanged or modestly in- the immunetarget were reduced 89fold compared with hibited by TnsE. insertions into the nonimmune target. Transposition Thesedifferential responses suggest thatthe six activated bytwo of the TnsC mutantq, TnsC"''"'" and TnsC mutants arenot activating Tn7 transposition ~~~cl?!i:1K,was also sensitiveto the immunetarget, show- through a single mechanism. Instead, the mutants can ing 100- and 50-fold inhibition, respectively. Thus, a be segregated into two classes, based on their ability minimal transposition machinery involvingonly the to respond to TnsD and TnsE. Transposition activated TnsA+B transposase and TnsC can still identify and by the Class I mutants, TnsCA2'"'' and T~sC""~,can avoid immune targets. TnsD and TnsE are not required be stimulatedby TnsD and targeted toPOX-G by TnsE. for the recognition of this negative target signal. Transposition activated by the Class I1 mutants- Transposition activated by the four remaining TnsC Tn 7 Gain-of-Function Mutants 579

.I'ABLL z TIW(BC~'-~~)promotes intermolecular transposition

Tns functionsTns Transposition frequency TnsABC"' < 10" T~~A(BCAIS!N) 2.5 t 3.3 X 1O-6 ~~acA22.5\' 8.8 f 8.1 X lo-" TndC"'DE 5.5 t 1.1 x 1o-4 Frequencies of transposition of miniTn 7-KmK from a high copy plasmid to p0X-G were determined using the mating- out assay and are expressed as the number of KmK exconjugants/total exconjugants. Each value is the average of three independent measurements.

in the process of activating the TnsA+B transposase. SMITHand JONES (1984) created a Tn 7 element that produced TnsA and a deletion-fusion protein, in which the amino-terminal half of TnsC (including theWalker A and B motifs) was deleted and the remainder fused in frame to TnsB; this deletion-fusion is hereafter re- ferred to as Tns(BCA"m2"3).TnsA(BCA"mR) was capable of promoting transposition, although at a frequency 1000-foldless than native Tn7. Unlike TnsABC', T~~A(BCAI-Z~R) transposition did not require TnsE or intact TnsD. Thus,the Tns(BCA1-mJ) deletion-fusion protein appears to be a gain-of-function mutant, with the same phenotype as the TnsC point mutantsisolated in this work.However, interpretation of SMITHand FIGURE6.-The Tns(BCA"%") deletion-fusion mutant. (A) JONES' results is complicated by ambiguities in their Construction of tmA(Be'-293)from a tmABC expression plav experimental design: it is unclear whether these trans- mid. Shortarrows labeled "Bum" indicate BumHI sites present position events were targeted to uttTn7 or not, or in tmBand tnsC; deletion of the intervening fragment resulted whether TnsA(BCA1-z9') activityrequired the nearly full- in the fusion of the 3' half of lmC to the end of tnsB. The molecular weightsof the protein roducts are indicated below length TnsD polypeptide. their respective genes;tm(BCd.'2R) encodes a protein of - 112 We reconstructed the Tns(BCA."m2gJ)deletion-fusion kD. Hatched boxes represent TnsC's ATP motifs, which are mutant using our tnsABCplasmid (Figure6A), to confirm deleted from the Tns(BCA"%") mutant. (B) Papillation phe- and extend the observations of SMITHand JONES. We notypes of cells containing TnsA(BCA"?!"'),as observed on found that TnsA(BCA'-m3)promoted as many Lac+ rear- MacConkey lactose plates after a May incubation at 30". rangements as did TnsABC"' + E in the papillation assay (Figure 6B). Therefore, TnsA(BCA"m') can, in fact, exe- mutants was not dramatically inhibited by the immune cute transposition in the absence of TnsD or TnsE. The target. Therefore,the ability to respond to immune amount of transposition activated by the Tns(BCA1-''') targets segregates the TnsC mutants into the same two mutant was highly dependent on the transposition assay classes that were proposed based on the responses of used. TnsA(BCA'-mS)could promote intermolecular trans- the TnsC mutant-activated reactions to TnsD and TnsE. location of miniTn7-KmRfrom a high copy plasmid to Transposition activated by the Class I mutants, pOXG, at a frequency -200-fold less than TnsABC'DE TnsC""5\\' and TnsC"'", maintains the ability to re- (Table 2). However,TnsA(BCA'-mJ)-activated transposi- spond to both positive and negative target signals char- tion of miniTn ?KmR from the chromosome or from a A acteristic of wild-type Tn7 transposition. By contrast, phage was undetectable (data not shown). These varia- the Class I1 TnsC mutants appear to promote transposi- tions suggestthat transposition activatedby Tns(BCA""") tion into any target DNA they encounter. may have a greater dependence on the donor site or the A TnsC deletion-fusionmutant that can activatetrans- donor copy number than transposition activated by the position: TnsC is a multifunctional protein that can TnsC point mutants. interact with both the target DNA and the transposase We also investigated whether TnsA(BCA"m') transpo- (CAMASand CRAIG1992; BAINTONet dl. 1993; A. STELL sition reactions were responsive to positive or negative WAGEN and N. L. CRAIG. unpublished results). An early target signals. The frequency of TnsA(BCA."2"S)transpo- report in the Tn 7 literature,which predates the identi- sition was not affected by the presence of TnsD + uWTn7 fication of the tnr genes, implicates a domain of TnsC or TnsE + POX-C in either the papillation or mating- 580 A. E. Stellwagen and N. L. Craig out assays (data not shown). Thus, T~s(BC*'"'~)mu- site and miniTn7-KmRelsewhere in the chromosome, tant reactions do not appearto be influenced by these experienced more transposition events and had more target selectors. We were unable to determine whether ZacZ expression. The chromosomal location of the the T~s(BC*""~)reactions could recognize and avoid miniTn 7-KmR element was important: SOS induction immune targets, due to high levels of recombination was assayed in strains with miniTn 7-KmKelements in 12 between the miniTn7-dhfr on the immune target plas- different locations, and the amount of lacZ expression mid and miniTn 7-KmRon the high copy donor plasmid varied considerably (data not shown).This could reflect that confounded the detectionof transposition events. different accessibilities of these DNA damage sites to HOWhas Tns ( BCn"29s)acquired theability to activate the SOS-inducing machinery or the DNA repair appara- transposition? Other experiments have revealed that it tus, or the variations could reflect different transposi- is the removal of the amino-terminal domain of TnsC, tion frequencies from these different donor sites. The not the fusion to TnsB, which is responsible for this observed Lac+ phenotypes were dependent on RecA, gain-of-activity (A.STELLWAGEN, F. Lu, and N. L. CRAIG, the master regulator of the SOS response (WALKER unpublished results). An attractive hypothesis is that 1996), and RecBCD, a DNA unwinding enzyme which the carboxy-terminal half of the protein interacts with is required to convert the double-strand breaks caused the transposase to activate transposition, whereas the by some DNA damaging agents into SOS inducing sig- amino-terminal half is required for the recognition of nals (KARU and BELK 1982; CHAUDHURYand SMITH target DNA signals and/or the coupling of transposi- 1985) (data not shown).Cells undergoing Tn 7transpo- tion activation to those signals. sition also had a fivefold higher frequency of spontane- Target DNA regulates the initiation of transposition ous induction of a wild-type X lysogen, another standard in vivo: The order of events in the Tn7 transposition measure of SOS induction (data not shown). reaction has been studiedin detail in vitro. Tn 7 transpo- All of the components necessary for high frequency sition uses a cut-and-paste mechanism, in which the Tn7 transposition -a transposon (line 2), the Tnspro- element is first excised from the donorDNA by double- teins (line 3), and an attTn7 target (line 7) -were re- strand breaks, and then inserted into attTn7 (BAINTON quired to trigger the SOS response. TnsE-mediated et al. 1991, 1993).The attTn7 target is requiredfor transposition to POX-G did not induce a detectable both steps in thereaction pathway: neither excision nor SOS response, probably because these transposition insertion is observed unless an attTn7 target is present. events occurredat 1000-foldlower frequency than This suggests that the nature of the target DNA regu- TnsD-mediated transposition to attTn7. Taken to- lates the initiation of Tn7 transposition in vitro. Here gether, these results suggest that the process of Tn7 we have investigated whether targetDNA plays a similar transposition induces the cellular SOS response, and role in controlling transposition initiation in vivo, by the strength of that SOS induction correlates with the evaluating the effects of different target DNAs on the amount of ongoing transposition. process of Tn 7 excision. What step in the transposition pathway is responsible Chromosomal damage caused by Tn 7 excision could for SOS induction? An attractive model is that the dou- potentially induce the SOS response, the cellular re- ble-strand breaks left in the chromosomeafter transpo- sponse to DNA damage (WALKER1996), as has been son excision trigger the SOS response. SOS induction observed for other transposable elements (ROBERTS is also observed as a consequenceof Tn 10 transposition and KLECKNER 1988; LANE et al. 1994). Tn 7 transposi- (ROBERTSand KLECKNER 1988), and there is strong evi- tion was evaluated in a strain in which lacZ expression dence that theexcision of TnlO from the chromosome served as a reporter of the SOS state of the cell. This is the event that induces theSOS response: TnlO trans- strain contains a transcriptional fusion of lacZ to the posase mutants thatcan promote excision but not inser- pL promoter of a lysis-defective A prophage (ELESPURU tion maintain the ability to induce SOS (HANIFORD et and YARMOLINSKY1979); thus,ZacZ expression is inhib- al. 1989). If the breaks in the chromosome which result ited by A repressor. In cells that have experienced DNA from Tn7 excision are similarly responsible for SOS damage, A repressor is cleaved as part of the SOS re- induction, then the observation that attTn7 targets are sponse (ROBERTSand ROBERTS1975), and the subse- required for SOS induction suggests that Tn7 target quent increase in lacZ expression can be detected on sites are being evaluated before transposon excision in MacConkey lactose color indicator plates or in bacterial vivo. This would be consistent with results in vitro, in extracts. which breaks at the transposon ends are not observed We observed that cells that were undergoing high unless attTn7 is present (BAINTONet al. 1991). There- frequency Tn 7 transposition into attTn7 became Lac+ fore we propose that targetDNA regulates Tn 7excision (Table 3). Mobilization of a single copy of Tn 7 resulted in vivo, and that this feature of the reaction mechanism in a small but reproducible increase in lacZ expression, has been reproduced in vitro. detectable as a change in color of these cells on Mac- We alsoused the SOS induction assay to examine Conkey lactose plates. However,cells containing two when negative target signals are evaluated. An immune transposable elements, Tn 7in the chromosomalattTn7 target that contained anattTn 7sequence and a miniTn 7- Tn 7 Gain-of-Function Mutants 581

TABLE 3 Tn7 transposition induces the cellular SOS response

Transposition frequency' LucZ expressiond Transposon" TnTargetb miniTn 7 7-KmR MacLac phenotype Miller units

~~ - - - - White 64 t 4 - pox-uttTn7 - - White ND miniTn 7-KmR pox-uttTn7 - - White ND Tn 7 - - - White ND Tn 7 pox-uttTn7 8.9 2 3.7 X - Pink 79 ? 11 Tn 7 + miniTn 7-KmR POX-uttTn7 6.8 2 7.1 X lo-' 1.7 2 1.4 X Red 218 2 69 Tn 7 + miniTn 7-KmR POX-G 1.5 f 1.8 X 1.8 ? 1.9 X lo-' White 64 ? 9 Tn 7 + miniTn 7-KmR POX-attTn7; EP- 2.3 ? 0.8 X 8.4 2 2.0 X White ND 1::miniTn 7-CmR '' Tn 7 was located in the chromosomal attTn7 site and miniTn 7-hR(which does not encode the Tns proteins) was located in the chromosomal position EG2 (KUBO and CRAIG 1990) of the pLlucZ transcriptional fusion strain BR293. 'Nonimmune targets were POX-uttTn7 and POX-G (which does not contain attTn7); the immune target was POX-uttTn7 containing a miniTn 7-CmRlocated in a position designated EP-1. Transposition frequency was measured by mating-out assays, with each value representing theaverage of at least five independent measurements. LucZexpression was evaluated on MacConkey lactose (MacLac) color indicator plates, or quantitatedusing the assay described by MILLER(1972), with each value representing the average of at least six independent measurements.

CmR element elsewhere in the plasmid was introduced DISCUSSION into the SOS reporter strain. The frequency of insertion into this target was reduced more than 200-fold in com- This work has investigated when and how target DNA parison to anonimmune attTn7 target (Table 3). regulates Tn 7 transposition in vivo. We have presented Tsansposon excision also appeared to be inhibited: no experiments that suggest that positive and negative tar- SOS response was observed in strains containing the im- get signals are evaluated before Tn 7 excision, providing mune target. Thus, negative features as well as positive inputs that control theinitiation of transposition. Multi- features of a potential target molecule appear to be eval- ple transposition proteins are involved in target evalua- uated prior to the initiation of Tn 7 transposition in vivo. tion; here we have focused onthe contributions of Finally, we examined whether theTnsC mutant reac- TnsC. We have isolated TnsC gain-of-function mutants tions triggered the SOS response. In theory, TnsC mu- that can activate the TnsA+B transposase without the tants might exist that primarily stimulate transposon target selection factors TnsD and TnsE. The TnsC mu- excision, perhaps by facilitating the assemblyof the tant-activated transposition machinery can promote in- TnsA+B transposase with the transposon ends or by sertions into avariety of targets and, in some cases, still uncoupling the initiation of transposition from target avoid unfavorable targets. This work supports the view regulation. The activity ofsuch excision-promoting mu- that TnsC plays a key role in target evaluation and utili- tants might be underrepresented by insertion-based zation. assays such as X hops or mating-outs. TnlO transposase Targetevaluation occurs early in Tn7 transposi- mutants that specifically activateexcision have been de- tion: Tn 7 transposition is accomplished by two chemi- scribed (HANIFORD et al. 1989).However, no SOS induc- cal steps, the excision of the element from its donor tion was detected in transposition reactions activated site and the insertion of the element into atarget DNA by any of the TnsC mutants (data not shown). This (BAINTONet al. 1991). The nature of the target DNA underscores the limited sensitivity ofthe SOS induction could, in theory, regulate either or both of these steps: assay for Tn 7 transposition: DNA damage apparently target DNA could provide a signal that allows the trans- needs to occur at TnsD-activated frequencies to be de- position reaction to initiate, and/or target DNA could tected. None of the TnsABCmUmntreactions generate as be involved in late events such as the capture of the much insertion as TnsABC"' + D reactions (Figure 3); excised transposon. We have demonstrated here that the mutant reactions also appear to be promoting less Tn 7 transposition induces the cellular SOS response, excision than TnsABC"' + D. Therefore, the six gain- and we speculate that theprimary event responsible for of-function TnsC mutantsidentified in thiswork do that induction is the excision of the transposon from not appear to be hyper-excision mutants, but instead the chromosome. If so, then the nature of the target promote both theexcision and insertion of Tn 7. Future DNA influences whether transposon excision will occur: studies may be able to exploit the SOS induction assay favorable attTn7 targets appear to provoke the double- as a screen to identify hyper-excision mutants among strand breaks at the transposon ends that initiate Tn7 the Tns proteins. transposition, whereas unfavorable targets, such as im- 582 A. E. Stellwagen and N. L. Craig mune plasmids, do notpromote these breaks. Thus, we sults). However, wild-type TnsC is not sufficient to acti- propose that target DNAs provide both positive and vate transposition. Instead,Tn 7 transposition is negative signals that control theinitiation of Tn 7 trans- dependent on TnsD or TnsE to activate the TnsABC"' position in vivo. Similar conclusions have been reached machinery and select a target site. TnsD is an attTn7 from studies of the Tn7 transposition reaction in vitro, bindingprotein (BAINTON et al. 1993) that recruits in which theinitiating double-strand breaks atthe TnsC to this target. The resulting TnsC-TnsD-attTn7 transposon ends can be examined directly. No breaks complex can then attract the transposase in vitro (BAIN- occur unless the transposition proteins have hada TON et al. 1993). The mechanism by which TnsE acti- chance to interact with a nonimmune attTn7 target vates transposition is not yet known. TnsE might be (BAINTONet al. 1991, 1993). Thus thereaction pathway preferentially localized to conjugating plasmids and in vitro appears to accurately reflect the role of target subsequently recruit TnsC to those molecules, or TnsE DNA in vivo in regulating transposition initiation. might modify TnsC so that TnsC's binding activity is Coupling the initiation of transposition to the identi- now directed to those targets. Alternatively, TnsEmight fication of a favorable target DNA could be advanta- modify the transposase directly, without proceeding geous for the transposon and its host. If transposition through TnsC. However, we are attractedto the hypoth- only initiated when it could be successfully completed, esis that TnsD and TnsE provide alternative inputs into fewer copies of the transposon would be unproductively TnsC, which in turn recruits the TnsA+B transposase excised and lost, and less damage to the host chromo- to the target DNA. some would occur. However, this form of target regula- The successful isolation of TnsC gain-of-function mu- tion is not universally observed. The bacterial transpo- tants reveals that the TnsABC machinery is capable of son TnlO, for example,follows a dramatically different engaging target DNA and promoting insertions without reaction pathway in vitro. TnlO transposase can synapse TnsD or TnsE. However, the mutant reactions have not the transposon ends and initiate cleavage at those ends mimicked the abilities of TnsD or TnsE to direct trans- before interactions with target DNA occur (SAKAIet al. position into particular targets: transposition activated 1995). In fact, the TnlO transposase cannot capture a by the TnsC mutants does not show the preferential target DNA unless the transposon end cleavages have insertion into conjugable plasmids seen with TnsE-acti- occurred (KLECKNER et al. 1996). Bacteriophage Mu vated reactions, nor the attTn7specificity of TnsD-acti- transposase (MuA) can also synapse and nick the Mu vated reactions. Therefore, TnsD and TnsE are essential transposon ends before the introductionof target DNA to recognize these positive target signals. However, we into the reaction in vitro (CRAIGIEand MIZUUCHI1987; have demonstratedthat TnsD and TnsE are not re- SURETTEet al. 1987). Itwill be interesting to determine quired to recognize the negative signal contained on whether these assembly and cleavage steps remain un- immune targets. Transposition activated by a subset of coupled from target interactions in vivo. Recently, MuA the gain-of-function TnsC mutants maintains the ability transposase mutants have been identified that impose of wild-type Tn 7 transposition to recognize and avoid a dependence upon target DNA interactions on Mu targets that already contain acopy of Tn 7. This strongly transposition in vitro (MIZUUCHIet al. 1995). Thesemu- suggests that the ability to evaluate immune targets lies tants cannot interact with the Mu enhancer sequence, in TnsC and the transposase, not in the positive target which normally promotes the assembly of the synaptic selectors TnsD and TnsE. complex. Instead, the transposase mutants require an TnsC is therefore hypothesized to receive a variety of appropriate target DNA and the target-binding protein inputs, from TnsD, TnsE and from immune targets, MuB to initiate transposition. The MuB-target complex that control its activity.The activity of TnsC can also be has been proposed to serve as a scaffold that directs the influenced by mutation. Six gain-of-function TnsC assembly of the synaptic complex. Thus, Mu transposi- point mutants have been described in this work, which tion as executed by these transposase mutants has be- segregate into two classes. The fact that we recovered come mechanistically more similar to Tn7 transposi- different classes ofTnsC mutants with different transpo- tion, in which transposition initiation is dependent on sition activities is consistent with the hypothesis that inputs from target DNA both in vivo and in vitro. there are multiple routes to activating TnsC. The Class Proteins involved in target evaluation:How is an ap- mutants, I TnsCMZ5"and enablethe TnsABC propriate target for Tn7 transposition identified? We machinery to execute transposition without sacrificing have hypothesized that TnsC may serve as a "connec- its abilityto respond to both positive and negative target tor" or "matchmaker", linking the transposase and the signals. Both are substantial gain-of-function mutants, target DNA in a manner regulated by the ATP state of with TnsABCAZz5"promoting eight-fold more transposi- TnsC (BAINTONet al. 1993; SANCARand HEARST 1993). tion to the chromosome than TnsABC"' + E (Figure TnsC has the biochemical properties necessary for that 3). Transposition activated by these Class I mutants can connection: it can directly interact with target DNA be profoundly stimulated by TnsD + attTn7or directed (GAMASand CRAIG1992) and with the TnsA+B transpo- to conjugable plasmids by TnsE, as well asbeing able to sase (A. STELLWAGENand N. L. CRAIG,unpublished re- discriminate between immune and nonimmunetargets. Tn 7 Gain-of-Function Mutants 583

Thus, the gain-of-function phenotypes seen with the position is also profoundly influenced by its ATP-utiliz- Class I mutants have been achieved while preserving ing proteinMuB. MuB isan ATPdependentDNA bind- the ability of these TnsCs to transduce information being protein (CHACONAS 1985; MAXWELL et al. 1987), tween the target DNA and the transposase. which is required for efficient transposition invivo The TnsC mutants that fall into the second class be- (FAELENet al. 1978; O'DAYet al. 1978). In vitro, the MuA have much more like constitutively activated versions transposase preferentially directs insertions into targets of TnsC. Some of these mutants also promote consider- that are bound by MuB (CRAIGIEand MIZUUCHI1987; able amounts of transposition: TnsABCS401YA40' results SURETTEet al. 1987; ADZUMA and MIZUUCHI1988). Al- in 50-fold more transposition to the chromosome than though there is no particular sequence specificity to TnsABC"' + E (Figure 3). However, the nature of the MuB binding, its distribution on DNA is not random: transposition reactions promoted by the Class I1 TnsC MuB binding to target molecules that already contain mutants is quite different than those seen with the Class Mu sequences is specifically destabilized through an I mutants. Immune and nonimmune targets are used ATP-dependent mechanism (ADZUMA and MIZUUCHI essentiallyequivalently in reactions with the ClassI1 1988). Therefore Mu, like Tn 7, recognizes and avoids mutants, and TnsD and TnsE are notable to profoundly immune targets; moreover, MuB and TnsCAZz5"appear influence the frequency or distribution of these trans- to play functionally similar roles in regulating transposi- position events. Intriguingly, a similar loss of respon- tion. siveness to target signals is seen when Tn 7transposition Mu and Tn 7 belong to a family of transposons that is activated by nonhydrolyzable ATP analogs invitro. encodes proteins with ATP binding/hydrolysis motifs; Transposition can still occur when TnsC's ATPase activ- other members include IS21 (REIMMANN et al. 1989; ity is blocked with AMP-PNP, but those transposition KOONIN 1992),Tn552 (ROWLAND and DYKE 1990), events no longer require TnsD and are no longer tar- Tn5053 (KHOLODII et al. 1995), and Tn5090(RADSTROM geted to attTn7 (BAINTONet al. 1993). Instead, any DNA et al. 1994). Therefore, the strategy of using an ATP molecule, including immune targets, can serve as a tar- binding protein to regulate target site selection may get for Tn7 insertion. Thus, TnsABC transposition can extend to the entirefamily. Tn5053 is particularly inter- be constitutively activated by AMP-PNP or by the Class esting, since it encodes three proteins that are required I1 TnsC mutants. It will be interesting to explore for its transposition: a presumptive transposase con- whether theATP interactions of the TnsC mutants have taining a D, D (35)E motif characteristic of transposases been altered. In that regard, it is provocative that the and integrases, a potential regulatory protein con- amino acid affected in TnsCE233K lies inone of TnsC's taining Walker A and B motifs, and a third protein of ATP motifs. unknown function (KHOLODII et al. 1995).Tn5053 To be an effective communicator of multiple target shows some degree of target site specificity, inserting DNA signals, TnsC is probably a multidomain protein. predominantly into the parlocusof the conjugable plas- Some parts of TnsC may be involved in contacting tar- mid FW4. It is tantalizing to speculate that the third get DNAs, other parts of TnsC may contact the transpo- protein of Tn5053 is a target selector, like TnsD or sase, and still other domains may bind and hydrolyze TnsE, directing insertions into the par locus. ATP. The TnsC point mutants are scattered across the This work has illustrated the role of target DNA in TnsC protein sequence and could affect any or all of controlling Tn 7 transposition in vivo and has strongly these activities. We have also analyzed a TnsC deletion implicated TnsC as a central player in this regulation. mutant that may identify a domain in TnsC that con- Single amino acid changes in TnsC can disrupt the tacts the TnsA+B transposase. Based on the observa- communication between the transposon and the target tions of SMITH and JONES (1984), we created a site, reducing thestringency of Tn 7's target site selectiv- T~S(BC~"''~)deletion-fusion mutant inwhich the ity. However, close relatives of Tn7 containing func- amino-terminal half of TnsC (including the Walker A tional equivalents of these TnsC gain-of-function mu- and B motifs) is removed and the remainder of TnsC tants have yet to be identified. It will be interesting to is fused to TnsB. Unlike TnsABC"', T~SA(BC*"~~~)can see if such elements exist or whether the precise tar- activate transposition in the absence of TnsD or TnsE geting conferred by TnsD and TnsE is more advanta- and without the ability torespond to these factors. geous for Tn7's survival and dissemination. TnsD pro- Thus, T~S(BC~"'~~)behaves like a ClassI1 TnsC mu- motes Tn 7 insertion at high frequency into attTn7, a tant. The deletion may have exposed a domain that safe haven in the bacterial chromosome, whereas TnsE interacts with (and isnow covalently linked to)the allows Tn 7 access to conjugable plasmids, and thus a transposase, whereas the activity of that domain may means to spread through bacterial populations. Avoid- normally be regulated by ATP and/or inputs from tar- ance of immune targets also promotes the spread of get DNAs or target selection factors. the element, rather than local hopping, and prevents Comparison to other elements: The use of an ATP- one Tn 7 element from inserting into another. We hy- dependent protein such as TnsC to regulate target site pothesize that TnsC integrates all of these target signals selection is not unique to Tn 7. Bacteriophage Mu trans- and communicates that information to the transposase. 584 A. E. Stellwagen and N. L. Craig

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