Isolation and characterization of conjugation-deficient ... · PDF filemutants of Escherichia...

1976, 126(3):1194. J. Bacteriol.

J O Falkinham 3rd and R Curtiss 3rd K-12.mutants of Escherichia coli conjugation-deficientcharacterization of Isolation and

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JOURNAL OF BACTERIOLOGY, June 1976, p. 1194-1206Copyright C) 1976 American Society for Microbiology

Vol. 126, No. 3Printed in U.SA.

Isolation and Characterization of Conjugation-DeficientMutants of Escherichia coli K-12JOSEPH 0. FALKINHAM III* AND ROY CURTISS III

Department ofBiology, Virginia Polytechnic Institute, Blacksburg, Virginia 24061,* and Department ofMicrobiology, University ofAlabama Medical Center, Birmingham, Alabama 35294

Received for publication 19 March 1976

Conjugation-deficient mutants (Con-) of Escherichia coli K-12 have beenisolated by a variety of indirect selective techniques. Mutants with mutationsconferring ampicillin resistance, fosfomycin resistance, an alanine requirement,and a failure to ferment a number of carbohydrates were selected because theimpaired functions occur in association with cell wall and cell membrane de-fects. The integrity ofthese catalytic or structural elements is postulated to havea role in conjugation. The mutants could be divided into at least six generalcategories corresponding to their defectiveness in the following postulated recip-ient cell functions: (i) specific-union formation, (ii) effective-union formation,(iii) deoxyribonucleic acid transfer, (iv) plasmid establishment, (v) plasmidmaintenance, and (vi) recombination. The availability of these mutants shouldcontribute to the description of the molecular events involved in each of theseconjugation steps and the elucidation of the genetic control over the inheritanceof conjugationally transferred deoxyribonucleic acid.

A description ofthe functional steps of recipi-ent cells in conjugation can be accomplished byeither biophysical or genetic techniques. If therecipient cell has specific roles during conjuga-tion, there must exist recipient genes that spec-ify and regulate these functional steps. Becausethe stages of conjugation are not synchronous(based upon the kinetics ofdonor marker trans-mission), characterization of the intermediatesteps of gene transmission in populations ofcells is difficult. In contrast, mutants blocked atspecific stages of conjugation should demon-strate intermediate steps unambiguously. Ourapproach, therefore, has been to isolate a largenumber of conjugation-deficient (Con-) mu-tants by various techniques with the hope ofobtaining mutants with defects in each stage ofconjugation.The maintenance ofviable transconjugants is

not the only function of the recipient cell inconjugation. Recipient cells have other specificroles in conjugational gene transmission (15).The functional roles are postulated as: (i) spe-cific-union formation, (ii) effective-union for-mation, (iii) deoxyribonucleic acid (DNA)transfer and (iv) donor and recipient DNA asso-ciation resulting in recombination or (v) stableinheritance of an extrachromosomal plasmid(establishment and maintenance) (15). It is alsopossible that a recipient cell structure and/orproduct triggers the initiation of conjugationaltransmission events in the donor cell (chromo-

some or plasmid mobilization) (16; Falkinham,unpublished data).

Specific-union formation between donor andrecipient cells is mediated by donor pili (9), thesynthesis ofwhich is encoded by genes on conju-gative plasmids (1, 33). Since it is the uniquestructure of the donor pilus tip that interactswith the recipient cell surface to form a specificunion (34), it follows that the recipient cellshould have a specific receptor for the pilus tipand that one class of Con- mutants should havealterations in this receptor and therefore bedefective in specific-union formation.The conversion of specific unions to effective

unions has been postulated to involve thoseevents needed to establish a connection be-tween donor and recipient cells through whichDNA can pass (15). Ou and Anderson (35) ob-served that close mating pairs were more fertilein yielding recombinants than loose matingpairs. Goldschmidt and Curtiss (unpublisheddata) observed an exchange of the outer mem-brane A receptor protein as a consequence ofconjugation. These observations therefore sug-gest that close cell-cell contact with membranefusion may be required for effective-union for-mation (20). Retraction of donor pili, which hasbeen shown to occur during donor phage infec-tion in Pseudomonas aeruginosa (7, 8) andEscherichia coli (28, 32), could provide themechanism whereby donor and recipient cellsare brought together to establish effective un-

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VOL. 126, 1976

ions. It is possible that some of the donor trans-fer-deficient (Tra-) mutants (2) are both resist-ant to donor-specific phage and conjugation de-ficient (as donors) because they fail to retracttheir donor pili since mutants ofP. aeruginosawhose pili fail to retract upon infection withpili-dependent phage are resistant to thosephage (8). If effective-union formation requiresa specific recipient cell function and/or surfacereceptor necessary for membrane fusion, itwould be anticipated that another class of Con-recipient mutants would form specific unionsbut not undergo the transition to effective un-ions.

Initiation of plasmid or chromosome transferis controlled by plasmid genes but may be trig-gered by a product or structure of the recipient(16; Falkinham, unpublished data). Conjuga-tional DNA replication to replace the strand ofdonor DNA being transferred to the recipientutilizes enzymes supplied by the donor (19) andmay act as the sole driving force for transfer ofshort plasmids that are transferred in toto toDNA-deficient minicells (13, 24) and do not re-quire any known recipient cell functions or ac-tivities (18). Transfer of long plasmid and chro-mosomal DNA, however, requires homologouspairing between the proximally transferred re-gion of the donor DNA and the recipient chro-mosome (19, 36) and an expenditure of energyby the recipient that somehow regulates therate of chromosome transfer (18). It would thusbe expected that certain recipient Con- mu-tants should be defective with respect to trans-fer of long plasmids and chromosomal DNA.Some ofthese might contain deletion mutationsin which that portion of the recipient chromo-some corresponding to the leading end of thetransferred donor DNA had been deleted.

Recombination leading to inheritance of do-nor chromosomal information or inheritance ofa transferred plasmid (establishment andmaintenance) are recipient functions. Recombi-nation and plasmid inheritance have been dis-tinguished as functional processes by the char-acteristics of the recombination-deficient (Rec-)mutants of E. coli K-12 (10). These mutants,although unable to inherit DNA from Hfr do-nors, are proficient in the inheritance of plas-mids. These Rec- mutants are probably recom-bination deficient because of a block after thesynaptic-like association of donor and recipientDNA noted by Paul and Riley (36). Isolationand characterization of recipient Con- mutantsmight reveal some that are defective in synap-sis or recombination functions other than thosein the Rec- mutants already isolated (11). Syn-apsis-defective mutants may appear to be defec-tive in the transfer of long plasmid and chromo-

CONJUGATION-DEFICIENT MUTANTS 1195

somal DNA, however. We would also expectCon- mutants that are specifically unable toinherit both long and short plasmids. Thesemutants might be defective in membrane at-tachment of the transferred single strand ofplasmid DNA (39) or in some later stage ofconjugational replication, resulting in the ref-ormation of the circular plasmid molecule (23).Others might be normal in these initial estab-lishment functions but be defective in mainte-nance of plasmids.

Recipient Con- mutants (other than the Rec-mutants initially described by Clark and Mar-gulies [12]) were first described by Monner etal. (31), who isolated high-level ampicillin-re-sistant (AmpR) mutants that displayed a Con-phenotype. These mutants were presumablydefective in union formation as are the Con-mutants isolated more recently by Skurray etal. (40) and by Reiner (37). Skurray et al. (40)isolated their mutants on the basis ofresistanceto phage K3, whereas Reiner selected mutantsresistant to phage ST-1.We describe recipient mutants in this report

that were selected for defects in cell wall andinner and outer membrane structure and func-tion and which have been found to display aCon- phenotype. These mutants have muta-tions that effect the various stages of conjuga-tion and which, in many cases, result in pleio-tropic phenotypes. Attempts to isolate Con-mutants by selecting directly for their inabilityto undergo conjugation with a donor strain bykilling the conjugation-proficient recipient cellsthat receive and express a donor character thatis lethal for the partial zygote were unsuccess-ful.

MATERIALS AND METHODSBacterial strains and bacteriophages. The bacte-

rial strains employed in this study are described inTable 1. Bacteriophages T7, T3, II, fl, and P1L4came from the collection of R. Curtiss. Bacterio-phages ST-1 and OW were the kind gifts of Albey M.Reiner and Hans G. Bowman, respectively.Media and chemicals. The synthetic media em-

ployed were ML (liquid) and MA (agar) (14). Themedia were supplemented with amino acids, pu-rines, pyrimidines, and vitamins at optimal concen-trations (18). Carbohydrates were added to a finalconcentration of 0.5%, and streptomycin sulfate wasadded to 100 Ag/ml, final concentration. Penassaybroth (PB), Penassay broth agar (PBA), and Penas-say agar (PA) (Difco) were employed as completemedia and supplemented with 100 lug of L-alanineper ml for growth of the alanine-requiring mutants.N-methyl-N'-nitro-N'-nitrosoguanidine (NTG) waspurchased from the Aldrich Chemical Co., Milwau-kee, Wis. Fosfomycin was a gift of Merck, Sharp andDohme, Rahway, N.J. Ampicillin was a gift of Bris-tol Laboratories, Syracuse, N.Y.


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1196 FALKINHAM AND CURTISS

TABLE 1. Bacterial strains

Strain Mating type Genotype Derivation/reference

X833 F- thr-16 1acZ76 proC24 tsx-63 purE41 supE42 42A- pyrF30 his-53 strA97 xyl-14 cycAl cycB2

X503 Hfr OR21 Prototrophic supE42 X- 0-proC tax purE --. 21lac F

X573 F'ORF4 0-purE tsx proC lac FlAiac-purE supE42 X- 5serA12

X1301 F'F42 0-iac FlApro-lac supE42 X- cycAl X314 (18); X354 (18)x1009 R64-11 R64-11 (drd Tc SmR)Ithr-1 ara-13 leu-6 azi-8 38

tonA2 lacYl minAk supE44 gal-6 X- minB2strA135 maITi xyl-7 mtl-2 thi-1

X289 F- Prototrophic supE42 X- 17x489 F- leu-6 tonA2 lacYl tsx-i supE44 gal-6 X- his-1 JC1553 (12)

recAl argG6 strA104 malTi xyl-7 mtl-2metBI

X935 F- thr-I ara-14 leu-6 proA2 iacYl tsx-33 supE44 AB247 (27)galK2 X- his-4 recB21 strA31 xyl-5 mtl-iargE3 thi-i

JF131 F'F390 O-thr leu pyrB F/leu recAl argG strA pyrB JC182 x JF4 (22)a Allele designations are those used by Coli Genetic Stock Center.

Conjugation methods. (i) Screening putativeCon- mutants for conjugation proficiency. The con-jugation proficiency of isolated recipient clones wasnormally tested by replica plating 6-h grown patchesof such clones from PBA plates to selective platesspread with a donor culture. With plates selectivefor transmission of Pro+ and spread either with X503(Hfr) or X573 (primary F'), Cont recipient clonesgave confluent patches ofrecombinant growth. Con-bacterial patches could be easily scored.

(ii) Quantitative measurement of conjugationproficiency of Con- mutants. Methods for conjuga-tional crosses were those described by Curtiss et al.(18). The ratio of donor to recipient cells was 1:10and the length of mating was 60 min. The valuesreported in Tables 3, 4, 5, and 7 are averages of atleast two determinations, with the individual deter-minations of transconjugant formation varying byless than a factor of two. The average recombinantfrequencies for X833 with the five donors used inconjugation are given in the footnote to Table 3.Chemotaxis measurements. Measurement of the

ability of the Con- mutants and their parent todemonstrate chemotaxis to glucose and mannitolwas done by a modification of the method of Arm-strong et al. (4) as suggested by G. Hazelbauer. Aloopful of a culture grown overnight in PB mediumwas applied to the center of the surface of a semi-solid agar medium (MA) containing 0.25% agar and50 puM carbohydrate. The diameter of the ring ofgrowth after 48 h of incubation at 37 C was mea-sured. Those Con- mutants demonstrating less than10% the diameter of the parent were designated asnonchemotactic.Union formation. Specific-union formation was

measured by particle counting with a Coultercounter (1; N. Achtman, personal communication).Recipient strains were grown in PB medium to logphase with shaking and mixed with a donor strain(x503 or x1009) grown in PB medium to log phasewithout shaking. The donor/recipient ratio was 1:1

and the final bacterial concentration was 2 x 108/ml.The mating culture and separate donor and recipi-ent cultures were incubated at 37 C for 15 min. A0.10-ml volume of the mating culture was gentlyadded to 10 ml of 0.85% saline (filtered through a0.22-pim pore diameter Millipore filter) and countedin a Coulter counter. To a second counting vial(containing 10 ml of 0.85% saline), 0.05 ml each ofthe separate donor and recipient cultures was addedand the total particles were counted. The percentageof union formation reported in Tables 3, 4, 5, and 7was derived from the equation:

% union formation =

[2(l mated donor + recipient count 100unmated donor + recipient count/i

Accuracy of this method is limited such that de-creases in union formation to 10% or below are notmeasurable. Some strains displayed better unionformation than the X833 parent strain.

Ultraviolet light sensitivity. Colonies of strains tobe tested for ultraviolet light sensitivity were inocu-lated on PBA plates and incubated overnight. Rep-lica plates were inoculated from this master andirradiated for various lengths of time under a Gen-eral Electric germicidal lamp. Based upon overnightgrowth after irradiation, patches could be scored asUVR or UV; upon comparison with recA- (x489),recB- (X935), and rec+ (x833) controls.P1 transduction. Methods for transduction using

P1L4 phage are the same as those described byCurtiss et al. (17). P1L4 was propagated on strainX289. The possibility was eliminated that failure toundergo transduction was not through an inabilityof P1L4 to grow or adsorb to the strains used in thisstudy by enumerating the number of infectious cen-ters after adsorption of phage to bacteria. Theseexperiments were done in conjunction with P1transduction. After the adsorption period and theremoval of cells for plating on selective medium, the

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remaining culture was washed twice in bufferedsaline with gelatin (14) and serial dilutions were

plated on strain X289 for infectious centers.Phage sensitivity. (i) Donor-specific phage. A

loopful of a turbid suspension of a clone to be testedfor sensitivity to the donor-specific phage fl was

streaked across another streak of the bacteriophagelysate. It was also found that clones picked with thebroad end of a flat toothpick would provide an ade-quate inoculum. Bacteria and phage were streakedon eosin methylene blue agar base (EMB; Difco)plates containing 0.1% glucose, 0.5% NaCl, 0.015 MCaCl2, and 0.03 M MgSO4. In all cases, the lysates ofphage equalled 1010 plaque-forming units per ml.

(ii) Recipient-specific phage. The efficiency ofplating of the phages II, qbW, T3, T7, and ST-1 was

performed by plating dilutions of high-titer lysateswith suspensions of the conjugation-deficient mu-tants and their parent on PBA plates.NTG mutagenesis. Mutagenesis by NTG followed

the procedure outlined by Adelberg et al. (3). Sur-vival of cells of strain X833 to the 60-min treatmentwith 50 jig of NTG per ml was 10%. This was themutagenesis procedure used to isolate all the mu-tants reported in this study.

RESULTS

In presenting the results of the isolation andcharacterization of Con- mutants, it will beuseful to describe first the rationale for andmethods ofmutant isolation and the phenotypiccharacteristics of the mutants. Following thisdiscussion is presented the description of thevarious classes of Con- mutants grouped ac-

cording to their postulated defect in conjuga-tion.

Isolation of glutamic acid- and alanine-re-quiring mutants. Glutamic acid- and alanine-requiring mutants of Bacillus subtilis are de-fective in transformation (41) probably throughan inability to achieve full levels of compe-tence. Although conjugation and transforma-tion may not be strictly analogous processes,elements common to cell wall structure (as areglutamic acid and alanine) may function inboth processes. Three alanine-requiring mu-tants (Ala-) and nine glutamic acid-requiringmutants of X833 have been isolated (Ala- mu-

tants and nine GIu- mutants among 107 surviv-ing cells). The three alanine-requiring mutants(JF23, JF24, and JF25) were found to be Con-(L-alanine was added to media when measuringtransconjugant formation proficiency). The mu-tants were recovered as survivors of NTG mu-tagenesis which formed small colonies on mini-mal media to which was added 5 ,ug of either L-

alanine or L-glutamate per ml. Three of 234small colonies growing on alanine-supple-mented media proved to be alanine-requiringmutants and 9 of 200 small colonies growing onglutamate-supplemented media proved to be


glutamate-requiring auxotrophs. The charac-teristics of the alanine-requiring mutants,other than their conjugation deficiency, will bereported (Falkinham and Curtiss, manuscriptin preparation).

Isolation of ampicillin-resistant mutants.Double mutants resistant to high levels of am-picillin (80 ug/ml) isolated from a mutant re-sistant to low levels of ampicillin (20 ,ug/ml)have been shown to be conjugation defective(31). These strains were demonstrated to havechanges in cell wall lipopolysaccharide.Changes in lipopolysaccharide have beenshown to affect the yield of transconjugantsafter conjugation in Salmonella typhimurium(44). With this in mind, we isolated mutantsresistant to high levels of ampicillin on PA orPBA media from a mutant (JF35) that is resist-ant to low levels (20 ,ug/ml) of ampicillin (24mutants among 107 cells surviving mutagene-sis). Two percent of those resistant to high lev-els of ampicillin were characterized as beingCon-. Unlike x833, strain JF35 is mucoid andits descendants (JF39, JF41, JF47, JF49, JF57,and JF58) are mucoid also. Strains JF51, JF52,and JF55, although descendants of JF35, arenot mucoid. In addition, strains JF47 and 49 areultraviolet light sensitive. Although isolated onPA medium containing 80 gg of ampicillin perml, the Con- mutants are not resistant to thisconcentration when tested for growth in ampi-cillin-containing PB medium. Furthermore,strain JF35 is not as resistant to ampicillin inPB as on PA medium. Table 2 includes data onthe maximum concentrations of ampicillinwhich permit growth in PB medium and sev-eral additional characteristics of the mutants.The reason for the discrepancy between anti-biotic resistance in PB medium and that in PAmedium has not been investigated.

Isolation of fosfomycin-resistant mutants.Because of the necessity of maintaining theintegrity of the recipient cell wall and mem-brane for proficient conjugational gene trans-mission, any alteration in those structures isexpected to have pleiotropic effects. An exam-ple is the class of mutants refractory to colicinE2 which are also sensitive to ultraviolet lightand are recombination deficient, form fila-ments, and demonstrate abortive growth ofbacteriophage (26). Since the enzymes for thepermeation and transport of carbohydrates areassociated with the cell wall and membrane (6),some mutants defective in carbohydrate trans-port might also be conjugation deficient (Con-).Mutants postulated to be defective in the L-a-glycerophosphate and glucose-6-phosphatetransport systems (30) were isolated to deter-mine whether changes in this system have ef-


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fects on the conjugation process. Mutants defec-tive in this transport system were recovered bytheir resistance to the antibiotic fosfomycin(43). Preliminary experiments established thatstrain X833 was sensitive to 1 mg of fosfomycinper ml in complex (PB) medium. After muta-genesis and growth of X833 in PB containing 1mg of fosfomycin per ml, 22 fosfomycin-resist-ant mutants (FosR) were recovered. Thirty-fivepercent of the FosR mutants were found to beCon-. The four mutants reported here are JF16,JF19, JF83, and JF89. One mutant, JF16, isunable to grow on PA medium (Table 2). Two ofthe strains (JF83 and JF89) fail to grow onglucose minimal medium, yet grow on eitherglycerol or pyruvate as the sole carbon sourceand on PB or PA medium. The nature of thedefect in these four FosR strains has not beeninvestigated. Resistance to fosfomycin is de-pendent upon the cultural condition, especiallyphosphate concentration (Moody and Curtiss,unpublished data). The four strains are resist-ant to 1 mg of fosfomycin per ml in PB or MAmedium owing to the phosphate in those media.In contrast, strains JF19, JF83, and JF89 areresistant to only 100 ug of fosfomycin per ml inPA medium which contains no phosphate.

Isolation of carbohydrate fermentation-de-ficient mutants. The phosphotransferasetransport system (24) ofE. coli K-12 responsiblefor the transport of a variety of carbohydrateshas components that are membrane bound (6).Mutations in these membrane-bound compo-nents may affect the integrity of the cell mem-brane and render the cell conjugation defective.Any membrane-associated function of conjuga-tion could be altered. Selection for mutants thathave lost simultaneously the ability to fermenttwo or more sugars transported by the phospho-transferase system should result in an enrich-ment for phosphotransferase system mutantsrather than those with defects in the steps fol-lowing transport which would be unique foreach carbohydrate. Mutants unable to fermentboth glucose and mannitol by loss of a func-tional phosphoenolpyruvate-coupled transportsystem might be Con-. Ten percent ofthe NTG-induced mutants forming white colonies onEMB agar containing 0.5% glucose and 0.5%mannitol were Con-. Although the mutants(Sug-, for sugar) were selected for their inabil-ity to form dark-centered colonies on glucose-mannitol containing EMB (20% of approxi-mately 107 cells surviving mutagenesis), mostof the mutants can still grow on glucose ormannitol. One reason why the mutants cangrow on glucose could be that the original selec-tion resulted in the recovery of clones unable to


form enough acid to turn the colony centerdark. Strain JF69 failed to grow on mannitol ora combination of glucose and mannitol minimalmedium. This strain did grow well on glucoseminimal, suggesting some toxicity of mannitolfor this strain.Other phenotypic properties of Con- mu-

tants. Although all ofthe strains were resistantto streptomycin and retained the amino acidauxotrophic and carbohydrate fermentationmarkers of x833, most Con- mutants displayedother mutant phenotypes. Some of the Con-mutants have changes in their sensitivity tosodium dodecyl sulfate and bile salts and lossesin their capacity to utilize carbohydrates (Table2).

Classes of conjugation-deficient mutants.The following discussion and tables describethe properties of various classes of conjugation-deficient mutants blocked at different stages ofconjugation. Care has been taken to rule outslow growth as a reason for conjugation defi-ciency. Although some strains grew moreslowly than the parent, recombinants werecounted only after a sufficient interval forgrowth to occur. All ofthe strains except for thealanine-requiring mutants JF23, JF24, andJF25 grew in minimal media containing glu-cose or pyruvate and supplements necessary forx833. As mentioned above, all crosses were per-formed in media containing alanine. No differ-ence in yield of recombinants was noted withX833 in media with or without alanine.The separation into classes is based upon the

union-forming ability of the mutants as mea-sured with a Coulter counter and the behaviorof the mutants in matings with Hfr OR21(x503), donors transferring the short plasmidsF-lac+ (x1301) and R64-11 (x1009), and donorstransferring the long plasmids ORF-4 (X573)and F390 (JF131). It is possible, however, thatsingle mutants are defective in more than onestage. For example, a strain unable to formspecific unions may be defective in a subse-quent stage of conjugation. However, any sec-ond defect will be obscured by the phenotypicexpression of the first.

Conjugation-deficient mutants defective inspecific-union formation. Table 3 displays thecharacteristics of those mutants we provision-ally designate as defective in specific-union for-mation. The inclusion of the strains into thisgroup is arbitrary, based upon a union forma-tion index of less than 25% and their inability toform transconjugants with either the Hfr, longF', or short F' donors. The accuracy of measur-ing union formation with a Coulter counter islimited, and probably decreases in union forma-


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TABLE 3. Specific-union formation-defective mutants

Transconjugant formationa Union forma-Strain Selection ciency of

Prof ~~Pro+ Thr claiencyoPro+ Hfr F'ORF4 F390 Lac+ F-lac+ TcR R64-11 Hfr R6411 plating

X833b Parent 1.00 1.00 1.00 1.00 1.00 70 16 1.00JF35 AmpRCon+ 0.80 0.62 0.76 0.42 0.63 58 8 0.46JF39 AmpR <0.01C <0.01 0.22 <0.01ndc 0.43 18 40 <0.OindJF41 AmpR <0.01 <0.01 0.15 <0.Olnd <0.Olnd 8 2 <0.OindJF57 AmpR 0.04 0.06 0.05 <0.Olnd 0.33 10 20 0.37JF71 Sug- <0.01 0.01 0.06 <0.Olnd 0.02 24 6 0.15

a The numbers in column 3 refer to the yield of Pro+[StrR] transcoDjugants with donor strainsX503 (an Hfrdonor) normalized to the yield with the parent strain x833. The numbers in column 4 are for Pro+[StrR]transconjugants in crosses with the donor strain X573 (an F' donor) normalized to the frequency of suchtransconjugants with x833. Column 5 displays the frequency of Thr+[Arg+] transconjugants in crosses withthe mutants and JF131 (an F-thr+ pyrB+ donor) normalized to the number obtained with x833. Column 6displays the frequency of Lac+[Str'II transconjugants in crosses of the mutants and X1301 (an F-lac+ donor)normalized to the number obtained with x833. Column 7 reports the yield ofTce (Leu+) transconjugants fromcrosses between the mutants and X1009, the R64-11 donor, normalized to the yield obtained with x833.Columns 8 &nd 9 display the percentage of pair formation for each mutant and the parent strain with X503(column 8) and x1009 (column 9). Note that the numbers are derived from the equation given in Materialsand Methods and are not normalized. Column 10 displays the efficiency of plating ofphage ST-1 for mutantscompared to their parent, x833.

b Frequency of transconjugants for strain X833 - for donors X503 (Hfr OR21), X573 (F'ORF4), JF131 (F390),X1301 (F-lac+), and X1009 (R64-11) the selection and frequency were: Pro+[Stri], 2.2%; Pro+[StrR], 11.2%;Thr+[Arg+], 2.1%; Lac+[StrR], 62.0%; and Tce[Leu+], 12.5%, respectively.

c <0.01, Transconjugants detected but at less than 0.01 the number with x833. <0.Olnd, No transconju-gants or ST-1 plaques detected.

tion to 10% or below are not measurable. Therep -oducibility of pair formation is influencedby the stage of growth of the recipient anddonor cells, although, even if this variable iskept constant, measurements for a single strainvary over a range of 10% union formation. Be-cause of the lack of accuracy of this method,division of the strains into those proficient inspecific-union formation and those deficient inspecific-union formation (Table 3) is tentative.Three mutants isolated for resistance to highlevels of ampicillin and one for defectiveness insugar fermentation are represented. These mu-tants give few, if any, recombinants with HfrOR21 or the donors of either long or short F'plasmids and display a reduced capacity to formpairs with the Hfr donor x503. The weak corre-lation between recombinant yields and unionformation represents the inaccuracy of assess-ing union formation. Alternatively these mu-tants would be defective in functions other thanspecific-union formation.The fact that two of the mutants (strains

JF41 and JF71) show decreased ability to in-herit the R64-11 plasmid could indicate eitherthat they fail to form specific unions with thisplasmid donor (indicating some functionaloverlap of pilus receptors) or that the strainsare unable to establish or maintain this plas-mid. The inability of the two strains to form

unions with the R64-11 donor (Table 3) indi-cates that there is a defect in union formationand there exists a functional overlap of F- and I-like pilus receptors.Two of the mutants (strains JF39 and JF41)

are resistant to bacteriophage ST-1. These mu-tant strains resemble members of one class ofthe ST-1-resistant Con- mutants isolated byReiner (37). Both strains JF39 and JF41 showno adsorption of ST-1 bacteriophage and no un-ion formation with the Hfr donor X503 as ob-served in a light microscope (Fiore and Falkin-ham, unpublished data). Strains JF57 and JF71are sensitive to ST-1 and demonstrate adsorp-tion of ST-1 at rates similar to those of strainX833 and form unstable pairs with the Hfrstrain X503 as observed with a light microscope(Fiore and Falkinham, unpublished data). It ispossible that these latter two mutants are de-fective in effective-union formation. An inabil-ity to form effective unions might be manifestedby an instability in the formation of specificunions and low union formation as measuredby a Coulter counter.

In an attempt to correlate the changes ob-served in union formation with changes inother phage receptors, the efficiency of platingof four recipient-specific phage was measured(data not shown). From the efficiency of platingphages 4II, qW, T3, and T7 no correlations

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could be drawn between alterations in the ca-pacity to form plaques and changes in conjuga-tion properties.The frequencies of Pro+ and Lac+ transduc-

tants after P1L4 infection was determined forJF39, JF41, JF57, and JF71 (data not shown).One strain, JF71, appears to be defective in thecapacity either to support the growth of P1 or toallow adsorption of phage (infectious centersare 10% wild type), but the other three strains,JF39, JF41, and JF57, produce wild-type levelsof P1 infectious centers. Strains JF39 and JF41are unable, however, to form Lac+ transduc-tants. This failure cannot be ascribed to a fail-ure to adsorb bacteriophage P1L4 because Pro+transductants are formed, and the number ofinfectious centers recovered after transductiondoes not differ substantially from the numberformed by strain X833 (data not shown). Thisbehavior suggests that strains JF39 and JF41are unable to express a lac+ gene and preventsany conclusions concerning the inheritance ofF-lac+ by these two strains.

Conjugation-deficient mutants defective ineffective-union formation and/or DNA trans-fer. Mutants of this second class fail to formtransconjugants with donor bacteria yet areproficient at forming unions as measured bythe Coulter counter technique. Table 4 showsthat two fosfomycin-resistant, three alanine-requiring, one ampicillin-resistant, and threecarbohydrate fermentation-defective mutantsare defective in effective-union formation and/or DNA transfer. At present we cannot distin-guish between inability to form effective unionsand inability to transfer donor DNA to the

recipient cell. We can, however, make somepredictions based upon data on inheritance ofthe I-like plasmid R64-11. Two classes of mu-tants can be discerned: those able to inheritR64-11 and those unable to do so. If effective-union formation is at least partially plasmidspecific, mutants blocked in effective-union for-mation for F-like plasmids might be competentfor I-like plasmid inheritance. Strains JF25,JF58, JF81, and JF83 are thus assumed to beproficient in the transfer of DNA through thecell wall and membrane since they are compe-tent in inheriting the I-like plasmid R64-11.They therefore are presumably defective in ef-fective-union formation, with donors possessingF-like plasmids.Mutants deficient in the capacity to inherit

both F-like and I-like plasmids yet proficient inspecific-union formation could possibly fall intotwo classes: (i) defective in effective-union for-mation for both F- and I-like plasmids; and (ii)defective in DNA transfer. Although we areunable to separate the two processes experi-mentally, we suggest that there is a secondunion formation step (close-union formation,see Introduction) occurring at the cell surface,which takes place before DNA transfer cantranspire. The strains falling into this class ofmutants unable to form effective unions and/orto permit DNA transfer with both F- and I-likeplasmid-carrying strains are JF19, JF23, JF24,JF59, and JF69.

Conjugation-deficient mutants defective inthe inheritance of plasmids. Characteristics ofthe mutants able to form transconjugants withan Hfr donor and with donors possessing long

TABLE 4. Effective union formation or DNA transfer-deficient mutantsa

Transconjugant formation Union formationStrain Selection

Pro+ Hfr F'ORF4 Thr+ F390 Lac+ F-iac+ TcR R64-11 Hfr R64-11

X833 Parent 1.00 1.00 1.00 1.00 1.00 70 16JF35 AmpRCon+ 0.80 0.62 0.76 0.42 0.63 58 8JF19 FosH 0.23 0.03 0.11 0.09 0.12 43 8JF23b Ala- 0.20 <0.01 0.16 <0.01 0.03 86 10JF24b Ala- 0.11 <0.01 0.16 <0.01 0.11 90 40JF25' Ala- 0.27 0.27 0.09 <0.01 0.50 86 22JF58 AmpR 0.03 0.08 0.13 <0.Olnd 0.45 38 22JF59 Sug- 0.03 0.06 <0.01 <0.Olnd 0.15 59 30JF69 Sug- <0.01 <0.01 0.53 <0.Olnd <0.01 36 4JF81 Sug- <0.01 <0.01 0.53 <0.Olnd 1.75 88 26JF83c FosH 0.17 0.34 0.34 0.03 0.42 74 6

a Refer to footnotes of Table 3.bTransconjugant yields measured on plates containing 100 gg of L-alanine per ml and compared to yields

ofX833 transcornugants on identical media. There was no difference in transconjugant yield ofX833 whetheralanine was present or absent.

c Transconjugant formation measured on plates supplemented with pyruvate and compared with X833transconjugant yields on identical media.

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F' plasmids yet unable to inherit the smallF-lac+ plasmid are displayed in Table 5. Thestrains are also unable to inherit the smallF-his+ plasmid (J. Falkinham, Genetics 80:s29,1975). All six strains form specific unions withboth the F-like and I-like plasmid-carrying do-nors. Three ampicillin-resistant mutants(JF51, JF52, and JF55), two carbohydrate fer-mentation-deficient mutants (JF67 and JF73),and one fosfomycin-resistant mutant (JF89)comprise this class. One mutant, JF51, formsTcR transconjugants with the R64-11 donor,whereas the remaining strains fail to form (orform at a reduced frequency) such transconju-gants. This result suggests that the inheritanceof R64-11 does not require the function lost bythe mutant JF51 but does require the functionslost in the other five mutants. These other func-tions it shares with the F plasmid and arerequired for inheritance of both F-lac+ and R64-11.

If the strains in this class are unable to formplasmid-carrying derivatives by conjugation, itfollows that transconjugants ofmembers of thisgroup sired by X573, which contains a long F',will arise through recombination and notthrough inheritance of the intact ORF-4 plas-mid, carried by X573. Experimental support forthis contention follows. Transconjugants ob-tained by mating X833 and JF35, the Con+ pro-genitors of the six mutants, with X573 wereanalyzed for the inheritance of the unselectedmarkers Pro+, Ade+, and Lac+ and sensitivityto the donor-specific phage fl. Two classes oftransconjugant colonies among Pro+ and Ade+transconjugant colonies could be distinguishedon the basis of size. The inheritance of unse-lected markers by large and small Pro+ andAde+ transconjugant colonies of X833 and JF35is given in Table 6. The data are compatiblewith the concept that the small colonies of both

strains, regardless of the marker selected, arisefrom transconjugants, most of which have in-herited the plasmid (high inheritance of all fourmarkers). Conversely, the large colony typesarise from recombinants with low frequenciesof inheritance of the lac+ donor marker andsensitivity to the donor-specific phage. In con-

trast to the behavior ofthe Con+ strains, the six

TABLE 6. Inheritance of unselected markers andsensitivity to donor-specific phage by mutants defec-

tive in plasmid inheritance and their parents

Recip- Selec-ient ted

strain marker

X833 Pro+

Ade+

Colonytype

LargeSmallLargeSmall

JF35 Pro+ LargeSmall

Ade+ LargeSmall

JF51 Pro+ LargeAde+ Large



JF67 Pro+ LargeSmall

Ade+ LargeSmall



Small

Per-cent

8614973

Percent inheritance

Pro+ Ade+ Lac+ fls100 90 29 23100 98 98 5178 100 53 083 100 65 29

83 100 80 64 017 100 96 100 6590 100 100 58 010 96 100 96 32

100 100 92 0 0100 92 100 0 0

100 100 88 0 0100 84 100 0 0

100 100 35 10 0100 67 100 34 0

72 100 100 40 028 100 100 13 8080 90 100 47 020 84 100 80 67

100 100 96 35 10100 100 100 50 0

100 100 41 34 1586 32 100 4 014 47 100 6 59

TABLE 5. Mutants unable to inherit plasmidsa

Transconjugant formation Union formation

Strain Selection PM+ Thr+Pro+ Hfir F'ORF4 F390 Lac+ F-lac+ Tc" R64-11 Hfr R64-11

x833 Parent 1.00 1.00 1.00 1.00 1.00 70 16JF35 AmpVCon+ 0.80 0.62 0.76 0.42 0.63 58 8JF51 AmpR 0.70 2.90 0.48 <0.01 0.95 54 28JF52 AmpR 0.54 0.42 0.13 <0.Olnd 0.08 34 28JF55 AmpR 0.50 0.31 0.12 <0.01 <0.Olnd 52 80JF67 Sug- 0.32 0.37 0.26 0.01 0.19 71 78JF73 Sug- 0.17 0.17 0.26 <0.Olnd <0.Olnd 36 14JF89b FosR 0.22 0.72 0.26 <0.01 <0.01 40 8a Refer to footnotes of Table 3.b Transconjugant formation measured on plates supplemented with pyruvate and compared to X833 yields

on identical media.

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Con- mutant strains demonstrated a decreasedcapacity to form plasmid-carrying transconju-gants. Strains JF51, JF52, JF55, and JF73 formonly large Pro+ or Ade+ transconjugant coloniesin crosses with X573. The inheritance of unse-lected markers by these four mutants is dis-played in Table 6 and supports the contentionthat the transconjugants formed by these mu-tants are haploid recombinants. The transcon-jugants have a low frequency of inheritance ofthe Lac+ marker, and none (or a few) are sensi-tive to donor-specific phage. Two strains, JF67and JF89, are still able to form some plasmid-carrying derivatives based on their ability toform large and small transconjugant colonies.The data in Table 6 demonstrate that the smallAde+ transconjugant colonies arise by inherit-ance and maintenance of the autonomous plas-mid ORF4, whereas the large Ade+ transconju-gant colonies arise from haploid recombinants.One possible explanation for these results is

that the failure to inherit the Lac+ marker andthe sensitivity to fl phage is through an inabil-ity to express the lac+ and tra+ genes of theplasmid. Because Lac+ transductants of thesemutants have been recovered at frequenciessimilar to those of X833 and some F plasmidderivatives of the mutants can be isolated thatare sensitive to fl phage, we feel that thesepossibilities are unlikely.The mutants deficient in plasmid inheritance

can be divided into two classes based upon theirability or inability to stably maintain a conju-gationally transferred plasmid (Falkinham,Genetics 80:s29, 1975; Falkinham, manuscriptin preparation). All of the mutants in Table 5appear to be defective in the establishment ofplasmids transferred by conjugation based uponthe decreased frequency of inheritance of F-lac+or F-his+ (Table 5; Falkinham, Genetics 80:s29,1975; Falkinham, in preparation). In addition,several of the mutants, strains JF51, JF67,JF73, and JF89, demonstrate instability inmaintenance of infrequently inherited F plas-


mids. F plasmid-carrying derivatives sponta-neously segregate F- derivatives at high fre-quencies (Falkinham, Genetics 80:s29, 1975;Falkinham, in preparation). Because plasmidmaintenance can be separated functionallyfrom establishment, the Con- phenotype usedhere refers only to defects in plasmid establish-ment.

Conjugation-deficient ultraviolet light-sen-sitive mutants. Four mutants that are conjuga-tion deficient and sensitive to ultraviolet lightwere recovered (Table 7). One mutant is fosfo-mycin resistant (JF16), two are ampicillin re-sistant (JF47 and JF49), and one is carbohy-drate fermentation deficient (JF75). Basedupon ultraviolet light sensitivity, two types canbe identified, those with high and those withlow sensitivity. Strains JF16 and JF75 werevery ultraviolet light sensitive (data notshown) and resemble a recA - strain, x489, inultraviolet light sensitivity. Strains JF47 andJF49 are mildly ultraviolet light sensitive andresemble the sensitivity of the recB- strainX935. Both JF47 and JF49 form mucoid colonieslike their parent JF35. Unlike Rec- mutants,JF16, JF47, and JF49 form Lac+ transconju-gants at reduced frequencies with the F-lac+donor strain X1301.We suggest that these UVS mutants carry

mutations affecting the inheritance of plas-mids. Strains JF47, JF49, and JF75 form unsta-ble plasmid-carrying derivatives (Falkinham,Genetics 80:s29, 1975; Falkinham, in prepara-tion). This evidence suggests the strains carryadditional mutations affecting plasmid estab-lishment and (in three strains) maintenance.

All four conjugation-deficient ultravioletlight-sensitive mutants are proficient in theformation of TcR transconjugants with the Iplasmid R64-11-containing donor. This datawould indicate that the functions deficient inthe strains JF16, JF47, JF49, and JF75 re-quired for F plasmid inheritance are not re-quired for the inheritance for the I plasmid.

TABLE 7. Conjugation-deficient ultraviolet light-sensitive mutantsa

Transcorjugant formation Union formation

Strain SelectionPro+ Hfr Pro Thr+ F390 Lac+ F-Iac+ TcR R64 Hfr R64-11

F'ORF4 11X833 Parent 1.00 1.00 1.00 1.00 1.00 70 16JF35 AmpRCon+ 0.80 0.62 0.76 0.42 0.63 58 8JF16 FosR 0.26 0.08 0.16 <0.01 1.38 57 24JF47 AmpR 0.01 0.09 <0.01 0.03 0.63 20 8JF49 AmpR 0.02 0.08 <0.01 0.10 0.63 80 14JF75 Sug- 0.32 0.08 0.12 0.75 0.50 66 16

a Refer to footnotes of Table 3.


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DISCUSSIONThe isolation of the various classes of conju-

gation-deficient mutants demonstrates that re-cipient cells have specific roles in conjugation.In addition, the separation of mutants isolatedby the same selection technique into classeswith different functional defects demonstratesthat the procedures for mutant isolation did notresult in recovery ofone phenotype. This classi-fication is tentative and preliminary and servesonly to reduce the number of strains with whichprimary mapping experiments will be done.Two provisions must be made. Although themutants are conjugation defective and alteredin another function (AmpR, FosR, Ala-, etc.),this does not prove that the two altered func-tions have a single defect common to two func-tions. The mutants could have arisen throughdouble linked mutational events especiallycommon with NTG (25). In addition, the mu-tants display pleiotropic deficiencies that couldbe due to single or multiple mutations. Becausethese mutants were selected on the basis ofdefects in cell wall or cell membrane functions,it is not surprising that many characteristics ofthe parent strain are different in the mutants.The data represent comparisons made betweenmutant and parent grown under conditions tominimize the differences, not having a specificrole in conjugation, that could possibly contrib-ute to transconjugant yield. Under differentconditions these mutants might not be Con-.Certain strains cannot express a lac+ gene and,thus, Lac+ transconjugant formation cannot beused as a measure of conjugation proficiency.Some of the Con- mutants do not grow asrapidly as X833 and, in experiments (20 strainsdone simultaneously) for which the data arereported here, the ratios of donor to recipientfor mutants were different from those of theparent x833. Rather than a ratio of 1 donor per10 recipients for x833, the ratio of donor tomutant was closer to 1:1. To rule out the possi-bility that this contributed to conjugation defi-ciency, the effect of different ratios of donor torecipient upon transconjugant yield for crossesbetween X503 and X833 was measured. Over arange of donor/recipient ratios of 0.05:1 to 3.0:1,the recombination frequency of Pro+ [StrR]transconjugants changes by a factor of 4. Re-combination frequencies were highest at lowdonor/recipient ratios (4x). Because the conju-gation deficiency for the slow-growing mutants(strains JF39, JF41, JF51, JF59, and JF71)were less than 25% of that exhibited by wild-type recipients, we conclude that differences inconjugation proficiency associated with slow

growth or changes in donor/recipient ratios didnot result in inclusion of Con+ strains amongthe mutants reported here.Because the mutation(s) responsible for the

conjugation-deficient phenotype affects the fit-ness of the strains (Table 2), it is not surprisingthat Con' revertants appear in storage cul-tures. Although we have not measured the fre-quency of Con+ revertants, it is our experiencethat they appear at appreciable frequencies,requiring isolation and growth and storage un-der controlled conditions to make comparisonsvalid.

Bacterial conjugation requires both donorand recipient cell functions to achieve genetransmission by cell-to-cell pairing. The con-cept that membranes contain structural or or-ganizational proteins and catalytic proteinsthat function during conjugation might be dem-onstrated in a rigorous fashion by using theconjugation-deficient mutants reported in thestudy. Presently we are using the collection ofmutants to separate and identify the processesof specific- and effective-union formation andDNA transfer. Analysis of the establishmentof plasmid DNA in the mutants deficient inplasmid inheritance will demonstrate someof the sequence of events required for plasmidinheritance and their temporal order. Thesepostulated steps are: association of the linear,single-stranded plasmid DNA to the inner cellmembrane; conversion of the single-strandedDNA to a linear, double-stranded form; andcircularization of plasmid DNA.Further work is proposed to identify the

membrane and cell wall proteins altered in themutants and to ascertain the mechanism of thedual roles of these proteins in conjugation andeither alanine metabolism, ampicillin resist-ance, fosfomycin resistance, and carbohydratecatabolism.

ACKNOWLEDGMENTS

This work was supported by National Science Founda-tion grant GB-37546 to Roy Curtiss III and by funds fromthe Department of Biology, Virginia Polytechnic Instituteand State University. Part of the work was undertakenduring the tenure of a University of Alabama Medical Cen-ter Postdoctoral Fellowship from the Department of Micro-biology.We would particularly like to thank John Lancaster for

his stimulating discussions during the early portions of thiswork.

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