The adsorption of Pseudomonas aeruginosa pilus-dependent bacteriophages to a host mutant with nonretractile pili

VIROLOGY

68,

The

149-163

(1974)

Adsorption

of Pseudomonas

Bacteriophages

to

a Host DAVID

Department

o,f Zoology,

University

aeruginosa Mutant

Nonretractile

Pili

E. BRADLEY

of Edinburgh, Accepted

with

Pilus-Dependent

West

Mains Road,

Edinburgh

EH9

SJT,

Scotland

November 16, 1973

Pseudomonas aeruginosa strain K is the host for two pilus-dependent bacteriophages (Pf, filamentous, and P04, long noncontractile tail). This paper describes the isolation of a strain K mutant, which is resistant to both phages and yet bears many pili serologically similar to those of its parent. The efficiency of adsorption by the mutant was found to be close to that of strain K for each phage. The location of adsorbed PO4 virions along the sides of the mutant’s pili was consistent with the hypothesis that the pili were nonretractile. Pilus length measurements on strain K and the mutant showed no change in distribution before and after phage adsorption. This result is discussed in detail with reference to possible models for PO4 penetration.

INTRODUCTION

It has now been well-established that Pseudomonas aeruginosa, like Escherichia co&, has a number of pilus-dependent bacteriophages including both RNA-containing types (Feary et al., 1964; Bradley, 1966) and DNA-containing fllamentous forms (Takeya and Amako, 1966). In addition, a pilus phage with a long noncontractile tail (P04) has been isolated for P. aeruginosa only (Bradley, 1973a). These bacteriophages are valuable in studying the mode of action of their receptor pili. It has been found that the pili of two strains which are sensitive to the RNA phage PP7 (PAOl, PA038) retract on phage adsorption, but the serologically similar pili of two resistant strains (PA068, PA01264), which are also able to adsorb PP7, do not retract (Bradley, 1972b,c). These four strains of P. aeruginosa (PA01 and derivatives) are in a serological group different to that of the host for the filamentous phage Pf and the tailed pilus phage PO4 (P. aeruginosa strain K). PA01 pili are also serologically different to those of strain K. There is no cross-infection by Pf and the RNA phage PP7, and PO4 grows only at low efficiency on strain PAOl. While it is

thought that pilus retraction in strain K is essential for DNA penetration and infection by Pf and P04, it has not been demonstrated as described above for PA01 because no RNA phage has been isolated for strain K. Also a mutant with nonretractile pili has not been found. However, electron microscope observations have suggested models for the adsorption and penetration stages of both Pf and PO4 (Bradley, 1973b,d). With Pf, the filamentous virions are thought to adsorb to the tips of the pili which then retract and pull them into contact with, or perhaps right through, the cell envelope. With P04, the virions adsorb to the sides of the pili tail first, and retraction pulls them to the cell surface. It is suggested that the bases of the pili, which seem t’o be small holes in the cell wall (Bradley, 1972c), are secondary receptors triggering DNA injection (Bradley, 1973d). Clearly these models could be verified, and further evidence in support of pilus retraction in strain K obtained, if a K mutant with the characteristics of nonretractile pili could be isolated. The present paper describes such a mutant and some experiments on the adsorption of phages PO4 and Pf to it. 149

Copyright All rights

@ 1974 by Academic Press, Inc. of reproduction in any form reserved.

150

DAVID

E.

Certain predictions can be made regarding the behavior of this mutant when mixed with virions of Pf or P04. In both cases, phage particles should be adsorbed by the mutant but should not infect it; adsorption should be demonstrable by a decrease in phage titer after incubation with the mutant. In the electron microscope, phage Pf should be visible attached to the tips of the pili. Phage PO4 should be adsorbed along the lengths of the pili, not preferentially to the poles, as is the case with the sensitive strain K. In addition, there should be no change in the mean pilus length of the mutant after PO4 adsorption. The experiments to be described are aimed at testing these predictions. It is of interest that pilus retraction has been suggested for a star-forming strain of Pseudomonas echinoides (Heumann and Marx, 1964; Mayer and Schmidt, 1971), but the proposed mechanism is quite different to that of P. aeruginosa. It involves a structural change in the P. echinoides pilus rather than a withdrawal into the cell as with P. aeruginosa. MATERIALS

AND

METHODS

The following procedures have been described in greater detail elsewhere (Bradley, 1972b,c; 1973b,d). Bacteria and bacteriophages. P. aeruginosa strain K, ATCC 25102, was streaked, and a single clone (numbered KSC) was used. The mutant under study, K/BPfS, was derived from strain K/SPf, a “carrier” for the filamentous phage Pf (Bradley, 197313). Strain K/12Pf2 was originally numbered 25102/12Pf (Bradley, 1972a) and is a pilusless host mutant of strain K resistant to Pf. Strain K/IPO4 is a pilus-less mutant of strain K resistant to phage PO4 (Bradley, 1973b,d). Bacteriophage PP7 is an RNA pilus phage of P. aeruginosa strain PA01 Bradley, 1966). Phage PBl is a contractile phage (Bradley and Robertson, 1968) with the same host. Phage Pf (ATCC 25102B) is a filamentous phage (Takeya and Amako, 1966) for P. aeruginosa strain K. Phage PO4 is a tailed pilus phage (Bradley, 1973a,d), host strain K.

BRADLEY

Culture media and methods. Oxoid nutrient broth was used for liquid and 2 % agar plate cultures. Bacteriophages were grown by confluent lysis of the host strain on double agar layer plates, which were extracted with broth. Phage Pf was prepared by growing an infected culture of strain K with shaking at 37” for 24 hr as described elsewhere (Bradley, 197313). Standard methods were used for phage titration. Bacterial cultures were grown with shaking at 37”. Cell concentration was determined by precalibrated optical density measurement. Anti-p&s serum was prepared by inoculating rabbits subcutaneously with a pilus suspension + complete Freund’s adjuvant (Bradley, 1972c, 1973b). Some flagella were also present in the inoculum. The spot test was used to determine the sensitivity or otherwise of a bacterial strain to a phage (Bradley, 1973b). A loopful of high-titer phage placed on a double agar layer plate of bacteria showed clearing or otherwise after overnight incubation. Phageresistant mutants were isolated by streaking the cleared agar and selecting colonies. Electron microscopy . Bacteria were mounted for electron microscopy by holding a carbon-coated specimen grid vertically beneath the surface of a culture sample, and agitating the liquid back and forth for 2 min. Grids were washed by immersion in water. If required, they were immersed in anti-pilus serum for 10 set and again washed Negative staining was carried out by irnmersing the grid in neutral 0.67 % sodium phosphotungstate solution. Excess liquid was removed with a filter paper before drying. RESULTS

Isolation and Characterization of the Piliated Host Mutant Resistant to Pilus Phages The isolation of the mutant of P. aeruginosa strain K numbered K/2PfS was fortuitous. It originated from the previously described strain K/BPf (Bradley, 1973b), which was a “carrier” for phage Pf, that is, an active producer of the virus. In the course of storage over 2 years (on slopes at 4”, subcultured every 6 months), it lost the ability to produce Pf though it remained

-----YHACiJi

'-"^-"""'"1 1 1UlY

ALKSWKY

resistant to it. When stored by freezing at -2O”, the same strain retained the ability to produce Pf, and i% was concluded that some form of selection had been inadvertently carried out in the course of storage at 4”. On streaking, K/BPf produced two different colony types: large (rough) and small (smooth). They remained pure on subculturing from single colonies. The large form, numbered K/2PfL, was found to be pilusless in the electron microscope, and the small form, K/2PfS, was heavily piliated. The present work concerns K/2PfS. The following tests were carried out to verify that both K/BPf and K/BPfS were true strain K mutants and not contaminants. First, the cells were labeled for electron microscopy with antiserum prepared against the pili + flagella of strain K, using the method of Lawn (1967). Figure 1 shows the parent strain K/2Pf after it had lost the ability to produce Pf. Here the antibodies have coated the pili and flagella and have taken up the sodium phosphotungstate as a positive rather than a negative stain. The overall effect is to render the masses of polar pili more clearly visible. Some nonpiliated cells are included in the field (arrowed), presumably belonging to the large colony form K/BPfL. At higher magnification (Fig. 2), K/BPfS pili and flagella can be seen clearly labeled with strain K antibodies. The appearance of K/2PfS without antibodies is similar to the cell in Fig. 4 without adsorbed phage particles. It was subsequently confirmed that strain K/SPfS belonged to the serological group 6 of P. aeruginosa, the same as that of strain K (T. L. Pitt, personal communication). This serological typing was with reference to cell wall and heat-stable surface antigens. Both the parent strain K/BPf and the two components K/2PfS and K/BPfL were tested for susceptibility to various bacteriophages using the spot test method. The results are given in Table 1 and compared with the susceptibilities of strains K and K/lP04, which is a pilus-less host mutant resistant to phage PO4 (Bradley, 1973b,d). K/2Pf and its piliated constituent K/2PfS behave similarly to nonpiliated strains. In

TO

P. AERUGINOSA

151

addition, it was found that, using the standard typing phages of the Cross-Infection Reference Laboratory (London), K/2PfS corresponded in its sensitivity pattern to the strain of origin, K. There was one exception which is thought to be another pilus phage (T. L. Pitt, personal communication). It is concluded that strain K/BPfS is a true mutant of strain K, not a contaminant. Adsorption of Pilus Phages PO4 and Pf to Strain K/dPjS To establish whether the resistance of K/BPfS to pilus phages was due to failure to adsorb, phage suspensions were mixed with an excess of exponential phase bacteria (m.o.i. 0.04 phages/bacterium) and incubated with shaking for 20 min. After centrifugation, the supernatant was titrated (Bradley, 1973d). The percent phage adsorbed was calculated with reference to a blank sample in which the bacterial culture was replaced with broth, and which was processed simultaneously. In Table 2 it can be seen that K/2PfS does adsorb phage P04, but somewhat less efficiently than the host strain K. The pilus-less mutant K/ lP04 does not adsorb significantly due to lack of pili (Bradley, 1973d). Essentially similar results were obtained with the filamentous phage Pf (Table 3), although it is adsorbed less efficiently by K/2PfS. The pilus-less host mutant K/12Pf2, which was isolated from strain K using Pf (Bradley, 1973b), shows insignificant adsorption. The ability to adsorb pilus phages and yet be resistant to infection, as shown in these results, is consistent with the concept of nonretractile pili in K/BPfS. Since these experiments were done under the same conditions, it can be said that K/BPfS is significantly less efficient at adsorbing both pilus phages than the sensitive parent strain K. The adsorption of PO4 is not strictly comparable with that of Pf since the virions are attached to different sites on the pilus (sides and tips, respectively). However, it seems probable that adsorption to pili is reversible for both phages and that irreversible adsorption occurs only when the virions reach the pole of the sensitive strain (Fig. 3). This is further

152

I)AVII)

13. BItADLISY

labeled with antibodies I ?IG. 1. Cells of strain K/2Pf used has acted as a positive stain. Arrows ho ltungstate X’i ‘000.

to strain K pili and flagella. The sodium phosmark nonpiliated cells (component K/LPfL).

PHAGE

FIG.

2. Pole

of

strain

K/BPfS

ADSORPTION

labeled

with

TO

antibodies

P. AERUGZNOSA

to

strain

K

pili

153

and

flagella.

X 46,000.

154

DAVID TABLE

E.

1

SENSITIVITY OF MUTANTS OF P. aeruginosa STRAIN K TO VARIOUS BACTERIOPHAGES AS SHOWN I)Y THE SPOT TESTY Strain

Pili

K K/lPO4 K/ZPf K/PPfS K/PPfL

Sensitivity

+ + + -

PP7

PO4

Pf

PBl

-

+ -

+ -

+ + + + +

a A loopful of phage was agar layer plate of bacteria. cated sensitivity and none phage. The presence of pili is their absence by -. TABLE ADSORPTION STRAIN Sample

Titer

2

of superna(PFU)

4.00 x 107 1.88

3.55 3.83

S

x x x

TABLE ADSORPTION STRAIN Sample

placed on a double Clearing (f) indi(-) resistance to a indicated by + and

OF PHAGE PO4 TO P. aeruginosa K AND RESISTANT MUTANTS tant

Blank K K/PPf K/lP04

to phages

106 106 107

% PO4 adsorbed 0 99.5 91.1

4.3

3

OF PHAGE PF TO P. aeruginosa K AND RESISTANT MUTANTS Titer tant

Blank K K/2PfS K/12Pf2

of superna(PFU)

3.75 x 4.15 7.93

3.60

x x x

107 105 106 lo7

% Pf adsorbed 0 98.9 78.9

4.0

illustrated in electron micrographs (Figs. 4 and 6) where some PO4 virions can be seen lying beside, but not fixed to, the pili, suggesting that they have become detached on drying. The apparent irreversible adsorption at the cell surface could be due to attachment to secondary receptors or some interaction with the cell wall. Electron Microscopy Pilus Phages

of Cells with Adsorbed

The electron microscope was used to study PO4 and Pf virions adsorbed to K/2PfS

BItADLEk-

pili, and to compare Dhe appearance of these cells with thost> of the sensitive strain TC; 3.5hr cultures were diluted t’o 2 X lo8 cells/ml with broth and mixed with an equal volume of phage PO4 at 6 X log PE’U/ml. After gentle shaking at 37” for 10 min, bacteria were negatively stained for electron microscopy. Figure 3 shows a strain K cell with many empty virions adsorbed to the pole. This may be contrasted with Fig. 4, a K/BPfS cell, where virions are either attached to, or lie very near the pili, but are not preferentially adsorbed to the pole. The only two virions at t,he pole (arrowed) still contain their DlYA, in contrast to those in Fig. 3. It was possible to find empty virions adsorbed to K/2PfS poles only on rare occasions (Fig. 6, arrowed). In some cases the phages were so firmly attached to K/BPfS pili that they caused them to bend (Fig. 5). The difference in appearance between the sensitive and resistant strains suggests that K/BPfS pili are nonretractile, as will be discussed. In order to place this visual data on a quantitative basis, 187 poles of each strain were scored for adsorbed virions, all those within about one tail length of the cell surface being counted as attached. The results are shown as histograms in Fig. 7. For strain K there are numerous poles with many adsorbed virions, but with K/BPfS most of the poles have less than two. The average number of virions per pole for the host strain K was 9.16 and for K/2PfS, 1.25. Obviously some phage particles would be located close to the poles of K/BPfS since that is where the pili are at their greatest density. These results were checked in a second experiment with about twice the m.o.i.: strain Ii had 18 virions per pole and K/2PfS 1.6 virions per pole. The observation that K/BPfS has many more pili than strain K (22.6 and 1.6 pili/ pole, respectively, in the first experiment) is discussed below. The electron microscopy of K/BPfS cells with adsorbed antibody-labeled filamentous phage Pf failed to produce convincing examples of virions attached to the pilus tips. This was probably due to the excessive tangling of the pili with the long Pf filaments (1915 nm; Bradley, 1973c).

PHAGE

ADSORPTION

TO

P. AERUGINOSA

FIG. 3. Strain K (PO4 sensitive) with adsorbed PO4 virions at the pole. FIG. 4. Strain K/PPfS cell (PO4 resistant) with PO4 virions lying beside, *row marks two phages containing DNA close to a pole. X 21,000.

X 50,000. or adsorbed

to

pili.

156

FIG. attached

DAVID

6. Pole to the

E.

BRADLEY

FIG. 5. PO4 virions attached tail-first to K/BPfS pili. X 110,000. of K/2PfS cell showing numerous pili with adsorbed virions. One PO4 polar surface and has lost its DNA (arrowed). X 90,000.

phage

particle

is

PHAGE

ADSORPTION

TO P. AERUGZNOSA

157

K/2PfS

K

10.

i

0 0

5

10

0

Adsorbed

5

PO4

10 virions

15

per

20 25 pole

30

35

FIG. 7. Distribution of PO4 virions adsorbed to the poles of strains K/SPfS were scored for adsorbed virions in the electron microscope.

Pilus Length Measurements The measurement of pilus lengths before and after PO4 adsorption should indicate whether or not retraction occurs in strains K and K/2PfS in a similar manner to that described for the PP7/PA038 phage/host system (Bradley, 197213). Cells of strains K and K/BPfS were each mixed with phage P04. The m.o.i. was 8 by plaque count and 36 by electron microscope count, the difference being doubtless due to inactive virions. After incubation for 10 minutes with gentle shaking, cells from the mixture and those from a similar sample with broth instead of phage were mounted for electron microscopy. Cell poles were photographed at random, and pilus lengths were measured. The figures obtained were plotted as length distribution curves (Fig. 8, solid lines). Theoretical curves representing distributions after retraction were obtained as follows. Assuming that PO4 virions adsorbed along the pili at random (Fig. 4), and that a single virion could stop the retraction of a pilus at the point of adsorption, a mean length reduction of 50% would be predicted after phage attachment. Theoretical length distributions representing this amount of retraction are obtained from cells without adsorbed PO4

40

and K. 187 poles of each

by halving each pilus length measurement and are plotted in the lower graphs of Fig. 8 (broken lines). If retraction had occurred, theoretical and experimental distributions should coincide, which they do not. Conversely, if the pili with adsorbed PO4 virions had retracted, then doubling each measurement should produce a distribution corresponding to the pilus lengths in cells with no adsorbed virions (Fig. 8, top graphs, broken lines). Again the curves do not match. The interpretation of these results is made easier by comparing them to pilus length distributions obtained with a phage/host system where retraction is known to take place. The curves in Fig. 9 show data obtained from similar experiments using phage PP7 adsorbed to strains PA01 and PA068 (Bradley, 1972c) and processed in the same way. It can be seen that, with the retractile pili of strain PAOl, there is a good match between the theoretical and experimental curves (broken and solid lines, respectively). With the nonretractile pili of strain PA068, the curves differ as they do with strains K and K/2PfS. Indeed, the four experimental curves obtained with these two strains are all very similar to one another. The significance of these results is discussed below.

15S

DAVII)

0

1

2

3

4-

-5

E.

6

BRADLEY

-7

-0 LENGTH

2

1 (pm)

3-

4

5

6

7

FIG. 8. Frequency distributions of pilus lengths on strains K and K/ZPfS before and after phage PO4 adsorption. A---A, Experimental curves obtained from the strains as marked in each graph. n-----n, theoretical curves obtained by halving each pilus length without adsorbed phage (plotted on lower graphs), or doubling each measurement from preparations with adsorbed phage (top graphs). Theoretical curves are predicted distributions if a 507, retraction of the pili occurred, so the curves should match, Since they do not, there is little or no apparent retraction.

50

,

,

,

,

1 I

I

,

I

I

PA01

40RetracttIe

I

,

I

I

I

I

I

__

I

I

PA068

Pili

Non-retract#le

PA01 + PP7

-:

; 1 ,

LENGTH

The data from this experiment are summarized in Table 4. It can be seen that the 50% reduction in average pilus length obtained with strains PA01 (Bradley, 1972c)

PA068

Pili

+ PP7

-

_

(pm )

9. Frequency distribut,ions of pilus lengths on strains phage PP7 adsorption (numerical data from Bradley, 1972c). Fig. 8. Predicted curves for a 50% retraction (broken lines) which has nonretractile pili (Bradley, 1972c). FIG.

,I,

PA01 Curves match

and PA068 are plotted with PA01

before and after RNA similarly to those in but not with PAO68,

and PA038 (Bradley, 1972b) has not been observed here. Likewise there has been no reduction in modal length after PO4 adsorption.

PHAGE TABLE

ADSORPTION

TO

4

AVERAGE PILUS LENGTHS AND THE NUMBER OF PILI/POLE FOR P. aeruginosa STRAINS K AND K/2PFS BEFORE AND AFTER PHAGE PO4 ADSORPTION Specimen

K K,

PO4 sorbed K/2PfS K/2PfS, adsorbed a Pili/pole

Pili/ pole"

Mean l?gtf

Modal

Pili

Poles

1.21 1.12

2033 1840

900 900

158 135

46 49

1616 1542

500 750

167 169

14 14

ad-

22.8 19.7

PO4

was

The Numbers

l~gtf IIIXCI- III;;:mll mn

counted

of Pili

on 70-100

poles.

on Strains

K and

K/.%‘PfS Table 4 shows that the number of pili on strain K is not greatly affected by PO4 adsorption at the m.o.i. used in the determination (8 by plaque count, 36 by electron microscopy). However, it seemed possible that the effect of PO4 adsorption at a much higher m.o.i. might be greater. Exponential phase cells of strain K were therefore mixed with PO4 suspension to give an m.o.i. of 200 (by plaque counts), and incubated with shaking for 10 min. The cells were then mounted on grids and negatively stained. The pili were counted on random cells (100 poles). A blank sample, in which the phage suspension was replaced with a similar volume of broth, was treated identically. The results in Table 5 show that the number of pili on strain K after PO4 adsorption is only one-fifteenth of the normal level. The significance of this will be discussed in the context of the length measurements described above. The value of 0.87 pili/pole for the cells without adsorbed phage is rather lower than that given in Table 4 for the previous determination, but it represents the normal variation in pilus counts obtained from Merent cultures. As has already been mentioned, and ab shown in Table 4, strain K/BPfS has many more pili than strain K although the filaments are serologically similar. This situation was also found with strains PA01 and PA068 (Bradl.ey, 1972a). Only a few PA01 retractile pili were visible in the electron microscope (c 0.1 pili/pole), but the non-

P. AERUGINOSA

159

retractile pili of strain PA068 numbered about 2.25 pili/pole. PA01 piliation increased to 2 pili/pole if cells were mounted on grids which were then treated with antipilus serum (1: 1 dilution in water) for 10, set, preventing the pili retracting but not allowing more to grow (Bradley, 1972d). With the nonretractile pili of PA068 there was no such increase. An identical experiment was carried out with strains K and K/BPfS. The results in Table 6 show an increased piliation after antibody-labeling with strain K, indicating the retractile nature of the pili (Bradley, 1973b), but with strain K/SPfS there is a decrease, probably due to the tangling of the more numerous antibody-coated pili. The value for K/SPfS unlabeled pili is less than in Table 4 because of the normal variations in pilus counts and because a 4.5 hr rather than a 3.5 hr culture was used. The numbers of pili present on K/2PfS varied greatly from 0 to a maximum of 130 pili on a single pole. DISCUSSION

Evidence for pilus retraction in P. aeruginosa strain K. It is important to consider whether strain K pili retract, and whether those of K/2PfS do not. The effect of antibody-labeling on piliation will be discussed first. If pili retract under external influences such as pH changes, drying, or chemical action (Bradley, 1972c), all of which occur TABLE

5

PILIATIONOF P. aeruginosaS~~~~~ AFTER ADSORPTION OFPHAGE PLICITY OF INFECTION Sample

Pili/pole

Without PO4 PO4 adsorbed

0.87 0.06

TABLE PILIATION K/2PFS

Pili/pole unlabeled

Km K/PPfS

1.44 16.3 from

Bradley

AND

% pole piliated 33 6

6

OF P. aeruginosa STRAINS BEFORE AND AFTER LABELING ANTI-PILUS ANTIBODIES

Strain

o Results

K BEFORE PO4 ATAMULTIOF Xl0

K

AND WITH

Pili/pole antibody-labeled 3.53 13.7 (1973b).

160

DAVID

E.

in negative staining, any treatment which might prevent retraction and which is applied before negative staining, should cause an increase in the number of pili/pole which would not be apparent with nonretractile pili. The only treatment not involving such influences is antibody-labeling. Since the antibodies coat the pili thickly and almost instantaneously, they might reasonably be .expected to prevent the filaments being drawn through the cell wall (Bradley, 1972d). We have seen that strain K piliation increases from 1.44 to 3.53 pili/pole after antibody-labeling, and that strain K/2PfS shows no increase. It is difficult to conceive of any explanation other than the respective retraction and non-retraction of Ii and K/2PfS pili. The use of bacteriophages provides completely separate evidence regarding pilus retraction. When PO4 is adsorbed to strains K and K/BPfS, valuable numerical support data is obtained (Fig. 7). Before discussing the interaction of PO4 with pili, it must be established that the phage uses them at least as the primary adsorption organelle. Although this has been demonstrated elsewhere (Bradley, 1973a,d), the present cxperiments with K/BPfS are valuable confirmation. Figs. 4-6 leave no doubt that the virions adsorb to K/BPfS pili, which are serologically similar to those of strain K. This visual evidence is supported by the ‘91.1% adsorption using titration methods. Add to this the fact that PO4 cannot infect nor adsorb to pilus-less host mutants (Bradley, 1973d; Table 3) and there can be little doubt that it is a true pilus phage. The most significant observation described here is t’he adsorption of PO4 virions at random along the lengths of K/BPfS pili (Fig. 4) in complete contrast to their attachment to the poles of the sensitive strain K (Fig. 3). This is very similar to observations with phage PP7 which was adsorbed at random points along the nonretractile pili of strains PA068 and PA01264. With the sensitive strains PA01 and PA038, it was attached preferentially to the polar surface at the pilus bases (Bradley, 1972b,c). By analogy, this strongly suggests that strain K pili are retractile and that K/SPfS pili are not. It is thought that PO4 virions adsorb

BRADLEY

t,o the sides of strain K pili and arc drawn to the cell surface by retraction. With K/2PfS they remain where they adsorb and cannot reach the surface to effect DXA injection. It is obvious that this visual evidence of pilus retraction in strain K is in direct conflict with the pilus length measurements. After PO4 adsorption to st,rains K and K/’ 2PfS at m.o.i. 8 (36 by electron microscopy), there is no change (a) in the average number of pili/pole, (b) in the mean length, (c) in the modal length, and (d) in the overall distribution of pilus lengths. Since both phage adsorption and antibody-labeling indicate in two different ways that strain K pili only do retract, a satisfactory explanation for this anomaly must be sought. The only possibility is that’ the pili disappear, either by complete retraction into the cell, or by breaking off. In both cases, the measurements obtained on strain K will be derived from pili which have not retracted (or broken off) because they are defective or have not received a stimulus from an adsorbed phage virion (Bradley, 1972c). It follows that the length distributions will correspond to those for nonretractile pili. The pilus length measurement procedure thus appears to be limited in that it will only detect partial retraction, and then only when phage virions attach firmly enough to prevent the completion of retraction when they reach the cell surface. The electron microscopv of a new pilus phage (M6) with a tail similar to that of PO4 shows that attachment to the pilus is by means of the delicate bar-shaped tail structure (Bradley and Pitt, in preparation) ; it is thought that this may not give a strong enough linkage to prevent complete retraction. This explanation is supported by the loss of piliation observed after PO4 was adsorbed to strain K at high m.o.i. (Table 5). Under these circumstances, if complete retraction occurred, each pilus would disappear as a phage particle adsorbed to it. The filament would then be replaced by another growing out from a different position. Eventually, with a high enough m.o.i., all the available growth sites would be blocked by adsorbed virions, preventing further outgrowth and lowering the pili/pole. In the length measure-

PHAGE

ADSORPTION

ment experiment, the m.o.i. (by electron microscopy) of 18 virions per pole was close to the potential number of pili as indicated by K/SPfS (22.8 pili/pole) but below saturation level. Unless strain K was completely saturated, a residual piliation equal to that observed without adsorbed phage would always be present as was the case here (Table 4). The drop in piliation at higher m.o.i. is very large and it is suggested that the few remaining pili are defective and cannot retract. If the blockage of growth sites did not occur, replacement pili would be free to grow and no change in piliation would be observed regardless of the m.o.i. used. An alternative explanation is that each phage virion breaks off the pilus to which it adsorbs when it reaches the cell surface. The question of pilus breakage will be discussed below. It is clear that in both cases there would be no change in the mean length, modal length, and length distributions of the pili. Both antibody-labeling and phage adsorption provide results indicating pilus retraction in strain K, and the observations are consistent with the concept of an equilibrium between pilus outgrowth and retraction. If the retraction process is prevented either by coating the pili with antibodies, or by the mutation of an. appropriate gene, the equilibrium shifts in favor of outgrowth, resulting in a net increase in piliation. This situation involves what might be termed “spontaneous” retraction in which the pili would be continuously growing in and out. If retraction required stimulation, they would normally be e:xtended until a suitable stimulus caused them to withdraw. Model for the adsorption and penetration of P04. It seems fairly certain that PO4 is a pilus phage a,dsorbing to its retractile receptors which pull the virions to the cell surface. The pili probably continue to withdraw until they disappear because the virions are unable to stop retraction. In addition, it has been shown that virions adsorbed to the pili of strains K and K/2PfS are full, whereas those at the cell pole of strain K are empty. The implication is that the active receptor which triggers DNA penetration lies at the cell surface. Also there are more PO4 virions at the pole of strain K after

TO

P. AERUGZNOSA

161

infection than there are pili. If the virions stopped pilus retraction, one would expect approximately one phage per pilus at the pole. For a count of 1.12 pili/pole (Table 4), 18 PO4 virions per pole were found on the same grid. This supports complete retraction because several virions could be adsorbed to one pilus, which could then draw them to the surface in succession. In addition, more pili could be stimulated to grow as suggested elsewhere (Bradley, 1973a). Each new pilus would draw a virion to the surface then disappear. An alternative to complete retraction would be pilus breakage. This is considered less likely since it would involve an additional function for the phage tail tip at the cell wall, namely the release of a proteolytic enzyme to break the pilus. This would not be necessary if the pilus withdrew completely. In addition, the enzyme would have to remain inactive until the cell wall was contacted or virions would appear preferentially at the pilus tips, which they do not. The most favored model for PO4 penetration is shown in Fig. 10. The sequence suggests that the pilus filament only assists in and that its absence would not prevent the phage from adsorbing and injecting via the cell wall. The fact that PO4 cannot infect nonpiliated cells, and must be considered pilus-dependent, conflicts with this hypothesis. However, a complete pilus assembly must include both filament and base, which appears to be a small hole in the cell wall (Bradley, 1972c) perhaps incorporating specific proteins. These holes seem to be absent in pilus-less mutants (Bradley, unpublished). If pili were completely removed from a sensitive cell leaving no trace of pilin at the cell surface, a virion might still be able to infect via this receptor, though at a reduced efficiency. This could be the explanation for the rare empty virions found at the poles of K/BPfS, where all the avail1 able pilin might have been assembled into filaments, but leaving the occasional surplus hole without any associated pilus protein. It has been indicated that pilus outgrowth is blocked by the PO4 virions after they reach the cell surface (drop in piliation after PO4 adsorption at high m.o.i.). This suggests that the phage tail attaches directly to the

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very strong evidence in favor of F-pilus retraction in E’. coli. The similarities of this system with that of P. aeruginosa are also particularly noticeable with respect to RNA phage infection. The nature of strain K/ZPfS. Unfortunately there was no indication in the isolat,ion procedure for P. aeruginosa strains PA068 and PA01264 as to why their pili should be nonretractile (Holloway, personal communication). However, with K/BPfS, C d some informed speculation is possible. The parent strain K/2Pf was a Pf “carrier” and it seems reasonable to suppose that storage Fs=c_ at 4” caused all the phage-bearing clones to z die so that resistant strains could grow prefe erentially on subculturing. One of these strains (K/2PfL) had lost its pili, and the FIG. 10. Proposed model for the adsorption and other (K/BPfS) had increased its pili. It is penetration of phage P04. (a) pole of a sensitive cell with pili and phage virions; (b) virions adsorb thought that K/BPfS could be a spontaneous to the sides of the pili without ejecting their DNA; mutation of a Pf “carrier” clone which had (c) pili begin to retract drawing the virions to the ceased to produce Pf and changed to the cell surface, at the same time new pili appear; production of defective pili. These variants (d) the virions at the cell surface inject their provide a very good defense for bacterial DNA, others adsorb to the new pili, and more species against pilus phages. Not only are continue to appear; (e) the newly adsorbed phages they phage resistant, but they also remove are drawn to the cell and inject their DNA. Some the viruses from an environment. They pili remain available for further adsorption. would be most valuable in studying both pilus base, and not to some point near it,. pili and pilus phages in other genera, and could be detected by their ability to adsorb The hole in the cell wall could thus provide a route of entry for the viral DNA or a such viruses though being resistant to them. They also seem to have many more pili means of access for the tip of the phage tail. .The possibility of more than one virion in- than their parent strains. Indeed, K/2PfS jecting its DNA through a single hole can- probably produces the maximum number of pili possible for the strain, and there should not be entirely ruled out. be no pilin remaining within the cell. The adsorption of phage Pj. The characterConclusion. While the nature of PA01 and ization of Pf as a pilus phage is confirmed by K pili (whether plasmid controlled) is still the ability of strain K/BPfS to adsorb but unknown, it is interesting to compare them not to be infected by it. Visual demonstration of Pf adsorption to K/BPfS pili is evi- with pili associated with the P. aeruginosa R factors R1822 and RPl (Olsen and Shipdently very difficult. In the suggested model ley, 1973, and personal communication). for the Pf penetration process (Bradley, 1973b), the virions are adsorbed to the pilus PA01 and K pili are apparently serologically tips which draw them to the cell surface, or different to the R factor pili as shown by antibody-labeling for electron microscopy, perhaps right into the cell, thus effecting but it seems that PA01 pili can coexist DNA penetration. This is much the same as the model for filamentous coliphages as sug- with the R factor pili in the same cell (Bradley, unpublished). In addition, PAOl- and gested by Jacobson (1972), who noted an K-specific phages including PO4 do not accumulation of virions at the cell surface. This could very well correspond to the Pf grow on RPl+ and R1822+ strains in the filaments found in a similar situation (Bradabsence of PA01 or K pili. Conversely, the ley, 1973b). Indeed, Jacobson’s data provide R1822-specific RNA phage PRRl (Olsen

PHAGE

ADSORPTION

and Shipley, 1973) will not infect strains PA01 or K in the absence of the R factor. While the pili associated with the R factors are thus quite different to those of strains PA01 and K, the existence of an R-specific RNA phage does suggest that the R-associated pili may be retractile like those of PA01 and K, assuming they are the receptors for PRRl. The isolation of a mutant with nonretractile R factor pili similar to strain K/SPfS would clearly be valuable in studying the mechanism of R factor transfer by conjugation. ACKNOWLEDGMENTS I am grateful to Mr. T. L. Pitt, Cross-Infection Reference Laboratory, Colindale Avenue, London, for the serological and phage typing of strain K/ 2PfS, and to Miss R. Pringle for helping with the manuscript. I also thank Dr. R. H. Olsen (University of Michigan) for phage PRRl and host strains. Mr. G. Duncan provided able technical assistance. REFERENCES D. E. (1966). The structure and infective process of a Pseudomonas aeruginosa bacteriophage containing ribonucleic acid. J. Gen. Microbial. 45,83-96. BRADLEY, D. E. (1972a). A study of pili on Pseudomonas aeru.ginosa. Genet. Res. 19,39-51. BRADLEY, D. E. (1972b). Evidence for the retraction of Pseudomonas aeruginosa RNA phage pili. Biochem. Biophys. Res. Commun. 47, 142 149. BRADLEY, D. E. (1972c). Shortening of Pseudomonas aeruginosa pili after RNA-phage adsorption. J. Gen. Microbial. 72, 303319. BRADLEY, D. :E. (1972d). Stimulation of pilus formation in Pseudomonas aeruginosa by RNA bacteriophage adsorption. Biochem. Biophys. Res. Commun. 47, 1086-1087. BRADLEY, D. E. (1973a). A pilus-dependent PseuBRADLEY,

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domonas aeruginosa bacteriophage with a long noncontractile tail. Virology 51, 489492. BRADLEY, D. E. (1973b). The adsorption of the Pseudomonas aeruginosa filamentous bacteriophage Pf to its host. Can. J. Microbial. 19, 623-631. BRADLEY, D. E. (1973c). The length of the filamentous Pseudomonas aeruginosa bacteriophage Pf. J. Gen. Viral. 20,249-252. BRADLEY, D. E. (1973d). Basic characterization of a Pseudomonas aeruginosa pilus-dependent bacteriophage with a long non-contractile tail. J. Viral. 12, 1139-1148. BRADLEY, D. E., and ROBERTSON, D. (1968). The structure and infective process of a contractile Pseudomonas aeruginosa bacteriophage. J. Gen. FEARY,

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T. W., FISHER, E., and FISHER, T. N. (1964). Isolation and preliminary characteristics of three bacteriophages associated with a lysogenic strain of Pseudomonas aeruginosa. J. Bacterial. 87, 196-208. HEUMANN, W., and MARX, R. (1964). Feinstruktur und Funktion der Fimbrien bei dem sternbildenden Bakterium Pseudomonas echinoides. Arch. Mikrobiol. 47, 325-337. JACOBSON, A. (1972). Role of F pili tion of bacteriophage fl. J. Viral. LAWN, A. M. (1967). Simple

in the penetra10,835-843. immunological labelling method for electron microscopy and its application to the study of filamentous appendages of bacteria. Nature (London) 214, 1151-1152. MAYER, F., and SCHMITT, R. (1971). Elektronenmikroskopische, diffraktometrische und discelektrophoretische Untersuchungen an Fimbrien des sternbildenden Bodenbakteriums Pseudomonas echinoides und einer nicht-sternbildenden Mutante. Arch. Mikrobiol. 79, 311326. OLSEN, R. H., and SHIPLEY, P. (1973). Host range and properties of the Pseudomonas aeruginosa R factor R1822. J. Bacterial. 113, 772-780. TAKEYA, K., and AMAKO, K. (1966). A rod-shaped Pseudomonas phage. Virology 28, 163-165.