3206 Proteomics 2012, 12, 3206–3218DOI 10.1002/pmic.201200157

RESEARCH ARTICLE

Metabolic and proteomic adaptation of Lactobacillus

rhamnosus strains during growth under cheese-like

environmental conditions compared to de Man, Rogosa,

and Sharpe medium

Claudio Giorgio Bove1, Maria De Angelis2, Monica Gatti1, Maria Calasso2, Erasmo Neviani1

and Marco Gobbetti2

1 Department of Food Science, University of Parma, Parma, Italy2 Department of Soil, Plant and Food Science, University of Bari Aldo Moro, Bari, Italy

The aim of this study was to demonstrate the metabolic and proteomic adaptation of Lactobacillusrhamnosus strains, which were isolated at different stages of Parmigiano Reggiano cheeseripening. Compared to de Man, Rogosa, and Sharpe (MRS) broth, cultivation under cheese-like conditions (cheese broth, CB) increased the number of free amino acids used as carbonsources. Compared with growth on MRS or pasteurized and microfiltrated milk, all strainscultivated in CB showed a low synthesis of D,L-lactic acid and elevated levels of acetic acid.The proteomic maps of the five representative strains, showing different metabolic traits, werecomparatively determined after growth on MRS and CB media. The amount of intracellularand cell-associated proteins was affected by culture conditions and diversity between strains,depending on their time of isolation. Protein spots showing decreased (62 spots) or increased (59spot) amounts during growth on CB were identified using MALDI-TOF-MS/MS or LC-nano-ESI-MS/MS. Compared with cultivation on MRS broth, the L. rhamnosus strains cultivatedunder cheese-like conditions had modified amounts of some proteins responsible for proteinbiosynthesis, nucleotide, and carbohydrate metabolisms, the glycolysis pathway, proteolyticactivity, cell wall, and exopolysaccharide biosynthesis, cell regulation, amino acid, and citratemetabolism, oxidation/reduction processes, and stress responses.

Keywords:

Cheese / Lactobacillus rhamnosus / Metabolic and proteomic adaptation / Micro-biology

Received: April 16, 2012Revised: August 20, 2012

Accepted: August 22, 2012

1 Introduction

During ripening, the biota of several varieties of long-ripenedcheese are dominated by starter and nonstarter lactic acidbacteria (SLAB and NSLAB, respectively) [1]. Usually, thesemicrobial populations exhibit opposite kinetics of growth

Correspondence: Dr. Maria De Angelis, Department of Soil, Plantand Food Science, University of Bari, Via G. Amendola 165/a,70126 Bari, ItalyE-mail: m.deangelis@agr.uniba.itFax: +39-080-5442911

Abbreviations: CB, cheese broth; FAA, free amino acids; MRS, deMan, Rogosa, and Sharpe; NSLAB, nonstarter lactic acid bacteria;PR, Parmigiano Reggiano; SLAB, starter lactic acid bacteria

and death [1,2]. SLAB is present at elevated numbers (ca. 9.0log CFU/g) during early cheese ripening, rapidly consumethe residual lactose, and quickly undergo autolysis [3, 4].Previously, five functional groups of microbial proteins wereidentified in Emmental cheese [3]. These proteins wereinvolved in proteolysis, glycolysis, oxidoreduction, DNA andRNA repair, and stress responses. During early cheese ripen-ing, contaminating NSLAB is present at low numbers (ca.2.0 log CFU/g), which markedly increase to approximately8.0 log CFU/g during late maturation. The adaptation ofNSLAB to the hostile environmental conditions of the cheeseduring ripening was previously described [1, 2]. None ofthese studies considered a proteomic approach to investigate

Colour Online: See the article online to view Figs. 1 and 2 in colour.

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Proteomics 2012, 12, 3206–3218 3207

the metabolic versatility and diversity of NSLAB. The value ofsuch an innovative approach would exceed the specific nicheand microbial population, increasing knowledge concerningbacterial physiology and biochemistry.

Lactobacillus rhamnosus is predominantly found within thepopulation of NSLAB of several cheeses [4–6], includingParmigiano Reggiano (PR) cheese and persists throughoutthe duration of PR cheese ripening (1–20 months), indicat-ing the capacity of L. rhamnosus to adapt to changing envi-ronmental conditions. Thus, L. rhamnosus is considered asa valuable model microorganism. Despite the extensive useand frequent isolation from foods, little is known concerningthe global gene expression of L. rhamnosus under differentenvironmental conditions [7–9]. Together with Lactobacillusplantarum, L. rhamnosus has the largest genome of the lacticacid bacteria (LAB) currently studied [10, 11]. The compara-tive genome analysis of Lactobacillus species showed that thecombination of gene gain and gene loss frequently occurredduring adaptation and in dairy niches [10, 12]. The adapta-tion of L. rhamnosus would also depend on the capacity touse nonconventional energy sources and regulate differentpathways [13, 14]. Overall, the basic knowledge concerningthe global protein patterns of L. rhamnosus in response toenvironmental adaptation should enhance the selection anduse of starters for food processing through the optimizationof growth, acidification, and proteolysis [15].

The aim of this study was to characterize the metabolic andproteomic adaptation of L. rhamnosus strains that were previ-ously isolated at different stages of PR cheese ripening. Thestrains were cultivated under optimal conditions (MRS broth)or under environmental conditions (cheese broth, CB) thatmimic those of PR cheese during ripening. The proteins wereidentified using MALDI-TOF-MS/MS and multidimensionalliquid chromatography (MDLC) coupled to nano-ESI-MS/MSto obtain comprehensive understanding of the mechanism ofenvironmental adaptation in L. rhamnosus strains.

2 Materials and methods

2.1 Bacterial strains and culture condition

L. rhamnosus strains were previously identified using 16SrRNA gene sequence analysis. The strains were isolated [6]after one (PR825, PR826, and PR830), four (PR1019), 12(PR1215 and PR1224), and 20 months (PR1473, PR1479,PR1484, and PR1489) of PR cheese ripening [16]. L. rham-nosus ATCC 53103 (GG) was purchased from American TypeCulture Collection (ATCC) (Milan, Italy). The strains werecultivated in MRS broth (Oxoid Ltd., Basingstoke, UnitedKingdom), pasteurized and microfiltrated milk or CB at 30�Cunder anerobiosis for 24, 36, or 48 h, respectively. The CB wasprepared as previously described [16] with several modifica-tions [17]. The freeze-dried cell lysate of Lactobacillus helveticusPR775 (isolated from PR cheese) was added to each medium

at the final cell density, which corresponded to approximately8.0 log CFU/mL.

2.2 Fermentative profiling by the Biolog system

Before inoculation of Biolog AN plates (Biolog, Inc., Hay-ward, CA, USA), strains grown in MRS broth or CB werestreaked twice on MRS agar or cheese agar plates, respec-tively. The plates were incubated at 30�C for 48 h and sub-sequently the cells were used for Biolog assays [18]. Threeseparate experiments were performed in triplicate. The sim-ilarities between the fermentation profiles were calculatedusing the Jaccard coefficient and the Unweighted Pair GroupMethod with Arithmetic Mean (UPGMA) analysis [18]. Thedata were also analyzed using PCA.

2.3 Growth, acidification, and proteolysis

The growth and acidification of L. rhamnosus strains were as-sayed using MRS broth, pasteurized and microfiltrated milk,and CB. The cells were harvested and washed in 50 mMphosphate buffer pH 7.0. Subsequently, the cells were cen-trifuged, resuspended at a cell density of approximately 6.8log CFU/mL and used to inoculate the various media types.After 48 h of incubation (30�C), the cell numbers were deter-mined through plating on MRS agar at 30�C for 72 h. Theacidification kinetics was monitored on-line using a pH me-ter (Model 507, Crison, Milan, Italy). After centrifugation at10 000 × g for 10 min, the cell-free supernatants were usedto determine the organic and free amino acids (FAA). Theconcentration of organic acids was determined using HPLCon a AKTApurifierTM system (GE Healthcare Life Sciences,Uppsala, Sweden) equipped with a 300 mm × 7.8 mm idcation exchange column (Aminex HPX-87H, BioRad Labora-tories, Hercules, CA). The fractions were eluted isocraticallyat 60�C with a 0.3 mL/min flow rate using 10 mM of H2SO4

as a mobile phase. A UV detector, operating at 210 nm, wasused to detect organic acids. The organic acid standards werepurchased from the Sigma Chemical Co. (Milan, Italy). Todetermine the FAA concentration, the proteins and peptidesfrom the cell-free supernatants were precipitated using 5%(vol/vol) cold solid sulfosalicylic acid, followed by incubationat 4�C for 1 h and centrifugation at 15 000 × g for 15 min.The FAA contents were analyzed using a Biochrom 30 seriesAmino Acid Analyzer (Biochrom Ltd., Cambridge SciencePark, England) [19]. Three separate experiments were per-formed in triplicate.

2.4 Protein extraction and 2DE analysis

For 2DE analysis, the harvested stationary-phase cells fromthe MRS broth or CB were washed in 50 mM Tris-HCl, pH7.5, and the protein extracts were produced as previously

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3208 C. G. Bove et al. Proteomics 2012, 12, 3206–3218

described [20]. The concentration of protein in the cell ex-tracts was determined using the Bradford method [21]. A60- (analytical runs) or 200-�g (preparative runs for proteinidentification) sample of total protein was used for each elec-trophoretic run. The 2DE was conducted according to themethods of Gorg et al. [22] and Hochstrasser et al. [23], using aPharmacia 2-D-EF system (GE Healthcare) [20]. The gels werestained using Brilliant Blue G-Colloidal Concentrate (Sigma)or an MS-compatible silver method [24]. The protein mapswere scanned using LabScan software on an ImageScanner(GE Healthcare) and analyzed with the ImageMaster 2D Plat-inum v6.0 computer software (GE Healthcare). Three gelsfrom three independent experiments were analyzed, and thespot intensities were normalized as previously reported [25].In particular, the spot quantification for each gel was calcu-lated as the relative intensity (% INT), which corresponded tothe INT of each spot divided by the total INT over the wholeimage [20]. The induction factor was defined as the ratio be-tween the spot intensity of a protein from cells cultivatedin CB and the spot intensity of the same protein from cellsgrown in MRS broth. The reduction factor was defined as theratio between the spot intensity of protein from cells grown inMRS and the spot intensity of the same protein from cells cul-tivated in CB. The comparison between strains and media forthe amount of the same spot was calculated as the percentageof relative intensity, attributing the value of 100 to the condi-tion wherein the protein was synthesized at the highest level.The PCA was calculated in accordance with the methods ofJacobsen et al. [26], using the PermutMatrixEN software [27].

2.5 Protein identification

The spots were excised from the gels and transferred to pierceV-bottom 96-well polypropylene microplates loaded with ul-trapure water. The samples were digested automatically us-ing a Proteineer DP robot (Bruker Daltonik, Bremen, Ger-many) with the Control 1.2 software according to the protocolof Shevchenko et al. [28] with minor modifications. Modi-fied porcine trypsin (sequencing grade; Promega, Madison,WI, USA) was added at a final concentration of 8 ng/�L in50 mM ammonium bicarbonate to the dried gel pieces, andthe digestion proceeded at 37�C for 8 h. Finally, 0.5% TFAwas added for peptide extraction, and the resulting digestionsolution was transferred to V-bottom 96-well polypropylenemicroplates using centrifugation. To prepare the MALDIsamples, equal volumes of the above mentioned digestionsolution and a matrix solution composed of CHCA were com-bined in 50% aqueous ACN and 0.25% TFA. This mixture wasdeposited onto a 600 �m AnchorChip prestructured MALDIprobe (Bruker Daltonik) [29]. The samples were automaticallyanalyzed through an Ultraflex MALDI-TOF/TOF mass spec-trometer (Bruker Daltonik) [30]. For MALDI-TOF-MS/MS,the calibrations were conducted with a fragment ion spectraobtained for the proton adducts of a peptide mixture cover-ing the 800–3200 m/z region. The peak lists of all samples

were generated using flex Analysis 2.2 software (Bruker Dal-tonik) [7]. MALDI-TOF-MS and MS/MS spectra were man-ually inspected in detail and reacquired, recalibrated and/orrelabeled when necessary, using commercial and customizedsoftware programs (Unidad de Proteomica, Centro Nacionalde Investigaciones Cardiovasculares, Madrid, Spain). TheMALDI-TOF-MS and MS/MS data were combined using theBioTools 3.0 program (Bruker Daltonik) to search the nonre-dundant protein database (NCBInr; ∼107 entries; or Swis-sProt; ∼5 × 105 entries), using the Mascot software v.2.0(Matrix Science, London, UK). The other relevant search pa-rameters were programed as follows: enzyme, trypsin; fixedmodifications, carbamidomethyl; one missed cleavage per di-gest; peptide tolerance ± 20 ppm and MS/MS tolerance ±0.5 Da.

The protein identification was also conducted using aMDLC coupled with nano-ESI-MS. The HPLC apparatus con-sisted of an Ettan MDLC (GE Healthcare) equipped witha Zorbax 300 SD C18 precolumn and a Thermo ElectronBioBasic-8 column. The MDLC was connected to a FinninganLCQ Deca XP Max ion trap mass spectrometer (Thermo Elec-tron Co., San Jose, CA) through the nano-ESI interface [31].The MS spectra were automatically recorded using Xcalibursoftware (Thermo Electron) in positive ion mode accordingto the manufacturer’s instructions. The MS/MS spectra wereprocessed using BioWorks 3.2 software (Thermo Electron),generating peak lists suitable for database searches. The pep-tides were identified using MS/MS ion searches of the Mas-cot search engine and non-redundant NCBI database [31].Confidence in peptide identification was assessed using aMASCOT sequence assignment score and visual inspectionof the MS/MS spectra. A minimum of two peptides withan ion score of at least 40 was required to obtain a reliableidentification [7].

2.6 Statistical analysis

The data (three replicates) from the microbial growth, acid-ification, organic acids, and FAA were subjected to one-wayANOVA (SAS, 1985), and a pair-wise comparison of the treat-ment means was achieved using Tukey’s procedure at p <

0.05. UPGMA and PCA were performed using Statistica 6.0per Windows 1998 software (StatSoft, Vigonza, Italy).

3 Results

3.1 Biolog profiles of fermentation

The fermentation profiles of the L. rhamnosus strains weredetermined using cells cultivated on both MRS and cheeseagar media (Supporting Information Table S1). Comparedto cultivation on MRS agar, the strains grown on cheeseagar medium demonstrated changed (p < 0.05) fermenta-tion profiles. A higher number of FAA was metabolized,

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particularly in strains isolated after 12 (PR1215 and PR1224)and 20 (PR1473, PR1479, PR1484, and PR1489) months ofPR cheese ripening. The fermentation profiles were ana-lyzed using PCA (Supporting Information Fig. S1). Whencultivated on MRS agar, the strains were grouped together(group I). Two groups (II and III) of distinguished strainswere cultivated on cheese agar. Group II only containedstrains isolated from 1 or 4 months of PR cheese ripen-ing. According to UPGMA clustering (data not shown),the strains isolated from early cheese ripening (PR825,PR826, PR830, and PR1019) showed similar profiles offermentation during cultivation on MRS and cheese agarmedia.

3.2 Kinetics of growth and acidification

The stationary growth phase was obtained after approximately24, 36, or 48 h, depending on the culture media (MRS broth,pasteurized and microfiltrated milk, or CB, respectively). Dur-ing cultivation on MRS broth, the final cell count varied from8.79 ± 0.09 (PR1215) to 9.45 ± 0.11 (PR826) log CFU/mL(Fig. 1A). The median value was 9.25 log CFU/mL. The cellgrowth was reduced on pasteurized and microfiltrated milkand CB media (median value of 8.70 and 8.49 log CFU/mL,respectively). Compared with growth on pasteurized and mi-crofiltrated milk, only PR825, PR1019, and PR1479 showedhigher cell numbers during cultivation on CB. The strainsPR1019 and PR1473 showed the highest and lowest final cellnumbers, respectively, on CB.

During growth on MRS broth, the �pH values varied fromapproximately 1.67 (PR1215) to 1.95 (PR1473) (Fig. 1B). Themedian value was 1.88. During growth in pasteurized andmicrofiltrated milk, the median value was 1.20. The pH valuesincreased during growth on CB. The negative variation of the�pH ranged from ca. −0.06 to −0.40. The median valueof �pH was −0.13. Strains PR1215, PR1479, and PR1484showed the highest increases of pH.

During cultivation on MRS broth, the concentration ofD,L-lactic acid ranged from ca. 43.3 to 151.7 mM (medianvalue of 103.5 mM) (Fig. 1C). Almost similar results wereobserved during growth on pasteurized and microfiltratedmilk. As expected, the concentration of D,L-lactic acid waslowest during cultivation in CB (median value of 4.5 mM).No synthesis of acetic acid was observed when the strainswere cultivated on MRS broth (Fig. 1D). Only strains PR825,PR826, and PR1489 synthesized acetic acid during growth onpasteurized and microfiltrated milk. All strains when culti-vated on CB synthesized acetic acid. The median value was39.7 mM.

3.3 Proteolysis during growth in CB

Compared with the control (CB without bacterial inoculums),the FAA concentration was increased in all strains (Sup-

porting Information Table S2). Only PR1473 showed a de-crease in FAA (the only exception was Orn). The increaseof FAA varied from approximately 17.1 (PR1489) to 2657.8mg/kg (PR825). Compared with control, CB inoculated withPR830 and PR1215 showed a marked decrease of Ser andArg.

The L. rhamnosus strains PR825 (group II of PCA, highestconcentration of FAA and isolated after 1 month of cheeseripening), PR830 (group II of PCA, high synthesis of aceticacid, and isolated after 1 month), PR1019 (group II of PCA,highest cell density, high synthesis of acetic acid, and isolatedafter 4 months), PR1215 (group III of PCA, highest synthesisof acetic acid, highest increase of pH during growth in CBand isolated after 12 months), and PR1473 (group III of PCA,lowest growth and concentration of FAA, and isolated after 20months) were considered as the representative strains, whichshowed diversity during growth on CB. These strains wereselected for further characterization.

3.4 2DE analysis and protein identification using

MALDI-TOF-MS/MS and nano-ESI-MS/MS

After the stationary phase of growth was obtained, the cytoso-lic proteins of L. rhamnosus PR825, PR830, PR1019, PR1215,and PR1473 grown in MRS broth were used to construct2DE reference maps. Most proteins had pI values that rangedfrom ca. 4.35 to 9.0 and Mr from ca. 11 to 88 kDa. StrainsPR825 and PR830, and strains PR1019 and PR1215 had al-most similar 2DE maps. Only strains PR825, PR1019, andPR1473 are shown in Fig. 2. A total of 121 proteins differedbetween strains (Fig. 2A–C, Supporting Information Fig. S2Aand C and Supporting Information Table S3). The 2DE mapsof strains cultivated on CB were compared with those con-structed after growth on MRS broth. Compared with cultiva-tion on MRS broth, the amount of 54 (PR825), 55 (PR830),52 (PR1019), 49 (PR1215), and 33 (PR1473) protein spotsdecreased (Supporting Information Table S3, Supporting In-formation Fig. S2A–D and Fig. 2A–F). The decrease of 26proteins was common between strains. Compared with culti-vation on MRS broth, the amount 42 (PR825 and PR830), 45(PR1019), 37 (PR1215), and 44 (PR1473) protein spots wereincreased. The increase of 20 proteins was common betweenthe strains. The PCA was used to visualize the dimensionalitydata of the 2DE maps. PC1 and PC2 contained 70% of the totalvariance of data, showing the amount of each protein and thetotal number of proteins, respectively (Supporting Informa-tion Fig. S3). The PCA analysis confirmed that the differencesin amount of proteins depended either on the culture mediaor on the time of isolation of the strains. The L. rhamnosusstrain, PR1473, which was isolated after 20 months of PRcheese ripening, behaved differently from the other strainsin both culture media. Overall, the effect of the time of isola-tion was more pronounced when the strains were cultivatedon MRS. All proteins (except one) that showed a decrease(61 spots) or an increase (59 spots) during growth on the

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Figure 1. Cell numbers (log CFU/mL) (A), acid-ification (�pH) (B), synthesis (mM) of lactic(C), and acetic (D) acids of Lactobacillus rham-nosus PR825, PR826, PR830, PR1019, PR1215,PR1224, PR1473, PR1479, PR1484, and PR1489during growth on MRS broth ( ), pasteurizedand microfiltered milk ( ), or CB ( ) at 30�C for24, 36, or 48 h, respectively. �pH was the dif-ferences between the pH of the medium at thebeginning (pHTo) and end of incubation (pHTf).Data are presented as the means ± SD of threeseparate experiments conducted in triplicate.Box plots are shown also. The line in the cen-ter of each box represents the median (�), andthe top and bottom of the box represent the75th and 25th percentile of the data, respec-tively. The top and bottom of the error barsrepresent the 5th and 95th percentile of thedata, respectively. The circles in each box plotextend to the outliers (*).

CB, were identified using MALDI-TOF-MS/MS or LC-nano-ESI-MS/MS. Changes in the amount of proteins between L.rhamnosus strains cultivated on MRS and CB media were an-alyzed using PermutMatrixEN software (Fig. 3A). Proteinsthat decreased/increased during growth on CB (Table 1, Sup-

porting Information Fig. 3S and S4 and Fig. 3A) were re-lated to protein synthesis (PS, six/five proteins); nucleotidemetabolism (NM, 11/four); carbohydrate metabolism, andglycolysis (CMG, 13/seven); cell wall biosynthesis (CWB,three/one); proteolytic enzyme system (PES, two/nine);

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Figure 2. 2DE analysis of intra-cellular and cell-associated pro-teins synthesized by Lactobacil-lus rhamnosus PR825 (A, D),PR1019 (B, E), and PR1473 (C, F)grown on MRS (A, B, C) and CB(D, E, F) media until the station-ary phase of growth (30�C for24 and 48 h, respectively) wasreached. Compared with culti-vation on MRS, numbered tri-angles and circles refer to pro-teins whose amount decreasedor increased during growth onCB. The numbered rectanglesrefer to proteins with decreasedor increased expression duringgrowth on CB for at least one ofthe other strains.

amino acid catabolism (AMC, one/14); exopolysaccharidebiosynthesis (EPSB, nine/zero); citrate metabolic process(CMP, zero/three); cell regulation (CR, one/one); metabolicprocess (MP, two/one); oxidation/reduction processes (OR,zero/five); stress response (SR, three/four); miscellaneous

(M, two/one); and hypothetical proteins (HP, eight/four). Us-ing KEGG database (www.genome.jp/kegg/pathway.html),the changes in the carbohydrate metabolism and glycolysis,and proteolytic enzyme system and amino acid catabolismwere described (Fig. 3B, C).

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Table 1. Putative function of proteins showing a decreased level (p ≤ 0.05) of concentration in Lactobacillus rhamnosus strains whengrown on CB compared with cultivation on MRS broth

Spota) Estimated Homologous protein Function A.N.Mr (kDa)and pI

1 88.0/5.30 Phenylalanyl-tRNA synthetase subunit beta (FARSB) PS ZP_032106282 77.0/4.70 Protein translation elongation factor (EF-G) PS YP_003172239.1.4 74.4/6.20 Dipeptidyl aminopeptidase/acylaminoacyl-peptidase (DAP/AAP) PES C7TDR6-15 74.3/4.60 Phosphoribosylformylglycinamidine synthase II (FGAM II) NM YP_0031715556 73.7/5.90 Fructose-1.6-bisphosphatase (FBPase) CMG LRH_046947 70.0/6.15 Beta-glucoside-specific PTS system IIABC component (PTS II glc) CMG LRH_024528 67.2/4.65 Chaperone protein dnaK (DnaK) SR C2JXM99 57.4/5.30 Dihydrolipoamide acetyltransferase (DLAT) CMG B5QMX110 57.4/5.40 GMP synthase (GMP S) NM O8519211 57.0/4.35 GroEL protein (GroEL) SR C7TLI312 54.6/4.95 H(+)transorting two-sector ATPase, alpha subunit (AtpA) M C7TBT313 54.6/4.90 H(+)transorting two-sector ATPase, beta subunit (AtpB) M C7TBT614 54.5/4.70 Glucose-6-phosphate 1-dehydrogenase (G6PDH) CMG C7THA915 54.5/5.80 Hypothetical protein (HP) HP C7T8A216 54.5/6.75 CTP synthetase (CTPS) NM ZP_0321222917 54.2/4.65 Putative glycosyltransferase (EpsC) EPSB Q9FDJ520 54.1/6.65 CTP synthase (CTPS) NM C7TMF921 54.0/6.00 Glycogen synthase (GS) CMG C7TKX922 54.0/7.40 Lipopolysaccharide synthesis sugar transferase (LPS-Sugar-tfrase) EPSB B5QQG523 51.5/4.40 Transcription elongation factor NusA (NusA) PS ZP_0321053224 51.5/5.95 Amidophosphoribosyltransferase (PurF) NM C7TK9525 51.0/4.80 Dipeptidase (PepV) PES ZP_0444195728 49.5/4.95 Glucose-6-phosphate isomerase (G6PI) CMG C7TBI730 48.8/5.75 Serine–tRNA ligase (SerRS) PS ZP_0444204531 48.8/7.00 UDP-N-acetylmuramoylalanine-D-glutamate ligase (MurD) CWB C7TC3432 48.7/4.74 Phosphomannomutase (PMM) CMG ZP_0321074933 48.7/7.60 N.I.34 48.6/6.90 Hypothetical protein (HP) HP Q1WVU035 48.5/5.95 Hypothetical protein (HP) HP Q38Z8643 43.0/4.45 Inorganic pyrophosphatase/exopolyphosphatase (PPase/PPX) NM ZP_0321266045 42.5/6.10 UDP-N-acetylmuramate-L-alanine ligase (MurC) CWB C7TK5346 42.5/6.15 UDP-N-acetylglucosamine 2-epimerase (UDP-GlcNAc-2-epimerase) CWB YP_00317468847 42.0/5.00 UDP-galactopyranose mutase (Glf) EPSB C1J9I548 41.8/5.15 DNA-directed DNA polymerase III, beta chain (DnaN) NM C7TF5749 41.8/5.95 Glucose-1-phosphate adenylyltransferase (ADP-Glc PPase) CMG ZP_0321247250 40.6/5.30 dTDP-glucose 4,6-dehydratase (RmlB) EPSB C7TE9051 40.5/5.50 Carbamoyl-phosphate synthase, small subunit (CPSase) NM C7TJC954 40.0/6.10 Alcohol-acetaldehyde dehydrogenase (ADH ALDH) CMG C7TEH655 40.0/6.75 Phosphomannose isomerase (PMI) CMG ZP_0321270256 40.0/7.40 dTDP-glucose 4,6-dehydratase (RmlB) EPSB C7TE4957 39.2/5.95 Aryl-alcohol dehydrogenase related enzyme (BADH) AMC ZP_0321123166 35.2/5.95 Putative galactofuranosyltransferase (WelG) EPSB C7TE9768 34.6/7.50 Phosphoribosylpyrophosphate synthetase (RPPK) NM YP_80774869 34.5/5.50 Aspartate carbamoyltransferase (ATCase) NM C2JX7972 27.9/4.60 Hypothetical protein LRH_05549 (HP) HP ZP_0321075374 27.8/5.30 Capsule polysaccharide biosynthetic process (Wze) EPSB Q58Z2780 24.5/5.30 30S ribosomal protein S3 (RpsC) PS C2JYI682 24.5/5.15 Hypothetical protein (HP) HP B5QJH183 24.4/5.50 Exopolysaccharide tyrosine-protein kinase (PTK) EPSB C2JZY485 24.4/5.30 Phosphoglycerate mutase 1 (PGAM1) CMG ZP_0321087586 24.3/5.50 Hypothetical phage-like protein (HP) HP B5QQ0792 20.5/5.55 dTDP-4-dehydrorhamnose 3.5-epimerase (RmlC) EPSB YP_00317472994 20.4/6.10 Pyrimidine regulatory protein PyrR (PyrR) NM ZP_03211791104 18.3/4.95 Hypothetical protein (HP) HP C7TLJ7107 18.0/4.66 Small heat shock protein (sHSP) SR YP_002221594109 17.5/5.75 Arginine repressor 1 (ArgR1) CR B5QJX6110 17.3/6.90 PTS system transporter subunit IIB (PTS IIB) CMG YP_003173174

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Table 1. Continued

Spota) Estimated Homologous protein Function A.N.Mr (kDa)and pI

114 16.2/4.50 N-acetylglucosamine and glucose PTS (EIICBA) CMG C2K0R3115 14.5/4.65 Hypothetical protein LRH_11899 (HP) MP ZP_03211087116 14.5/5.10 Pyridoxine 5’-phosphate oxidase V (Pnpo) MP ZP_03211323117 14.0/5.15 Hypothetical protein (HP) HP B5RSF8121 10.7/4.90 30S ribosomal protein S6 (RpsF) PS C7T8G3

a) Spot designation corresponds with those of the gels shown in Fig. 3 A–F.Protein synthesis, PS; proteolytic enzyme system, PES; nucleotide metabolism, NM; carbohydrate metabolism and glycolysis, CMG; stressresponse, SR; miscellaneous, M; hypothetical protein, HP; exopolysaccharide biosynthesis, EPSB; cell wall biosynthesis, CWB; amino acidcatabolism, AMC; cell regulation, CR; metabolic process, MP; accession number, A.N.

4 Discussion

The majority of L. rhamnosus strains catabolize a numberof compounds, which are usually found in cheese dur-ing ripening (e.g. N-acetyl-D-galactosamine, D,L-lactic acid,thymidine, uridine, L-alanyl-L-histidine, glycyl-L-methionine,and FAA) [6,16]. The strains isolated during late cheese ripen-ing showed an increased capacity for FAA use.

Due to the high levels of glucose and lactose, lactic acidwas the primary end product during growth on MRS brothand pasteurized and microfiltrated milk (Fig. 1C). A low con-centration of lactic acid and a high level of acetic acid wereobserved during growth on Cheese broth (Fig. 1D), whichcontained a low concentration of carbohydrates and high lev-els of FAA [16, 17].

The media used for cultivation and strain diversity (time ofisolation) influenced the capacity of the metabolic adaptation.The proteomic data confirmed this hypothesis. The amountof EF-G, NusA, RpsC, and RpsF, which are responsible forprotein biosynthesis, was the highest in strains isolated fromearly cheese ripening and grown on MRS broth. The amountof other proteins with similar functions (RPL1, EF-P, RP 50S-L5, Rps10, and Adt) increased in strains (e.g. PR1473) grownon CB (Fig. 3A). Compared with whey medium, L. rhamnosusGG upregulated the genes for protein biosynthesis duringgrowth on MRS broth [7]. This reference strain was also usedin this study, but it grew poorly on cheese agar medium.

Compared with MRS broth, L. rhamnosus downregulatedthe enzymes responsible for pyrimidine biosynthesis duringgrowth on whey medium [7]. In this study, the amount ofproteins responsible for the nucleotide metabolism varieddepending on the strains and especially the time of isolation.During growth on MRS broth, FGAM II, GMPS, CTPS, PurF,DnaN, and CPSase were synthetized at the highest levels instrains isolated from early cheese ripening. On the contrary,the amount of ATCase and UPRTase was highest in PR1473(isolated after 20 months). Furthermore, the amount of theenzymes of the Nudix hydrolase (NUDIX) and haloacid de-halogenase (HAD) (YqeK) superfamilies was highest duringgrowth on CB. The NUDIX substrates include nucleoside

triphosphates, nucleotide sugars, and diadenosine polyphos-phates. NUDIX hydrolases perform vital cellular functions,which include the modulation of natural metabolites toregulate signaling or biochemical pathways [32]. The HADsuperfamily catalyzes the hydrolysis of a P-group, which isesterified at the 5’-C of ribose and deoxyribose moiety [33]. Ac-cording to the fermentation profiles, the high level of NUDIXand YqeK enzymes suggests that the L. rhamnosus strainsmay use nucleotides as carbon sources during growth on CB.

Due to the high level of glucose, the amount of proteinsresponsible for the carbohydrate transport system (PTS IIglc and EIICBA) was the highest during growth on MRSbroth. The only exception was observed for strain PR1473(isolated after 20 months), which showed low amounts ofPTS II glc and EIICBA, regardless of the culture media. Theamount of PTS IIB increased only in strain PR825. Duringgrowth on MRS broth, L. rhamnosus strains showed an in-crease of the amount of proteins involved in glycolysis (PMI),and pentose phosphate pathway (G6PDH and ADH ALDH)(Fig. 3B). According to metabolic data (Fig. 1C), the increasedamount of these proteins favored the synthesis of lactic acid(G6PDH) and ethanol (ADH ALDH). The amount of phos-photransacetylase (PTA) and acetate kinase (AckA), whichare involved in the synthesis of acetic acid, increased duringgrowth on CB (Fig. 1A, D). The amount of AckA varied be-tween L. rhamnosus strains and mirrored the concentration ofacetic acid. Overall, the cultivation on CB determined the in-crease of the amount of several proteins with different phys-iological role. RpiA (catabolism of pentoses), glucosamine6-phosphate deaminase/isomerase (GlcN6P), citrate lyases(CitC and CitF), and citrate lyase acyl carrier protein (CitD)were upregulated during growth on CB. The growth of L.rhamnosus on citrate and the concomitant synthesis of lac-tate and acetate were previously described [34]. Strain PR830showed an increased amount of Dhak (Fig. 3B), which isresponsible for glycerolipid metabolism and leads to the syn-thesis of glycerone-P, which is metabolized via glycolysis.When cultivated on MRS, L. rhamnosus strains (especiallythose isolated after 1–4 months) increased the amount of en-zymes (PMM, FBPase, ADP-Glu-PPase, and GS) responsible

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3214 C. G. Bove et al. Proteomics 2012, 12, 3206–3218

Figure 3. PermutMatrixEN analysis of the amount of proteins (grouped based on KEGG functions) (A), and changes in the carbohydratemetabolism and glycolysis (B), and proteolytic enzyme system and amino acid catabolism (C) of Lactobacillus rhamnosus PR825 and PR830(isolated after 1 month of cheese ripening), PR1019 (4 months), PR1215 (12 months), and PR1473 (20 months) grown on MRS (PR825M,PR830M, PR1019M, PR1215M, and PR1473M) or CB (PR825C, PR830C, PR1019C, PR1215C, and PR1473C) media until the stationary phaseof growth (30�C for 24 and 48 h, respectively) was reached. (A) Changes in the protein amount (average of three replicates) are representedcolorimetrically, with red and green indicating the highest and lowest values of the standardized data, respectively, for each protein ofdifferent strains and medium. Each spot was quantified as a percentage of the relative volume, attributing the value of 100 to the conditionwherein the protein had been synthesized at the highest level. All data were shown as a percentage of dissimilarity using Euclideandistance. A–C, protein names correspond to those of Tables 1 and 2. B–C, enzymes, which showed the highest protein amount in strainsisolated at early cheese ripening and/or during cultivation on MRS broth, are represented in blue. B–C, enzymes, which showed the highestprotein amount in strains isolated at late cheese ripening and/or during cultivation in CB, are represented in red. The spot designation(numbers in parenthesis) corresponds with those of Tables 1 and 2.

for the synthesis of glycogen. Glycogen, a large �-glucan, is aubiquitous energy storage molecule among bacteria [35]. Tothe best of our knowledge, no published data have reportedthe synthesis of glycogen in LAB. When grown on glucose-,fructose-, or sucrose-containing media, the amino acid pro-ducer Corynebacterium glutamicum transiently accumulateslarge amounts of glycogen. Only a low amount of glycogenwas found during growth on acetate [36].

Variations in the carbon/nitrogen ratio, sugar composi-tion, and growth rate markedly affected the synthesis of EPSin LAB [7]. The amount of proteins responsible for EPSbiosynthesis (EpsC, Glf, RmlB, WelG, Wze, and RmlC) wasthe highest during the cultivation of L. rhamnosus strains onMRS broth (Fig. 3A). However, the amount of exopolysac-charide tyrosine-protein kinase (PTK) was the highest in thestrain PR1473 grown on CB.

During growth on CB, L. rhamnosus strains (especiallythose isolated after 1–12 months) showed a decrease of theamount of proteins responsible for cell wall biosynthesis(MurD, MurC, and UDP-GlcNAc-2-epimerase) (Fig. 3B). Onthe contrary, the amount of MurF was the highest in PR1473during growth on CB.

During cheese ripening, the proteolytic system of LAB de-grades caseins into small peptides and FAA, which fulfillthe nutritional requirements and concomitantly contributeto cheese flavor [1, 3]. The results obtained in this studyshowed that the amount of protease (PepS16), aminopepti-dases (PepA and MAP), endopeptidases (PepS, PepS24, andPepM16) and proline-specific peptidases (PIP and PepQ) wasincreased during cultivation on CB (Fig. 3C).

During the last decade, the catabolism of FAA was con-sidered to be the major event that characterizes the flavor of

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Figure 3. Continued

ripened cheeses. The simplest method for improving and/oraccelerating the flavor of cheeses is the supplementation ofconventional starters with strains isolated from high qualityaged cheeses [37]. The L. rhamnosus strains also showed anincrease of the amount of proteins that are responsible forthe catabolism of amino acids (e.g. MetC, SDH), favoring the

synthesis of lactic acid, acetic acid, and ATP via the pyruvatepathway. Consistent with the Biolog data (e.g. fermentationof amino acids), the amount of the enzymes responsible forthe catabolism of aspartic acid (AspAT and OadA), glutamicacid (GDH), arginine (ADI), aromatic amino acids (ArAT),methionine and cystathionine (MetC), branched-chain amino

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3216 C. G. Bove et al. Proteomics 2012, 12, 3206–3218

Table 2. Putative function of proteins that showing an increased level (p ≤ 0.05) of concentration in Lactobacillus rhamnosus strains whengrown on CB compared with cultivation on MRS broth

Spota) Estimated Homologous protein Function A.N.Mr (kDa) and pI

3 75.0/5.30 Putative uncharacterized protein (HP) HP B5QP0618 54.2/5.65 Citrate (pro-3S)-lyase (CitC) CMP ZP_0444202019 54.2/5.70 Citrate lyase alpha subunit (CitF) CMP ZP_0321189226 51.0/5.60 Oxaloacetate decarboxylase alpha chain (OadA) AMC C7TDW427 50.0/5.70 Dihydrolipoyl dehydrogenase (DLDH) OR C7TC7529 49.4/6.80 Pyridine nucleotide-disulphide oxidoreductase family protein

(PNDR)OR YP_002221633

36 48.4/6.00 Zn-dependent Peptidase, M16 family (Pep M16) PES C7THT837 45.5/6.10 Aspartate aminotransferase (AspAT) AMC C7TJG238 45.5/6.15 Glutamate dehydrogenase (GDH) AMC C2K15739 45.2/5.10 Arginine deiminase (ADI) AMC ZP_0060411940 45.2/5.65 UDP-N-acetylmuramoyl-tripeptide–D-alanyl-D-alanine ligase

(MurF)CWB YP_003175234

41 45.1/5.15 Aminopeptidase PepS (PepS) PES YP_003170796.1.42 45.1/5.65 Aromatic amino acid aminotransferase (ArAT) AMC C7THH744 43.0/5.20 Putative uncharacterized protein (HP) HP B5QQ4652 38.4/5.20 Glutamyl aminopeptidase (PepA) PES C2JV8653 38.4/5.30 Proline dipeptidase (PepQ) PES YP_003170538.1.58 39.0/7.00 Transcriptional regulator (TR) CR ZP_0321266159 39.0/6.00 Alcohol dehydrogenase (ADH) AMC/OR C7T7Q160 38.8/6.50 Acetate kinase (AckA) CMG/MP B5QKR461 38.6/5.25 S16 family peptidase (Pep S16) PES C2JWU662 38.5/4.70 NADH dehydrogenase, FAD-containing subunit (NADH FAD) OR B5QM2763 38.5/5.15 Cystathionine beta-lyase/cystathionine gamma-synthase

(MetC)AMC LRH_02562

64 35.2/5.25 Phosphotransacetylase (PTA) CMG C7TB6165 35.2/5.30 Branched-chain-amino-acid aminotransferasi (BcaT) AMC B5QQE267 35.1/6.15 Glycerol-3-phosphate acyltransferase (GPAT) CMG C7TJU570 34.4/4.70 Prolyl aminopeptidase (PIP) PES C7TAI871 30.0/4.85 Bifunctional S24 family peptidase/transcriptional regulator

(Pep S24)PES C2JVF6

73 27.8/5.15 Glutamine amidotransferase (GATase) AMC YP_003170280.1.75 27.8/5.25 Cysteine synthase (CysK) AMC C7TA2076 27.8/8.30 Aldose 1-epimerase (GalM) CMG ZP_0444093977 26.9/5.80 Methionine aminopeptidase (MAP) PES C7TBW878 24.8/4.60 Putative uncharacterized protein (HP) HP B5QQ0679 24.5/5.90 L-serine dehydratase beta subunit (SDH) AMC C7TBW881 24.5/9.30 Ribosomal protein L1 (RP L1) PS-SR ZP_0321295184 24.4/5.95 Branched-chain alpha-keto acid dehydrogenase E1 component

(BCKDHE)AMC Q039A0

87 24.2/4.85 Esterase M C7TL9488 24.0/5.65 Putative uncharacterized protein (HP) HP B5QRR189 23.9/4.70 Ribose-5-phosphate isomerase A (RpiA) CMG C2JUN790 20.6/4.45 Hypoxanthine phosphoribosyltransferase (HPRT) NM ZP_0444034891 20.6/4.70 Translation elongation factor P (EF-P) PS ZP_0321059993 20.5/6.00 Glucosamine-6-phosphate deaminase/isomerase (GlcN6P) CMG YP_003172659.1.95 20.3/7.35 Uracil phosphoribosyltransferase (UPRTase) NM ZP_0321101296 20.2/5.30 Glycine betaine/carnitine/choline ABC transporter permease

(LRH_10015)SR B5QQQ9

97 20.0/4.50 Glycerone kinase (DhaK) CMG ZP_0482604098 19.8/5.05 Heat shock protein Hsp20 (HSP20) SR C2JUC999 19.8/7.80 50S ribosomal protein L5 (RP 50S-L5) PS-SR ZP_03212255100 18.5/4.80 Thiol peroxidase (Tpx) OR ZP_04442480101 18.4/4.70 Hydrolase, NUDIX family protein (NUDIX) NM C7TGW2102 18.4/4.75 4-Methyl-5(B-hydroxyethyl)-thiazole monophosphate

biosynthesis enzyme, amidase family protein (LRH_10090)PES ZP_03211639

103 18.4/5.10 Peptide methionine sulfoxide reductase msrA 2 (MsrA) OR/SR C7TJ74105 18.3/5.90 Phospholipid-binding protein (LRH_11292) M ZP_03211261

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Table 2. Continued

Spota) Estimated Homologous protein Function A.N.Mr (kDa) and pI

106 18.2/5.95 Hydrolase (YqeK) NM C7TDD7108 18.0/5.20 D-tyrosyl-tRNA(Tyr) deacylase (Dtd) AMC C2JXJ5111 17.2/4.60 Acetyltransferase, GNAT family protein (AT GNAT) AMC B5QMD1112 17.0/5.05 Universal stress protein UspA (UspA) SR B5QKT5113 16.3/5.30 Universal stress protein (Usp) SR ZP_04441763118 12.0/9.00 30S ribosomal protein S10 (Rps10) PS-SR B5QPR4119 11.0/5.05 Citrate lyase acyl carrier protein (CitD) CMP B5QNR2120 10.8/5.90 Asp-tRNA-Asn/Glu-tRNA-Gln amidotransferase C subunit (AdT) PS B5QKK8

a) Spot designation correspond with those of the gels shown in Fig. 3 A–F.Hypothetical protein, HP; citrate metabolic process, CMP; amino acid catabolism, AMC; oxidation/reduction processes, OR; proteolyticenzyme system, PES; cell wall biosynthesis, CWB; cell regulation, CR; carbohydrate metabolism and glycolysis, CMG; metabolic process,MP; protein synthesis, PS; stress response, SR; miscellaneous, M; nucleotide metabolism, NM; accession number, A.N.

acids (BcaT), and serine (SDH) increased during cultivationof L. rhamnosus strains on CB. The level of these enzymes dif-fered between strains. The highest amount of AspAT, GDH,ADI, and ArAT was observed in strain PR1473, which wasisolated after 20 months of cheese ripening. The induction ofGDH was previously reported for L. plantarum under cheese-like conditions and was positively related to the catabolism ofFAA and survival of LAB [19]. The synthesis of NH3 via thecatabolism of FAA increased the pH value during fermen-tation of CB, and consequently the amount of H(+) trans-porting two-sector ATPase (AtpA and AtpB) of L. rhamnosusstrains decreased under cultivation on CB. The catabolism ofFAA also generates ATP via proton-motive force-driven tran-shydrogenase reaction. The final concentration of FAA of CBresulted from the balance between peptidase activities andcatabolism of FAA [1].

The amount of some enzymes related to oxida-tion/reduction processes (DLDH, PNDR, NADH FAD,MsrA) also increased under these conditions. This findingwas probably related to the requirement for maintaining thebalance between NAD(P)+ and NAD(P)H + H+, which isindispensable for the catabolic reactions that allow the syn-thesis of ATP [19]. These reactions were involved in prote-olysis, glycolysis, oxidoreduction, DNA and RNA repair, andstress response. The amount of chaperones (DnaK, GroEL)and sHSP was the highest under cultivation in MRS brothcompared with CB. The osmotic stress protein, HSP20 anduniversal stress proteins were upregulated under cheese-likeconditions.

The adaptation of L. rhamnosus to cheese-like conditionsis a complex process. Compared with cultivation on MRSbroth, L. rhamnosus strains cultivated under cheese-like con-ditions increased the amount of proteins responsible for cit-rate catabolism, acetate production, proteolytic activity, andamino acid catabolism and decreased the amount of pro-teins responsible for sugar transport, glycogen biosynthesis,pentose phosphate pathway, EPS biosynthesis, and cell wallbiosynthesis. The changes of the metabolism are primarily

associated with the composition of culture media. The vari-ability of adaptation to changing environmental conditionsand the diversity between strains suggested a spectrum of di-verse physiological responses in L. rhamnosus strains duringcheese under ripening.

The authors have declared no conflict of interest.

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