Vol. 175, No. 2JOURNAL OF BACTERIOLOGY, Jan. 1993, p. 448-4560021-9193/93/020448-09$02.00/0Copyright © 1993, American Society for Microbiology

Identification and Characterization of RP1 Tral CistronsInvolved in Pilus Function and Plasmid Mobilization

S. T. FONGt AND VILMA A. STANISICH*

Department ofMicrobiology, La Trobe University, Bundoora 3083, Australia

Received 21 January 1992/Accepted 6 November 1992

Transfer-defective mutants of the Tral region of RP1 were isolated. Complementation studies involvingstable heterozygotes combined with the mapping of TnS insertion mutations revealed two pilus cistrons, pi4and pilB, at positions 46.9 to 48.2 kb and 46.0 to 46.4 kb, respectively. All pilB mutants were Dps- (i.e.,resistant to donor-specific phages PR4 and PRR1), whereas piL4 mutants were Dps- (promoter-proximalmutations), Dps+'- (sensitive only to PR4 [more centrally located mutations]), or Dps' (sensitive to bothphages [promoter-distal mutations]). The correlation between the site mutated and the Dps phenotype, togetherwith the finding that certain Dps+ piL4 mutants continued to mobilize nonconjugative plasmids, suggested thatpiL4 is bifunctional, contributing both to pilus function (at the promoter-proximal end) and to RP1mobilization. It was also shown that the 43.5- to 49.5-kb region that includes pilA and pilB encodes all of theTral pilus functions required for propagation of donor-specific phages and hence, probably, for pili that are

active in conjugation. Finally, three cistrons that specifically affect RP1 mobilization were identified. Two ofthese, mobA and mobB, occur immediately anticlockwise to oriT and probably correspond to the traJ and traIgenes characterized by other workers. The third cistron, mobC, occurs clockwise to oriT and may be a newmobilization gene, since its function can be substituted by IncPp plasmids, a feature different from that of thetraK mobilization gene which occurs in the same region but is RP1 specific. None of the mob cistrons was

required for mobilization of nonconjugative plasmids, except for mobB, which was required by pVS99.

The broad-host-range IncPa plasmid RP1 (60 kb; synony-mous with R18, RP4, and RK2) carries two regions (Tral andTra2) that encode conjugal functions (22, 37, 45). Mutationsin these regions affect transfer proficiency (Tra) and canhave concomitant effects on surface exclusion (Sfx) and onresponse to donor (Dps) or female-specific (Phi) phages. TheTra2 region occupies map positions from 18.0 to 29.3 kb (27,37) and therefore includes the regions previously designatedTra2 and Tra3 by Barth et al. (2, 3). Six genes within thisregion (traA, -B, -E, -R, -P. and -Q) encode pilus functions,and mutations in some of these have pleiotropic effects onsurface exclusion and response to female-specific phage (37,49). Additional genes may have exclusive roles in surfaceexclusion (eexA and eexB [28] and traS [29]).The Tral region occurs entirely between 39.1 and 54.5 kb

(22) and includes the origin of conjugal transfer, oriT (at 51.0kb [18, 23]), and a primase gene(s) (at about 42 kb) that isrequired for efficient conjugal transfer to certain hosts (31,33). Barth et al. (3) used RP4 insertion mutants to identifyfour complementation groups within Tral (groups 2 to 5), butwhether these were equivalent to cistrons was not estab-lished. In a separate study, Watson et al. (53) and Schmidt etal. (43) used point mutants of RP1 to define four cistrons(traA and traB [not synonymous with the similarly namedTra2 cistrons] and traC and traD). However, their locationswere not determined. The possible equivalence of the groupsand cistrons defined in these two studies was never estab-lished. The phenotypes displayed by the Tral mutantsimplicated this region in two aspects of conjugation: group 3,traC, and traD mutants have the phenotype typical of pilusmutants (i.e., Tra- Dps-), while group 2, group 5, and traA

* Corresponding author.t Present address: Department of Genetics, University of Mel-

bourne, Parkville 3052, Australia.

mutants are typical of mobilization mutants (i.e., Tra-Dps+). A third set of mutants (group 4) included Dps' andDps- representatives as well as those sensitive to only somephages (i.e., Dps+-). It is possible that this set also includesthe two DpsW- traB mutants.More extensive studies of the Tral region have now been

undertaken (22), and particular attention has been focusedon a 2.2-kb region (the Tral core) which contains onTstraddled by traK and by traJ and the promoter-proximalportion of traL. These three genes are required for theRP1-specific interactions that initiate DNA mobilization (18,21, 38). The precise role of traK is not known (21); however,traJ and traI constitute a relaxase operon that also includestraH, a gene that occurs outside the Tral core but is withinthe promoter-distal portion of traf. The TraJ, -I, and -Hproteins are believed to interact to form a nucleoproteincomplex at onT (39).

In the present report, we describe a genetic analysis of theTral region in which two pilus-associated cistrons are de-fined and localized. One cistron is bifunctional, having a rolein both pilus and mobilization activities. Moreover, all of thepilus functions required for propagation of donor-specificphages occur within a 6.0-kb portion of Tral. Three addi-tional cistrons that are required only for plasmid mobiliza-tion are identified, one of which may be a new gene commonto both IncPa and IncPP plasmids (45). These studies bothconfirm and extend previous genetic and molecular analysesof Tral.

MATERIALS AND METHODS

Bacterial strains, plasmids, and phages. Apart fromPseudomonas aeruginosa PA09505 (argB18 chl-2 Rif' [47])and PA02637 (prototroph [48]), the bacteria used werederivatives of Eschenchia coli K-12. Other strains andplasmids used are shown in Table 1. The map positions of

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TABLE 1. Bacterial strains and plasmids used

Strain or plasmid Relevant features and/or derivation Reference or source

recA13 Res- Mod- StrrRif derivative of HB101Nalr derivative of HB101pro met Nalr; carries TnS in chromosomePrototroph Rif'pro met recA56 Nalr

63737371942

Conjugative plasmids and their derivativesRP1pKM101pWS142pUB307R388pVS520pVS664

R751R772pVS791pVS793R906pUB1601

Nonconjugative plasmids and clonespUC19pVS97pVS99pBR322pBR325pVS657

pVS658

pVS659

pVS661

pVS727pVS729

pVS751

pVS987

RSF1010

Km' Tcr Apr Tra+ Dps' IncPotApr fip+ Tra+ IncNTnl725 (Cmr)+ derivative of pUB307Aps deletant of RP1Sur Tpr fisB+ Tra' IncWIn vitro Km' mutant of pUB1601HindIII (38.9-kb)-ApaI (54.5-kb) ligation derivative of pUB1601;

Tral- Tra2+Tpr Tra+ IncPpKmr Tra+ IncPpfisB mutant of R388fip::Tnl725 (Cmr) mutant of pKM101Apr Sur Hgr Tra+ IncP,3In vitro PstI Ap5 deletant of RP1

Apr plasmid vectorCryptic plasmid mobilizable by RP1Sur; mobilizable by RP1 in the presence of pVS97Apr Tcr plasmid vectorApr Tcr Cmr plasmid vectorpSC101 carrying the 2.1-kb HindIII-HincII Cmr fragment frompBR325; Tcr Cmr

pUC19 carrying the HindIII (38.9-kb)-EcoRI (60.0-kb) region ofRP1 (from pUB1601); the cloned DNA has sustained a 9-kbspontaneous deletion; Apr TralA oriTi trfB+

pVS987 carrying Tral on the ClaI (37.2-kb)-EcoRI (60.0-kb) regionof RP1 (from pUB307); Apr Kmr Tral oriT+

ColEl derivative carrying the Kmr determinant from Tn5 and theCmr determinant from pBR325; Kmr Cmr

SstII ligation derivative of pVS987; AprSstII clone of pVS727 carrying the 43.5- to 49.5-kb region of RP1

(from pVS588) and part of TnS; carries all Tral pildeterminants; Apr

pVS987 carrying the 51.1- to 54.5-kb region of RP1 (from pVS626)and part of TnS; Apr mobC+ oriT+

pBR322tet::Tn5O4 (Smr); the insert occurs between the Sall (0.6-kb) and BglI (0.9-kb) sites; Apr Smr

Sur Smr IncQ; mobilizable by RP1

portions of RP1 carried by clones and, in parentheses, theplasmids from which the DNAs were obtained are listed. Ap5R18tra mutants (pMO435 [traC54], pMO431 [traF49], andpMO480 [traHlOS]) (49) were also used. Additional Tra-mutants of pUB1601 (52) and pVS520 (37) are described inthe text and in Table 2, as are mob and pil mutants of theTral+ clone, pVS659 (Table 3). The IncP donor-specificphages used were PRR1 (36) and PR4 (46); these werepropagated on PA02637(RP1) in soft-agar overlays.Media and antibiotics. Nutrient broth, nutrient agar (NA),

and diagnostic sensitivity agar have been described previ-ously (37). Supplements (in micrograms per milliliter) in NAwere as follows: ampicillin, 300; chloramphenicol, 10; mer-curic chloride, 10; kanamycin, 10; nalidixic acid, 10; ri-fampin, 150; streptomycin, 100; and tetracycline, 5. Sulfa-thiazole (80 ,ug/ml) and trimethoprim (1 ,ug/ml) were used indiagnostic sensitivity agar.

Conjugation procedures. Nutrient broth cultures in late-exponential growth phase were used. Quantitative assess-ments of conjugal transfer were carried out by spot mating(17) or filter mating (37), and qualitative assessments weremade by cross-streak mating (29).

Determination of complementation. Stable heterozygoteswere constructed by transforming HB101 sublines carrying atransfer-defective mutant of pUB1601, pVS520, or R18 (Aps)with the Tral+ plasmid clone (Apr). Selection for the twoplasmids was imposed (i.e., on NA containing ampicillin andtetracycline). Complementation of the transfer mutation wasassessed from the recovery of Tcr Rif transconjugants ofLT101 in spot matings. In most matings, the recovery of AprRif transconjugants was also monitored, since the clones are

oriT+ and hence mobilizable from Tra+ cells.Isolation of plasmid mutants. The final test applied to the

RP1 mutants isolated in the first two procedures described

E. coliHB101LT101LT102LT104UB1301UB5201

1957W. Schilf4153729

2612This studyThis study2452

6011111340This study

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450 FONG AND STANISICH

TABLE 2. RP1 mutants used

Plasmid mutant"bMutagen No.MutagentNo. pVS (or other) designation

Tra- Dps' mutantscTnS 12 570, 574, 575, 576, 582, 583,

585, 588, 625, 626, 640, 642NTG 12 647, 649, 651, 653, 654, 745-

750, pMO431HA 1 pMO480

Tra- Dps+' mutantscTnS 15 577, 580, 587, 590, 591, 618-

624, 643-646NTG 2 648, 752

Tra- Dps- mutantscTnS 10 688, 753-761NTG 5 762-766None 1 pMO435

a RP1 and R18 are probably identical plasmids (45). The pMO plasmids aremutants of R18 and include the spontaneous Dps- mutant pMO435 (49); theTnS mutants and pVS766 are derivatives of pVS520; the NTG mutants arederivatives of pUB1601. pUB1601 and pVS520 are RP1 deletion mutants (37,52).

b pVS659 (Tral+) complemented all of the plasmid mutations; pVS658(carrying part of Tral) complemented only mutations in plasmids in boldfacetype. Plasmid transfer was determined by spot mating with donors carryingpVS658 or pVS659 together with an RP1 mutant; the recipient was LT101. TcrRif` or Apr Rif transconjugants were selected, thereby monitoring comple-mentation of the RP1 mutation or mobilization of the clone.

Dps+ and Dps- indicate, respectively, sensitivity (efficiency of plating[EOP) = ca. 0.5) and resistance (EOP < 10-8) to phages PR4 and PRR1;Dps+ - indicates sensitivity to PR4 but resistance to PRR1. The EOP wasdetermined by spot phage assays (48) on HB101 carrying RP1 (EOP = 1) orthe RP1 mutant.

below was to determine whether their mutations were com-plemented by pVS659 (Tral). Only mutants in which apositive response was obtained were used in the study.

(i) The procedure for mutagenesis of P. aeruginosaPA09505(pUB1601) by N-methyl-N'-nitro-N-nitrosoguani-dine (NTG) followed by filter mating of the treated donorswith HB101 has been described previously (37). To isolateTra- Dps- mutants, samples of the mated bacteria wereplated on NA containing tetracycline and streptomycinseeded with PR4 (ca. 108 PFU). The colonies obtained were

TABLE 3. Mutants of the Tral clone pVS659 andtheir derivations

RP1 mutant (DpsPlasmid Genotypea Mutagen phenotype) used toisolate pVS659

mutant

pVS768 mobAl NTG pVS625 (Dps')pVS769 mobBI HA pVSS88 (Dps')pVS770 mobB2 NTG pVS576 (Dps')pVS771 mobCl HA pVS649 (Dps')pVS773b piL43 HA pVS651 (Dps')pVS774b piL4l HA pVS648 (Dps+'-)pVS775 pilBI NTG pVS766 (Dps-)

a The genotypes were assigned retrospectively and reflect the cistronsidentified when these mutants were used in complementation studies (Table4).

b These two plasmids carry mutations in the same gene (see Table 4),despite their derivation using RP1 mutants with different Dps phenotypes.

purified twice on the same medium and then tested by spotmating with LT101 to confirm their Tra- phenotype. Tra-Dps+ or Tra- Dps+' mutants were isolated by screeningtransconjugant colonies obtained on unseeded selective me-dium. The colonies were cross-streak mated with LT101 todetect Tra- mutants. The Dps phenotypes of all mutantswere then determined.

(ii) pVS520::TnS mutants were isolated from filter matingsbetween LT104(pVS520) and HB101 as described previously(37). Kmr StW transconjugants were recovered, and thosethat had inherited Tra- mutants were identified by cross-streak mating with LT101 on NA containing tetracycline andrifampin. The Dps phenotypes of such mutants were thendetermined.

(iii) Mutants (mob or pil) of the Tral clone, pVS659,were obtained either by hydroxylamine (HA) treatment ofplasmid DNA or by NTG treatment of a UB1301(pVS659,pVS644) culture as described previously (37). The muta-genized pVS659 plasmids were then transferred to HB101sublines carrying a particular mob (Tra- Dps') or pil (Tra-Dps- or Tra- Dps'l-) mutant of pUB1601 or pVS520.HA-treated DNA was transferred by transformation; NTG-treated pVS659 (oriT+) was transferred by conjugal mobili-zation. The colonies obtained were cross-streak mated withLT101 to detect ones in which complementation failed tooccur. The putative pVS659 mutant plasmids carried bythese bacteria were then recovered by transformation, andtheir phenotypes were confirmed.DNA procedures. Standard DNA isolation and molecular

cloning techniques were performed as described previously(29, 37, 41).

Isolation of R388fisB mutants. The fertility of R388 (fisB+[sensitivity to fertility inhibition by RP1]) is inhibited bypUB1601 (fiwB+) (17). NTG-treated cultures of UB1301(R388) were filter mated with UB5201(pUB1601), and TprNalr transconjugants were isolated. These were cross-streakmated with UB1301 on diagnostic sensitivity agar containingtrimethoprim and rifampin to detect ones which transferredR388 at high frequency, i.e., that carried putative R388fisBmutants. Such a mutant plasmid was recovered from aUB1301 transconjugant, and its fisB mutant phenotype wasconfirmed.

Isolation of pKM101fip mutants. pKM101 (fip+ [fertilityinhibition of IncP plasmids]) can inhibit the fertility of RP1(58). A UB1301(pKM101, pWS142) culture was filter matedwith HB1O1(pVS520), and Cmr Apr (Strr) transconjugantswere isolated. These were cross-streak mated with LT101 toidentify ones which transferred pVS520 at high frequency.Such bacteria carried putative pKMlOlfip::Tnl725 mutantswhich were separated from pVS520 by conjugal transfer.Their fip mutant phenotype was then confirmed.

Nomenclature. Transfer-defective mutants of R18 (synon-ymous with RP1 [51]) and of the RP1 deletants, pUB1601and pVS520, are all referred to as RP1 mutants. The genedesignationspil and mob (rather than tra) are applied to Traltransfer genes believed to have specific roles in pilus andmobilization function, respectively. This is done to facilitatecomparison with previously described mutants for whichthree similar tra nomenclature schemes have been described(22, 49, 53).

RESULTS

Construction of clones carrying Tral DNA. Two cloneswere constructed to allow the identification of Tral mutantsof RP1 (Fig. 1B). Plasmid pVS659 carries the entire Tral

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E

Ss

p

s s

A Ss

s s s s Ss sC IHI W5Kmr

B

Tral ori T

pVS659

VpVS729

pVS751

FIG. 1. (A) Map of the 60-kb plasmid RP1 (outer circle), basedon that of Thomas and Smith (51). The RP1 deletion derivativespUB307 (54.8 kb [4]) and pUB1601 (and its derivative pVS520; both49.2 kb [37, 52]) are represented by the inner circles. The positionsof the transfer (Tra) regions (in black) and the resistance functions(to ampicillin, tetracycline, and kanamycin) are indicated. (B) Lin-ear map of RP1 from the ClaI (36.0-kb) to the EcoRI (60.0-kb) sites,showing the Tral region (in black) and the locations of the transferorigin, oriT (at 51 kb [23]), and the incompatibility locus, incC, in theadjoining trfB maintenance region (32, 50). The lines beneath themap show the regions of RP1 contained in various clones. PlasmidpVS658 has sustained a spontaneous deletion of part of the Tralregion (dotted line). The cloned DNA in pVS729 and pVS751originated from pVS520::TnS mutants; the triangles indicate thelocations of TnS sequences in these clones. Cleavage sites forvarious restriction enzymes are as follows: A, ApaI; C, ClaI; E,EcoRI; H, HindIII; P. PstI; S, SphI; and Ss, SstII. In panel A, onlythe ApaI, ClaI, and SstII sites that occur in the second half of RP1are shown.

region on the ClaI (37.2-kb)-ApaI (54.5-kb) fragment in-serted in pVS987. In contrast, pVS658 carries the HindIII(38.9-kb)-EcoRI (60.0-kb) region in which a spontaneousinternal deletion from about 39.1 to 48.6 kb has occurred so

that, for practical purposes, it carries only the oniT end ofTral. In all studies involving heterozygotes carrying eitherof these Apr clones together with an Aps Tcr derivative ofRP1, double selection was imposed on NA containing ampi-cillin and tetracycline to ensure the retention of both plas-

mids. This was particularly relevant for pVS658, since thisplasmid carries the low-level incompatibility locus IncP-1(II)/IncC (32, 50).

Origin and preliminary characterization of Tral mutants.Table 2 lists the 58 transfer-defective mutants of RP1 thatwere investigated. Three were from a previous study (theyare prefixed with pMO [49]) and included a spontaneousDps- mutant, pMO435; all of the remaining mutants wereinduced by either TnS, NTG, or HA. With the exception ofpMO480, which carries a cis-dominant mutation (49; seebelow), the transfer defects of all of the plasmids could becomplemented by pVS659, indicating that the respectivemutations are in Tral. A similar analysis of heterozygotescarrying pVS658 revealed complementation in only ninecases (i.e., with the plasmids shown in boldface in Table 2).In these instances, the mutations must occur in the 48.6- to54.5-kb region of Tral that is common to both pVS658 andpVS659. The last mutant, pMO480, carries a mutation that isnot complemented by either pVS658 or pVS659 but alsooccurs in the 48.6- to 54.5-kb region. This conclusion stemsfrom the finding that pMO480 shares with the nine comple-mentable mutants the ability to mobilize pVS658 or pVS659(both oriT+) from the respective heterozygotes. Thus, themutation in pMO480 is both cis dominant and likely to occurwithin oniT as previously suggested (49).The various RP1 mutants exhibited differences in their

responses to pilus-specific phages: some retained sensitivityto both PR4 and PRR1 (Dps+); some retained sensitivityonly to PR4 (Dps'l-), while others became resistant to bothphages (Dps-). It is evident from Table 2 (footnote b) thatchanges from the Dps+ phenotype result only from muta-tions that are complemented by pVS659 but not by pVS658,i.e., among mutations that are proximal to the 48.6- to54.5-kb region.

Insertion mapping of TnS+ mutants of RP1. The locationsof TnS in the various insertion mutants were determined byagarose gel electrophoresis of SstII and SstII-ApaI digests orof XhoI and XhoI-ApaI digests. This utilized the SstII(43.5-kb) andApal (54.5-kb) sites in the Tral region (Fig. 1),together with the SstII andXhoI sites that occur 0.45 and 0.5kb, respectively, from each terminus of TnS (5.9 kb [1]). Thedistribution of insertion sites is shown in Fig. 2 and spans a5-kb region up to and apparently including oriT. The 12 Tra-Dps+ mutations map in two regions (49.5 to 51.1 kb and 46.9to 47.3 kb), as do the 10 Tra- Dps- mutations (48.0 to 48.2kb and 46.0 to 46.4 kb). In contrast, the 15 Tra- Dps'/-mutations map in a single region (47.2 to 48.0 kb) thatoverlaps the flanking Tra- Dps+ and Tra- Dps- mutations.The mutations in pVS625, pVS626, and pVS642 do not, infact, occur within otiT but are close to it (the initial place-ment reflects minor mapping errors). Complementationaltransfer of these plasmids (Table 2, footnote b) clearlydemonstrates that they are oriT. Moreover, their mutationsoccur anticlockwise to oriT. This was evident from thefinding that pVS751, a clone carrying DNA clockwise to theinsertion in pVS626, is oriT+ (see below). Thus, all of theinsertion mutations studied occur anticlockwise to the oniTlocus.

Identification of Tral cistrons. The identification of cis-trons likely to affect pilus function was commenced byisolating mutants of pVS659 that failed to complement thetransfer defect of a Dps'/- or Dps- mutant of RP1 (i.e.,pVS659pilUI and pVS659pilBJ, respectively; Table 3). Fromthe behavior of these mutants in complementation tests(Table 4), it was evident that all 17 of the Dps'/- mutationsoccur in a single cistron, piL4 (Table 4, column 3; footnote

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pilA

Dps +/ -

9 g r.

pilBDps Dps Dps

SstII

Im 143 44 45 46 47 48

mobC

Dps +

mobB mobADps DpS

I i 1

49 50 51

PL2

Apal

52 53 54PR2PR3

PL1 PR1

20 [DI 85 [El 18 [F] 72 [G] 22 [H] 11 [J] 15 [K] 12 [M] 52 [N]

IEIZIz 81 [I] =Z1 I IZ II I

I oriT 26 [L]

FIG. 2. A map of the Tral region of RP1 from the SstII (43.5-kb) to the ApaI (54.5-kb) sites, showing the locations of various tra::TnSinsertion mutations. The respective plasmids have been distinguished according to their Dps phenotypes (see Table 2) and the cistronsaffected (see Table 4) as follows (from right to left): Dps' and mobA or mobB, pVS625/626, -642, -585, -576, -570, -575, and -588; Dps- andpiIA, pVS754/755 and -753; Dps'/- and piU4, pVS587/590, -646, -643/645, -591, -577/621/622, -620, and 580/618/619/623/624; Dps' and piLA,-640, -574, -582, and -583; Dps- and pilB, pVS756/761 and -668/757/758/759/760. The location of a fifth Dps' cistron, mobC, is not knownprecisely but occurs to the right of oriT in the 51.1- to 54.5-kb region. The boxed areas below the map indicate the extents of genes (traD totraN) defined by E. Lanka and coworkers and the estimated sizes (in kilodaltons) of the corresponding gene products (22, 26a, 38, 39, 61).The horizontal arrows represent transcriptional units and the directions of transcription arising from promoters (P) situated to the right (R)or left (L) of oriT (22, 26a, 38, 39, 61). Transcription from PL2 (and probably PL1) extends beyond traD and through the adjacent primase gene(traC [22]) (region not shown).

TABLE 4. Complementation of RP1 mutant allelesof the Tral region

RP1 mutant Dps Transfer frequency'w4 of RP1 mutants fromcarried by pheno- heterozygotes carrying the pVS659 allele

gotea trie piLUI piIU3 pilBI mobAl mobBI mobB2 mobCI

pVS625e Dps+ 110 130 150 7 75 140 180pVS642e Dps+ 222 119 200 2 166 94 72pVS576e Dps+ 78 56 128 122 <0.1 0.5 417pVS588e Dps+ 42 40 63 79 <0.1 <0.1 63pVS649 Dps+ 38 51 27 164 18 20 0.1pVS647 Dps+ <0.1 0.2 222 155 166 188 444pVS651 Dps+ <0.1 <0.1 125 188 150 138 150pVS648 Dps'/- <0.1 <0.1 58 83 75 63 58pVS752 Dps'l- <0.1 <0.1 115 100 65 92 500pVS753e Dps- <0.1 <0.1 41 64 64 91 68pVS754e Dps- <0.1 <0.1 69 156 106 63 131pVS763 Dps- 64 80 <0.1 122 64 68 84pVS766 Dps- 109 218 <0.1 500 77 77 127

a Donors were sublines of HB101 carrying pVS659 or a mutant of it togetherwith an RP1 mutant.

b See Table 2, footnote c.c Numbers are percentages determined relative to the level of complemen-

tation obtained with pVS659. Transfer frequencies were determined from thenumber of Tcr Rif' transconjugants recovered in spot matings with LT101.Transfer frequencies from heterozygotes carrying pVS659 and from all othercombinations in which complementation occurred were about 10-1 per donor.

d The cistron assignments and phenotypes of other plasmids studied were asfollows: pVS626, mobA; pVSS70, -575, and -585, mobB; pVS577, -580, -587,-590, -591, -618 to -624, -643, -645, and -646, piL4 and Dps'/-; pVS574, -582,-583, -640, -653, -654, and -745 to -750 and pMO431, piLA and Dps+; pVS755,pilA and Dps-; pVS668, -756 to -762, -764, and -765 and pMO435, pilB.

' TnS insertion mutant; all other plasmids listed are NTG mutants.

d). In contrast, 13 of 16 of the Dps- mutations occur in asecond cistron, pilB, while 3 are pilA mutations (Table 4,columns 5 and 3, respectively; footnote d). Moreover, thepilA cistron also includes 16 of 25 of the Dps+ mutations(Table 4, column 3; footnote d). To confirm these assign-ments, a third pVS659 mutant (pVS659piUA3) was isolatedthat was unable to complement one such Dps+ mutant (i.e.,pVS651 [Table 3]); its complementation behavior paralleledthat of pVS659piUIl (Table 4, columns 3 and 4). Thepleiotropy of piLU mutations detected in this analysis is inkeeping with the overlap observed between Dps+'- insertionmutations and both Dps- and Dps+ mutations in the 46.9- to48.2-kb region (Fig. 2). Nevertheless, it is noteworthy that astrong correlation exists between the location of the inser-tion mutation and the Dps phenotype that ensues, i.e., Dps+to Dps+'- to Dps- (proceeding clockwise in RP1).To complete the investigation of the remaining Dps+

mutants, four additional mutants of pVS659 were isolated(pVS768 to pVS771; Table 3). As expected, these comple-mented the various piL4 and pilB mutations (Table 4, col-umns 6 to 9). The remaining RP1 mutations could beassigned to three further cistrons; mobC, defined by thesingle NTG-induced mutation in pVS649 (Table 4, column9), and two others, mobA and mobB. Each of the last twomay represent more than a single cistron, since they arecomposed only of insertion mutations (three and five, re-spectively) (Table 4, columns 6 to 8; footnote d). Finally, inkeeping with the suggestion that pMO480 (Dps+) is an oniTmutant, this plasmid permitted mobilization of pVS659 andits mutants from the respective heterozygotes (data notshown).

Mobilization of nonconjugative plasmids by the RP1 mu-tants. RP1 can mobilize the nonconjugative plasmids

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TABLE 5. Mobilization of nonconjugative plasmidsby Tral mutants of RP1

RP1 deriv- Mobilization frequency of:ative Genotype RSF1010 pSClOlcam ColElcam pVS99

pUB1601b Wild type 7.5 x 10-1 7.5 x 10-1 1.1 9.0 x 10-1pVS570 mobB 1.0 7.0 x 10-1 6.0 x 10-1 <5 x 10-7pVS647 piIA 1.2 x 10-2 5.5 x 10-3 5.5 X 10-3 7.0 x 10-2pVS749 piL4 6.5 x 10-3 5.5 x 10-2 6.5 x 10-2 1.3 x 10-1pVS750 pilA 5.5 x 10-2 <5 x 10-7 6.5 x 10-3 <5 x 10-7

a Determined from the number of transconjugants from spot matings withLT101. The HB101 donors carried an RP1 mutant together with a nonconju-gative plasmid; donors carrying pVS99 also carried pVS97, a cryptic plasmidrequired for mobilization of pVS99 by RP1 (11). Selections imposed were TcrRif' for the RP1 mutants, Sur Rif' for RSF1010 and pVS99, and Cmr Rif' forpSClOlcam (= pVS657) and ColElcam (= pVS661). No transfer was de-tected when each nonconjugative plasmid was carried alone (<5 x 10-7 per

donor); this was also true when most of the RP1 mutants were carried alone,although some had residual transfer (up to 5 x 10-4 per donor).

b pUB1601 (Tra+ Dps+) served as the wild-type control. The mobilizationfrequencies found for all of the Tra- Dps+ mutants listed in Table 2 were

similar to that of the control, except for those of the mobB mutants (whichbehaved like pVS570) and the three pil4 mutants shown above. None of theTra- Dps'/- and Tra- Dps- mutants listed in Table 2 mobilized any

nonconjugative plasmid (<5 x 10-7 per donor).

RSF1010, pSC101, ColEL, and pVS99 (11, 54, 56), but itsspecific contributions to these interactions have not beendetermined. We anticipated a broad correlation between Dpsphenotype and mobilization ability but were particularlyinterested in that involving Dps+' and other mutants ofpilA. The Dps+ plasmids with mutations in mobA, mobB, or

mobC (as well as pMO480OoiT) continued to mobilize thevarious nonconjugative plasmids, except that mobB mu-

tants, such as pVS750, did not mobilize pVS99 (Table 5).These results indicate that the pili encoded by these plasmidsare normal in terms of their roles in phage adsorption,penetration of the cells by phage DNA or RNA, and theexiting, during conjugation, of nonconjugative plasmidDNA. The various mob mutations therefore seem to specif-ically affect transfer of RP1 DNA or, in the case of mobBmutants, of both RP1 and pVS99 DNAs. This conclusion istherefore consistent with the demonstration by other work-ers that genes immediately adjacent to oriT serve in the RP1mobilization process (38, 39).

In contrast, only 3 of 13 of the Dps+ plasmids withmutations inpiU4 permitted mobilization of the nonconjuga-tive plasmids (i.e., pVS647 and pVS749) or of only RSF1010and ColEl (i.e., pVS750). However, the frequencies ob-tained were reduced 10- to 100-fold (Table 5). No mobiliza-tion from any of the remaining mutants (Dps+'- or Dps-)was detected. Therefore, it seems that most piLA and all ofthe pilB mutants tested are unable to transfer plasmid DNAbecause their mutations specifically affect pilus function,either by failure to elaborate a pilus or by production of a

defective structure. However, rare Dps+ pilA mutants seem

able to produce a pilus with moderate conjugal activity,suggesting that these mutations, like those in mob, specifi-cally affect DNA transfer.

Distribution of pilus functions within Tral. The two pro-

posed pilus cistrons,piL4 andpilB, occur at positions 46.0 to48.2 kb. The 43.5- to 49.5-kb region of Tral was cloned fromthe mobB::TnS mutant pVS588 (Fig. 2) into pVS987 (on an

SstII fragment). The plasmid obtained (pVS729) was thenintroduced into an HB101 subline carrying pVS664 (Tral-Tra2+). Phages PR4 and PRR1, which attach to the pilus tipand shaft, respectively (7, 9), produced plaques as efficiently

TABLE 6. Mobilization of pVS659 and its mutantsby IncPI3 plasmids

Transfer frequencya of pVS659 or its derivatives fromMutation in donors' also carrying:pVS659

pVS520 pVS751 pVS772 R906

None 0.9 0.6 3.7 x 10-2 1.3mobAl 1.8 3.1 x 10-3 3.1 x 10-4 8.0 x 10-3mobBI 1.2 4.5 x 10-5 5.0 X 10-7 5.5 X 10-4mobB2 1.2 8.0 x 10-4 8.0 x 10-5 1.5 x 10-2mobCl 1.6 1.4 x 10-1 3.0 x 10-2 8.5 x 10-2pilA3 1.5 0.5 5.5 x 10-2 1.5pilBi 0.9 0.7 3.2 x 10-2 0.9

a Determined from the number of transconjugants in matings betweendonor sublines of HB101 and either LT102 (for matings involving R906) orLT101 (all other matings). The donors carried an IncP plasmid together withpVS659 or a mutant of it. Filter matings were used, except in the case ofpVS520 donors, which were spot mated. Selection was for Apr (pVS659 andmutants), Tcr (pVS520), Tpr (R751), Kmr (R772), or Hgr (R906) transcon-jugants on either nalidixic acid- or rifampin-supplemented medium as appro-priate.

b Donors were constructed by transforming HB101 sublines carrying theIncP plasmid with DNA of the appropriate clone. Selection was for bothplasmids (Apr and those shown in footnote a above), except in the case ofR772, for which only Apr could be used (R772 and the clones both carry Kmr).

on pVS729+ pVS664+ bacteria as on those carrying pVS520(Tra+). Thus, the 6-kb region encodes all of the Tral genesthat are required for the elaboration of a pilus that functionsnormally in phage propagation.

Localization and characterization of mobC. Only a singlemobC mutant (pVS649) was isolated, and its point mutationwas mapped to the 48.6- to 54.5-kb region by complementa-tion with pVS658 (Table 2, footnote b). The mutation wasfurther localized to the 51.1- to 54.5-kb region by cloning thisDNA from pVS626 (a mobA::TnS mutant; Fig. 2) intopVS987 (on a HindIII-ApaI fragment). The resulting clone,pVS751, complements the mobC mutation but none of theother RP1 mutations (data not shown). More importantly,pVS751 is mobilized by pVS649 and hence is oniT+. Thus,mobC must occur clockwise to oriT, whereas the remainingfour cistrons are anticlockwise to this locus at the locationsshown in Fig. 2.Other workers have defined the Tral core as the oriT

region flanked by traJ, -I, and -K and further shown thatthese three genes are RP4 specific and cannot be substitutedby functions from IncPj plasmids (18, 21, 38, 59; Fig. 2). Weused the latter observation to further characterize the mobcistrons and found that pVS659mobA or pVS659mobB mu-tants were poorly mobilized by the IncPB plasmids (ca. 10-2to 10-5 per donor), whereas the mobC mutant was efficientlymobilized (Table 6). This suggests that mobA and mobB areequivalent to traJ and traI, respectively, in keeping with themapping data (Fig. 2). In contrast, the ability of IncPPplasmids to substitute for mobC suggests that this is a newmobilization function that occurs in the vicinity of traK butis distinct from it. For completeness, piL4 and pilB mutantsof pVS659 were also studied and found to be efficientlymobilized by the IncPPi plasmids, supporting previous ob-servations of the interchangeability of Tral pilus functions(21, 45).

Functional similarity between IncPca, IncPfl, IncN, andIncW plasmids. The IncPa subgroup of plasmids to whichRP1 belongs probably represents an evolutionary branchdifferent from that of the smaller IncPB subgroup (45, 59).The data described above show that of the five cistronstested, only two (mobA and mobB) cannot be adequately

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replaced by IncPP plasmids. We also asked whether muta-tions of the five Tral cistrons of RP1 could be complementedby pKM101 (IncN) and R388 (IncW). This was a possibility,since members of all three Inc groups confer various cross-sensitivities to donor-specific phages (7, 8, 25) and interact interms of fertility inhibition (17, 35). However, when anappropriate pKM101fip or R388fis mutant (see Materials andMethods) was introduced into HB101 sublines carryingpVS576, pVS618, pVS625, pVS649, or pVS668, no enhance-ment in the transfer of the pVS plasmids to LT101 wasobserved (data not shown), i.e., none of the five functionsstudied can be replaced by the IncN or IncW plasmids.

DISCUSSION

In this study of Tral mutants of RP1, two cistrons, pilAand pilB, that are required for pilus function in E. coli K-12have been localized to the 46.0- to 49.2-kb region anticlock-wise to oriT (Fig. 2). All pilB mutations resulted in a Dps-phenotype, probably reflecting the cell's inability to elabo-rate a pilus. In contrast, piLA mutations were pleiotropic,affecting propagation of both PR4 and PRR1 (Dps-), of onlyPRR1 (Dps+ -), or of neither phage (Dps+). Barth et al. (3)observed such pleiotropy among Tn7+ mutants of comple-mentation group 4 but could not eliminate the possibility thatmore than one gene was involved. We have clarified thisissue by using chemically induced pilA mutants of RP1 andpVS659 (a Tral+ clone), i.e., presumed point mutants. Asingle pilA cistron was indicated by the lack of complemen-tation in the alternative combinations (Table 4).

Interestingly, the Dps phenotype of pilA mutants is notrandom; mutations at one or the other end confer a Dps- orDps+ phenotype, while more central mutations are Dps'/-(Fig. 2). This is not due to polar effects on downstreamcistrons, since pVS659pilU mutants complement all butpilAmutations. Thus, the pleiotropy must reflect direct effects ofthe mutations onpilA itself. Given that PR4 adsorbs to thepilus tip and PRR1 adsorbs to the pilus shaft (7, 9), thevarious piU4 phenotypes apparently reflect the absence ofpili, the presence of pilus tips, and the presence of pilusshafts. Therefore, the piLA cistron may contribute to pilusfunction in two ways: by the provision of pilus tips encodedby the promoter-proximal portion of the gene and by theprovision of pilus shafts encoded by the distal portion (Fig.2). Furthermore, piLA seems to also be required for themobilization of RP1, since three exceptional Dps+ mutantsthat continue to mobilize nonconjugative plasmids (andhence have conjugally active pili; Table 5) nevertheless failto transfer RP1.Our mapping ofpiU4 andpilB insertion mutations suggests

that these genes correspond to traF and traG identified bynucleotide sequence analysis (61) (Fig. 2). If this is so, pilUbears similarities to FtraG both in the duality of its functionsand in its relatively large size (116 versus 72 kDa; 30, 61).However, there is no evidence that pilA plays a role inmating pair stabilization, as is the case for FtraG. Indeed, itis possible that RP1 entirely lacks such a function, since itcan effect conjugation on solid surfaces but apparently not inliquids (as is the case with IncN and IncW plasmids [10, 44]).Our demonstration that the 43.5- to 49.5-kb region of Tral issufficient, in conjunction with the Tra2 region, to permitpropagation of the donor-specific phages localizes all of theTral pilus genes required for this process to the DNAsegment that carries piLA and pilB. The third gene in thissegment, i.e., traE (61) (Fig. 2), may also be a pilus gene,

given that Watson et al. (53) reported two Dps- cistrons inTral in addition to a pleiotropic cistron.Apart frompilA and pilB, we defined three other cistrons

that are required for RP1 mobilization, i.e., mobA, -B, and-C. Mutants of these cistrons are Dps+ and further displaynormal pilus function by promoting mobilization of variousnonconjugative plasmids (Table 5). None, however, allowsmobilization of RP1, although mobB is exceptional in alsobeing required for the mobilization of pVS99. The location ofmobA and mobB immediately anticlockwise to onT togetherwith the finding that neither of these functions can beefficiently substituted by IncPI3 plasmids suggests that theyare identical to the traJ and traI mobilization genes charac-terized by E. Lanka and coworkers (18, 21, 38, 39). How-ever, mobC, which is defined by only a single point mutationand occurs clockwise to oriT in the 51.1- to 54.5-kb region, isof interest because it can be substituted by IncPP plasmids.The only other known mobilization gene in this region, traK,is specific to IncPa plasmids (18, 21). Thus, mobC may be anew mobilization function common to both IncPax and IncPPplasmids; it is presumably equivalent to traL, -M, or -N (61)(Fig. 2).Like mobC,pilA andpilB can also be substituted by IncPB

plasmids but not by either pKM101 (IncN) or R388 (IncW)(Table 6). This is consistent with the known homology of theIncPa and IncPP transfer regions (45) and also with recentstudies by Bolland et al. (5), who showed that the R388 pilusgenes could be entirely substituted by those of pCU1 (IncN)but not by those of RP1. Other similar studies (46a) have alsofailed to detect substitution of Tra2 pilus functions (i.e.,traA, -B, -E, -R, -P. and -O [37]) by pKM101 or R388.Therefore, it appears that the similarity among IncP, -N, and-W plasmids on the basis of plating of PR4, similar pilusmorphology, and fertility inhibition interactions does notreflect functional homology, which occurs between plasmidF and its relatives (55).

In conclusion, we note two instances in which Tralinsertion mutations apparently have no effect on down-stream genes. Ziegelin et al. (61) have reported transcriptionof the Tral region as indicated in Fig. 2. We found thatmobB::TnS mutations prevent mobilization of pVS99; how-ever, this occurs normally if the insertions are in mobA, i.e.,mobB remains functional. Second, both TnS and Tn7 inser-tions in pilA (equivalent to group 4 of Barth et al. [3]) canresult in a Dps+ phenotype, even though mutations in pilB(possibly equivalent to group 3 of Barth et al. [3]) confer aDps- phenotype, i.e., pilB remains functional. These obser-vations are similar to those made by Palombo et al. (37) forthe RP1 Tra2 region (with TnS and Tn504) and by Winansand Walker (57) for the Tra regions of pKM101 (with TnS).Either these transposons are not strongly polar, contradict-ing other findings (14, 16), or transcription can occur from anumber of sites in Tral and Tra2 (perhaps at weak promotersor at cryptic levels [34]) permitting sufficient gene expressionto satisfy certain of the transfer requirements.

ACKNOWLEDGMENTS

We thank Rhonda McCaw for assistance in the preliminarymapping of mob mutants, Wolfgang Schilf for plasmid pWS142, andErich Lanka for providing details of the molecular map of RP4 andthe structure of the Tral region.

Financial support for this work was provided by recurrent fundsto the Department of Microbiology and by the Australian ResearchCouncil. S.T.F. was the recipient of a Commonwealth PostgraduateResearch award.

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