JOURNAL OF BACTERIOLOGY, OCt. 1984, p. 192-198 Vol. 160, No. 10021-9193/84/100192-07$02.00/0Copyright C 1984, American Society for Microbiology

Regulation of the F-Factor pif Operon: pifO, a Site Required in cisfor Autoregulation, Titrates the pifC Product in trans

JEFF F. MILLER AND MICHAEL H. MALAMY*Department of Molecular Biology and Microbiology, Tufts University Schools of Medicine, Dental Medicine and

Veterinary Medicine, Boston, Massachusetts 02111

Received 17 April 1984/Accepted 25 July 1984

F factor pijC, pifA, and pi{B gene expression is subject to negative regulation by the product of the pifC locus(J. F. Miller and M. H. Malamy, J. Bacteriol. 156:338-347, 1983). In this paper, we describe the properties ofa new regulatory site in the pif region, pifO, which is required in cis for autoregulation of pif gene expression.Spontaneous pifO mutations were isolated that allow expression of a pifJC-acZ protein fusion in the presence ofpifC product in trans. Recombination of these pifO mutations onto F'lac results in increased pifA and pijBactivity. Thus, a single regulatory element, pifO, regulates pigC, pifA, and piJB expression in cis. The presenceof multiple copies of a fragment from the pif region carrying wild-type pifO sequences (F coordinates 42.9 to42.43 kilobases) in trans to F'Iac results in an increase in pifA and pi.B activity as measured by inhibition of T7plating. When the pifO mutations are recombined onto a plasmid carrying pifO, the resulting recombinants aregreatly decreased in the ability to increase F'lac pif expression. These results suggest that increased F'lac pifAand pi.B expression caused by pifO sequences in trans is a consequence of titration of pifC product andderepression of the pif operon.

The pif genes of the Escherichia coli F factor provide aninteresting system for the analysis of the control of geneexpression by autoregulation. The pif region consists of atleast three genes, pifC, pifA, and pijB, located near theprimary origin of mini-F replication, oriVI (1, 10, 12, 15, 17).The products of pifA and pijfB inhibit bacteriophage T7development (1, 15, 17), and the pifC locus codes for anegative regulator of its own synthesis as well as that of pifAand pifB (12).A 5.2-kilobase (kb) PstI fragment from the pif region of F

has been cloned into plasmid pSC101, and the resultingplasmid, pGS103, is fully Pif+ (17). Through the use of pif-lacZ protein fusions we have determined the approximateposition of the N termini of pifC, pifA, and piJB (12). Theestimated boundaries of the pifC gene are shown in Fig. 1with the assumption that the N terminus of pifA is contigu-ous with the C terminus of pifC. As can be seen in Fig. 1,oriVI (F coordinate 42.6 kb [4, 5]) lies approximately 200base pairs upstream of the N-terminal coding region of pifC.Recombination of the pifC-lacZ, pifA-lacZ, and pifB-lacZ

translational fusions onto F'lac, thereby joining the fusionswith the wild-type pif transcriptional control sequences, hasallowed us to study the regulation of the synthesis of each ofthe fusion products (12). We have shown that the product ofthe pifC gene, supplied in trans by pGS103 or pif deletionderivatives retaining the pifC gene, inhibits the synthesis ofthe pifC-lacZ, pifA-lacZ, and pifB-lacZ fusion proteins. Inaddition, pifC+ plasmids deleted for pifA and pifB decreasepif expression of F'lac when present in trans as determinedby T7 efficiency of plating and plaque morphology (12). Inthis paper, we report the location and properties of a site,which we have named pifO, that is required in cis forautoregulation of pif gene expression. Spontaneous muta-tions in pifO result in decreased sensitivity to the inhibitoryeffect of the pifC product on expression of pifC, pifA, andpifl, showing that these genes make up an operon. We havealso analyzed the effect of the presence of multiple copies of

* Corresponding author.

pifO in trans to the pif operon. A dramatic increase in F'lacpifA and pijB product activity results from the presence ofpifO supplied in trans on plasmid pSC101 and pBR322replicons. Spontaneous mutations in pifO result in the loss ofthis ability to elevate F'lac pif expression.

MATERIALS AND METHODSBacterial strains, plasmids, bacteriophage, and growth me-

dia. E. coli K-12 strains used were JF270 (/lacX74 recArpsL), RV101 (AlacX74 gyrA101), and RV200 (AlacX74rpsL200). Plasmids used and constructed for this study aredescribed in Table 1. Phage T7 was originally from F. W.Studier. Solid and liquid growth media (ML) have beenpreviously described (12, 14).DNA manipulations and analysis. Isolation of plasmid

DNA, use of restriction endonucleases, DNA ligation, andcalcium chloride transformation procedures have been de-scribed (12).T7 efficiency of plating. The plating efficiency of T7 on

plasmid-containing strains was determined as previouslydescribed (17) and is reported as the ratio of the T7 titerobtained on plasmid-containing cells divided by the T7 titerobtained on isogenic, plasmid-free cells.

I-Galactosidase assay. f3-Galactosidase levels were deter-mined as described by Miller (13) with the following modifi-cations, Cell extracts were prepared from 2 ml of cells byadding 3 drops of 0.1% sodium dodecyl sulfate and 4 drops ofchloroform, followed by vigorous agitation for 10 s followedby incubation for 15 min at 37°C on a tissue culture rollerdrum. Units are as described by Miller (13) and have beennormalized to an initial cell density (absorbancy at 600 nm)of 1.0.

Isolation ofpifO mutations in F'C521 resulting in decreasedsensitivity to the pifC product in trans. Strain JF270, contain-ing both F'C521 and pGS103 (phenotypically Lac- due tothe inhibition of F'C521 pifC-lacZ expression by the pifCproduct supplied in trans by pGS103 [12]), was streaked ontoseveral MacConkey lactose plates and incubated for 48 h at37°C, after which time numerous Lac' papillae were visible.Sixty-five tubes of ML broth were inoculated with 5 to 10

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pif GENE EXPRESSION 193

F----pi ----

oriVlIs

PstI BamHI Nco I PvuII43-6 43.0 42.9 42-43

pi C pi AItv v

BgIJI Hindlil BamHI41.5 4&9 40.5

pit B3 3 38.9Eoll Hindnl Pstr39.3 39.2 38.9

H-

FIG. 1. Partial physical map of pif region and the structure of pif plasmids used in this study. Restriction sites shown in the figure havebeen assigned F factor kb coordinates as previously described (12). This figure is drawn to scale except where interruptions are indicated byslashed lines. The NcoI and PvuII coordinates are from H. E. D. Lane (personal communication). The top line presents the DNA content ofpGS103 (17), the Pif+ plasmid used to construct the other plasmids shown. pGS103 contains F-factor sequences spanning the f5-f7 junctionwith coordinates 44.1 to 38.9 kb. pRC201 and pRC201.N1 are pBR322-derived replicons. Other plasmids shown are pSC101 replicons. oriVI(4, 5) is the primary origin of mini-F replication. The approximate boundaries of the PifC coding sequences are indicated by vertical lines,based on the size of PifC-LacZ and PifA-LacZ fusion proteins and the assumption that pifC and pifA are contiguous (12). The pifgenotypes ofthe plasmids used are indicated. pifO designates the sequences required in cis for PifC regulation ofpifexpression which are also capable of el-evating F'lac pifexpression in trans (see text). Since the exact size and location ofpifO has not been determined, a 0.47-kb interval containingpifO is shown. Although pifO and oriVi map to the same region, the relationship between these loci is not yet known (see text).

papillae per tube and were incubated at 37°C until cellsentered the log phase. Each of these cultures was mated withRV101(pGS103) by a standard mating protocol (9). Afterincubation at 37°C for 2 h with gentle aeration, each matingmixture was streaked onto a MacConkey lactose platecontaining 50 pg of nalidixic acid per ml to select forrecipient cells. Out of 65 plates, 28 were found to contain 1 to10 Lac' colonies among about 100 isolated colonies. Asingle Lac' colony was purified from each of these plates.Three independent Lac' isolates obtained in this manner,containing F'C521-36 (pifO36) plus pGS103, F'C521-55(pifOSS) plus pGS103, and F'C521-70 (pifO70) plus pGS103,were chosen for further study. F'C521-36, -55, and -70 weretransferred into JF270 and JF270(pGS103) by mating formeasurement of ,B-galactosidase levels and further charac-terization.

Construction of pVU14 pifO mutants. The pifO36, pifOSS,and pifO7O mutations isolated in F'C521 as described abovewere recombined onto plasmid pVU14 by mobilization-associated recombination (9, 12). Cultures of F'C521-36, -55,and -70 in the JF270 background were mated with RV101(Nalr) containing pVU14 (Tcr; Fig. 1), and Lac' Nalr Tcrrecipients were isolated. pVU14 was then mobilized byF'C521-36, -55, and -70 from this background into RV200(Smr) containing pJM1383 (Ampr mini-F derivative; Table1), and Lac- Ampr Tcr recipients were obtained (see below).In this situation, homology-mediated recA dependent cointe-

grate formation is responsible for the majority of mobiliza-tion events (9). F'C521 (wild type or -36, -55, -70) cointe-grates with pVU14 are highly unstable and resolve quicklyowing to the presence of two class III mobilization sites (9).Reciprocal exchange of sequences can occur if a cointegratemolecule resolves at a different position from that used forits formation. The presence of plasmid pJM1383 in theRV200 recipients has two consequences. First, pJM1383 isan ampicillin-resistant mini-F replicon and is incompatiblewith F'C521 and the mutant derivatives of F'C521 used inthis study. Selection for Ampr Tcr results in loss of F'C521after cointegrate resolution, and the resulting colonies arepredominately Lac-, some with small Lac' sectors on theappropriate MacConkey lactose plates. Second, pJM1383 isPifr owing to the addition of the f fragment from pGS103 tothe f5 replicon pSC138 (Table 1). pif expression by pJM1383is elevated by the presence of wild type pifO sequences intrans, as is F'lac pif expression (see below). Thirty RV200isolates containing pJM1383 and pVU14 plasmids derivedfrom cointegrates with F'C521-36, -55, and -70 (10 isolatesper mutant) were plated with phage T7 to differentiatebetween wild-type pVU14 and mutant pVU14 plasmidsunable to increase pJM1383 pif expression. When platedwith approximately 2 x 103 PFU of T7, RV200(pJM1383)plus wild-type pVU14 yielded about 25 barely detectableplaques. RV200(pJM1383) plus pVU14-36 (pifO36), pVU14-55 (pifO55), or pVU14-70 (pifO7O), however, gave 150 to 250

pGS 103

0 fO C A B

pGS211

pGS221

pVU14

pRC 201

pRC 201. Ni

+ .e.A A

+ A A A

4. AA AZ

.g AA AA

+ A AA

pGS204 A A A + - \I

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194 MILLER AND MALAMY

distinct clear small plaques, a plating result similar to thatseen with pJM1383 alone. When results from all threemutant crosses were combined, the frequency of recombina-tion among mobilized pVU14 plasmids was about 15%.

Plasmid DNA was isolated from RV200(pJM1383) pluspVU14-36, -55, and -70, and both JF270(F'lac) and RV200were transformed. Tcr Amps colonies, containing the pVU14derivative only, were obtained and used for further study.

Recombination of pifO mutations onto F'lac. The pifO36,pifOSS, and pifO7O mutations were recombined onto F'lacby mobilization-associated recombination (9, 12). F'lac wasintroduced by conjugation into RV200 carrying pVU14-36,pVU14-55, or pVU14-70. The pVU14 pifO mutants werethen mobilized by F'lac into RV101 (Nalr) by using homolo-gy-mediated, recA-dependent cointegrate formation. FifteenLac' Nalr Tcr colonies were isolated from each mating.F'Iac was then transferred from each isolate into JF270(Smr). Lac' Smr Tcs colonies obtained from these matingscontained F'lac factors that were the products of cointegrateresolution. One such isolate from each mating (45 total) wasplated with bacteriophage T7. Eleven percent of the isolateshad elevated levels of pifA and pifB products, as shown byreduced T7 efficiency of plating (see Table 5). F'lac-36(pifO36), F'lac-55 (pifOSS), and F'lac-70 (pifO7O) were thusobtained after mobilization of pVU14 pifO mutants by F'lac.For further analysis, the F'lac pifO mutants were transferredinto JF270 containing pGS211.

RESULTSMutations within F'C521 resulting in decreased sensitivity

to the pifC product supplied in trans. The F factor derivative

F'C521 carries a fusion of the pifC gene to lacZ. Expressionof pifC-lacZ activity by this plasmid is controlled by wild-type pif transcription and translation signals. F'C521 alsocontains the site required in cis for inhibition by the pifCproduct. The 3-galactosidase activity of F'C521 is reduced inthe presence of plasmid pGS103 (Table 2). pGS103 (17) is a

pSC101 replicon which carrys the entire pifregion containedon a 5.2-kb PstI fragment with coordinates 38,9 through 44.1kb (F coordinates have been described [17, 12]). pGS103 ispifC+ and is therefore able to decrease expression of pifC,pifA, and pifB when present in trans (12; Table 2). pGS103inhibits T7 infection more effectively than the F factor (17).To isolate mutations in F'C521 at the site of action of PifC,

we took advantage of the fact that cells containing bothF'C521 and pGS103 are phenotypically Lac- on MacConkeylactose plates. However, during incubation at 37°C for 36 to48 h, Lac' papillae arose in regions of heavy growth as wellas from individual colonies. Upon further analysis (seeabove), these spontaneous Lac+ mutants were found tocontain either pGS103 mutations, resulting in the inability tosynthesize functional PifC, or F'C521 mutations conferringinsensitivity to PifC. This was the expected phenotype ofoperator-constitutive mutations, and we designated the ge-notype of these F'C521 derivatives as pifO. pifC- mutants ofpGS103 were isolated about 20 times more frequently thanF'C521 mutants by this procedure.Table 2 shows ,B-galactosidase levels of three independent-

ly isolated pifO mutants of F'C521. Each of the mutationsresulted in altered expression of the fusion protein in boththe presence and absence of a pifC+ locus in trans. In theabsence of pifC, the level of ,-galactosidase activity pro-

TABLE 1. Plasmids used in this study

Plasmid Description, construction, and genotype Phenotype referencepif plasmids and deletionspGS103 5.2 kb (f5 through f7, 44.1 to 38.9 kb), 0.46 kb Pif+a PifC+b Tcr 17

(f6), 0.1 kb (f3); 9.1-kb pSC101 repliconpGS211 1.9-kb HindIlI deletion of pGS103 Pif- PifC+ Tcr 17pGS221 2.45-kb BgIII deletion of pGS103 Pif- PifC- Tcr 17pVU14 6.4-kb PvuII deletion of pGS103 Pif- PifC- Tcr R. CooneypGS204 2.7-kb BamHI deletion of pGS103 Pif- PifC- Tcr 17pRC201 0.57 kb (fS, 43.0 to 42.43 kb); 9.9-kb pMC1403 (3) Pif- PifC- Ampr R. Cooney

derived from pBR322 repliconpRC201.N1 0.47 kb (f5, 42.9 to 42.43 kb); 4.5-kb NcoI dele- Pif- PifC- Ampr G. Terranova

tion of pRC201F replicons

F' lac pifC+ pifA+ pifB+ Lac+ Mel+ Pif+ Pasteur InstituteF'C521 pifC-1acZ protein fusion from pJM521 (12), Lac+ Mel' Pif- 12

A(pifAB), fusion protein expressed from pif reg-ulatory signals

pJM1383 pSC138 (f5 replicon, 49.3 to 40.3 kb [22]), f7 (40.3 Pif+ IncF+ Ampr This workto 38.9 kb), and 0.1-kb f3 linker from pGS103

Mutant plasmidsF'C521-36,-55, -70 Spontaneous F'C521 pifO36, pifOSS, and pifO7O Lac+ Mel' Pif- This work

mutants with decreased sensitivity to PifCpVU14-36, -55, -70 pVU14 pifO36, pifOSS, and pifO7O mutants, de- Pif- PifC- Tcr This work

rived from recombination with F'C521 pifOmutants, showing decreased ability to increaseF' lac pif expression (see text)

F'lac-36, -55, -70 F'lac pifO36, pifOSS, and pifO7O mutants, derived Lac' Mel+ This workfrom recombination with pVU14 pifO mutants, Pif+c+showing increased pifA and pifB expression

F'lac C21 pifC21 pifA+ pifB+ Lac+ Mel+ Pif++ 12a Pift phenotype is inhibition of T7 plating.PifC+ phenotype is inhibition of pif expression (12).

c Pift+ phenotype designates greater inhibition of T7 plating than F'lac.

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pif GENE EXPRESSION 195

TABLE 2. Effect of pifC in trans on p-galactosidase expression by F'C521 and F'C521 pifO mutants"

Il-Galactosidase units'F' factor pif genotype of F' factor Absence of Presence Repression

pGS103" pGS103 ratio'

F'C521 plfO±e pifC-IacZ A(pifAB) 214.2 ± 5.8 9.0 ± 1.8 23.8F'C521-36 pifO36 pifC-lacZ A(pifAB) 141.8 ± 1.5 115.8 ± 2.5 1.2F'C521-55 pifO55 pifC-lacZ A(pifAB) 353.6 ± 8.2 171.6 ± 10.6 2.1F'C521-70 pifO7O pifC-IacZ A(pifAB) 344.5 ± 4.5 79.9 ± 1.2 4.3

" All assays were performed in the strain background JF2 70 (Alac recA) grown in M63 with 0.4% glucose, 0.2% Casamino Acids, and 2 pg of vitamin Bi per ml.b Results are expressed in units of ,-galactosidase measured and defined as described by Miller (13). All results are normalized to an initial cell absorbancy at

600 nm of 1.0. Levels reported are averages of at least two independent determinations.' Repression ratio is defined as the units of ,B-galactosidase produced in cells containing F'C521 or F'C521 pifO mutants divided by the units of ,B-galactosidase

produced by the same F' factors in the presence of pifC product supplied in trans by pGS103.d pGS103 is pifO+C+A+B' (Table 1, Fig. 1).e pifO designates the sequences required in cis for sensitivity to inhibition by PifC.

duced by F'C521-36 (pifO36) was decreased 1.5-fold as

compared with that of the parental F'C521; the activities ofF'C521-55 (pifO55) and F'C521-70 (pifO7O) were increasedabout 1.6-fold. Decreased sensitivity to pifC in trans was

also reflected by a decreased repression ratio (Table 2). Thelevel of P-galactosidase expressed by F'C521 is normallyreduced by a factor of about 24 when the pifC gene is presentin trans on a pSC101 vector (12; Table 2). The F'C521 pifOmutants were much less sensitive to the pifC product in transbut did show a low level of inhibition. The three mutantplasmids showed different repression ratios. F'C521-36, witha repression ratio of 1.2, was least sensitive to PifC, whereasF'C521-55 was repressed by a factor of 2. F'C521-70 showedthe greatest response to PifC, as indicated by a repressionratio of 4.3.

Effect of pif sequences in trans on T7 inhibition by F'lac.While investigating the effect of cloned fragments from thepif region on F'lac pif gene expression, we observed thatcertain plasmids when present in trans were capable ofdramatically enhancing the ability of F'lac to inhibit T7development. The presence of the pif deletion plasmidpGS221 resulted in an increase in F'Iac pifactivity (Table 3),as shown by a 140-fold decrease in the efficiency of T7plating with a corresponding alteration in plaque morpholo-gy. (T7 plaques on F'lac-containing cells are pinpoint,whereas those seen on cells containing pGS221 and F'lac are

barely visible. Differences in plaque size and morphology area reflection of burst size [15]). pGS221 is deleted for pifA andpifB as well as the distal portion ofpifC and is phenotypicallyPifC- (12; Fig. 1).

We have localized the sequences responsible for themarked increase in F'Iac inhibition of T7 plating by examin-ing additional deletion derivatives of pGS103 and plasmidscontaining fragments from the pif region. Plasmids pVU14,pRC201, and pRC201.N1 were also capable of elevating thelevel of F'lac inhibition of T7, whereas pGS204 showed no

effect (Table 3). The structures of these plasmids are shownin Fig. 1.The smallest pif fragment so far tested that is capable of

increasing F'Iac pif activity is the NcoI-PvuII fragment withF coordinates 43.14 to 42.7 kb, contained in plasmidpRC201.N1. Cells containing pRC201 and F'Iac or

pRC201.N1 and F'lac showed a greater degree of T7 inhibi-tion than did cells containing pGS221 and F'Iac or pVU14and F'lac (Table 3). This difference may reflect the increasedcopy number of pRC201 and pRC201.N1 (pBR322-derivedreplicons) over that of GS221 and pVU14 (pSC101 repli-cons).Two possibilities might explain the observed results. It

may be that pGS221, pVU14, pRC201, and pRC201.N1supply a positive factor involved in pif expression or activi-ty. Alternatively, an increase in pif expression could resultfrom titration of pif repressor by additional PifC bindingsequences present in trans (see below). To differentiatebetween these possibilities, we tested whether the pifOmutations of F'C521 could be crossed onto pVU14, givingmutant pVU14 plasmids affected in the ability to increase pifexpression in trans.pifO mutations result in a decreased ability to alter F'Iac pif

expression in trans. The effect of the pifO36, pifOSS, and

TABLE 3. pifO sequences elevate F'Iac pif activity when present in trans'

F' factor pif genotype of Plasmid in pifgenotypeb of T7 efficiency PlaqueF' factor trans plasmid of plating morphology'

None None (F-) None 1.0 Pif-F'lac pifO+dC+A+B+ None 0.10 Pif+F'lac pifO+C+A+B+ pGS221 pifO+A(pifCAB) 7 x 10-4 Pif++F'lac pifO+C+A+B+ pVU14 pifO+ A(pifCAB) 7 x 10-4 Pfi++F'lac pifO+C+A+B+ pRC201 pifO+ A(pifCAB) <10-5 Pif++F'lac pifO+C+A+B+ pRC201.N1 pifO+ A(pifCAB) lo-I Pif++F'lac pifO+C+A+B+ pGS204 A(pifOCA) pifB+ 0.11 Pif+

aAll plasmids were carried by strain JF2 70 (Alac recA). T7 efficiency of plating was determined as detailed in the text. T7 efficiency of plating on the F- strainJF2 70 is assigned a value of 1.0, and all other determinations are normalized to this value.bpif genotype is based on the pif structural map as shown in Fig. 1.c The wild-type Pif (F-) plaque is characterized by a large center surrounded by a large halo. PifF plaques are pinpoint. Pif+ + plaques are irregular and barely

visible.d pifO designates the sequences required in cis for sensitivity to inhibition by PifC.

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TABLE 4. Effect of pifO+ and pifO mutations in trans on T7 inhibition by F'Iac"

F' factor pif genotype of Plasmid in pif genotype of T7 efficiency PlaqueF' factor trans plasmid' of plating morphology'

None None (F-) None 1.0 Pif-F'lac pifO+dC+A+B+ None 0.26 Pif+F'lac pifO+C+A+B+ pVU14 pifO+A(pifCAB) 7 x 10-4 Pif++F'lac pifO+C+A+B+ pVU14-36 pifO36Lt(pif-CAB) 0.17 Pif+F'lac pifO+C+A+B+ pVU14-55 pifO55 A(pifCAB) 0.14 Pif+F'lac pifO+C+A+B+ pVU14-70 pifO 70 A (pifCAB) 0.09 Pif+'a See Table 3, footnote a.bSee Table 3, footnote b.c See Table 3, footnote c.d pifO designates the sequences required in cis for sensitivity to inhibition by PifC.e T7 plaques on cells containing F'lac and pVU14-70 were smaller and more irregular then those seen with Flac alone.

pifO7O mutations on the ability of plasmids containing the pifregulatory region to increase F'lac pifexpression was tested.Each of the F'C521 pifO mutations analyzed in Table 2 was

crossed onto pVU14 by a procedure involving mobilization-associated recombination as described above. The frequen-cy of pifO recombinants among mobilized plasmids was

about 15%. The resulting plasmids, pVU14-36 (pifO36),pVU14-55 (pifOS5), and pVU14-70 (pifO7O), did not appear

to have suffered deletions or rearrangements on the basis ofrestriction analysis (data not shown).The pVU14 derivatives containing the pifO mutations had

a greatly reduced ability to stimulate F'lac pif expression(Table 4). The fact that the pifO mutations could be crossedonto pVU14 indicates that they lie within the part of the pifregion common to pVU14 and F'C521. The increase in F'Iacpifexpression caused by PVU14 and related plasmids carry-

ing a pifO region is therefore dependent on the sensitivity ofthe pifO locus to PifC. We therefore extend our definition ofpifO (Fig. 1) to include the sequences which are required incis for sensitivity to inhibition by PifC and are capable ofelevating F'lac pif expression in trans. Each of the pVU14plasmids carrying the pifO mutations did, however, show alow but detectable alteration in the ability of F'lac to inhibitT7 development. The efficiency of plating of T7 on cells,containing F'lac and pVU14-36 or F'lac and pVU14-55 was

slightly less than that on cells containing F'lac alone (Table4). pVU14-70, however, caused a threefold decrease in theT7 efficiency of plating, and the resulting plaques weresmaller and more irregular than those produced by cellscarrying only F'lac. This effect was much less than the 370-fold decrease in efficiency of plating seen with the parentalplasmid pVU14. Consistent with this result is the observa-tion that F'C521-70 showed a repression ratio of 4.3 (Table

2), a higher value than that seen with F'C521-36 or F'C521-55. Therefore, the repression ratios of the pifO mutants seemto correlate with their effect on F'lac pif expression.

cis-dominant pifO mutations result in increased F'lac pifAand pipl product activity. Each of the pifO mutations was

recombined onto F'lac by mobilization-associated recombi-nation between F'lac and pVU14-36, pVU14-55, or pVU14-70 (see above). Approximately 11% of the F'lac factors thathad mobilized the pVU14 pifO mutants showed a largeincrease in pifA and pifB product activity as measured by T7efficiency of plating. These F'lac derivatives are assumed tocarry the respective pifO mutations.The efficiency of bacteriophage T7 plating on cells con-

taining F'lac-36 (pifO36), F'lac-55 (pifO55), or F'Iac-70(pifO7O) was reduced 10,000-fold in comparison with F'lac,and the resulting plaques were barely visible (Table 5). Thepresence of pGS211 (pifC+ ApifAB) (Table 1, Fig. 1) in transto the F'Iac pifO mutants had only a small effect that was

seen when large numbers of T7 (106/ml) were plated. pGS211decreased expression of the wild-type pif region of F'lac, as

shown by a threefold increase in T7 efficiency of plating witha corresponding alteration in plaque morphology (Table 5).Differences between F'lac-36, -55, and -70 were not detectedeither in the absence or presence of the pifC product intrans, probably owing to decreased sensitivity of the T7efficiency of plating assay on strains containing high levels ofthe pifA and pifB gene products.These results demonstrate that the pifO mutations have a

cis effect on F'lac pifA and pi4B expression and are dominantto the wild-type pif sequences supplied by pGS211 in trans.This should be contrasted with F'Iac C21, which carries a

pifC mutation (pifC21; Table 1) that is recessive to thewildtype pifC gene (12). The greatly increased pif activity of

TABLE 5. T7 inhibition by F'lac pifO and pifC mutants in the presence and absence of pifC product in transa

Absence of pGS211b Presence of pGS211

F' factor pif genotype ein7 Plaque T7 efficiency of Plaqueefficiency of morphology" plating morphologyplating

None None (pi}) 1.0 Pif- 1.0 Pif-F' lac pifO+d pifC+A+B+ 0.16 Pif+ 0.50 Pif-'F' lac-36 pifO36 pifC+A+B+ slO-s Pif++ 10-4-_0-5 Pif++F' lac-55 pifO55 pifC+A+B+ _10-5 Pif++ 10-4_10-5 Pif++F' lac-70 pifO7O pifC+A+B+ <10-5 Pif++ 10-41-0-5 Pif++F' lacC21 pifO+ pifC21 pifA+B+ s10-6 Pif++ 0.45 pif-e

a See Table 3, footnote a.b pGS211 is pifO+ pifC+ A(pifAB); see Table 1 and Fig. 1.c See Table 3, footnote c.d See Table 3, footnote d.These plaques were similar to Pif plaques but had smaller halos.

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pif GENE EXPRESSION 197

F'lac C21 was markedly reduced by the presence of pGS211in trans (Table 5). The T7 efficiency of plating on cellscontaining F'lac C21 plus pGS211 was nearly indistinguish-able from that seen on cells carrying wild-type F'lac pluspGS211.

DISCUSSIONThe product of the pifC gene acts in trans to decrease

expression of pifC, pifA, and pifB (12). We have nowidentified an additional regulatory element, pifO, locatednear the N-terminal coding sequences of pifC (Fig. 1). pifOmutations in cis result in increased expression of the F'C521pifC-IacZ protein in the presence of pifC product in trans.pifO mutations in cis also increase F'lac pifA and pif1expression in the presence or absence of additional pifCproduct supplied in trans. Since pifC, pifA, and pifB expres-sion is subject to regulation by the same cis-acting site, weconclude that these genes form an operon. An interestingfeature of this operon is that the structural gene for thenegative control product is part of the operon it regulates;therefore, the pif operon is autoregulated. The polarityproperties of Tn5 insertions and pifC amber mutations areconsistent with cotranscription of pifC, pifA, and pifB (17;J. F. Miller, E. Buchert, and M. H. Malamy, manuscript inpreparation).pif expression, measured by F'Iac pifA and pifB product

activity, can be increased by the presence of certain pifsequences in trans. The region responsible for this effect hasbeen localized to the NcoI-PvuII fragment (F coordinates42.9 to 42.43 kb) present on pRC201.N1 (Fig. 1). Thisincrease in pif operon expression results from the presenceof wild-type pifO sequences on the fragments provided intrans. Spontaneous mutations in F'C521 (pifC-IacZ transla-tional fusion) result in decreased sensitivity to inhibition byPifC. F'C521-36 (pifO36), F'C521-55 (pifOS5), and F'C521-70 (pifO7O) contain cis-dominant constitutive mutationsshowing repression ratios ranging from 1.2 to 4.3, as com-pared with 24 for the parental plasmid (Table 3). When thesemutations are recombined onto pVU14, a pifO+ plasmiddeleted for pifC, the resulting plasmids, pVU14-36 (pifO36),pVU14-55 (pifOSS), and pVU14-70 (pifO7O), are no longercapable of causing the dramatic increase in pif expressionthat is normally seen with the parental plasmid, pVU14. Inaddition, recombination of each of the pifO mutations ontoF'lac results in increased pifA and pifB expression as shownby decreased T7 efficiency of plating in the presence andabsence of pifC product in trans (Table 5).Thus, there is a relationship between the sensitivity of a

site within the pif operon to the pifC product and the abilityof this site to increase pifoperon expression when present intrans to F'Iac. We conclude that a single genetic element,pifO (Fig. 1), is responsible for pifoperon regulation by PifCand also efficiently titrates PifC in trans as a result of itsability to bind PifC.

Several laboratories have shown that expression of lacZfrom the wild-type lac operon is increased to constitutivelevels by the addition of lac operator sequences in trans onhigh-copy-number plasmid vectors (8, 11, 18). The explana-tion for this increase is based on the disparity between thesteady-state level of lac repressor, estimated at approxi-mately 10 molecules per cell (6, 16), and the 20 or so operatorsequences being supplied by high-copy-number ColEl deriv-atives (7). The result is a decrease in the ratio of repressormolecules to operator sequences and a concomitant increasein lacZ expression.

Although the consequence of the addition of multiplecopies of pifO in trans to pif genes is similar to repressortitration in the lac operon, the specific regulatory features ofthe two systems are quite different. The pif operon, unlikelac, is negatively autoregulated. It is therefore possible toexplain the elevation in pif expression by pifO+ plasmids intrans by postulating that the presence of additional pifOsequences results in increased expression of pifC as a resultof autoregulation. The increase in pifC expression would beaccompanied by an increase in pifA and pifB products sincethese genes are coregulated. The actual level of pif expres-sion may be determined by the total number of pifO lociwithin the cell. This could maintain a constant ratio of PifCto pifO sequences as long as the concentration of pifOsequences does not exceed the capacity to synthesize PifC.The exact nature of the pifO sequence is not yet known. It

is most probable that pif regulation is at the transcriptionallevel, but it is also possible that the pifC product couldinhibit translation of pifmRNA, perhaps by binding to the 5'region containing the ribosome binding site. To determinethe nature of pif regulation by PifC we must locate the exactposition of transcription initiation and the sequence alter-ations of the pifO36, pifO55, and pifO7O mutations. Recentresults with operon fusions in pifA suggest a transcriptionalmode of control (Miller et al., manuscript in preparation).The pifO36, pifO55, and pifO7O mutations affect F'C521

pifC-lacZ expression in at least two ways. The sensitivity toPifC is reduced severalfold, and the level of expression in theabsence of PifC is altered. Each of the mutants differs in theamount of inhibition by PifC, as shown by individual differ-ences in the repression ratio (Table 2). In the absence ofPifC, the levels of ,-galactosidase activity produced byF'C521-55 and F'C521-70 are increased to a similar extentrelative to F'C521. F'C521-36 expression, however, is ap-proximately 1.5-fold less than that of the parental plasmid.Alterations in the fully induced level of lacZ expression werealso seen in the majority of lacOc mutations analyzed bySmith and Sadler (19). The pifO36, pifO55, and pifO7Omutations, when crossed onto pVU14, do not result indetectable DNA alterations on the basis of restriction map-ping. This, in addition to the partial sensitivity to PifC,suggests that these mutations are point substitutions orperhaps very small deletions.The small and characteristic amount of elevation of pif

expression in the presence of pVU14-36, pVU14-55, andpVU14-70 (Table 4) correlates with the repression ratiosseen with F'C521-36, F'C521-55, and F'C521-70, respective-ly (Table 3). The pifO36 mutation results in the greatestdecrease in sensitivity to PifC since F'C521-36 has thelowest repression ratio and the T7 efficiency of plating oncells containing pVU14-36 and F'Iac is greater than that seenon cells containing F'lac and pVU14-55 or pVU14-70. ThepifO70 mutation seems to have a severalfold greater sensitiv-ity to PifC than pifO36, and the pifO55 mutation is intermedi-ate in its response to this product in trans. F'lac-36, -55, and-70, in the presence or absence of pifC product in trans,appear nearly identical with respect to T7 efficiency ofplating. This may be due to a lack of sensitivity of the T7plating assay in situations in which the level ofpifA and pifBproducts is abnormally high.The ability to maintain a constant amount of repression of

unlinked genes in the presence of multiple or variableoperator sequences seems to be a consequence of autoge-nous regulation of repressor synthesis. Bogosian and Somer-ville (2) have recently reported an in vivo analysis of the trpRgene, an unlinked autorepressor that negatively controls

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198 MILLER AND MALAMY

transcription of the trp operon. Their results show that a

single-copy trpR gene, in the presence of excess tryptophan,is capable of repressing trp-lacZ and trpR-lacZ operonfusions in strains carrying multiple copies of the trp operonoperator sequence. These workers have also shown that a20-fold increase in the number of trpR genes has only a smalleffect on the actual level of repression, as long as the wild-type trpR transcriptional control sequences are regulatingexpression of repressor.As stated above, it is possible that autoregulation of the pif

operon results in a constant ratio of PifC to pifO sequences.An increase in the cellular concentration of pifO sequencesresults in a corresponding alteration in pifC expression. Itwould be expected, however, that the increased concentra-tion of PifC could regulate the additional pifO loci. The pifsystem may have evolved this control feature to allowregulation of genes other than pifC, pifA, and pifB. Recently,Tanimoto and lino (21) have shown that the F factor iscapable of reducing the frequency of transfer of the broadhost range plasmid RP4 by a factor of 500- to 1,000-fold.They have localized the gene responsible for this inhibitionto the region we assign to the pifC gene (12). We have foundthat RP4 plasmids contain PifC binding sequences (manu-script in preparation). The efficiency of plating of T7 on cellscontaining RP4 and F'lac is decreased to a similar level as

that seen on cells containing pVU14 and F'lac. The ability ofRP4 to titrate PifC when present in trans to the wild-type pifoperon suggests that F-factor inhibition of RP4 transfer is a

result of the activity of the pifC product.The relationship between the pifoperon and the regulation

of the initiation of F-factor DNA replication has not yet beendetermined. We have previously suggested, however, thatpif gene expression may be involved in the control of F-factor replication at oriVI (12). It is possible that the pifCproduct exerts negative control by decreasing expression ofan RNA primer required for DNA replication or by decreas-ing expression of a required protein or both. Alternatively, itis also possible that PifC protein has a dual role, functioningas both an autorepressor and as a positive factor required forinitiation at oriVi. This regulatory circuit would be analo-gous to that which has been proposed for the regulation ofreplication of oriV2-dependent mini-F replicons by the au-toregulated 29-kilodalton E protein (20). Analysis of thestructural features of PifC binding sequences and the regula-tory consequences of the interaction of PifC with thesesequences will be important in determining whether PifCdoes indeed have a role in replication control at oriVI.

ACKNOWLEDGMENTS

We thank E. Buchert, P. Rahaim, M. B. O'Connor, M. Challberg,and A. L. Sonenshein for critically reading the manuscript.

This work has been supported by Public Health Service grant Al-15840 from the National Institutes of Health.

LITERATURE CITED

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2. Bogosian, G., and R. L. Somerville. 1984. Analysis in vivo offactors affecting the control of transcription initiation at promot-ers containing target sites for Trp repressor. Mol. Gen. Genet.193:110-118.

3. Casadaban, M. J., J. Chou, and S. N. Cohen. 1980. In vitro genefusions that join an enzymatically active ,B-galactosidase seg-ment to amino terminal fragments of exogenous proteins: Es-cherishia coli plasmid vectors for the detection and cloning oftranslation initiation signals. J. Bacteriol. 143:971-980.

4. Eichenlaub, R., D. Figurski, and D. R. Helinski. 1977. Bidirec-tional replication from a unique origin in a mini-F plasmid. Proc.Natl. Acad. Sci. U.S.A. 74:1138-1141.

5. Figurski, D., R. Kolter, R. Meyer, M. Kahn, R. Eichenlaub, andD. R. Helinski. 1978. Replication regions of plasmids ColEl, F,R6K, and RK2, p. 105-109. In D. Schlessinger (ed.), Microbiol-ogy-1978. American Society for Microbiology, Washington,D.C.

6. Gilbert, W., and B. Muller-Hill. 1966. Isolation of the lacrepressor. Proc. Natl. Acad. Sci. U.S.A. 56:1891-1898.

7. Hershfield, V., H. W. Boyer, C. Yanofsky, M. A. Lovett, andD. R. Helinski. 1974. Plasmid ColEl as a molecular vehicle forcloning and amplification of DNA. Proc. Natl. Acad. Sci.U.S.A. 71:3455-3459.

8. Heyneker, H. L., J. Shine, H. M. Goodman, H. W. Boyer, J.Rosenberg, R. E. Dickerson, S. A. Narang, K. Itakura, S. Lin,and A. D. Riggs. 1976. Synthetic lac operator DNA is functionalin vivo. Nature (London) 263:748-752.

9. Kilbane, J. J., and M. H. Malamy. 1980. F factor mobilization ofnonconjugative chimeric plasmids in Escherichia coli: generalmechanisms and a role for site specific recA independentrecombination at oriVl. J. Mol. Biol. 143:73-93.

10. Makela, D., P. Makela, and S. Soilkeli. 1964. Sex specificity ofthe bacteriophage T7. Ann. Med. Exp. Biol. Fenn. 42:188-195.

11. Marians, K. J., R. Wu, J. Stawinski, T. Hozumi, and S. A.Narang. 1976. Cloned synthetic lac operator DNA is biological-ly active. Nature (London) 263:744-748.

12. Miller, J. F., and M. H. Malamy. 1983. Identification of the pifCgene and its role in negative control of F factor pif geneexpression. J. Bacteriol. 156:338-347.

13. Miller, J. H. 1972. Experiments in molecular genetics. ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y.

14. Morrison, T. G., and M. H. Malamy. 1970. Comparisons of Ffactors and R factors: existence of independent regulationgroups in F factors. J. Bacteriol. 103:81-88.

15. Morrison, T. G., and M. H. Malamy. 1971. T7 translationalcontrol mechanisms and their inhibition by F factors. Nature(London) New Biol. 231:37-42.

16. Muller-Hill, B., L. Crapo, and W. Gilbert. 1968. Mutants thatmake more lac repressor. Proc. Natl. Acad. Sci. U.S.A.59:1259-1264.

17. Rotman, G. S., R. Cooney, and M. H. Malamy. 1983. Cloning ofthe pif region of the F sex factor and identification of a pifprotein product. J. Bacteriol. 155:254-264.

18. Sadler, J. R., M. Tecklenburg, J. L. Betz, D. V. Goeddel, D. G.Yansura, and M. H. Caruthers. 1977. Cloning of chemicallysynthesized lactose operators. Gene 1:305-321.

19. Smith, T. F., and J. R. Sadler. 1971. The nature of lactoseoperator constitutive mutations. J. Mol. Biol. 59:273-305.

20. Sogaard-Anderson, L., L. A. Rokeach, and S. Molin. 1984.Regulated expression of a gene important for replication ofplasmid F in E. coli. EMBO J. 3:257-262.

21. Tanimoto, K., and T. Iino. 1983. Transfer inhibition of RP4 by Ffactor. Mol. Gen. Genet. 192:104-109.

22. Timmis, K., F. Cabello, and S. H. Cohen. 1975. Cloning,isolation and characterization of replication regions of complexplasmid genomes. Proc. Natl. Acad. Sci. U.S.A. 72:2242-2246.

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