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Autolytic systems in propionic acid bacteriaHilde M. 0stlie, Gerd Vegarud, Thor Langsrud

To cite this version:Hilde M. 0stlie, Gerd Vegarud, Thor Langsrud. Autolytic systems in propionic acid bacteria. Le Lait,INRA Editions, 1999, 79 (1), pp.105-112. �hal-00929641�

Lait (1999) 79,105-112© Inra/Elsevier, Paris

105

Review

Autolytic systems in propionic acid bacteria

Hilde M. 0stlie*, Gerd Vegarud, Thor Langsrud

Department of Food Science, Agricultural University of Norway,P.O. Box 5036, N-1432 Âs, Norway

Abstract - This review summarises the present knowledge of autolysis and autolytic systems of dairypropionibacteria. Details of physical and biochemical parameters affecting autolysis in media and butferare presented, The effect of strain variation, temperature of incubation, growth phase, pH, ioniestrength and cations is discussed. In addition, different methods to follow autolysis and specificity stud-ies of autolytic enzymes are described, Autolytic enzymes in specifie cell fractions were studied byrenaturing sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE); ail strainsshowed Iytic activity by this method. © Inra/Elsevier, Paris.

Propionibacterium / autolysis / buffer studies / SDS-PAGE

Résumé - Systèmes autolytiques des bactéries propioniques. Cet article a pour but de faire unrésumé des connaissances actuelles en ce qui concerne l'autolyse et les systèmes autolytiques des bac-téries propioniques utilisées en laiterie. Les différents paramètres physiques et biochimiques quiaffectent l'autolyse en bouillon et en solution tampon sont présentés en détail. Il est question del'effet des paramètres suivants: variations de souches, température, phase de croissance, pH, forceionique et cations. En outre, différentes méthodes pour suivre l'autolyse, ainsi que des études despécificité des enzymes autolytiques, sont mentionnées. La technique d'électrophorèse renaturantesur sodium dodécyl sulfate gel de polyacrylamide (SDS-PAGE) a été utilisée pour caractériser lesenzymes autolytiques dans des fractions cellulaires spécifiques. Toutes les souches s'avèrent mon-trer une activité autolytique par électrophorèse renaturante SDS-PAGE. © Inra/Elsevier, Paris.

Propionibacterium / autolyse / solution tampon / SDS-PAGE

1. INTRODUCTION involve the formation of peptides and aminoacids from caseins, fatty acids from milkfat, and the conversion of lactose to lactateor other fermentation products, such as COz'ethanol, acetate and other aroma compo-

Ripening of cheese is a complex processwhich involves the degradation of carbo-hydrate, fat and protein. The main changes

Oral communication at the 2nd Symposium on Propionibacteria, Cork, Ireland, June 25-27, 1998.* Correspondence and reprints. hilde.ostlie@inf.nlh.no

106 H.M. 0stlie et al.

nents. Ithas been known for a long time thatproteolysis of milk casein is a key factor inthe ripening of cheese. Autolysis of bacterialcells has attracted interest owing to the pos-sibility of accelerating proteolysis.

Bacterial autolysins are defined asendogenous enzymes that hydrolyse cova-lent bonds in the peptidoglycan causing selflysis of intact bacterial cells [21, 23]. Thephysiological role of these potentially lethalenzymes is not fully understood. It is sug-gested that the autolytic enzymes areinvolved in important biological processessuch as cell division and separation, cellwall turnover, competence in genetic trans-formation, formation of flagella, and sporu-lation [21, 24, 26, 29]. Generally, autolyticenzymes have been considered to be syn-onymous with the peptidoglycan hydrolases.However, peptidoglycan hydrolases whichhydrolyse bonds in the peptidoglycan thatare not relevant for the mechanical stabil-

ity of the cell wall, or enzyme activities thathydrolyse only relatively few of the struc-turally essential bonds of the wall peptido-glycan, without release of soluble products,are not considered to be autolysins. Theenzymes are classified on the basis of theircleavage specificities as N-acetylmurami-dases, N-acetylglucosaminidases, N-acetyl-muramyl-L-alanine amidases and endopep-tidases (figure 1). The dairy propionibacteria(PAB) contain mainly L-diaminopimelicacid (L-DAP) as the diamino acid in thepeptidoglycan of the cell wall; however,Propionibacterium freudenreichii containsmeso-diaminopimelic acid (m-DAP) [4].In strains with L-DAP, glycine is the bridg-ing amino acid between DAP and the D-ala-nine of the adjacent chain. Most bacteriacontain multiple autolytic enzymes. The pres-ence of multiple autolysins within one organ-ism have complicated the determination ofthe exact function of these enzymes [24].

1 IIJ- J-

...GleNA - MurNAc - GleNAc ...I1I~ 1

L-Ala1

D-Glu IV IVIv~1 J- J-

dAA - [~] - D-AlaIv~1 1

D-Ala dAA1

n-oi«1

L-Ala1

...GleN - MurNAc - GleNAc ...

Figure 1. Schematic presentation of the sites of hydrolysis of the peptidoglycan by peptidoglycanhydrolases present in bacteria. I, N-acetylglucosaminidase; II, N-acetylmuramidase; III, N-acetyl-muramyl-L-alanine amidase; IV, endopeptidase. Abbreviations: GleNAc, N-acetylglucosamine;MurNAc, N-acetylmuramic acid; dAA = diamino acid; [R4J = interpeptide bridge.Figure 1. Présentation schématique des sites d'hydrolyse du peptidoglycane par les hydrolases pep-tidoglycaniques présentes dans les bactéries. I, N-acetylglucosaminidase ; II, N-acetylmuramidase ;III, N-acetylmuramyl-L-alanine amidase ; IV, endopeptidase. Abréviations: GleNAc, N-acetyl-glucosamine ; MurNAc, acide N-acetylmuramique ; dAA = diaminoacide ; [R4J = pont interpeptidique.

Autolysis of propionibacteria

Dairy PAB are important organisms inEmmental and other Swiss-type cheeses,where they con vert lactate to propionate,acetate and CO2. The acids and other volatilecompounds contribute significantly to thecharacteristic flavour and aroma, and CO2 isresponsible for the characteristic eyes inthese cheeses [7, 8]. In addition, aminoacids, particularly proline, and sm ail pep-tides are also assumed to contribute to thesweet, nutty flavour [10]. Autolysis of PABmay be important for the release of intra-cellular peptidases which may influence theripening of Swiss-type cheeses.

The importance of autolysis in chee seripening is becoming evident [l, 3, 30, 31].However, the mechanisms of autolysisrequire further studies. In order to properlyevaluate the possible technological appli-cations to cheese ripening, the fundamen-tal properties of the autolytic systems of thebacteria being used must be understood.Autolytic studies of PAB were first con-ducted in aqueous systems, such as media orbuffer [12-14, 16, 17]. The main advantageof the se studies is that different parametersare more easily defined than in cheese. Thedisadvantages are that the aqueous systemslack the complexity of cheese. The inten-tion of this paper is to focus on recent workconducted on autolysis of the propionibac-teria, with the main focus on work con-ducted in the authors' laboratory.

107

2. MEASUREMENTOF AUTOLYSIS

Several methods, such as optical density(Ofr), enumeration, electron microscopy,and detection of released intracellular mark-ers, have been used to follow autolysis inbroth [II, 16,27,28].

Autolysis monitored by Ol) can be char-acterised by the following two parameters:the rate of autolysis, expressed as thedecrease in Ol) per minute during the first 60min; and the extent of autolysis, expressedas percentage decrease of optical densityafter a certain time.

2.1. Autolysis of propionibacteriain media

Data on the autolysis of PAB in mediaare summarised in table I. Growth and autol-ysis of 28 strains of PAB were measured insodium lactate broth (SLB) by Ol) mea-surements [16]. Large variations in growthand maximum autolysis occurred betweenstrains. Maximum growth varied from anOD6oo of 2.1 to 5.3 and maximum autoly-sis varied from lOto 90 %. Seventeen ofthe strains showed 70 to 90 % autolysis.Maximum autolysis was observed after 13 to72 d incubation at 30 oc. This observation isin contrast with the observation of Leméeet al. [13] who studied autolysis of 4 selected,

Table I.Autolysis of propionibacteria (PAB) in media.Tableau I.Autolyse des bactéries propioniques (PAB) en bouillon.

Strains Media Maximal autolysis

% dt temp

28 PAB SLB* 10-90 13-72 30°4PAB YEU 60-80 8-91 PAB YEL 80-88 8.3

Reference

[16][13][12]

t Days after inoculation / nombre de jours après l'inoculation.* Sodium lactate broth / bouillon de lactate de sodium.§ Yeast extract-sodium lactate / extrait de levure-lactate de sodium.

108 H.M. 0stlie et al.

good autolytic strains in growth mediumand observed spontaneous autolysis justafter reaching maximum optical density.Their observation corresponded to 60 to80 % autolysis after 8 to 9 d, respectively.These strains were also highly prone toautolysis in buffer. The spontaneous autol-ysis of Propionibacterium freudenreichiiCNRZ 725 was shown to occur at pH 6 to6.2, when the carbon source was depleted[l4]. P. freudenreichii CNRZ 725 showed88 % autolysis in yeast extract-sodium lac-tate broth (YEL) after 8.3 d of incubationat 30 -c [12].

Autolysis was monitored by measuringthe release of proline (Pro )-iminopeptidasefrom 6 strains [16]. Four strains showedmaximal Pro-iminopeptidase activity after52 to 59 d incubation and maximal autoly-sis, measured by OD, after 49 to 59 d. Twostrains showed an early uns table Pro-iminopeptidase peak after 8 to 9 d incuba-tion; however, a second high Pro-iminopep-tidase level was observed after 49 to 59 dincubation corresponding to maximumautolysis. This indicates a good correlationbetween Pro-iminopeptidase activity andmaximum autolysis, which makes Pro-iminopeptidase a good marker for autoly-sis of most of the strains tested. Autolysis oftwo of the strains were studied in more detailby using OD, Pro-iminopeptidase, DNAand RNA as markers of autolysis. In addi-tion, Pro production was followed. Resultsshowed that the change in OD is the bestmarker for following autolysis. The releaseof Pro-iminopeptidase activity could alsobe used as a marker; however, sorne limita-tions, such as enzyme stability, degradationby intracellular proteinases, or the regula-tion of the lytic enzyme system duringgrowth were noted. Both RNA and DNAwere good markers for measuring initiationof autolysis but not for measuring autoly-sis over a longer period of time, probablybecause of degradation by RNAses andDNAases. A major disadvantage of moni-toring autolysis in broth is that it is verytime consuming.

The influence of storage temperature onautolysis of 13 strains has also been tested.Maximum autolysis was observed at 30 "C,Three of the strains were little influencedby different storage temperatures and twoof the strains showed high autolysis at 20 "Cas well as 30 oc. These results may be ofsignificance during chee se ripening as Swiss-type chee se is held at 20 to 25 "C in thewarm room and at 10 to 12 "C in the coldroom during the ripening process.

Langsrud et al. [9, 10] and 0stlie et al.[16] found that propionibacteria releasedlarge amounts of proline when grown inmedia containing peptides. The release ofproline from peptides coincided with theautolysis of propionibacteria.

2.2. Autolysis of propionibacteriain buffer systems

Propionibacterial cells autolysed spon-taneously when they were transferred fromSLB to a buffer solution. This phenomenonhas been observed in many Gram-positiveorganisms [2, 15, 17,22]. Autolysis in bufferoccurs much faster than in broth.

Results regarding autolysis of propioni-bacteria in buffer are summarised in table II.0stlie et al. [17] studied autolysis of21 strains of propionibacteria in buffer sys-tems. Optimal conditions of autolysis weredetermined in potassium phosphate buffer(50 mmol-I.", pH 7.0). The effect of growthphase, temperature, pH, ionie strength andcations was investigated. The influence ofthe growth phase was tested on 6 strains andautolysis varied depending on the age of thecells. AlI strains showed maximum autoly-sis in the exponential growth phase at anOD of 0.2 to 1.4. The ability of the cells toautolyse decreased sharply at the end of theexponential growth phase and during thestationary growth phase. The influence oftemperature on autolysis was tested for13 strains. Autolysis was maximal at 30 "Cfor 5 of the strains and at 40 "C for the

Autolysis of propionibacteria 109

Table II. Autolysis of propionibacteria (PAB) in buffer.Tableau II. Autolyse des bactéries propioniques (PAB) en solution tampon.

Strains Buffer Maximal autolysis Specificity Reference

% ht temp pH

21PAB PPB* 20-80 41-51 30-40° 7.2 0.3--0.5 glycosidase [17]amidaselendopeptidase

57 PAB PPB§ 38-86 24 [13]

IPAB PPBI 65 3 40° 5.8 NAG1 [12]

t Hours of incubation / heures d'incubation.j Potassium phosphate buffer (50 mmol-L", pH 7,0) / tampon de phosphate de potassium (50 mrnol-L -l, pH 7.0).§ Potassium phosphate buffer (0.1 mol-L", pH 6.2) / tampon de phosphate de potassium (0.1 mol-L-I, pH 6,2).J Potassium phosphate buffer (0.1 mol-L-I, pH 5.8) / tampon de phosphate de potassium (0.1 mol-L:", pH 5,8).'H N-acetylglucosaminidase / N-acetylglucosaminidase.

remaining strains. Maximum autolysis var-ied from 20 to 80 % after 41 to 51 h of incu-bation. Seven strains autolysed between 50to 80 % under the conditions of the experi-ment. The rate of autolysis was highest forail strains at 40 and 50 "C during the first2 to 5 h of incubation. Autolysis was optimalat an ionie strength (1) of 0.3 to 0.5 depend-ing on the age of the cells. This observationmay be important when related to cheeseripening because 1 in the chee se usually isabout 0.3. The effect of pH on autolysis wasstudied and an optimum pH of 7.2 wasfound. In addition, there was indication of asecond optimal pH at 6.0. Autolysis wasstimulated by Na", K+ and NH4 + and wasinhibited by most of the divalent cationstested.

Lemée et al. [13] have studied autolysisof 57 strains of dairy PAB in potassiumphosphate buffer (0.1 mol·L-l,pH 6.2,37 "C) harvested during exponential growth.They found that both the rate and extent ofautolysis appeared to be strain-dependent.Two distinct clusters were observed. Onecluster, containing 8 P. freudenreichii strainsautolysed by 86 ± 9.4 %, and the other elus-ter, containing 49 strains, autolysed by38.3 ± 9.4 % after 24 h. Optimal autolysis of

P. freudenreichii CNRZ 725 cells and iso-lated cell walls was observed in phosphatebuffer at pH 5.8 and 40 "C [12]. The highestautolytic activity was observed inthe earlyexponential growth phase at an OD of 0.3.The effect of various salts on autolysis ofP. freudenreichii CNRZ 725 was also stud-ied, and the presence of potassium andsodium salts led to significant increases inboth the rate and the extent of autolysis.

The specificity of the autolytic enzymesfrom 5 strains of PAB was studied by 0stlieet al. [17]. Muramidase or a N-acetylglu-cosaminidase would lead to an increase inthe number of reducing sugar groups, whilean increase in free amino groups wouldresult from amidase or endopeptidase activ-ity. Increases in both reducing sugars andfree amino groups were observed for allstrains, but to various extents. Lysis of thepeptidoglycan seemed to result from a gly-cosidase and a N-acetylmuramyl-L-ala-nine amidase or an endopeptidase. Leméeet al. [12] reported only N-acetylglu-cosaminidase activity in the autolytic sys-tem of P. freudenreichii CNRZ 725. Thisresult is different from our findings and mayindicate strain variations.

110 H.M. 0stlie et al.

3. CHARACTERISATIONOF AUTOLYTIC ENZYMESBY RENATURINGPOL YACRYLAMIDEGEL ELECTROPHORESIS

The ability of autolytic enzymes to rena-ture after sodium dodecyl sulfate polyacry-lamide gel electrophoresis (SDS-PAGE) hasmade it possible to study their activity, sub-strate specificity and effect of physical andchemical treatments on activity [6,20,25].Characteristics of autolytic enzymes of PABstudied by SDS-PAGE are summarised intable Ill. The use of renaturing gel elec-trophoresis to determine the autolyticenzyme profiles in specifie cell fractions ofthe PAB have been studied by 0stlie et al.[18]. Inactivated Propionibacterium cellswere routinely used as substrate in the gels.The autolytic enzymes of 5 good autolysingstrains (70-90 %) of PAB were studied byrenaturing SDS-PAGE. Two differentautolytic profiles were observed among thestrains tested. P. acidipropionici ATCC4965 showed 8 autolytic bands of molecularmass 25, 31, 39,43,55,71,97, and 122 kDain the different cell fractions. The other4 strains, P. freudenreichii INF-a, P. freu-denreichii ISU P-59, P.freudenreichii ISUP-24, P. jensenii ISU P-50, showed onemain autolytic band of molecular mass inthe range 123-143 kDa in the different cell

fractions. In addition, a larger molecularweight band of weak intensity was oftenfound in the cell wall fractions. Identicalnumber of Iytic bands were observed wheninactivated cell walls were used as substrateinstead of inactivated cells; however, lowercontrast was observed in these gels. Micro-coccus luteus cells were also tested as a sub-strate but no lytic activity was observed fromany of the strains. Routinely, the gels wereincubated in Tris-HCl buffer (0.025 rnol-L -1,

pH 7.5) containing 1 % Triton X-100.

Lemée et al. [14] reported 81ytic enzymebands in P. freudenreichii CNRZ 725. Themost intense band of this strain had anapparent molecular mass of 121 kDa; 6 otheractivities had molecular masses of 81, 87,92, 100, 109 and 118 kDa; and a weak activ-ity band at 34 kDa. Lemée et al. [14] triedseveral solutions for renaturating lytic activ-ities. The optimum conditions were to incu-bate the gels in phosphate buffer (0.1 mol-L -l,

pH 5.8), KCl (0.1 mol-L'") or Emmentaljuice, all containing 1 % Triton X-100.

Both P. freudenreichii CNRZ 725 andP. acidipropionici ATCC 4965 had 8 lyticenzyme bands; however, the enzyme pro-file of P. freudenreichii CNRZ 725 was dif-ferent from the lytic enzyme pattern ofP. acidipropionici ATCC 4965. The multi-plicity of the lytic bands does not necessar-ily reflect the exact number of cell wallhydrolases. Proteolytic processing of cell

Table III. Characteristics of autolytic enzymes of propionibacteria (PAB) by SDS-PAGE.Tableau III. Caractéristiques des enzymes au toi ytiques des bactéries propioniques (PAB) parSDS-PAGE.

Strains Substrate" Samples! No. of bands Mw (kDa) Reference

1 PAB cells, cw if, cw, cm, s 8 25-122 [18]4PAB cells, cw if, cw, cm, s 1 123-143 [18]1 PAB cells cru de extract 8 34-121 [14]

t Substrate in the gel: propionibacteria cells or cell wall / substrat dans le gel: cellules ou paroi cellulaire debactéries propioniques.1 if, intracellular fraction; cw, cell wall fraction; cm, cell membrane fraction; s, supernatant fraction / if, fractionintracellulaire; cw, fraction de paroi cellulaire; cm, fraction de membrane cellulaire; s, fraction de surnageant.

Autolysis of propionibacteria

wall hydrolases may generate more than onepolypeptide that may or may not retain enzy-matie activity [5, 20]. Proteolysis ofautolysins has been described as a method ofautolysin regulation [19]. Proteolytic activ-ity of 5 propionibacteria strains has beentested and preliminary results demonstratedthat P. acidipropionici ATCC 4965 had thehighest proteolytic activity (0stlie, unpub-lished results). The 8 lytic bands ofP. acidipropionici ATCC 4965 may be gen-erated by proteolytic processing of one ormore autolytic enzymes.

4. CONCLUSION

The focus of this paper was the funda-mental aspects of the autolytic systems ofPAB. Conditions for growth and autolysis inmedia and buffers have been discussed.These conditions are very different from thecomplex conditions in a cheese. Therefore,further studies of autolysis of PAB in con-trolled model systems and in chee se arerequired to understand their behaviour incheese during ripening. To what extent strainvariations in autolysis are related to differ-ent enzymes or to different regulation mech-anisms is not known. Further studies areneeded to get a better understanding of thesevariations. The results described, especiallythe SOS-PAGE studies and specificity anal-ysis, suggest that more than one autolyticenzyme are involved in the autolysis of dairyPAB. Purification and characterisation ofautolysins need to be done to clarify thenumber and type of enzyme(s) involved inautolysis of PAB, and also to provide aninsight into the function and regulation ofautolysins, especially related to cheese ripen-ing. PAB grow in synergism with lacticstarter bacteria in the cheese, and thus, it isdifficult to distinguish their individu al con-tributions to proteolysis and the release ofpeptides and amino acids.

111

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