Morphology of pili determined by the N incompatibility group plasmid N3 and interaction with bacteriophages PR4 and IKe

Morphology of pili determined by the N incompatibility group plasmid N3 and interaction with bacteriophages PR4 and IKe

PLASMID 2, 632-636 (1979) SHORT COMMUNICATION Morphology of Pili Determined by the N Incompatibility Group Plasmid N3 and Interaction with Bacterio...

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PLASMID

2,

632-636 (1979)

SHORT COMMUNICATION Morphology of Pili Determined by the N Incompatibility Group Plasmid N3 and Interaction with Bacteriophages PR4 and IKe DAVID Faculty

E. BRADLEY

of Medicine, Memorial University of Newfoundland, St. John’s, Newfoundland AIB 3V6, Canada

Received April 2, 1979 The IncN plasmid N3 was transferred to bald strains of Salmonella typhimurium LT2 and co/i K-12. In both cases, transconjugants were found to carry short pili, which were designated N pili. They were very easily detached from cells and readily broken into short pieces. N pili were found to be straight inflexible rods 9.5 nm thick and sharply pointed at the distal end. The N-specific filamentous bacteriophage IKe and the lipid-containingphage PR4 both adsorbed to the pointed tips when mixed with cell-free suspensions of N pili.

Escherichia

For some years it was thought that plasmids of the incompatibility group N (IncN) did not determine any pili ((I); E. Meynell, personal communication; D. E. Bradley, unpublished), and were able to conjugate without the assistance of such organelles. However, a low-magnification electron micrograph was published (C. M. To, in Ref. (2)) showing very thin filaments on a cell of Salmonella typhimurium LT2 (N3). No experimental details were given, nor was a description of the pili included, hence the present investigation in which the existence of N pili is confirmed and their morphology is described in detail. The IncN plasmid N3 (3) was transferred by conventional mating techniques from Escherichia coli K-12 strain J53(N3), supplied by N. Datta, to the bald strains S. typhimurium LT2 SQ1139 (from R. Bradley) and E. co/i JE2571 (from R. Iyer). Transconjugants were checked for sensitivity to the filamentous phage IKe (from R. Iyer), which forms plaques on strains carrying IncN plasmids (4). Bacteria were heavily streaked onto selective plates (BBL brain heart infusion agar containing 10 pg/ml tetracycline) and grown overnight at 37°C. For electron microscopy, they were suspended with a loop in a drop of 0.1 M ammonium acetate solution. A carbon0147-619)(/79/040632-05$02.00/O Copyright 0 1979 by Academic Press, Inc. All rights of reproduction in any form reserved.

coated specimen support grid was touched onto the surface of the suspension, and after washing in 0.1 M ammonium acetate, the mounted cells were negatively stained with 0.67% sodium phosphotungstate solution and dried. For the preparation of a pilus suspension, bacteria were transferred with a loop to a Thomas Tellon tissue grinder containing 2 ml of M9 minimum medium salts solution for each plate harvested. They were then ground at about 150 rpm for 1.5 min as described in detail elsewhere (5). After the removal of bacterial cells by centrifugation, the suspension, which was remarkably free from debris, was used for studying the interaction of free N pili with bacteriophages IKe and PR4 (6,7). The suspension was incubated with an equal volume of phage suspension, mounted, and negatively stained for electron microscopy as above. N pili, which were found at variable frequencies on cells of E coli JE2571(N3) and S. typhimurium SQ1139(N3), are illustrated in Figs. 1 and 3, respectively. The highest frequencies observed from several preparations of each strain were 0.4 pili/cell for JE2571(N3) and 0.8 pili/cell for SQ1139(N3), based on counts of 50 cells in the electron microscope. Very considerable frequency variation was found from one

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electron microscope specimen support grid to another, even when they were prepared from the same plate; sometimes no pili were visible on the cells. However, in all cases short lengths of detached pili could be found (Fig. 2). It seemed clear that they had been broken from the cells by the vigorous agitation required to suspend the bacteria. This hypothesis was borne out by the large numbers of pili found in pilus suspensions prepared by shearing, as described above (see Fig. 6). These observations probably account for earlier difficulties in visualizing the pili. N pili are sharply pointed at their distal ends (Figs. l-3) and are about 9.5 nm thick. Their protruding lengths vary between 0.1 and about 0.5 pm, but longer rods (~1.0 km) are present in cell-free suspensions (Fig. 6). Their most distinctive morphological characteristic is their inflexibility; they are straight rods rather like the thinner Type I pili (8) and break rather than bend, apparently being very brittle, during preparation procedures for electron microscopy. IncN plasmids, as well as IncP and IncW plasmids, confer sensitivity to several isolates of a lipid-containing phage, the one used here being PR4. It has been demonstrated elsewhere that PR4 adsorbs to the tips of P- 1 and W pili (9 JO), not by the phage tail, but by an apex of the icosahedral head (II). After the incubation for 20 min of a mixture of PR4 and isolated N pili, virions could be seen adsorbed to the pointed ends of one or more rods. The point of attachment was apparently an apex of the head, and certainly not the tail (Figs. 4, 5). No PR4 virions could be found attached to pili on whole cells; they were at the cell surface. IncN plasmids also confer sensitivity to the filamentous phage IKe at a high level (12), turbid plaques being formed. The apparent absence of a pilus-type receptor for IKe has led to speculation about the nature of its adsorption (1,13). This question is to some extent resolved by the following experiment. After a mixture of IKe and an N pilus suspension had been

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incubated for 30 min, electron microscopy revealed very few examples of virions adsorbed to pili (Figs. 7, 8). The distinct morphology of the N pilus enabled it to be clearly distinguished from the long flexible IKe vu-ions. Since the phage + pilus complexes shown seemed to represent a genuine adsorption rather than a nonspecific random attachment, another mixture was incubated for 4 h. Here, 20 of the IKe virions in a 50virion sample were found attached to pili at the distal end. Assuming that N pili are indeed IKe receptors (this is not certain at present), adsorption appears to be unusually slow. Brodt et al. (I) reported that IKe adsorption was directly to the bacterial cell wall, an observation most readily explainable by the possible ability of N pili to retract, pulling attached virions to the surface as has been suggested for other filamentous phages (14,15). While the results described here do not indicate that N pili perform a role in conjugation, some speculation is of interest. Dennison and Baumberg (26) have shown that bacteria carrying IncN plasmids are very poor donors in liquid cultures, but that they can transfer at derepressed levels on solid media. This may well be explained by the fragile nature of N pili should they prove to be required for mating: Brownian motion and motility would tend to shear them off in a liquid, whereas on a solid surface such forces would be reduced. The role of N pili as bacteriophage receptors is more certain. It has been clearly demonstrated here that both PR4 and IKe adsorb to N pilus tips. Precedent suggests that this represents the specific phage receptor although confirmation is obviously required. The ease with which N pili are removed from cells is likely to mean that they will be most readily detected in suspension rather than attached to cells. Hopefully this approach will reveal that pili are determined by other IncN plasmids and cultures with conjugative plasmids of other groups which do not appear to determine pili; for example the IncX type plasmid R6K (17,

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FIGS. 7.8. Vu-ions of the N-specific filamentous phage IKe attached to the tips of N pili from a cellfree suspension. Bars = 100 nm.

18) should also be examined for detachable pili. Hitherto, IncN plasmids were thought to be examples of a conjugal system not requiring pili as mating organelles. The discovery of N pili means that the existence of such systems must be questioned. The unusual brittle nature of N pili suggests that they may have a different chemical structure from other plasmid-determined pili which are more flexible. Further experiments in this and other areas are continuing. ACKNOWLEDGMENTS I thank those who kindly provided bacterial strains and bacteriophages, and also Doris Cohen for valuable technical assistance. The work was supported by the Medical Research Council of Canada (Grant MA5608).

REFERENCES 1. BRODT, P., LEGGETT, F., AND DYER,R., Nature (London) 249, 856-858 (1974). 2. ACHTMAN, M., AND SKURRAY,R., In “Microbial Interactions,” Series B, Vol. 3: “Receptors and Recognition,” pp. 233-279. Chapman and Hall, London (1977). 3. WATANABE, T., NISHIDA, H., OGATA, C., ARAI, T., AND SATO, S., .I. Bacterial. 88, 716-726 (1964). 4. KHATOON, H., IYER, R. V., AND IYER, V. N., Virology 48, 144- 15.5(1972). 5. BRADLEY, D. E., J. Gen. Microbial. 102, 349-363 (1977). 6. STANISICH, V., J. Gen. Microbial.

84, 332-342

(1974). 7. BRADLEY, D. E., AND RUTHERFORD, E. L., Canad. J. Microbial. 21, 152-163 (1975).

FIG. 1. N pilus on a cell of E. coli JE257l(N3). Bar = 100 nm. FIG. 2. Fragment of an N pilus from a cell suspension ofS. typhimurium

SQll39(N3) showing pointed tip. Bar = 100 nm. FIG. 3. N pilus on a cell of S. typhimurium SQll39(N3). Bar = 100 nm. FIG. 4. Bacteriophage PR4 virion with adsorbed N pili: empty virion with tail. Bar = 100 nm. FIG. 5. Single PR4 virion (full) with adsorbed N pilus: tail not visible. Bar = 100 nm. FIG. 6. N pili from a suspension prepared by shearing cells ofS. typhimurium SQll39(N3) in a tissue grinder. Bar = 100 nm.

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C. C., Trans. N. Y. Acad. Sci. 27, 1003-1054 (1965). BRADLEY, D. E., AND COHEN, D. R., .I. Gen. Microbial. 98, 619-623 (1977). BRADLEY, D. E., J. Gen. Microbial. 95, 181-185 (1976). BRADLEY, D. E., In “Pili,” pp. 339-353. International Conferences on Pili, Washington, D. C. (1978). DENNISON, S., AND BAUMBERG, S., Proc. Sot. Gen. Microbial. 1, 10 (1973). BRINTON,

13. DENNISON, S., AND BAUMBERG, S., Genet. Res. 25, 275-284 (1975). 14. JACOBSON, A., J. Virol.

10, 835-843 (1972).

15. BRADLEY, D. E., Canad. J. Microbial. 19, 623631 (1973). 16. DENNISON, S., AND BAUMBERG, S., Mol. Gen. Gene?. 138, 323-331 (1975). 17. KONTOMICHALOU, P., MITANI, M., AND CLOWES, R. C., J. Bacterid. 104, 34-44 (1970). 18. BRADLEY, D. E., Plasmid

1, 376-387 (1978).