Relationships between arginine degradation, pH and survival in Lactobacillus sakei

Relationships between arginine degradation, pH and survival inLactobacillus sakei

Marie-Christine Champomier Verge©s a;*, Manuel Zun¬iga b,Franc°oise Morel-Deville a;1, Gaspar Perez-Mart|nez b, Monique Zagorec a,

S. Dusko Ehrlich c

a Laboratoire de Recherches sur la Viande, Institut National de la Recherche Agronomique, Domaine de Vilvert,78352 Jouy en Josas Cedex, France

b Departamento de Biotecnologia, Instituto de Agroquimica y Technologia de Alimentos (C.S.I.C.), Poligono de la coma s/n,Apartado de Correos 73, 46100 Burjassot, Valencia, Spain

c Laboratoire de Genetique Microbienne, Institut National de la Recherche Agronomique, Domaine de Vilvert,78352 Jouy en Josas Cedex, France

Received 8 August 1999; received in revised form 20 September 1999; accepted 21 September 1999

Abstract

Lactobacillus sakei is one of the most important lactic acid bacteria of meat and fermented meat products. It is able todegrade arginine with ammonia and ATP production by the arginine deiminase pathway (ADI). This pathway is composed ofthree enzymes: arginine deiminase, ornithine transcarbamoylase and carbamate kinase, and an arginine transport system. Thetranscription of the ADI pathway is induced by arginine and subjected to catabolite repression. In order to understand thephysiological role of the degradation of this amino acid we investigated the growth of bacteria under various conditions. Weshow that arginine degradation is responsible for an enhanced viability during the stationary phase when cells are grown underanaerobiosis. Arginine is necessary for the induction of the ADI pathway but in association with another environmental signal.Using a mutant of the L-lactate dehydrogenase unable to lower the pH we could clearly demonstrate that (i) low pH is notresponsible for cell death during the stationary phase, so survival is due to another factor than elevated pH, (ii) neither low pHnor oxygen limitation is responsible for the induction of the ADI pathway together with arginine since the ldhL mutant is ableto degrade arginine under aerobiosis. ß 1999 Federation of European Microbiological Societies. Published by ElsevierScience B.V. All rights reserved.

Keywords: Lactic acid bacteria; L-LDH; arc operon; arcA (ADI) pathway; Phosphoenol pyruvate phosphotransferase system;

Catabolyte repression; Acid stress; Meat

1. Introduction

The arginine deiminase pathway of arginine deg-radation (Fig. 1) is composed of three enzymes: ar-ginine deiminase (ADI, EC 3.5.3.6), catabolic orni-

0378-1097 / 99 / $20.00 ß 1999 Federation of European Microbiological Societies. Published by Elsevier Science B.V. All rights reserved.PII: S 0 3 7 8 - 1 0 9 7 ( 9 9 ) 0 0 4 9 2 - 9

* Corresponding author;E-mail: [email protected]

1 Present address: Laboratoire de reproduction et devel-oppement des plantes ENS-Lyon, 46 allee d'Italie, 69364Lyon cedex 07, France.

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FEMS Microbiology Letters 180 (1999) 297^304

thine transcarbamoylase (cOTC, EC 2.1.3.3) and car-bamate kinase (CK, EC 2.7.2.2). A fourth proteinimportant for this pathway is a membrane argininetransport system (arginine/ornithine antiporter). Thispathway, leading to ATP and ammonia production,is widely present among bacterial and archaebacteri-al species such as Bacillus, Streptococcus, Clostridi-um, Lactococcus, Pseudomonas, Mycoplasma andHalobacterium [1^3] and can ful¢l various roles. InPseudomonas aeruginosa it is used exclusively underanaerobiosis as sole energy source for growth [1]. InHalobacterium salinarum it is involved in the fermen-tative degradation of arginine [3] whereas in Bacilluslicheniformis it is involved in nitrogen regulationunder anaerobiosis [4].

Several types of complex regulations of the argi-nine degradation pathway have been observedamong bacteria. In P. aeruginosa and B. licheniformisoxygen is the main factor controlling its expression.In some lactic acid bacteria such as Carnobacteriumand Streptococcus, relationships with sugar metabo-lism and pH have been investigated, and repressionby glucose was observed [5^8].

The genes encoding the enzymes of the ADI path-way have been most extensively studied in P. aeru-ginosa [9], H. salinarum [3] and Rhizobium etli [10].

In Gram-positive bacteria the genetic organisation isknown for Clostridium perfringens [2] and B. lichen-iformis [4]. The genes from Streptococcus sanguishave also been cloned [11] but their sequence hasnot yet been reported.

The four genes involved in arginine degradation:arcA (ADI), arcB (cOTC), arcC (CK) and arcD (theantiporter), usually form an operon, the organisationand transcription of which vary in di¡erent species.In P. aeruginosa the genes are cotranscribed in asingle polycistronic transcript arcDABC, which isthen processed by a RNase [12]. The transcriptionof the arc operon is under the control of an Anr-dependent promoter belonging to the Fnr-dependentpromoter family [9]. In H. salinarum the gene organ-isation is arcRACB. Oxygen limitation yields di¡er-ential induction of transcripts controlled by ArcR[3]. In B. licheniformis, the gene cluster is arc-RABDC. arcR encodes an ArgR protein with func-tions similar to the Escherichia coli ArgR [4]

Among lactic acid bacteria Lactobacillus sakei,formerly called L. sake [13], is one of the main spe-cies belonging to the natural £ora of fresh vacuum-packed meat. It is also used as starter in the meatfermentation process mainly for its ability to rapidlymetabolise sugars resulting in a pH drop that pre-

Fig. 1. Schematic representation of the arginine deiminase pathway (ADI). ADI: arginine deiminase; cOTC: catabolic ornithine transcar-bamoylase; CK: carbamate kinase.

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vents the development of spoilage micro-organismsand ensures a right ¢rmness of the products. Theability of L. sakei to degrade arginine by the ADIpathway was reported many years ago but this prop-erty was mainly used as a di¡erentiating feature be-tween L. sakei and Lactobacillus curvatus, the closelyrelated species also found in meat and meat products[14]. The presence of the ¢rst two enzymes (ADI andcOTC) in L. sakei was shown [15] but the physiolog-ical role of this pathway has not been extensivelystudied yet.

In a previous work we have cloned and sequencedthe arc operon of L. sakei [16]. The gene order isarcABCTD. Mutants in the four genes involved inthe ADI pathway were constructed [16]. Upstream ofthe arcA gene a CRE (catabolite responsive element)sequence was found indicating a regulation by catab-olite repression. However, neither Fnr-like nor Anr-like boxes were found upstream of arcA. In thepresent study we investigated the physiological roleof arginine degradation by the ADI pathway. In or-der to explain the role of this pathway and its regu-lations we used mutants in the arc operon along witha ptsI mutant and an ldhL mutant.

2. Materials and methods

2.1. Strains and growth conditions

The origin of strains is listed in Table 1. All L.sakei strains were routinely grown at 30³C on MRSmedium [17]. MCD medium [18] containing 0.05 or3 g l31 arginine and 1 g l31 sugar was used forphysiological studies. The media were supplemented

with erythromycin 5 Wg ml31 for the propagation ofthe ptsI and arc mutant strains. Aerobiosis was ob-tained by shaking £asks at 150 rpm and anaerobiosiswas obtained by blowing nitrogen according to Hun-gate [19]. Growth was followed by measuring opticaldensity at 600 nm and by the determination of col-ony forming units after plating diluted aliquots onMRS agar plates. The initial pH of the MCD me-dium is 6.50. Values obtained after growth and re-ported in the ¢gures represent the mean þ S.D. of atleast three independent experiments.

2.2. Arginine deiminase activities

These were determined as previously described[16].

3. Results

Previous studies have shown that L. sakei is aux-otrophic for arginine and is unable to grow on argi-nine as sole carbon source [15]. Thus the degradationof arginine needs the presence of glucose as a carbonsource but high glucose concentrations repress argi-nine degradation. Arginine degradation can be ob-served only when arginine is present in the growthmedium at 3 g l31 [15]. However, these studies madeuse of complex media, making detailed interpreta-tions di¤cult.

In the present study we used a chemically de¢nedmedium [18] supplemented with two carbon sourcespresent in meat (glucose and ribose), at two arginineconcentrations (0.05 g l31 is the minimal concentra-tion required for growth; 3 g l31 is the concentrationthat fully induces the ADI pathway). In addition, thee¡ects of aerobiosis and anaerobiosis were investi-gated. We also tested the behaviour of RV2000, amutant of the L-lactate dehydrogenase (L-LDH),which is unable to lower the pH of the medium[20]. The growth and the viability of bacteria werefollowed as well as the pH variations re£ecting argi-nine degradation via the ADI pathway.

We tested di¡erent growth conditions regardingarginine concentration (0.05 g l31 and 3 g l31), aero-biosis or anaerobiosis, with glucose or ribose as thecarbon source. The results are presented in Fig. 2.Under aerobiosis on glucose (Fig. 2A) viable cells

Table 1Origin of L. sakei strains

Strain Description Reference

23K Wild-type strain Laboratory collectionplasmid-cured [23]

RV2000 23K ldhL : :Cma [20]RV1000 23K ptsI : :pRV10 [22]RV4000 23K acaA : :pRV401 [16]RV4010 23K arcB : :pRV402 [16]RV4020 23K arcC : :pRV403 [16]RV4030 23K arcD : :pRV404 [16]

aCm: chloramphenicol resistance gene of pC194.

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diminished soon after growth stopped, whatever thearginine concentration. The ¢nal pH was 5.50 re£ect-ing the production of acid from glucose fermenta-tion. On ribose under aerobiosis, the growth ratewas slower and survival was observed during station-ary phase. The ¢nal pH reached 6.88, indicating that

after the pH drop due to the ribose degradation,ammonia production led to a pH rise.

Under anaerobiosis (Fig. 2B), both on glucose andon ribose, the e¡ect of inducing concentration ofarginine was more pronounced. Survival was betterboth on ribose and on glucose when arginine 3 g l31

Fig. 2. Growth of L. sakei strain 23K in MCD medium containing glucose 1 g l31 (circles) or ribose 1 g l31 (triangles) as carbon sources.A low concentration of arginine (0.05 g l31, ¢lled symbols) or an inducing concentration (3 g l31, open symbols) was added. Bacteriawere grown under aerobiosis (A) or anaerobiosis (B). Experiments were repeated several times and a representative experiment is shown.Standard deviation was less than 5% of the mean for experiments under anaerobiosis and less than 10% under aerobiosis. The ¢nal pHmeasured after 48 h cultivation is indicated.

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was added. In both cases, the ¢nal pH was higherthan the initial pH of the medium, suggesting ammo-nia production resulting from arginine degradation.As under aerobiosis, survival was better on ribose

than on glucose and was correlated with the higher¢nal pH (7.38 on ribose versus 6.73 on glucose).These results suggest that a better survival in thestationary phase is linked to the degradation of ar-

Fig. 3. Growth of L. sakei strain RV4000 in MCD medium containing ribose 1 g l31 under anaerobiosis. A low arginine (0.05 g l31, ¢lledsymbols) or an inducing concentration (3 g l31, open symbols) was added. Experiments were repeated several times and a representativeexperiment is shown. Standard deviation was less than 5%. The ¢nal pH measured after 48 h cultivation is indicated.

Fig. 4. Growth of L. sakei strain RV1000 in MCD medium containing glucose 1 g l31 under anaerobiosis. A low concentration of argi-nine (0.05 g l31, ¢lled symbols) or an inducing concentration (3 g l31, open symbols) was added. Experiments were repeated several timesand a representative experiment is shown. Standard deviation was less than 5% of the mean. The ¢nal pH measured after 48 h cultivationis indicated.

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ginine correlated with an increased pH value. Indeedall the mutants in the arc operon were impaired insurvival during the stationary phase whether argininewas added or not as shown for the arcA mutant(RV4000) in Fig. 3.

A better survival on ribose than on glucose mightre£ect a repression of the ADI pathway on glucose.The presence of a CRE element upstream of arcA,responsible for the catabolite repression of the tran-scription of the arc operon, might explain why onglucose the ADI pathway is not active [16]. Carboncatabolite repression requires the CcpA protein andthe phosphoenol pyruvate phosphotransferase sys-tem (PTS) [21]. A mutant of enzyme I of the PTSwas previously constructed. Although this mutant(RV1000) does not possess a functional PTS, it isable to grow on glucose by a PTS-independent trans-port system [18,22]. RV1000 was then tested for itsgrowth and survival on glucose. We could expect, aswas shown in several other bacteria, that in RV1000catabolite repression is abolished. Indeed, RV1000seemed to have a higher arginine degradation levelthan the wild-type strain in the presence of glucose(Fig. 4). The ¢nal pH in the medium with argininewas 7.20, which suggests that arginine degradation is

higher than in the wild-type strain, for which pH was6.73 when grown in the same conditions. These re-sults support a catabolite repression e¡ect of glucosewhich is abolished in the ptsI mutant. Furthermore,no drop in population with arginine 0.05 g l31 wasobserved but the pH was still low (5.50), suggesting adi¡erent metabolism of glucose in the ptsI mutant.

All these results clearly indicate that the degrada-tion of arginine via the ADI pathway is correlatedwith an enhanced viability under anaerobiosis. Thisrole has been mentioned previously for other speciesbut it was not clearly demonstrated whether elevatedpH per se (due to ammonia production by the ADIpathway) was responsible for this enhanced viability.In order to investigate the putative role of pH in theregulation of the ADI pathway and the survival ofL. sakei during stationary phase, we used a mutantde¢cient in L-LDH activity. This mutant does notproduce lactate and does not lower the pH of themedium below 6.20 [20]. As shown in Fig. 5, whenan inducing arginine concentration was omitted, thecfu observed during stationary phase strongly de-creased although the pH was high (6.90). This sug-gests that the pH drop leading to the low pH valueafter glucose consumption is not the factor respon-

Fig. 5. Growth of L. sakei strain RV2000 in MCD medium containing glucose 1 g l31 under aerobiosis. A low concentration of arginine(0.05 g l31, ¢lled symbols) or an inducing concentration (3 g l31, open symbols) was added. Experiments were repeated several times anda representative experiment is shown. Standard deviation was less than 10% of the mean. The ¢nal pH measured after 48 h cultivation isindicated.

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sible for cell death after exponential growth. More-over, when an inducing arginine concentration wasadded, the ldhL mutant, like the wild-type, has anincreased cell viability during the stationary phasecertainly linked to arginine degradation as can bededuced from the high pH value (8.35). These dataseem to indicate that the low pH reached after ex-ponential growth is not responsible per se for celldeath. The protective e¡ect of arginine degradationis not due to elevated pH since this strain, whichdoes not lower the pH, has a low viability duringthe stationary phase and needs to degrade arginineto survive. Moreover, in mutant RV2000, this argi-nine degradation also took place during growthunder aerobiosis revealing that lack of oxygen isnot the factor responsible for the induction of theADI pathway, but rather another signal that mightbe linked to carbohydrate metabolism. The behav-iour of this strain indicates that the pH drop mightnot be this signal. Furthermore, ADI measurementscon¢rmed that arginine degradation was associatedwith survival. For the wild-type, maximum activitieswere obtained when cells were grown under anaero-biosis with arginine 3.19 þ 0.03 U under anaerobiosisversus 0 under aerobiosis, whereas RV2000 had ac-tivity under both aerobiosis (2.55 þ 0.31 U) andanaerobiosis (8.88 þ 0.88 U).

4. Discussion

We have previously shown that transcription ofthe arc genes encoding the ADI pathway is inducedby arginine and is repressed by carbon cataboliterepression [16]. In this work we show that argininedegradation by L. sakei is clearly associated with ahigher survival during the stationary phase since (i)the survival is correlated with the conditions of ex-pression of the ADI pathway and (ii) survival isabolished in the arc mutants. This degradation oc-curs through the ADI pathway and no other argininedegradation route could be detected, since arc mu-tants could no longer degrade arginine and survive.The presence of arginine is necessary to induce thissurvival but another environmental factor is neces-sary to promote this degradation. Catabolite repres-sion of the arc operon was con¢rmed by the use of aptsI mutant in which arginine catabolism was no

longer repressed by glucose. Such a repression byglucose has long been reported for various speciesexcept for some Carnobacterium strains which arereported to degrade arginine by the ADI pathwayeven at high glucose concentrations [6]. In L. sakeiit lightens the role of the PTS in the catabolite re-pression mechanism. Concerning anaerobiosis wecould demonstrate that the lack of oxygen per se isnot responsible for induction of the arc operonwhich is di¡erent from what was observed in P. aer-uginosa [9], R. etli [10] or B. licheniformis [4]. In H.salinarum, the induction by oxygen limitation wasdi¡erent for the di¡erent transcripts [3]. In L. sakeiarginine degradation could take place under aerobio-sis but only when cells were grown in the presence ofribose as the carbon source or in a ldhL mutant. Inthis mutant, the end products resulting from glucosecatabolism are di¡erent than in the wild-type strain[20]. Furthermore, ribose metabolism also leads todi¡erent intracellular compounds and end productssince ribose is catabolised through the phosphoceto-lase pathway. The signal responsible for ADI induc-tion might thus be linked rather to the metabolicstate of the cells. Intracellular ATP, NADH or otherintermediate metabolites pools might be good candi-date signals.

The enhanced viability might be attributed to thepH rise due to ammonia production by the ADIpathway as mentioned for S. sanguis [8]. These au-thors showed that the enzymes of the ADI pathwayhave their optimal pH of activity at lower valuesthan other intracellular enzymes. The activity ofthe ADI catabolic pathway was supposed to protectstreptococci of the oral cavity from an acid environ-ment. Nevertheless, these authors could demonstratethat the beginning of arginine degradation was notpart of the acid-adaptive response [5]. In L. sakei,this protective role against cell death cannot be re-lated only to pH protection, since the ldhL mutantthat was unable to lower the pH below 6.20 alsoneeds arginine degradation to survive. Moreover, ar-ginine degradation can take place even when the pHis still high and no clear-cut correlation between lowpH and cell death was observed. Since ATP is pro-duced from arginine degradation this indicates thatthe energy level is the triggering factor for survivalrather than the pH.

The environmental conditions leading to the ex-

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pression of the ADI pathway in L. sakei are: lowoxygen concentration, low glucose concentration orpresence of ribose, and arginine. They all seem to becompatible with its natural meat environment and itis likely that L. sakei has developed regulatory mech-anisms particularly well adapted to this environment.

Acknowledgements

This work was supported by a Spanish-French co-operation project PICASSO Action No. 96026. Wethank V. Davant for her technical assistance.

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Relationships between arginine degradation, pH and survival in Lactobacillus sakei