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.