Phage Tail-Like (High-Molecular-Weight) Bacteriocins of Budvicia aquatica and Pragia fontium (Enterobacteriaceae)

Electron microscopic analysis of contractile phage tail-likebacteriocins of three Pragia fontium strains and one Budviciaaquatica strain was performed. Fonticin and aquaticin are remarkablyheat sensitive but trypsin resistant. Simultaneous productionof contractile and flexible phage tail-like bacteriocins inthe P. fontium 64613 strain is shown for the first time.

INTRODUCTION

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Introduction

Phage tail-like (high-molecular-weight [HMW]) bacteriocins havebeen described in many species of gram-negative and gram-positivebacteria (4, 8, 11, 12, 15, 18, 21). Generally, phage tail-likebacteriocins belong to two different families: contractile phagetail-like (R-type) and noncontractile but flexible ones (F-type)(17). The former family is similar to tails of P2 or T-evenphages of Escherichia coli (virus family Myoviridae), whilethe morphology of the latter family resembles the flexible tailsof ${lambda}$ phage of E. coli (virus family Siphoviridae). Nakayama etal. (19) confirmed the relationship of R-type pyocin to P2 phageand F-type pyocin to coliphage ${lambda}$ . Simultaneous production ofR-type and F-type bacteriocins was described in Pseudomonasaeruginosa strains (10, 16). The narrow specificity of phagetail-like bacteriocins and the absence of nucleic acid in theirparticles may play an important role in the construction ofnew therapeutic agents with directed action against bacterialstrains (7, 23).

$S$ marda (22) analyzed five strains of Pragia fontium and ninestrains of Budvicia aquatica, the hydrogen sulfide-producingspecies of Enterobacteriaceae (1, 2, 5), for production of bacteriocin-likeagents. All strains produced contractile phage tail-like particles,each killing two to six strains of the same set. Here, we confirmand extend these data and present a detailed comparative studyof the morphology of these elements.

Culture conditions.

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Culture conditions.

Strains of P. fontium 24613, 24647, and 25240 and B. aquatica24522, kindly donated by E. Aldová (National Instituteof Public Health, Prague, Czech Republic), were cultured inTY broth (8 g casein enzymatic hydrolysate type I, 5 g yeastextract powder [HI Media Laboratories, India], 5 g NaCl per1,000 ml H₂O). The maximum spontaneous production of phage tail-likeparticles was after 12 h at 30°C and heavy shaking. Theiractivity usually ranged from 10³ to 10⁶ arbitrary units. Theproduction of phage tail-like particles could be raised by 1to 2 orders of magnitude by UV light (Philips TUV 254-nm 15-Wgermicidal tube; 60 s from 50-cm distance) or mitomycin C (1µg/ml) induction. Enhanced titers were used in all experiments.None of the phage tail-like bacteriocins we examined is plasmidencoded; no plasmid could be detected in cells of any of thestrains.

Activity of isolated phage tail-like particles.

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Activity of isolated phage...

The phage tail-like bacteriocins of both Budvicia and Pragiakilled sensitive strains of both species (Table 1), and thelethal effect was not transmissible in series. The standardagar double layer procedure was used in cross-experiments. Inpositive combinations, very narrow (0.7 to 2.5 mm wide) inhibitionzones were formed: they were mostly clear, partly turbid, withisolated tiny colonies. No strain was sensitive to its own bacteriocin.All phage tail-like bacteriocins were temperature sensitive(Table 2) and lost their activity completely after 10 min attemperatures of 45°C to 60°C. On the other hand, theyshowed a high degree of resistance to trypsin (Table 3). Intergenericactivity was tested on seven Klebsiella spp. strains, five bacteriophage-and low-molecular-weight colicin-sensitive indicator strainsof E. coli and one Shigella sonnei colicin indicator strain.In the 52 combinations tested, only 3 were positive: fonticinof the strain P. fontium 24342 inhibited one Klebsiella sp.strain plus the strain of E. coli ${varphi}$ , while that of the strainP. fontium 24613 inhibited the strain E. coli B. The other 49combinations were negative. All bacterial strains under studyoriginated from the collection at the Faculty of Medicine, MasarykUniversity, Brno, Czech Republic.

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TABLE 1. Strain-specific lethal action of HMW bacteriocins

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TABLE 2. Heat sensitivity of HMW bacteriocins

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TABLE 3. Trypsin sensitivity of HMW bacteriocins^a

Morphology of phage tail-like particles.

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Morphology of phage tail-like...

The negatively stained samples (see Fig. 1, 2, and 4 below)were prepared as follows: drops of phage tail-like bacteriocinsuspension were applied onto glow discharge-activated (3) Formvar-carbon-coatedgrids. After adsorption (30 s), the grids were floated on 1-mldrops of 1% (wt/vol) ammonium molybdate, pH 7.1, for 10 min.Finally, the grids were negatively stained with 2% (wt/vol)uranyl acetate. The samples were analyzed in a Philips CM100electron microscope.

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FIG. 1. Contractile phage tail-like particles, aquaticin, produced by B. aquatica strain 24522: a mixture of native and contracted particles and their released components. Some bullet-like intact particles with base plate and attached tail fibers are marked with arrows. Intact bacteriocin particles show a typical cross-striation pattern. Contracted particles with a protruding core and shortened sheath with clearly visible tail fibers are marked with asterisks. Some of the polysheaths formed by at least two contracted sheaths are marked "ps," and contracted sheath monomers are marked "cs." The sample was prepared from cultivation media concentrated by ultracentrifugation. The image was recorded on Kodak 4489 EM film at a primary magnification of x46,000. The negative was scanned with a Minolta Multi Pro film scanner in TEM mode at 2,400 pixels per inch. Bar, 200 nm.

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FIG. 2. Set of typical structures found in cultivation media of P. fontium strain 24647. (A) Native form; (B) contracted form of R-type particle; (C) "polycore" composed of two units; (D) polysheath composed of five contracted sheaths (pentamer). In panel B, the remainders of the basal plate are clearly visible on the distal end of the contracted sheath. Bar, 50 nm.

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FIG. 4. Simultaneous production of contractile phage tail-like particles (R-type) and flexible F-type particles by P. fontium strain 24613. (Inserts) Higher magnifications of marked areas with intact F-type particles. The image was recorded using a MegaView II digital slow scan camera. Nine full-frame images (12 bits; 1,280 by 1,024 pixels; 3-by-3 matrix), which covered about 15 µm² of the negatively stained sample on a support film, were recorded and stitched together using the MIA module functions of the Analysis 3.2 software. Only part of the recorded area is shown. The F-type particles were found at low frequencies (less than 5%) compared with the occurrence of intact contractile R-type particles (arrows) and contracted sheaths (asterisks). The sample was prepared from cultivation medium concentrated by an Ultrafree-15 centrifugal device (Millipore). The images were recorded at a primary magnification of x46,000, which gives a pixel size of 1.37 nm, resulting in an image resolution of 2.7 nm, according to the Nyqiust criterion. Bars: main image, 500 nm; inserts, 50 nm.

The cultivation media of all tested bacterial strains (P. fontium24613, 24647, and 25240 and B. aquatica 24522) contain phagetail-like particles resembling isolated tails of T-even bacteriophagesof E. coli. A narrow, hollow inner core encompassed by a widerouter sheath, which is contractile but not flexible, forms theirtypical structure. A perpendicular basal plate endowed withspikes and several tail fibers complements this structure. Twosuperimposed streams of spiral coiling of the contractile protein,including an angle of some 45°, forming the sheath, arerecognizable by a general regular superficial cross-striationpattern, oblique to the length (Fig. 1).

Contracted tail-like forms show two structural components: anative narrow full-length core protrudes from the contractedsheath through the central aperture of the base plate or onboth ends. The spiral coiling of the sheath disappears withits contraction. Two- or severalfold longitudinal polymers ofcontracted sheaths show "polysheath" formations. Interestinglyenough, native sheaths never show analogous polysheath formations.Apparently, they first become contracted and eject their cores;only after that do they bind with other ones, forming polysheathsof varied numbers of monomers (Fig. 1 and 2D). Inner cores frequentlyslip out of the contracted sheaths completely; also, freed nativecores form polymers, called polycores (Fig. 2C).

The native and contracted forms of four types of contractilephage tail-like particles were measured in detail (Table 4).We did not find any significant morphological difference betweenthem. For P. fontium fonticin, the calculated length (Fig. 3,dimension A) of a native particle was 115 nm, and the width(dimension B) was 20 nm. B. aquatica aquaticin length was 107nm, and the width was 18 nm. The difference between the lengthsof native forms of fonticin and aquaticin was statisticallysignificant on the 95% level. Table 5 compares all the dimensionsof phage tail-like bacteriocins published for gram-negativebacteria with those found here for P. fontium and B. aquatica.

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TABLE 4. Dimensions of HMW bacteriocin particles produced by strains of P. fontium and B. aquatica

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FIG. 3. Dimensions measured in extended and contracted forms of phage tail-like bacteriocins. For an explanation of the symbols, see Tables 4 and 5.

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TABLE 5. Dimensions of HMW bacteriocins of gram-negative producers

Besides the contractile-type bacteriocins, the samples of cultivationmedia of P. fontium strain 24613 contained typical F-type bacteriocin(Fig. 4) at low frequencies. The presence of F-type bacteriocinparticles in P. fontium strain 24613 was a surprise. It hasnever been observed before in this strain nor in the other examinedstrains of P. fontium or B. aquatica.

ACKNOWLEDGMENTS

This research was supported by grants 310/01/0013 and 310/03/1091from the Grant Agency of the Czech Republic and by InstitutionalResearch Concept no. AV0Z50200510.

FOOTNOTES

* Corresponding author. Mailing address: Institute of Microbiology, Academy of Sciences of the Czech Republic, Víde $n$ ská 1083, CZ-142 20 Praha 4-Kr $c$ , Czech Republic. Phone: 420-241062399. Fax: 420-241722347. E-mail: benada{at}biomed.cas.cz.

REFERENCES

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