Adsorption of bacteriophages specific for Pseudomonas aeruginosa R factors RP1 and R1822

Vol. 57, No. 3, 1974

BIOCHEMICAL

ADSORPTION

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

OF BACTERIOPHAGES AERUGINOSA

SPECIFIC

R FACTORS RPl David

FOR PSEUDOMONAS

AND RJ.822

E. Bradley

Department of Zoology, Univeraity of Edinburgh, West Maine Road, Edinburgh EH9 3JT, Scotland. Received

February

13,1974

SUMMARY Short, thick pill were found by eleotron microscopy on bacteria carrying the P group drug resistance plaamids RPl and The Rl822-specifio phage PRFU was seen to adsorb to Rl822. the bases of the pili. Three RPl-specific phagea, one filamentous (Pf3), and two with very thick capsids (PR3, PR4), were seen to attach all around the surface of P. aeruginosa celle, and were thought to be somatic, since pilus phagee appear to be strictly polar on this species. PR3 and PR4 also lysed a strain of E. coli containing an N group plasmid, suggesting a relationship between the N and P group plasmids. INTRODUCTION Following specific (12), for

for three

the

the

the

to

RPl

subject

nm) filamentous

diameter

capsid

lipid-containing

of this

thought

to

have

usually

visible;

Pf3

being

hexagonal

with

a thickened

a short this

PM2 (8). (60 nm) remains

in

a brief unusually

PR3 and PR4 are outline

inner Unlike easily

and having

layer PM2, detached

to be confirmed.

is

structure

communication,

Isolates

obtained

University,

their

Is a typical,

R1822

been

(Monash

While

(PRRl)

R factor

have

by V. Stanisich

type.

phage

P group phages

communication).

is necessary.

one another,

of an RNA phage

different

plasmld

personal

description

Isolation aeruainosa

morphologically

primarily

(760

recent

Pseudomonas

related

Australia, not

the

(Fig.

short similar a 65 nm

8) like

the

PR3 and PR4 are tail,

not

Preliminary

Vol. 57, No. 3, 1974

electron

BIOCHEMICAL

microscope

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

observations

phages to sensitive

bacteria

on the adsorption

are described

of all

four

here.

MATERIALS AND METHODS Bacteria follows:

and phages were very kindly

E. coli

PR3 and PR4 by Dr V. Stanisich;

AS19 and AS19 RPl (15), Mr T. L. Pitt, E. coli

Public

Health

E. coli

18s and 18s RPl by

Laboratory,

London;

55-3 and 55-3 Rl.99 by Mr R. Thompson (Edinburgh). K/lPO4-RPl

The spot test activity.

plicity

by

with mixing

was used for

log.

was prepared from double

inoculating

phase bacteria After

grids

(5).

from agar

rabbits.

phages were prepared

of between 5 and 20.

were mounted on support

extract

K

bacteriophage

RPl pill

free

adsorbed

mutant of strain

determining

against

a cell

of bacteria

cells

microscopy

was used for

1SS RPl:

plates

Bacterial

is a pilus-less

Antiserum

P. aeruginosa layer

and P. aeruglnosa

Central

P. aeruginosa

lytic

as

CR34 R1822 and phage PRRl by Dr R. H. Olsen

phages Pf3,

(12);

supplied

for

electron

with phage to a multi-

20 mfnutes

at 3'i"C,

(5) and negatively

cells

stained.

RESULTS AND DISCUSSION The lytic Table

activities

1, showing their

both P. aeruginosa limited an

activity

on E. coli

plaques extracted particular

for

they

electron

interest

carrying

the R factor Confirmation

of PR4 on E. coli

were obtained;

contained

microscopy

because

on strains

of

PR3 and PR4 also have some

group plasmid.

dilutions

phages are compared in

RPl- and RlS22-specificity

and E. coli.

N oompatlbillty

by plating

of the four

(1).

RI-99 (ll), was obtained

55-3 Rl99. viriona

(as Big.

This result

the DNA of RPl (P group) a94

Very turbid 8) when

is of has a 5C$

Vol. 57, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

TABLE 1 LYTIC ACTIVITY OF BACTERIOPHAGESAS SHOWNBY THE SPOT TEST

Sensitivity

to phages*

Strain PR3

PR4

Pi3

PRRl

18s RPl

+

+

+

+

K/lPO4-RPl

+

+

+

+

AS19 RPl

+

+

+

+

CR34 Rl822

+

+

+

+

(+I

(+I

-

-

P. aeruginosa 18s

E. co11 AS19

55-3 55-3 R199

* + indicates clearing and sensitivity to a phage, (+) lndiaates partial olearing and sensitivity, Phages were and - no clearing and resistance. grown on strains not carrying bacteriocins active on the test bacteria.

homology with

DNA of the N group plasmid

R-specific bodiesr five After

one part

parts

of log.

mounting

for

pili

were identified

of antiserum

eought on E. co11 strains.

by labeling

(see above)

phase baoterial electron

390 (10).

oulture

miaroscopy, Fig.

anti-

was incubated for

with

two hours.

R-srpecifla

1 showe a heavily

895

with

plli

were

labeled

pilus

Vol. 57, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Fig. 1. Antibody-labeled RPl pilua on E. coli AS19 RPl with uncoated wcommonW pili and a flagellum (top). X 125,000. E. co11 CR34 Rl822 preparecl as for Fig. 1. X 125,000. Fig. 2. (arrowed) E. 001i CR34 R1822 with PRIU viriona Figs. 3, 4. attached to R1822 pili, the cell surface, and flagella. NAN is a bacteriocln rod. X 125,000. P. aeruainoaa Fig. 5. 18s RPl with PRRl adsorbed strictly to the pole. X 125,000. 896

Vol. 57, No. 3, 1974

BIOCHEMICAL

on an AS19 RPl unlabeled strain

it

"common"

plli

CRS4 R1822

(no

obvious, the

cell;

and

seemed

apparent

labeling

polar

of

relationship,

to the

the

plli

PRRl

of the

PAOl.

It

was shown that

virions

to

the

being

at

3 was

from

the

graphs

cell

Fig.

the

pili

It

PRRl

adsorbs

The unusually

short

F-pili),

polar With

preferentially

which

plli

18s RPl,

to the

surface

tailed

pili,

like

it

that

they

and disappear

RPl pull

PRRl

the

phages

897

cell.

virions an example

attached

wall

these

in away

micro-

other

known

6 nm of the

(Fig.

5) as do a

7, 14). at

to the

(4).

attached

cell (6,

RNA

9 nm thick

virions

located

bases

the

P. aeruginosa

phages are

the

the

of the

pili,

the

pulling

cell

about

with

pilus

into

the

than

associated

polar

also

that

are

to

pili,

as do all

thicker

similar

is

The cell

at

R pili

This

on P. aeruginosa

a flagellum.

P. aeruginosa

P. aeruginosa

retract

are

pili

CRY4

few Purl

the

considered to

is

usually

of P. aeruginosa

thought

A very

(9).

E. 0011

to adsorb

away from

is

4).

had retracted,

(3,4).

to

to

plasmid)

some particles

pilus.

show that

nm for

9,

any known

The

same plasmid

adsorbed

was found

to

an expected

may be the

and had no virions

single

wnormal"

they

whioh

surface

4;

typical,

phages.

ls

indicates

(Figs.

more

as opposed

anti-PPl

generally

surface

cell

on P. aeruginosa.

non-specifically)

Fig.

number

the

the

With

2) were

(Fig.

around

the

covered.

is

of those

with

cell

shown in

(presumably

(9.5

associated

from

strain

R pili

R pili

PP7 (2),

(not

found

this

all

with

of pill

were

which

RESEARCH COMMUNICATIONS

distinguishable

F@l and Rl822

bases

RNA phage

with

to appear

since

at

clearly

common pili),

The RNA phage XL822

la

location

R1822

AND BIOPHYSICAL

the

cell

Alternatively

"Normal" pole,

surface

and as the

Vol. 57, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

Fig. 6. Phage PR4 adsorbed to P. aeru&nosa K/lPO4%Pl. X 36,000. Fig. 7. Phage PR3 adsorbed to E. coli CR34 RPl. Some of the virions are arrowed. X 36,000. Fig. 8. PR4 virions extracted from a spot test "plaque" on E. ooli Suspeated tails may have become detached. 55-3 R199. P. aeruginosa K/lPO4'RPl with adsorbed x 200,000. Fig. 9. filamentous phage Pf3. X 50,000.

898

Vol. 57, No. 3, 1974

pili

could

R pili

break

with

seem to

BIOCHEMICAL

Phage

pili E.

the

pole

would

(Fig.

expect are

(Fig.

entous

Phage

Pf3 Pi3

R factor

results

thus

somatic

receptors be that

fully

or disappear

very

short

be more

in

pill

agreement

communication), to any

which

one of the

suggesting

a common

on P. aeruglnosa virions the

four

(peripheral) synthesis

of which

like

the

is

that

receptors:

a

off,

for

PRRl

or perhaps

they

adsorb

results

of Stanlsich

mutants always

However,

explanation

selected

two different the

kinds

would

as resistant to

different

and adsorbed

to

(personal

resistant the

and

it retract

the

899

and R1822

which

with

may be under

RPl

to pili

This

(polar)

K/lPO4-RPl

Alternatively

visible.

are

filam-

N group

pill

not

suggest

of

communication).

are

of R pili

the

and In

strain

personal

results

receptor.

wall

pole,

obtained

PR3 and PR4.

phages

one

is where

cell

P. aeruginoea

show that

pili,

when the

PR3 and PR4 adsorb

which

for

were

(Iyer,

by breaking

strain

of any visible

phage

Pf3,

as

independently

of these

for

Pf3,

Ike

this

the

cells,

to

to RPl

for

(13).

preference

to the

to be somatic

phage

two different

could

to

appears

The Implication for

phages

was adsorbed

18s RPl

since

no preference

somatic

filamentous

can code

location

PR3 attached

Similar

7).

9).

with

of

phage

(Fig.

polar

around

adsorbed

does not

and Thomas

had a slight

phage

coli

26 nm in

PR4 adsorbed

frequently

a strictly

typical

isolate

this

about

of Olsen all

to E.

or breakage

RNA phage

adsorbed

If

different

retraction

estimate

similar

6).

CR34 R1822

manner

the

virions

located.

coli

pili

the

is

a typical

PR3 virions

case with

.K/lPo4-RPl;

where

is

near

is

situation

PRRl,

PRRl

whloh

was the

This

adsorbed

occur.

diameter,

the

off.

AND BIOPHYSICAL RESEARCH COMMUNICATIONS

Pf3, of

same control.

the

rest,

locations PR3 and PR4 receptor,

Vol. 57, No. 3, 1974

BIOCHEMICAL

AND BIOPHYSICAL

RESEARCH COMMUNICATIONS

REFERENCES 1. 2. 3. ihi: ;: 8.

9. 10. 11. 12. 13. 14.

15.

Bradley, D. E. J. Roy. Yioroscop. Sac. 84, 257 (1965). 45, 83 (1966). Bradley, D. E. J. Gen. liorobiol. Bradley, D. E. Bioohem. Biophys. Res. Commun. 47, 142 (1972). Bradley, D. E. J. Gen. Microbial. 72, 303 (1972). Bradley, D. E. Can. J. Microbial. 1 , 623 (1973). Bradley, D. E. J. Virol. l2, 1139 P1973). In press. Bradley, D. E. and Pitt, T. II. J. Gen. Virol. Espejo, R. T. and Canelo, E. S. Virology 34, 738 (1968). Genet. Res. Camb. Holloway, B. W. and Richmond, M. H. 21, 103 (1973). Ingram, L. C. J. Bacterial. z, 1130 (1973). Meynell, E. and Datta, N. Genet. Res. Camb.

1,134 (1966).

Olsen, R. H. and Shipley, P. J. Baoteriol. 12, 772 (1973). Olsen, R. H. and Thomas, D. D. J. Virol. 12, 1560 (1973). Pemberton, J. M. Virology 22, 558 (1973). Nat. Acad. Sskiguchi, M. and Iida, S. Proc. z, 2315 (1967).

900

Soi.

U.S.A.