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Wl'l1H THE APPEARANC}; OF SLOW VAilIAN'I'S IN CULTURES OF STREP1
.rococcus
LACTISA. thesis preson ted in partial fulfih1ent of tho requirements fer the deg.reo of
M:.istor of Sci(,nce in ll~icrobiolc,gy at Y...::i.ssoy l\1:i.vcrsl t;<{, Ne,; Zealand
NicoJ Armst1·onB' SJdppe-,r
1972
(ii)
A
B S T RA
C TStreptococcus lactis C10 Slow, a variant of the normal 'fast' strain of S.J.actis
c10 ,
was only capable of rapid and extensive growth in skim milk if casein hydrolysates were added. It was postulated, therefore, that the slow variant is defectively proteolytic.A sensitive assay of proteolysis, based on the release of radio- activity from iodinated casein, was developed, checked for use-
fulness with known proteinases, then used to assay the streptococcal enzymes.
Fractionation of the two strains, by either mechanical cell disruption and differential centrifugation or by cell-wall digestion with a muraminidase, established that most of the cell-bound proteinases of the paront strain were surface-bound. Tb.is activity was virtually absent in the slow variant.
Partial characterization of the surface-proteinase(s) showed that maximum activity was exhibited at pH 6.o-6.8, in 0.05 M phosphate buffer, at 30-32°C. It 1-,as rapidJ.y inactivated at 37°c, both when cell-bound and when free of the cells. Examination of a
second pair of strains, s.1actis H1 Fast and S.,lactis H1 Slou, indicated a difference in protoinaso activity and localization similar to that found between the two S.lact.is C10 stra.inso
It was concluded, on the basis of both nutritional evidence and enzymatic analyses, that the slow variant of S.lactis C10 is limited in skim milk by the supply of amino acids and that this is due to a defective surfGce-bound proteolytic activity.
(iv) A C K N O W L E D G E M E N T S
I am indebted to the New Zealand Dairy .Research Insti·tute and the Department of Microbiology and_ Genetics, :Massey University, for providing the opportunity and facilities for this investigation.
In particular I would like to thank:
Dr
B.D.W. Jarvis for his guidance and constant encouragement.Dr
L.E. Pearce for his discussions and for the hospitality of his laboratory.Dr T0D. Thomas for advice with fractionation experiments, and for the gift of phage ml3 lysin.
Dr
R. C0 Lawre11ce for useful criticism.The Applied Biochemistry Division, Departmont of Scientific and Industrial Research, for the uEie of the Packard scintilla- tion counter.
Mr C6M., Rofe for the photographs ..
Mrs L. M. Foste:r for the typing.
P
RE F A C E
Rapid growth in milk, with the concomitant production of lactic acid, is the primary attribute of the single-strain
Cheddar cheese starter. Variations in a strain's activity may be due to changes in the culture's environment or to changes in its capabilities. A clear example of the latter is seen in those strains which segregato slow variants. The variants are stable genetic derivations of the normal 'fast' cells and, when cultured as pure isolates, exhibit limi tod growth in milk. Their
segregation at high frequency, together with their nutritional support by the parental cells9 enables accumulation to occur to the point where they contribute the majority of the cells in the culture.
It bas been suggested that slow variants are proteolytically deficient in comparison to the parental type. The relationship between starter proteolysis and activity in milk is unclear, but many strains are stimulated by the addition of nitrogenous hydrolysates to the mecliurno
This investigation was und0rta.ken to examine the hypothesis that slow variants are defectively proteolytic. It was anticipated that the work would also provide useful in:formation concerning the properties and cellular location of starter proteinases in general.,
C O N T
E
NT S
ABSTRACT • • • •
• • • • • 0 • • • • • • • • • • ••
••
• ACKNOWLEDGEMENTS • • • • • • • • • • • • • • • • • • • • • PREF.A.CE • • • • • • • • • • f> • • • • • • • • • • • • • •LIST OF
FIGURES
LIST OF TABLES LIST OF PLATES• • • • • • • • • • • • • • • • • • • • •
• • ;I • • • • • • • •
• • • • 0 • • • • • • INTRODucrrroN •
General
• • • • • • • • e
•
•Carbohydrate metabolism Nutritional requirements Milk as a medium
• • • • • •
•
•• • • • • • • •
•
• • • • • • •Starter proteinasos and ' slowness'
AIM
OFTHE INVESTIGATION.
• • • •. . ,. • • • • • •
•
• ••
•
•• • •
•
•
• METHODS • • • • c i o e • • • e - • • • • • • • • • • • • • •(vi)
Page ii
iv
V
ix xi xi 1 1
1 2
4
5 7
8
Microbiological methods 8
Media 8
Bacteria and bacter iophage 10
Culture maintenance 12
Preparation of cells for assay and fractionation 14
Determination of viable count 14
Determination of phage titre 15
Analytical methods 15
Lactate determination 15
Protein determination 15
Dry-weight do-termination 17
Pro
teinase a.s say 17Introduction 17
Preparation of proteinase substrate Measuroment of iodine-125
The substrate blank Standard assay procedure Assay conditions
Units
Measui~ement of trypsin and subtil isin Fractionation procedures
Introduction The Mickle method
The spheroplast method.
RESULTS • • O • 0 • e • C e o • e e e • e • 6 • • • • • • Growth of S.,la.ctis C10
Growth in skim milk, the symptoms of slowness Growth in T
5 brot~
Growth in mo<lifi ed skim milk
Preliminary experiments to localize Sola,ctis C'10 protei11ases
Proteinase assays
EnzY1J1e dis tri bu ti on: Mickle method Enzymo distribution: spheroplast method Characterization of (crude) S~ lac tis
cm
Fastsurface proteinase(s) Effect of pH
Effect of buffer strength Effect of temperature
Effect of ED'rA and other reagents
Page 18
20 20
23
24 24 2427 27 27
28
31 31 31 31 3439 39
4040
43
43 43 43 43
Standardization of conditions of fractionation and assay
Localization of S.lactis C10 proteinases Enzyme localization: Mickle method Enzyme localization: spheroplast method
0 .
Instability of S.lactis C10 proteinases st
37
C Whole-cell proteinasesCell-wall digest proteinasos
Localization of S,lactis H1 proteinases
DISCUSSION. • • • •
. . . . . . . . . . . . . . . . . . .
Proteinase localization
The importance of proteolysis to acid production REFERENCES • • • 0 • • • • • • • • • • • • • • 0 • • • • •
(viii)
48 48
49 51
58 58
60 60
6565
70
74
L I S T O F F I G U R E S
1. Colorimetric measurement of lactateo 16
2. Effect of trichloroacetic acid (TCA) concentration
on the substrate blank. 21
3.
Non enzymatic release of radioactivity fromiodinated casein. 22
4. Proteolysis of iodinated casein by trypsin. 25
5.
Proteolysis of iodinated casein by subtilisin. 26 6. Cell fractionation by the Mickle method. 297.
Growth of s.1actis c10 Fast and S.lactis c10 Slowin skim milk., 32
8.
Acid production by S.lactis C10 Fast anQ S.lactisC10 Slow in skim milk. 32
9. Relationship between growth and acid production
in skim milk
(30°c). 33
10. Growth of S.lactis C10 Fast and s.1actis
cm
Slow in T5
brotho35
11. Relationship between growth and acid production in
T
5
broth(30°c) . 35
12. Effect of casein bydrolycut.es on S.la.ctis C10 Slow in skim milk. 1. Hyd.rolysates a.d.ded before inoculation.,
13.
15.
16.,
17.
18 ..
Effect of casein hydrolysates on S~lactis C10 Slow in skim milk. 2. Hydrolysa tes added 7 hours after inoculation.
Effect of casein byd.roly0ates on S.lactis C10 Fast in skim milk.,
Effect of pH on Solactis C10 surface proteinase(s)o Effect of buffer strength on
s.
la.ctis C10 Fastsurface proteinase(s).
Effect of temperature on S~lactis C10 Fast surface proteinase(s)o
Time course of release of proteinase(s) from osmotically stabilised cells of S.lactis c10 Fa.st and S., lac tis c,rn Slow.
37
37
38 44
45
46
53
19. Inactivation of S.lactis C10 Fast whole cell proteinase(s) at
3 7° c.
20. Inactivation of S.lactis C10 Fast surface proteinase(s) at
37°c.
21. Inactivation of Solactis H1 Fast surface proteinase(s) at
37°c.
22. The assimilation of exogenous amino acids by
s.1actis c10: an ovorviewo
(x)
59
61
69
1.
3.
L I S T 0 F' T A B L E S
Culture maintenance..,
Preparations of proteinasa substrate. Proteinase localization in Solactis C10
(prelimina:cy data).
4.
Proteinase localization in S.lactis C10: enzyme distribution in fractio11s prepared by the Mickle method.5.
Release of cell-bound proteinn.se from osmotically sta:bilized cells of S.laetis C10 Fas·t and S.lactis C10 Slow.6. Proteinasa localization in Ss lac tis C10: enzyme distribution in fractions prepared by the spheroplast method.
7.
8.
Proteinase localization in S.lactis C10: comparison of results from the two fractionation methods.
Proteinase localizati.on in S.,Jactis H1.
L I S T 0 F
P L
AT E S
1. Differentiation of S0 lactis C10 Fast and S.lactis C10 Slow on citra.tod-milk-agar.
13 19
42
50
54
56
57
6411
I N T R O D U C T I O N
A small number of strains of Streptococcus lactis and Streptococcus cremoris (the lactic streptococci) are used as ' starters' in the manufacture of Cheddal.' cheese. Their primary role is to produce lactic acid; they also contribute to cheese flavour (Lowrie, Lawrencc9 Pearce & Richards, 1972 ).
1
A clear-cut difference in activity (rate and extent of acid production in milk) between the cells in somG pure cultures of S.lactis was reported by Harriman & Hammer (1931 ). About 2% of the cells were ' slow co3.gulators' .. The slow characteristic was stable and the variant culturei, never segregated the parental
'fast' cells,. Analysis of the non-protein nitrogen fraction of 'fa.st' and 'slow' milk cultures indicated less proteolysis in tho latter, al though proteolytic activity por cell was not detormined., Subsequent uork has confirmed: ( 1) the high fre- quency with which slow ,ra:riants appoar in susceptible cul tlll'es
(Garvie & Mabbitt, 1956; Citti, Sandine & Elliker, 1965; West- hoff, Co,-rman & Spack9 '1971); (2) the one-way nature of the
transition (Garvie & Tfabbitt, 1956; Cit-ti et alQ, 1965); and (3) the correlation between 'slowness' and relatively low
proteolytic acti.vi ty (Ci tti et
f-1-.•,
1965; Wo,.:ithoff et al., 1971 ).'l'he last point has been tho_ lea.st convincingly shown and its examination forms the basis of the present investigation.
CAHBOHYDRA 'J1E T'-ETABOLISM
The production of lactic acid by starters is the direct
result of energy-yielding carbohydrate catabolism and is the growth function usually measured. ,-ih en starter 'activity' is assessed.
The lactic streptococci are part of a large group of micro organisms that carry out the homolactic fermentation. This process, analagous to muscle glycolysis, has been elucidated. largely through the use of radioactive tracers and has been thoroughly reviewed (Wood,
1961,
Kandler,1961;
Yarth,1962;
Busse,1966).
The fate of lactose is as follows:~bat lactic streptococci (including S.lactis C10) accumulate lactose by a phonphoenolpyruvat.e-dependent phosphotransferase system
(McKay, Miller, Sandine & Elliker,
1970),
a.nalagous to that of Staphylococcus aureu:::. The sugar, transported as a phosphate derivative, is split by a 6-phospho-/-galactosidase into glucose and galactose-phonphate, 1rhich then enter the &nbden-1leyerof-Parnas pathway. The latter is sunmarized as follows: (lfood,1961)
glucose + 2ADP +· 2P
1
->
2 lactate+ 2ATP + 2 H 20The proclucts from one mole of lactose are, theoretically, four moles of lactate and four moles of ATP. Analysis of the products of glucose metabolism by hornolactic stroptococci has sho\om that small amounts of acetate, format.o,
co
2, ethanol, acetoin, and 2,3-butanediol may be formed in addition to lactic acid, their relative proportions depending on culture conditions, particularly
the pH. At the loast, lactate accounted for
90%
of the recovered glucose carbon (Platt & Fostor,1958).
NUTRJ TJONAL REQ,UIREF.ENTS
Tho rate and extent of acid production by a given single- strain starter reflects its ov0rall ability- to grow in the
3
medium, an ability determined by a complex interaction between the available nutrients, the culture conditions, and the strain's capabilities. In the absence of constraints, such as inhibitors
(e.g. penicillin in milk), suboptimal temperatures, and virulent bacteriophage, the activity is probably a function of the strain's ability to obtain essential amino acids from the medium.
Niven (1944) reported. that S.lactis required niacin, biotin and pantothenate for growth in a medium complete but for the B vitamins. Thiamin and riboflavin were essential for 18 and 10 strains, respectively, of the 21 examined, while pyridoxine stimulated most. Amino acid requirements were extensive. All strains required valine, loucine, i soleucine, arginine and
methionine·for growth, but a response comparable to that obtained in the presence of casein hydrolysates required a minimum of 16 amino acids. The 0111;,, acids without effect \-1ere tyrosine, norleucjne, aspartic acid, threonine, hydro:xyproline and
tryptophane. Tho absolute requirement of all strains for glutamine and asparagine was contro-vo:r.3ial, Pollack & Lindner (1944) having reported that several strains of S.lactis could use the equival ent acids if they were included at sufficiont concentration. Sub- sequent investigations have substantiated the findings of Niven, minor discrepancies being attributed to the use of different basal media, experimental conditions, and strain3. Husain &
McDonald ( 1957) found the.t s.1actis and
s.
cremoris required histidine in addition to the acids reported by Niven; however,excellent grouth was obtained in the presence of only 12 amino acids, roflocting the basal lltedium which was improved by the inclusion of Tween 80 (a source of oleate) and acetate. Their finding that
s.
c,~cmoris was more fastidious than S. lactis foramj_no acids was conf:i.rmod by ReJ ter & Oram ( 1962).
:MILK AS A :MEDIUM
Bovino milk, the stock substrate for starter maintenance and cheesemaking, appears to contain ample concentrations of protein, lactose, and the essential vitamins (average levels in whole milk9 per l i tre: protein, 35 g; lactose, 49 g; niacin, 0.94 mg; biotin, 31 f g; pantothenate, 3.46 mg; thiamin, 0.44 mg;
riboflavin, 1o 75 mg) (Wel1b & Johnson, 1965; Hartman & Dryden, 1965). All the essential amino acids, except methionine, have been found in the non-pr-otein nitrogen fraction of milk (:Block,
1951; Van der Zant & Nelson, 1954; Deutsch & Samuelson, 1959;
Schwartz & Pallansch, 1962), but tha concentrations are generally considered to be insufficient for optimal starter development.
'I'he evidence for this contenticn is indirect and generally un- satisfactOTy but forms tho basis of the "prote:inase-dependency"
hypothesis. Tb.ere have berm numerous reports of the stimulation of lactic streptococci in milk, particularly of 'slow' strains, by nitrogenous extracts (pancrens extract, yeast extract, corn steep liquor , liver fraction L; seo reviow by Reiter & Ii/'1011 er- Madsen, 1963 )o The effect of the~:;e additives has usually been attributed to peptid0n but the only complex whose stimulatory component ho,s been identified. ("pancreas extract") was effective because of its content of inosino9 hypoxan-thine, and adenine
(Ko burger, Speck & Aurand9 1963). 'Iba only convincing evidence for stimulation specifically by amino acids and/or peptides was given by Garvie & }fabbi tt
(1 956 )
who demonstrated that peptone, casamino acids, and acid--hydrolysed casein stimulated both S., crernoris 5)24. and its slow va,:i.·ia.nt in milk.,5
STARTER PROTEINASES A.ND 'SLOWNESS'
T'.11ere is a sound a-priori basis for the hypothesis that starter growth in milk i s dependent 011 milk ·protein degradation, and that slow strains and slow variants are special cases of cultures limited by this constraint. The evidence for the importance of starter proteinases in this process is scarce and difficult to assess, due mainly to the very low proteolytic activity of even the fastest of strains.
Proteinasea have been purified from the 'solubl e' and 'particulate' fractions of sonicated cell suspensions of both S.lactis 3 and its slow variant (Cowman, 1966; Westhoff, Cowman
& Swai sgood, 1971; Westhoff, Cowman & Speck, 1971). Quantitative
differences in proteolytic activity between the two strains, whether assessed from whole cells or from the fractions, were very low (ca. 1.
5-f'o
ldt
The variant ' intra.cellular' enzyme did, however, differ in rna.ny properties from the equivalent enzyme of the parent strain, and the authors were forced to conclude that the difference in specificity of the proteinase was responsible for the in1111ili ty of the variant to grow exten-sively in milk ('Westhoff & Cowman, 1971 ). :Much of the published work concerning S.,la,ctis
3.
is unsatisfactory, al though there can be no doubt that the protcinases have been thoroughly characterized as enzymes. Tb.a proteinase assay used was not tested for its sensitivity or reproducibility and specific activities of assayed fractions a.re rarely stated. In addition to this lack of data for the comparison of activities, the evidence for the localization of the proteinases, essential to a meaningful int0rpratation of their physiological function, is insuffi.cient, consisting m6rely of whother or not the activityremains with
the
pellet after centrifugationof
disruptedcells
(Cowman, 1966). The major criticismof
the work, however,concerns
thelack of
essential information relevantto the
procedures, a deficiency which
precludesboth the interpretation
of the results and the extension of the
methodsto other strains.
A I M 0 F THE I N V E S T I G A T I O N
The aim of this s tucly vas to examine the hypothesis that slow variants are relatively non-proteolytic in comparison to normal 'fast' strains. It uas evident from assessment of previous attempts to characterize the proteinase enzyme systems of S. lactin that the uork would require a very sensitive assay for proteinaae and subsoq_uently a critical determination of the locali~ation of relevant enzymes in the organisms.
7