Isolation and characterization of conjugation-deficient mutants of ...

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

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

Isolation and Characterization of Conjugation-Deficient Mutants of Escherichia coli K-12 JOSEPH 0. FALKINHAM III* AND ROY CURTISS III Department of Biology, Virginia Polytechnic Institute, Blacksburg, Virginia 24061,* and Department of Microbiology, University of Alabama Medical Center, Birmingham, Alabama 35294 Received for publication 19 March 1976

Conjugation-deficient mutants (Con-) of Escherichia coli K-12 have been isolated by a variety of indirect selective techniques. Mutants with mutations conferring ampicillin resistance, fosfomycin resistance, an alanine requirement, and a failure to ferment a number of carbohydrates were selected because the impaired functions occur in association with cell wall and cell membrane defects. The integrity ofthese catalytic or structural elements is postulated to have a role in conjugation. The mutants could be divided into at least six general categories corresponding to their defectiveness in the following postulated recipient cell functions: (i) specific-union formation, (ii) effective-union formation, (iii) deoxyribonucleic acid transfer, (iv) plasmid establishment, (v) plasmid maintenance, and (vi) recombination. The availability of these mutants should contribute to the description of the molecular events involved in each of these conjugation steps and the elucidation of the genetic control over the inheritance of conjugationally transferred deoxyribonucleic acid. A description ofthe functional steps of recipi- some or plasmid mobilization) (16; Falkinham, ent cells in conjugation can be accomplished by unpublished data). either biophysical or genetic techniques. If the Specific-union formation between donor and recipient cell has specific roles during conjuga- recipient cells is mediated by donor pili (9), the tion, there must exist recipient genes that spec- synthesis of which is encoded by genes on conjuify and regulate these functional steps. Because gative plasmids (1, 33). Since it is the unique the stages of conjugation are not synchronous structure of the donor pilus tip that interacts (based upon the kinetics of donor marker trans- with the recipient cell surface to form a specific mission), characterization of the intermediate union (34), it follows that the recipient cell steps of gene transmission in populations of should have a specific receptor for the pilus tip cells is difficult. In contrast, mutants blocked at and that one class of Con- mutants should have specific stages of conjugation should demon- alterations in this receptor and therefore be strate intermediate steps unambiguously. Our defective in specific-union formation. The conversion of specific unions to effective approach, therefore, has been to isolate a large number of conjugation-deficient (Con-) mu- unions has been postulated to involve those tants by various techniques with the hope of events needed to establish a connection beobtaining mutants with defects in each stage of tween donor and recipient cells through which DNA can pass (15). Ou and Anderson (35) obconjugation. The maintenance of viable transconjugants is served that close mating pairs were more fertile not the only function of the recipient cell in in yielding recombinants than loose mating conjugation. Recipient cells have other specific pairs. Goldschmidt and Curtiss (unpublished roles in conjugational gene transmission (15). data) observed an exchange of the outer memThe functional roles are postulated as: (i) spe- brane A receptor protein as a consequence of cific-union formation, (ii) effective-union for- conjugation. These observations therefore sugmation, (iii) deoxyribonucleic acid (DNA) gest that close cell-cell contact with membrane transfer and (iv) donor and recipient DNA asso- fusion may be required for effective-union forciation resulting in recombination or (v) stable mation (20). Retraction of donor pili, which has inheritance of an extrachromosomal plasmid been shown to occur during donor phage infec(establishment and maintenance) (15). It is also tion in Pseudomonas aeruginosa (7, 8) and possible that a recipient cell structure and/or Escherichia coli (28, 32), could provide the product triggers the initiation of conjugational mechanism whereby donor and recipient cells transmission events in the donor cell (chromo- are brought together to establish effective un1194

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ions. It is possible that some of the donor transfer-deficient (Tra-) mutants (2) are both resistant to donor-specific phage and conjugation deficient (as donors) because they fail to retract their donor pili since mutants of P. aeruginosa whose pili fail to retract upon infection with pili-dependent phage are resistant to those phage (8). If effective-union formation requires a specific recipient cell function and/or surface receptor necessary for membrane fusion, it would be anticipated that another class of Conrecipient mutants would form specific unions but not undergo the transition to effective unions. Initiation of plasmid or chromosome transfer is controlled by plasmid genes but may be triggered by a product or structure of the recipient (16; Falkinham, unpublished data). Conjugational DNA replication to replace the strand of donor DNA being transferred to the recipient utilizes enzymes supplied by the donor (19) and may act as the sole driving force for transfer of short plasmids that are transferred in toto to DNA-deficient minicells (13, 24) and do not require any known recipient cell functions or activities (18). Transfer of long plasmid and chromosomal DNA, however, requires homologous pairing between the proximally transferred region of the donor DNA and the recipient chromosome (19, 36) and an expenditure of energy by the recipient that somehow regulates the rate of chromosome transfer (18). It would thus be expected that certain recipient Con- mutants should be defective with respect to transfer of long plasmids and chromosomal DNA. Some of these might contain deletion mutations in which that portion of the recipient chromosome corresponding to the leading end of the transferred donor DNA had been deleted. Recombination leading to inheritance of donor chromosomal information or inheritance of a transferred plasmid (establishment and maintenance) are recipient functions. Recombination and plasmid inheritance have been distinguished as functional processes by the characteristics of the recombination-deficient (Rec-) mutants of E. coli K-12 (10). These mutants, although unable to inherit DNA from Hfr donors, are proficient in the inheritance of plasmids. These Rec- mutants are probably recombination deficient because of a block after the synaptic-like association of donor and recipient DNA noted by Paul and Riley (36). Isolation and characterization of recipient Con- mutants might reveal some that are defective in synapsis or recombination functions other than those in the Rec- mutants already isolated (11). Synapsis-defective mutants may appear to be defective in the transfer of long plasmid and chromo-

CONJUGATION-DEFICIENT MUTANTS

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somal DNA, however. We would also expect Con- mutants that are specifically unable to inherit both long and short plasmids. These mutants might be defective in membrane attachment of the transferred single strand of plasmid DNA (39) or in some later stage of conjugational replication, resulting in the reformation of the circular plasmid molecule (23). Others might be normal in these initial establishment functions but be defective in maintenance of plasmids. Recipient Con- mutants (other than the Recmutants initially described by Clark and Margulies [12]) were first described by Monner et al. (31), who isolated high-level ampicillin-resistant (AmpR) mutants that displayed a Conphenotype. These mutants were presumably defective in union formation as are the Conmutants isolated more recently by Skurray et al. (40) and by Reiner (37). Skurray et al. (40) isolated their mutants on the basis of resistance to phage K3, whereas Reiner selected mutants resistant to phage ST-1. We describe recipient mutants in this report that were selected for defects in cell wall and inner and outer membrane structure and function and which have been found to display a Con- phenotype. These mutants have mutations that effect the various stages of conjugation and which, in many cases, result in pleiotropic phenotypes. Attempts to isolate Conmutants by selecting directly for their inability to undergo conjugation with a donor strain by killing the conjugation-proficient recipient cells that receive and express a donor character that is lethal for the partial zygote were unsuccessful. MATERIALS AND METHODS Bacterial strains and bacteriophages. The bacterial strains employed in this study are described in Table 1. Bacteriophages T7, T3, II, fl, and P1L4 came from the collection of R. Curtiss. Bacteriophages ST-1 and OW were the kind gifts of Albey M. Reiner and Hans G. Bowman, respectively. Media and chemicals. The synthetic media employed were ML (liquid) and MA (agar) (14). The media were supplemented with amino acids, purines, pyrimidines, and vitamins at optimal concentrations (18). Carbohydrates were added to a final concentration of 0.5%, and streptomycin sulfate was added to 100 Ag/ml, final concentration. Penassay broth (PB), Penassay broth agar (PBA), and Penassay agar (PA) (Difco) were employed as complete media and supplemented with 100 lug of L-alanine per ml for growth of the alanine-requiring mutants. N-methyl-N'-nitro-N'-nitrosoguanidine (NTG) was purchased from the Aldrich Chemical Co., Milwaukee, Wis. Fosfomycin was a gift of Merck, Sharp and Dohme, Rahway, N.J. Ampicillin was a gift of Bristol Laboratories, Syracuse, N.Y.

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TABLE 1. Bacterial strains Strain

X833

Mating type F-

X503 X573 X1301 x1009

Hfr OR21

X289 x489

FF-

X935

F-

F'ORF4 F'F42 R64-11

Genotype

thr-16 1acZ76 proC24 tsx-63 purE41 supE42 A- pyrF30 his-53 strA97 xyl-14 cycAl cycB2 Prototrophic supE42 X- 0-proC tax purE --. lac F 0-purE tsx proC lac FlAiac-purE supE42 XserA12 0-iac FlApro-lac supE42 X- cycAl R64-11 (drd Tc SmR)Ithr-1 ara-13 leu-6 azi-8 tonA2 lacYl minAk supE44 gal-6 X- minB2 strA135 maITi xyl-7 mtl-2 thi-1 Prototrophic supE42 Xleu-6 tonA2 lacYl tsx-i supE44 gal-6 X- his-1 recAl argG6 strA104 malTi xyl-7 mtl-2

Derivation/reference

42 21 5

X314 (18); X354 (18) 38 17 JC1553 (12)

metBI

thr-I ara-14 leu-6 proA2 iacYl tsx-33 supE44

galK2 X- his-4 recB21 strA31 xyl-5 mtl-i argE3 thi-i JF131 F'F390 O-thr leu pyrB F/leu recAl argG strA pyrB a Allele designations are those used by Coli Genetic Stock Center.

AB247 (27) JC182 x JF4 (22)

Conjugation methods. (i) Screening putative and the final bacterial concentration was 2 x 108/ml. Con- mutants for conjugation proficiency. The con- The mating culture and separate donor and recipijugation proficiency of isolated recipient clones was ent cultures were incubated at 37 C for 15 min. A normally tested by replica plating 6-h grown patches 0.10-ml volume of the mating culture was gently of such clones from PBA plates to selective plates added to 10 ml of 0.85% saline (filtered through a spread with a donor culture. With plates selective 0.22-pim pore diameter Millipore filter) and counted for transmission of Pro+ and spread either with X503 in a Coulter counter. To a second counting vial (Hfr) or X573 (primary F'), Cont recipient clones (containing 10 ml of 0.85% saline), 0.05 ml each of gave confluent patches of recombinant growth. Con- the separate donor and recipient cultures was added bacterial patches could be easily scored. and the total particles were counted. The percentage (ii) Quantitative measurement of conjugation of union formation reported in Tables 3, 4, 5, and 7 proficiency of Con- mutants. Methods for conjuga- was derived from the equation: tional crosses were those described by Curtiss et al. (18). The ratio of donor to recipient cells was 1:10 % union formation = and the length of mating was 60 min. The values mated donor + recipient count 100 [2(l unmated reported in Tables 3, 4, 5, and 7 are averages of at donor + recipient count/i least two determinations, with the individual determinations of transconjugant formation varying by Accuracy of this method is limited such that deless than a factor of two. The average recombinant creases in union formation to 10% or below are not frequencies for X833 with the five donors used in measurable. Some strains displayed better union formation than the X833 parent strain. conjugation are given in the footnote to Table 3. Ultraviolet light sensitivity. Colonies of strains to Chemotaxis measurements. Measurement of the ability of the Con- mutants and their parent to be tested for ultraviolet light sensitivity were inocudemonstrate chemotaxis to glucose and mannitol lated on PBA plates and incubated overnight. Repwas done by a modification of the method of Arm- lica plates were inoculated from this master and strong et al. (4) as suggested by G. Hazelbauer. A irradiated for various lengths of time under a Genloopful of a culture grown overnight in PB medium eral Electric germicidal lamp. Based upon overnight was applied to the center of the surface of a semi- growth after irradiation, patches could be scored as solid agar medium (MA) containing 0.25% agar and UVR or UV; upon comparison with recA- (x489), 50 puM carbohydrate. The diameter of the ring of recB- (X935), and rec+ (x833) controls. P1 transduction. Methods for transduction using growth after 48 h of incubation at 37 C was measured. Those Con- mutants demonstrating less than P1L4 phage are the same as those described by 10% the diameter of the parent were designated as Curtiss et al. (17). P1L4 was propagated on strain nonchemotactic. X289. The possibility was eliminated that failure to Union formation. Specific-union formation was undergo transduction was not through an inability measured by particle counting with a Coulter of P1L4 to grow or adsorb to the strains used in this counter (1; N. Achtman, personal communication). study by enumerating the number of infectious cenRecipient strains were grown in PB medium to log ters after adsorption of phage to bacteria. These phase with shaking and mixed with a donor strain experiments were done in conjunction with P1 (x503 or x1009) grown in PB medium to log phase transduction. After the adsorption period and the without shaking. The donor/recipient ratio was 1:1 removal of cells for plating on selective medium, the

CONJUGATION-DEFICIENT MUTANTS

VOL. 126, 1976 remaining culture was washed twice in buffered saline 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 tested for sensitivity to the donor-specific phage fl was streaked across another streak of the bacteriophage lysate. It was also found that clones picked with the broad end of a flat toothpick would provide an adequate inoculum. Bacteria and phage were streaked on eosin methylene blue agar base (EMB; Difco) plates containing 0.1% glucose, 0.5% NaCl, 0.015 M CaCl2, and 0.03 M MgSO4. In all cases, the lysates of phage equalled 1010 plaque-forming units per ml. (ii) Recipient-specific phage. The efficiency of plating of the phages II, qbW, T3, T7, and ST-1 was performed by plating dilutions of high-titer lysates with suspensions of the conjugation-deficient mutants and their parent on PBA plates. NTG mutagenesis. Mutagenesis by NTG followed the procedure outlined by Adelberg et al. (3). Survival of cells of strain X833 to the 60-min treatment with 50 jig of NTG per ml was 10%. This was the mutagenesis procedure used to isolate all the mutants reported in this study.

RESULTS

In presenting the results of the isolation and characterization of Con- mutants, it will be useful to describe first the rationale for and methods of mutant isolation and the phenotypic characteristics of the mutants. Following this discussion is presented the description of the various classes of Con- mutants grouped according to their postulated defect in conjugation. Isolation of glutamic acid- and alanine-requiring mutants. Glutamic acid- and alaninerequiring mutants of Bacillus subtilis are defective in transformation (41) probably through an inability to achieve full levels of competence. Although conjugation and transformation may not be strictly analogous processes, elements common to cell wall structure (as are glutamic acid and alanine) may function in both processes. Three alanine-requiring mutants (Ala-) and nine glutamic acid-requiring mutants of X833 have been isolated (Ala- mutants and nine GIu- mutants among 107 surviving cells). The three alanine-requiring mutants (JF23, JF24, and JF25) were found to be Con(L-alanine was added to media when measuring transconjugant formation proficiency). The mutants were recovered as survivors of NTG mutagenesis which formed small colonies on minimal media to which was added 5 ,ug of either Lalanine or L-glutamate per ml. Three of 234 small colonies growing on alanine-supplemented media proved to be alanine-requiring mutants and 9 of 200 small colonies growing on glutamate-supplemented media proved to be

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glutamate-requiring auxotrophs. The characteristics of the alanine-requiring mutants, other than their conjugation deficiency, will be reported (Falkinham and Curtiss, manuscript in preparation). Isolation of ampicillin-resistant mutants. Double mutants resistant to high levels of ampicillin (80 ug/ml) isolated from a mutant resistant to low levels of ampicillin (20 ,ug/ml) have been shown to be conjugation defective (31). These strains were demonstrated to have changes in cell wall lipopolysaccharide. Changes in lipopolysaccharide have been shown to affect the yield of transconjugants after conjugation in Salmonella typhimurium (44). With this in mind, we isolated mutants resistant to high levels of ampicillin on PA or PBA media from a mutant (JF35) that is resistant to low levels (20 ,ug/ml) of ampicillin (24 mutants among 107 cells surviving mutagenesis). Two percent of those resistant to high levels of ampicillin were characterized as being Con-. Unlike x833, strain JF35 is mucoid and its descendants (JF39, JF41, JF47, JF49, JF57, and JF58) are mucoid also. Strains JF51, JF52, and JF55, although descendants of JF35, are not mucoid. In addition, strains JF47 and 49 are ultraviolet light sensitive. Although isolated on PA medium containing 80 gg of ampicillin per ml, the Con- mutants are not resistant to this concentration when tested for growth in ampicillin-containing PB medium. Furthermore, strain JF35 is not as resistant to ampicillin in PB as on PA medium. Table 2 includes data on the maximum concentrations of ampicillin which permit growth in PB medium and several additional characteristics of the mutants. The reason for the discrepancy between antibiotic resistance in PB medium and that in PA medium has not been investigated. Isolation of fosfomycin-resistant mutants. Because of the necessity of maintaining the integrity of the recipient cell wall and membrane for proficient conjugational gene transmission, any alteration in those structures is expected to have pleiotropic effects. An example is the class of mutants refractory to colicin E2 which are also sensitive to ultraviolet light and are recombination deficient, form filaments, and demonstrate abortive growth of bacteriophage (26). Since the enzymes for the permeation and transport of carbohydrates are associated with the cell wall and membrane (6), some mutants defective in carbohydrate transport might also be conjugation deficient (Con-). Mutants postulated to be defective in the L-aglycerophosphate and glucose-6-phosphate transport systems (30) were isolated to determine whether changes in this system have ef-

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FALKINHAM AND CURTISS

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