Cell surface differences of lactococcal strains

Inr. Dairy Journal 5 (1995) 4548 Q 1994 Elsevier Science Limited Printed in Ireland. All rights reserved 0958-6946/95/$7.00 ELSEVIER

Cell Surface Differences of Lactococcal Strains

Vaughan New Zealand

L. Crow, Pramod

Dairy Research

Institute,

Anthony School of Microbiology,

(Received

17 August

University

K. Gopal

Palmerston

North,

New Zealand

J. Wicken

of New South Wales, New South Wales 2033, Australia

1993; revised version

accepted

26 November

1993)

ABSTRACT A number of cell surface properties were compared in 1.5pairs of lactococcal strains in order to gain an understanding of cell surface diversity and the relationship between the acquisition of the phage-resistance phenotype and alteration of cell surface properties. Each pair comprised a parent strain and a derivative resistant to a phage (erR) or a number of phages. Three cell surface hydrophobicity patterns were found: (I) three parent strains were more hydrophobic than their BR derivatives; (2) five 0R derivatives were more hydrophobic than their parent strains; (3) there were no differences for seven strain pairs. Loosely associated cell surface material was removed without cell lysis, and concentration dtfferences between 28 strains of 40-, 23- and II-fold were found for the extracted protein, hexose and rhamnose, respectively. These three surface components were extracted in higher concentrations from the BR derivative for seven strain pairs and from the parent strain for three strain pairs, and no differences were observed for four strain pairs. Intracellular and extracellular lipoteichoic acid concentrations varied in four of six strain pairs studied. The extracted protein profiles determined on polyacrylamide gels and by Superose 12 chromatography and the compositions of the extracted polysaccharide were different between most of the strain pairs. In addition, the surface properties, particularly cell hydrophobicity, varied according to growth conditions for some strains. The cell-surface components showed considerable diversity within the 30 lactococcal strains studied, with multiple differences between many of the strain pairs. For 45

46

Vaughan L. Crow, Pramod K. Gopal, Anthony J. Wicken example, d[fferences in hydrophobicity. the extracellular lipoteichoic acid concentration, molecular weight profile of proteins and the amount of protein, hexose and rhamnose extracted as loosely associated cell surface material were observed between the strains of pair E8/398. No unifying theme was evident to describe the basis of changes to the cell surface in the phage-resistant derivative strains.

INTRODUCTION The lactococcal cell surface has a number of properties that are important to dairy fermentations, including phage adsorption (Oram & Reiter, 1968; Thomas rt al., 1980; Sanders, 1988; Sijtsma et al., 1990a, b), roles in exopolysaccharide production (Brooker, 1976; Macura & Townsley, 1984; Forsen et al., 1985; Cerning, 1990) and cell autolysis (Ohmiya & Sato, 1970; Bie & Sjostrom, 1975; Vegarud ef al., 1983) and the location site for the proteinase required for rapid coagulation of milk (for reviews, see Thomas & Pritchard, 1987; Kok, 1990). Knowledge of the cell surface would also assist in improving DNA transformation studies and in the sedimentation of some lactococcal strains (starters) for commercial production. These properties have usually been studied in a limited number of strains only, with emphasis on phage adsorption, and a comprehensive understanding of the starter cell surface is still a long way off. A variety of lactococcal surface components have been implicated as possible phage receptors. A lipoprotein fraction of the plasma membrane in Lactococcus lactis subsp. Iactis has been proposed as a receptor in ML3 for phage ml3 (Oram, D-glucosamine and D-galactosamine inhibit interaction 1971). Rhamnose, between L. lactis subsp. cremoris EB7 and its phage, eb7, suggesting that a polysaccharide component is involved in the cell wall phage receptor (Keogh & Pettingill, 1983). The phage skl1G binds poorly to the resistant strain L. lactis subsp. cremoris SK1 10 compared with the phage-sensitive derivative SK1 12, due to a blocking of the receptor site. The resistance is encoded on the plasmid pSK112. The blocking galactose-containing component has been removed from cells of strain SK1 10, thus rendering the cells sensitive to the phage (Sijtsma et al., 1988). The cell surface of strain SK1 12 is more hydrophobic and negatively charged than that of strain SK1 10, the pelleted cells of SK1 10 resist suspension more than those of SK112 (Sijtsma et al., 1990a) and only SK1 12 cells were agglutinated with Concanavalin A (Sijtsma et al., 1988). Galactose is present in the lipoteichoic acid (LTA) only from the phage-resistant strain SK1 10, and a likely mechanism for phage resistance is that the galactosylated LTA causes steric shielding of the phage receptor on strain SK1 10 (Sijtsma et al., 19906). Two recent studies have shown that non-covalently bound extracellular material has a role in phage adsorption (Lucey et al., 1992; Gopal & Crow, 1993). Cell wall galactose and rhamnose components of L. lactis subsp. cremoris KH are involved with the binding of different phages (Valyasevi et al., 1990). Phageresistance mechanisms involving interference of phage adsorption have been related to plasmids in L. lactis subsp. cremoris (de Vos et al., 1984; Vlegels et al., 1986) and L. lactis subsp. lactis (Sanders & Klaenhammer, 1983, 1984). A large conjugative plasmid from L. lactis subsp. lactis strain 334 has been associated with phage resistance, lactose fermentation, conjugation and cell wall polypeptide antigens. Defective phage adsorption is not the resistance mechanism, but

Cell surfaces

oJlactococcal

41

strains

another cell surface function involving prevention of DNA injection has not been ruled out. The plasmid-encoded surface antigens may be important in conjugation and/or phage resistance (Dunny et al., 1988). The cell surface structure of lactococcal cells is complicated and poorly understood. In this study of up to 15 pairs of lactococcal strains, the diversity of cell surface properties was studied as an initial step to understanding the structure and function of lactococcal cell surfaces, including phage receptors. Each pair of lactococcal strains comprised a parent strain and a derivative resistant to a phage or a number of phages. The resistant strains were derived by either selecting a spontaneous mutant after phage challenge or by transconjugation (Table 1).

TABLE 1 Parent Strains and Their Phage-Resistant Strain pairs (parent/O’ L. lads

Variants

variant)

subsp. luctis HI/T.Hl

Notes

T.H 1 is a transconjugant containing pAJ33 1, resistant to H 1 0623 and 0923 (Jarvis et cd., 1989)

LM2302/T.LM2302

T.LM2302 is a transconjugant containing pAJllO6, resistant to LM2302 0C2 and 01039 (Jarvis, 1988)

ML3/T.ML3

T.ML3 is a transconjugant containing pAJ33 I, resistant to ML3 0643 (Jarvis et al., 1989)

var. dacetylactis

L. lads

D6/T.D6

subsp. cremoris 4854/T.4854

1041578 2661566

T.D6 is a transconjugant containing pAJllO6, resistant to D6 (and Lac-derivative, D6A) 01459 (Jarvis, 1988) T.4854 is a transconjugant containing pAJ2074, resistant to 01440 and 01496 but sensitive to 01483. All three phages attack the parent strain, 4854, a plasmid-cured derivative of strain 584 (Jarvis, 1988) The three variants were whey-derived phageresistant strains (Jarvis, 1981)

4481512

AM21134 ES/398 108/546 158/402 58412146 2176/2176R17 2178/2178R7

Variant Variant Variant Variant Variant phase Variant Variant

insensitive insensitive insensitive insensitive insensitive

to to to to to

parent parent parent parent parent

insensitive to 01746 insensitive to 01727

0602 0618, 701, 833, 852 01140 0874 factory-derived

48

Vaughan L. Crow, Pramod K. Gopal, Anthony J. Wicken MATERIALS

Organisms

AND

METHODS

and culture conditions

All strains were from the collection held at the New Zealand Dairy Research Institute, and the relationships between strains of the pairs studied are shown in Table 1. Static batch cultures were grown at 30°C in three media, as specified. For most experiments, T5 complex broth (Thomas et al., 1974), containing 0.5% (w/v) of galactose, glucose or lactose, was used and the initial pH was 7.2. Autoclaved reconstituted skim milk (RSM) was buffered (11.5 g /?-glycerophosphate/30 ml HZO, solution autoclaved and added to 500 ml autoclaved RSM) and used in one series of experiments. Extracellular lipoteichoic acid, protein and cellular lipoteichoic acid were measured as described below using cultures grown on the lowmolecular-weight components of complex medium, denoted ‘LM’, prepared by hollow-fiber filtration, as previously described (Jacques et al., 1979~) and containing 0.5% (w/v) of glucose or lactose. Secreted lipoteichoic

acid and protein in culture fluid

Cells were removed by centrifugation and the supernatant from each culture fluid was filtered, dialysed, freeze dried and dissolved in 0.5-1.0 ml filtered distilled water, as previously described (Knox et al., 1985). Analysis was then carried out on IO-50 ~1 of the concentrate for the protein components, and on 5 ~1 of the concentrate (diluted up to l/5 in distilled water) for lipoteichoic acid. Extraction

of cellular lipoteichoic

acid

Cells harvested (13 000 g for 10 min) from 200 ml LM medium were washed (2 x 100 ml) with 0.85% (w/v) NaCl, freeze dried and reconstituted in water to give 40 mg (dry wt) of cells/ml. The cell suspension was mixed with an equal volume of 90% (w/w) aqueous phenol, pre-equilibrated to 68°C for 10 min and then stirred for 30 min. After centrifugation, the aqueous layer was removed and the phenol layer was washed with an equal volume of water, using the same pre-equilibration and stirring treatment. The combined aqueous layers were dialysed against three changes of distilled water (5 litres), concentrated by rotary evaporation and finally freeze dried. The dried material was taken up in 2-5 ml water and 5 ~1 samples (diluted up to I/ 10 in distilled water) were analysed for lipoteichoic acid. Lipoteichoic

acid analysis

The concentrations of lipoteichoic acid in both cell extracts and culture fluids were measured by rocket immunoelectrophoresis using rabbit antiserum prepared against purified lipoteichoic acid from Lactobacillus casei (Jacques et al., 19796). Rocket heights were calculated in cm/50 pg (dry wt) of cells after standardization against a standard L. casei lipoteichoic acid. Rocket immunoelectrophoresis detects both acylated and deacylated lipoteichoic acid. Lipoteichoic acid from L. lactis subsp. lactis cross-reacts with antisera to L. casei lipoteichoic acid (Wicken & Knox, 1971, 1975).

49

Cell surfaces of lactococcal strains

Hydrophobicity

studies

The cell surface hydrophobicity of different strains was determined by a modification of the phase-partitioning assay described by Rosenberg et al. (1980). Cells were harvested (generally late log from T5 medium), washed in cold 50 mM Trishydrochloride buffer (pH 7.8) and resuspended in pH 7.1 buffer (16.95 g K2HP04, 7.26 g KH2P04, 1.8 g urea, 0.2 g MgS047H20 and water to 1 litre) to give a cell suspension with an absorbance (600 nm) between 1.2 and 1.6. The suspension was mixed with various volumes of xylene (up to 1.0 ml), incubated for 10 min at 30°C and then vigorously mixed on a vortex for 2 min at ambient temperature. The absorbance (600 nm) of the aqueous phase was measured after phase separation (standing for 20 min at ambient temperature). Polyacrylamide

gel electrophoresis

High pH, discontinuous (stacks at pH 8.3, separates at pH 9.5), non-dissociating gel electrophoresis was performed in l-mm thick slab gels using the Protean II vertical gel system (Bio-Rad Laboratories, Richmond, CA). A stacking gel containing 7.5% (w/v) acrylamide (acrylamide to bisacrylamide 3O:O.S) and a resolving gel containing 12.5% (w/v) acrylamide were used routinely (Laemmli, 1970). Occasionally, a resolving gel containing either 7.5 or 15% (w/v) acrylamide was used for better visualization of the slower- and faster-migrating protein bands, respectively. Separated proteins were routinely fixed and stained using the silver stain kit and procedure of Bio-Rad Laboratories. Occasionally, the separated proteins were fixed and stained with 0.1% Coomassie brilliant blue R-250 dissolved in 40% (v/v) methanol and 10% (v/ v ) acetic acid, and were destained with the same solvent mixture. For sodium dodecyl sulphate (SDS)-gel electrophoresis, preparation of concentrated culture fluid samples in SDS and gel electrophoresis were performed essentially as described previously (Hardy et al., 1986) except that the fixing and staining used the silver-stain method rather than Coomassie brilliant blue R-250. Extraction

of loosely associated cell surface material

Cells harvested (13 OOOg for 10 min) from 1.2 litres of T5 medium grown for 1416 h were washed with 2 x 200 ml ice-cold 50 mM sodium acetate/phosphate buffer (pH 6.4) and then incubated in 200 ml 50 mM Tris-hydrochloride buffer (pH 7.5) at 30°C for 30 min and shaking gently every 5 min before the cells were collected by centrifugation (13 000 g for 10 min) and washed four times more with the extraction buffer. The supernatants were assayed within 1 h for the cytoplasmic enzyme markers fructose 1,6_bisphosphate aldolase (Crow & Thomas, 1982) and lactate dehydrogenase (Thomas et al., 1980) dialysed against water (2 x 4 litres) for 48 h, freeze dried and reconstituted in 0.5-2.0 ml of water for later analysis of protein and polysaccharide. Cell wall preparation Cell walls were prepared by mechanical precooled Bead-Beater (Biospec Products,

disruption Bartlesville,

of organisms OK), followed

using a by treat-

50

Vaughan L. Crow, Pramod K. Gopal, Anthony J. Wicken

ment with boiling SDS (Campbell et al., 1978). Cell wall preparations were heated at 60°C in 0.1 N H2S04 for 24 h to acid-solubilize cell wall polysaccharide (Campbell et al., 1978). The cell wall preparations and the acid-solubilized cell wall polysaccharides were hydrolysed in sealed ampoules with 1 M HCl for 15 h at 100°C. The hydrolysates were neutralized with Ba(OH)* and centrifuged, and the supernatants lyophilized. Chromatography Polysaccharide-containing material extracted under mild conditions (i.e. loosely associated cell surface material) from the cells was separated by gel-permeation chromatography on a column (80 x 2 cm*) of Sephacryl S-300 pre-equilibrated and developed (0.2 ml/min) in 1% (w/v) NaCl containing 0.01 M NaN3. The protein material extracted from the cell surface was rapidly (- 1 h) separated on a column (3.1 x 0.6 cm2) of Superose 12 pre-equilibrated and developed (0.5 ml/ min) in 50 mM MES [2-(N-morpholino)ethanesulfonic acid] buffer (pH 6.5) containing 0.15 M NaCl. An FPLC (fast protein liquid chromatography) system from Pharmacia (Uppsula, Sweden), consisting of a liquid chromatography controller LCC-500 plus, pump P-500 and monitor UV-M, was used. Other procedures Enzymatic analyses were routinely used to assay for galactose (Kurz & Wallenfels, 1974) and glucose (Glucostat reagents, Worthington Diagnostics, Freehold, NJ). Hexoses were determined by the primary cysteine method (Dische, 1955) with glucose as the standard, while methylpentoses were determined by the method of Gibbons (1955) with rhamnose as the standard. Protein was estimated by the method of Bradford (1976) with bovine serum albumin as the standard.

RESULTS Lipoteichoic

acids

The cellular and extracellular lipoteichoic acid concentrations were determined in six strain pairs (Table 2). The extracellular lipoteichoic acid concentration showed considerable variation, ranging from undetectable (< 0.01) to 1.91 cm/ 50 pg (dry wt) of cells for strain ES. The extracellular lipoteichoic acid concentration in one pair was over 1Cfold higher in the parent strain (E8/398), while in another pair (158/402) it was over 6-fold higher in the OR strain. The concentration of cellular lipoteichoic acid was more constant for the 12 strains, ranging from 1.8 to 7.5; however, two strain pairs showed marked differences, with the concentration of the lZIR strain being over 2-fold higher than that of the parent strain. Extracellular

proteins

The strains that were analysed for lipoteichoic acid (as in Table 2) were also analysed for extracellular protein present in the culture fluid after growth on LM

Cell surfaces of lactococcal strains

Cellular and Extracellular

Strain pair (parenllOR)

TABLE 2 Lipoteichoic Acid Concentrations Six Strain Pairs” Lipoteichoic Extracellular

ML3/T.ML3 AM21134 E8/398 1041578 1581402 4481572

51

O.OSjO.06 0~18/0~18 1.91/0.13 ND’ 0.03/0,20 0.29/0.28

for

acid concentrationh Cellular 2.313.1 3.Oi6.4 4.9i4.1 2.511.8 3.7jl.5 3.917.1

“Strains were grown on LM medium containing 0.5% lactose at 30°C and cellular and extracellular lipoteichoic acids were obtained and measured as described in Materials and Methods. ‘Concentration expressed as rocket height (cm/50 ,ug (dry wt) of cells). ‘ND, not detectable, co.01 cm/50 pg (dry wt) of cells.

medium (data not shown). The protein profiles on SDS gels were identical for the parent and 0R strains of the six pairs studied, with each strain pair having its own characteristic protein profile of 8-12 bands. The protein profile did not change when glucose replaced lactose as the growth sugar. Hydrophobicity

studies

Three hydrophobicity patterns were evident for the 15 strain pairs studied (Fig. 1). For the strain pair 448/572 (Fig. l(A)), the OR strain was more hydrophobic than the parent strain (similar data were obtained for four other strain pairs - 266/ 566, E8/398, Hl/T.Hl and ML3/T.ML3). In contrast, no hydrophobicity differences were observed between the parent and RIR strains of the 158/402 pair (Fig. l(B)) or six other strain pairs (2178/2178R7, 108/546, 104/578, 584/2146, D6/T.D6 and 4854/T.4854: data not shown). Three strain pairs D6/T.D6, 108/ 546 and 4854lT.4854 showed no evidence of hydrophobicity at all with up to 1 ml xylene. In the third hydrophobicity pattern, the parent strain (AM2) was more hydrophobic than the IZIRstrain (134) (Fig. l(C)). This same pattern was observed for two other strain pairs (LM2302/T.LM2302 and 2176/2176R17: data not shown). These hydrophobicity studies were performed with cells harvested after 16 h of growth on the same medium (T5 medium containing 0.5% lactose, where the pH at harvest was N 5.2), except for the strain pairs LM2302/T.LM2302 and 4854/ T.4854, which were grown on glucose because of the inability of the parent strain to grow on lactose. Similar growth and harvest conditions are essential for comparative purposes, as the conditions were shown to influence the hydrophobicity of some strains. For example, when the growth sugar was changed from glucose to lactose to galactose, the hydrophobicity increased for strain ML3 (Fig. 2(A)) and strain T.ML3 (data not shown). In contrast, for the strain pair

Vaughan

52

L. Crow, Pramod

K. Gopal, Anthony

J. Wickm

100

50

0 100 E ifi iii aI 50 E B b $ m s

0 100

402 (0

B

I

I

I

I

I

c-

A

T-+

(a)/

134 (0")

50

\ C 0

AM2

I

I

I

I

I

0.2

0.4

0.6

0.8

1.0

Xylens (ml)

Fig. 1. Hydrophobicity of strain pairs using cells harvested after 16 h of growth at 30°C on TS medium containing 0.5% lactose. The absorbance of cells with no added xylene was used as the control value (100% absorbance at 600 nm).

108/546 (where no hydrophobicity was observed with up to 1 ml of xylene), no hydrophobicity changes were observed when the cells were grown on the three sugars. The hydrophobicities of cells grown on lactose or glucose were checked for seven strain pairs; the results are shown only for the pair 266/566 (Fig. 2(B)). The hydrophobicity of strain 266 was independent of the growth sugar, in contrast with that of strain 566 which was affected to such an extent that the relative hydrophobicity of the pair was reversed on changing the growth sugar. The hydrophobicity was greater in the lactose-grown cells than in the glucosegrown cells for both strains of the pairs E8/398 and 584/2146 (data not shown). The hydrophobicities of four strain pairs (AM2/134, 158/402, 104/578 and Hl/ T.Hl) were identical in glucose- and lactose-grown cells (data not shown). Cells of both strains of the pair E8/398 were more hydrophobic when harvested from

53

Cell surfaces of lactococcai strains

0

8 0 100

0.4

0.8

0.6 Xylene

1 .o

(ml)

Fig. 2. Effect of growth conditions

on the hydrophobicity of cells from strain ML3 grown on T.5 medium containing 0.5% glucose, lactose or galactose (A); strain pair 266/566 grown on T5 medium containing 0.5% glucose or lactose (B); and strain pair E8/398 grown on TS medium containing 0.5% lactose and on RSM (C). Cells were harvested after 16 h of growth at 30°C.

RSM than when harvested from T5 medium (Fig. 2(C)). from two strain pairs (266/566, E8/398) were not altered shifted from 5.2 to 6.2 whereas for two other strain pairs cells harvested at the higher pH were more hydrophobic

Hydrophobicity of cells by the harvest pH when (584/2146, 448/572), the (data not shown).

Loosely associated cell surface material Material from the cell surface of 14 strain conditions using 50 mM Tris-hydrochloride

pairs was extracted under identical (pH 7.5) as the extraction buffer

54

Vaughun L. Crow, Pramod K. Gopal, Anthony J. Wicken

(Table 3). Three patterns were observed. In the first seven strain pairs, more protein, hexose and rhamnose was extracted from the RIR strains than from the parent strains. In the next three strain pairs, more material was extracted from the parent strains. In the last four strain pairs, the quantity of material was similar for the parent and IZIR strains. There was a considerable range in concentration of extracted material between the 28 strains, with differences of 40-, 23- and 1 l-fold between the extreme values for protein, hexose and rhamnose, respectively. Effect of different growth and extraction surface material

conditions on the loosely associated cell

Experiments were carried out in triplicate for four strain pairs (i.e. extractions from three different harvests), with very similar results. For example, for strain pair AM2/134, the replicate data for protein (1.3/4.6, 1.8/4.5), hexose (2.6/8.2, 1.9/8.3) and rhamnose (0.2/0.6, 0.3/0.9) concentration ratios (mg extracted from 100 mg dry wt bacteria) were similar to the results shown in Table 3. Furthermore, the growth conditions influenced the concentration of material extracted from some of the strains. Although the protein ratio difference between the pairs AM2/134 and 584/2146 remained the same (as shown in Table 3), whether grown on glucose or lactose, the protein extracted from the glucose-grown cells was l/6 and l/2, respectively, of that extracted from the lactose-grown cells. When cells

Extraction

TABLE 3 of Loosely Associated Cell Surface Material

Strain pair (parent/OR)

Material extracted,from 100 mg (dry wt) hucteria” (mg) Protein

Hexose

Rhamnose

AM211 34 2178/2178R7 E8/398 2661566 HI/T.Hl 4854/T.4854 ML3/T.ML3

1.414.4 2.6112.0 0.814.2 5.217.2 0.613.3 0.6/ 1.6 0.2jO.9

2.617.8 7.8126.0 2.7130.1 17.8126.6 2.1/11.7 3.1/11.1 1.315.7

0.210.6 0.611.9 0.2/1.1 1.1/2.0 0.311.4 0.2/0.9 0.3/1.2

104/578 4481572 D6/T.D6

2.210.3 3.9/l .8 5.510.3

17.6114.2 23.5113.7 18.2/5.7

0.7lO.3 0.6/0.5 1.0/0.3

108/546 2176/2176Rl7 1581402 58412146

2.012.3 5.616.0 1.5/1.0 6.718.0

16.1/18.0 22.212 1.4 4.413.7 7.9110.2

0.5lO.4 1.5jl.O 1.912.2 0.510.4

“Total material from five 30-min extractions using 50 mM Trishydrochloride buffer (pH 7.5) at 30°C (for details see Material and Methods). Bacteria were harvested from T5 medium containing 0.5% (w/v) lactose.

Cell surfaces qf lactococcal strains

55

from three of the five strain pairs tested were grown on LM medium (data not shown), decreases of from 2- to 5-fold in both the protein and hexose concentrations (the ratio for the pairs was similar) extracted were observed compared with growth on T5 medium (Table 3). Extraction of the loosely associated cell surface material was achieved without significant lysis (data not shown). For 21 of the 28 strains studied (Table 3) neither of the cytoplasmic markers (aldolase and lactate dehydrogenase) was detected (
56

Vaughan L. Crow, Pramod K. Gopal, Anthony J. Wicken

578 (I+“) cells

104 (parent) cells

se EL .m ti$

2.0

-

1.0

-

.E ‘u

B z F .o, g ? 0,

O14

-

F 8 $ ti g

12 -

10 -

2

12345

123

45

12

Extraction

345

Ill l-l 123

45

Fig. 3. Cells of the strain pair 104/578 were harvested from T5 medium containing 0.5% lactose after 16 h of growth and were extracted five times with either 50 tIIM Tris hydrochloride buffer (pH 7.5) (light histograms) or 50 mM bis-Tris-propane hydrochloride buffer (pH 9.5) (dark histograms). Each extract was concentrated and analysed for hexose and protein.

the strain pair. Other strains may give different patterns; however, for four other strains and using Tris-hydrochloride.buffer, the majority of the protein or hexose was extracted within 15 min and there was only a gradual increase thereafter. From the combined results, it can be concluded that for routine comparison, five extractions, each of 30 min, and using the Tris-hydrochloride buffer (pH 7.5) was the most suitable procedure. Although a buffer at higher pH (pH 9.5) appeared to extract more material in the two strains investigated, this was considered to be the extreme of physiological conditions and even a reasonably

Cell swfaces

1.0

c

158

30

(parent)

60

qf lactococcal

strains

402

cells

90

120 Time

30

57

(4”)

60

cells

90

120

(min)

Fig. 4. Cells of the strain pair I%/402 were harvested

from T5 medium containing 0.5% lactose after 16 h of growth and were incubated with different extraction buffers and sampled at three times. Each extract was concentrated and analysed for hexose and protein. The four different 50 mM buffers used are bis-Tris-propane hydrochloride buffer

(pH 9.5) (0); Tris hydrochloride buffer (pH 7.5) (0); MES buffer (pH 5.8) (A); and Tris hydrochloride buffer (pH 7.5) containing 10 mM CaClz (0). stable marker, lysis reliably. Composition

such as aldolase,

required

rapid analysis

in order to estimate

cell

of loosely associated cell surface proteins

The cell surface proteins, extracted from cells under conditions where no significant cell lysis was observed, were analysed on non-dissociating polyacrylamide electrophoresis gels. The profile pattern differences in protein for 14 strain pairs are summarized in Table 4. The first three strain pairs showed no differences in protein profile between the parents and their IZIR derivatives. The next six strain pairs showed either one or two extra bands in the OR strain of the pair. In contrast, the next four strain pairs showed an extra one or two bands in the

58

Vaughan L. Crow, Pramod K. Gopal, Anthony J. Wicken

Summary

of Polyacrylamide

TABLE 4 Gel Electrophoretic

Analysis

of

Loosely Associated Cell Surface Protein Strain pair (parent/OR)

Number qfextra protein silverstaining bands in” Parent

4481572 4854/T.4854 E8/398 1041578 108/546 1581402 2661566 58412146 2176/2176Rl7 2178/2178R7 D6/T.D6 Hl/T.Hl ML3/T.ML3 AM21134 “Protein silver-staining bands on 12.5% polyacrylamide (w/v) gels. Sample added (5-20 ~1) to the gel was from the first extraction of loosely associated material (50 IIIM Tris hydrochloride buffer, pH 7.5, 30°C for 30 min) from cells without lysis. ‘No extra protein bands observed.

parent strains. In the last strain pair, unique protein-staining bands were observed both in the parent (AM2) and in the IZIR strain (134). For all strains listed in Table 4, between N 24 and 30 bands were observed on 12.5% polyacrylamide gels when silver-stained compared with only from 6 to 10 bands when stained with Coomassie blue. For 23 of the 28 strains, the protein profile pattern was similar in the five successive extractions using 50 mM Trishydrochloride buffer (data not shown). For five strains, the protein profile pattern on the gels suggested that one or two proteins were selectively extracted in either the earlier or the later extractions. The protein profile patterns of five strain pairs were not influenced by the growth sugar (glucose or lactose) in T5 medium (data not shown). Molecular weight profiles of the loosely associated cell surface proteins from 10 strain pairs were compared on a Superose 12 column using a Pharmacia FPLC system. Since eluted material was monitored at 280 nm, it is possible that some of the material absorbing at this wavelength was not protein. For the strain pair 448/572, a high-molecular-weight protein peak (eluting at 29 min) was present as a major peak in the parent strain and was either absent or present at a relatively

Cell surfaces of lactococcal strains

59

low concentration in the IZIR strain (Fig. 5(A)). No differences were observed between these two strains on polyacrylamide gel analysis of the same extracts (Table 4). Contrasting pattern differences between the polyacrylamide gel analyses (Table 4) and molecular weight profiles (data not shown) were also observed for four other strain pairs: for strain pairs 2178/2178R7 and 266/566, no differences were observed in the molecular weight profiles whereas an extra band was present in the polyacrylamide gel profiles for the parent strain and the OR strain, respectively; an extra peak was observed in the molecular weight profile of the parent strain 108 compared with an extra stained band on polyacrylamide gels for

0.04 0.03

0.04

0.02

- 006

0.03

0.01

0.02

0.0

- 0.05

001 - 004 0.0

- 003

006

0 0:

0 08

0.06

0.04

002

0.0‘

0.0:

0.0:

0.0’

0.0

Time (min)

Fig. 5. Molecular weight profiles (absorbance at 280 nm) of the loosely associated material extracted from the strain pairs 448/572 (A), 584/2146 (B) and 104/578 (C) using 50 mM Tris hydrochloride buffer (pH 7.5, 30°C for 30 min) and separated on a Superose 12 column with an FPLC system.

60

Vaughan

L. Crow, Pramod

K. Gopal, Anthony

J. Wicken

the IZIR strain 546; no differences were observed between the staining pattern on polyacrylamide gels for E8/398, whereas the molecular weight profile indicated an extra peak for strain 398. Differences in the molecular weight profile between the pair 584/2146 (Fig. 5(B)) indicated that a high-molecular-weight protein (eluting at 26.4 min) was either absent or present at a relatively low concentration in the parent strain but was a major peak in the IZIR strain. The gel electrophoresis pattern also indicated extra bands in the RIR strain of this pair. Extra bands on the gels (Table 4) and molecular weight species by gel-permeation chromatography (data not shown) were also observed in the OR strain of the pair 1.58/402. Major differences in the molecular weight profiles were observed between the strains of the pair 104/578 (Fig. 5(C)): two extra peaks were present in both strains and higher proportions of three molecular weight peaks (eluting at 34, 35 and 37 min) were present in the IZIR strain. The gel patterns for this strain pair indicated one extra band in the 0R strain (Table 4). The strain pair AM2/134, which had extra gel bands in the extracts from both the parent and OR strains, also gave multiple protein peak profile pattern differences when the extracts were separated by gelpermeation chromatography (data not shown). Composition

of loosely associated cell surface polysaccharides

The composition of the loosely associated cell surface polysaccharides, retained after dialysis, from the parent and IZIR strains was similar for the first six of the nine strain pairs listed in Table 5, although the sixth strain pair, E8/398, showed a difference in galactose content. The last three strain pairs showed differences in the molar ratios of both galactose and rhamnose, relative to glucose, being higher in the three parent strains. The composition pattern was reproducible when two of the strain pairs were grown on separate occasions. The molar ratio of monosaccharides for the 18 strains was similar, whether extracted with 50 mM Trishydrochloride (pH 7.5) (Table 5) or 50 mM bis-Tris-propane buffer (pH 9.5) (data not shown). The molar ratios of the monosaccharides varied considerably between strains, with the galactose ratio ranging from 1 to 4 and the rhamnose ratio from 2 to 14, indicating considerable diversity in the composition of the loosely associated cell surface polysaccharides. These differences may reflect differences in intact polysaccharide and/or may result from variable and strainspecific loss of particular polysaccharides during extraction or dialysis (Fig. 4). The molecular weight distribution patterns of the polysaccharides eluted from a Sephacryl S-300 column varied between the parent and MR strains of three strain pairs studied; the results are shown for the pair 158/402 (Fig. 6). Properties of a variant with poor sedimentation The OIR strain 402 also has a variant (402s) that exhibited poor sedimentation properties compared with all other strains studied. On harvesting cells from either LM or T5 medium (at 13 OOOg for 10 min), strain 402s formed a fluffy pellet that was easily lost when the supernatant was poured off; all other strains formed a compact pellet The strain pair 158/402 was one of three out of the five strain pairs studied that showed a decrease in the extracted material when cells were grown on LM medium rather than on T5 medium. The LM medium was chosen to investigate

Cell surfaces

Composition

of Loosely

of lactococcal

TABLE 5 Associated Cell Surface Polysaccharides” Molar

Strain pair (parent/OR)

Glucose AM21

61

strains

ratio of monosaccharides Galactose

Rhamnose

1.0 1.0

1.3

2.8

1.0

2.4

566

1.0 1.0

3.1 (3.O)h 3.1 (3.3)h

7.3 (7.2)’ 9.2 (8.8)h

5841 2146

I.0 1.0

3.0 3.7

11.6 12.2

21761 2176R17

1.0 1.0

2.5 2.1

6.0 4.9

2178/ 2178R7

1.0 1.0

2.3 2.2

4.2 4.0

E8/ 398

1.0 1.0

1.8 1.0

7.3 78

104/ 578

I.0 1.0

4.3 1.3

13.2 3.2

lOS/ 546

1.0 1.0

2.0 (3.3)h 1.1 (1.3p

4481 572

1.0 1.0

3-4 1.5

134 2661

7.7 (7.5)h 2.4 (2.3p 14.1 3.9

“The five extractions (30 min at 30°C) with 50 tTIM Tris hydrochloride buffer (pH 7.5) from cells grown for 16 h on T5 medium were combined, dialysed, concentrated and hydrolysed in 4 M HCI at 100°C for 60 min, and the monosaccharides were analysed. hValues in parentheses represent results from separately grown cells.

the cell surface of the variant, as any changes in composition of the loosely associated material should have been more detectable. Clearly, more loosely associated cell surface protein and hexose (lo- and 50-fold, respectively) were extracted from 402s than the RIR strain 402 (Table 6(A)). The concentrations of protein and hexose extracted from 402 cells was less than found for the parent strain, 158. The two extra protein bands observed in strain 402, compared with strain 158 (Table 4), were also observed in the extracted material of strain 402s (data not shown). Analysis of the loosely associated cell surface polysaccharides (Table 6(B)) showed that the 402s polysaccharide(s) was enriched - 10 fold in rhamnose compared with the material extracted from strains 158 and 402. Slight differences in the galactose and rhamnose contents were observed for 158/402. The purified cell walls of strains 158 and 402 were similar in their rhamnose content (50-55% of cell wall dry wt), of which 3OO35% was released after mild

62

Vaughan L. Crow, Pramod K. Gopal, Anthony J. Wicken

0.16

0.04

40

60 Fraction

80

100

120

number

Fig. 6 The first three extractions (30 min at 30°C in 50 mM Tris hydrochloride buffer (pH 7.5) from strain pair 158 (A)/402 (0) were combined and a sample (I ml) from each strain was added to a Sephacryl S-300 column. Fractions (3.8 ml) were analysed for hexose.

(0.1 N H2S04 at 60°C for 24 h). In contrast, the cell wall of 402 S contained only 2530% of the cell wall (dry wt) as rhamnose and 95-100% was released after mild acid hydrolysis. acid hydrolysis

DISCUSSION There were a number of cell surface differences between the parent strain and the O-resistant derivative for all of the strain pairs studied. The possibility that defective phage adsorption was the resistance mechanism was identified for only some of the strains. In these strain pairs, the cell surface differences identified can not be clearly related to phage resistance. Where particular phage attacked the parent but not the OR variant, defective phage binding to the 0’ variant was

Cell swfaces

Properties

A Strain

158 402 402s B Strain 158 402 402s

of

63

qf lactococcal strains

TABLE 6 a Variant with Poor Properties

Sedimentation

Material e.utracted,from 100 mg (dry wt) bacteria” (mg) Protein

Hexose

0.25 0.12 5.10

0.41 0.29 3.10

Molar ratio qf monosaccharidesh Glucose

Galactose

Rhamnose

I.0 I.0 I.0

1.4 2.0 I.6

6.2 4.5 45.0

LILoosely associated cell surface material was extracted as for Table 3, except that cells were grown on an LM medium containing 0.5% (w/v) lactose. h Loosely associated cell surface polysaccharides were extracted and analysed as described in Table 5, except that cells were grown on an LM medium containing 0.5% (w/v) lactose.

found for pairs E8/398 (0618) 2176/2176R17 (01860), 584/2146 (01827) and 158/402 (0874). Phage binding was not impaired between strain pairs 2178/ 2178R7 (01727) AM2/134 (0602) (G. Davey, 1990, unpublished data) and 4854/ T.4854 (01440, 01496) (Jarvis, 1988). The latter group may still have cell surface differences associated with phage resistance. Resistance mechanisms involving the prevention of DNA injection and associated changes of cell wall antigens have been suggested but are not yet well established (Dunny et al., 1988). Alternatively, some of the cell surface differences observed between strain pairs may not be directly related to phage resistance. Recently, Jarvis (1993) found that the phage-resistance mechanism encoded by pAJ2074 prevented phage DNA replication and that its presence in strain LM0230 had no significant effect on phage adsorption or DNA injection. However, cell surface differences were observed between the strain pair 4854/T.4854 in the absence and presence of this plasmid in this different background. Whether the cell surface differences in this pair are related to phage resistance or to other changes imparted by the plasmid would require further investigations. The patterns of cell surface differences observed between the strain pairs were diverse. Although a number of strain pairs did not show differences in cell surface hydrophobicity, in other strain pairs either the parent or the OR variant was more hydrophobic. The phage-sensitive SK112 strain has a more hydrophobic cell surface and a higher negative charge than its phage-resistant derivative (Sijtsma

64

Vaughan L. Crow, Pramod K. Gopal, Anthorq

J. Wicken

et al., 1990~~). Surface lipoteichoic acids have been purported to be involved in cell surface hydrophobicity in group A streptococci (Miorner et al., 1983). Only two of the six strain pairs studied showed differences in cellular lipoteichoic acid concentration and the parent strain of one pair (AM2/134) had a lower lipoteichoic acid concentration but was more hydrophobic. No hydrophobicity differences were observed in the other strain pair (158/402), even though strain 402 had a higher concentration of cellular lipoteichoic acid. Either lipoteichoic acid is not important in determining hydrophobicity, or the cell surface location, specific composition or structure rather than just concentration of lipoteichoic acid is important. The concentration and composition of both the proteins and the polysaccharides removed from the cell surface by gentle procedures showed differences between all the pairs studied. One pair (104/578) showed differences in all these properties, whereas in another pair (2176/2176Rl7) only the protein profile pattern on polyacrylamide gel electrophoresis was different. Proteins and carbohydrates may be associated covalently or non-covalently with the bacterial cell wall (Sleyr et a/., 1988). The loosely associated cell surface material characterized in this study is likely to be a composite of non-covalent, freshly released covalent material plus their breakdown products and has the potential to influence surface properties due to its variability and location at the interface of the cell wall and internal environment. For example, it has recently been demonstrated that the loosely associated material from strain E8 can bind phage (Gopal & Crow, 1993). The loosely associated cell material in the present study was strain-dependent, differed between strain pairs and could be reproducibly extracted from the cell surface under mild conditions (physiological pH and mild temperature). The - 20 loosely associated proteins may have structural and/or enzymatic functions. The only cell surface associated protein that has been well characterized in lactococcal strains is proteinase (Thomas & Pritchard, 1987; Kok, 1990) and, even in this case, the cell surface architectural position and role of the proteinase requires further study. That many of the strain pairs showed a number of cell surface differences suggests that either the interlocking structural and functional reliance between surface components can produce a cascade effect when a single entity is altered or that a global regulator is altered. In the long term, it will be important to establish if the alteration in the phage receptor initiates the changes in cell surface. Therefore, it is necessary to be highly demanding of scientific evidence when attempting to assign a physiological function such as phage receptor to a cell component. The excess production of a rhamnose-rich polysaccharide(s) located loosely and covalently-bound to the cell surface in one strain (402s) may be responsible for the poor sedimentation properties of this strain compared with its parent strain, though with the proviso that further evidence is required. Varying the growth sugar, time of harvest (thus, pH at harvest) or medium changed the cell surface hydrophobicity for some, but not all, strains studied. Growth conditions influenced the concentrations of the loosely associated protein and polysaccharide material extracted from the surface of some strains. However, the pattern of protein bands of both the extracellular and the loosely associated material did not change in the strains studied when lactose was replaced by glucose as the growth sugar. Growth conditions alter the concentration and types of the extracellular lipoteichoic acids (Jacques et al., 1979rr,b) and the protein

Cell surfaces qf lactococcal strains

65

profiles (Hardy et al., 1986) of Streptococcus mutans strains. Other properties, such as compositions of polysaccharide, peptidoglycan or proportion of polysaccharide in the cell wall, were stable under different growth conditions of S. mutans Ingbritt (Knox et al., 1979). In general, the cell surface properties of bacteria change in response to the environment (Tempest & Wouters, 1981; Knox & Wicken, 1985). Lactococcal strains are no exception, and thus it is important in comparative work, such as between strain pairs, to culture and harvest under similar conditions. This study has highlighted the complex and variable nature of some lactococcal cell surface components and properties. The strain pair, E8/398, has been selected for further study (Gopal & Crow, 1993). From the present study this strain pair has a number of cell surface differences, with the IZIR variant defective in phage adsorption. Further, mechanistic studies on the structure, function and interactions of lactococcal cell surface components of selected strains are needed to define its role in dairy fermentations.

ACKNOWLEDGMENTS We are grateful to Graham Davey, Audrey Jarvis and Gaetan Limsowtin for the strains used in this study and for helpful discussion. Thanks are also due to Jillian Smith for excellent technical assistance, to Tim Coolbear and John Smart for helpful discussion and to Tim Coolbear for the FPLC analysis. Part of this work was carried out at the School of Microbiology, University of New South Wales while V. L. Crow was on staff development leave; the help and hospitality of the school, and in particular Lucy Dagostino and Mary Souflias, are gratefully acknowledged. This research was supported in part by the Foundation for Research, Science and Technology, New Zealand.

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Dische, Z. (19.55). New colour reactions for determinations of sugars in polysaccharides. In Methods of Biochemical Analysis, Vol II, ed. D. Glick. Interscience, New York, pp. 313358. Dunny, G. M., Krug, D. A., Pan, C.-L. & Ledford, R. A. (1988). Identification of cell wall antigens associated with a large conjugative plasmid encoding phage resistance and lactose fermentation ability in lactic streptococci. Biochitnie, 70, 4433.50. Forsen, R., Niskasaari, K. & Niemitalo, S. (1985). Immunochemical demonstration of lipoteichoic acid as a surface-exposed plasma membrane antigen of slime-forming, encapsulated Streptococcus cremoris from the fermented milk product viili. FEMS Microhiol. Lett., 26, 249-53. Gibbons, M. N. (1955). The determination of methylpentose. Analyst (London), 80,268876. Gopal, P. K. & Crow, V. L. (1993). Characterization of loosely associated material from the cell surface of Lactococcus lactis subsp. cremoris strain E8 and its phage resistant variant strain 398. Appl. Environ. Microhiol.. 59, 3177-182. Hardy, L. N., Knox, K. W., Brown, R. A., Wicken, A. J. & Fitzgerald, R. J. (1986). Comparison of extracellular protein profiles of seven serotypes of mutans streptococci grown under controlled conditions. J. Gen. Microbial., 132, 13899400. Jacques, N. A., Hardy, L., Campbell, L. K., Knox, K. W., Evans, J. D. & Wicken, A. J. (1979a). Effect of carbohydrate source and growth conditions on the production of lipoteichoic acid by Streptococcus mutans Ingbritt. Infect. Immun., 26, 1079-87. Jacques, N. A., Hardy, L., Knox, K. W. & Wicken, A. J. (19796). Effect of growth conditions on the formation of extracellular lipoteichoic acid by Streptococcus mutans BHT. Infect. Immun., 25, 75-84. Jarvis, A. W. (1981). The use of whey-derived phage-resistant starter strains in New Zealand cheese plants. NZJ Dairy Sci. Technol., 16, 25-3 I. Jarvis, A. W. (1988). Conjugal transfer in lactic streptococci of plasmid-encoded insensitivity to prolate- and small isometric-headed bacteriophages. Appl. Environ. Microhiol., 54,777-83. Jarvis, A. W. (1993). Analysis of phage resistance mechanisms encoded by lactococcal plasmid pAJ2074. Can. J. Microbial., 39, 252-g. Jarvis, A. W., Heap, H. A. & Limsowtin, G. K. Y. (1989). Resistance against industrial bacteriophages conferred on lactococci by plasmid pAJllO6 and related plasmids. Appl. Environ. Microbial., 55, 1537-43. Keogh, B. P. & Pettingill, G. (1983). Adsorption of bacteriophage eb7 on Streptococcus cremoris EB7. Appl. Environ. Microbial., 45, 19468. Knox, K. W. & Wicken, A. J. (1985). Environmentally induced changes in the surfaces of oral streptococci and lactobacilli. In Molecular Basis of Oral Microbial Adhesion, ed. S. E. Mergenhagen & B. Rosen. American Society for Microbiology, Washington, DC, pp. 212-19. Knox, K. W., Jacques, N. A., Campbell, L. K., Wicken, A. J., Hurst, S. F. & Bleiweis, A. S. (1979). Phenotypic stability of the cell wall of Streptococcus mutans Ingbritt grown under various conditions. Infect. Zmmun., 26, 1071-8. Knox, K. W., Hardy. L. N., Markevics, L. J., Evans, J. D. & Wicken, A. J. (1985). Comparative studies on the effect of growth conditions on adhesion, hydrophobicity, and extracellular protein profile of Streptococcus sanguis G9B Infect. Immun., 50,545554. Kok, J. (1990). Genetics of the proteolytic system of lactic acid bacteria. FEMS Microbial. Rev., 87, 1542. Kurz, G. & Wallenfels. K. (1974). D-Galactose UV-assay with galactose dehydrogenase. In Methods of Enzymatic Analysis, Vol. 3, 2nd edn, ed. H. U. Bergmeyer. Verlag Chemie, Weinheim, pp. 1279-82. Laemmli, U. K. (1970). Cleavage of structural proteins during the assembly of the head of bacteriophage T4. Nature (London), 227, 680-5. Lucey, M., Daly, C. & Fitzgerald, G. F. (1992). Cell surface characteristic of Lactococcus lactis harbouring pCI528, a 46 kb plasmid encoding inhibition of bacteriophage adsorption. J. Gen. Microbial.. 138, 213743.

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Wicken, A. J. & Knox, K. W. (1971). A serological comparison of the membrane teichoic acids from lactobacilli of different serological groups. J. Gen. Microbial., 67, 2514. Wicken, A. J. & Knox, K. W. (1975). Characterization of group N streptococcus lipoteichoic acid. Infect. Immun., 11, 973-81.