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Analysis of exopolysaccharide (EPS) production mediated by the bacteriophage adsorption blocking plasmid, pCI658, isolated from Lactococcus lactis ssp. cremoris HO2
International Dairy Journal 9 (1999) 465}472
Analysis of exopolysaccharide (EPS) production mediated by the bacteriophage adsorption blocking plasmid, pCI658, isolated from Lactococcus lactis ssp. cremoris HO2 Amanda Forde , Gerald F. Fitzgerald * Department of Microbiology, National Food Biotechnology Centre, University College, Cork, Ireland Departments of Microbiology and Food Science and Technology, National Food Biotechnology Centre, University College, Cork, Ireland Received 18 November 1998; accepted 10 April 1999
Abstract Lactococcus lactis ssp. cremoris MG1363 harbouring pCI658, a 58 kb plasmid originating in L. lactis ssp. cremoris HO2, adsorbs phages 712 (936 phage species) and c2 (c2 species) less e$ciently than the plasmid-free phage sensitive strain. The presence of an alkali-soluble loose &#u!y' pellet following centrifugation of the phage resistant derivative was established. In addition this pCI658-containing strain possessed a more hydrophilic cell surface and did not agglutinate with the glucosyl-speci"c lectin concanavalin A. Furthermore, electron micrographs also illustrated signi"cant plasmid-mediated alterations of the cell surface. HPLC analysis of the loosely associated extracellular material revealed that galactose and glucuronic acid appeared to be its major components. It was concluded that pCI658 encodes the production of a hydrophilic exopolysaccharide which masks cell surface receptors causing a dramatic decrease in bacteriophage adsorption. 1999 Published by Elsevier Science Ltd. All rights reserved. Keywords: Exopolysaccharide; Plasmid; Bacteriophage resistance; Adsorption inhibition
1. Introduction Lactococcal starter cultures used in industrial practice are constantly threatened by lytic bacteriophage attack, a phenomenon which poses serious consequences for a variety of dairy fermentation processes (Neve, 1996). Research has demonstrated that many lactococci possess inherent resistance to phage infection due to the presence of native phage resistance plasmids. These antiphage systems have been organised into four groups depending on the manner in which they operate: adsorption inhibition, DNA penetration blocking, restriction/modi"cation and abortive infection (reviewed by Dinsmore & Klaenhammer, 1995; Garvey, van Sinderen, Twomey, Hill & Fitzgerald, 1995; Daly, Fitzgerald & Davis, 1996; Allison & Klaenhammer, 1998). Mechanisms which hinder phage attachment to cell surfaces have previously been associated with the development of bacteriophageinsensitive mutants which exhibited reduced receptor * Corresponding author. Tel.:#353-21-902730; fax:#353-21903101/276318. E-mail address: g."[email protected] (G.F. Fitzgerald)
availability (Vlegels, Hazeleger, Helmerborst & Wouters, 1988) or with plasmids encoding the production of exopolysaccharides (EPS) which coat the cell surface (Sijtsma, Sterkenburg & Wouters, 1988; Sijtsma, Jansen, Hazeleger, Wouters & Hellingwerf, 1990; Lucey, Daly & Fitzgerald, 1992). Several investigations have demonstrated the role of non-covalently bound extracelluar material in mediating defective phage adsorption to lactococcal cells (Sijtsma et al., 1988; Lucey et al., 1992; Gopal & Crow, 1993). Other bacterial exopolysaccharides have also been proven to be bene"cial by conferring antigenic speci"city (Wu & Park, 1971; Forsen, Niskasaari & Niemitalo, 1985), protection against dessication and phagocytosis (Ophir & Gutnick, 1994; Pasquier, Marty, Dournes, Chabanon & Pipy, 1997) and some o!er biological advantages such as antiviral and anti-tumour activities (Oda, Hasegawa, Komatsu & Tsuchiya, 1983). Furthermore, microbial polysaccharides have actual or potential applications as thickening agents leading to improved texture, viscosity and smoothness of mouthfeel in a wide variety of fermented dairy products and some biopolymers have non-food industrial usages such as in the
0958-6946/99/$ - see front matter 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 1 1 5 - 6
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oil industry (Sutherland, 1990, 1998). A growing interest has developed in the manipulation of exopolysaccharides of food-grade lactic acid bacteria for use in the food industry where they may provide a new range of thickening, gelling and stabilising agents. Several structural studies have already been performed on exopolysaccharides produced by these organisms (Doco, Wieruszeski, Fournet, Carcano, Ramos & Loones, 1990; Nakajima, Hirota, Toba & Adachi, 1992; Gruter, Lee#ang, Kuiper, Kamerling & Vliegenthart, 1993; Robijn, Thomas, Haas, van den Berg, Kamerling & Vliegenthart, 1995a; Robijn, van den Berg, Haas, Kamerling & Vliegenthart, 1995b; Bubb, Urashima, Fujiwara, Shinnai & Ariga, 1997; Lemoine, Chirat, Wieruszeski, Strecker, Favre & Neeser, 1997) and this aspect continues to be an expanding area of research in the light of their industrial importance. de Vos (1996) also addressed the topic of &metabolic engineering' which could improve the yield or alter the structure of exopolysaccharides produced by lactic acid bacteria. This work presents the characterisation of the exopolysaccharide produced by a strain harbouring the adsorption blocking phage resistance plasmid pCI658. This 58 kb molecule was initially identi"ed following conjugal co-transfer with the lactose plasmid from the parent Cheddar cheese starter culture Lactococcus lactis ssp. cremoris HO2 to the laboratory host L. lactis ssp. cremoris MG1363. A derivative designated Tc-AF021 Lac\, which was cured of the lactose plasmid but still contained pCI658, exhibited the production of a hydrophilic exopolysaccharide which acted as a potent barrier against bacteriophage infection. A remarkable reduction in the ability of the small isometric-headed
712 to adhere to this derivative was previously observed and while the prolate-headed c2 had the capacity to adsorb to the strain, it was noted that the cell remained partially insensitive based on the production of hazy plaques of decreased diameter (Forde, Daly & Fitzgerald, 1999). The system bears some similarity to that mediated by the adsorption inhibition plasmid pCI528 formerly described by Lucey et al. (1992). However, the current study has allowed the identi"cation of features which distinguish between the two plasmids and the exopolysaccharides they encode.
2. Materials and methods Bacterial strains and bacteriophages. The bacterial strains and bacteriophage used in this study are listed in Table 1. Lactococcal strains were grown at 303C in M17 medium (Terzaghi & Sandine, 1975) containing 0.5% glucose (GM17) or lactose (LM17) as required. Escherichia coli V517 was grown at 373C with aeration in Luria Bertani (LB) medium (Sambrook, Fritsch & Maniatis, 1989). Stocks of all cultures were maintained at !203C in 40% glycerol. Phage 712 was propagated on L. lactis ssp. cremoris MG1363 at 303C in GM17. Bacteriophage plaque assays. Bacteriophage plaque assays were performed by adding 0.1 ml of an overnight culture, 0.1 ml of 0.185 M CaCl and 1 ml of the appropri ate phage dilution to 3 ml of sloppy GM17 agar (0.7%) and overlaying onto prepared GM17 agar (1.5%) plates. Plates were incubated at 303C. Plasmid proxle analysis. Plasmid DNA was extracted using the method of Anderson and McKay (1983) and separated by electrophoresis on 0.7% agarose gels in TAE bu!er (40 mM Tris}Acetate, 2 mM EDTA, pH 8.0) at 100 V for 3 h. Resuspension rate of pelleted cells. Cells from 1 ml of fully grown GM17 cultures were pelleted in a bench centrifuge at 13,000 g for 30 s. Pellets were resuspended in GM17 broth by shaking on a vortex at speci"c time periods (0, 2, 5 10, 20, 30 and 40 s). The OD was then immediately measured as a function of vortexing time. Hydrophobicity studies. The cell surface hydrophobicity of strains was determined by measuring their adsorption to n-octane in a two-phase partitioning system based on the technique of Rosenberg, Gutnick and Rosenberg (1980). Cells (50 ml) from an 18 h culture were centrifuged at 13,000 g for 10 min and resuspended in 200 ml PUM bu!er (per litre, 22.2 g K HPO ) 3H O, 7.26 g KH PO , 1.8 g urea and 0.2 g MgSO ) 7H O, pH 7.1) to give an OD of 0.5. A 4 ml fraction of the cell suspension was added to test tubes containing various volumes (0.0, 0.5, 1.0, 1.5 and 2.0 ml) of n-octane. Following vigorous vortexing for 2 min at room temperature the samples were allowed to stand for 30 min to permit separation of the aqueous and hydrocarbon layers. The aqueous layer was
Table 1 Bacterial strains and bacteriophages Strain/phage
Plasmid content (kb)
Comment/source
L. lactis ssp. cremoris HO2 MG1363 Tc-AF021 Lac\
58, 46, 42, 22.5, 8.9, 4.5 Plasmid free 58
Wild-type pCI658-containing host, UCC Culture Collection FP> Derivative of L. lactis ssp. cremoris 712 (Gasson, 1983) FP\, phage sensitive Transconjugant containing pCI658, derived from HO2;MG1363 mating and cured of the co-transferred lactose plasmid FP>, phage resistant (Forde et al., 1999)
Phages
712 (936 phage species) FP"&Flu!y' pellet.
Small isometric-headed phage lytic for MG1363
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carefully extracted and examined spectrophotometrically at 600 nm. A decrease in the optical density was used as a measure of cell surface hydrophobicity. Chemical treatment of cell surfaces. 1.5 ml portions of cultures (containing approximately 10 cfu ml\) were washed and resuspended in one of the following: Triton X-100 (1%, w/v, in distilled H O, 30 min at 453C); SDS (1%, w/v, in distilled H O, 30 min at 453C); HCl (0.1 M, 30 min at 213C); NaOH (0.05 and 0.025 M, 30 min at 213C); pronase E (1.5 mg ml\ in distilled H O, 30 min at 373C); trypsin (1.5 mg ml\ in distilled H O, 30 min at 373C) (Sijtsma et al., 1988); trichloroacetic acid (5%, w/v, 5 min at 903C) (Chatterjee, 1969). Cells were assayed for loss of the &#u!y' pellet and phage resistance phenotypes. Agglutination of cells with lectins. A 1.5 ml aliquot of pelleted cells was washed and resuspended in 100 ll bu!er with concanavalin A (0.1% concanavalin A, 1.9% b-glycerophosphate, 0.025% MgSO ) 7H O, 10 mM cal cium borogluconate, pH 6.8) or Ptilota plumosa lectin (1 mg ml\ in 10 mM potassium phosphate bu!er, pH 7.3), each for 30 min at 223C. Agglutination was detected both visually in an eppendorf tube and by microscopic examination. Preparation of cell surface polysaccharides for HPLC analysis. Isolation of extracelluar material for chromatographic analysis was based on the procedure employed by Wallace (1980) and Sijtsma et al. (1988) with some modi"cations. Pelleted cells (3 ml) were washed twice in sterile H O (without disturbing the &#u!y' pellet during decanting steps) and resuspended in 120 ll of 30% w/v NaOH. Samples were boiled for 15 min, centrifuged at 13,000 g and the supernatants were precipitated with 60% ethanol. 15 ll samples were hydrolysed with an equivalent volume of 1 M H SO for 15 min at 1003C, and 20 ll were injected directly into an LKB 2150 HPLC apparatus (Bromma, Sweden) with a refractive index detector. The column was an Aminex HPX-87H ion exclusion column (Bio-Rad Laboratories, Richmond, California, USA) maintained at 653C using 0.01 N H SO as the elution #uid at a #ow rate of 0.6 ml min\. 1 mM sugar standards (glucose, galactose, rhamnose, arabinose, fructose and glucuronic acid) were passed through the column for comparative analysis. Electron microscopy of cell surfaces. A 1.5 ml portion of each culture (pre-grown for 18 h) was centrifuged, washed twice and resuspended in quarter strength Ringers solution. 100 ll of each suspension was mounted on a Formvar-coated copper grid and negatively stained with 100 ll 2% phosphotungstic acid for 3 min. Samples were dessicated and examined under a JEM 1200EX TESCAN transmission electron microscope at an accelerating voltage of 80 kV. Ewect of growth conditions on exopolysaccharide (EPS) production. The e!ects of various parameters on the production of EPS by Tc-AF021 Lac\ were investigated using conditions of altered pH (pH 5.0, 6.0, 7.0 and 9.0),
467
temperature (21, 30, 373C) and medium composition. For each parameter, cells were harvested at the same OD and EPS concentrations (mM) were determined by HPLC analysis as described above. Stability studies of plasmid pCI658. pCI658-containing cells were subcultured for 50 and 100 generations in GM17 broth, diluted in Ringers solution and spread plated on GM17 agar. In each case, 100 individual colonies were selected randomly and the percentage of the population which had maintained or lost pCI658 was estimated by screening for the presence or absence of the &#u!y' pellet and/or phage insensitivity phenotypes.
3. Results 3.1. Analysis of a derivative of L. lactis ssp. cremoris MG1363 harbouring the phage resistance plasmid pCI658 A conjugal derivative of L. lactis ssp. cremoris MG1363, designated Tc-AF021 Lac\, containing the phage resistance plasmid pCI658, demonstrated a soft, easily suspended &#u!y' pellet following centrifugation at 13,000 g, while the equivalent plasmid-free, phage sensitive strain (MG1363) produced a normal hard pellet. The phenomenon was estimated quantitatively in a cell resuspension assay where the times required for resuspension of pelleted cells of L. lactis ssp. cremoris MG1363 and its phage resistant variant Tc-AF021 Lac\ were compared. It was noted that almost total resuspension of Tc-AF021 Lac\ had occurred after 10 s while the time needed for complete resuspension of MG1363 extended to 20 s (Fig. 1). In order to further investigate the di!erences in cell surface composition between these strains, they were tested for their adsorption to n-octane in a two-phase partitioning system. It was essential that these hydrophobicity studies were conducted under similar growth and harvest conditions as any variations could a!ect the resulting hydrophobicity pattern obtained (Crow, Gopal & Wicken, 1995). Under stringently controlled conditions L. lactis ssp. cremoris MG1363 was found to adhere readily to octane implying that its cell surface is hydrophobic while Tc-AF021 Lac\ appeared to possess a more hydrophilic cell surface (Fig. 2). In addition, this latter strain did not agglutinate upon incubation with the lectins concanavalin A (con A) or Ptilota plumosa (Pt p) which have a$nities for glucosyl and a-D-galactosyl residues, respectively (Goldstein, Hollerman & Smith, 1965; Sijtsma et al., 1990). However, while L. lactis ssp. cremoris MG1363 also did not react with Pt p, it did aggregate with con A suggesting that the presence of pCI658 does lead to cell surface alterations. These "ndings were reinforced following electron microscopic examination of each strain where L. lactis ssp. cremoris MG1363 was shown to possess a regular
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Fig. 3. Electron micrographs of Tc-AF021 Lac\ demonstrating the presence of a layer of extracelluar material on the cell surface. Occasional "lamentous extensions of this layer make contact with adjacent cells (Magni"cation 89,000;). Fig. 1. Resuspension rates of pelleted cells of Lactococcus lactis ssp. cremoris MG1363 and Tc-AF021 Lac\ following centrifugation at 13,000 g. The results are the average of three separate independent experiments.
Fig. 2. Surface hydrophobicity of cells of Lactococcus lactis ssp. cremoris MG1363 and Tc-AF021 Lac\, as measured by partitioning of cells into the octane phase. A fall in OD indicates an increase in cell surface hydrophobicity. Each value represents the average obtained from triplicate experiments.
compact cell surface while in contrast, Tc-AF021 Lac\ showed the presence of strands or "bril-like material which emanated from the surface of the cell. Another noteworthy feature is the manner in which these occasional "lamentous extensions projected outwards from the cell and made contact with neighbouring cells (Fig. 3). 3.2. Chemical treatment of cells In order to determine the composition of the &#u!y' pellet produced by cells harbouring pCI658, freshly grown cultures of Tc-AF021 Lac\ were treated with a range of chemicals (Table 2). It was demonstrated that incubation of the pCI658-containing cells with trypsin,
Table 2 Chemical treatment and phage testing of Tc-AF021 Lac\ to determine the nature of the pCI658-encoded exopolysaccharide Treatment
&Flu!y' pellet
No treatment Triton X-100 (1%) SDA (1%) HCl (0.1 M) TCA (5%, 43C) TCA (5%, 903C) NaOH (0.025 M) Pronase E (1.5 mg ml\) Trypsin (1.5 mg ml\)
# # # # # ! ! # #
pronase E, SDS and Triton X-100 did not a!ect the loose &#u!y' pellet, suggesting that its components are neither proteinaceous nor lipid in nature. Treatment of the cells with 0.05 N NaOH at 213C for 30 min resulted in elimination of the &#u!y' pellet, implying the presence of polysaccharide material. Milder alkali treatment (0.025 M NaOH) also resulted in reversion of the culture to phage sensitivity; high 712 titres ('10 pfu ml\) generated visible plaques (however, these were not completely clear probably due to the inhibitory e!ects of NaOH on the growth of the test strain in the overlay). Loss of the &#u!y' pellet and phage resistance phenotypes provided evidence that the extracelluar material is involved in mediating reduced susceptibility to phage infection (NaOH treatment did not alter the sensitivity of plasmid-free MG1363). Treatment with trichloroacetic acid (TCA) for 5 min at 903C was also shown to remove the loose pellet phenotype from the pCI658-containing cell, but this solution did not have the same e!ect when tested at 43C. The &hot' and &cold' TCA treatments gave rise to di!erent results possibly due to hydrolysis of the EPS by the &hot' TCA treatment, while no hydrolysis took place at the lower temperature. It was not possible to determine if the &hot' TCA treatment also resulted in restoration of phage sensitivity since it caused death of the culture.
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3.3. HPLC analysis of extracted cell surface polysaccharides In order to further investigate the nature of the pCI658-encoded polymer, preparations of cell surface material were isolated and evaluated quantitatively by HPLC using 1 mM concentrations of glucose, galactose, rhamnose, arabinose, fructose and glucuronic acid as standards. Analysis of hydrolysates of crude EPS samples of Tc-AF021 Lac\ (Table 3, Fig. 4) indicated the presence of elevated levels of galactose and glucuronic acid (ratio of 1 : 3 for glucose-grown cells) when compared with the plasmid-free host MG1363. The results of preliminary experiments indicate that EPS levels are a!ected to some extent by growth conditions, being optimal between 30 and 373C and between pH 6 and 7. 3.4. Stability studies on pCI658 When the stability of pCI658 in TC-AF021 Lac\ was investigated it was discovered to be quite unstable. Following standard plaque assays with 712, 35% of the population had reverted to phage sensitivity and lost the characteristic &#u!y' pellet phenotype after 50 generations. Plasmid pro"le analysis con"rmed that pCI658 had been lost from these strains. After 100 generations, 50% of the population lacked the plasmid. Also noteworthy was the plaque forming ability of 712 on this host. Following overnight incubation, no plaques were seen on the Tc-AF021 Lac\ lawn when compared with plasmid-free L. lactis ssp. cremoris MG1363. However, when the plates were left at room temperature for another 4 days, hazy plaques ('10 pfu ml\) were apparent and by day 10, these plaques had become clearly evident.
4. Discussion Despite considerable technological advances in the control of culture inhibition caused by lactococcal bacTable 3 HPLC analysis demonstrating composition of the cell surface material extracted from L. lactis ssp. cremoris MG1363 and Tc-AF021 Lac\ (containing pCI658) grown in GM17 at pH 7 and at 303C Component
MG1363 (mM)
Tc-AF021 Lac\ (mM)
Glucuronic acid Glucose Galactose Rhamnose Formate Ethanol
0.035 0.047 0.037 0.080 2.141 29.22
1.324 0.0 0.476 0.0 2.029 5.422
May be an end-product of fermentation. May be an end-product of fermentation or carried over from ethanol precipitation of EPS.
Fig. 4. HPLC pro"le of extracelluar material extracted from Tc-AF021 Lac\. Glucuronic acid and galactose elute from the column at retention times of 8.51 and 10.59 min, respectively. Under the HPLC conditions used, other signi"cant peaks on the chromatogram which were detected may have been end-products of culture fermentation (formic acid and ethanol, eluting at 14.41 and 22.32 min, respectively).
teriophages, problems of this nature continue to interfere with a variety of milk-fermentation processes. This has provided a stimulus for persisting in the search for additional tools to eliminate bacteriophage infection in the factory environment. Since the pre-requisite to any e!ective infection is the speci"c attachment of the phage particle to the host cell surface, this therefore presents a highly attractive target for disrupting the propagation process. Two research avenues leading to interference of phage adherence to the exterior of the cell have been pursued. Firstly, the study of bacteriophage insensitive mutants (BIMs) has given some insight into the nature of phage receptors and this has shown that a carbohydrate and/or protein moiety might play a key role in adsorption (Oram & Reiter, 1968; Oram, 1971; Schafer, Geis, Neve & Teuber, 1991; 1991; Valeyasevi, Sandine & Geller, 1990, 1991, 1994). Geller, Ivey, Trempy and HettingerSmith (1993) successfully isolated mutants defective in the Pip protein which did not permit phage adsorption. Gopal and Crow (1993) found that the loosely associated material (LAM) extracted from the surfaces of L. lactis ssp. cremoris E8 and its phage resistant derivative 398 di!ered signi"cantly both in structure and composition. The LAM from strain 398 was more abundant, contained augmented levels of rhamnose and galactose and lacked at 21 kDa protein which was present in the material of the parent strain E8. The role of plasmid DNA in mediating adsorption inhibition in lactococci has also been reported (Sanders & Klaenhammer, 1983; de Vos, Underwood & Davies, 1984; Tortorello, Chang, Ledford & Dunny, 1990; Akcelik & Tunail, 1992; Lucey et al., 1992; Harrington & Hill, 1992). Various plasmid molecules have been demonstrated to direct the synthesis of cell surface antigens (Tortorello et al., 1990; Akcelik & Tunail, 1992) or to furnish the cell with an extracelluar carbohydrate coating to mask the phage receptors (Sijtsma et al., 1988, 1990; Lucey et al., 1992). In general, it appears that exopolysaccharide production is plasmid linked in mesophilic lactic acid bacteria (Vedamuthu & Neville, 1986; von Wright
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& Tynkkynen, 1987; Neve, Geis & Teuber, 1988) but not in thermophilic strains such as Streptococcus thermophilus (Stingele, Neeser & Mollet, 1996). Harrington and Hill (1992) reported the identi"cation of an adsorption inhibition plasmid resulting from a co-integration event between two endogenous plasmids in L. lactis ssp. lactis var. diacetylactis DPC220. Previous studies (Forde et al., 1999) indicated that pCI658, originally isolated from L. lactis ssp. cremoris HO2, encodes complete insensitivity to the small isometric-headed 712 and partial resistance to the prolateheaded c2. This report describes the mode of action of pCI658, revealing that it mediates the synthesis of a hydrophilic exopolysaccharide which shields the cell surface thereby inhibiting phage attachment. The presence of an alkali-soluble &#u!y' pellet in Tc-AF021 Lac\, a phage resistant derivative of L. lactis ssp. cremoris MG1363 harbouring pCI658, was demonstrated. Incubation with trichloroacetic acid (TCA) for 5 min at 903C also resulted in removal of the loose pellet; however, the solution did not have a similar e!ect at 43C. &Hot' trichloroacetic acid is reputed to extract teichoic acid and to remove O-acetyl groups whereas mild alkali is more speci"c in that it removes only ester-linked groups without removing the remainder of the teichoic acid (Chatterjee, 1969). The results indicate that the &#u!y' pellet material is largely composed of an acidic carbohydrate which may involve O-acetyl or ester-linked groups. TcAF021 Lac\ also displayed a hydrophilic cell surface and it resuspended more rapidly following centrifugation compared to the plasmid-free phage sensitive variant, MG1363. However, unlike MG1363, Tc-AF021 Lac\ failed to agglutinate with the glucosyl-speci"c lectin concanavalin A, which was also observed by Sijtsma et al. (1988) for the phage resistant strain L. lactis ssp. cremoris SK110. The system described here also bears some similarity to the well-characterised adsorption blocking plasmid pCI528, formerly identi"ed by Costello (1988) in L. lactis ssp. cremoris UC503. However, the results of a number of experiments permitted the identi"cation of features which distinguish between these two plasmids and the polysaccharides produced in each case. The &hard' colony morphology on agar media reported for pCI528-containing cells (Lucey et al., 1992) was not observed for Tc-AF021 Lac\. In addition, electron micrographs of material isolated from cells carrying pCI528 depicted the presence of a thick irregularly distributed substance on the cell surface while Tc-AF021 Lac\ cell surfaces exhibited extracelluar "brils extending outwards to adjacent cells. Similar features were previously recorded for lactococcal cultures by Brooker (1976) and Gopal and Reilly (1995). Brooker (1976) speculated that these "lamentous extensions might permit adhesion of cheese starter cultures to the curd, minimising their expulsion with the whey on syneresis (whey separation). Restriction
analysis of pCI658 and pCI528 revealed that both plasmids are also genetically distinct (result not shown), and the results of HPLC analysis provided evidence that the composition of the EPS encoded by the two plasmids was di!erent. pCI528 for example, mediates the production of a polymer rich in galactose and rhamnose, whereas the putative components of the pCI658-encoded polysaccharide include galactose and glucuronic acid. Preliminary investigations suggest that the levels of EPS produced by Tc-AF021 Lac\ appear to vary slightly in accordance with alterations in physical (pH and temperature) and chemical (medium composition) factors. Previous reports have also described the e!ects of varying growth conditions on the concentration and types of extracelluar material produced by microorganisms (Grobben, Sikkema, Smith & de Bont, 1995; Roberts, Fett, Osman, Wijey, O'Connor & Hoover, 1995; de Vuyst, Vanderveken, van de Ven & Degeest, 1998). Grobben et al. (1995) found that extracelluar polysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus NCFB 2772 in chemically de"ned medium was growth related, increasing with temperatures up to 473C and decreasing thereafter. No EPS was detected after growth had ceased and excess carbohydrate in the medium did not lead to increased EPS production. According to Cerning (1990), an excess of carbohydrate in combination with nutrient (nitrogen or phosphorus) limitation did stimulate EPS production in other lactic acid bacteria. The instability of pCI658 was recognised when 50% of the population had lost the plasmid after 100 generations. Also noteworthy was the formation of hazy plaques in a 712 overlay after four days, which by day 10 became more clearly visible. This suggested that EPS depolymerising enzymes had been synthesised during this prolonged exposure to 712. Sutherland (1990) reported that bacteriophage particles are actually a rich source of polysaccharases (EPS degrading enzymes) which form part of the particle structure itself, usually in the form of small spikes attached to the base-plate of the phage. Despite extensive investigations, the lactococcal cell surface and the mechanisms involved in the inhibition of phage adsorption remain poorly understood at the molecular level. The recent upsurge in the genetic characterisation of the determinants responsible for EPS production in lactic acid bacteria (Stingele et al., 1996; van Kranenburg, Marugg, van Swam, Willem & de Vos, 1997) has generated some information which may have relevance to EPS-mediated adsorption blocking mechanisms. Nevertheless, a more thorough analysis of the biological mechanisms which either regulate or interfere with the phage adsorption process is necessary if these types of resistance systems are to be fully comprehended. The use of pCI658 in strain improvement programmes may be limited given its unstable nature and the activity of EPS degradative enzymes in the presence of phage, but
A. Forde, G.F. Fitzgerald / International Dairy Journal 9 (1999) 465}472
the plasmid could successfully confer an additive e!ect on a strain if combined with one or more other suitable phage resistance plasmids.
Acknowledgements This work was supported by the Irish Department of Agriculture and Food (DAF) under the Food Industry Sub-Programme of EU Structural Funds, 1994-9 and by the Irish Co-operative Organisation Society (ICOS) Ltd., Dublin, Ireland. The authors thank Dan Walsh and Mary Heapes for their assistance with HPLC and electron microscopy, and acknowledge Daniella Guldimann for her contribution to the plasmid stability studies.
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Ophir, T., & Gutnick, D. L. (1994). A role for exopolysaccharides in the protection of microorganisms from dessication. Applied and Environmental Microbiology, 60, 740}745. Oram, J. D. (1971). Isolation and properties of a phage receptor substance from the plasma membrane of Streptococcus lactis ML3. Journal of General Virology, 13, 59}71. Oram, J. D., & Reiter, B. (1968). The adsorption of phage to group N streptococci. The speci"city of adsorption and the location of phage receptor substances in cell wall and plasma membrane fractions. Journal of General Virology, 3, 103}119. Pasquier, C., Marty, N., Dournes, J. L., Chabanon, G., & Pipy, B. (1997). Implication of neutral polysaccharides associated to alginate in inhibition of murine macrophage response to Pseudomonas aeruginosa. FEMS Microbiology Letters, 147, 195}202. Roberts, C. M., Fett, W. F., Osman, S. F., Wijey, C., O'Connor, J. V., & Hoover, D. G. (1995). Exopolysaccharide production by Bixdobacterium longum. Journal of Applied Bacteriology, 76, 463}468. Robijn, G. W., Thomas, J. R., Haas, H., van den Berg, D. J. C., Kamerling, J. P., & Vliegenthart, J. F. G. (1995a). The structure of the exopolysaccharide produced by Lactobacilus helveticus 766. Carbohydrate Research, 276, 137}154. Robijn, G. W., van den Berg, D. J. C., Haas, H., Kamerling, J. P., & Vliegenthart, J. F. G. (1995b). Determination of the structure of the exopolysaccharide produced by Lactobacillus sake 0-1. Carbohydrate Research, 276, 117}136. Rosenberg, M., Gutnick, D., & Rosenberg, E. (1980). Adherence of bacteria to hydrocarbons: a simple method for measuring cell-surface hydrophobicity. FEMS Microbiology Letters, 9, 29}33. Sambrook, J., Fritsch, E. F., & Maniatis, T. (1989). Molecular cloning: a laboratory manual (2nd ed.). Cold Spring Harbor, New York: Cold Spring Harbor Laboratory. Sanders, M. E., & Klaenhammer, T. R. (1983). Characterisation of phage-sensitive mutants from a phage-insensitive strain of Streptococcus lactis: evidence for a plasmid determinant that prevents phage adsorption. Applied and Environmental Microbiology, 46, 1125}1133. Schafer, A., Geis, A., Neve, H., & Teuber, M. (1991). Bacteriophage receptors of Lactococcus lactis ssp. diacetylactis F7/2 and Lactococcus lactis ssp. cremoris Wg2-1. FEMS Microbiology Letters, 78, 69}74. Sijtsma, L., Jansen, N., Hazeleger, W. C., Wouters, J. T. M., & Hellingwerf, K. J. (1990). Cell surface characteristics of bacteriophage resistant Lactococcus lactis ssp. cremoris SK110 and its bacteriophage sensitive variant SK112. Applied and Environmental Microbiology, 56, 3230}3233. Sijtsma, L., Sterkenburg, A., & Wouters, J. T. M. (1988). Properties of the cell walls of Lactococcus lactis ssp. cremoris SK110 and their relation to bacteriophage resistance. Applied and Environmental Microbiology, 54, 2808}2811.
Stingele, F., Neeser, J. R., & Mollet, B. (1996). Identi"cation and characterisation of the eps (exopolysaccharide) gene cluster from Streptococcus thermophilus S"6. Journal of bacteriology, 178, 1680}1690. Sutherland, I. W. (1990). Biotechnology of microbial exopolysaccharides. Cambridge: Cambridge University Press. Sutherland, I. W. (1998). Novel and established applications of microbial polysaccharides. Trends in Biotechnology, 16, 41}46. Terzaghi, B. E., & Sandine, W. E. (1975). Improved medium for lactic streptococci and their bacteriophages. Applied Microbiology, 29, 807}813. Tortorello, M. L., Chang, P. K., Ledford, R. A., & Dunny, G. M. (1990). Plasmid associated antigens associated with resistance to phage adsorption in Lactococcus lactis. In Abstracts of the third international ASM conference on streptococcal genetics, A/50. Valeyasevi, R., Sandine, W. E., & Geller, B. L. (1990). Bacteriophage kh receptor of Lactococcus lactis ssp. cremoris KH is rhamnose of extracelluar wall polysaccharide. Applied and Environmental Microbiology, 56, 1882}1889. Valeyasevi, R., Sandine, W. E., & Geller, B. L. (1991). A membrane protein is required for bacteriophage c2 infection of Lactococcus lactis ssp. lactis C2. Journal of Bacteriology, 173, 6095}6100. Valeyasevi, R., Sandine, W. E., & Geller, B. L. (1994). Lactococcus lactis ssp. lactis C2 bacteriophage sk1 receptor involves rhamnose and glucose moieties in the cell wall. Journal of Dairy Science, 77, 1}6. van Kranenburg, R., Marugg, J. D., van Swam, I. I., Willem, N. J., & de Vos, W. M. (1997). Molecular characterisation of the plasmidencoded eps gene cluster essential for exopolysaccharide biosynthesis in Lactococcus lactis. Molecular Microbiology, 24, 387}397. Vedamuthu, E. R., & Neville, J. M. (1986). Involvement of plasmid in production of ropiness (mucoidness) in milk cultures by Streptococcus cremoris MS. Applied and Environmental Microbiology, 51, 677}682. Vlegels, P. A. P., Hazeleger, W. C., Helmerborst, T. H., & Wouters, J. T. M. (1988). Phage resistance of Streptococcus cremoris due to low adsorption e$ciency. Netherlands Milk and Dairy Journal, 42, 195}206. von Wright, A., & Tynkkynen, S. (1987). Construction of a Streptococcus lactis ssp. lactis strain with a single plasmid associated with mucoid phenotype. Applied and Environmental Microbiology, 53, 1385}1386. Wallace, R. J. (1980). Cytoplasmic reserve polysaccharide of Selenomonas ruminantium. Applied and Environmental Microbiology, 39, 630}634. Wu, T. C. M., & Park, J. T. (1971). Chemical characterisation of a new surface antigenic polysaccharide from a mutant of Staphylococcus aureus. Journal of Bacteriology, 108, 874}884.
Analysis of exopolysaccharide (EPS) production mediated by the bacteriophage adsorption blocking plasmid, pCI658, isolated from Lactococcus lactis ssp. cremoris HO2 Amanda Forde , Gerald F. Fitzgerald * Department of Microbiology, National Food Biotechnology Centre, University College, Cork, Ireland Departments of Microbiology and Food Science and Technology, National Food Biotechnology Centre, University College, Cork, Ireland Received 18 November 1998; accepted 10 April 1999
Abstract Lactococcus lactis ssp. cremoris MG1363 harbouring pCI658, a 58 kb plasmid originating in L. lactis ssp. cremoris HO2, adsorbs phages 712 (936 phage species) and c2 (c2 species) less e$ciently than the plasmid-free phage sensitive strain. The presence of an alkali-soluble loose &#u!y' pellet following centrifugation of the phage resistant derivative was established. In addition this pCI658-containing strain possessed a more hydrophilic cell surface and did not agglutinate with the glucosyl-speci"c lectin concanavalin A. Furthermore, electron micrographs also illustrated signi"cant plasmid-mediated alterations of the cell surface. HPLC analysis of the loosely associated extracellular material revealed that galactose and glucuronic acid appeared to be its major components. It was concluded that pCI658 encodes the production of a hydrophilic exopolysaccharide which masks cell surface receptors causing a dramatic decrease in bacteriophage adsorption. 1999 Published by Elsevier Science Ltd. All rights reserved. Keywords: Exopolysaccharide; Plasmid; Bacteriophage resistance; Adsorption inhibition
1. Introduction Lactococcal starter cultures used in industrial practice are constantly threatened by lytic bacteriophage attack, a phenomenon which poses serious consequences for a variety of dairy fermentation processes (Neve, 1996). Research has demonstrated that many lactococci possess inherent resistance to phage infection due to the presence of native phage resistance plasmids. These antiphage systems have been organised into four groups depending on the manner in which they operate: adsorption inhibition, DNA penetration blocking, restriction/modi"cation and abortive infection (reviewed by Dinsmore & Klaenhammer, 1995; Garvey, van Sinderen, Twomey, Hill & Fitzgerald, 1995; Daly, Fitzgerald & Davis, 1996; Allison & Klaenhammer, 1998). Mechanisms which hinder phage attachment to cell surfaces have previously been associated with the development of bacteriophageinsensitive mutants which exhibited reduced receptor * Corresponding author. Tel.:#353-21-902730; fax:#353-21903101/276318. E-mail address: g."[email protected] (G.F. Fitzgerald)
availability (Vlegels, Hazeleger, Helmerborst & Wouters, 1988) or with plasmids encoding the production of exopolysaccharides (EPS) which coat the cell surface (Sijtsma, Sterkenburg & Wouters, 1988; Sijtsma, Jansen, Hazeleger, Wouters & Hellingwerf, 1990; Lucey, Daly & Fitzgerald, 1992). Several investigations have demonstrated the role of non-covalently bound extracelluar material in mediating defective phage adsorption to lactococcal cells (Sijtsma et al., 1988; Lucey et al., 1992; Gopal & Crow, 1993). Other bacterial exopolysaccharides have also been proven to be bene"cial by conferring antigenic speci"city (Wu & Park, 1971; Forsen, Niskasaari & Niemitalo, 1985), protection against dessication and phagocytosis (Ophir & Gutnick, 1994; Pasquier, Marty, Dournes, Chabanon & Pipy, 1997) and some o!er biological advantages such as antiviral and anti-tumour activities (Oda, Hasegawa, Komatsu & Tsuchiya, 1983). Furthermore, microbial polysaccharides have actual or potential applications as thickening agents leading to improved texture, viscosity and smoothness of mouthfeel in a wide variety of fermented dairy products and some biopolymers have non-food industrial usages such as in the
0958-6946/99/$ - see front matter 1999 Published by Elsevier Science Ltd. All rights reserved. PII: S 0 9 5 8 - 6 9 4 6 ( 9 9 ) 0 0 1 1 5 - 6
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oil industry (Sutherland, 1990, 1998). A growing interest has developed in the manipulation of exopolysaccharides of food-grade lactic acid bacteria for use in the food industry where they may provide a new range of thickening, gelling and stabilising agents. Several structural studies have already been performed on exopolysaccharides produced by these organisms (Doco, Wieruszeski, Fournet, Carcano, Ramos & Loones, 1990; Nakajima, Hirota, Toba & Adachi, 1992; Gruter, Lee#ang, Kuiper, Kamerling & Vliegenthart, 1993; Robijn, Thomas, Haas, van den Berg, Kamerling & Vliegenthart, 1995a; Robijn, van den Berg, Haas, Kamerling & Vliegenthart, 1995b; Bubb, Urashima, Fujiwara, Shinnai & Ariga, 1997; Lemoine, Chirat, Wieruszeski, Strecker, Favre & Neeser, 1997) and this aspect continues to be an expanding area of research in the light of their industrial importance. de Vos (1996) also addressed the topic of &metabolic engineering' which could improve the yield or alter the structure of exopolysaccharides produced by lactic acid bacteria. This work presents the characterisation of the exopolysaccharide produced by a strain harbouring the adsorption blocking phage resistance plasmid pCI658. This 58 kb molecule was initially identi"ed following conjugal co-transfer with the lactose plasmid from the parent Cheddar cheese starter culture Lactococcus lactis ssp. cremoris HO2 to the laboratory host L. lactis ssp. cremoris MG1363. A derivative designated Tc-AF021 Lac\, which was cured of the lactose plasmid but still contained pCI658, exhibited the production of a hydrophilic exopolysaccharide which acted as a potent barrier against bacteriophage infection. A remarkable reduction in the ability of the small isometric-headed
712 to adhere to this derivative was previously observed and while the prolate-headed c2 had the capacity to adsorb to the strain, it was noted that the cell remained partially insensitive based on the production of hazy plaques of decreased diameter (Forde, Daly & Fitzgerald, 1999). The system bears some similarity to that mediated by the adsorption inhibition plasmid pCI528 formerly described by Lucey et al. (1992). However, the current study has allowed the identi"cation of features which distinguish between the two plasmids and the exopolysaccharides they encode.
2. Materials and methods Bacterial strains and bacteriophages. The bacterial strains and bacteriophage used in this study are listed in Table 1. Lactococcal strains were grown at 303C in M17 medium (Terzaghi & Sandine, 1975) containing 0.5% glucose (GM17) or lactose (LM17) as required. Escherichia coli V517 was grown at 373C with aeration in Luria Bertani (LB) medium (Sambrook, Fritsch & Maniatis, 1989). Stocks of all cultures were maintained at !203C in 40% glycerol. Phage 712 was propagated on L. lactis ssp. cremoris MG1363 at 303C in GM17. Bacteriophage plaque assays. Bacteriophage plaque assays were performed by adding 0.1 ml of an overnight culture, 0.1 ml of 0.185 M CaCl and 1 ml of the appropri ate phage dilution to 3 ml of sloppy GM17 agar (0.7%) and overlaying onto prepared GM17 agar (1.5%) plates. Plates were incubated at 303C. Plasmid proxle analysis. Plasmid DNA was extracted using the method of Anderson and McKay (1983) and separated by electrophoresis on 0.7% agarose gels in TAE bu!er (40 mM Tris}Acetate, 2 mM EDTA, pH 8.0) at 100 V for 3 h. Resuspension rate of pelleted cells. Cells from 1 ml of fully grown GM17 cultures were pelleted in a bench centrifuge at 13,000 g for 30 s. Pellets were resuspended in GM17 broth by shaking on a vortex at speci"c time periods (0, 2, 5 10, 20, 30 and 40 s). The OD was then immediately measured as a function of vortexing time. Hydrophobicity studies. The cell surface hydrophobicity of strains was determined by measuring their adsorption to n-octane in a two-phase partitioning system based on the technique of Rosenberg, Gutnick and Rosenberg (1980). Cells (50 ml) from an 18 h culture were centrifuged at 13,000 g for 10 min and resuspended in 200 ml PUM bu!er (per litre, 22.2 g K HPO ) 3H O, 7.26 g KH PO , 1.8 g urea and 0.2 g MgSO ) 7H O, pH 7.1) to give an OD of 0.5. A 4 ml fraction of the cell suspension was added to test tubes containing various volumes (0.0, 0.5, 1.0, 1.5 and 2.0 ml) of n-octane. Following vigorous vortexing for 2 min at room temperature the samples were allowed to stand for 30 min to permit separation of the aqueous and hydrocarbon layers. The aqueous layer was
Table 1 Bacterial strains and bacteriophages Strain/phage
Plasmid content (kb)
Comment/source
L. lactis ssp. cremoris HO2 MG1363 Tc-AF021 Lac\
58, 46, 42, 22.5, 8.9, 4.5 Plasmid free 58
Wild-type pCI658-containing host, UCC Culture Collection FP> Derivative of L. lactis ssp. cremoris 712 (Gasson, 1983) FP\, phage sensitive Transconjugant containing pCI658, derived from HO2;MG1363 mating and cured of the co-transferred lactose plasmid FP>, phage resistant (Forde et al., 1999)
Phages
712 (936 phage species) FP"&Flu!y' pellet.
Small isometric-headed phage lytic for MG1363
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carefully extracted and examined spectrophotometrically at 600 nm. A decrease in the optical density was used as a measure of cell surface hydrophobicity. Chemical treatment of cell surfaces. 1.5 ml portions of cultures (containing approximately 10 cfu ml\) were washed and resuspended in one of the following: Triton X-100 (1%, w/v, in distilled H O, 30 min at 453C); SDS (1%, w/v, in distilled H O, 30 min at 453C); HCl (0.1 M, 30 min at 213C); NaOH (0.05 and 0.025 M, 30 min at 213C); pronase E (1.5 mg ml\ in distilled H O, 30 min at 373C); trypsin (1.5 mg ml\ in distilled H O, 30 min at 373C) (Sijtsma et al., 1988); trichloroacetic acid (5%, w/v, 5 min at 903C) (Chatterjee, 1969). Cells were assayed for loss of the &#u!y' pellet and phage resistance phenotypes. Agglutination of cells with lectins. A 1.5 ml aliquot of pelleted cells was washed and resuspended in 100 ll bu!er with concanavalin A (0.1% concanavalin A, 1.9% b-glycerophosphate, 0.025% MgSO ) 7H O, 10 mM cal cium borogluconate, pH 6.8) or Ptilota plumosa lectin (1 mg ml\ in 10 mM potassium phosphate bu!er, pH 7.3), each for 30 min at 223C. Agglutination was detected both visually in an eppendorf tube and by microscopic examination. Preparation of cell surface polysaccharides for HPLC analysis. Isolation of extracelluar material for chromatographic analysis was based on the procedure employed by Wallace (1980) and Sijtsma et al. (1988) with some modi"cations. Pelleted cells (3 ml) were washed twice in sterile H O (without disturbing the &#u!y' pellet during decanting steps) and resuspended in 120 ll of 30% w/v NaOH. Samples were boiled for 15 min, centrifuged at 13,000 g and the supernatants were precipitated with 60% ethanol. 15 ll samples were hydrolysed with an equivalent volume of 1 M H SO for 15 min at 1003C, and 20 ll were injected directly into an LKB 2150 HPLC apparatus (Bromma, Sweden) with a refractive index detector. The column was an Aminex HPX-87H ion exclusion column (Bio-Rad Laboratories, Richmond, California, USA) maintained at 653C using 0.01 N H SO as the elution #uid at a #ow rate of 0.6 ml min\. 1 mM sugar standards (glucose, galactose, rhamnose, arabinose, fructose and glucuronic acid) were passed through the column for comparative analysis. Electron microscopy of cell surfaces. A 1.5 ml portion of each culture (pre-grown for 18 h) was centrifuged, washed twice and resuspended in quarter strength Ringers solution. 100 ll of each suspension was mounted on a Formvar-coated copper grid and negatively stained with 100 ll 2% phosphotungstic acid for 3 min. Samples were dessicated and examined under a JEM 1200EX TESCAN transmission electron microscope at an accelerating voltage of 80 kV. Ewect of growth conditions on exopolysaccharide (EPS) production. The e!ects of various parameters on the production of EPS by Tc-AF021 Lac\ were investigated using conditions of altered pH (pH 5.0, 6.0, 7.0 and 9.0),
467
temperature (21, 30, 373C) and medium composition. For each parameter, cells were harvested at the same OD and EPS concentrations (mM) were determined by HPLC analysis as described above. Stability studies of plasmid pCI658. pCI658-containing cells were subcultured for 50 and 100 generations in GM17 broth, diluted in Ringers solution and spread plated on GM17 agar. In each case, 100 individual colonies were selected randomly and the percentage of the population which had maintained or lost pCI658 was estimated by screening for the presence or absence of the &#u!y' pellet and/or phage insensitivity phenotypes.
3. Results 3.1. Analysis of a derivative of L. lactis ssp. cremoris MG1363 harbouring the phage resistance plasmid pCI658 A conjugal derivative of L. lactis ssp. cremoris MG1363, designated Tc-AF021 Lac\, containing the phage resistance plasmid pCI658, demonstrated a soft, easily suspended &#u!y' pellet following centrifugation at 13,000 g, while the equivalent plasmid-free, phage sensitive strain (MG1363) produced a normal hard pellet. The phenomenon was estimated quantitatively in a cell resuspension assay where the times required for resuspension of pelleted cells of L. lactis ssp. cremoris MG1363 and its phage resistant variant Tc-AF021 Lac\ were compared. It was noted that almost total resuspension of Tc-AF021 Lac\ had occurred after 10 s while the time needed for complete resuspension of MG1363 extended to 20 s (Fig. 1). In order to further investigate the di!erences in cell surface composition between these strains, they were tested for their adsorption to n-octane in a two-phase partitioning system. It was essential that these hydrophobicity studies were conducted under similar growth and harvest conditions as any variations could a!ect the resulting hydrophobicity pattern obtained (Crow, Gopal & Wicken, 1995). Under stringently controlled conditions L. lactis ssp. cremoris MG1363 was found to adhere readily to octane implying that its cell surface is hydrophobic while Tc-AF021 Lac\ appeared to possess a more hydrophilic cell surface (Fig. 2). In addition, this latter strain did not agglutinate upon incubation with the lectins concanavalin A (con A) or Ptilota plumosa (Pt p) which have a$nities for glucosyl and a-D-galactosyl residues, respectively (Goldstein, Hollerman & Smith, 1965; Sijtsma et al., 1990). However, while L. lactis ssp. cremoris MG1363 also did not react with Pt p, it did aggregate with con A suggesting that the presence of pCI658 does lead to cell surface alterations. These "ndings were reinforced following electron microscopic examination of each strain where L. lactis ssp. cremoris MG1363 was shown to possess a regular
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Fig. 3. Electron micrographs of Tc-AF021 Lac\ demonstrating the presence of a layer of extracelluar material on the cell surface. Occasional "lamentous extensions of this layer make contact with adjacent cells (Magni"cation 89,000;). Fig. 1. Resuspension rates of pelleted cells of Lactococcus lactis ssp. cremoris MG1363 and Tc-AF021 Lac\ following centrifugation at 13,000 g. The results are the average of three separate independent experiments.
Fig. 2. Surface hydrophobicity of cells of Lactococcus lactis ssp. cremoris MG1363 and Tc-AF021 Lac\, as measured by partitioning of cells into the octane phase. A fall in OD indicates an increase in cell surface hydrophobicity. Each value represents the average obtained from triplicate experiments.
compact cell surface while in contrast, Tc-AF021 Lac\ showed the presence of strands or "bril-like material which emanated from the surface of the cell. Another noteworthy feature is the manner in which these occasional "lamentous extensions projected outwards from the cell and made contact with neighbouring cells (Fig. 3). 3.2. Chemical treatment of cells In order to determine the composition of the &#u!y' pellet produced by cells harbouring pCI658, freshly grown cultures of Tc-AF021 Lac\ were treated with a range of chemicals (Table 2). It was demonstrated that incubation of the pCI658-containing cells with trypsin,
Table 2 Chemical treatment and phage testing of Tc-AF021 Lac\ to determine the nature of the pCI658-encoded exopolysaccharide Treatment
&Flu!y' pellet
No treatment Triton X-100 (1%) SDA (1%) HCl (0.1 M) TCA (5%, 43C) TCA (5%, 903C) NaOH (0.025 M) Pronase E (1.5 mg ml\) Trypsin (1.5 mg ml\)
# # # # # ! ! # #
pronase E, SDS and Triton X-100 did not a!ect the loose &#u!y' pellet, suggesting that its components are neither proteinaceous nor lipid in nature. Treatment of the cells with 0.05 N NaOH at 213C for 30 min resulted in elimination of the &#u!y' pellet, implying the presence of polysaccharide material. Milder alkali treatment (0.025 M NaOH) also resulted in reversion of the culture to phage sensitivity; high 712 titres ('10 pfu ml\) generated visible plaques (however, these were not completely clear probably due to the inhibitory e!ects of NaOH on the growth of the test strain in the overlay). Loss of the &#u!y' pellet and phage resistance phenotypes provided evidence that the extracelluar material is involved in mediating reduced susceptibility to phage infection (NaOH treatment did not alter the sensitivity of plasmid-free MG1363). Treatment with trichloroacetic acid (TCA) for 5 min at 903C was also shown to remove the loose pellet phenotype from the pCI658-containing cell, but this solution did not have the same e!ect when tested at 43C. The &hot' and &cold' TCA treatments gave rise to di!erent results possibly due to hydrolysis of the EPS by the &hot' TCA treatment, while no hydrolysis took place at the lower temperature. It was not possible to determine if the &hot' TCA treatment also resulted in restoration of phage sensitivity since it caused death of the culture.
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3.3. HPLC analysis of extracted cell surface polysaccharides In order to further investigate the nature of the pCI658-encoded polymer, preparations of cell surface material were isolated and evaluated quantitatively by HPLC using 1 mM concentrations of glucose, galactose, rhamnose, arabinose, fructose and glucuronic acid as standards. Analysis of hydrolysates of crude EPS samples of Tc-AF021 Lac\ (Table 3, Fig. 4) indicated the presence of elevated levels of galactose and glucuronic acid (ratio of 1 : 3 for glucose-grown cells) when compared with the plasmid-free host MG1363. The results of preliminary experiments indicate that EPS levels are a!ected to some extent by growth conditions, being optimal between 30 and 373C and between pH 6 and 7. 3.4. Stability studies on pCI658 When the stability of pCI658 in TC-AF021 Lac\ was investigated it was discovered to be quite unstable. Following standard plaque assays with 712, 35% of the population had reverted to phage sensitivity and lost the characteristic &#u!y' pellet phenotype after 50 generations. Plasmid pro"le analysis con"rmed that pCI658 had been lost from these strains. After 100 generations, 50% of the population lacked the plasmid. Also noteworthy was the plaque forming ability of 712 on this host. Following overnight incubation, no plaques were seen on the Tc-AF021 Lac\ lawn when compared with plasmid-free L. lactis ssp. cremoris MG1363. However, when the plates were left at room temperature for another 4 days, hazy plaques ('10 pfu ml\) were apparent and by day 10, these plaques had become clearly evident.
4. Discussion Despite considerable technological advances in the control of culture inhibition caused by lactococcal bacTable 3 HPLC analysis demonstrating composition of the cell surface material extracted from L. lactis ssp. cremoris MG1363 and Tc-AF021 Lac\ (containing pCI658) grown in GM17 at pH 7 and at 303C Component
MG1363 (mM)
Tc-AF021 Lac\ (mM)
Glucuronic acid Glucose Galactose Rhamnose Formate Ethanol
0.035 0.047 0.037 0.080 2.141 29.22
1.324 0.0 0.476 0.0 2.029 5.422
May be an end-product of fermentation. May be an end-product of fermentation or carried over from ethanol precipitation of EPS.
Fig. 4. HPLC pro"le of extracelluar material extracted from Tc-AF021 Lac\. Glucuronic acid and galactose elute from the column at retention times of 8.51 and 10.59 min, respectively. Under the HPLC conditions used, other signi"cant peaks on the chromatogram which were detected may have been end-products of culture fermentation (formic acid and ethanol, eluting at 14.41 and 22.32 min, respectively).
teriophages, problems of this nature continue to interfere with a variety of milk-fermentation processes. This has provided a stimulus for persisting in the search for additional tools to eliminate bacteriophage infection in the factory environment. Since the pre-requisite to any e!ective infection is the speci"c attachment of the phage particle to the host cell surface, this therefore presents a highly attractive target for disrupting the propagation process. Two research avenues leading to interference of phage adherence to the exterior of the cell have been pursued. Firstly, the study of bacteriophage insensitive mutants (BIMs) has given some insight into the nature of phage receptors and this has shown that a carbohydrate and/or protein moiety might play a key role in adsorption (Oram & Reiter, 1968; Oram, 1971; Schafer, Geis, Neve & Teuber, 1991; 1991; Valeyasevi, Sandine & Geller, 1990, 1991, 1994). Geller, Ivey, Trempy and HettingerSmith (1993) successfully isolated mutants defective in the Pip protein which did not permit phage adsorption. Gopal and Crow (1993) found that the loosely associated material (LAM) extracted from the surfaces of L. lactis ssp. cremoris E8 and its phage resistant derivative 398 di!ered signi"cantly both in structure and composition. The LAM from strain 398 was more abundant, contained augmented levels of rhamnose and galactose and lacked at 21 kDa protein which was present in the material of the parent strain E8. The role of plasmid DNA in mediating adsorption inhibition in lactococci has also been reported (Sanders & Klaenhammer, 1983; de Vos, Underwood & Davies, 1984; Tortorello, Chang, Ledford & Dunny, 1990; Akcelik & Tunail, 1992; Lucey et al., 1992; Harrington & Hill, 1992). Various plasmid molecules have been demonstrated to direct the synthesis of cell surface antigens (Tortorello et al., 1990; Akcelik & Tunail, 1992) or to furnish the cell with an extracelluar carbohydrate coating to mask the phage receptors (Sijtsma et al., 1988, 1990; Lucey et al., 1992). In general, it appears that exopolysaccharide production is plasmid linked in mesophilic lactic acid bacteria (Vedamuthu & Neville, 1986; von Wright
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& Tynkkynen, 1987; Neve, Geis & Teuber, 1988) but not in thermophilic strains such as Streptococcus thermophilus (Stingele, Neeser & Mollet, 1996). Harrington and Hill (1992) reported the identi"cation of an adsorption inhibition plasmid resulting from a co-integration event between two endogenous plasmids in L. lactis ssp. lactis var. diacetylactis DPC220. Previous studies (Forde et al., 1999) indicated that pCI658, originally isolated from L. lactis ssp. cremoris HO2, encodes complete insensitivity to the small isometric-headed 712 and partial resistance to the prolateheaded c2. This report describes the mode of action of pCI658, revealing that it mediates the synthesis of a hydrophilic exopolysaccharide which shields the cell surface thereby inhibiting phage attachment. The presence of an alkali-soluble &#u!y' pellet in Tc-AF021 Lac\, a phage resistant derivative of L. lactis ssp. cremoris MG1363 harbouring pCI658, was demonstrated. Incubation with trichloroacetic acid (TCA) for 5 min at 903C also resulted in removal of the loose pellet; however, the solution did not have a similar e!ect at 43C. &Hot' trichloroacetic acid is reputed to extract teichoic acid and to remove O-acetyl groups whereas mild alkali is more speci"c in that it removes only ester-linked groups without removing the remainder of the teichoic acid (Chatterjee, 1969). The results indicate that the &#u!y' pellet material is largely composed of an acidic carbohydrate which may involve O-acetyl or ester-linked groups. TcAF021 Lac\ also displayed a hydrophilic cell surface and it resuspended more rapidly following centrifugation compared to the plasmid-free phage sensitive variant, MG1363. However, unlike MG1363, Tc-AF021 Lac\ failed to agglutinate with the glucosyl-speci"c lectin concanavalin A, which was also observed by Sijtsma et al. (1988) for the phage resistant strain L. lactis ssp. cremoris SK110. The system described here also bears some similarity to the well-characterised adsorption blocking plasmid pCI528, formerly identi"ed by Costello (1988) in L. lactis ssp. cremoris UC503. However, the results of a number of experiments permitted the identi"cation of features which distinguish between these two plasmids and the polysaccharides produced in each case. The &hard' colony morphology on agar media reported for pCI528-containing cells (Lucey et al., 1992) was not observed for Tc-AF021 Lac\. In addition, electron micrographs of material isolated from cells carrying pCI528 depicted the presence of a thick irregularly distributed substance on the cell surface while Tc-AF021 Lac\ cell surfaces exhibited extracelluar "brils extending outwards to adjacent cells. Similar features were previously recorded for lactococcal cultures by Brooker (1976) and Gopal and Reilly (1995). Brooker (1976) speculated that these "lamentous extensions might permit adhesion of cheese starter cultures to the curd, minimising their expulsion with the whey on syneresis (whey separation). Restriction
analysis of pCI658 and pCI528 revealed that both plasmids are also genetically distinct (result not shown), and the results of HPLC analysis provided evidence that the composition of the EPS encoded by the two plasmids was di!erent. pCI528 for example, mediates the production of a polymer rich in galactose and rhamnose, whereas the putative components of the pCI658-encoded polysaccharide include galactose and glucuronic acid. Preliminary investigations suggest that the levels of EPS produced by Tc-AF021 Lac\ appear to vary slightly in accordance with alterations in physical (pH and temperature) and chemical (medium composition) factors. Previous reports have also described the e!ects of varying growth conditions on the concentration and types of extracelluar material produced by microorganisms (Grobben, Sikkema, Smith & de Bont, 1995; Roberts, Fett, Osman, Wijey, O'Connor & Hoover, 1995; de Vuyst, Vanderveken, van de Ven & Degeest, 1998). Grobben et al. (1995) found that extracelluar polysaccharide production by Lactobacillus delbrueckii ssp. bulgaricus NCFB 2772 in chemically de"ned medium was growth related, increasing with temperatures up to 473C and decreasing thereafter. No EPS was detected after growth had ceased and excess carbohydrate in the medium did not lead to increased EPS production. According to Cerning (1990), an excess of carbohydrate in combination with nutrient (nitrogen or phosphorus) limitation did stimulate EPS production in other lactic acid bacteria. The instability of pCI658 was recognised when 50% of the population had lost the plasmid after 100 generations. Also noteworthy was the formation of hazy plaques in a 712 overlay after four days, which by day 10 became more clearly visible. This suggested that EPS depolymerising enzymes had been synthesised during this prolonged exposure to 712. Sutherland (1990) reported that bacteriophage particles are actually a rich source of polysaccharases (EPS degrading enzymes) which form part of the particle structure itself, usually in the form of small spikes attached to the base-plate of the phage. Despite extensive investigations, the lactococcal cell surface and the mechanisms involved in the inhibition of phage adsorption remain poorly understood at the molecular level. The recent upsurge in the genetic characterisation of the determinants responsible for EPS production in lactic acid bacteria (Stingele et al., 1996; van Kranenburg, Marugg, van Swam, Willem & de Vos, 1997) has generated some information which may have relevance to EPS-mediated adsorption blocking mechanisms. Nevertheless, a more thorough analysis of the biological mechanisms which either regulate or interfere with the phage adsorption process is necessary if these types of resistance systems are to be fully comprehended. The use of pCI658 in strain improvement programmes may be limited given its unstable nature and the activity of EPS degradative enzymes in the presence of phage, but
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the plasmid could successfully confer an additive e!ect on a strain if combined with one or more other suitable phage resistance plasmids.
Acknowledgements This work was supported by the Irish Department of Agriculture and Food (DAF) under the Food Industry Sub-Programme of EU Structural Funds, 1994-9 and by the Irish Co-operative Organisation Society (ICOS) Ltd., Dublin, Ireland. The authors thank Dan Walsh and Mary Heapes for their assistance with HPLC and electron microscopy, and acknowledge Daniella Guldimann for her contribution to the plasmid stability studies.
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