Identification and Characterization of RP1 Tra1 Cistrons Involved in Pilus Function and Plasmid Mobilization

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JOURNAL OF BACTERIOLOGY, Jan. 1993, p. 448-456 Vol. 175, No. 2 0021-9193/93/020448-09$02.00/0 Copyright © 1993, American Society for Microbiology Identification and Characterization of RP1 Tral Cistrons Involved 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 involving stable heterozygotes combined with the mapping of TnS insertion mutations revealed two pilus cistrons, pi4 and 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-proximal mutations), Dps+'- (sensitive only to PR4 [more centrally located mutations]), or Dps' (sensitive to both phages [promoter-distal mutations]). The correlation between the site mutated and the Dps phenotype, together with the finding that certain Dps+ piL4 mutants continued to mobilize nonconjugative plasmids, suggested that piL4 is bifunctional, contributing both to pilus function (at the promoter-proximal end) and to RP1 mobilization. It was also shown that the 43.5- to 49.5-kb region that includes pilA and pilB encodes all of the Tral 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 of these, mobA and mobB, occur immediately anticlockwise to oriT and probably correspond to the traJ and traI genes characterized by other workers. The third cistron, mobC, occurs clockwise to oriT and may be a new mobilization gene, since its function can be substituted by IncPp plasmids, a feature different from that of the traK 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- mutants are typical of mobilization mutants (i.e., Tra- mous with R18, RP4, and RK2) carries two regions (Tral and Dps+). A third set of mutants (group 4) included Dps' and Tra2) that encode conjugal functions (22, 37, 45). Mutations Dps- representatives as well as those sensitive to only some in these regions affect transfer proficiency (Tra) and can phages (i.e., Dps+-). It is possible that this set also includes have concomitant effects on surface exclusion (Sfx) and on the two DpsW- traB mutants. response to donor (Dps) or female-specific (Phi) phages. The More extensive studies of the Tral region have now been Tra2 region occupies map positions from 18.0 to 29.3 kb (27, undertaken (22), and particular attention has been focused 37) and therefore includes the regions previously designated on a 2.2-kb region (the Tral core) which contains onT Tra2 and Tra3 by Barth et al. (2, 3). Six genes within this straddled by traK and by traJ and the promoter-proximal region (traA, -B, -E, -R, -P. and -Q) encode pilus functions, portion of traL. These three genes are required for the and mutations in some of these have pleiotropic effects on RP1-specific interactions that initiate DNA mobilization (18, surface exclusion and response to female-specific phage (37, 21, 38). The precise role of traK is not known (21); however, 49). Additional genes may have exclusive roles in surface traJ and traI constitute a relaxase operon that also includes exclusion (eexA and eexB [28] and traS [29]). traH, a gene that occurs outside the Tral core but is within The Tral region occurs entirely between 39.1 and 54.5 kb the promoter-distal portion of traf. The TraJ, -I, and -H (22) and includes the origin of conjugal transfer, oriT (at 51.0 proteins are believed to interact to form a nucleoprotein kb [18, 23]), and a primase gene(s) (at about 42 kb) that is complex at onT (39). required for efficient conjugal transfer to certain hosts (31, In the present report, we describe a genetic analysis of the 33). Barth et al. (3) used RP4 insertion mutants to identify Tral region in which two pilus-associated cistrons are de- four complementation groups within Tral (groups 2 to 5), but fined and localized. One cistron is bifunctional, having a role whether these were equivalent to cistrons was not estab- in both pilus and mobilization activities. Moreover, all of the lished. In a separate study, Watson et al. (53) and Schmidt et pilus functions required for propagation of donor-specific al. (43) used point mutants of RP1 to define four cistrons phages occur within a 6.0-kb portion of Tral. Three addi- (traA and traB [not synonymous with the similarly named tional cistrons that are required only for plasmid mobiliza- Tra2 cistrons] and traC and traD). However, their locations tion are identified, one of which may be a new gene common were not determined. The possible equivalence of the groups to both IncPa and IncPP plasmids (45). These studies both and cistrons defined in these two studies was never estab- confirm and extend previous genetic and molecular analyses lished. The phenotypes displayed by the Tral mutants of Tral. implicated this region in two aspects of conjugation: group 3, traC, and traD mutants have the phenotype typical of pilus mutants (i.e., Tra- Dps-), while group 2, group 5, and traA MATERIALS AND METHODS Bacterial strains, plasmids, and phages. Apart from Pseudomonas aeruginosa PA09505 (argB18 chl-2 Rif' [47]) * Corresponding author. and PA02637 (prototroph [48]), the bacteria used were t Present address: Department of Genetics, University of Mel- derivatives of Eschenchia coli K-12. Other strains and bourne, Parkville 3052, Australia. plasmids used are shown in Table 1. The map positions of 448 VOL. 175, 1993 RP1 tra CISTRONS 449

TABLE 1. Bacterial strains and plasmids used Strain or plasmid Relevant features and/or derivation Reference or source E. coli HB101 recA13 Res- Mod- Strr 6 LT101 Rif derivative of HB101 37 LT102 Nalr derivative of HB101 37 LT104 pro met Nalr; carries TnS in chromosome 37 UB1301 Prototroph Rif' 19 UB5201 pro met recA56 Nalr 42 Conjugative plasmids and their derivatives RP1 Km' Tcr Apr Tra+ Dps' IncPot 19 pKM101 Apr fip+ Tra+ IncN 57 pWS142 Tnl725 (Cmr)+ derivative of pUB307 W. Schilf pUB307 Aps deletant of RP1 4 R388 Sur Tpr fisB+ Tra' IncW 15 pVS520 In vitro Km' mutant of pUB1601 37 pVS664 HindIII (38.9-kb)-ApaI (54.5-kb) ligation derivative of pUB1601; 29 Tral- Tra2+ R751 Tpr Tra+ IncPp 26 R772 Kmr Tra+ IncPp 12 pVS791 fisB mutant of R388 This study pVS793 fip::Tnl725 (Cmr) mutant of pKM101 This study R906 Apr Sur Hgr Tra+ IncP,3 24 pUB1601 In vitro PstI Ap5 deletant of RP1 52 Nonconjugative plasmids and clones pUC19 Apr plasmid vector 60 pVS97 Cryptic plasmid mobilizable by RP1 11 pVS99 Sur; mobilizable by RP1 in the presence of pVS97 11 pBR322 Apr Tcr plasmid vector 13 pBR325 Apr Tcr Cmr plasmid vector 40 pVS657 pSC101 carrying the 2.1-kb HindIII-HincII Cmr fragment from This study pBR325; Tcr Cmr pVS658 pUC19 carrying the HindIII (38.9-kb)-EcoRI (60.0-kb) region of This study RP1 (from pUB1601); the cloned DNA has sustained a 9-kb spontaneous deletion; Apr TralA oriTi trfB+ pVS659 pVS987 carrying Tral on the ClaI (37.2-kb)-EcoRI (60.0-kb) region This study of RP1 (from pUB307); Apr Kmr Tral oriT+ pVS661 ColEl derivative carrying the Kmr determinant from Tn5 and the This study Cmr determinant from pBR325; Kmr Cmr pVS727 SstII ligation derivative of pVS987; Apr This study pVS729 SstII clone of pVS727 carrying the 43.5- to 49.5-kb region of RP1 This study (from pVS588) and part of TnS; carries all Tral pil determinants; Apr pVS751 pVS987 carrying the 51.1- to 54.5-kb region of RP1 (from pVS626) This study and part of TnS; Apr mobC+ oriT+ pVS987 pBR322tet::Tn5O4 (Smr); the insert occurs between the Sall (0.6- This study kb) and BglI (0.9-kb) sites; Apr Smr RSF1010 Sur Smr IncQ; mobilizable by RP1 20 portions of RP1 carried by clones and, in parentheses, the Conjugation procedures. Nutrient broth cultures in late- plasmids from which the DNAs were obtained are listed. Ap5 exponential growth phase were used. Quantitative assess- R18tra mutants (pMO435 [traC54], pMO431 [traF49], and ments of conjugal transfer were carried out by spot mating pMO480 [traHlOS]) (49) were also used. Additional Tra- (17) or filter mating (37), and qualitative assessments were mutants of pUB1601 (52) and pVS520 (37) are described in made by cross-streak mating (29). the text and in Table 2, as are mob and pil mutants of the Determination of complementation. Stable heterozygotes Tral+ clone, pVS659 (Table 3). The IncP donor-specific were constructed by transforming HB101 sublines carrying a phages used were PRR1 (36) and PR4 (46); these were transfer-defective mutant of pUB1601, pVS520, or R18 (Aps) propagated on PA02637(RP1) in soft-agar overlays. with the Tral+ plasmid clone (Apr). Selection for the two Media and antibiotics. Nutrient broth, nutrient agar (NA), plasmids was imposed (i.e., on NA containing ampicillin and and diagnostic sensitivity agar have been described previ- tetracycline). Complementation of the transfer mutation was ously (37). Supplements (in micrograms per milliliter) in NA assessed from the recovery of Tcr Rif transconjugants of were as follows: ampicillin, 300; chloramphenicol, 10; mer- LT101 in spot matings. In most matings, the recovery of Apr curic chloride, 10; kanamycin, 10; nalidixic acid, 10; ri- Rif transconjugants was also monitored, since the clones are fampin, 150; streptomycin, 100; and tetracycline, 5. Sulfa- oriT+ and hence mobilizable from Tra+ cells. thiazole (80 ,ug/ml) and trimethoprim (1 ,ug/ml) were used in Isolation of plasmid mutants. The final test applied to the diagnostic sensitivity agar. RP1 mutants isolated in the first two procedures described 450 FONG AND STANISICH J. BACTERIOL.

TABLE 2. RP1 mutants used purified twice on the same medium and then tested by spot mating with LT101 to confirm their Tra- phenotype. Tra- Plasmid mutant"b Dps+ or Tra- Dps+' mutants were isolated by screening Mutagen No. transconjugant colonies obtained on unseeded selective me- MutagentNo. pVS (or other) designation dium. The colonies were cross-streak mated with LT101 to Tra- Dps' mutantsc detect Tra- mutants. The Dps phenotypes of all mutants TnS 12 570, 574, 575, 576, 582, 583, were then determined. 585, 588, 625, 626, 640, 642 (ii) pVS520::TnS mutants were isolated from filter matings NTG 12 647, 649, 651, 653, 654, 745- between LT104(pVS520) and HB101 as described previously 750, pMO431 (37). Kmr StW transconjugants were recovered, and those HA 1 pMO480 that had inherited Tra- mutants were identified by cross- streak mating with LT101 on NA containing tetracycline and Tra- Dps+' mutantsc TnS 15 577, 580, 587, 590, 591, 618- rifampin. The Dps phenotypes of such mutants were then 624, 643-646 determined. NTG 2 648, 752 (iii) Mutants (mob or pil) of the Tral clone, pVS659, were obtained either by hydroxylamine (HA) treatment of Tra- Dps- mutantsc plasmid DNA or by NTG treatment of a UB1301(pVS659, TnS 10 688, 753-761 pVS644) culture as described previously (37). The muta- NTG 5 762-766 genized pVS659 plasmids were then transferred to HB101 None 1 pMO435 sublines carrying a particular mob (Tra- Dps') orpil (Tra- a RP1 and R18 are probably identical plasmids (45). The pMO plasmids are Dps- or Tra- Dps'l-) mutant of pUB1601 or pVS520. mutants of R18 and include the spontaneous Dps- mutant pMO435 (49); the HA-treated DNA was transferred by transformation; NTG- TnS mutants and pVS766 are derivatives of pVS520; the NTG mutants are treated pVS659 (oriT+) was transferred by conjugal mobili- derivatives of pUB1601. pUB1601 and pVS520 are RP1 deletion mutants (37, The were 52). zation. colonies obtained cross-streak mated with b pVS659 (Tral+) complemented all of the plasmid mutations; pVS658 LT101 to detect ones in which complementation failed to (carrying part of Tral) complemented only mutations in plasmids in boldface occur. The putative pVS659 mutant plasmids carried by type. Plasmid transfer was determined by spot mating with donors carrying these bacteria were then recovered by transformation, and pVS658 or pVS659 together with an RP1 mutant; the recipient was LT101. Tcr their phenotypes were confirmed. Rif` or Apr Rif transconjugants were selected, thereby monitoring complementation of the RP1 mutation or mobilization of the clone. DNA procedures. Standard DNA isolation and molecular Dps+ and Dps- indicate, respectively, sensitivity (efficiency of plating cloning techniques were performed as described previously [EOP) = ca. 0.5) and resistance (EOP < 10-8) to phages PR4 and PRR1; (29, 37, 41). Dps+ - indicates sensitivity to PR4 but resistance to PRR1. The EOP was Isolation of mutants. The fertility of R388 (fisB+ determined by spot phage assays (48) on HB101 carrying RP1 (EOP = 1) or R388fisB the RP1 mutant. [sensitivity to fertility inhibition by RP1]) is inhibited by pUB1601 (fiwB+) (17). NTG-treated cultures of UB1301 (R388) were filter mated with UB5201(pUB1601), and Tpr Nalr transconjugants were isolated. These were cross-streak below was to determine whether their mutations were com- mated with UB1301 on diagnostic sensitivity agar containing plemented by pVS659 (Tral). Only mutants in which a trimethoprim and rifampin to detect ones which transferred positive response was obtained were used in the study. R388 at high frequency, i.e., that carried putative R388fisB (i) The procedure for mutagenesis of P. aeruginosa mutants. Such a mutant plasmid was recovered from a PA09505(pUB1601) by N-methyl-N'-nitro-N-nitrosoguani- UB1301 transconjugant, and its fisB mutant phenotype was dine (NTG) followed by filter mating of the treated donors confirmed. with HB101 has been described previously (37). To isolate Isolation of pKM101fip mutants. pKM101 (fip+ [fertility Tra- Dps- mutants, samples of the mated bacteria were inhibition of IncP plasmids]) can inhibit the fertility of RP1 plated on NA containing tetracycline and streptomycin (58). A UB1301(pKM101, pWS142) culture was filter mated seeded with PR4 (ca. 108 PFU). The colonies obtained were with HB1O1(pVS520), and Cmr Apr (Strr) transconjugants were isolated. These were cross-streak mated with LT101 to identify ones which transferred pVS520 at high frequency. Such bacteria carried putative pKMlOlfip::Tnl725 mutants TABLE 3. Mutants of the Tral clone pVS659 and which were separated from pVS520 by conjugal transfer. their derivations Their fip mutant phenotype was then confirmed. RP1 mutant (Dps Nomenclature. Transfer-defective mutants of R18 (synon- Plasmid Genotypea Mutagen phenotype) used to ymous with RP1 [51]) and of the RP1 deletants, pUB1601 isolate pVS659 and pVS520, are all referred to as RP1 mutants. The gene mutant designationspil and mob (rather than tra) are applied to Tral pVS768 mobAl NTG pVS625 (Dps') transfer genes believed to have specific roles in pilus and pVS769 mobBI HA pVSS88 (Dps') mobilization function, respectively. This is done to facilitate pVS770 mobB2 NTG pVS576 (Dps') comparison with previously described mutants for which pVS771 mobCl HA pVS649 (Dps') three similar tra nomenclature schemes have been described pVS773b piL43 HA pVS651 (Dps') pVS774b piL4l HA pVS648 (Dps+'-) (22, 49, 53). pVS775 pilBI NTG pVS766 (Dps-) RESULTS a The genotypes were assigned retrospectively and reflect the cistrons identified when these mutants were used in complementation studies (Table Tral DNA. Two clones 4). Construction of clones carrying b These two plasmids carry mutations in the same gene (see Table 4), were constructed to allow the identification of Tral mutants despite their derivation using RP1 mutants with different Dps phenotypes. of RP1 (Fig. 1B). Plasmid pVS659 carries the entire Tral VOL. 175, 1993 RP1 tra CISTRONS 451

E mids. This was particularly relevant for pVS658, since this plasmid 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 that were investigated. Three were from a previous study (they are prefixed with pMO [49]) and included a spontaneous Dps- mutant, pMO435; all of the remaining mutants were induced by either TnS, NTG, or HA. With the exception of pMO480, which carries a cis-dominant mutation (49; see below), the transfer defects of all of the plasmids could be complemented by pVS659, indicating that the respective mutations are in Tral. A similar analysis of heterozygotes Ss carrying pVS658 revealed complementation in only nine cases (i.e., with the plasmids shown in boldface in Table 2). In these instances, the mutations must occur in the 48.6- to 54.5-kb region of Tral that is common to both pVS658 and pVS659. The last mutant, pMO480, carries a mutation that is not complemented by either pVS658 or pVS659 but also occurs in the 48.6- to 54.5-kb region. This conclusion stems from the finding that pMO480 shares with the nine comple- mentable mutants the ability to mobilize pVS658 or pVS659 p (both oriT+) from the respective heterozygotes. Thus, the mutation in pMO480 is both cis dominant and likely to occur s s s s Ss s s s within oniT as previously suggested (49). The various RP1 mutants exhibited differences in their C IHI W5 A Ss responses to pilus-specific phages: some retained sensitivity I 0 I to both PR4 and PRR1 (Dps+); some retained sensitivity only to PR4 (Dps'l-), while others became resistant to both Kmr Tral ori T phages (Dps-). It is evident from Table 2 (footnote b) that pVS659 changes from the Dps+ phenotype result only from mutations that are complemented by pVS659 but not by pVS658, i.e., among mutations that are proximal to the 48.6- to 54.5-kb region. B VpVS729 Insertion mapping of TnS+ mutants of RP1. The locations of TnS in the various insertion mutants were determined by pVS751 agarose gel electrophoresis of SstII and SstII-ApaI digests or FIG. 1. (A) Map of the 60-kb plasmid RP1 (outer circle), based of XhoI and XhoI-ApaI digests. This utilized the SstII on that of Thomas and Smith (51). The RP1 deletion derivatives (43.5-kb) andApal (54.5-kb) sites in the Tral region (Fig. 1), pUB307 (54.8 kb [4]) and pUB1601 (and its derivative pVS520; both together with the SstII and XhoI sites that occur 0.45 and 0.5 49.2 kb [37, 52]) are represented by the inner circles. The positions kb, respectively, from each terminus of TnS (5.9 kb [1]). The of the transfer (Tra) regions (in black) and the resistance functions distribution of insertion sites is shown in Fig. 2 and spans a (to ampicillin, tetracycline, and kanamycin) are indicated. (B) Lin- 5-kb region up to and apparently including oriT. The 12 Tra- 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 transfer Dps+ mutations map in two regions (49.5 to 51.1 kb and 46.9 origin, oriT (at 51 kb [23]), and the incompatibility locus, incC, in the to 47.3 kb), as do the 10 Tra- Dps- mutations (48.0 to 48.2 adjoining trfB maintenance region (32, 50). The lines beneath the kb and 46.0 to 46.4 kb). In contrast, the 15 Tra- Dps'/- map show the regions of RP1 contained in various clones. Plasmid mutations map in a single region (47.2 to 48.0 kb) that pVS658 has sustained a spontaneous deletion of part of the Tral overlaps the flanking Tra- Dps+ and Tra- Dps- mutations. region (dotted line). The cloned DNA in pVS729 and pVS751 The mutations in pVS625, pVS626, and pVS642 do not, in originated from pVS520::TnS mutants; the triangles indicate the fact, occur within otiT but are close to it (the initial place- locations of TnS sequences in these clones. Cleavage sites for ment reflects minor mapping errors). Complementational various restriction enzymes are as follows: A, ApaI; C, ClaI; E, transfer of these plasmids (Table 2, footnote b) clearly EcoRI; H, HindIII; P. PstI; S, SphI; and Ss, SstII. In panel A, only demonstrates that are the ApaI, ClaI, and SstII sites that occur in the second half of RP1 they oriT. Moreover, their mutations are shown. occur anticlockwise to oriT. This was evident from the finding that pVS751, a clone carrying DNA clockwise to the insertion in pVS626, is oriT+ (see below). Thus, all of the insertion mutations studied occur anticlockwise to the oniT region on the ClaI (37.2-kb)-ApaI (54.5-kb) fragment in- locus. serted in pVS987. In contrast, pVS658 carries the HindIII Identification of Tral cistrons. The identification of cis- (38.9-kb)-EcoRI (60.0-kb) region in which a spontaneous trons likely to affect pilus function was commenced by internal deletion from about 39.1 to 48.6 kb has occurred so isolating mutants of pVS659 that failed to complement the that, for practical purposes, it carries only the oniT end of transfer defect of a Dps'/- or Dps- mutant of RP1 (i.e., Tral. In all studies involving heterozygotes carrying either pVS659pilUI and pVS659pilBJ, respectively; Table 3). From of these Apr clones together with an Aps Tcr derivative of the behavior of these mutants in complementation tests RP1, double selection was imposed on NA containing ampi- (Table 4), it was evident that all 17 of the Dps'/- mutations cillin and tetracycline to ensure the retention of both plas- occur in a single cistron, piL4 (Table 4, column 3; footnote 452 FONG AND STANISICH J. BACTERIOL.

pilA mobC

Dps +/ - Dps +

9 g r.

pilB mobB mobA Dps Dps Dps Dps DpS

SstII Apal

Im 1 I i 1 43 44 45 46 47 48 49 50 51 52 53 54 PR2PR3 PL2 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 Z I 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::TnS insertion mutations. The respective plasmids have been distinguished according to their Dps phenotypes (see Table 2) and the cistrons affected (see Table 4) as follows (from right to left): Dps' and mobA or mobB, pVS625/626, -642, -585, -576, -570, -575, and -588; Dps- and piIA, 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 known precisely 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 to traN) 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).

d). In contrast, 13 of 16 of the Dps- mutations occur in a second cistron, pilB, while 3 are pilA mutations (Table 4, columns 5 and 3, respectively; footnote d). Moreover, the TABLE 4. Complementation of RP1 mutant alleles pilA cistron also includes 16 of 25 of the Dps+ mutations of the Tral region (Table 4, column 3; footnote d). To confirm these assignments, a third pVS659 mutant (pVS659piUA3) was isolated RP1 mutant Dps Transfer frequency'w4 of RP1 mutants from carried by pheno- heterozygotes carrying the pVS659 allele that was unable to complement one such Dps+ mutant (i.e., pVS651 [Table 3]); its complementation behavior paralleled gotea trie piLUI piIU3 pilBI mobAl mobBI mobB2 mobCI that of pVS659piUIl (Table 4, columns 3 and 4). The pVS625e Dps+ 110 130 150 7 75 140 180 pleiotropy of piLU mutations detected in this analysis is in pVS642e Dps+ 222 119 200 2 166 94 72 keeping with the overlap observed between Dps+'- insertion pVS576e Dps+ 78 56 128 122 <0.1 0.5 417 mutations and both Dps- and Dps+ mutations in the 46.9- to pVS588e Dps+ 42 40 63 79 <0.1 <0.1 63 48.2-kb region (Fig. 2). Nevertheless, it is noteworthy that a pVS649 Dps+ 38 51 27 164 18 20 0.1 strong correlation exists between the location of the inser- pVS647 Dps+ <0.1 0.2 222 155 166 188 444 tion mutation and the Dps phenotype that ensues, i.e., Dps+ pVS651 Dps+ <0.1 <0.1 125 188 150 138 150 to Dps+'- to Dps- (proceeding clockwise in RP1). pVS648 Dps'/- <0.1 <0.1 58 83 75 63 58 To complete the investigation of the remaining Dps+ pVS752 Dps'l- <0.1 <0.1 115 100 65 92 500 pVS753e Dps- <0.1 <0.1 41 64 64 91 68 mutants, four additional mutants of pVS659 were isolated pVS754e Dps- <0.1 <0.1 69 156 106 63 131 (pVS768 to pVS771; Table 3). As expected, these comple- pVS763 Dps- 64 80 <0.1 122 64 68 84 mented the various piL4 and pilB mutations (Table 4, col- pVS766 Dps- 109 218 <0.1 500 77 77 127 umns 6 to 9). The remaining RP1 mutations could be assigned to three further cistrons; mobC, defined by the a Donors were sublines of HB101 carrying pVS659 or a mutant of it together in with an RP1 mutant. single NTG-induced mutation pVS649 (Table 4, column b See Table 2, footnote c. 9), and two others, mobA and mobB. Each of the last two c Numbers are percentages determined relative to the level of complemen- may represent more than a single cistron, since they are tation obtained with pVS659. Transfer frequencies were determined from the composed only of insertion mutations (three and five, re- number of Tcr Rif' transconjugants recovered in spot matings with LT101. Transfer frequencies from heterozygotes carrying pVS659 and from all other spectively) (Table 4, columns 6 to 8; footnote d). Finally, in combinations in which complementation occurred were about 10-1 per donor. keeping with the suggestion that pMO480 (Dps+) is an oniT d The cistron assignments and phenotypes of other plasmids studied were as mutant, this plasmid permitted mobilization of pVS659 and follows: pVS626, mobA; pVSS70, -575, and -585, mobB; pVS577, -580, -587, its mutants from the respective heterozygotes (data not -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, shown). pilA and Dps-; pVS668, -756 to -762, -764, and -765 and pMO435, pilB. Mobilization of nonconjugative plasmids by the RP1 mu- ' TnS insertion mutant; all other plasmids listed are NTG mutants. tants. RP1 can mobilize the nonconjugative plasmids VOL. 175, 1993 RP1 tra CISTRONS 453

TABLE 5. Mobilization of nonconjugative plasmids TABLE 6. Mobilization of pVS659 and its mutants by Tral mutants of RP1 by IncPI3 plasmids

RP1 deriv- Mobilization frequency of: Transfer frequencya of pVS659 or its derivatives from Genotype Mutation in donors' also carrying: ative RSF1010 pSClOlcam ColElcam pVS99 pVS659 pVS520 pVS751 pVS772 R906 pUB1601b Wild type 7.5 x 10-1 7.5 x 10-1 1.1 9.0 x 10-1 pVS570 mobB 1.0 7.0 x 10-1 6.0 x 10-1 <5 x 10-7 None 0.9 0.6 3.7 x 10-2 1.3 pVS647 piIA 1.2 x 10-2 5.5 x 10-3 5.5 X 10-3 7.0 x 10-2 mobAl 1.8 3.1 x 10-3 3.1 x 10-4 8.0 x 10-3 pVS749 piL4 6.5 x 10-3 5.5 x 10-2 6.5 x 10-2 1.3 x 10-1 mobBI 1.2 4.5 x 10-5 5.0 X 10-7 5.5 X 10-4 pVS750 pilA 5.5 x 10-2 <5 x 10-7 6.5 x 10-3 <5 x 10-7 mobB2 1.2 8.0 x 10-4 8.0 x 10-5 1.5 x 10-2 mobCl 1.6 1.4 x 10-1 3.0 x 10-2 8.5 x 10-2 a Determined from the number of transconjugants from spot matings with pilA3 1.5 0.5 5.5 x 10-2 1.5 LT101. The HB101 donors carried an RP1 mutant together with a nonconju- pilBi 0.9 0.7 3.2 x 10-2 0.9 gative plasmid; donors carrying pVS99 also carried pVS97, a cryptic plasmid required for mobilization of pVS99 by RP1 (11). Selections imposed were Tcr a Determined from the number of transconjugants in matings between Rif' for the RP1 mutants, Sur Rif' for RSF1010 and pVS99, and Cmr Rif' for donor sublines of HB101 and either LT102 (for matings involving R906) or pSClOlcam (= pVS657) and ColElcam (= pVS661). No transfer was de- LT101 (all other matings). The donors carried an IncP plasmid together with tected when each nonconjugative plasmid was carried alone (<5 x 10-7 per pVS659 or a mutant of it. Filter matings were used, except in the case of donor); this was also true when most of the RP1 mutants were carried alone, pVS520 donors, which were spot mated. Selection was for Apr (pVS659 and although some had residual transfer (up to 5 x 10-4 per donor). mutants), Tcr (pVS520), Tpr (R751), Kmr (R772), or Hgr (R906) transcon- b pUB1601 (Tra+ Dps+) served as the wild-type control. The mobilization jugants on either nalidixic acid- or rifampin-supplemented medium as appro- frequencies found for all of the Tra- Dps+ mutants listed in Table 2 were priate. similar to that of the control, except for those of the mobB mutants (which b Donors were constructed by transforming HB101 sublines carrying the behaved like pVS570) and the three pil4 mutants shown above. None of the IncP plasmid with DNA of the appropriate clone. Selection was for both Tra- Dps'/- and Tra- Dps- mutants listed in Table 2 mobilized any plasmids (Apr and those shown in footnote a above), except in the case of nonconjugative plasmid (<5 x 10-7 per donor). R772, for which only Apr could be used (R772 and the clones both carry Kmr).

RSF1010, pSC101, ColEL, and pVS99 (11, 54, 56), but its on pVS729+ pVS664+ bacteria as on those carrying pVS520 specific contributions to these interactions have not been (Tra+). Thus, the 6-kb region encodes all of the Tral genes determined. We anticipated a broad correlation between Dps that are required for the elaboration of a pilus that functions phenotype and mobilization ability but were particularly normally in phage propagation. interested in that involving Dps+' and other mutants of Localization and characterization of mobC. Only a single pilA. The Dps+ plasmids with mutations in mobA, mobB, or mobC mutant (pVS649) was isolated, and its point mutation mobC (as well as pMO480OoiT) continued to mobilize the was mapped to the 48.6- to 54.5-kb region by complementa- various nonconjugative plasmids, except that mobB mu- tion with pVS658 (Table 2, footnote b). The mutation was tants, such as pVS750, did not mobilize pVS99 (Table 5). further localized to the 51.1- to 54.5-kb region by cloning this These results indicate that the pili encoded by these plasmids DNA from pVS626 (a mobA::TnS mutant; Fig. 2) into are normal in terms of their roles in phage adsorption, pVS987 (on a HindIII-ApaI fragment). The resulting clone, penetration of the cells by phage DNA or RNA, and the pVS751, complements the mobC mutation but none of the exiting, during conjugation, of nonconjugative plasmid other RP1 mutations (data not shown). More importantly, DNA. The various mob mutations therefore seem to specif- pVS751 is mobilized by pVS649 and hence is oniT+. Thus, ically affect transfer of RP1 DNA or, in the case of mobB mobC must occur clockwise to oriT, whereas the remaining mutants, of both RP1 and pVS99 DNAs. This conclusion is four cistrons are anticlockwise to this locus at the locations therefore consistent with the demonstration by other work- shown in Fig. 2. ers that genes immediately adjacent to oriT serve in the RP1 Other workers have defined the Tral core as the oriT mobilization process (38, 39). region flanked by traJ, -I, and -K and further shown that In contrast, only 3 of 13 of the Dps+ plasmids with these three genes are RP4 specific and cannot be substituted mutations inpiU4 permitted mobilization of the nonconjuga- by functions from IncPj plasmids (18, 21, 38, 59; Fig. 2). We tive plasmids (i.e., pVS647 and pVS749) or of only RSF1010 used the latter observation to further characterize the mob and ColEl (i.e., pVS750). However, the frequencies ob- cistrons and found that pVS659mobA or pVS659mobB mu- tained were reduced 10- to 100-fold (Table 5). No mobiliza- tants were poorly mobilized by the IncPB plasmids (ca. 10-2 tion from any of the remaining mutants (Dps+'- or Dps-) to 10-5 per donor), whereas the mobC mutant was efficiently was detected. Therefore, it seems that most piLA and all of mobilized (Table 6). This suggests that mobA and mobB are the pilB mutants tested are unable to transfer plasmid DNA equivalent to traJ and traI, respectively, in keeping with the because their mutations specifically affect pilus function, mapping data (Fig. 2). In contrast, the ability of IncPP either by failure to elaborate a pilus or by production of a plasmids to substitute for mobC suggests that this is a new defective structure. However, rare Dps+ pilA mutants seem mobilization function that occurs in the vicinity of traK but able to produce a pilus with moderate conjugal activity, is distinct from it. For completeness, piL4 and pilB mutants suggesting that these mutations, like those in mob, specifi- of pVS659 were also studied and found to be efficiently cally affect DNA transfer. mobilized by the IncPPi plasmids, supporting previous ob- Distribution of pilus functions within Tral. The two pro- servations of the interchangeability of Tral pilus functions posed pilus cistrons,piL4 andpilB, occur at positions 46.0 to (21, 45). 48.2 kb. The 43.5- to 49.5-kb region of Tral was cloned from Functional similarity between IncPca, IncPfl, IncN, and the mobB::TnS mutant pVS588 (Fig. 2) into pVS987 (on an IncW plasmids. The IncPa subgroup of plasmids to which SstII fragment). The plasmid obtained (pVS729) was then RP1 belongs probably represents an evolutionary branch introduced into an HB101 subline carrying pVS664 (Tral- different from that of the smaller IncPB subgroup (45, 59). Tra2+). Phages PR4 and PRR1, which attach to the pilus tip The data described above show that of the five cistrons and shaft, respectively (7, 9), produced plaques as efficiently tested, only two (mobA and mobB) cannot be adequately 454 FONG AND STANISICH J. BACTERIOL. replaced by IncPP plasmids. We also asked whether muta- given that Watson et al. (53) reported two Dps- cistrons in tions of the five Tral cistrons of RP1 could be complemented Tral in addition to a pleiotropic cistron. by pKM101 (IncN) and R388 (IncW). This was a possibility, Apart frompilA and pilB, we defined three other cistrons since members of all three Inc groups confer various cross- that are required for RP1 mobilization, i.e., mobA, -B, and sensitivities to donor-specific phages (7, 8, 25) and interact in -C. Mutants of these cistrons are Dps+ and further display terms of fertility inhibition (17, 35). However, when an normal pilus function by promoting mobilization of various appropriate pKM101fip or R388fis mutant (see Materials and nonconjugative plasmids (Table 5). None, however, allows Methods) was introduced into HB101 sublines carrying mobilization of RP1, although mobB is exceptional in also pVS576, pVS618, pVS625, pVS649, or pVS668, no enhance- being required for the mobilization of pVS99. The location of ment in the transfer of the pVS plasmids to LT101 was mobA and mobB immediately anticlockwise to onT together observed (data not shown), i.e., none of the five functions with the finding that neither of these functions can be studied can be replaced by the IncN or IncW plasmids. efficiently substituted by IncPI3 plasmids suggests that they are identical to the traJ and traI mobilization genes characterized by E. Lanka and coworkers (18, 21, 38, 39). How- DISCUSSION ever, mobC, which is defined by only a single point mutation and occurs clockwise to oriT in the 51.1- to 54.5-kb region, is In this study of Tral mutants of RP1, two cistrons, pilA of interest because it can be substituted by IncPP plasmids. andpilB, that are required for pilus function in E. coli K-12 The only other known mobilization gene in this region, traK, have been localized to the 46.0- to 49.2-kb region anticlock- is specific to IncPa plasmids (18, 21). Thus, mobC may be a wise to oriT (Fig. 2). All pilB mutations resulted in a Dps- new mobilization function common to both IncPax and IncPP phenotype, probably reflecting the cell's inability to elabo- plasmids; it is presumably equivalent to traL, -M, or -N (61) rate a pilus. In contrast, piLA mutations were pleiotropic, (Fig. 2). affecting propagation of both PR4 and PRR1 (Dps-), of only Like mobC,pilA andpilB can also be substituted by IncPB PRR1 (Dps+ -), or of neither phage (Dps+). Barth et al. (3) plasmids but not by either pKM101 (IncN) or R388 (IncW) observed such pleiotropy among Tn7+ mutants of comple- (Table 6). This is consistent with the known homology of the mentation group 4 but could not eliminate the possibility that IncPa and IncPP transfer regions (45) and also with recent more than one gene was involved. We have clarified this studies by Bolland et al. (5), who showed that the R388 pilus issue by using chemically induced pilA mutants of RP1 and genes could be entirely substituted by those of pCU1 (IncN) pVS659 (a Tral+ clone), i.e., presumed point mutants. A but not by those of RP1. Other similar studies (46a) have also single pilA cistron was indicated by the lack of complemen- failed to detect substitution of Tra2 pilus functions (i.e., tation in the alternative combinations (Table 4). traA, -B, -E, -R, -P. and -O [37]) by pKM101 or R388. Interestingly, the Dps phenotype of pilA mutants is not Therefore, it appears that the similarity among IncP, -N, and random; mutations at one or the other end confer a Dps- or -W plasmids on the basis of plating of PR4, similar pilus Dps+ phenotype, while more central mutations are Dps'/- morphology, and fertility inhibition interactions does not (Fig. 2). This is not due to polar effects on downstream reflect functional homology, which occurs between plasmid cistrons, since pVS659pilU mutants complement all butpilA F and its relatives (55). mutations. Thus, the pleiotropy must reflect direct effects of In conclusion, we note two instances in which Tral the mutations onpilA itself. Given that PR4 adsorbs to the insertion mutations apparently have no effect on down- pilus tip and PRR1 adsorbs to the pilus shaft (7, 9), the stream genes. Ziegelin et al. (61) have reported transcription various piU4 phenotypes apparently reflect the absence of of the Tral region as indicated in Fig. 2. We found that pili, the presence of pilus tips, and the presence of pilus mobB::TnS mutations prevent mobilization of pVS99; how- shafts. Therefore, the piLA cistron may contribute to pilus ever, this occurs normally if the insertions are in mobA, i.e., function in two ways: by the provision of pilus tips encoded mobB remains functional. Second, both TnS and Tn7 inser- by the promoter-proximal portion of the gene and by the tions in pilA (equivalent to group 4 of Barth et al. [3]) can provision of pilus shafts encoded by the distal portion (Fig. result in a Dps+ phenotype, even though mutations in pilB 2). Furthermore, piLA seems to also be required for the (possibly equivalent to group 3 of Barth et al. [3]) confer a mobilization of RP1, since three exceptional Dps+ mutants Dps- phenotype, i.e.,pilB remains functional. These obser- that continue to mobilize nonconjugative plasmids (and vations are similar to those made by Palombo et al. (37) for hence have conjugally active pili; Table 5) nevertheless fail the RP1 Tra2 region (with TnS and Tn504) and by Winans to transfer RP1. and Walker (57) for the Tra regions of pKM101 (with TnS). Our mapping ofpiU4 andpilB insertion mutations suggests Either these transposons are not strongly polar, contradict- that these genes correspond to traF and traG identified by ing other findings (14, 16), or transcription can occur from a nucleotide sequence analysis (61) (Fig. 2). If this is so, pilU number of sites in Tral and Tra2 (perhaps at weak promoters bears similarities to FtraG both in the duality of its functions or at cryptic levels [34]) permitting sufficient gene expression and in its relatively large size (116 versus 72 kDa; 30, 61). to satisfy certain of the transfer requirements. However, there is no evidence that pilA plays a role in mating pair stabilization, as is the case for FtraG. Indeed, it is possible that RP1 entirely lacks such a function, since it ACKNOWLEDGMENTS can effect conjugation on solid surfaces but apparently not in is the case with IncN and IncW plasmids [10, 44]). We thank Rhonda McCaw for assistance in the preliminary liquids (as mapping of mob mutants, Wolfgang Schilf for plasmid pWS142, and Our demonstration that the 43.5- to 49.5-kb region of Tral is Erich Lanka for providing details of the molecular map of RP4 and sufficient, in conjunction with the Tra2 region, to permit the structure of the Tral region. propagation of the donor-specific phages localizes all of the Financial support for this work was provided by recurrent funds Tral pilus genes required for this process to the DNA to the Department of Microbiology and by the Australian Research segment that carries piLA and pilB. The third gene in this Council. S.T.F. was the recipient of a Commonwealth Postgraduate segment, i.e., traE (61) (Fig. 2), may also be a pilus gene, Research award. VOL. 175, 1993 RP1 tra CISTRONS 455

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