Covert R Factors in fi+ R+ Strains of Bacteria

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1 JOURNAL OP BACreRIOLOOY, Feb. 1969, p Copyright 1969 American Society for Microbiology Vol. 97, No. 2 Printed In U.S.A. Covert R Factors in fi+ R+ Strains of Bacteria E. ROMERO1 AND ELINOR MEYNELL Medical Research Council Microbial Genetics Research Unit, Hammersmith Hospital, London W12, and Department of Microbiology, Lister Institute of Preventive Medicine, London SWI, England Received for publication 19 September 1968 The presence of an ft sex factor can be detected by propagation of the I-specific phage Ifl. By use of this method of detection, a high proportion of strains with fi+ R factors were shown also to carry an ft factor which was frequently a second R factor. In some doubly R+ strains, the fi+ and the fh factor were observed to be transferred independently at conjugation. Many bacterial characters, such as colicinogeny (9), antibiotic resistance (25), and production of a hemolysin (21) or an enterotoxin (22), are determined by genes carried on extrachromosomal genetic elements that are transmissible from one bacterium to another by conjugation. Two classes of sex factor responsible for conjugation have been recognized in the Enterobacteriaceae (11). The first, or F-like, class includes F itself (10) and determines the production of sex pili resembling the F pilus which acts as receptor for F-specific phages (2, 5). The second, or I-like, class determines a different kind of sex pilus typified by that detennined by the colicin factor Coll (16). This pilus does not adsorb F-specific phages but acts as receptor and confers sensitivity to I-specific phages (17). F-like sex factors can also be recognized, when they are introduced into an F+ cell, by the fi+ character (25), i.e., the ability to inhibit fertility by repressing F pilus formation. The repressor of I-like factors does not act on F, and these factors are therefore fi-. Since transmissible drug resistance due to R factors is now very common, one might well find R+ bacteria carrying more than one R factor. However, any R factor, whether fi+ or fi-, generally excludes another of the same type from a given strain (15). Doubly infected R+ strains would therefore be expected to carry one fi+ and one fi- factor, not two of the same kind. Thus, strains recorded as fi+ could also carry an fifactor without its being immediately apparent. It can, however, be detected by propagation of the filamentous I phage Ifl, which specifically attacks cells forming the I sex pili determined by fi factors (17). Strains with a maskedfi- factor proved to occur relatively frequently, and usually carried two R factors which were shown to coexist stably and often transfer independently at conjugation. MATERIALS AND METHODS Bacterial strains. The following derivatives of Escherichia coli K-12 were used: J5-3, pro- mer (4); J6-2 pro his trp- lac- (4); W945 thr- leu- Bl- lac- (13); and mutant derivatives of these resistant to high levels of streptomycin or to 50 pg of nalidixic acid per ml. Media. Oxoid Blood Agar Base was used for routine cultivation, for examination of antibiotic resistance with Oxoid Multodisks, and for tests for colicinogeny. L-agar (12) was used for preparing and titrating transducing stocks of phage Plkc. Minimal agar was the medium described by Tatum and Lederberg (24), except that it contained no asparagine and was solidified with 1.5% Davis (N.Z.) agar. Nutrient broth was either L-broth (12) or Oxoid Nutrient Broth no. 2. Stock cultures were stored on Dorset egg medium at room temperature. R factors. Those R factors which have not previously been described (11) were isolated from clinical specimens in northern Italy. R-factor transfer. The donor, freshly grown in broth to 2 X 108 to 5 X 108 bacteria/ml, and the recipient, grown overnight in static broth culture, were mixed, usually in a ratio of 1:10, and were held at 37 C for 20 min. The mixture was then blended to separate mating pairs, diluted, and plated on media selective for the donor and for the recipient strains. Either nutritional characters or the presence of 200 Ag of streptomycin per ml or 50,ug of nalidixic acid per ml was used to distinguish between them. R-factor transfer was measured by adding to the medium one or more of the antibiotics to which it conferred resistance. The rate of transfer was expressed as the number of resistant colonies of the recipient strain divided by the count of the R+ donor. Transduction. Stocks of phage Plkc were made on the R+ strains in overlays on L-agar supplemented with M CaCl2 and 0.2% glucose. Phage stocks were sterilized with chloroform and were controlled ' Present address; Istituto di Microbiologia, Universita di for bacterial sterility with each transduction. Transduction was performed by mixing phage and bacteria Pavia, Pavia, Italy. 780

2 VOL. 97, 1969 at concentrations of ca. 5 X 107/ml and 5 X 108/ml, respectively, in L-broth containing M CaC12. After 20 min, sodium citrate was added to a concentration of 1%, and the mixture was centrifuged to deposit the bacteria. These were afterwards suspended in fresh L-broth with citrate and plated on the selective medium in an overlay containing citrate. Strains which had received an R factor by transduction or conjugational transfer were purified by two single-colony isolations on the same medium and were examined with Oxoid Multodisks for their resistances. To determine the fi character, the R factor was transferred to an F+ strain, which was then tested for visible lysis by phage MS2 on nutrient agar. The presence of F in a strain, as opposed to its expression, was detected by restriction of phage T3 (19), and transduction of R factors from F+ strains was shown to be independent of F by the failure of the transductants to restrict T3. Donor-specific phages. The F-specific isometric ribonucleic acid phage MS2 (6) and the I-specific filamentous deoxyribonucleic acid phage Ifl (17) were used to detect F-like and I-like pili, respectively. Wild-type factors were tested by measuring the ability of the bacteria to propagate the phage in broth; derepressed factors, by the presence or absence of visible lysis in lawns on nutrient agar. Colicinogeny. Colicinogeny was detected by standard methods (9), with strain J5-3 and a mutant specifically resistant to colicin I as indicators. RESULTS The presence of two sex factors first became apparent during the examination of cultures of E. coli K-12 which had acquiredfi+ R factors from naturally occurring strains of E. coli and Salmonella sp. In 7 of 43 (16.3%) and 9 of 22 (41%) instances, respectively, the strains propagated the I-specific phage I11, indicating that they had received an fi- sex factor in addition to the F-like factor responsible for thefi+ character. This could reflect the presence of an fi+ and an fi- R factor, or, alternatively, of an fi+ R factor plus a Coll factor or some other transmissible I-like plasmid which perhaps carried no marker. Accordingly, eight of the exceptional strains were chosen for detailed analysis on the basis of differences in the resistances transferred (Table 1) or of failure to confer sensitivity to F-specific phage MS2 (11). These strains are referred to by their original numbers (538, 522, 114, 541, 62, 547, and 196), given before their composite nature became apparent. Unlinked plasmids can be separated in two COVERT fi- R FACTORS ways: they can be separated at conjugation, or they can be separated by transduction by phage P1, since this phage generally transmits a plasmid in its entirety (25) and, at the same time, transfers several markers jointly only when they are linked (12). The latter was confirmed here by the separation of R factors from F when an F+R+ strain was used in preparing transducing stocks of phage. With strains 538, 522, and 114, conjugation led to transfer of different sets of resistance determinants, depending on which was selected. (See Table 1 for explanation of symbols for resistance determinants.) Thus, 538 and 522 could each be separated into two linkage groups, Cm Sm Su and Tc Sm, and 114 could be separated into Tc and Sm Su (Table 1). In each case, the first group mentioned was an fi+ R factor which allowed an F- host to propagate F-specific, but not I-specific, phage, and the second was an fi- R factor which gave an increase of I-specific, but not of F-specific, phage; the fi- R factor in 522 also determined the production of colicin I (Table 2). With strains 73 and 541, however, all the resistance determinants were transferred with selection for Cm and Tc, respectively, whereas selection for Sm apparently led to segregation; transduction showed these anomalous patterns of resistance transfer to be due to the presence of two R factors, one of which conferred all the resistance determinants of the other which therefore possessed no markers by which it could be separately identified (Table 3). With the remaining three strains, 62, 547, and 196, the resistance markers again could not be separated by conjuga- TABLE 1. Transfer of resistance by conjugationa R Resistances tion for Gives Cm Tc Sm Su Cm Cm Sm Su Tc Tc Sm Sm Cm Sm Su or, rarely, Tc Sm 522 Cm Tc Sm Su Cm Cm Sm Su Tc Tc Sm Sm Cm Sm Su or Tc Sm 114 Tc Sm Su Tc Tc Sm Sm Su 73 Tc Sm Su Tc Tc Sm Su Sm Tc Sm Su or Sm Su 541 Cm Sm Su Cm Cm Sm Su Sm Cm Sm Su or Sm 62 Tc Ap Sm Su Tc Tc Ap Sm Su Sm Tc Ap Sm Su 547 Km Sm Km Km Sm Sm Km Sm 196 Cm Tc Sm Su Cm Cm Tc Sm Su Tc Cm Tc Sm Su Sm Cm Tc Sm Su Su Cm Tc Sm Su a Symbols: Cm, chloramphenicol; Tc, tetracycline; Sm, streptomycin; Km, karamycin; Ap, ampicillin; Su, sulphonamides.

3 782 7ROMERO AND MEYNELL J. BACTLqOL. tion (Table 1); nevertheless, transduction yielded two sex factors from 62, each with the same resistance determinants (Table 3), and showed that 547 and 196 each contained one R factor and a second sex factor without any identifying marker (Tables 3-5). R538, R522, and R114. Characteristics of these factors are shown in Table 2. TABLE 2. Characters of538, 522, and 114, and their component fi+ andfi- R factors as separated in transfer by conjugation or by phage Plkc transduction Propaga- No. of tion of Frequency of transfer to transfi phage Coli- duc- R factor Resistances char- cino- tants acter geny fully MS2 Ifl R- R+ (2) R+ (1) exam- R538 Complex Cm Tc Sm Su by Cm, 1.8 X 12-' by Tc, 3 X 10-4 by Cm Tc, 5 X 10-1 Cm Sm Su X X Tc Sm _ X X 10-4 R522 Complex Cm Tc Sm Su (I) by Cm, 1.4 X 12-' by Tc, 1.1 X 10-2 by Cm Tc, 2.2 X 10O4 1 Cm Sm Su X X Tc Sm (I) 2 X X 1-1 R114 Complex Tc Sm Su by Tc, I X 10-' by Sm, 9 X 10- by Tc Sm, 1.6 X Tc + + _ 6 X X Sm Su _ X lo- 5 X 10-2 a Several more were also tested for a few individual characters. TABLE 3. Characters of 73, 541, 62, and 547, and their component factors as separated by phage Plkc transduction Propagation No. of R factor Resistances Colicino 'ny char fi - of phage by Transfer conju- Frequency of transfer ductants transgeny acter gation fully MS2 If 1 examineda R73 Complex Tc Sm Su X 10-3 (Tc or Sm) 1 Tc Sm Su X 10-' (Tc or Sm) 4 2 Sm Su - _ X 10-' (Sm) 6 3 Tc _ 5 R541 Complex Cm Sm Su + (I) Cm Sm Su _ + + b + 1 X 10-2 (Cm or Sm) 2 Sm + (I) _ X 10-2 R62 Complex Tc Ap Sm Su + (I) X lo- 1 Tc Ap Sm Su + (I) X lo- 3 2 Tc Ap Sm Su + 7 X 10-' 2 R547 Complex Km Sm + (I) X 1-' (Km) Km Sm + (I) X 10-4 (Km) 2 a Several more were also tested for one or more individual characters. b I-like sex factor frequently cotransferred.

4 VOL. VO.7,19COVERT 97, 1969 fi R FACITORS 783 R73. R73 yielded three classes of transductant (Table 3), fi+ Tc Sm Su, fi- Sm Su, and a third with Tc only which could neither transmit its resistance by conjugation nor propagate phages MS2 or Ifl. The nature of these Tc transductants, which constituted about 8% of the total, is not clear, because segregants with Tc alone were never observed in transfer by conjugation. Perhaps 73 included a third, nontransmissible plasmid carrying only Tc; or possibly the solitary Tc determinant was ordinarily part of the fi+ factor, Tc Sm Su, but could occasionally be isolated in transduction by phage P1 in the same way that R factors are commonly transduced only in part by phage P22 (7, 25). R541. R541 contained an fi+ plasmid, Cm Sm Su, and an fi-, Sm (Table 3). R62. As shown in Table 3, R62, like a minority of other fi+ factors, did not lead to propagation of phage MS2 by the R+ bacteria (11). The negative result cannot have been due to exceptionally severe repression of pilus formation, in view of the high rates of transfer observed. It yielded two kinds of transductant: one coli+ and giving an increase of phage Ifl, and the other col- and phage Ifl-insensitive. Surprisingly, it was the coli+ factor determining I-like sex pili which was fi+, whereas the sex factor of the cot transductants appeared to be neither I-like nor fi+. These plasmids may therefore contain the reassortment of typical F-like and I-like characters envisaged previously (15). R547. R547 had an fi+ plasmid, not associated with any resistance determinants, which was recognized by the propagation of phage MS2 (Table 3). Therefore, although the strain was at first thought to carry an fi+ R factor, it was, in fact, the fi- factor and not the fi+ which was responsible for the R+ character of the bacteria. R196. R196 carried Cm Tc Sm Su, but these could not be separated by conjugation (Table 1), TABLE 4. and, whichever was selected, two sex factors were always transferred, one F-like and fi+ and the other I-like (Table 4). Unfortunately, transduction could not be used because the bacteria restricted phage Pl to such an extent (1) that stocks could not be prepared. Other methods were therefore needed. In crosses with R196 and another fi- R factor, R144 (KmColI), the results differed greatly according to whether it was the donor or the recipient that carried R196; a recipient carrying R144 accepted all the resistance markers of R196 as efficiently as an R- strain, but in the reverse cross R144 was donated at only % of the frequency found with an R- recipient. Thus, R144 was evidently excluded by the fi- component of 196, whereas the ready acceptance by strains carrying R144 of all the resistances of R196 implied that they were linked to its fi+ component. Restriction of phage P1 by R196 was not determined by the fi+ element, for with transfer to a recipient carrying R144, the P1 restriction disappeared. The fi+ component was finally separated from R144 in a cross in which the donor carrying both plasmids was used and selection was made for transfer of Cm without Km and colicinogeny (Table 4). Attempts to isolate the fi- component of R196 were unsuccessful, for when R196 was transferred to a recipient carrying the fi+ factor Rl (Km), recombinant fi+ R factors were produced with all or some of the resistances of the two factors in various combinations (Table 5). In addition, the fi- sex factor was demonstrated in two clones (Km Cm Tc Sm Su) which propagated both phage MS2 and phage Ifl. During these transfers, restriction of phage P1 again disappeared, so that phage could be grown on one of these two clones Transfer of196 to an R- recipient and isolation ofits fi+ component by transfer to a strain already carrying an fi- R factor to test for linkage between the fi+ and fi- factors. All of the transductants received the entire resistance factor (Km Cm Tc Sm Su), but both of two propagated phage MS2 and not Ifl. How- Propaga- Cofi- Phage tion of Determination Selection Resistances Cio- P1 phae Probabl geytion 1152 IfI R196 Cm Tc Sm Su _ fi+ 196 fi- Transfer to an R- recipient Cm, Sm, Tc, or Su Cm Tc Sm Su fi+ 196 fi Transfer to a host with Cm Km Cm Tc Sm Su Km fi+ R144 (Km) fi 144 fi- Transfer from the double Cm+ Km- col- Cm Tc Sm Su _ fi+ R+ host to an R- strain

5 784 ROMERO AND MEYNELL J. BAarmoL. TABLE 5. Transfer of196 to a recipient carrying an fi+ R factor Propaga- Phage tion of Determination Selection Resistances Pi phage Probable factors restric tion MS2 if Cm Tc Sm Su fi+, 196fl- Transfer to a host Cm Km, Sm Unstable combinations Rl/R196 recombiwith Rl (Km) Km, Tc Km, segregating to give: nant, 196 f or Su Km 28% (20/72) Km Cm Tc Sm Sua 200% (14/72) Km Su 14% (10/72) Km 12% (9/72) Km Cm Sm Su 26% other combinations Transfer from a colony to an R- strain By conjugation Cm Km Cm Tc Sm Su R1/R196 recombinant By P1 transduc- Cm Km Cm Tc Sm Su Rl/R196 recombition nant Refers to "Transfer from a colony to an R- strain" (column 1). ever, this clone did not behave like the original R196+ bacteria in conjugation, for resistance transfer was now accompanied only by the fi+ factor, as again shown by the ability of both of two recipients tested to propagate phage MS2 but not phage Ifl. It may be concluded that all the resistance determinants of R196 were linked to its fi+ sex factor, whose transfer was habitually accompanied by an fi- factor unless this was specifically excluded by the presence of another fi- sex factor in the recipient. When the recipient carried an fi+ R factor, selection for the combined resistance of the two led to the production of a recombinant fi+ R factor, unlinked to, and now transferred independently of, 196 fi-. The combined action of R196fi+ andfji restricted phage P1, but the location of the genes concerned was not apparent, for restriction disappeared, both with loss of the fisex factor and with the formation of the recombinant R factor derived from R196 fi+ and Rl fi+. Superinfection immunity and stability in doubly R+ strains. Superinfection immunity was tested with strains 538, 522, and 114. There was no exclusion of thefi+ or theffi by the accompanying R factor, for the rate of transmission of either factor was not affected by the presence of the other in the recipient (Table 2). Stability was also tested with 538, 522, and 114, growing exponentially in broth. No segregants lost either the fi+ or the fl R factor (as measured by loss of either Cm or Tc from 538 and 522, or of Tc or Sm from 114), judging from several thousandcolonies from broth cultures which had been grown for over 30 generations. These cultures had been kept at a concentration below 5 x 106 bacteria/ml by repeated dilution to avoid cell contacts, because these might lead to cross-transmission of either factor and consequent reinfection of a segregant, which might be mistaken for stable inheritance. Although 114 was inherited stably under these conditions, a number of stock cultures were found to contain only the fi+ or the fi- factor after storage on dorset egg medium, a treatment recognized as leading to loss of plasmids or prophage (8). Joint transfer of two sex factors. The transfer by a transmissible plasmid of another lacking sex-factor activity cannot be a simple consequence of conjugation, for the frequency of joint transfer depends on the particular pair of transmissible and nontransmissible plasmids involved (3, 23). In the present case, where the donors carried a pair of transmissible plasmids, these might or might not be transferred together, again depending on whether they interacted specifically with each other. Conjugation is ordinarily repressed with wild-type factors, and only 102 to 104 of the bacteria produce sex pili at any one time; therefore, unless the individual host bacterium is exceptional in allowing expression of conjugation, there will only be a small probability that both conjugation systems will operate simultaneously. In 538, thefi- R factor was transferred about 20 times less frequently than the fi+, and the fre-

6 VOL. 97, 1969 COVERT fi- R FACTORS 785 quencies of transfer of each were only slightly, if at all, increased by the presence of the other in the donor (Tables 2 and 6). Moreover, simultaneous transfer of Cm and Tc, with selection on agar containing both chloramphenicol and tetracycline, was extremely rare, as though each factor was almost always transmitted independently of the other, and thus used only the transfer mechanism which it itself determined. This could be more precisely investigated by use of mutant R factors in which conjugation was derepressed; derepressed mutants of both fi+ andfi- factors of 538 were therefore isolated by replica-plating. Rates of resistance transfer with these mutants and the wildtype factors, separately and in combination, are given in Table 6 and show that the rate for fi+ and fi- together was almost equal to the product of their individual rates. Thus, when one of the factors was derepressed, the joint rate was limited by the transfer rate of the repressed factor. In some experiments, transfer of one factor or the other did appear to be slightly increased by the presence of the other, but the irregularity with which this occurred suggested that it might be a nonspecific effect due to persistence of cell clumps formed in the initial mating. Rates of transfer could be determined for 522 in the same way as for 538, with selection for Cm for the fi+ and for the Tc for the fi-, and again the two factors generally appeared to be transferred individually (Table 2). On the other hand, the fi+ and fi- factors of 114, although readily separated from each other, seemed to be transferred together rather more frequently than if each was completely independent; although the rate of joint transfer was less than that for each factor alone, it was not so small as the product of the individual rates (Table 2). A number of recipients of both Donor carrying Wild fi+ Wild fi+, Wild fi Wild fi+, drd fi- Wild ft Wild fi-, drd fi+ drd fi+ drd f drd fi+, drd fi- Sm and Tc were tested and were found to propagate both phage MS2 and phage Ifl, showing that they had, in fact, received the two factors and that transfer of both resistances was therefore not due to occasional recombination between the R factors in the donor. With 541, 73, and 62, it was not practicable to measure conjugational transfer of the component R factors in this way, since the fi- factor did not have a resistance determinant which distinguished it from the fi+. No resistance determinants were identified on either the fi+ sex factor of 547 or the f- sex factor of 196. The latter, although evidently not part of the same linkage group as R196fi+, was nevertheless regularly transferred in association with it. DISCUSSION Sixteen of sixty-five fi+ R factors present in E. coli K-12 after growth overnight in mixed culture with wild strains of Escherichia or Salmonella concealed the additional presence of an fi- sex factor determining an I-like sex pilus and susceptibility to I-specific phage. The real incidence of doubly R+ strains in nature is likely to be higher than the figure obtained here, which is simply that for double transfer in the initial screening; indeed, many of the wild R+ donors had more resistances than they donated (Romero, unpublished data). One of the constituent factors in 522, 541, 547, and 62 determiined the production of colicin I, and the association of colicinogeny with drug resistance in phage P1 transductants showed that the colicin determinant was an integral part of the R factor. R factors that also determine colicin I production are not uncommon (15, 20) and may constitute even as many as 30 to 40% of all fi- TABLE 6. Independent transfer offi+ and fi components of R538 Wildfi+ 2.3 X l0' 8 X 10r' 1.8 X 102 Wild fi- 3 X X 10-6 X 1O4 Wile.fi+, wildfi- 5 X 10' Frequency of transfer of drd fi 5 X X 1O1 5 X 10r drdf 3.3 X X 10-2 X 10r drd f+, wild.fi- 3.1 X 104 Wild fi+, drd.fi- 3.3 X 10- drdfi+, drd fi 8 X 1O--'

7 786 ROMERO AND MEYNELL J. BAcTERJoL. R factors isolated (E. Meynell, unpublished data), which would suggest that they may have been formed by linkage of resistance determinants with Coll. In several strains, both R factors determined resistance to the same drug, but whether their genetic determinants were identical is open to question. In 538, 522, 73, and 62, both factors carried Sm, but the level of resistance did not differ; the same applied to the Su markers of 73 and 62. The presence of two different Sm (or Su) determinants might have resulted from superinfection of an Sm+ (or Su+) strain; indeed, when an R factor confers a particular resistance such as Sm, there is no certainty that it carries only a single Sm determinant. A second Sm gene will not be directly selected unless it produces a sizable increase in resistance, either by virtue of its own activity or by acting synergistically with the other Sm determinant already in the cell. The degree of streptomycin resistance conferred by R factors is generally low, but when a bacterium carries an R factor with Sm, in addition to a chromosomal mutation of the sort also conferring only a low level of streptomycin-resistance, its resistance is increased to a disproportionately high degree (18). A comparable enhancement of resistance does not occur with the combined presence of two R-factor Sm genes; these, on the contrary, continue to give a degree of resistance little if at all greater than that conferred by either R factor alone (E. Meynell, unpublished data). Conjugational transfer of the fi+ and fi- R factors of 538 or 522 occurred almost independently of one another, indicating that each factor was transmitted virtually exclusively by its own mechanism for conjugation. This behavior is in striking contrast to the high rate at which some nontransmissible plasmids are transferred by sex factors to which they are unlinked, as in the transfer of ColE2 by ColI (3, 23). When a sex factor is repressed, its occasional spontaneous transfer is due to pilus formation by a small proportion of the bacterial population (14). The independent transfer of fi+ and fi- wild types in 538 and 522 shows that the rare conditions leading to expression of pilus genes and conjugation function are related specifically to the particular sex factor and not to a general change in the properties of an exceptional cell. LITERATURE CITED 1. Bannister, D., and S. W. Glover Restriction and modification of bacteriophages by R+ strains of Escherichia coil K-12. Biochem. Biophys. Res. Commun. 30: Brinton, C. C The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram negative bacteria. Trans. N.Y. Acad. Sci. 27: Clowes, R. C Transfert genetique des facteurs colicinogenes. Ann. Inst. Pasteur 107(Suppl.): Clowes, R. C., and D. 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