Comparative Study on F-Type Pyocins of Pseudomonas-aeruginosa

J. Biochem. 89, 1721-1736 (1981)

Comparative Study on F-Type Pyocins of Pseudomonas-aeruginosa

Kazufumi KURODA and Makoto KAGEYAMA

Mitsubishi-Kasei Institute of Life Sciences, Minamiooya, Machida, Tokyo 194

Received for publication, November 28, 1980

Pseudomonas aeruginosa strain PAF41 was found to produce a new F-type pyocin,

pyocin F3, the action spectrum of which was different from those of previously reported pyocins Fl and F2. These three F-type pyocins were compared with respect to their structure and biological properties. These pyocins were almost the same with regard to the structure and the dimensions, and have similar amino acid compositions and S values. The particle weights of these pyocins were also suggested to be similar. Analyses of subunit proteins by SDS-polyacrylamide slab gel electro

phoresis showed that these pyocins were composed of 5 major (bands 1, 2, 3, 4, and 6) and 2 minor (bands 5 and 7) subunit proteins and that no difference in the mobilities of these subunit proteins could be detected among the pyocins except that of the second major subunit protein (band 4), which did differ.

Pyocins Fl, F2, and F3 were immunologically cross-reactive, and carried com-

mon antigens as well as specific ones. It was shown that band 6 was a common

antigen among the three pyocins and that band 4 was antigenically different in

pyocins Fl and F3 by immunological reaction after protein blotting. Electron

microscopic observation of pyocin particles treated with anti-sera revealed that the

common antigens were located on the rod part and the specific ones were on the

fiber part.

Pyocin F3 was neutralized by both anti-F3 and anti-Fl sera showing apparent

first order rate kinetics, whereas the neutralization for pyocin Fl by these sera did

not show such kinetics, but a considerable increment of pyocin Fl activity was

observed when small amounts of the sera were added. This increment seemed to

be due to the antibodies common to pyocins Fl, F2, and F3.

A phage, which had a flexuous rod-like tail, was found to be immunologically

cross-reactive with the three pyocins and was named KFI.

Flexuous pyocins are a class of bacteriocins pro

duced by many strains of Pseudomonas aeruginosa

and have a structure similar to the tail of non-

contractile bacteriophages, such as bacteriophage

Abbreviations: SDS, sodium dodecyl sulfate; BSA,

bovine serum albumin.

2 (1, 2), and are generically named F-type pyocins (3). Among F-type pyocins, pyocin 28 was first reported by Takeya et al. (4) followed by some flexuous pyocins such as the 430f particle by Govan (5), and pyocins Fl and F2 by us (6). However, only a few biological and biophysical studies have been done, and only two F-type

Vol. 89, No. 6, 1981 1721

1722 K. KURODA and M. KAGEYAMA

pyocins (pyocin 28 and pyocin Fl) were investigated with regard to the dimensions (4, 7, 8). As we found three F-type pyocins, we attempted to

purify them to compare their dimensions, and other biochemical and biophysical properties of them.

The subunit compositions of R-type pyocins, the structure of which resembles the tail of con-tractile bacteriophages such as bacteriophage T4, were reported by Shinomiya (9) and Ohsumi et al.

(10). They found that four R-type pyocins were composed of essentially 22 similar subunit proteins, but a subunit protein supposed to be a main constituent of the fiber was a little different in molecular weight among these pyocins. It was also reported that specific antigens of R-type

pyocins were located on the distal portion of the fibers.

In contrast to R-type pyocins, few studies of subunit proteins of F-type pyocins have been reported except for our previous one (6). We reported that pyocins Fl and F2 were composed of only about 6 subunit proteins, and that they were clearly different from each other in the relative mobility of the second major band, band 4, whereas the other 5 bands were the same.

Pyocin F2 producer strain PRD125 used in the previous study (6) was a recombinant of two

parents, PML14 and PAO3012 (Kageyama, M., unpublished data). Therefore, it is of interest to see from which strain pyocin F2 is derived. PAF41 is known to produce an S-type pyocin, AP41 (11), which was recently purified by Sano and Kageyama

(unpublished). Another killing activity was found with PAF41, which was attributed to a new F-type pyocin, named pyocin F3. Pyocins Fl, F2, and F3 were cross-reactive immunologically. Therefore, it is of interest to elucidate which subunit proteins are antigenically common to or specific for these pyocins, and on what part each subunit protein is located.

The present paper reports the comparison of

biochemical and biophysical properties of pyocins

F2 and F3 with pyocin Fl, an attempt to assign

the subunit proteins to the antigens common to

and specific for these F-type pyocins, and the

locations of the antigens on the pyocin particles.

MATERIALS AND METHODS

Bacterial Strains-Pseudomonas aeruginosa strains PML1540 (formerly P15-40 (6)), PRD125

(formerly M12-E5 (6)), and PAF41 (11) were used as pyocinogenic strains for pyocin Fl, F2, and F3, respectively. As indicator strains, Pseudomonas aeruginosa strain GG8 (for pyocins Fl and F2) and PML14 (for pyocin F3, formerly P14 (12)) were used. Govan and Gillies' strains for pyocin typing and their pyocin producer strains (13), NIH typing strains (14), and PAOI and PAO3012

(15), and PML4, 1505, 1516, 1516d, 1516f, and 15163 (formerly P4, etc. (16, 17)) were also used as indicator strains.

Preparation and Assay of Pyocins F1, F2, and F3-The procedures of induction and purification, and the media were described previously (6). All

pyocin samples were finally purified by sucrose density gradient centrifugation except where other-wise noted (6). Pyocin activity was assayed by the serial dilution method (18). To measure the killing activity of pyocin more precisely, the effect of pyocin on the colony formation of sensitive cells was determined (6).

Amino Acid Analysis, Analytical Ultracentrifu

gation, and SDS-Polyacrylamide Gel Electropho

resis-Amino acid analysis, analytical ultracen

trifugation, and SDS-polyacrylamide gel electro

phoresis were described previously (6, 8). The

standard proteins used for molecular weight cali

bration were as follows: rabbit muscle phospho

rylase b (94,000), bovine serum albumin (BSA)

(67,000), hen ovalbumin (43,000), bovine eryth

rocyte carbonic anhydrase (30,000), soybean tryp

sin inhibitor (20,100), and bovine milk ƒ¿-lactal

bumin (14,400). These were all products of

Pharmacia Fine Chemicals. SDS-polyacrylamide

gel electrophoresis was done with 4 M urea, that

is, the stacking gel and the separating gel contained

4 M urea (19).

Electron Microscopy-All specimens were negatively stained with 2% uranyl acetate (6, 8). Pyosin-antibody complexes were observed by the following two procedures; A) a carbon-coated collodion grid was floated on a drop of pyocin-antibody reaction mixture for about 1 min and then washed with distilled water. B) a carbon-coated collodion grid was first floated on a drop

J. Biochem.

COMPARATIVE STUDY ON F-TYPE PYOCINS 1723

of pyocin solution and then washed with distilled

water. The grid was then floated on a drop of

appropriately diluted antiserum and incubated at

37•Ž for 2 h or more. Finally the grid was washed

with distilled water (10).

Detection of Subunit Proteins Antigenically

Common to or Specific for Pyocins F1, F2, and

F3-The separation of subunit proteins was done

by SDS-polyacrylamide gel electrophoresis with

4M urea. The transfer of proteins from the gel

to nitrocellulose filters (BA85, Scheicher and

Schull) was done as follows according to the

procedure described by Bowen et al. (19). After

electrophoresis, a gel strip (about 12 x 5 cm2) was

immersed in about 300 ml of urea-containing buffer

(transfer buffer containing 4 M urea, see below)

and gently agitated for 3 h at room temperature.

The gel was sandwiched between two strips of

nitrocellulose, and the "sandwich" apparatus was

submerged in 2 liters of transfer buffer (0.05M

NaCl, 2mM Na-EDTA, 0.1mM dithiothreitol, 10

mM Tris-HCl, pH 7.0) for about 40h (three or

four changes, total). Transfer of proteins from

the gel to the nitrocellulose filters occurred during

this time by diffusion. Immunological detection

of proteins with horseradish peroxidase-conjugated

anti-rabbit IgG goat serum (Miles-Yeda Otd.) on

nitrocellulose filters was performed according to

the procedure described by Towbin et al. (20),

except that no carrier serum was used. Each

blotted nitrocellulose filter was washed with 50 ml

of saline buffer (0.01M Tris-HCl, pH 7.4, contain-

ing 0.9% NaCl) at room temperature (three or

more changes for 30min), and soaked in 50ml of

3 % BSA in saline for 1 h at 37•Ž. After washing

with 50ml of saline for 10 min, it was incubated

with 30ml of anti-pyocin serum (rabbit) appro

priately diluted with 3 % BSA in saline for 2 h at

37•Ž. The filter was washed with 50ml of saline

(five or more changes during 30min, total), then

incubated with 30ml of horseradish peroxidase

conjugated anti-rabbit IgG goat serum appro

priately diluted with 3 % BSA in saline for 2 h

at 37•Ž. For the color reaction, the filter was

soaked in 30 ml of 0.01M Tris-HCl, pH 7.4, con

taining 0.01 % H2O2, and 0.75 mg of o-dianisidine

(21). The reaction was terminated after 30 min

by washing with distilled water.

Serological Methods-Antisera against pyocins

Fl and F3 were prepared as described previously

(6). Pyocins Fl and F3 purified by DEAE-cellu

lose column chromatography (0.70 mg of pyocin

Fl or 0.91 mg of F3 for the first injection, and

1.10 mg of F1 or 0.77mg of F3 for the second

injection) were used for preparations. The IgG

fraction was prepared as follows. Crude rabbit

IgG was precipitated with 40% saturated (NH4)2-

SO4, dissolved in a small volume of distilled water,

and dialyzed against 0.01M KH2PO4-Na2HPO4

buffer, pH 7.3, at 4•Ž. The IgG fraction was

pooled after passing through a column of DEAE-

cellulose equilibrated with the above buffer. Anti

phage sera against PS3, PS10, and PS17 were

prepared as previously (16, 6, 22).

Neutralization of pyocins with antisera was

done as follows. A sample was mixed with an

equal volume of antiserum appropriately diluted

with dilution buffer (DB: 10mm Tris-HCl, 85mM

NaCl, 1mM MgCl2, pH 7.6) at 37•Ž, and samples

were withdrawn at appropriate intervals and di

luted to 1 : 50 to stop the reaction, and remaining

pyocin activity was assayed by the serial dilution

method (18). Neutralization activity of a serum

was estimated by the method described before (16)

and was expressed by the K value calculated ac-

cording to the formula; K=2.3 x (D/t) x log(P0/

P), where D is the final dilution of antiserum in

the pyocin-serum mixture, Po the pyocin activity

at 0 time, and P the activity at t min in the mix

ture. Calculation of the K value was made only

when it was verified that the reaction obeyed

apparent first order kinetics, and was done by the

following two procedure; (a) varying the sampling

time t at constant D and (b) varying the dilution

D at constant t. The K value shown in this paper

is the mean value of those obtained by the above

two procedures, for which almost the same K

values of an antiserum were given in every case.

Ouchterlony's precipitation reaction was carried out as described previously (6). Precipitin bands were stained as follows. After immersing in about 2 liters of 0.9%. NaCl solution for 2-3 days (4-6 changes, total), the gels were stained with 0.5 % Amide black 10B-5 % acetic acid for 30 min at room temperature and destained in 2% acetic acid.

Preparation of Anti-F-Type Pyocin IgG Aborbed with Heterologous F-Type Pyosin-Anti-F

type pyocin IgG absorbed with heterologous F-type pyocin was prepared as follows (10). An

Vol. 89, No. 6, 1981


anti-pyocin IgG solution in 0.01M KH2PO4-

Na2HPO4 buffer, pH 7.3, was mixed with a suffi

cient amount of heterologous pyocin solution of

0.01M Tris-HCl, 0.1M NaCl buffer, pH 7.5, and

incubated overnight or more at 37°C. The pres

ence of remaining pyocin activity was checked by

spot tests with PML14 (for pyocin F3) or with

GG8 (for pyocin Fl). Centrifugation at 156,000

x g for 3 h at 4•Ž in a Hitachi 65P ultracentrifuge

was done to remove the residual pyocins and the

antigen-antibody complexes. The absorbed IgG

solutions obtained still had neutralizing activity

against the homologous pyocins, but no activity

against the pyocin used for absorption.

RESULTS

Origin and Specificity of Three F- Type Pyocins

-Pseudomonas aeruginosa strains PML15 and

PRD125 have been found to produce F-type

pyocins, pyocin Fl and pyocin F2, respectively

(6). Pseudomonas aeruginosa strain PAF41 was

also found to produce a new F-type pyocin, named

pyocin F3. Pyocin F3 could be purified by the

same procedure as that for pyocin F1 (6). These

F-type pyocins were produced most effectively at

34•Ž. When cultured at 37-40•Ž, F-type pyocin

activity of the mitomycin C lysate was only about

1/10-1/100 of that obtained at 34•Ž.

PRD125, the pyocin F2 producer, was a recombinant derived from PML14 and PAO3012. Both PML14 and PAO3012 were found to produce flexuous rod-like particles by electron microscopic observation of the mitomycin C lysates of the strains. The flexuous rod-like particles could be

purified by the same procedure as that used for the purification of pyocin FI (6). The purified

particles from both PML14 and PAO3012 showed killing activity. These two F-type pyocins showed the same action spectra as that of pyocin F2 of PRD125 against the 54 indicator strains tested. Namely, they killed the following strains; NIH1, 2, 5, 6, 8, 13, 14, 17, 19, 22, 25, and 27, GG1, 2, 3, 4, 7, 8, A, D, Fl, and 2285, PML4, 15, and 1505, but did not kill the following; NIH strains other than those described above of 27 strains, GG5, 6, B, C, E, 21, and 430, PML14, 1516, 1516d, 1516f, and PRD125, and PAO3012. This shows that both PML14 and PAO3012 produce the same F-type pyocin, pyocin F2. This was

confirmed by the fact that the electrophoretic

patterns of their subunit proteins were the same as that of pyocin F2 of PRD125 on SDS-polyacrylamide slab gel electrophoresis with and without 4 M urea (data not shown). Thus, it was concluded that two independent strains produced the same F-type pyocin.

Pyosins Fl, F2, and F3 showed different action spectra (Table I). Govan reported five F-type pyocin activities detected by the modified pyocin typing technique using cellulose acetate membranes (5). The action spectra of pyocins Fl, F2, and F3 were different from any of Govan's five F-type pyocins. The specific activity, the activity divided by the A280 value of the purified

pyocin sample, of pyocin F3 against PML14 was about 106 units per A280. This specific activity was about 10 times higher than those of pyocins Fl and F2 against the best indicators tested for them (GGE for pyocin Fl, and GG8 for pyocin F2), and was almost the same as that of pyocin Ri.

TABLE I. Action spectra of pyocins Fl, F2, and F3.

+: Inhibition of the indicator strain was observed. -: no inhibition was observed .

J. Biochem.


Killing Action-The time courses and the extent of killing with various amounts of pyocin F3 are shown in Fig. Ia. The initial velocity of killing and the final levels of survival depended on the dose of pyocin. A linear correlation was obtained between the logarithm of the final survival and pyocin dose down to a survival ratio of about 0.2% (Fig. 1b), indicating that the killing action of pyocin F3 was a single-hit process. As reported previously, pyocin F1 required some cofactor for killing to occur (6). Pyocin F3 as well as pyocin Fl kills the sensitive cells in DB containing 1 % polypeptone, but not in DB alone (which was composed of Tris, NaCl, and MgCl2).

Structure of Pyocins F1, F2, and F3-The structure and dimensions of pyocin Fl have been reported previously (8). To compare the struc-

tures and dimensions of pyocins F2 and F3 with pyocin Fl, electron microscopic observations of these pyocins were made. The structures of pyocins F2 and F3 (Fig. 2a and b) were very similar to that of pyocin F1 (8). One end of the rod seemed to be square, but the other end tapered off to a point at which a fiber-like structure was observed. Regular striations were also present in the rod part.

The length distributions of the rod and the

fiber parts of the three pyocins were investigated.

The length of the rod parts were almost the same;

for 87 % of a total of 66 rods (FI), for 96 % of 77

rods (F2), and for 88 % of 146 rods (F3), it was

105.0•}9.7nm. In the case of the fiber part, the

longest filament was measured. 88 % of a total

of 66 fibers (Fl), 94% of 83 fibers (F2), and 86

Fig. 1. Mode of killing action of pyocin F3. (a) Time course of the killing action in DB +1 % polypeptone. The relative pyocin F3 doses were 1.5 (•), 2.0 (A), 3.0 (O), and 4.0 ([]). One relative pyocin F3 dose was equivalent to 1.48 x 10-4 (absorbance at 280 nm) and the number, of cells (strain PMLI4) was 1.0 x 108 per ml at zero time in the reaction mixture. The percentage survival was the ratio of the number of surviving cells to that obtained in the control experiment at each time. A control experiment was carried out simultaneously adding 0.1 ml of 0.01M Tris-Cl-0.1M NaCl buffer, pH 7.5, to 0.9 ml of cell suspension, instead of 0.1 ml of the pyocin F3 solution. (b) Relation of survival ratio to pyocin dose. The final levels of survival percentage were obtained from (a).

Vol. 89, No. 6, 1981


Fig. 2. Electron microscopy of pyocins F2 and F3. (a)

pyocin F2, (b) pyocin F3. The bars represent 100 nm.

of 153 fibers (F3) showed a length of 50.1 +9.7

nm.

The distributions of width and striations of the three pyocins were as follows. The widths seemed to be uniform; 75 % of a total of 174 striations (Fl), 83% of 183 striations (F2), and 85 % of 160 striations (F3) were 9.6 f 1.0 nm. The most frequent number of striations in the rod part of the three pyocins was found to be 23 excluding the distal part; 54% of 111 rods (Fl), 49% of 74 rods (F2), and 51 % of 161 rods (F3) gave the number of 23.

Results obtained here for the size and the number of striations of pyocin F1 are in good agreement with the previous observation (8) (rod, 105.5+9.5 nm x 10.0±1.4 nm, and 23 striations: fiber, 43.0+12.0 nm). Some differences in the

TABLE II. Amino acid compositions of pyocins F], F2, and F3. Values are expressed as mol percent and

are averages of the values obtained with 24- and 48-h hydrolysates except where otherwise noted. The data

of pyocin FI are quoted from our previous report (7).

a Values estimated by linear extrapolation to zero time

of hydrolysis. b Values obtained with 18-h hydrolysate

of the performic acid oxidized sample. c Values esti

mated spectrophotometrically.

fiber length may be due to observational errors

because the filament of the fiber was very thin

and not well resolved. Thus, not only the shapes

but also the sizes of pyocins Fl, F2, and F3 are

very similar.

Amino Acid Analysis and Analytical Ultracentrifugation-The amino acid compositions of pyocins F2 and F3 are shown in Table II. That of

pyocin Fl reported previously (8) is also shown in the table for comparison. Pyocins Fl, F2, and F3 showed a close resemblance in the composition. A characteristic feature of the F-type pyocins is the high contents of Gly, Glu (Gin), Asp (Asn), Ser, Thr, and hydrophobic amino acids, which was

J. Biochem.


pointed out by Yui to be common to the proteins forming the quartenary structure (23). Another feature is the high Pro contents of these F-type

pyocins. These values are much higher than those of pyocin RI sheath, pyocin RI core, and tail-core of T-even bacteriophage (24), but similar to that of bacteriophage 2 tail (25). The partial specific volumes of pyocins F2 and F3 were calculated to be 0.73 ml/g using the values in Table 11 by the method of Cohn and Edsall (26).

The S20,w values of pyocins F2 and F3 were calculated to be 34.45 and 34.7S, respectively, by analytical ultracentrifugation using the above values of partial specific volume. Centrifugation was performed at 25,619 rpm for pyocin F2 (A280=0.484) and at 25,606 rpm for pyocin F2 (A280=0.454). The values obtained were almost the same as the S20,w value (34.4S) of pyocin Fl (A280= 0.511). Therefore, the S020 values of pyocins F2 and F3 should also be very similar to pyocin F1 (35.15) (8).

Serological Properties-When the killing ac

tivities of pyocins Fl, F2, and F3 were measured

by spotting on a lawn of indicator strains contain-

ing anti-Fl or anti-F3 serum, every pyocin was

Fig. 3. Neutralization of pyocin F3 by anti-F1 and

anti-F3 sera. Dependence on the serum concentration

of the neutralization by anti-F1 (a) and anti-F3 (b)

sera is shown. Pyocin activity was measured at 5 min

and one relative serum concentration was equivalent to

1/800 dilution (a) and to 1/2,000 dilution (b) of each

original serum in the reaction mixture. Pyocin activity

in the reaction mixture was 6.4•~103 units at 0 time in

the reaction mixture.

Fig. 4. Neutralization of pyocin Fl by anti-Fl and anti-F3 sera. Depend

ence on the serum concentration of the neutralization by anti-F1 (a) and anti-F3 (b) sera is shown. Pyocin activity was measured at 5 min, and one

relative concentration was equivalent to 1/400 dilution of each original serum,

and pyocin Fl activity was 3.2 x 103 units at 0 time in the reaction mixture.

Vol. 89, No. 6, 1981


neutralized. Thus, pyocins Fl, F2, and F3 were immunologically cross-reactive. The neutralizing activities of these antisera against pyocins Fl and F3 were further studied quantitatively. As shown in Fig. 3, pyocin F3 was neutralized apparently by first order kinetics not only with anti-F3 (Fig. 3b) but also with anti-Fl (Fig. 3a) serum. The K values of anti-Fl and anti-F3 sera against pyocin F3 were calculated to be 780min-1 and 540min-1, respectively.

Figure 4 shows the neutralization of pyocin

F1 by the antisera. The neutralization did not

show such a simple exponential dependence on

the antiserum concentration as was the case with

pyocin F3. Instead, the pyocin Fl activity in-creased by two to four times in the presence of

small amounts of anti-Fl or anti-F3 serum, and

then the exponential neutralization began with

increasing amounts of the antiserum. This phe

nomenon was also observed with purified anti-Fl

Fig. 5. Neutralization of pyocins Fl and F3 by the absorbed IgG solutions with heterologous pyocins.

(a) Dependence on the IgG concentration of the neutralization of pyocin Fl by anti-Fl IgG absorbed with

pyocin F3. Pyocin activity was measured at I min (0) and at 2min (A). One relative IgG concentration was equivalent to 1/20 dilution of the original absorbed IgG solution in the reaction mixture. (b) Dependence on the IgG concentration of the neutralization of

pyocin F3 by anti-F3 IgG absorbed with pyocin Fl. Pyocin activity was measured at 5 min. One relative IgG concentration was equivalent to 1/20 dilution of the original absorbed IgG solution in the reaction mixture. Pyocin Fl and F3 activities were 3.2 x 103 units and 6.4 x 103 units in the reaction mixture, respectively.

IgG, but the IgG fraction treated for 10 min at

100•Ž, or the antisera against phages independent

of F-type pyocin such as anti-PS3, anti-PS10, and

anti-PS17 sera showed neither an increment nor

neutralization. The neutralization of pyocins Fl

and F3 by antisera absorbed with heterologous

pyocins was investigated. As shown in Fig. 5,

the anti-Fl IgG absorbed with pyocin F3 or anti-

F3 IgG absorbed with pyocin Fl could still neu

tralize pyocin Fl or pyocin F3, respectively. Not

only pyocin F3 but also pyocin FI was neutralized

by first order kinetics. These results suggest that

the increment of pyocin Fl activity must be due

to some components of anti-F type pyocin sera,

probably to a common antibody against pyocins

Fl and F3.

Figure 6 shows the results of Ouchterlony's immunoprecipitation test. Either anti-Fl or anti-F3 serum gave a precipitin band with every pyocin. When the central well contained anti-Fl IgG (Fig.

Fig. 6. Ouchterlony's immunoprecipitation reaction on agarose gel. Wells numbered 1, 2, and 3 contained

pyocins Fl, F2, and F3, respectively. The central wells contained, (a) the purified anti-Fl IgG (A280= 2.02), (b) the anti-Fl IgG absorbed with pyocin F3 (concentrated with an AMICON concentrator using a UM2 membrane and corresponded to about 4.1 times enrichment of the anti-F1 IgG used in (a)). (c) the original anti-F3 serum, and (d) the anti-F3 serum absorbed with pyocin Fl (concentrated by the same method as in (b) and corresponded to about 0.9 times enrichment of the original serum used in (c)). Absorbance at 280 nm of each pyocin sample was about 4.2

(pyocin Fl), about 3.9 (F2), and about 4.0 (F3) in (a), (c), and (d), and was 0.366 (Fl), 0.262 (F2), and 0.214 (F3) in (b). About 20 pl of sample was put into each well.

J. Biochem.


6a), spurs were seen at the fusion points between pyocin F1-F2 and F1-F3. In the case of anti-F3 serum in the central well (Fig. 6c), spurs were seen between pyocin F3-F1 and F3-F2. The anti-Fl IgG absorbed with pyocin F3 still formed a precipitin band with pyocin Fl, but not with

pyocin F2 or F3 (Fig. 6b). The anti-F3 IgG absorbed with pyocin FI showed the reverse situation (Fig. 6d). These results show that pyocins Fl, F2, and F3 have some antigenically common components, and that pyocins Fl and F3 have some specific ones.

Subunit Proteins of Pyocins Fl, F2, and F3-In a previous paper (6) it was shown that pyocins Fl and F2 were composed mainly of 6 subunit

proteins. By SDS-slab gel electrophoresis, the

mobility of band 4 was different though those of the other five bands were the same in the two

pyocins. The subunit proteins of pyocin F3 were compared with those of pyocins Fl and F2 by SDS-slab gel electrophoresis with or without 4m urea. As shown in Fig. 7a, the electrophoretic

pattern of pyocin F3 was similar to those of pyocins Fl and F2; 5 major bands (bands 1, 2, 3, 4, and 6) and 2 minor bands (bands 5 and 7) were observed in the system without 4M urea. In the figure, several weak bands can be seen between band I and band 2. But their mobilities and intensities are not always reproducible. They might be due to insufficient dissociation. The mobility of band 4 of pyocin F3 was different from either that of pyocin Fl or pyocin F2, and

Fig. 7. SDS-polyacrylamide slab-gel electrophoresis of pyocins Fl, F2, F3, and calibra

tion proteins. Fl, Pyocin Fl; F2, pyocin F2; F3, pyocin F3; cal, calibration proteins.

About 0.7ƒÊg (phosphorylase b), 0.8ƒÊg (bovine serum albumin), 1.5ƒÊg (ovalbumin), 0.8ƒÊ

g (carbonic anhydrase, indicated by "car"), 0.8 ƒÊg (trypsin inhibitor), and 1.2ƒÊg (ƒ¿-

lactalbumin) were applied. (a) SDS-polyacrylamide slab-gel electrophoresis without

4M urea. About 15 beg (Fl), 14ƒÊg (F2), and 15ƒÊg (F3) were applied. (b) SDS-poly

acrylamide slab-gel electrophoresis with 4M urea. About 10ƒÊg (Fl), 9ƒÊg (F2), and

10ƒÊg (F3) were applied. (c) A gel strip stained after protein transfer. After SDS-poly

acrylamide slab-gel electrophoresis with 4 M urea of the same amounts of samples de

scribed in (b) was performed, the proteins on the gel strip were transferred to nitrocellulose

filters. Then the gel strip was stained by the same procedure used in (a) and (b). See " MATERIALS AND METHODS ."

Vol. 89, No. 6, 1981


no differences in the mobilities of the other 6 bands were detected among the three pyocins. The molecular weight of band 4 of pyocin F3 was estimated to be 42,500. Figure 7b shows the electrophoretic patterns of the three pyocins on SDS-slab gel electrophoresis with 4M urea. The

patterns were similar to those obtained for the system without 4M urea, and the molecular weights of corresponding bands were the same as estimated with both systems (bands 1-6). In the system with 4M urea, band 4* (M.W. 36,000) appeared in pyocin F2, and some smaller proteins were better resolved (6*, 7*, 8*). A band below band 6* of pyocin F3 (Fig. 7b) was not usually observed and was regarded as an impurity.

TABLE III. Molecular weights of subunits of pyocins Fl, F2, and F3 and number of each subunit per pyocin

particle.

Column a; Percent amounts of subunit proteins, which were roughly estimated from the areas of each peak on

densitograms. Column b; The numbers of subunit proteins per pyocin particle, which were calculated from the

quantitative ratios assuming that the molecular weight of the F-type pyocin was 3.2 x 106 daltons. SDS-polyacrylamide slab gel electrophoreses were performed without and with 4M urea (see "MATERIALS AND METHODS").

Molecular weights were calculated from several slab gels of each sample. The molecular weights of bands 1, 2, 3,

4, 5, and 6 of pyocin F1 and F2 were the same as reported previously (5). Because the peak of band 7 was broad, the molecular weight of it is an approximate value. The bands marked * were observable only in the system with

4 m urea.

The ratios of the amounts of subunit proteins were roughly estimated from the area of each peak on the densitogram of the stained gel (Table III). The ratios of corresponding subunit proteins of

pyocins Fl, F2, and F3 were approximately the same in the system without 4 m urea (Table III). Even in the system with 4 m urea, the ratios were approximately the same assuming that the amount of band 4 of pyocin F2 was the sum of those of bands 4 and 4* (Fig. 7b). Band 6 is probably a component of the rod part because of its highest content and the largest number in the pyocin

particle. The ratio of band 4, the molecular weight of which was different among the three

pyocins, was second in quantity.

J. Biochem.

CO

MPA

RA

TIV

E S

TU

DY

ON

F T

YPE

PY

OC

INS

1731

Fig.

8.

D

etec

tion

of

subu

nit

prot

eins

of

py

ocin

s F1

, F2

, an

d F3

w

ith

hors

erad

ish

pero

xida

se-c

onju

gate

d an

ti-ra

bbit

IgG

go

at

seru

m

afte

r pr

otei

n bl

ottin

g.

Aft

er

elec

trop

hore

sis,

a

slab

ge

l w

as

sand

wic

hed

by

nitr

ocel

lulo

se

filte

rs

so

that

pr

otei

ns

in

the

gel

wer

e tr

ansf

erre

d to

th

e tw

o fi

lters

. (a

) an

d (b

) ar

e th

e fi

lters

, th

e pr

otei

ns

on

whi

ch

wer

e tr

ansf

erre

d fr

om

one

gel,

and

(c)

and

(d)

from

an

othe

r,

resp

ectiv

ely.

T

here

fore

, th

e pa

ttern

s of

pr

otei

ns

on

the

set

of

filte

rs

are

sym

met

rica

l. E

ach

filte

r w

as

trea

ted

with

ab

out

30m

l of

an

ti-Fl

Ig

G

solu

tion

(A28

0 0.0

10)

(a),

of

an

ti-F3

Ig

G

solu

tion

(A28

0 0.0

12)

(b),

of

an

ti-Fl

IgG

ab

sorb

ed

with

py

ocin

F3

(c

),

of

anti-

F3

IgG

ab

sorb

ed

with

py

ocin

Fl

(d

),

and

phag

e PS

10

antis

erum

(e

).

The

am

ount

s of

th

e ab

sorb

ed

IgG

us

ed

for

(c)

and

(d)

wer

e ad

just

ed

such

th

at

the

conc

entr

atio

ns

of

spec

ific

an

tibod

ies

for

pyoc

in

Fl

and

F3

wer

e co

mpa

rabl

e to

th

ose

used

in

(a

) an

d (b

),

resp

ectiv

ely.

T

hen,

ea

ch

filte

r w

as

trea

ted

with

abo

ut

30m

l of

hor

sera

dish

pe

roxi

dase

-con

juga

ted

anti-

rabb

it Ig

G

goat

se

rum

di

lute

d to

1/

500

(1/2

50

for

(e))

of

the

orig

inal

se

rum

, an

d co

lor

reac

tion

was

pe

rfor

med

. Fl

: py

ocin

F1

(a

bout

8l

ig),

F2

: py

ocin

F2

(ab

out

8 pg

),

F3:

pyoc

in

F3 (

abou

t 4

pg),

an

d ca

l: ca

libra

tion

prot

eins

(t

he

sam

e am

ount

s as

sho

wn

in t

he l

egen

d to

Fig

. 7)

wer

e ap

plie

d.

1, 2

, 3,

4,

and

6 sh

ow

the

band

nu

mbe

rs.

"car

" sh

ows

carb

onic

an

hydr

ase.

B

ands

4

of p

yoci

ns

Fl

and

F3 a

re s

how

n by

arr

ows.

M

arks

(o

) sh

ow

the

posi

tions

of

ban

d 6

and

band

4

dete

cted

.

Vol

. 89,

No.

198

1


The Detection of Subunit Proteins Antigenically Common to and Specific for Pyocins F1 and F3-Pyocins Fl, F2, and F3 have some common antigens and specific ones as revealed by the neutralization test with anti-F type pyocin Sera (see above). Attempts were made to elucidate which subunit proteins are antigenically common or specific. For this purpose the method of protein blotting described by Bowen et al. (19) was employed together with an immunological method using horseradish peroxidase-conjugated anti-rabbit IgG goat serum (20).

Figures 7b and c show gels stained with Coo

massie brillant blue before and after blotting with

nitrocellulose filters, respectively. The efficiency

of transfer to nitrocellulose seems to vary with

proteins as roughly estimated from the staining of

gels before and after blotting. In general, proteins

of high molecular weight tended to be transferred

less efficiently, but bands 4 of pyocins Fl, F2, and

F3 and carbonic anhydrase were not transferred

efficiently although their molecular weights were

relatively low. Band 6 of every pyocin as well

as ovalbumin, trypsin inhibitor and ƒ¿-lactalbumin

disappeared completely after blotting.

Subunit proteins were detected on the nitrocellulose filters with a horseradish peroxidaseconjugated goat serum specific for rabbit IgG. Figures 8a, b, c, d, and e show the nitrocellulose filters, to which the subunit proteins of pyocins Fl, F2, F3, and calibration proteins were transferred from the gels, and treated with anti-F1 IgG, anti-F3 IgG, anti-F1 IgG absorbed with pyocin F3, anti-F3 IgG absorbed with pyocin Fl, and anti-PS10 serum, respectively. The bands ap

peared as the result of interactions between the subunit proteins and the anti-F type pyocin IgG, because treatment of a nitrocellulose filter with anti-phage PS10 serum (the phage is independent of F-type pyocins) gave no bands (Fig. 8e).

Band 1, 2, 3, 4, and 6 can be seen in Fig. 8a

and b. The reason why band 5 was not detected

is not clear. Besides the above bands, several

bands which were not seen on staining the acryl

amide gel were also seen. This is probably at

tributable to the higher sensitivity of this detection

method than that of staining with Coomassie

brilliant blue. Band 4 of pyocin F3 was seen

more clearly in the case with anti-F3 IgG than

with anti-F1 IgG. This result was confirmed using

much larger quantities of pyocins Fl, F3, and anti-sera (Fig. 9). Furthermore, band 4 of F3 was detected by treatment with the anti-F3 IgG absorbed with pyocin F1 (Fig. 8d), but not with the anti-Fl IgG absorbed with pyocin F3 (Fig. 8c). Band 4 of pyocin Fl showed the reverse situation (Fig. 8, c and d). These results suggest that bands 4 of pyocins F1 and F3 contain the antigen specific for each pyocin. No clear-cut results were obtained for bands 1, 2, and 3 of

pyocin Fl, as these were observed in both c and d. The reason for these is not known.

Band 6 was detected to be the major com

ponent of the three F-type pyocins by both direct staining of the gels with Coomassie brilliant blue and treatment with the horseradish peroxidaseconjugated anti-rabbit IgG goat serum of the

Fig. 9. Detection of subunit proteins of pyocins Fl

and F3. The procedures of detection were the same

as in the legend to Fig. 8. Each filter was treated with

about 30ml of anti-F1 (a) and anti-F3 (b) sera . Each

serum was diluted to 1/100 of the original one. The

filter was treated with about 30ml of the goat serum

diluted to 1/150. Fl: pyocin Fl (about 15ƒÊg), F3:

pyocin F3 (about 7ƒÊg), and cal: calibration proteins

(the same amounts as shown in the legend to Fig. 7)

were applied. "car" shows carbonic anhydrase.

J. Biochem.


Fig. 10

Fig. 11. Electron microscopy of the complex between

pyocin F3 and anti-pyocin F3 IgG absorbed with

pyocin Fl. Electron micrographs were obtained by

procedure A described in "MATERIALS AND METHODS," or solutions of pyocin F3 and the anti-

body were mixed and the reaction mixture was put on

a collodion grid. The bars represent 100nm.

Fig. 10. Electron microscopy of pyocin-antibody com

plexes. (a) Pyocin F3 treated with anti-pyocin F3 IgG. Protrusions can sometimes be seen as shown by

arrows. (b) Pyocin Fl treated with anti-pyocin F3 IgG. (c) Pyocin F3 treated with anti-pyocin F3 IgG

absorbed with pyocin Fl. The bars represent 200 nm

(a, b) and 100 nm (c). Electron micrographs were obtained by procedure B described in " MATERIALS

AND METHODS," or pyocins fixed on a collodion

grid were reacted with a solution of antibody.

Vol. 89, No. 6, 1981


filters treated with non-absorbed antisera. But it

was not detected at all by the same treatment of

the filters treated with the absorbed antibodies

with the heterologous pyocin (Fig. 8, c and d).

Band 6 of pyocin F2 was not detected by the

procedure using the absorbed antisera, either. These results show that the antibodies against

band 6 were completely absorbed with the heter

ologous pyocin, or band 6 was antigenically

common to the three F-type pyocins.

Electron Microscopic Observation of Pyocin-Antibody Complexes-In order to estimate the location of the common and the specific antigens on the structures of pyocins F1 and F3, electron microscopic observations were made with pyocin-

Fig. 12. Electron microscopy of phage KF1. An

electron micrograph of the confluent lysate obtained

from strain PML28 infected by KF1 is shown. The

bar represents 200nm.

antibody complexes. The images of the com

plexes obtained by procedure B are shown in Fig. 10 (see " MATERIALS AND METHODS"). When pyocin F3 or F1 fixed on the grids was treated with anti-F3 IgG, both pyocin particles were covered with antibody molecules (Fig. 10, a and b). The differences between the complex of pyocin F3 and that of pyocin F1 were not clear, but protrusions can sometimes be seen at one end of the pyocin F3-anti-F3 IgG complexes. Figure 10c shows the complexes between pyocin F3 and anti-F3 IgG absorbed with pyocin Fl, indicating that no antibodies can be seen on the rod part, though the fiber part was not clear. Figure 11 shows the complexes between pyocin F3 and the pyocin F3 IgG absorbed with pyocin Fl obtained by procedure A. No antibodies can be seen on the rod parts, and most of the pyocin

particles appear to be attached to each other at the fiber part, and antibody molecules can be seen on the fiber parts of some particles. It may be concluded that the common antigens are located on the rod part and that the specific antigens are involved in the fiber parts.

A Phage Neutralized with Anti-F Type Pyocin

Sera-A search was made for phages which were

immunologically cross-reactive with the F-type

pyocins. Only one out of 56 phages tested was neutralized with anti-pyocin F1 and anti-pyocin

F3 sera, and it was named phage KF1. Phage

KF1 was produced by strain NIHS of Pseudomonas

aeruginosa, and had a flexuous rod-like tail, resem

bling F-type pyocins (Fig. 12).

DISCUSSION

The structures of pyocins Fl, F2, and F3 were

very similar to one another, and no appreciable

difference was detected in the length distribution

and in the striation number among the pyocins.

The amino acid compositions and S values of

these pyocins were also similar. Therefore, the

particle weights of pyocins F2 and F3 should be comparable to that of pyocin Fl (3.32 x 106 daltons

(8)). The electrophoretic patterns of subunit pro

teins of the pyocins were similar, and no differences

in the relative mobilities of corresponding bands

were detected among these pyocins except those of

band 4 (Fig. 7). Pyocins Fl, F2, and F3 were

immunologically cross-reactive, and band 6 was

J. Biochem.


found to be immunologically common among the

pyocins (Fig. 8). The quantitative ratios of corresponding subunit proteins were also approximately the same. Thus, these F-type pyocins were very similar with regard to the structure, the dimensions, and the chemical composition.

As shown in Table III, the numbers of each subunit protein in one pyocin particle were calculated assuming that the particle weights of pyocins F2 and F3 were equal to that of pyocin Fl. As band 6 was found to be a common antigen and the rod part appeared to be composed of a com-mon antigen, the main protein band 6 seems to be a component of the rod part. The number of band 6 in one pyocin particle was calculated to be from 108 to 122, and its molecular weight was 19,500. On the rod part of each pyocin, 23 annuli were observed by electron microscopy. Therefore, one annulus seems to be composed of 5 subunit proteins. Assuming that the subunit

protein was spherical and the hydration ratio was 1.3, the diameter of the protein was calculated to be 4.7nm using the value of partial specific volume

(0.73 ml/g). This shows the length of the rod part to be 108.1 nm (23 x 4.7nm), which agrees well with the 105.0+9.7 nm obtained from electron microscopic observations. However, a de-tailed study is required for the analysis of architecture of F-type pyocins.

Band 4 appeared to be specific antigens and specific antigens were located on the fiber part. Therefore, the second major band, band 4, may be a component of the fiber part.

The specific activity, the pyocin activity di-vided by the A280 of the pyocin sample, of pyocin F1 or pyocin F2 was about one tenth of that of

pyocin F3 (about 106 units/A280). One killing unit of pyocin F3 was calculated to be about 100 molecules by the procedure described previously

(6), and was about one third of that of pyocin Fl (6). The reason for the higher specific activity of pyocin F3 is not known.

Pyocin Fl activity was enhanced 2 to 4 times

in the presence of small amounts of anti-F1 serum,

anti-F3 serum, or anti-F1 IgG, but pyocin F3

activity was not. This enhancement of pyocin

Fl activity was not observed on addition of non-

specific sera, anti-Fl IgG, heated at 100°C for 10

min, or anti-Fl IgG absorbed with pyocin F3.

This suggests that the enhancement is caused by

interactions between the pyocin FI particle and the antibodies common to pyocins Fl and F3. It is of interest to elucidate the mechanism of the enhancement of pyocin Fl activity with anti-F type pyocin antibodies. Jerne reported that some component of anti-T4 antibodies stabilized permanently the active state of phage T4 which had been activated by tryptophan (27). This component was contained in small concentrations in the sera from some normal nonimmunized animals, but it could be increased by specific immunization against T4. The amount of this component increased enormously within a week after a single injection of phage T4 antigen into the animal, i.e. before the serum acquired any appreciable

phage inactivating power. Though he did not describe what component served as the stabilizer, the phenomenon was similar to the activation of pyocin F1 with antibodies common to the F-type pyocins.

The protein blotting method described by Bowen et al. (19) was a useful and simple technique, but care must be taken for quantitative analysis, because the efficiency of transfer from a

gel strip to nitrocellulose filters was not always sufficient and varied with proteins used. The detection with horseradish peroxidase-conjugated anti-rabbit IgG goat serum showed several bands besides those detected by staining with Coomassie brilliant blue. As control experiments with treatment with anti-phage PS10 rabbit serum or with-out rabbit serum did not show these bands, they may be antigens of pyocins Fl, F2, and F3 which were detected because of the higher sensitivity of the method using the goat serum. Carbonic anhydrase was detected, though the other five calibration proteins were not, with the two different lots of samples (S631 and S380) of horseradish

peroxidase-conjugated goat serum specific for rabbit IgG not only after the pretreatment with anti-pyocin or phage sera (Figs. 8e and 9) but also without rabbit sera (Figs. 8e, 9a, and 9b). It was not known whether carbonic anhydrase had some structure which could bind the goat serum, or anti-carbonic anhydrase antibodies were accidentally contained in the goat serum used. Care must be taken to detect proteins with the

goat serum.

Vol. 89, No. 6, 1981


We thank M. Kobayashi for her important contribu

tion in finding pyocin F3, Dr. T. Maeda for calculation

of the diameter of the protein, band 6, and Dr. A.

Kikuchi for his useful discussion on the protein blotting

method.

REFERENCES

1. Bradley, D.E. (1967) Bacteriol. Rev. 31, 230-3142. Amako, K. & Yasunaka, K. (1979) Tampakushitsu

Kakusan Koso (in Japanese) 24, 719-7263. Kageyama, M. (1979) Tampakushitsu Kakusan Koso

(in Japanese) 24, 871-8804. Takeya, K., Minamishima, Y., Amako, K., &

Ohnishi, Y. (1967) Virology 31, 166-168

5. Govan, J.R.W. (1974) J. Gen. Microbiol. 80, 17-30

6. Kuroda, K. & Kageyama, M. (1979) J. Biochem. 85,749

7. Takeya, K., Minamishima, Y., Ohnishi, Y., &

Amako, K. (1969) J. Gen. Virol. 4,145-149

8. Kuroda, K., Kageyama, M., Maeda, T., & Fujime, S. (1979) J. Biochem. 85, 21-28

9. Shinomiya, T. (1972) J. Biochem. 72,499-510

10. Ohsumi, M., Shinomiya, T., & Kageyama, M.

(1980) J. Biochem. 87, 1119-112611. Holloway, B.W., Rossiter, H., Burgess, D., &

Dodge, J. (1973) Genet. Res., Comb. 22, 239-253

12. Kageyama, M. (1974) J. Gen. Appl. Microbiol. 16, 269-2751

3. Govan, J.R.W. & Gillies, R.R. (1969) J. Med. Microbiol. 2,17-25

14. Farmer, J.J. & Herman, L.G. (1969) Appl. Micro

biol. 18,760-765

15. Kageyama, M., Sano, Y., & Shinomiya, T. (1979) J. Bacteriol. 138, 748-755

16. Ito, S. & Kageyama, M. (1970) J. Gen. Appl. Microbiol. 16, 231-240

17. Ito, S., Kageyama, M., & Egami, F. (1970) J. Gen.

Appl. Microbiol. 16, 205-214

18. Kageyama, M. & Egami, F. (1962) Life Science No. 9, 471-476

19. Bowen, B., Steinberg, J., Laemmli, U.K., & Weintraub, H. (1980) Nucl. Acids Res. 8,1-20

20. Towbin, H., Staehelin, T., & Gordon, J. (1979) Proc. Natl. Acad. Sci. U.S. 76, 4350-4354

21. Avrameas, S. & Guklbert, B. (1971) Eur. J. Immu

nol. 1, 394-39622. Kageyama, M., Shinomiya, T., Aihara, Y., and

Kobayashi, M. (1979) J. Virol. 32, 951-95723. Yui-Furihata, C. (1972) Tampakushitsu Kakusan

Koso (in Japanese) 17, 165-17524. Hasegawa, T. & Ishi, S. (1979) J. Biochem. 85,

403-41125. Buchwald, M., Steed-Claister, P., & Simmno

vitch, L. (1970) Virology42, 375-38926. Schachman, H.K. (1957) in Methods in Enzymology

(Colowick, S.P. & Kaplan, N.O., eds.) Vol. 4, pp. 32-103, Academic Press, New York

27. Jerne, N.K. (1956) J. Immunol. 76, 209-216

J. Biochem.

Documents

Comparative Study on F-Type Pyocins of Pseudomonas-aeruginosa