Specialized Transduction of Pseudomonas aeruginosa PAO by Bacteriophage D3

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1 JOURNAL OF BACTEROLOGY, Feb. 1986, p /86/2448-5$2./ Copyright X3 1986, Americn Society for Microbiology Vol. 165, No. 2 Specilized Trnsduction of Pseudomons eruginos PAO by Bcteriophge D3 MARGARET M. CAVENAGH AND ROBERT V. MLLER* Deprtment of Biochemistry nd Biophysics nd The Progrm in Moleculr Biology, Stritch School of Medicine, Loyol University of Chicgo, Mywood, llinois 6153 Received 26 July 1985/Accepted 18 November 1985 D3, temperte bcteriophge of Pseudomons er*ginos PAO, ws found to specificlly trnsduce the lleles met-49 nd. nduction of estblished lysogens with UV light ws necessry for the production of trnsducing lystes. Trnsduced cells were immune to superinfection by phge D3 nd could give rise to high-frequency trnsducing lystes. Cotrnsduction of these two lleles could not be demonstrted. ws mpped to 26 min on the PAO genetic mp. Complementtion studies using thp generlized trnsducing phge F116L indicted tht met-49 is n llele of met-911 which mps t 55 min. The integrted D3 prophge ws shown to be coinherited with nd with met-49. Although there re mny temperte phges of Pseudomons eruginos nd the frequency of lysogeny mong P. eruginos strins is probbly close to 1%, no cses of prophge-linked specilized trnsduction in this species hve been reported (9, 1, 13). D3, first isolted in 196 by Hollowy et l. (11), is temperte bcteriophge of P. eruginos PAO which hs polyhedrl hed nd prominent til with six knoblike projections (2). t contins liner DNA molecule of moleculr weight 4.4 x 17 (2). The D3 prophge cn be induced to lytic growth with UV light (14). Genetic recombintion hs been demonstrted in mixed infections of plque morphology mutnts of D3 (3). The dt presented here suggest tht D3 cts s specilized trnsducing phge of P. eruginos nd tht the D3 prophge integrtes into the P. eruginos PAO chromosome t t lest two sites. MATERALS AND METHODS Bcteri nd bcteriophges. The strins used in this study re described in Tble 1. All re derivtives of P. eruginos PAO. Bcteriophges F116 nd D3 were obtined from B. W. Hollowy. F116L ws obtined from J. A. Shpiro. Medi. Luri broth ws purchsed from GBCO Lbortories, Mdison, Wis. Pseudomons miniml medium (PMM [18]) ws supplemented with.2% glucose nd mino cids s pproprite t 5 plg/ml. Ornston-Stnier medium ws s described (21), except nitrolocetic cid ws omitted. To mke pltes of ny of these medi, gr ws dded to 1.5%. Preprtion of trnsducing lystes. F116 nd F116L phges were prepred by the plte lyste method of Miller nd Ku (18). For use in trnsduction experiments, F116 or F116L ws pssed twice consecutively through the donor strin. D3 ws obtined by induction of the prophges from lysogenic P. eruginos strins by exposure to 3.5 J/m2 of UV light. Conjugtions. Time-of-entry experiments were performed ccording to the method of Hs nd Hollowy (6). They were crried out on miniml medium pltes, supplemented s needed with mino cids. PMM ws used when selecting for mino cid mrkers, nd Ornston-Stnier medium ws used for crbon source mrkers. Mtings were interrupted * Corresponding uthor. TABLE 1. Bcteril strins Strin Genotype Source or reference PAO1 Prototroph 9 PA25 rgf leu-1 22 PA283 his-i ys-12 met-28 trpc ese-i 18 PA33 rgb21 18 PA381 leu-38 str-2; FP2 22 PAO515 met-911 mie2 nla5 D. Hs PA881 rg-164 leu-8 pur-136 ese PAO1 trpa,b::pme319 his-31 str-i 6 PA167 leu-8 pur-136 rif-i ese-21; pmo171 B. W. Hollowy PA2375 met-92 ctal mtu-92 nr-911 B. W. Hollowy PT615 leu-1 rec-i2 stra; pme134b D. Hs RM17 leu-38 str-2 [D3]; FP2 D3 lysogen of PA381 RM34 cys-72 met-28 D. H. Clhoun RM4 lys-12 met-28 trpc6 pur-6 str RM42 trpb D. H. Clhoun RM44 trpa 1 RM45 met-49 1 RM46 1 RM48 met-16 1 RM49 met417 1 RM51 cys411 1 RM172 leu-38 [D3]; FP2 RM17 x RM512 RM23 rgf leu-1 nl-95 Nlr derivtive of PA25 RM268 met-92 ctal mtu-92 nr-911 Nlr derivtive nl-96 of PA2375 RM46 ys-12 met-28 trpc6 pur-6 Nlr derivtive nl-91 str-91 of RM4 RM512 Prototroph [D3] D3 lysogen of PAO1 RM23 ser-3; FP11 B. W. Hollowy RM262 met49 nl-93 dtr-91 D3r derivtive of RM45 RM263 nl-94 dtr-92 D3r derivtive of RM46 Nomenclture ccording to Royle et l. (24), with the ddition of dtr, which indictes D3 resistnce nd [D3] which indictes tht the strin is lysogenic for prophge D3. Nlr indictes resistnce to nlidixic cid, nd DY indictes resistnce to infection by phge D3. b ntegrted into the chromosome t 72 min. 448

2 VOL. 165, 1986 TABLE 2. D3-medited trnsduction of vrious lleles Recipient Mrker Mp MOb ductnts/ strin tested (min) PFU PA33 rgb <1-9 PA881 pur <1-9 RM x 1-7 RM42 trpb <1-9 RM44 trpa 27.4 < 1-9 PA283 met <1-9 PA167 leu <1-9 RM45 met x 1-7 PA283 his <1-9 RM34 cys-72? 1. <1-9 RM48 met-16? 2. <1-9 RM49 met <1-9 RM51 cys <1-9 Mp positions re from Royle et l. (24) except met49 nd, which were determined in this study. b MO, multiplicity of infection. by spreding the pltes with nlidixic cid to finl concentrtion of 25,ug/ml. For coinheritnce studies, uninterrupted mtings were crried out s described by Miller nd Ku (18). Trnsconju- SPECALZED TRANSDUCTON N P. AERUGNOSA 449 gnts inheriting donor prototrophic mrkers were selected on ppropritely supplemented miniml medi nd purified. Purified trnsconjugnts were scored for coinheritnce of the lysogeny/nonlysogeny phenotype by testing for spontneous relese of phge on n indictor strin (PAG1). Trnsductions. Cells to be trnsduced were grown in 1 ml of Luri broth to erly log phse. They were spun down nd resuspended in 5 ml of TNM buffer (.1 M Tris,.15 M NCl,.1 M MgSO4; ph 7.4). Cells were then mixed with phge nd incubted t 37 C for 1 min. The trnsduction mixtures were plted in duplicte on selective medi t desired dilutions. Trnsductnts ppered fter incubtion for 2 dys t 37 C. RESULTS Trnsductions of specific methionine lleles by phge D3. We tested number of genetic loci to determine whether they re trnsduced by bcteriophge D3 (Tble 2). Ech of the strins tested ws sensitive to phge D3, nd ech of the lleles tested ws trnsducible by the generlized trnsducing phge F116 (11, 18) t n equl efficiency (dt not shown). Only two methionine lleles, met49 nd, were found to be trnsduced by D3. While the frequency of trnsduction ws found to vry with the multiplicity of A 5s 4.PA381 met-28 B c 1 RM 23 l/ rpc o C O. E / ys c C E o m 2' 4b eb 4 8 Mting Time (min) Mting Time (min) FG. 1. nterrupted mtings. Plte mtings were interrupted with nlidixic cid fter vrious time intervls. Results for ech selected llele re presented s the frequency of recombinnts observed s function of mting time. The strins used re listed below, followed by the lleles selected. No recombinnts were recovered for lleles tht re listed s hving been selected but tht do not pper on the corresponding grph. (A) Mpping : PA381, n FP2 donor, ws crossed with RM262 (met49), RM263 (), nd RM46 (met-28 trpc). RM23, n FP11 donor, ws crossed with RM262 (met49), RM263 (), nd RM46 (met-28 lys-56). (B) Mpping met49: PAO1 nd PTO615 were crossed with RM262 (met49), RM263 (), RM23 (rgf), nd RM46 (met-28 trpc lys-s6).

3 45 CAVENAGH AND MLLER TABLE 3. Trnsductionl frequencies of met49 nd Experi- No. of Trns- Mrker ment Lyse MOb trns- ductnts/ no. no. ductntsc 17 PFU met , met-i , , , D3 ws induced by UV irrdition from RM512. b MO, multiplicity of infection. c Number of prototrophic colonies bove the level of spontneous revertnts. infection nd with the phge lyste used (Tble 3), only met49 nd met-i17 could be trnsduced by D3 lystes. The phge preprtions used for trnsductions were induced from D3 lysogens of strin PAO by irrdition with UV light. We found tht phge progeny collected fter induction of lysogen trnsduced t frequencies of 1- to 1-fold higher thn phge prepred from lytic infections of sensitive strins. Such enhnced production of trnsducing prticles by induction of lysogenic cells is chrcteristic of specilized trnsducing phges (16). At lest one trnsductnt colony ws chosen t rndom from ech of 26 seprte trnsductions nd tested for lysogeny by the methods of Miller et l. (19). All trnsductnts tested hd become lysogenic. n three seprte experiments, RM45 trnsductnts which hd been trnsduced to methionine prototrophy by D3 grown on PAO1 were induced J. BACTEROL. by exposure to UV light to relese phge. The D3 relesed ws then used to trnsduce RM45 to prototrophy. The trnsduction frequency in one of the three instnces ws incresed by fctor of 13, indicting tht high-frequency trnsducing (HFT) lystes of this phge cn be produced. Thus when D3 trnsduces cell, it usully converts tht cell to lysogen, nd the newly formed lysogens cn be used to produce HFT lystes by methods similr to those used to produce HFr lystes of phge lmbd (17). Clhoun nd Fery (1) found met-i 17 nd met49 to be in unlinked trnsductionl groups when phge F116 ws used s the trnsducing vector. We confirmed tht these mrkers re not cotrnsducible by F116. n ddition, we were unble to detect cotrnsduction of met49 with met-i 7 by D3. We conclude tht met49 nd re not tightly linked. Mp loction of nd met-49. Since neither D3 nor F116 will cotrnsduce nd met49, we wnted to determine the loction of these lleles on the P. eruginos PAO genetic mp (24). Through series of conjugtion experiments we were ble to mp met-i 1 7 to bout 27 min on the PAO chromosome (Fig. 1A). FP2 is chromosomemobilizing plsmid tht trnsfers clockwise from min on the Pseudomons chromosome mp (12). FP11 mobilizes counterclockwise from 26 min (23). n interrupted mtings medited by FP2, met-h17 ws trnsferred before met-28 nd trpc, while met49 ws not trnsferred t detectble levels (Fig. 1). FP11 did not trnsfer either or met49 s erly mrkers. This plces met-h17 between the FP11 origin of trnsfer nd the met-28 locus, nd met49 somewhere lter thn trpc (Fig. 2). PAO1 is n Hfr donor strin which mobilizes the chromosome clockwise from the trpa,b gene cluster (7). PAO1 did not mobilize, indicting tht lies between the FP11 origin of trnsfer nd the PAO1 origin, or t bout 26 min on the P. eruginos mp. PAO1 did not trnsfer met49. The met49 llele hs been locted in the mid-to-lte ys / 1 2 FP2 8 FP11 PA1 3 trpa,b met-28 trpc PT615 4 _7 6 * *- 5 * * mtu-9)2 Ct A rgf met-49 FG. 2. Genetic mp of P. eruginos PAO showing loctions of met49 nd reltive to previously mpped genes. Loctions of previously mpped genes re from Royle et l. (24) nd Fruh et l. (4).

4 VOL. 165, 1986 TABLE 4. Donor strin Trnsductionl complementtion of met49 nd met-911 Trnsductnts/18 PFU with recipient strinb: RM4 RM45 RM242 (met-28) (met49) (met-911) RM1 (prototrophic) RM45 (met49) 11. <.1 <.1 RM242 (met-911) 2.1 <.1 <.1 Strins on which F116L lystes were prepred. b Strins trnsduced by F116L to methionine prototrophy. region of the mp by conjugting with the donor PT615 (Fig. 1B). PT615 is n Hfr donor which ws constructed by D. Hs by the insertion of the chromosome-mobilizing plsmid pme134 in unique orienttion t 72 min (personl communiction). PT615 dontes chromosome in counterclockwise direction strting t 72 min on the PAO mp (24). n interrupted mtings between PT615 nd RM268, we observed essentilly complete trnsfer of mtu-92 nd ctal t our erliest time point (dt not shown). n other crosses (Fig. 1B), met49 ws trnsferred while rgf ws not. Therefore met49 lies between rgf (45 min) nd cta (63 min). F116L trnsductionl nlysis (Tble 4) indicted tht met49 is very tightly linked to met-911, which mps t 55 min (4), suggesting tht met49 nd met-911 re lleles of the sme gene. Site of integrtion of the D3 prophge. As the D3 prophge must be induced before significnt levels of trnsduction of specific lleles cn be observed, it seemed likely tht the D3 prophge integrtes ner the loci which it trnsduces. We tested this possibility by crrying out uninterrupted mtings between lysogenic donor strins nd recipients which were resistnt to the phge under study. The resistnce of the SPECALZED TRANSDUCTON N P. AERUGNOSA 451 recipient strin to the phge ensured tht lysogeniztion could not tke plce by dsorption of free phge which might be relesed by the lysogenic donor. Lysogeniztion ws restricted to cquisition of the phge through trnsfer of the prophge during conjugtion. Preliminry tests for zygotic induction of the D3 prophge indicted tht this phenomenon did not tke plce under the conditions used in our mting experiments (dt not shown). n FP2-medited conjugtions, the D3 prophge ws coinherited with. Donor strin RM17 ws mixed with recipient RM263, nd methionine prototrophs were selected. Of these, 317 were streked out for single colonies nd tested for cquisition of D3 prophge. Of the recipients tht hd cquired, 315 (99%) hd concurrently inherited the D3 prophge. Similr experiments were done using RM172 s donor nd selecting for vrious other genetic mrkers, including met49 (Fig. 3). The D3 prophge ws found to be coinherited with high frequency with genes in the 2- to 3-min region of the genetic mp nd with met49. DSCUSSON This is the first reported exmple of specilized trnsducing phge in P. eruginos PAO. The two genes which it trnsduces re widely seprted on the chromosome, nd the D3 prophge integrtes djcent to both these genes. Crey nd Krishnpilli (2) hve reported tht phge H9 integrtes into the P. eruginos PAO chromosome t 7 min. They were unble to demonstrte specilized trnsduction by this virus. Mny of the properties of phge D3 re similr to those of other specilized trnsducing phges (5, 16). The estblishment of lysogeny followed by the induction of estblished prophge is necessry for the production of trnsducing prticles. Like coliphge lmbd, this induction ppers to require tht the host hve Rec+ phenotype nd suggests tht protein similr to the reca gene product of Escherichi 1, pur-136 met-49 so cx - 6 C - 2^ or ys-12 trpb met -28 ilvb ttrpc Mp Position (minutes) FG. 3. Coinheritnce of selected lleles with the D3 prophge. See the text for experimentl detils.

5 452 CAVENAGH AND MLLER coli is required for UV-light-stimulted induction (15). This dependence is further emphsized by the fct tht severl muttions which reduce the recombintionl proficiency of P. eruginos lso reduce the bility of phge D3 to estblish lysogeny (8, 18, 25). The D3 prophge integrtes into the host chromosome t minimum of two sites which re closely linked to the loci which it trnsduces. Trnsductnts pper to be lysogens of prophge D3, nd HFT lystes cn be produced from trnsduced clones. These dt suggest tht trnsducing prticles contin both bcteril nd phge DNA nd tht the insertion of the bcteril frgment does not eliminte essentil phge genes (i.e., trnsducing prticles re not defective). The identifiction of specilized trnsducing phge of P. eruginos provides n opportunity to ddress myrid of questions concerning the genetics nd biochemistry of this orgnism. ACKNOWLEDGMENTS We thnk D. H. Clhoun, G. A. Jcoby, D. Hs, nd B. W. Hollowy for the kind gifts of mny of the strins used in this study. This work ws supported in prt by grnt from the Pott's Fund of the Stritch School of Medicine, Loyol University of Chicgo, nd by Public Helth Service grnt A from the Ntionl nstitute of Allergy nd nfectious Disese. LTERATURE CTED 1. Clhoun, D. H., nd T. W. Fery Trnsductionl nlysis of Pseudomons eruginos methionine uxotrophs. J. Bcteriol. 97: Crey, K. E., nd V. Krishnpilli Loction of prophge H9 on the chromosome of Pseudomons eruginos PAO. Genet. Res. 23: Egn, J. B., nd B. W. Hollowy Genetic studies on lysogeny in Pseudomons eruginos. Aust. J. Exp. Biol. 39: Fruh, R., J. M. Wtson, nd D. Hs Construction of recombintion-deficient strins of Pseudomons eruginos. Mol. Gen. Genet. 191: Gottesmn, M. E., nd R. A. Weisberg Prophge insertion nd excision, p n A. P. Hershey (ed.), The bcteriophge lmbd. Cold Spring Hrbor Lbortory, Cold Spring Hrbor, N.Y. 6. Hs, D., nd B. W. Hollowy R fctor vrints with enhnced sex fctor ctivity in Pseudomons eruginos. Mol. Gen. Genet. 144: Hs, D., J. Wtson, R. Krieg, nd T. Leisinger soltion of n Hfr donor of Pseudomons eruginos PAO by insertion of the plsmid RP1 into the tryptophn synthse gene. Mol. Gen. Genet. 182: J. BACTEROL. 8. Hollowy, B. W Mutnts of Pseudomons eruginos with reduced recombintion bility. Mutt. Res. 3: Hollowy, B. W Genetics of Pseudomons. Bcteriol. Rev. 33: Hollowy, B. W Genetic orgniztion of Pseudomons, p n P. H. Clrke nd M. H. Richmond (ed.), Genetics nd biochemistry of Pseudomons. John Wiley & Sons, Ltd., London. 11. Hollowy, B. W., J. B. Egn, nd M. Monk Lysogeny in Pseudomons eruginos. Aust. J. Exp. Biol. 38: Hollowy, B. W., nd P. A. Jennings An infectious fertility fctor for Pseudomons eruginos. Nture (London) 181: Hollowy, B. W., nd V. Krishnpilli Bcteriophges nd bcteriocins, p n P. H. Clrke nd M. H. Richmond (ed.), Genetics nd biochemistry of Pseudomons. John Wiley & Sons, Ltd., London. 14. Hollowy, B. W., nd P. vn de Putte Lysogeny nd bcteril recombintion, p n W. J. Pecock nd R. D. Brock (ed.), Repliction nd recombintion of genetic mteril. Austrlin Acdemy of Science, Cnberr. 15. Kokjohn, T. A., nd R. V. Miller Moleculr cloning nd chrcteriztion of the reca gene of Pseudomons eruginos PAO. J. Bcteriol. 163: Lewin, B Gene expression-3: plsmids nd phges. John Wiley & Sons, nc., New York. 17. Miller, J. H Experiments in moleculr genetics. Cold Spring Hrbor Lbortory, Cold Spring Hrbor, NY. 18. Miller, R. V., nd C.-M. C. Ku Chrcteriztion of Pseudomons eruginos mutnts deficient in the estblishment of lysogeny. J. Bcteriol. 134: Miller, R. V., J. M. Pemberton, nd A. J. Clrk Prophge F116: evidence for extrchromosoml loction in Pseudomons eruginos PAO. J. Virol. 22: Miller, R. V., J. M. Pemberton, nd K. E. Richrds F116, D3, nd G11: temperte bcteriophges of Pseudomons eruginos. Virology 59: Ornston, L. N., nd R. Y. Stnier The conversion of ctechol nd protoctechute to,-ketodipte by Pseudomons putid. J. Biol. Chem. 241: Pemberton, J. M., nd B. W. Hollowy Chromosome mpping in Pseudomons eruginos. Genet. Res. 19: Royle, P. L., nd B. W. Hollowy Reltionship between R nd FP plsmids in Pseudomons eruginos. Antimicrob. Agents Chemother. 17: Royle, P. L., H. Mtsumoto, nd B. W. Hollowy Genetic circulrity of the Pseudomons eruginos PAO chromosome. J. Bcteriol. 145: vn de Putte, P., nd B. W. Hollowy A thermosensitive recombintion deficient mutnt of Pseudomons eruginos. Mutt. Res. 6: