Basic Characterization of W-pili

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1 Journal of General Microbiology (I 976), 97,9 I -I 03 Printed in Great Britain Basic Characterization of W-pili By DAVID E. BRADLEY AND DORIS R. COHEN Faculty of Medicine, Memorial University of Newfoundland, St John s, Newfoundland, Canada AI C 5S7 (Received 10 May 1976) SUMMARY The bacterial drug-resistance plasmids Sa, R388 and R7K that comprise the W compatibility group were transferred by conjugation to species in the genera Escherichia, Salmonella, Shigella and Pseudomonas, chosen for their lack of common pili. On receiving the W plasmids, all strains produced pili, which were similar morphologically, at average frequencies of up to 3.0 pililcell. The pili determined by the W plasmids were also related serologically, but unrelated to those of other plasmids. R- segregant strains which had lost their plasmids showed a simultaneous loss of both pili and drug-resistance characteristics. W-pili are pointed flexible filaments 10 to 12 nm thick, with an average length of 450 nm. The best host found was SalmoneIIa typhimurium. INTRODUCTION Pili determined by bacterial drug-resistance plasmids have been identified for the compatibility groups F (Brinton, 1971), I (Meynell, Meynell & Datta, 1968) and T (To, To & Brinton, I 979, and for the recently designated Pseudomonas aeruginosa compatibility groups P-I (Bradley, 1974) and P-2 (Shahrabadi, Bryan & Van Den Elzen, 1975). Group P-I was originally called P (Datta et al., 1971). Preliminary observations (Bradley, 1975) have indicated that pili are also associated with a W group plasmid. Groups P-I and W are related through nucleic acid homology (Ingram, 1973), and their ability to transfer sensitivity to the same bacteriophages (Bradley & Rutherford, 1975 ; Olsen, Siak & Gray, 1974 ; Stanisich, I 974). They are of particular interest because they can transfer intergenerically. The W compatibility group, originally described by Hedges & Datta (1971)~ at present consists of three plasmids : Sa (formerly written S-a) confers resistance to chloramphenicol, sulphonamides and, at a low level, to streptomycin and kanamycin (Watanabe, Furuse & Sakaizumi, 1968) ; R388 (Datta & Hedges, 1972) confers resistance to trimethoprim and sulphonamides ; and R7K (Coetzee, Datta & Hedges, 1972) to ampicillin, carbenicillin and streptomycin (low level). Plasmid-associated pili have only been demonstrated for Sa (Bradley, 1975), which is the type plasmid of the group. The present work confirms these observations, and shows that similar pili are determined by R388 and R7K. The object of this paper is to describe the essential characteristics of the piii rather than to provide a detailed study of a particular aspect. METHODS Bacterial strains and growth media. The strains used are listed in Table I. Escherichia coli ~~34-3 is an R- segregant (a strain which has lost its plasmid) constructed from CR~~(RPI), previously CR~~(RI 822) described by Olsen & Shipley (1973). Pseudomonas aeruginosa

2 92 D. E. BRADLEY AND D. R. COHEN Donor strains Escherichia coli K I (Sa) 353(Sa-1) J53(R388) 353tR7K) c~34wr) Salmonella typhimurium L T~ SQ 1 139(R46) Pseudomonas aeruginosa pu2 I /~6(Sa) Recipient strains Compat i- bility group W w P- I N W Table I. Bacterial strains used Description* %fs, Strs at 800,ug ml-l Sa-I is a Tra- variant of Sa Rif RifS &fs, formerly c~34(r1822) Pil- Pil- mutant of ~uz~(sa), see text Source or reference Watanabe et al. (1968) Hedges & Jacob (I 974) Datta & Hedges (1972) Coetzee et al. (1972) Olsen & Shipley (I 973) Authors G. Jacoby and authors Escher ich ia co li CR34-3 c~34-3(rif-r) c~34-3(str-r) Saltnonella typhimurium LT2 SQI I38(leu) SQ I I 39(ade pro ilv) SQI I 39(rif-r ade projlv) Salmonella abony s~4o I -I (rif-r) Salmonella montevideo SQ3 83kif-r) Shigella flexneri ~3-2(rifr) Shigella sonnei w(rif-r) R- segregant from c~34(rpr) Rifr mutant of ~~34-3 Strr mutant of ~~34-3 Pil-, ChP, smooth Pil-, Str', rough Rif'mutant of s~r139 Pil- Pil- Pil- Rifr mutant of wild-type clinical isolate Authors Authors Authors R. Bradley R. Bradley Authors R. Bradley R. Bradley St John's General Hospital St John'srGeneral Hospital Pseudomonas aeruginosa pu2 I /~6(rif-r) Rifr mutant of R- segregant of Authors pu2 I /~6(Sa) * Chl, Chloramphenicol ; Rif, rifampicin ; Str, streptomycin ; Pi]-, no common pili ; Tra-, transfer-deficient. pu21/~6(sa) is a mutant of G. Jacoby's pu21(sa) lacking common pili (Bradley, 1975). Strain PU~I originated from OT47 (Loutit, Pearce & Marinus, 1968). Strains were grown routinely on Difco nutrient broth plus 2 % (w/v) agar. Mg minimal medium (NH,CI, I 00 g 1-1 ; KH2P04, 3.0 g 1-1 ; Na,HP04, 6.0 g 1-l) was supplemented with glucose (0.25 %), vitamin BI (2.0 pg ml-l), and other additives as appropriate. Bacteriophages. Phage PR4 is a lipid-containing, short-tailed, double-stranded DNA bacteriophage that grows on bacteria harbouring either P, N or W plasmids (Bradley, 1974 ; Stanisich, 1974). Phage PRRI (Olsen & Thomas, 1973) is an isometric, RNA-containing, P-I plasmid-specific bacteriophage, adsorbing to pili determined by P- I plasmids. Matings. Unless otherwise stated, the same mating procedure was used throughout. Shake cultures (nutrient broth) were inoculated from streaks grown overnight on nutrient agar. Plates for R+ donors (strains carrying drug-resistance plasmids) contained appropriate selective antibiotics : chloramphenicol for Sa, trimethoprim for R388, ampicillin for R7K, tetracycline or carbenicillin for RPI (at the concentrations given below). Cultures were grown at 37 "C to early exponential phase, then adjusted to an extinction of 0.2 at 620 nm.

3 Basic characterization of W-pili 93 For intergeneric crosses, the recipient was incubated for 30 min at 50 "C immediately before mating to reduce restriction and increase the yield of transconjugants (Mojica-a & Middleton, 1971). Equal volumes (ratio of donors to recipients I : I) were mixed and incubated statically at 37 "C for 1.5 h. Portions (0.1 ml) of the mating mixture and also serial dilutions in buffered saline or broth were spread on nutrient agar plates containing appropriate selective drugs (rifampicin was used for counterselection against donors in intergeneric crosses; selection against recipients was made with one of the drugs listed above). M9 minimal medium supplemented with amino acids according to the requirements of the strain (Table I) was also used. The drug and growth supplement concentrations were (pg ml-l) : rifampicin IOO ; chloramphenicol 25 ; sulphadimidine 200 ; trimethoprim roo [ roo0 pg ml-l for selection against P. aeruginosa pu2 I /M6(rzyr)]; ampicillin 400 ; carbenicillin 500; tetracycline 10; streptomycin 800; leucine 20; adenine 12.5; proline 20; isoleucine 20. Plasmids Sa and R7K confer resistance to streptomycin at a low level, but at this high concentration (800 pg ml-l) the drug can be used to counterselect against donors. After overnight incubation at 37 "C (48 h for minimal medium plates), transconjugants were purified. The presence of W plasmids in transconjugants was confirmed by sensitivity to phage PR4. Transfer frequencies were calculated by dividing the number of transconjugant colonies by the number of donor cells (by viable count) in 0-1 ml of mating mixture at the beginning of the mating. Because of the high levels of mating on the plates (see Results), comparisons of transfer frequencies were based on colony counts from spreads of the same dilutions of mating mixtures. The Tra- plasmid Sa-r was mobilized from E. coli J53(Sa-I), which bore common pili, into the non-piliated E. coli CR34-3 and examined by electron microscopy. The mobilizing P-I group plasmid RPI (Grinstead et al., 1972) was transferred to~53(sa-r) to form a 'double' (a strain carrying two compatible plasmids simultaneously). ~53(Sa-r, RPI) was then mated with E. coli CR34-3. Transconjugants were selected for chloramphenicol resistance (determined by Sa-r but not RPI) and patch tested for tetracycline sensitivity, which indicated a spontaneous loss of RPI, Further clones of CR34(Sa-I) were isolated by selecting RPI- segregants from transconjugants ('doubles') by the spot test method (see below) using the RPr-specific RNA phage PRRI (Olsen & Shipley, 1973). Transfer frequencies of < 6 x IO-~ transconjugants/donor were obtained for the resulting clones with E. coli c~34-3(str-r). They were considered to be Tra-. The spot test. This was used to demonstrate sensitivity to phages and to isolate spontaneous R- segregants, which were selected by resistance to R-specific phages (specific for drug-resistance plasmids). Agar from the 'clearings' of the spot test on soft agar plates was streaked. Colonies which showed complete resistance to the selecting phage by the spot test were purified and tested for loss of plasmid-determined drug-resistance characteristics. Electron microscopy. A thin-layer static overnight culture in nutrient broth at 37 "C was prepared from bacteria grown on a nutrient agar plate containing appropriate selective drugs. The culture was then agitated gently to resuspend any sedimented cells, and a carboncoated electron-microscope specimen support grid was touched on its surface. The grid was held, wet side uppermost, for 2 or 3 min, and then most of the liquid was removed with a filter paper, leaving a thin film over the grid. This was allowed to dry at the outside edge only, then was washed twice in 0.1 M-ammonium acetate solution, and negatively stained in 0.15 % (wlv) sodium phosphotungstate solution. The number of pilifcell was estimated using an electron microscope by counting the pili on roo random isolated cells. Pili were sometimes visible against the body of the bacterial cell because of the light negative staining, but were only scored if they extended beyond its edge. Samples of IOO cells of R- back-

4 94 D. E. BRADLEY AND D. R. COHEN ground strains (those used as recipients for the various plasmids) were checked in the same way. Incubation at the optimum bacterial growth temperature of 37 "C did not give the largest numbers of pili (maximum piliation was at 30 "C) but this temperature was used because there were fewer aberrant cells. Subsequent processing was carried out at room temperature rather than at 37 "C because negative staining was more reliable. Piliation was slightly higher, but the most reproducible results were obtained in this way. Antibody-labelling for electron microscopy was carried out as described previously (Bradley, 1972 ; Lawn, 1967). Rabbits were inoculated intravenously seven times in 2 weeks with a W-pilus suspension (4 x 0.5 ml, 3 x 2.0 ml). This suspension was prepared by blending an overnight static culture of Salmonella typhimurium SQI 138(Sa) in a Waring blender for 1-5 min, partly purifying the culture fluid by differential centrifugation (20000 g for 30 min and g for 24 h) and resuspending in about 0.01 of the original volume. Booster injections (0.1 ml) were given at 2, 6 and 12 weeks after the last injection, and bleeding was carried out 10 days after the last booster. Bacteria were labelled with antibodies by mixing 0.2 ml of bacterial culture with 0-1 ml of antiserum (complement removed by heating at 56 "C for 40 min) and incubating at 37 "C for I h. Organisms were mounted as follows : 10 ml broth was added to the mixture which was then centrifuged at 7000 g for 5 min ; the pellet was gently resuspended in a little broth and cells were mounted on grids and negatively stained (0.8 % sodium phosphotungstate solution) as described above. RESULTS Occurrence of W-pili in various bacterial genera To detect the presence of pili, the W 'type plasmid' Sa was transferred from E. coli (Sa), which carried common pili (Brinton, 1969, to various non-piliated species representing four different genera. The different levels of piliation, as revealed by electron microscopy, are shown in Table 2. With R- background strains, no pili were found in any of the loo-cell samples examined. Thus the plasmid Sa determines the production of pili in the different species, albeit at variable efficiency. This efficiency is reflected in the values for pili/ piliated cell, which show the level of pilus production in those cells definitely containing Sa (not spontaneous R- segregants). For example, ~u21/~6(sa) has I '0 pili/piliated cell, the lowest possible number, suggesting that pilus production is suppressed or inefficient in P. aeruginosa. This value can be compared to the 4-18 pililpiliated cell of S. tjphimurium SQI 13g(Sa), where pilus production is obviously efficient. The overall average number of pililcell together with the percentage of piliated cells in the sample indicate the condition of the culture as a whole. These values are more relevant to the present studies so are used in the rest of this paper. They varied greatly between different species of Sa+ (harbouring Sa) bacteria. With P. aeruginosa PU~I /~6(Sa), the numbers are comparable with those obtained previously (Bradley, 1g75), when cells from soft agar plates rather than static overnight broth cultures were used. The low level of piliation may be due to a continuous build-up of R- segregants resulting in a large majority of these at the end of the culture period ; Jacoby (personal communication) reports that W plasmids are unstable in P. aeruginosa. This would also explain the weak response to phage PR4 as shown by the spot test, in which very turbid 'clearings' were obtained. However, the plasmid Sa appears to be quite stable in Salmonellu abony SQ~OI-I(S~), hence such an explanation for its low level of piliation seems unlikely. A more likely reason is the presence of a thin mucoid-like layer on the surface of the cells, not observed in other species, which might affect the outgrowth or maintenance of pili. However, the repression of the plasmid Sa cannot be ruled out.

5 Basic characterization of W-pili 95 Table 2. Numbers of pili on cells of bacterial species carrying the plasmid Sa Species and strain Pi1 i/cell* Cells piliatedt Pili/piliated cell$ "/, E. coli c~34(sa) S. typhimurium SQI I 39(Sa) S. abony SQ~O I -I(Sa) S. montevideo s~383(sa) Sh. Jlexneri ~3-2(Sa) Sh. sonnei w(sa) 2'39 74 P. aeruginosa pu2 I /~6(Sa) * Average number of pili per cell from a pilus count on 100 cells. 7 Percentage of piliated cells in a sample of loo cells. $ Average number of pili per pilus-bearing cell from a sample of 100 cells. We attempted to study the piliation of cells from overnight static cultures containing selective drugs in order to eliminate the presence of R- segregants which contributed to the observed number of cells present that lacked pili. However, in some cases, notably P. aeruginosa PU~I /~6(sa), many morphologically abnormal cells were found. In others, such as S. typhimurium SQI 139(Sa), no increase in piliation was observed. We conclude that, in the first case, the abnormal cells resulted from drug action on R- segregants and, in the second, there were insignificant numbers of segregants present. This aspect was not studied in detail. Transfer frequencies Since we were concerned primarily with W-pili and not with the mating characteristics of W plasmids, quantitative observations on intergeneric crosses were not normally made. With S. abony SQ~OI-I(S~), however, the low level of W-pili (0.05 pililcell) gave an opportunity to test the hypothesis that W-pili are required for conjugation. Crosses of S. abony SQ~OI-I(S~) with S. typhimurium SQI 139 gave transfer frequencies of I x IO-~ and 2 x IO-~ transconjugants/donor for clones bearing 0.05 and o pililcell respectively. In the latter case, the fact that no pili were found does not necessarily mean that they did not occur at a frequency that was too low for detection in a loo-cell sample, though a further limited search did not reveal any. These low transfer frequencies suggested that S. abony SQ~OI-I(S~) was a poor donor, perhaps because it had few pili. Salmonella montevideo s~383(sa) was a more efficient donor (2 x 10- * transconjugants/donor when crossed with S. typhimurium SQI I 39) and had many more pili (0.64 pili/cell). With the majority of crosses, most of the mating occurred on the selective plates. When 0.1 ml of a 10-fold dilution of mating mixture was spread on a selective plate, the number of colonies obtained was very much less than one tenth of the number obtained from 0.1 ml of undiluted mating mixture. For example, the cross E. coli ~53(R7K) x Shigella flexneri ~3-2(rif-r) gave 360 colonies when 0-1 ml of a 10-fold dilution of mating mixture was spread on a plate, and only 12 colonies when a Ioo-fold dilution was used. With P-I and N compatibility group plasmids, Dennison & Baumberg (I 975) observed virtually no mating in liquid cultures, only plate mating. They suggest that this may be due to the inability of fragile mating pairs to remain attached in a liquid environment. There may be a similar explanation for W plasmids : W-pili appear to be easily detached from the cell, and they too could form fragile mating pairs. 7

6 96 D. E. BRADLEY AND D. R. COHEN Table 3. Piliation of transconjugants from intraspec8c matings of S. typhimurium strains SQT I 39(Sa)* and SQI I 38(Sa)T Cross Piliation of transconjugants Piliicell Cells piliated (%) ~53-1(Sa)z x SQI 139(rif--r) SQI I 39(Sa) x SQI I 38(leu) SQI I 38(Sa) x SQI I 39(adepro ih) I ~66 72 * Rough. t Smooth. $ E. coli. Table 4. Piliation of cells of bacterial species carrying the plasmids Sa, R7K and R388 Pili/cell (cells piliated, %) Recipient species I A > and strain* Plasmid... Sat R7K R388 E. coli ~~34-3 S. typhimurium SQ I I 39 Sh. flexneri ~3-2 P. aeruginosa pu2 I / ~ 6 * Piliation of recipient strains was 0. t From Table 2, included for comparison. Transfer of Sa between rough and smooth strains of S. typhimurium To ascertain whether a difference in wall characteristics, as exemplified by rough and smooth strains of S. typhimurium, affected pilus production, Sa was transferred from SQI 139 (rough) to SQI 138 (smooth) and then transferred back to SQI 139. Pilus counts for representative transconjugant clones (Table 3) were not affected by wall differences, the transfer of Sa coinciding with the transfer of pilus production. Salmonella typhimurium SQI 139(Sa) transconjugants from the third mating had fewer pili than those from the first, perhaps due to less efficient expression in a different clone. The Tra- plasmid Sa-I This plasmid resulted from the transposition of the ampicillin-resistance determinant of RP4(= RPI) on to the Sa replicon, which lost its ability to transfer (Hedges & Jacob, 1974). To ascertain whether or not pili were associated with such a plasmid, we examined three clones of E. coli c~3q(sa-1) : no pili were found. Determination of pili by W group plasmids R7K and R388 R7K and R388 were transferred from E. coli 553 R+ strains to the four species listed in Table 4, which shows the pilus counts. Both plasmids determined significant numbers of pili, although R7K gave fewer than R388 and Sa. The pili were all similar both morphologically and serologically (see below). Background strains of all species had no pili. Segregants presumed to be R- for all three plasmids were prepared from S. typhimuriurn SQI 139 R+ and E. coli CR34 Rf strains [by selecting for resistance to phage PR4 (high sensitivity is determined by R388 and R7K as well as by Sa) and were also found to have no pili. 3. >

7 Basic characterization of W-pili 97 Fig. I. Pilus on E. coli c~34(sa) labelled with antibodies against pilifrom S. typhirnuriurn SQI 138(Sa). Arrows mark groups of regularly-spaced antibodies. Fig. 2. Pili on P. aeruginosa PU~I/M~ (R7K) labelled with the same antibodies. Fig. 3. Cell of E. coli c~34(r388) labelled with the same antibodies. Pili are well-coated, the arrows marking short filaments which would not otherwise be visible. 7-2

8 98 D. E. BRADLEY AND D. R. COHEN Identijication of W-pili using immune electron microscopy A conclusive way of identifying a type of pilus is by immune electron microscopy (Lawn, 1967), in which the filaments are labelled with specific antibodies visible in the electron microscope. We used antiserum against S. typhimurium SQI 138(Sa) pili to treat cells of other bacterial genera harbouring W plasmids. The presence or absence of antibodies on pili was ascertained by electron microscopy. Figure I shows a pilus on E. coli c~3q(sa). The antibodies are attached in clusters, and some (arrowed) are regularly spaced, probably reflecting the arrangement of the pilus protein molecules. Thus the pili found on S. typhimurium SQI 138(Sa) are serologically related to those on E. coli c~34(sa). Figure 2 shows pili determined by R7K on P. aeruginosa PU~I/M~(R~K) treated with the same antiserum, and Fig. 3 shows a cell of E. coli c~34(r388). The pili produced by all three plasmid-bearing strains are similar regardless of genus. In Fig. 3, the antibodies show up many short pili, some of which are arrowed, which would not otherwise be visible against the body of the cell; the antibodies provide, in effect, a positive stain. It is possible that labelling the pili with antibodies may have stimulated their growth, as found with F-pili (Lawn & Meynell, 1972). The micrograph also reveals an even distribution of the pili over the cell surface, showing that W-pili do not cluster at the poles of E. coli. This is also seen with P. aeruginosa, on which strictly polar appendages are very common. No cross-labelling with antibodies was observed with F- or RPI-pili. Structure of W-pili The general appearance and dimensions of W-pili determined by the plasmid Sa were described previously (Bradley, I 975). Some additional ultrastructural features are mentioned here. Figure 4, which shows part of a cell of E. coli c~34(r388), demonstrates that R388- determined pili are morphologically similar to those determined by Sa. R~K-pili are also similar. The tips of the pili appear to be tapered to sharp points (Fig. 5). This is characteristic of W-pili ; RPI-pili have blunt ends (Bradley, 1974) and F-pili often have terminal knobs (Lawn, 1966). Another feature, shared with F-pili, is a dark line down the centre visible only after light negative staining (Fig. 6). This could be caused by penetration of the negative stain down the inside of the pilus, implying that it is tubular. Alternatively, the pilus could have an axial groove on the outside; this would probably be an artefact due to flattening on drying. When S. typhimurium SQI I 39(Sa) cells were blended, W-pili were readily detached but not broken up. A detailed study of this aspect was not made, but the method was used to prepare anti-pilus serum (see Methods). A partly-purified preparation, as used to inoculate rabbits, contained aggregates of pili (Fig. 7). The filaments may have adsorbed to cell debris, but the distal ends were pointed suggesting that adsorption would have taken place proximal end first. It is also possible that the region of the cytoplasm or plasma membrane in which pilus assembly had taken place was torn out of the cell. The micrograph illustrates the flexibility of the pili. Eflect of temperature on piliation Pilus counts were carried out on S. typhimurium SQI 13g(Sa) cultured at various temperatures within its growth range. Five thin-layer static cultures were grown at temperatures between 18 and 41 "C for 24 h to allow all cultures to produce sufficient cells for examination. Specimen preparation for electron microscopy was carried out at the specified temperatures. The largest numbers of pili were produced at 30 "C (Fig. 8) even though the optimum growth temperature for S. typhimurium is 37 "C. To et ul. (1975) reported that the synthesis of E-pili,

9 Busic charac ter izu t ion of W-p ili 99 Fig. 4. W-pili on E. coli c~34(r388). Pili determined by R7K (not illustrated) are similar Fig. 5. The pointed tips of W-pili on S. typhimrrr-iuni SQI 13g(Sa). Fig, 6. W-pilus on S. fyphimuriurn SQI 138(Sa) showing a dark line down its axis. Fig. 7. W-pili prepared by blending S. fyphirnuriim SQI I 38(Sa) and partly purifying the culture fluids by centrifugation. The preparation also contains many small molecules.

10 I00 D. E. BRADLEY AND D. R. COHEN Temperature ( C) Fig Time (min) Fig. 9 Fig. 8. The average number of pililcell (0) and the percentage of piliated cells (0) in different cultures of S. typhimurium SQI 139(Sa) grown at different temperatures. Fig. 9. Changes in the number of pililcell (0) and in the percentage of cells piliated (12) with respect to time, after rapidly cooling a 37 "C static culture of S. typhiniurium SQI 13g(Sa) to o "C. The piliation at o min is taken as the level at 37 "C just before cooling. which are determined by the T compatibility group plasmids, is temperature sensitive, and the same appears to be true to some extent for W-pili. It will be noted that a 37 "C culture of S. typhimurium SQI 13g(Sa) produced 1-83 pililcell (Fig. 8) when processed for electron microscopy at 37 "C, but about 3.0 pililcell (Table 2) when processed at room temperature (22.5 "C). This may be because the cells are cooled through 30 "C (the temperature of maximum piliation) sufficiently slowly to allow more filaments to be produced (total processing time about 2 min). Salmonella typhimurium does not grow at below I 5 "C, so the effect of low temperatures could only be studied by cooling cultures grown at higher temperatures. The 30 and 37 "C cultures used in the previous experiment were cooled in 50 ml flasks in ice-baths, held at o "C for about 5 h to ensure complete stabilization, and then processed for electron microscopy at 2 "C. The piliation of the 30 "C culture rose from 4-06 pililcell (70 % cells piliated) to 6.44 pililcell (84 % cells piliated). Piliation of the 37 "C culture rose from 1-83 pili/cell (44 % cells piliated) to 5-87 pililcell (87 % cells piliated). Surprisingly, these were the largest numbers of W-pili found in any preparation. To determine whether this increase occurred during cooling or at o "C, a 24 h static culture at 37 "C (chosen because cultures grown at this temperature showed the largest change in piliation in the first cooling experiment) was cooled rapidly, by placing single drops on metal dishes in an ice-bath located in the 37 "C incubator, and pilus counts were made immediately (processing time at o "C about 2 min) and at intervals for 250 min. In Fig. 9 the base level of piliation is taken as that at 37 "C; there was a sharp increase in the number of pili on rapid cooling, perhaps stimulated by cold shock. The rate of increase then slowed, probably because the enzyme reactions required to assemble the pilin (pilus protein) into filaments slow down. The piliation at 37 "C was lower than in the previous experiment. This degree of variation was normally experienced with 24 h cultures so that valid comparisons could oniy be made using results obtained for the same culture.

11 Basic characterization of W-pili I01 Eflects of growth conditions on piliatioii Previous studies (Bradley, 1975) on cells from exponential-phase cultures shaken at 100 oscillations min-l revealed no pili. This might have been due to shearing of the pili by the agitation, or to the rapid overgrowth of R- segregants. We reduced the latter possibility to a minimum by using bacteria from plates containing selective drugs, and by using clones of S. typhimuvium SQI 139(Sa) whose stability had been well-established over a long period. (Previously, strains which had been freshly constructed were used). The possibility of pilus loss due to shearing was investigated by taking samples from a shake culture and incubating them further under static conditions, pilus counts being taken at intervals. There was no significant change in the number of pili over a period of 40 min; this finding is consistent with the loss of pili due to shearing being small, although other explanations are not excluded. Bacteria from a culture shaken at 37 "C and IOO oscillations min-1 had about 1.4 pililcell and this level remained constant during the exponential phase of growth. However, after 7 h, when the culture reached the stationary phase, the level rose to 2-7 pili/cell, which is similar to that obtained from overnight static cultures. This increase might have been due to the natural increase in ph value as the culture aged (from ph 6.8 to ph 7.6 at 7 h), a higher ph favouring pilus formation. However, no change in piliation was found when bacteria were grown overnight in a static culture that had been adjusted from ph 5.8 to ph 7-6 then to ph 8-4 (using hydrochloric acid and sodium hydroxide). Thus the number of pili present depended apparently on the state of bacterial growth with a maximum in the stationary phase, although other environmental influences that have not been examined may be relevant. DISCUSSION We have shown that the three known W plasmids determine pili that are similar both morphologicaily and serologically. The similarities exist regardless of the bacterial host genus. The pili are different morphologically and serologically from other plasmid-determined pili. The existence of W-pili, as they can now be designated, is thus established. Since several types of plasmid-determined pili have now been discovered, their nomenclature must be clarified. With W-pili, we have adopted the premise that a pilus should be named using its compatibility group letter, as with F-pili. Pili determined by RPI would therefore be called P-I-pili, not D-pili (Brinton, unpublished), and the E-pili of group T plasmids (To et al., 1975) would be called T-pili. If a compatibility group can be designated by a letter, there is no reason why its specific pilus type should not have the same letter. Objections have been raised in the case of P-I-pili. Pili are apparently determined by a sex factor of Vibrio cholerae designated P (Bhaskaran, 1958) which has priority. In this case, the compatibility group P has been subdivided (Shahrabadi et al., 1975; Jacoby, unpublished) into groups P-I to P-8 with an appropriate change in pilus designation, which should be sufficient. Any other system would be confusing. P-I, N and W compatibility groups are related by genetic homology (Ingram, 1973). Fertility inhibition was described as a further criterion for their relatedness by Olsen & Shipley (1975). However, our results indicate that this relatedness does not extend to the type of pilus determined ; P-I-pili are morphologically and serologically distinct from W- pili. No pili have been found for N group plasmids (Brodt, Leggett & Iyer, 1g74), and we have substantiated this for strains carrying R46 (Datta & Hedges, 1971) grown overnight in static broth cultures (unpublished observation), conditions that favour the production of W-pili.

12 I02 D. E. BRADLEY AND D. R. COHEN W-pili may perform a dual role, as do o,her p1a;mid-determined pili, in that they play a part in conjugation and also act as bacteriophage receptors. With respect to conjugation, we have demonstrated qualitatively a correiation between low piliation and poor donor ability in S. abony SQL~I-I(S~) ; this is consistent with pilus involvement in conjugation. With regard to a possible function as phage receptors, it has been found (Bradley, 1976) that the lipid phage PR4 (Bradley & Rutherford, 1975 ; Stanisich, 1974) adsorbs to the tips of W-pili as well as to the walls of Sa+ strains. It is suggested that the pilus tips act as primary receptors : as soon as the phage is attached, the pili withdraw completely into the cell bringing the virion into contact with the outer membrane where a secondary receptor is thought to be located. We thank those mentioned in Table I for kindly supplying strains, and are grateful to Dr G. Jacoby, Harvard Medical School, Boston, Massachusetts, U.S.A., for strains 553- I (Sa), 553(R388) and ~53(R7K), to Dr N. Datta, Royal Postgraduate Medical School, London, for strain J53(Sa-I), and to Dr R. H. Olsen, University of Michigan Medical School, Ann Arbor, Michigan, U.S.A., for strain c~34(r1822). We also thank Dr N. Datta for valuable correspondence, and R. Bradley for reading the manuscript. The financial support of the Medical Research Council of Canada (grant no. MA5608) is gratefully acknowledged. REFERENCES BHASKARAN, K. (1 958). Genetic recombination in Vibrio cholerae. Journal of General Microbiology 19, BRADLEY, D. E. (1972). Shortening of Pseudomonas aeruginosa pili after RNA-phage adsorption. Journal of General Microbiology 72, BRADLEY, D. E. (1974). Adsorption of bacteriophages specific for Pseudomonas aeruginosa R factors RPI and RI 822. Biochemical and Biophysical Research Communications 57, BRADLEY, D. E. (1975). The occurrence of pili associated with a plasmid of the W compatibility group. Biochemical and Biophysical Research Communications 64, g I BRADLEY, D. E. (1976). Adsorption of the R-specific bacteriophage PRq to pili determined by a drug resistance plasmid of the W compatibility group. Journal of General Microbiology 95, BRADLEY, D. E. & RUTHERFORD, E. L. (1975). Basic characterization of a lipid-containing bacteriophage specific for plasmids of the P, N, and W compatibility groups. Canadian Journal of Microbiology 21, BRINTON, C. C. (1965). The structure, function, synthesis and genetic control of bacterial pili and a molecular model for DNA and RNA transport in gram-negative bacteria. Transactions of the New York Academy of Sciences 27, BRINTON, C. C. (1971). The properties of sex pili, the viral nature of conjugal genetic transfer systems, and some possible approaches to the control of bacterial drug resistance. Critical Reviews in Microbiology I, I BRODT, P., LEGGETT, F. & IYER, R. (1974). Absence of a pilus receptor for the filamentous phage IKe. Nature, London 249, COETZEE, J. N., DATTA, N. & HEDGES, R. W. (1972). R factors from Proteus rettgeri. Journal of General Microbiology 72, DATTA, N. & HEDGES, R. W. (1971). Compatibility groups amongfi- R factors. Nature, London 234, 222. DATTA, N. & HEDGES, R. W. (1972). Trimethoprim resistance conferred by W plasmids in Enterobacteriaceae. Journal of General Microbiology 72, DATTA, N., HEDGES, R. W., SHAW, E. J., SYKES, R. B. & RICHMOND, M. H. (1971). Properties of an R factor from Pseudomonas aeruginosa. Journal of Bacteriology 108, I DENNISON, S. & BAUMBERG, S. (1975). Conjugational behaviour of N plasmids in Escherichia coli ~12. Molecular and General Genetics 138, I. GRINSTEAD, J., SAUNDERS, J. R., INGRAM, L. C., SYKES, R. B. & RICHMOND M. H. (1972). Properties of an R factor which originated in Pseudomonas aeruginosa RI 822. Journal of Bacteriology 110, HEDGES, R. W. & DATTA, N. (1971). fi- R factors giving chloramphenicol resistance. Nature, London 234, HEDGES, R. W. & JACOB, A. E. (1974). Transposition of ampicillin resistance from RP4 to other replicons. Molecular and General Genetics 132,

13 Basic characterization of W-pili INGRAM, L. C. (1973). Deoxyribonucleic acid-deoxyribonucleic acid hybridization of R factors. Journal of Bacteriology 115, I LAWN, A. M. (1966). Morphological features of the pili associated with Escherichia coli K I carrying ~ R factors or the F factor. Journal of General Microbiology 45, LAWN, A. M. (1967). Simple immunological labelling method for electron microscopy and its application to the study of filamentous appendages of bacteria. Nature, London 214, I I LAWN, A. M. & MEYNELL, E. (1972). Antibody-stimulated increase in sex pili in R+ Enterobacteria. Nature, London 235,44 I LOUTIT, J. S., PEARCE, L. E. & MARINUS, M. G. (1968). Investigation of the mating system of Pseudomonas ueruginosa strain I. 1. Kinetic studies. Genetical Research 12, MEYNELL, E., MEYNELL, G. G. & DATTA, N. (1968). Phylogenetic relationships of drug resistance factors and other transmissable bacterial plasmids. Bacferiological Reviews 32, MOJICA-A, T. & MIDDLETON, R. B. (1971). Fertility of Salmonella typhimurium crosses with Escherichia coli. Journal of Bacteriology 108, I OLSEN, R. H. & SHIPLEY, P. (1973). Host range and properties of the Pseudomonas aeruginosa R factor RI 822. Journal of Bacteriology 113, OLSEN, R. H. & SHIPLEY, P. (1975). RPI properties and fertility inhibition among P, N, W, and X incompatibility group plasmids. Journal of Bacteriology 123, OLSEN, R. H., SIAK, J. & GRAY, R. H. (1974). Characteristics of PRDI, a plasmid-dependent broad host range DNA bacteriophage..journal of Virology 14, OLSEN, R. H. & THOMAS, D. D. (I 973). Characteristics and purification of PRRI, an RNA phage specific for the broad host range Pseudomonas R1822 drug resistance plasmid. Journal of Virology 12, SHAHRABADI, M. S., BRYAN, L. E. & VAN DEN ELZEN, H. M. (1975). Further properties of the P-2 R-factors of Pseudomonas aeruginosa and their relationship to other plasmid groups. Canadian Journal of Microbiology 21, STANISICH, V. (I 974). The properties and host range of male-specific bacteriophages of Pseudomonas aeruginosa. Journal of General Microbiology 84, To, C. M., To, A. & BRINTON, C. (1975). A new epiviral pilus, the E pilus, and a new RNA pilus phage pile/ri. Abstracts of the Annual Meeting of the American Society for Microbiology, p Washington : American Society for Microbiology. WATANABE, T., FURUSE, C. & SAKAIZUMI, S. (1968). Transduction of various R factors by phage PI in Estherichia coli and by phage P22 in Salmonella typhimurium. Journal of Bacteriology 96, I