Identification of Serotypes of Sex Pili

Transcription

1 JOURNAL OF BACTERIOLOGY, Mar. 1972, p Copyright American Society for Microbiology Vol. 109, No. 3 Printed in U.S.A. Inhibition of Gene Transfer by Antiserum and Identification of Serotypes of Sex Pili VALERIE HARDEN AND ELINOR MEYNELL Guinness-Lister Research Unit and Department of Microbiology, Lister Institute of Preventive Medicine, Chelsea Bridge Road, London SWl W 8RH, England Received for publication 29 October 1971 Gene transfer by conjugation due to F or R (drug resistance) factors is inhibited by antibody to the sex pili. Serological analysis is able to distinguish between the sex pili determined by closely related sex factors, and the specificity of inhibition of transfer agrees with that previously determined by direct electron microscopical observation of antibody bound to the sex pili (10). Inhibition of transfer can therefore be applied to the identification of wild-type R factors with repressed sex factors that determine too few pili to be examined directly. It can also be used to differentiate the activities of two unrelated sex factors in the same donor bacterium. The ability of a bacterial sex factor to bring about gene transfer depends on the formation of sex pili by the donor cells (3, 16). Although the exact function of these pili is not yet understood, experiments with F show that gene transfer is prevented when they adsorb F-specific phage, whether filamentous phage which adsorbs to their tips or isometric phage which adsorbs along their length (8, 9, 21). Moreover, when two sex factors are present in the same bacterium, one determining an F-like and the other determining an I-like sex pilus, transfer of either factor is specifically blocked by the homologous filamentous phage (23). The present paper reports that gene transfer by conjugation is also inhibited by antibody to the pilus and that, because pili which are closely related are nevertheless distinguishable by their serological reactions (10), the degree of inhibition can be used to identify the sex pili determined by closely related sex factors. F phages are capable of adsorbing to most, if not all, naturally-occurring sex pili within the F- like class, with minor differences in affinity sometimes showing as a difference in efficiency of plating or morphology of plaques (20). The only I phages yet isolated (18) do not discriminate between sex pili in the I-like class. However, by cross-absorption of antisera within either class, monospecific sera can be produced which react only with a single serotype of sex pilus (10). These sera specifically block conjugation brought about by the corresponding sex factor MATERIALS AND METHODS Bacterial strains. All experiments were carried out with derivatives of Escherichia coli K-12. Strains J53 (pro met) (4) and W945 (thr leu thi lac pil) [reference 14; this strain has previously been referred to as RC22 (10)] were used as R factor or Flac donors. The recipients were streptomycin-resistant (str-r) or nalidixic acid-resistant (nal-r) mutants of strain J62 (pro his trp lac) (4) and strain X478 (pro leu trp met lys pure thi ara xyl lacz tsx str-r), kindly provided by Claire Berg. Strain x478, when plated on lactose minimal medium, produced none of the background of tiny colonies seen with lacy-z+ recipients, which obscure the Lac+ colonies when the proportion of bacteria that have received the lac+ gene is very small. Plasmids. The Flac factor was originally derived from strain 200 PC (7) and kindly provided by Pearl Cooper. The R factors are listed in Table 1. Antisera. Antisera are listed in Table 2. Their preparation and the method of absorption of bacterial and cross-reacting pilus antibodies were described previously (10). Absorption was checked by electron microscopy of the serological reaction (10), and the sera were also checked for sterility. Culture media. Nutrient broth was Oxoid nutrient broth no. 2 and nutrient agar (NA) was Oxoid blood-agar base. Minimal agar (MM) was as described by Tatum and Lederberg (19), appropriately supplemented to distinguish between donor and recipient bacterial strains. Streptomycin at 200 Ag/ml was (Sm200) or nalidixic acid at 20 jag/ml (Nal 20) also used to eliminate the donor strain when the recipient was resistant. R+ recipients were selected on chloramphenicol, 20 Ag/ml (Cm2O); kanamycin, 20 Ag/ml (Km2O); tetracycline, 10 gg/ml (Tc10); or streptomycin, the concentration being 10, 15 or 20

2 1068 HARDEN AND MEYNELL J. BACTERIOL. TABLE 1. R factors Sex factor Wild type Derepressed Serotypea F-like Ri (Km Cm Ap Sm Su)b Rldrdl9 Ri R52 (Tc Sm Su) R82 (Tc Sm Su) R192 (Cm Tc Sm Su) R192drdF7 R100 R502 (Cm Tc Ap Sm Su) R504 (Cm Tc Sm Su) R538Fc (Cm Sm Su) R538Fdrdlc R538Fc I-like R56 (Tc Su) R64 (Tc Sm Su) R64drdll R64 R92 (Sm Su) R144 (Km) R144drd3 ColIbd R183 (Tc) R5381C (Tc Sm) R538Idrdl' ColIbd Unidentified fi+ R27 (Tc) R128 (Tc Ap Su) fi- R46 (Tc Ap Sm Su) R199 (Tc Su) areference 10. I Resistances conferred: kanamycin (Km), chloramphenicol (Cm), ampicillin (Ap), tetracycline (Tc), streptomycin (Sm), sulfonamide (Su). c Previously (10) referred to as R538-1 and R538-ldrd, R538-2 and R538-2drd, respectively. dpreviously known as ColI-R144 serotype (10); reciprocal tests with cross-absorbed antisera have since shown that the two are identical. TABLE 2. Antisera Antiserum Produced against Rabbit no. Absorbed with designation To F-like factors F W945 (Flac) 265 W945 F- R- Ri W945 (Rldrdl9) 264 W945 F- R- R192 W945 (Rl92drdF7) 99 W945 F- R- To F serotype F/Ri - R192 W945 (Flac) 265 W945 (Rldrdl9) + W945 (Rl92drdF7) To Ri serotype Rl/F-R192 W945 (Rldrdl9) 264 W945 (Flac) + W945 (Rl92drdF7) To R100 serotype R192/F-R1 W945 (Rl92drdF7) 96 or 99 W945 (Flac) + W945 (Rldrdl9) To I-like factors R144 W945 (R144drd3) 172 W945 F- R- R538I W945 (R538Idrdl) 108 W945 F- R- To R64 serotype R64/R5381 W945 (R64drdll) 113 W945 (R538Idrdl) To ColIb serotypea R538I/R64 W945 (R538Idrdl) 108 W945 (R64drdll) a Previously known as ColI-R144 serotype (10); reciprocal tests with cross-absorbed antisera have since shown that the two are identical. Ag/ml (Sm1O, Sm15, or Sm20) depending on the R factor and on whether the medium was nutrient agar or minimal medium. Experimental procedure. The experimental procedure was designed to avoid as far as possible any effects of the antisera other than on conjugation and transfer. The antisera caused no killing of donor or recipient bacteria, as shown by viable counts on each strain. Sex pili can cause bacteria carrying derepressed R factors to form clumps, and light microscopy of live cultures showed the average size of the clumps to be increased in anti-pilus serum. This complication was minimized by using young donor cultures which had not yet reached a concentration where they were significantly clumped, as seen by microscopy, and by agitating the mixtures on a Rotamixer (Hook & Tucker Ltd.) at the beginning and end of the mating period. This method dispersed the

3 VOL. 109, 1972 GENE TRANSFER INHIBITION BY ANTISERUM bacteria adequately, since viable counts of control cultures taken before and after addition of antiserum agreed with one another within a factor of two in either direction. The donor used in each experiment was tested for its antibiotic resistances and its sensitivity to the appropriate donor-specific phage, MS2 or Ifl (16), and the growth phase of the culture ensured optimal donor ability (5). To avoid epidemic spread of the R factor through the recipient, the recipient culture was in the stationary phase and only a short time was allowed for mating (6). The procedure with R factors with derepressed sex factors and with Flac was as follows. An overnight broth culture of the donor strain was diluted 1: 100 in 25 ml of fresh broth in a 50-ml flask and incubated in a 37 C water bath for about 2.5 hr with shaking at 80 strokes/min. One-milliliter samples then were added to 2 ml of either broth or a dilution of antiserum in broth, and, after continued incubation for 20 min, these donor cultures were shaken on the Rotamixer and 0.5 ml of a stationary-phase culture of the recipient strain concentrated 10-fold by centrifugation was added to each. This ensured a recipient-to-donor ratio of at least 10. The mating mixtures were incubated for 20 min at 37 C; shaken on the Rotamixer; serially diluted; plated on media selective for the donor strain, the recipient strain, and bacteria of this strain which had received the R, or Flac, factor; and then incubated at 37 C. Colonies on nutrient agar were counted after 24 hr and on minimal agar after 48 hr. The frequency of transfer is expressed as the number of R+ (or lac+) recipient colonies divided by the number of donor colonies, and transfer in the presence of antiserum is expressed as a percentage of that in its absence. A simplified test was used to examine R factors with wild-type sex factors which are transferred at 1% or less of the rate of the derepressed mutants. Here, 0.2-ml volumes of the donor cultures were mixed with 0.2-ml volumes of broth or antiserum dilution in the depressions of plastic hemagglutination trays and then, 20 min later, with 0.1 ml of 10- fold concentrated recipient culture. The mixtures were incubated for 1 hr, after which serial dilutions were plated on the selective media, using dropping pipettes delivering 0.02 ml (19). RESULTS Relation of transfer inhibition to antiserum concentration. When R+ donor cultures were mixed with antiserum reacting with their sex pili, the frequency of R factor transfer was decreased. Table 3 shows that with homologous serotype antisera the extent of the decrease was related to the serum concentration. Although the frequency was not always exactly reproducible, there was generally good agreement between experiments. Table 3 also shows that the highest antiserum concentrations had disproportionately less effect. The residual transfers might have resulted from mating of the bacteria on the plates after the antibody was diluted beyond 1069 the level at which it was active and new pili had been formed. But, if that were so, few colonies might have been expected when nalidixic acid was used to eliminate the donor (2), since its effect on R factor transfer, although less than on F (T. C. Salzman, unpublished data), is still considerable. Mutant sex factors producing altered sex pili have been obtained by selection with phage (17, 24); another possibility, therefore, was the occurrence of mutant sex factors determining sex pili with less affinity for the antibody. To test this, two isolates of Rldrdl9 and two of R538Idrdl which had been transferred from J53 nal-r in the presence of 1:20 homologous antiserum were returned to J53, and their transfer to J62 once again was compared in the presence and absence of the same antisera. There was no appreciable difference from the original tests: a 1:30 dilution of Rl antiserum decreased the frequencies of transfer of the two Rldrdl9 isolates to 0.89 and 1.14%, and the equivalent figures for the two R538Idrdl isolates with 1:30 R5381 antiserum were and 0.015%. The possibility that the residual transfers were due to noninherited differences in a fraction of the sex pili was not, however, excluded. Bacteria and culture media. An experiment on the effect of altering the strains used as donors or as recipients, or the medium used for isolating R+ recipients, is shown in Table 4. A relatively low frequency of transfer, with an exceptionally marked reduction by antiserum, was the rule when strain X478 was the recipient, particularly with F-like factors. Only small differences can be observed in this experiment between J53 and W945 as donors and between NA and MM as plating media, and these were not regularly found. Specificity of the reaction. Table 5 shows the specificity of inhibition as between the two major classes, F-like and I-like, whose pili show no cross-reactions when antibody binding is examined directly (10). Used even at 1:20, the highest concentration tested, antiserum to F-like pili never altered the frequency of transfer of any I-like factor. This was so whether or not antiserum to the I-like pili was also present. Equally, antiserum to I-like pili had no effect on transfer of any F-like factor. Specificities within each major class are shown in Tables 6 and 7. Although the pili within each class are antigenically related, cross-absorbed antisera can be prepared which react visibly with some pili but not with others. Thus, cross-absorbed antisera distinguish within the F-like class between the pili determined by F, Rl, and R192 and within the I-like class between those determined by R64

4 1070 HARDEN AND MEYNELL J. BACTERIOL. TABLE 3. Inhibition of R factor transfer by antiserum to the sex pili Donor Recipient Selected on. Concen- R factor transfer strain R factor Dostrain cstrain strainna NSe Antiserum plusn tration ~~~~~~~~~~Frequency % Rldrdl9 J53 J62 str-r Sm 200 Cm x RI 1: x Ri 1: x Ri 1: x x RI 1: x Ri 1: x Ri 1: x 10-s 0.8 R144drd3 W945 J62 nal-r Nal 20 Km x R538I 1: x R538I 1: x R538I 1: x R538Idrdl J53 J62 nal-r Nal 20 Sm x R538I 1: x R5381 1: x R5381 1: x a Abbreviations: NA, nutrient agar; Sm, streptomycin; Cm, chloramphenicol; Nal, nalidixic acid; Km, kanamycin. Numbers following abbreviations refer to concentrations in micrograms per milliliter. TABLE 4. Inhibition of R factor transfer by antiserum: alteration of donor and recipient bacteria and of selective media Recipient Selective medium Antiserum R factor transfer R factor Donor strain strain (+ Sm 200)a (1:60) Freuency (str-r)frqec Rldrdl9 (+ Cm 20) W945 J62 NA None 1.6 x Ri 7.0 x J53 J62 NA None 1.2 x Ri 3.0 x J NA None 7.4 x 10-l 100 Ri 6.2 x J53 J62 MMb None 1.0 x RI 3.0 x R144drd3 (+ Km 20) J53 J62 NA None 3.0 x R x W945 J62 NA None 1.7 x R x W NA None 1.6 x R x W945 J62 MMb None 8.5 x R x a Abbreviations: NA, nutrient agar; MM, minimal agar; Sm, streptomycin; kanamycin. Numbers refer to concentrations in micrograms per milliliter. O Supplemented to allow growth of J62 but not J53. Cm, chloramphenicol; Km, and all the remaining derepressed factors so far examined (10). Table 6, with F-like factors, and Table 7, with I-like factors, show that the ability of cross-absorbed antisera to inhibit transfer of particular factors agrees with their specificity as previously determined by electron microscopy of antibody attached to the corresponding sex pili. Wild-type repressed sex factors. With wild-type R factors whose sex factors are repressed, sex pili are only occasionally produced (16), and, because they are not sufficiently numerous to be examined directly, they have been allocated only to one or another of the major classes by the type of phage sensitivity which they confer on the bacterium (13). Table 8 shows the results of testing transfer of 17 wild-type R factors for inhibition

5 VOL. 109, 1972 GENE TRANSFER INHIBITION BY ANTISERUM 1071 TABLE 5. R factor Donor strain Recipient strain Specificity of serological reaction Antiserum (1: 60) R factor transfer F-like I-like Frequency % Rldrdl9 W945 J62 nal-r 7.0 x Ri 3.0 x R x Rl R538I 2.8 x R144drd3 W x 10' 100 F 1.0 x R x F R x TABLE 6. Specificity of reaction with cross-absorbed antisera in F-like class Antiserum (1: 20) Sex factor % Transfera F/R1 * R192 Flac 1.3 (F serotype) Rldrdl9 98 R192drdF7 84 R1/F*R192b Flac 19 (Rl serotype) Rldrdl9 0.2 R192drdF7 90 R192/F * Rlc Flac 47 (R192 serotype) Rldrdl9 50 R192drdF a Per cent of control without antiserum, the donor strain being W945 and the recipient strain 478. b Incompletely absorbed with F as seen by a reaction of degree + (10). c Rabbit no. 96. TABLE 7. Specificity of reaction with cross-absorbed antisera in I-like class Antiserum (1: 20) Sex factor % Transfera R64/R5381P (R64 serotype) R64drdl I R538Idrdl R5381/R64 R64drdll 70 (ColIb serotype) R538Idrdl 0.3 a Per cent of control without antiserum, the donor strain being J53 and the recipient, strain J62 nal-r. O Incompletely absorbed with R538I as seen by a reaction of degree - to + (10). by antisera of various specificities. The seven factors in the first section of the Table were known to belong to the F-like class, for all conferred sensitivity to phage MS2 and were fi+ (15). Rl, R192, and R538F corresponded to the derepressed mutants previously tested, and, when the same cross-absorbed antisera were used, each could readily be identified as falling into the same serotype as its derepressed mutant. Of the factors for which derepressed mutants have not yet been isolated, R502 could be allocated to the Rl serotype, and R82 to the R100 serotype. R52, and to an even greater extent R504, shared features of both the Rl and R192 serotypes, and perhaps also of F. For each R factor, transfer of several of its resistances was measured and since these were decreased to essentially the same extent, there was no evidence that more than one sex factor was concerned in the transfers. The second section of Table 8 shows the results with six wild-type R factors with I-like sex factors conferring sensitivity to phage Ifl (13). The relationship between serotypes is much closer in the I-like than in the F-like class, and cross-absorption considerably lowers the titer of an antiserum against the homologous factor, the decrease being more marked with antiserum to R538I than to R64 (10). This is reflected in the effect on transfer frequency, as can be seen by comparing the figures for unabsorbed sera in the first two columns with those for cross-absorbed sera in the second two. The reaction of R56, for which no derepressed mutant has been isolated, suggested that it belonged to the ColIb serotype. No other factor exactly resembled R64; R92 appeared as insensitive as R64 to the specific ColIb serotype antiserum but was also relatively insensitive to specific R64 antiserum. The remaining factor, R183, reacted to an intermediate degree with antisera to each serotype. The last two sections of Table 8 concern R factors which could not be allocated by phage reactions to either the F-like or the I-like class (13). R27 and R128, although fi+, could not be shown to lead to propagation of the F-specific phage MS2, but the results in Table 8 show that their pili are, in fact, F-like and belong to the R100 serotype; this serotype has a characteristically low affinity for F-specific phages (20) which therefore could explain the negative results of the phage tests. The fi- factors, R46

6 1072 HARDEN AND MEYNELL J. BACTERIOL. TABLE 8. Reactions of wild-type R factors with specific antisera % Transfer" in presence of antiserab R factor F/Rl1 R192 Ri/F R192 R192/F RlC (F serotype) s(r (Rlot [ serotype) serotype) F-like Rl R52 R82 R192 R502 R504 R538F I-like R56 R64 R92 R144 R183 R5381 Unknown fi+' R27 R128 Unknown fi-' R46 R < <1 16 <1 R64/R5381 R538I/R64 R64 R5381 (R64 (ColIb serotype) serotype) < < F/Ri * R192/ Pooled R192 R1/F * R192 FRi sero- serotype) serotype) type) F-like d (F (Rl (Rl00 like" R64/ R5381 R538I/R64 Pooled I-like (R64 (Collb Flk sero- serotype) F liked type) a Per cent of control without antiserum, the donor strain being J53 and the recipient, J62 nal-r. b 1:20. c Rabbit no. 99. d F, Rl, and R192. e R538I. tfi+ without propagation of phage MS2 (13). fi- but no propagation of phage Ifl, as with I-like factors (13). and R199, did not allow propagation of phage Ifl; their reactions, shown in Table 8, indicate that their pili are antigenically neither F-like nor I-like. Although only 1% or less of the numbers of pili are produced, it was perhaps remarkable that the concentrations of serum needed to produce a significant decrease in transfer frequency of repressed R factors were as high as with derepressed factors. What is more, a given concentration of serum appeared to have proportionately less effect on a wild-type repressed factor than on its derepressed mutant. The latter may have the same explanation as the observation with derepressed factors that alterations in the serum concentration had proportionately less effect at high serum concentrations where the frequencies of transfer were already low. If so, it supports the possibility that, under these conditions where the bacterial mixtures are plated at relatively high concentration, some of the R+ recipient colonies may be accounted for by transfers occurring on the plates. Donor bacteria carrying two sex factors. There is no bar to the stable coexistence of an F-like and an I-like R factor in the same bacterium, and, when both are de-repressed mutants, the bacterium produces both kinds of sex pili, shown by electron microscopy to be morphologically and antigenically distinct (12). Table 9 shows an experiment with strain J53 carrying both R538Fdrdl and R538Idrdl in which the bacteria were exposed (i) to Rl antiserum, which reacts strongly with the F-like pili of R538Fdrdl, and (ii) to R144 serum, which reacts strongly with the I-like pili of R538Idrdl. Transfer of R538Fdrdl was measured by transfer of Cm and of R538Idrdl by Tc, and, just as previously reported for these two R factors (22), when the recipient strain was greatly in excess of the donor and the frequencies of transfer were calculated in the usual way as number of R+ recipients divided by number of donors, the frequency of transfer of both factors was about equal to the product of the individual frequencies, as if each factor were transferred independently and were responsible for the act of conjugation leading to its own transfer. Transfer of R538Fdrdl was decreased by F- like antiserum to about the same extent as when it was the only sex factor in the donor, and was not appreciably affected by the I-like antiserum (Table 9). In the reciprocal test, the transfer of R538Idrdl was greatly decreased by the I-like antiserum; however, there was also a significant decrease with the F-like antiserum (Table 9). The reason for this is obscure. Electron microscopy does not show any evidence that the I-like pilus can incorporate occasional F-like antigenic subunits able to react with the antiserum (12). Nevertheless, if the alternative explanation that the decrease represented a corresponding proportion of R538Idrdl transfers occurring by the F-like pilus were true, the

7 VOL. 109, 1972 TABLE 9. Antiserum F-like0 IMlikec - GENE TRANSFER INHIBITION BY ANTISERUM Effects of specific antisera on donor bacteria carrying two unrelated R factors Transfer" from J53(R538Fdrdl) (R538Idrdl) of R538Fdrdld R5381drdle R538Fdrdl and RS38Idrdl' Frequency Frequency j Frequency % 1.4 x x 10-I x _ 1.1 x 10- ' x 10- ' x _ x x x x x x a To strain J62 nal-r. brl 1:60. CR144 1:60. d Selected with chloramphenicol, 20,g/ml. e Selected with tetracycline, 10 ug/ml. 'Selected with chloramphenicol (20,gg/ml)-tetracycline (10 Ag/ml). I-like antiserum should not have had the pronounced effect that it did. Neither should either antiserum alone have diminished transfer if this could occur by either kind of pilus indiscriminately. Thus, the transfer of both F-like and I-like factors depended predominantly on functional pili of the corresponding type. However, a minority of transfers of either sort could occur by pili of the opposite type, as shown by the greater decrease in the frequency of transfer with both antisera than with either the F-like or the I-like alone (Table 9). In this situation, transfer of the F-like or the I-like factor did not necessarily entail transfer of the other, as shown by the smaller figures for transfer of both factors than for either alone (Table 9). With figures as low as these, it is not possible to attach much significance to their actual values; nevertheless, the results in Table 9 are broadly representative of other experiments made with various pairs of F-like and I-like factors. DISCUSSION The assumption throughout this paper is that antibody to the sex pilus is responsible for inhibiting genetic transfer. The reasons are, firstly, that no antibody molecules are seen attached other than to sex pilus, once the bacterial antibodies have been removed from the serum by absorption with the strain used as bacterial host (10). Secondly, donor-specific phages whose sites of attachment are on the sex pili also block transfer (8, 9, 21, 23) although, here again, the same argument applies: that, although it is positively known that the phages attach to the pilus, there still remains an unlikely possibility that an occasional phage particle attaches to a different type of site on the bacterial surface, which is the critical one operating in conjugation. Fi nally, however, there are the conclusions to be drawn from the reactions of the cross-absorbed antisera, particularly those in the F-like class, where the presence or absence of ability to inhibit transfer is correlated with the presence or absence of antibody molecules attaching to the pilus, as seen by electron microscopy. If sex factors determining antigenically related sex pili also determined other antigens, which were those actually concemed in conjugation, these second antigens would have to bear the same degree of relationship to one another as the pilus antigens, and equivalent amounts of their antibodies and the pilus antibodies, in the case of each factor, would have to be present in its antiserum. The proportion of transfers that occurred in the presence of antiserum, and thus, by inference, the proportion of pili that were not so affected that the bacterium could no longer donate, was related to the concentration of the antiserum but scarcely, if at all, to the absolute numbers of pili. The concentrations of serum required with the wild-type repressed factors were as high as with their derepressed mutants, where at least a hundred times more pili were produced and correspondingly greater numbers of bacteria were able to act as donors. The situation thus formally resembles the neutralization of phage by concentrations of antibody that are not significantly depleted during the course of the reaction. As a result, whatever the actual phage concentration, the same proportion, whose value is govemed solely by the concentration of the antibody, is neutralized in a given time (1). Transfer was significantly inhibited by concentrations of antibody that did not completely coat the pili, as seen by electron microscopy. Thus, antibody need perhaps only block some critical site on the pilus; or perhaps the attachment of only a few molecules

8 1074 HARDEN AND MEYNELL J. BACTERIOL. anywhere on the pilus may prevent it from carrying out its required function. It is apparent that pilus antibody can affect the physiology of pilus production because incubation with antibody causes the appearance of large numbers of extruded pili on the bacteria (11). A practical problem in the isolation of R factors from natural sources lies in detecting recurrences of the same factor. For this purpose, a stable and relatively easily characterized plasmid marker is desirable, which is probably at present most readily provided by the type of sex pilus. The serological test in a simplified form has therefore been applied to sample numbers of wild-type R factors with repressed sex factors and has succeeded in revealing differences between them. The results with bacteria carrying two types of sex factor have reinforced the previous conclusion (22, 23) that each is mainly transferred by the type of pilus it determines. Nevertheless, the enhanced effect of the presence of both types of antisera suggested that a small proportion of transfers of either type could occur by means of the opposite type of pilus. The mechanism may be the same as that by which plasmids, which are in themselves nontransmissible, are transferred at conjugation mediated by second plasmid with sex factor activity (25). We cannot suggest any explanation for the remaining feature of these donor bacteria with two sex factors: the small, but significant, inhibition by F-like antiserum of transfer of the I-like factor. The inhibition cannot be the result of an antigenic relationship between the pili determined by the two types of factor, for F-like antiserum has no effect on donors carrying the I-like factor alone. Furthermore, electron microscope examination shows no evidence that I-like sex pili produced by bacteria also carrying an F- like sex factor contain any subunits of the F- like type (12). ADDENDUM IN PROOF Inhibition of F-mediated genetic transfer by F pilus antiserum has also been demonstrated by C. C. Brinton (Crit. Rev. Microbiol. 1: ). LITERATURE CITED 1. Andrewes, C. H., and W. J. Elford Observations on antiphage sera. L. "The percentage law." Brit. J. Exp. Pathol. 14: Bouck, N., and E. A. Adelberg Mechanism of action of nalidixic acid on conjugating bacteria. J. Bacteriol. 102: 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., and D. Rowley Some observations on linkage effects in genetic recombination in Escherichia coli K12. J. Gen. Microbiol. 11: Curtiss, R., L. G. Caro, D. P. Allison, and D. R. Stallions Early stages of conjugation in Escherichia coli. J. Bacteriol 100: Datta, N., A. M. Lawn, and E. Meynell The relationship of F type piliation and F phage sensitivity to drug resistance transfer in R+F- Escherichia coli K12. J. Gen. Microbiol. 45: Dubnau, E., and W. K. Maas Inhibition of replication of an F'lac episome in Hfr cells of Escherichia coli. J. Bacteriol. 95: Ippen, K. A., and R. C. Valentine 'Me sex hair of E. coli as sensory fiber, conjugation tube or mating arm? Biochem. Biophys. Res. Commun. 27: Knolle, P Evidence for the identity of the matingspecific site of male cells of Escherichia coli with the receptor site of an RNA phage. Biochem. Biophys. Res. Commun. 27: Lawn, A. M., and E. Meynell Serotypes of sex pili. J. Hyg. 68: Lawn, A. M., and E. Meynell Antibody-stimulated increase in sex pili in R+ enterobacteria. Nature (London) in press. 12. Lawn, A. M., E. Meynell, and M. Cooke Mixed infections with bacterial sex factors: sex pili of pure and mixed phenotype. Ann. Inst. Pasteur. 120: Lawn, A. M., E. Meynell, G. G. Meynell, and N. Datta Sex pili and the classification of sex factors in the Enterobacteriaceae. Nature (London) 216: Maccacaro, G. A., C. Colombo, and A. di Nardo Lo studio genetico delle fimbrie. Giom. Microbiol. 7: Meynell, E., and N. Datta The relation of resistance transfer factors to the F factor of Escherichia coli K12. Genet. Res. 7: Meynell, E., G. G. Meynell, and N. Datta Phylogenetic relationships of drug-resistance factors and other transmissible bacterial plasmids. Bacteriol. Rev. 32: Meynell, G. G., and E. Aufreiter Selection of mutant bacterial sex factors determining altered sex pili. J. Gen. Microbiol. 59: Meynell, G. G., and A. M. Lawn Filamentous phages specific for the I sex factor. Nature (London) 217: Meynell, G. G., and E. W. Meynell Theory and practice in experimental bacteriology, 2nd ed. Cambridge University Press. 20. Nishimura, Y., M. Ishibashi, E. Meynell, and Y. Hirota Specific piliation directed by a fertility factor and a resistance factor of Escherichia coli. J. Gen. Microbiol. 49: Novotny, D., W. S. Knight, and C. C. Brinton Inhibition of bacterial conjugation by ribonucleic acid and deoxyribonucleic acid male-specific bacteriophages. J. Bacteriol. 95: Romero, E., and'e. Meynell Covert fi- R factors in fi+ R+ strains of bacteria. J. Bacteriol. 97: Salzman, T. C Co-ordination of sex pili with their specifying R factors. Nature N. Biol. (London) 230: Silverman, P. M., H. W. Mobach, and R. C. Valentine Sex hair (F-pili) mutants of E. coli. Biochem. Biophys. Res. Commun. 27: Smith, S. M., H. Ozeki, and B. A. D. Stocker Transfer of ColEl and ColE2 during high-frequency transmission of ColI in Salmonella typhimurium. J. Gen. Microbiol. 33: