Proteome Analysis of Lactobacillus rhamnosus GG Using 2-D DIGE

Proteome Analysis of Lactobacillus rhamnosus GG Using 2-D DIGE and Mass Spectrometry Shows Differential Protein Production in Laboratory and Industrial-Type Growth Media Kerttu Koskenniemi,†,# Johanna Koponen,‡,# Matti Kankainen,‡ Kirsi Savijoki,‡ Soile Tynkkynen,§ Willem M. de Vos,† Nisse Kalkkinen,‡ and Pekka Varmanen*,†,| Department of Basic Veterinary Sciences, University of Helsinki, Finland, Institute of Biotechnology, University of Helsinki, Finland, Valio Ltd., Helsinki, Finland, and Department of Food Technology, University of Helsinki, Finland Received April 30, 2009

Lactobacillus rhamnosus GG (LGG) is one of the most extensively studied and widely used probiotic bacteria. While the benefits of LGG treatment in gastrointestinal disorders and immunomodulation are well-documented, functional genomics research of this bacterium has only recently been initiated. In the present study, a 2-D DIGE approach was used for the quantitative analysis of growth mediadependent changes in LGG protein abundance. Proteins were isolated from cells grown in industrialtype whey-based medium or in rich laboratory medium for subsequent 2-D DIGE. The analysis revealed patterns of protein abundance unique to each growth condition. In total, 196 quantitatively altered protein spots (at least 1.5-fold change in relative abundance, p < 0.05) representing approximately 13% of all protein spots in the gel were detected. From these protein spots, 157 were identified by mass spectrometry and were found to represent 100 distinct gene products. Collectively, these data show that growth of LGG in whey medium increased the relative abundance of proteins involved in purine biosynthesis, galactose metabolism, and fatty acid biosynthesis. In comparison, growth of LGG in laboratory medium resulted in an increase in the amount of proteins involved in translation and the general stress response, as well as pyrimidine and exopolysaccharide biosynthesis. Moreover, several enzymes of the proteolytic system of LGG demonstrated growth medium-dependent production. The present study demonstrates the fundamental effects of culture conditions on the proteome of LGG, which are likely to affect the functionality and characteristics of its use as a probiotic. Keywords: 2-D DIGE • probiotics • lactic acid bacteria • Lactobacillus rhamnosus GG • MRS medium • whey medium

Introduction Lactobacilli are a large and diverse group of lactic acid bacteria (LAB) that are naturally occurring and found in environments such as the surface of plant leaves, different food products, and the gastrointestinal tract of humans as well as other animals. They are of commercial and industrial importance and are widely used in the manufacturing of various fermented foods, beverages, and feed products.1 Moreover, some Lactobacillus strains, specifically belonging to species of Lactobacillus acidophilus, L. casei, L. gasseri, L. johnsonii, L. paracasei, L. plantarum, L. reuteri, and L. rhamnosus, have been reported to promote beneficial effects for human health.2 These * To whom correspondence should be addressed. Pekka Varmanen, Department of Food Technology, University of Helsinki, P.O. Box 66, FIN00014 University of Helsinki, Finland. Fax: +358-9-19157033. E-mail: [email protected]. † Department of Basic Veterinary Sciences, University of Helsinki. # These two authors contributed equally to this work. ‡ Institute of Biotechnology, University of Helsinki. § Valio Ltd. | Department of Food Technology, University of Helsinki. 10.1021/pr9003823 CCC: $40.75

 2009 American Chemical Society

probiotic LAB strains are under intensive research, and currently available genome sequences of these strains will advance functional genomics studies to unravel the molecular mechanisms behind their probiotic properties.3,4 In this research, proteomics plays a pivotal role in linking the genome and the transcriptome to potential biological functions. However, only a few proteomic studies on probiotic Lactobacillus strains (recently reviewed by Gagnaire et al.5), and, to our knowledge, only a single study utilizing 2-D DIGE6 have been reported to date. In the former studies, different kinds of stress responses have been studied in L. reuteri,7,8 L. rhamnosus,9 and L. gasseri.6 Kelly et al.10 have studied the proteome associated with the L. salivarius cell wall under different growth phases of this strain. In addition, Cohen et al.11 determined a proteome reference map of L. plantarum and analyzed the effect of growth phase. Furthermore, in a recent report, the proteomes of several probiotic lactobacilli were studied using [35S]-methionine labeling with the aid of newly defined growth media.12 L. rhamnosus GG (ATCC 53103, LGG) is one of the most intensively studied probiotic organisms,3 and the discovered Journal of Proteome Research 2009, 8, 4993–5007 4993 Published on Web 09/25/2009

research articles health-promoting effects associated with the consumption of LGG include a reduction in the risk for acute diarrhea in children13 and atopic diseases in infants,14 as well as relief for milk allergy/atopic dermatitis in infants,15-17 reduction of the risk of respiratory infections,18,19 and the occurrence of dental caries.20 Currently, however, the molecular mechanisms underlying the positive effects of LGG and other probiotic organisms on human health are poorly understood. In laboratory studies, the rich medium MRS21 is most commonly used to support the growth of lactobacilli. However, different types of media, for example, those containing milk proteins, are common in industrial large-scale cultivations22 and consequently probiotic cells grown in these media are used in clinical studies.23-29 The adhesion properties, which are important probiotic features, have been reported to be growth medium dependent in LGG.30 Consequently, studies of LGG performance with MRS may not correspond to the conditions under which LGG is actually grown for consumption. Thus, there is a surprising lack of information on the adaptive physiology of lactobacilli to the industrial media containing milk protein. There are a few functional genomics studies regarding the adaptation of certain lactic acid bacteria to cultivation in milk.31-33 However, in the present study, we used a whey-based medium, which is a common growth medium used in dairy industry. The components of the medium are processed from milk; the proteins as well as the lactose of whey are hydrolyzed, resulting in a peptide and sugar composition that is different from that of milk. The genome of LGG has recently been sequenced and annotated,34 which enables large-scale proteomic studies on this probiotic bacterium. In this work, using 2-D DIGE proteomic analysis with biological quadruplicate samples prepared from LGG cultivated in whey medium and the rich MRS laboratory medium, we quantified differences and identified proteins that are differentially abundant. Remarkable differences were observed between the proteomes of LGG grown on MRS and whey media, which clearly demonstrates the fundamental effects of culture conditions on the proteome of LGG.

Experimental Procedures Bacterial Strain and Growth Conditions. LGG (ATCC 53103) was propagated on MRS agar (BD, Sparks, MD) and cultivated anaerobically (using Anaerocult A, Merck KGaA, Darmstadt, Germany) at 37 °C. Thereafter, separate colonies were inoculated in quadruplicate in 1.3 mL of both MRS broth (BD) and hydrolyzed whey broth containing 5% hydrolyzed whey, 0.6% casein hydrolysate, and 0.0015% MnSO4 · H2O, and grown at 37 °C overnight. A 1 mL aliquot of culture was inoculated in 100 mL of MRS or whey broth, and the cultures were grown microaerobically at 37 °C. Samples were collected at an OD600 of 1.1 and 5 in MRS cultures and 0.25-0.28 and 1.3 in whey cultures, which corresponded to logarithmic and stationary growth phases, respectively (Figure 1). Sample volumes were 5 mL at logarithmic growth phases, and 1 and 1.8 mL at stationary growth phases for MRS and whey samples, respectively. Preparation of Protein Extracts. Cells were harvested by centrifugation at 4 °C and washed twice with ice-cold 50 mM Tris-HCl, pH 8. Bacterial cells were broken by bead beating with glass beads in 30 mM Tris with a FastPrep FP120 instrument (Qbiogene, Illkirch, France) at a speed of 6.5 m/s for 30 s, which was repeated three times. The samples were kept for 1 min on ice between bead beating treatments. The samples were then resuspended in urea buffer containing 7 M 4994

Journal of Proteome Research • Vol. 8, No. 11, 2009

Koskenniemi et al.

Figure 1. Growth (squares) and acidification (triangles, pH) curves of LGG in whey medium (black symbols) and MRS (white symbols). The error bars indicate standard deviations among four biological replicates. The sampling times are indicated with arrows.

urea, 2 M thiourea, 4% CHAPS, and 30 mM Tris, incubated for 60 min at room temperature, and frequently vortexed. Next, the samples were centrifuged at 16 000g for 30 min at room temperature, and the supernatant was collected and processed using a 2-D Clean-up kit (GE Healthcare, Buckinghamshire, U.K.). The protein samples were dissolved in 25-40 µL of urea buffer (see above). Protein concentrations of the samples were determined using a 2-D Quant Kit (GE Healthcare). Labeling of Samples. Protein samples were adjusted to pH 8.5 by addition of 2 M Tris. The samples were then labeled using Cy2, Cy3, and Cy5 dyes (CyDye DIGE Fluor minimal dyes; GE Healthcare), according to the Ettan 2-D DIGE protocol. The dyes were added in the protein samples as 280 µM solutions, dissolved in DMF, at a ratio of 50 µg of protein per 400 pmol dye. Labeling was performed reciprocally so that both the MRS and whey medium grown samples were labeled with Cy3 and Cy5 to account for any preferential protein labeling by the CyDyes. Cy2 was used for the internal pooled standard consisting of equal amounts of each sample. Samples of cells at logarithmic and stationary growth phases were analyzed in separate experiments (setup is shown in detail in Supplementary Table S1). Samples were incubated on ice for 30 min in the dark. The reaction was quenched by the addition of 1 µL of 10 mM lysine followed by incubation on ice for 10 min in the dark, after which the samples were pooled. Isoelectric Focusing (IEF). The labeled proteins were first separated by IEF. IPG strips (24 cm, pH 3-10 nonlinear, BioRad, Hercules, CA) were rehydrated in 500 µL of buffer, which contained 7 M urea, 2 M thiourea, 4% CHAPS, 50 mM DTT, 2 mM tributylphosphine, and 1% Bio-Lyte pH 3-10 (Bio-Rad), overnight at 20 °C using a Protean IEF Cell (Bio-Rad). Samples containing in total 105 µg of protein in 50 mM DTT, 4 mM tributylphosphine, and 1% Bio-Lyte pH 3-10 were applied to the IPG strips via cup-loading near the acidic end of the strips. IEF was performed using a Protean IEF Cell at 20 °C as follows: 15 min at 250 V, then linear ramping to 10 000 V for 40 000 Vh (using a limit of 50 µA/strip). After IEF, the strips were equilibrated in a buffer containing 50 mM Tris-HCl, pH 6.8, 6 M urea, 2% SDS, 20% glycerol, and alternatively either 2% DTT (buffer A) or 2.5% iodoacetamide (buffer B), first in buffer A for 25 min and then in buffer B for 25 min.

Proteome Analysis of L. rhamnosus GG Using 2-D DIGE and MS SDS-PAGE. The strips were loaded on 12% acrylamide gels that were subjected to electrophoresis in an Ettan DALTsix Electrophoresis Unit (GE Healthcare) at 80 V for 15 min, and then 400 V for approximately 3 h. The upper buffer was 2× TGS (50 mM Tris, 384 mM glycine, 0.2% (w/v) SDS; Bio-Rad), and the lower buffer was 1× TGS (25 mM Tris, 192 mM glycine, 0.1% (w/v) SDS). The gels were scanned between low fluorescence glass plates using a FLA-5100 laser scanner (Fujifilm, Tokyo, Japan) at wavelengths of 473 nm (for Cy2), 532 nm (Cy3), and 635 nm (Cy5) using voltages of 420, 410, and 400 V, accordingly. All gels were scanned at 100 µm resolution. The gel images were cropped to identical size by removing areas extraneous to the protein spots with ImageQuant TL 7.0 software (GE Healthcare). After scanning, the gels were fixed in 30% ethanol and 0.5% acetic acid for 60 min minimum and then silver stained.35 Image and statistical analyses for the cropped 2-D DIGE gels were performed using DeCyder 2D 6.5 software (GE Healthcare). With the use of a batch processor function, the gels were first automatically analyzed in a differential in-gel analysis (DIA) module, which normalized the Cy2, Cy3, and Cy5 image from each gel. Spot boundaries were detected, and spot volumes (protein abundances) were calculated. Then, the spot volumes of Cy3 and Cy5 samples were compared with the spot volumes of the Cy2 sample (internal standard) to generate standard spot volumes, thereby correcting intergel variations. In the biological variation analysis (BVA) module, the Cy2 images of four replicate gels were matched, and the standard spot volume ratios between all four gels were compared. Approximately 750 separate protein spots were detected on each gel. Protein spots demonstrating a minimum 1.5-fold difference in average spot volume ratios (average ratio g1.5 or e -1.5) between MRS and whey medium grown samples in at least three out of four separate biological replicates using a Student’s t test p-value value of less than 0.05 were picked and identified. Molecular weights and isoelectric points of protein spots were defined according to the instructions of DeCyder user manual. Protein Identification. 1. MALDI-TOF and MALDI-TOF/ TOF. From the silver-stained 2-D DIGE gels, protein spots of interest were excised manually, reduced with DTT, and alkylated with iodoacetamide, prior to digestion with trypsin (Sequencing grade Modified Trypsin V5111, Promega, Madison, WI) at 37 °C overnight.36 The peptides were extracted once with 3 µL of 25 mM ammonium bicarbonate and twice with 3 µL of 5% formic acid for 15 min at room temperature, and the extracts were pooled and desalted (ZipTip µ-C18; Millipore Corporation, Billerica, MA).37 Peptides were eluted directly from the tips onto a MTP 384 ground steel sample plate with saturated R-cyano-4-hydroxycinnamic acid (CHCA) in 0.1% trifluoroacetic acid (TFA) and 60% acetonitrile (ACN). MALDITOF mass spectra for peptide mass fingerprints (PMFs) and MALDI-TOF/TOF mass spectra for identification by fragment ion analysis were acquired in positive ion reflectron mode using an Ultraflex TOF/TOF instrument (Bruker Daltonik GmbH, Bremen, Germany). The spectra were externally calibrated with a peptide standard mixture (P/N 206195; Bruker Daltonik). The peak lists for all samples were generated using the FlexAnalysis 3.0 software (Bruker Daltonik). The data were then interpreted through the m/z software Biotools 3.0 (Bruker Daltonik) using a local MASCOT (MASCOT 2.2; Matrix Science, London, U.K.) server running on a Quad-Core Xeon 2.33 GHz processor. The protonated molecule ion “MH+” and “monoisotopic” were

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defined for the peak mass data input. Searches were restricted to an in-house database of the published ORF set of LGG.34 The database searches were performed using mass accuracy of 50 ppm, fixed carbamidomethyl cysteine modification, and variable modification due to methionine oxidation. One missed cleavage site was allowed. A successful identification was reported when a significant match (p < 0.05) was obtained according to the probability-based Mowse score in the Mascot search engine. For PMF identifications with low Mowse score, identifications were confirmed with MALDI TOF/TOF fragment ion analysis (data not shown). 2. LC-MS/MS. Separations of the protein digests were achieved on a C18 column (75 µm inner diameter × 15 cm, 100 Å, 5 µm, PepMap C18; LC Packings, Sunnyvale, CA) using a nanoscale HPLC system (Ultimate 3000; Dionex, Sunnyvale, CA). The samples (40 µL) were concentrated and desalted on a C18 precolumn (150 µm inner diameter × 10 mm, C18, 120 Å pore size, 3 µm particle size, PROTECOL; SGE Analytical Science, Griesheim, Germany) at a flow rate of 7 µL/min (0.1% TFA). Peptides were eluted with a linear gradient of acetonitrile (0-40% ACN in 50 min) in 0.1% formic acid at a flow rate of 200 nL/min. MS/MS of tryptic peptides was conducted on a hybrid quadrupole/TOF mass spectrometer with NanosprayII source (QSTAR Elite; Applied Biosystems, Foster City, CA). The nanoelectrospray was generated using a PicoTip needle (20 µm inner diameter with 10 µm tip, SilicaTip; New Objective, Ringoes, NJ) maintained at a voltage of 2300 V. TOF-MS and tandem mass spectral data were acquired using information-dependent acquisition (IDA), charge state selection from 2 to 4, an intensity threshold of 10 counts/s, and a collision energy automatically determined by the IDA based on the m/z values of each precursor ion. Following IDA data acquisition, precursor ions were excluded for 60 s using a window of 6 amu to minimize the redundancy in tandem mass spectra. The peak list was generated with ProteinPilot software (Applied Biosystem), and protein identifications were performed using the MASCOT database search engine (Matrix Science). Searches were performed against an in-house database of the published ORF set of LGG,34 using a mass tolerance of 0.2 Da. Confidence in peptide identifications was assessed by MASCOT sequence assignment score and visual inspection of the MS/MS spectra. To consider the identification reliable, a minimum of two peptides with an ions score at least 40 was required. Identification of Regulatory Elements. Putative PurR -binding elements (PurBoxes)38 in LGG genome were identified by screening for nucleotide sequences showing a similarity against the constructed PurBox weight matrix. Additionally, searches for elements matching the -10 and -35 elements were performed. All weight matrices were constructed similarly by, at first, manually aligning a list of binding sites and then converting that alignment onto weights by taking the natural logarithm of the ratio between the relative frequency (corrected for pseudoweight that was set to 1) and the prior residue probability of the genomic nucleotide frequencies.39 The PurBox weight matrix (Supplementary Figure S1A) was constructed from the elements listed in Prodoric40 and DBTBS41 databases and by Beyer et al.42 The -35 (Supplementary Figure S1B) and -10 (Supplementary Figure S1C) binding sites of Sigma factor 43 (SigA) on the other hand were created using elements listed only in Prodoric and DBTBS.40,41 Only binding elements of the Gram-positive bacteria were used. SupplemenJournal of Proteome Research • Vol. 8, No. 11, 2009 4995

research articles tary Table S2 shows matches having a score higher than 66% of the maximal score (score g9.61). Insignificant matches were next filtered out by manually evaluating the distance between the element and its surrounding genes (within the first 300 bp from the gene), the annotation of genes, and the existence of other PurBoxes (lower scoring motifs were here accepted) within the window of 100 bp from the first PurBox. Surrounding sequences, covering the intergenic region between the flanking genes and the first 50 bp of the genes, of the final set of elements (Supplementary Table S3) were obtained and used as a query to search for homologous regions in L. casei ATCC334 and BL23. Matching regions were aligned using Muscle.43 Supplementary Figures S2-S6 show the genomic regions and sequence alignments and highlight the locations of putative binding elements.

Results and Discussion In the present study, we exploited the fluorescence-based 2-D DIGE technique to investigate the effects of two different growth media conditions on protein production by LGG. To this end, LGG was cultivated in four biological replicate batch cultures in both hydrolyzed whey-based medium, a medium type widely used for industrial cultivations, and MRS broth, which is a nutritionally rich standard laboratory medium for lactobacilli. Protein samples from LGG cells were collected during both logarithmic and stationary growth phases (Figure 1). While the cell densities were different at the time points of sampling, the pH values of collected samples were the same for both media types from logarithmic and stationary growth phases with pH 5.3 and 4.1, respectively (Figure 1). The samples were subjected to CyDye labeling and 2-D DIGE analyses. Figure 2 shows a representative image of proteomes (pH range of 3-10) of LGG cultured in MRS compared to whey media, with the logarithmic phase in panel A and stationary phase in panel B. According to DeCyder analyses, between LGG cultivated in MRS and whey medium, a total of 157 protein spots with greater than 1.5-fold difference in volume (t test < 0.05) could be detected. Those spots were picked from the 2-D DIGE silver-stained gels and identified using MALDI-TOF mass spectrometry and/or LC-MS/MS (Tables 1and 2). Among these spots, 75 were detected more abundant during growth in whey medium, 44 of them from logarithmic and 31 from stationary growth phase. A total of 82 spots were detected more abundant during growth in MRS, and of these spots, 41 could be linked to logarithmic and 41 to stationary phase growth. The identified protein spots represented 100 distinct gene products (Tables 1 and 2). Proteins More Abundant during Growth in Whey Medium. Among the 45 proteins produced more during growth in whey medium, 15 were associated with nucleotide biosynthesis, and the majority (11) were proteins involved in purine biosynthesis (Table 1). The effect on the purine biosynthetic pathway was found to be extensive: all proteins, with the exception of PurN and PurA, from the biosynthetic pathway from phosphoribosyl pyrophosphate (PRPP) to adenosine monophosphate (AMP) and guanosine monophosphate (GMP) (PurBCDFHKLM and GuaAB) were identified as differentially abundant (Figure 3A). The relative abundance of all these proteins was increased during both logarithmic and stationary growth phases. Differences in protein abundances extended to 12-fold (on average 5-fold) changes during the logarithmic growth phase, while approximately 2-fold differences were found during stationary 4996


Koskenniemi et al. phase. Several proteins (GuaAB, PurDFHK) were identified migrating as horizontally adjacent proteins, suggesting that these proteins have undergone a charged post-translational modification. The organization of genes encoding PurCDFHKLM (Figure 3B) suggests that they are part of the same operon, whereas purB, guaB, and guaA are distinct transcriptional units (Figure 3B,C). This genetic organization of purine biosynthesis genes is conserved in other LAB.44 Furthermore, the comparison of the relevant genome sequences of LGG and L. casei (Supplementary Figures S2-S6) shows high level of conservation in the coding regions and to lesser extent in the regulatory regions. Whey medium does not supply LGG with purines as its components are processed from milk that is a poor source of purines.42 Thus, the observed increased amounts of the purine biosynthetic enzymes is in line with previous reports demonstrating that purine biosynthesis is required for the growth of LAB in milk.44,45 The up-regulation of purine biosynthesis in milk has also been observed for LAB species other than lactobacilli: Streptococcus thermophilus produced more PurBCDFHLM proteins,31 and Lactococcus lactis more PurABCDEFHLMQ and Xpt32 in milk than in a synthetic M17-lactose growth medium. In a transcriptomic study, expression of Lactobacillus helveticus purABCDFHKLMNQS, guaABC, and xpt in milk increased compared to that in MRS laboratory medium.33 Formyl-tetrahydrofolate synthetase (Fhs), which catalyzes the formation of 10-formyltetrahydrofolate, was more abundant during growth in whey, which was understandable, since 10formyltetrahydrofolate feeds purine biosynthetic pathway with formyl groups. Beyer et al.42 used a proteomics-based approach to demonstrate that Fhs was induced during purine starvation. Up-regulation of the Fhs protein and fhs gene was also observed during growth in milk in the studies of Gitton et al.32 and Smeianov et al.,33 respectively. Collectively, the induction of purine biosynthetic pathway at both RNA and protein level appears to be a common phenomenon in LAB cultivated in milk and milk-derived media. In L.lactis the expression of purine biosynthesis genes is regulated by a transcriptional activator PurR that binds to upstream located conserved DNA element designated as PurBox.38 We searched the LGG genome for presence of putative PurBoxes, which resulted in identification of putative PurR binding sites in the upstream region of purE, xpt, guaB, guaC and purA (Figure 3B, Supplementary Figures S2-S6, Supplementary Table S2-S3) suggesting that PurR-PurBox mediated regulation of purine biosynthesis genes applies also to L. rhamnosus. Remarkably, this PurBox is highly conserved in the regulatory region of L. casei (see Supplementary Figures S2-S6). In L.lactis,guaA and guaB do not belong to PurR-regulon,42,44 whereas in LGG,the presence of two putative PurBoxes in the upstream region of guaB (Figure 3B, Supplementary Figure S3) suggests PurR dependent regulation. Production of proteins related to carbohydrate metabolism was different during growth in whey medium compared to MRS. In whey medium, the galactose metabolizing enzymes were approximately 2-fold more abundant during logarithmic growth phase. There are two different galactose degradation pathways in lactic acid bacteria: (1) the Leloir pathway that converts galactose to glucose-6-phosphate and (2) the tagatose6-phosphate pathway that metabolizes galactose to glyceraldehyde-3-phosphate and dihydroxyacetone phosphate.46,47 In this study, the Leloir pathway enzymes of GalK, GalE, GalT, and Pgm and the tagatose-6-phosphate pathway enzyme of

Proteome Analysis of L. rhamnosus GG Using 2-D DIGE and MS

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Figure 2. Representative 2-D DIGE images of proteins extracted from L. rhamnosus GG cultivated in whey and MRS media at logarithmic (A) and stationary (B) growth phases. The total amount of protein used for CyDye labeling was 105 µg. The numbered protein spots (1-157) were cut from 2-D DIGE gels poststained with silver and subjected to MS or MS/MS identification (listed in Tables 1 and 2). The number of the spot is indicated above the particular spot or linked to the spot with a line. Protein spots that were more abundant in whey medium are shown in green and those more abundant in MRS are shown in purple. The protein spots with no differences in abundances between the treatments are white.

LacD were more significantly produced during growth in whey medium. The increased production of galactose-metabolizing enzymes is expected, since the main sugar in MRS is glucose, while whey medium contains both glucose and galactose (derived from hydrolyzed lactose). With the use of a transcriptome-level approach, Smeianov et al.33 similarly detected upregulation of pgm in L. helveticus grown in milk when compared to cells grown in MRS. Tsai and Lin48 demonstrated that L. rhamnosus strain TCELL-1 could use both the Leloir and tagatose-6-phosphate pathways for fermentation of lactose, but only the Leloir pathway for fermentation of galactose. LGG cannot ferment lactose,49 but it may have the capability to ferment the galactose moiety using either one of these pathways, as shown by the presence of the appropriate enzymes.

Several glycolytic enzymes (Eno, GapB, Pkg, and TpiA) were produced more in whey medium than in MRS broth, mostly during the stationary growth phase, but partly also during the logarithmic growth. The glycolytic enzymes enolase (Eno) and glyceraldehyde 3-phosphate dehydrogenase (GapB) have been found to be partly located on the cell surface of Lactobacillus crispatus at acidic conditions,50 and thus, they might be also in LGG as part of the surface proteome. Castaldo et al.51 have recently reported that enolase of Lactobacillus plantarum is a fibronectin binding protein, and hence, the LGG enolase could also be involved in the adhesion properties of this probiotic. Fatty acid biosynthesis was also accelerated in whey medium: five enzymes in fatty acid biosynthesis (FabD, FabF, FabG, FabZ, and AccD) were produced 2- to 3-fold more during Journal of Proteome Research • Vol. 8, No. 11, 2009 4997

4998

log log log log log log

log

log log

log

log

log log log stat stat stat stat stat stat stat stat stat stat

stat

stat

stat

stat stat stat

log log log

7

8 9

10

11c

12 13c 14 15c 16 17 18 19 20c 21 22 23 24

25

15c

26

27 28 29

30 31 32

growth phasea

1 2 3 4 5 6

spot no.


5.51 7.39

6.14

1.58

12.23 2.51 2.67 1.90 3.55 1.85 1.81 1.70 1.79 1.54 1.94 1.71 2.51

2.30

1.90

1.61

2.51 1.55 2.12

2.71 1.50 2.23

7.8 × 10-6 1.6 × 10-3

7.5 × 10-4

2.0 × 10-2

10-7 10-2 10-5 10-4 10-6 10-4 10-3 10-5 10-3 10-3 10-5 10-6 10-4

1.3 × 10-4

7.7 × 10-4

9.6 × 10-5

4.0 × 10-6 2.1 × 10-3 1.4 × 10-5

8.2 × 10-4 3.3 × 10-5 7.1 × 10-5

7.3 2.4 2.2 7.7 8.7 1.5 3.5 1.8 7.2 6.9 4.3 8.5 1.5

3.73

1.5 × 10-3

× × × × × × × × × × × × ×

× × × × × ×

2.23 1.89 7.20 2.13 2.44 8.39

fold change

10-4 10-7 10-8 10-7 10-4 10-7

2.5 3.3 9.1 1.4 2.2 2.0

t test

58.9/7.2 33.6/6.4 60.0/6.6

79.1/4.5 35.6/5.0 21.5/6.4

40.6/6.2

54.7/5.5

54.7/5.5

79.1/4.5 35.6/5.0 21.5/6.4 57.7/5.3 52.6/6.0 52.6/6.0 48.7/6.4 27.1/6.3 44.5/5.6 44.5/5.9 52.3/6.3 52.3/6.3 54.7/5.5

40.6/6.2

40.6/6.2

52.3/6.3 54.7/5.5

44.5/5.9

57.7/5.3 57.7/5.3 52.6/6.0 52.6/6.0 48.7/6.4 27.1/6.3

theoretical Mw (kDa)/pI

MS MS MS

MS MS MS

MS

MS

MS

MS MS/MS MS MS MS MS MS MS MS/MS MS MS MS/MS MS

MS/MS

MS

MS/MS MS

MS

MS MS MS MS MS MS

identif. typeb

134 68 80

224 51 81

70

55

82

65 474 98 73 66 228 74 117 463 78 121 147 246

214

91

147 124

84

106 118 233 197 74 135

Mowse score

no. of peptides

locus tag

18 17 12

22 15 19

11

16

12

7 22 21 15 8 35 11 39 28 12 15 9 46

10

17

9 26

10

19 20 36 28 11 42

LGG_01809 LGG_01807 LGG_01076 LGG_01465 LGG_02906 LGG_02546

Other 9 5 6

LGG_01813

LGG_01805

LGG_01805

LGG_01809 LGG_01807 LGG_01076 LGG_01968 LGG_00249 LGG_00249 LGG_01079 LGG_01812 LGG_01803 LGG_01803 LGG_01808 LGG_01808 LGG_01805

LGG_01813

LGG_01813

LGG_01808 LGG_01805

LGG_01803

16 5 6

4

6

5

5 4 7 9 4 17 5 10 11 6 8 2 17

4

6

2 12

7

Purine Biosynthesis 9 LGG_01968 12 LGG_01968 17 LGG_00249 14 LGG_00249 5 LGG_01079 8 LGG_01812

Nucleotide Metabolism

seq. cov. (%)

Fhs PrsA PyrG

PurL PurM Xpt

PurK

PurH

PurH

PurL PurM Xpt GuaA GuaB GuaB PurB PurC PurD PurD PurF PurF PurH

PurK

PurK

PurF PurH

PurD

GuaA GuaA GuaB GuaB PurB PurC

name

function

protein

Formyl-tetrahydrofolate synthetase Ribose-phosphate pyrophosphokinase CTP synthase

GMP synthase GMP synthase Inosine-5′-monophosphate dehydrogenase Inosine-5′-monophosphate dehydrogenase Adenylosuccinate lyase Phosphoribosylamidoimidazole-succinocarboxamide synthase Phosphoribosylamine-glycine ligase Amidophosphoribosyltransferase Phosphoribosylaminoimidazolecarboxamide formyltransferase/ IMP cyclohydrolase Phosphoribosylaminoimidazole carboxylase NCAIR mutase subunit Phosphoribosylaminoimidazole carboxylase NCAIR mutase subunit Phosphoribosylformylglycinamidine synthase Phosphoribosylformylglycinamidine cyclo-ligase Xanthine phosphoribosyltransferase GMP synthase Inosine-5′-monophosphate dehydrogenase Inosine-5′-monophosphate dehydrogenase Adenylosuccinate lyase Phosphoribosylamidoimidazole-succinocarboxamide synthase Phosphoribosylamine-glycine ligase Phosphoribosylamine-glycine ligase Amidophosphoribosyltransferase Amidophosphoribosyltransferase Phosphoribosylaminoimidazolecarboxamide formyltransferase/ IMP cyclohydrolase Phosphoribosylaminoimidazolecarboxamide formyltransferase/ IMP cyclohydrolase Phosphoribosylaminoimidazolecarboxamide formyltransferase/ IMP cyclohydrolase Phosphoribosylaminoimidazole carboxylase NCAIR mutase subunit Phosphoribosylformylglycinamidine synthase Phosphoribosylformylglycinamidine cyclo-ligase Xanthine phosphoribosyltransferase

Table 1. The Identified Proteins from LGG That Were More Abundant in Whey Medium than in MRS Medium (Fold Change g1.5, p e 0.05)

research articles Koskenniemi et al.

stat stat stat

log log log log log log stat

log log log log log stat stat stat stat stat stat stat stat stat stat

log log

log log log

log

log

log log

36 37 38 39 40c 41 42

43 44 45 46c 47 48 49 50 51 20c 52 53 54 55c 56c

13c 11c

57 58 59

60

61

62 63

growth phasea

33 34 35

spot no.

3.94 2.90 1.72

1.83 2.04 2.30 2.08 1.71 1.80 1.87

1.61 2.01 5.01 2.51 2.12 1.51 2.40 2.12 1.55 1.79 1.74 1.59 3.84 1.68 1.69

2.51 1.58

1.77 1.54 1.71

1.58

2.99

2.01 2.89

4.5 × 10-4 4.5 × 10-4 4.5 × 10-5

10-5 10-4 10-6 10-2 10-4 10-3 10-5

10-4 10-2 10-7 10-4 10-3 10-2 10-3 10-3 10-3 10-3 10-2 10-2 10-7 10-4 10-3

× × × × × × ×

× × × × × × × × × × × × × × ×

2.4 × 10-2 2.0 × 10-2

1.2 × 10-3 4.9 × 10-3 2.1 × 10-5

4.5 × 10-6

9.5 × 10-5

1.3 × 10-6 1.6 × 10-2

1.3 1.1 3.3 8.1 2.9 1.5 2.4 1.7 5.7 7.2 2.6 4.2 2.8 9.2 6.1

2.6 9.3 2.4 1.1 2.1 6.8 7.5

fold change

t test

Table 1. Continued

32.3/4.6 42.4/5.0

29.5/6.5

37.0/6.9

50.7/5.0 68.0/4.6 94.5/4.8

29.6/5.0 39.8/5.9

54.5/5.7 36.7/5.6 26.9/4.6 89.6/5.4 89.6/5.4 47.1/4.4 36.7/5.6 36.7/5.6 36.7/5.6 42.2/5.7 42.2/5.7 42.2/5.7 26.9/4.6 26.9/4.6 89.6/5.4

36.3/6.2 42.4/4.7 54.4/6.5 36.3/4.9 36.3/4.9 63.6/5.1 42.4/4.7

58.9/7.2 58.9/7.2 33.6/6.4


MS MS

MS

MS

MS MS MS

MS/MS MS/MS

MS MS MS/MS MS/MS MS MS MS MS MS MS/MS MS/MS MS/MS MS/MS MS/MS MS/MS

MS MS MS MS MS MS MS

MS/MS MS MS

identif. typeb

33 13 17

seq. cov. (%)

11 7 5

no. of peptides

LGG_01465 LGG_01465 LGG_02906

locus tag

73 72

103

65

101 119 147

376 355

101 87 95 301 111 139 90 142 97 348 179 102 95 129 833

105 72 70 153 276 196 72

AccD

Fatty Acid Biosynthesis 29 6 LGG_02111 24 14

FabD FabF

LysR

Regulation 4 LGG_01423 9

LGG_02117 LGG_02115

PepC PepF PepN

Proteolytic Enzyme System 17 9 LGG_02346 12 8 LGG_00732 16 12 LGG_00554

7 5

RpsB SelA

39 22

Protein Synthesis 5 LGG_01628 6 LGG_02708

GalE GalK GalT LacD LacD Pgm GalK CitF GapB TpiA XpkA XpkA Eno GapB GapB GapB Pgk Pgk Pgk TpiA TpiA XpkA

Other 8 6 2 4 11 10 6 11 7 6 3 2 2 2 11

name

Fhs Fhs PrsA

LGG_01914 LGG_00933 LGG_00935 LGG_00200 LGG_00200 LGG_00936 LGG_00933 LGG_00933 LGG_00933 LGG_00934 LGG_00934 LGG_00934 LGG_00935 LGG_00935 LGG_00200

20 22 9 8 13 28 21 28 27 26 22 22 9 9 18

Galactose Metabolism 25 7 LGG_00654 11 5 LGG_00653 10 5 LGG_00655 35 10 LGG_02575 71 21 LGG_02575 26 17 LGG_00921 11 5 LGG_00653

Carbohydrate Metabolism and Glycolysis

1125 95 68

Mowse score

Acetyl-coenzyme A carboxylase carboxyl transferase subunit beta Malonyl-CoA-[acyl-carrier-protein] transacylase 3-oxoacyl-[acyl-carrier-protein] synthase

Transcriptional regulators, LysR family

Aminopeptidase C Oligoendopeptidase F Aminopeptidase N

SSU ribosomal protein S2P selenium transferase family L-seryl-tRNA

Citrate lyase alpha chain/Citrate CoA-transferase Glyceraldehyde 3-phosphate dehydrogenase Triosephosphate isomerase Xylulose-5-phosphate/Fructose-6-phosphate phosphoketolase Xylulose-5-phosphate/Fructose-6-phosphate phosphoketolase Enolase Glyceraldehyde 3-phosphate dehydrogenase Glyceraldehyde 3-phosphate dehydrogenase Glyceraldehyde 3-phosphate dehydrogenase Phosphoglycerate kinase Phosphoglycerate kinase Phosphoglycerate kinase Triosephosphate isomerase Triosephosphate isomerase Xylulose-5-phosphate/Fructose-6-phosphate phosphoketolase

UDP-glucose 4-epimerase Galactokinase Galactose-1-phosphate uridylyltransferase Tagatose-bisphosphate aldolase Tagatose-bisphosphate aldolase Phosphoglucomutase/Phosphomannomutase Galactokinase

Formyl-tetrahydrofolate synthetase Formyl-tetrahydrofolate synthetase Ribose-phosphate pyrophosphokinase

function

protein


research articles

Journal of Proteome Research • Vol. 8, No. 11, 2009 4999


Two proteins were identified from these spots. It remains unclear which of these proteins was more c

MS, MALDI-MS; MS/MS, LC-MS/MS. b a Log, logarithmic growth phase; stat, stationary growth phase. abundant.

PncA SecA YcgG

ATP synthase gamma chain Glutamate racemase/Xanthosine triphosphate pyrophosphatase NADH dehydrogenase Protein translocase subunit secA Aldo/keto reductase family Hypothetical cytosolic protein NAD(P)H-dependent quinone reductase Pyrazinamidase/Nicotinamidase Protein translocase subunit secA Aldo/keto reductase family Hypothetical cytosolic protein Hypothetical cytosolic protein AtpG MurI Ndh SecA YcgG

Miscellaneous 9 LGG_01182 7 LGG_00782 5 LGG_02547 2 LGG_00899 8 LGG_00259 7 LGG_00226 9 LGG_01433 3 LGG_02764 4 LGG_00899 6 LGG_00259 7 LGG_00226 2 LGG_02373 35 20 17 5 28 59 34 15 6 25 59 13 85 114 74 134 131 119 123 447 255 99 119 173 33.7/4.9 52.3/5.3 42.6/7.4 89.5/5.2 31.6/6.4 14.7/4.9 23.1/6.0 20.6/5.9 89.5/5.2 31.6/5.9 14.7/4.9 20.1/4.6 10-4 10-4 10-5 10-4 10-5 10-5 10-3 10-4 10-3 10-4 10-4 10-4 log log log log log log log stat stat stat stat stat 40c 68 69 46c 70 71 72 73 56c 74 75 55c

2.1 8.1 5.7 8.1 6.9 6.5 1.8 5.4 6.1 4.6 2.9 9.2

× × × × × × × × × × × ×

1.71 1.63 1.75 2.51 1.78 2.00 1.74 1.74 1.69 1.56 2.07 1.68

MS MS MS MS/MS MS MS MS MS/MS MS/MS MS MS MS/MS

function

3-oxoacyl-[acyl-carrier-protein] synthase 3-oxoacyl-[acyl-carrier protein] reductase (3R)-hydroxyacyl-[acyl carrier protein] dehydratase (3R)-hydroxyacyl-[acyl carrier protein] dehydratase

name

FabF FabG FabZ FabZ LGG_02115 LGG_02116 LGG_02113 LGG_02113 42.4/5.0 25.3/6.8 15.9/8.5 15.9/8.5 10-4 10-6 10-4 10-4 log log log log 64 65 66 67

3.9 2.9 1.1 5.7

× × × ×

2.00 3.22 3.12 2.35

MS MS MS MS

133 76 51 81

28 17 26 38

11 4 3 7

locus tag theoretical Mw (kDa)/pI t test growth phasea spot no.

Table 1. Continued 5000

Koskenniemi et al.

fold change

identif. typeb

Mowse score

seq. cov. (%)

no. of peptides

protein

research articles

the logarithmic growth phase in whey medium. This is probably a consequence of differences in fatty acid types and contents of whey and MRS. MRS contains 0.1% Tween 80 (polyoxyethylenesorbitan monooleate), whereas the whey medium used contained 0.05% milk fat. Proteins More Abundant during Growth in MRS. Among the 58 LGG proteins detected in higher amounts under MRS conditions, 13 were involved in protein synthesis (Table 2). Eight were ribosomal proteins, and the other five were translation elongation factors and proteins related to tRNA synthesis. Higher protein synthesis rates in MRS are probably associated with the approximately 1.5-fold longer generation time of LGG in whey medium (∼115 min) than in MRS medium (∼75 min) (Figure 1). These results indicate that MRS fulfils the growth demands of LGG better than whey medium. Additionally, the general stress response proteins/chaperones GroEL and DnaK were more abundant in the MRS condition, especially during stationary growth phase. These proteins have been shown to be up-regulated at stationary growth phase in many LAB.52 Generally, when bacteria enter the stationary phase as a consequence of nutrient starvation, they develop a general stress-resistant state associated with the accelerated production of stress response proteins and chaperones.53 The increased production of chaperone proteins in MRS grown cells is potentially a response to the high protein synthesis rate and cell density that require the assistance of chaperones for de novo protein folding. The highest single up-regulated protein in MRS was a 95 kDa protein annotated as an alcohol dehydrogenase/acetaldehyde dehydrogenase,34 which is a bifunctional enzyme required for alcohol and acetaldehyde fermentation in pyruvate metabolism. This protein was represented in our analysis by four horizontally adjacent protein spots, indicating charged posttranslational modifications. The protein was present in an up to a 10-fold higher amount in MRS than in whey medium during logarithmic growth phase. At stationary phase, the fold change was five at the greatest. Thus, the alcohol or acetaldehyde fermentation is expected to be more intensive during growth in MRS than in whey medium. LGG has previously been detected to produce ethanol, but only as a minor fermentation product (produced 400-500-fold less than lactic acid).49,54 In addition, LGG has been shown to effciently metabolize acetaldehyde to acetate.55 Furthermore, two other proteins related to pyruvate metabolism were more abundant during growth in MRS: pyruvate dehydrogenase (PdhC, dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex) and acetolactate synthase (Als). These results suggest that LGG prefers mixed-acid fermentation during growth in MRS, while in whey medium, a homolactic acid fermentation is more significant. This might be a consequence of differences in redox balance of LGG under the two different growth media conditions.56 The enzymes of the pyrimidine biosynthesis pathway were moderately up-regulated in MRS compared to whey medium (genes CarB, PyrF, and PyrG at logarithmic phase, CarB, PyrB, and PyrC at stationary phase) (Table 2). Guillot et al.57 found that addition of lactose instead of glucose to M17-medium resulted in down-regulation of de novo pyrimidine biosynthetic pathway in L. lactis. Thus, the cultivation of LGG in whey medium containing hydrolyzed lactose might have caused decrease in pyrimidine biosynthesis compared to glucose containing MRS medium.

growth phasea

log log log log log log stat stat stat stat

log log log log stat stat stat stat stat

stat stat

log log log log log log log log log log log log log stat stat stat stat stat stat stat

spot no.

76 77 78 79 80 81 82 83 84 85

86 87 88 89 90 91 92 93c 94

95 96

97 98 99 100 101 102 103 104 105 106 107 108 109 110 111 112 113 114 115 116

1.63 1.50

1.69 1.79 1.83 1.89 3.97 7.06 5.18 3.97 1.91 1.51 1.53 3.21 1.68 1.60 1.62 2.02 3.08 1.96 1.53 1.83

1.6 × 10-4 1.3 × 10-5

10-3 10-5 10-5 10-4 10-5 10-5 10-6 10-4 10-4 10-3 10-2 10-5 10-3 10-4 10-5 10-5 10-4 10-4 10-4 10-5

1.5 5.9 5.6 2.0 4.3 2.2 2.5 2.8 7.7 2.1 1.7 4.4 5.8 1.8 3.6 3.2 2.0 4.7 5.8 2.9

× × × × × × × × × × × × × × × × × × × ×

9.88 5.29 3.66 2.47 4.90 3.18 1.96 1.78 1.65

10-7 10-6 10-5 10-5 10-6 10-6 10-4 10-4 10-4

× × × × × × × × ×

4.5 3.3 3.9 1.7 1.0 2.2 3.9 6.4 5.7

1.64 2.22 1.79 1.52 1.68 1.56 1.79 1.62 1.53 2.38

10-5 10-6 10-5 10-2 10-4 10-3 10-4 10-3 10-5 10-6

fold change

× × × × × × × × × ×

5.3 5.5 3.3 4.8 4.7 4.1 3.7 7.7 2.2 5.9

t test

67.7/4.9 76.9/4.5 47.8/7.3 40.3/6.0 24.5/9.9 19.3/10.2 14.9/9.6 16.6/10.2 11.3/10.0 47.2/4.9 29.6/5.0 14.8/10.1 100.7/6.2 76.9/4.5 40.3/6.0 24.5/9.9 14.9/9.6 16.6/10.2 47.2/4.9 14.8/10.1

63.6/5.1 63.3/4.8

94.6/6.8 94.6/6.8 94.6/6.8 94.6/6.8 94.6/6.8 94.6/6.8 94.6/6.8 60.4/4.7 46.8/4.8

23.7/6.5 23.8/7.0 116.2/4.9 25.5/9.8 60.0/6.6 60.0/6.6 116.2/4.9 35.0/5.5 45.1/6.6 82.5/6.5


MS MS MS/MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS MS

MS MS

MS MS MS/MS MS MS MS MS/MS MS MS

MS MS MS MS/MS MS MS/MS MS MS MS/MS MS/MS

identif. typeb

179 320 154 80 143 88 109 209 118 128 206 114 87 124 80 121 109 137 70 52

152 75

97 68 860 131 97 68 860 124 98

94 154 86 150 131 106 86 94 230 202

Mowse score

locus tag

12 5

Synthesis 16 LGG_01561 32 LGG_02493 3 LGG_01412 5 LGG_00754 11 LGG_02288 7 LGG_02472 8 LGG_02289 13 LGG_02448 6 LGG_01690 9 LGG_01389 17 LGG_01628 8 LGG_02473 11 LGG_01261 11 LGG_02493 5 LGG_00754 8 LGG_02288 8 LGG_02289 7 LGG_02448 5 LGG_01389 3 LGG_02473

15 10 Protein 24 47 10 13 54 26 53 58 43 22 68 59 10 18 13 30 53 39 11 22

LGG_00921 LGG_01820

Carbohydrate Metabolism 14 12 LGG_00757 5 5 LGG_00757 39 13 LGG_00757 24 11 LGG_00757 14 12 LGG_00757 5 5 LGG_00757 39 13 LGG_00757 25 15 LGG_01899 14 6 LGG_01322

Metabolism 9 LGG_02466 11 LGG_01674 12 LGG_01456 3 LGG_01454 11 LGG_02546 2 LGG_02546 12 LGG_01456 6 LGG_01459 4 LGG_01458 5 LGG_02296

no. of peptides

Nucleotide 40 40 11 27 18 10 11 17 8 8

seq. cov. (%)

AspS EF-G Gid QueA RplA RplF RplK RplM RplU RpsA RpsB RpsH ValS EF-G QueA RplA RplK RplM RpsA RpsH

Pgm PtsI

AdhE AdhE AdhE AdhE AdhE AdhE AdhE Als PdhC

Adk Gmk PyrAb PyrF PyrG PyrG PyrAb PyrB PyrC

name

function

protein

Aspartyl-tRNA synthetase Protein Translation Elongation Factor G Glucose inhibited division protein A S-adenosylmethionine:tRNA ribosyltransferase-isomerase LSU ribosomal protein L1P LSU ribosomal protein L6P LSU ribosomal protein L11P LSU ribosomal protein L13P LSU ribosomal protein L21P SSU ribosomal protein S1P SSU ribosomal protein S2P SSU ribosomal protein S8P Valyl-tRNA synthetase Protein Translation Elongation Factor G S-adenosylmethionine:tRNA ribosyltransferase-isomerase LSU ribosomal protein L1P LSU ribosomal protein L11P LSU ribosomal protein L13P SSU ribosomal protein S1P SSU ribosomal protein S8P

Alcohol dehydrogenase/Acetaldehyde dehydrogenase Alcohol dehydrogenase/Acetaldehyde dehydrogenase Alcohol dehydrogenase/Acetaldehyde dehydrogenase Alcohol dehydrogenase/Acetaldehyde dehydrogenase Alcohol dehydrogenase/Acetaldehyde dehydrogenase Alcohol dehydrogenase/Acetaldehyde dehydrogenase Alcohol dehydrogenase/Acetaldehyde dehydrogenase Acetolactate synthase Dihydrolipoamide acetyltransferase component of pyruvate dehydrogenase complex Phosphoglucomutase/Phosphomannomutase Phosphoenolpyruvate-protein phosphotransferase

Adenylate kinase/Nucleoside-diphosphate kinase Guanylate kinase Carbamoyl-phosphate synthase large chain Orotidine 5′-phosphate decarboxylase CTP synthase CTP synthase Carbamoyl-phosphate synthase large chain Aspartate carbamoyltransferase Dihydroorotase Anaerobic ribonucleoside-triphosphate reductase

Table 2. The Identified Proteins from LGG That Were More Abundant in MRS than in Whey Medium (Fold Change g1.5, p e 0.05)


research articles


5002

growth phasea

log log log stat stat stat stat

log log

log stat stat stat stat stat

log log log

log

stat stat stat stat

log log stat stat stat stat stat

log log log log

spot no.

117 118 119 120 121 122 123

124 125c

126 127 128 129 93c 130

131 132 133

134

135 136 137 138

139 125c 140 141c 141c 142c 142c

143 144 145 146

Table 2. Continued

1.83 1.93 2.28 1.95 1.78 1.66

1.57 1.92 1.51

1.55

2.22 1.82 1.54 1.57

2.43 2.77 1.70 1.53 1.53 1.50 1.50

1.50 1.58 1.89 1.55

10-2 10-7 10-5 10-3 10-4 10-3

2.0 × 10-4 1.2 × 10-3 7.9 × 10-4

1.8 × 10-4


10-6 10-5 10-4 10-2

10-6 10-5 10-5 10-3 10-3 10-4 10-4

10-4 10-3 10-4 10-3

× × × ×

× × × × × × ×

× × × ×

5.4 5.0 4.0 6.4

2.4 2.4 1.9 3.3 3.3 2.5 2.5

8.3 5.9 1.2 1.3

2.0 9.4 3.0 1.1 6.4 2.5

2.69 2.77

2.7 × 10-5 2.4 × 10-5

× × × × × ×

× × × × × × ×

1.77 1.67 1.64 1.57 1.58 1.55 1.72

fold change

10-4 10-5 10-7 10-3 10-4 10-2 10-4

6.2 6.6 2.9 7.6 5.0 1.9 6.0

t test

42.0/6.2 34.9/4.5 39.3/6.6 42.9/5.6

37.8/9.4 27.4/9.5 43.2/6.9 38.5/6.5 38.6/6.4 21.6/5.3 21.6/5.3

42.9/4.5 47.0/10.2 50.4/6.9 42.6/7.7

42.6/7.7

42.9/4.5 47.0/10.2 50.1/5.9

57.4/4.7 67.2/4.6 57.4/4.7 57.4/4.7 57.4/4.7 57.4/4.7

26.3/9.8 28.3/8.9

38.9/6.8 67.3/4.8 87.9/6.4 54.2/4.7 87.9/6.4 87.9/6.4 74.4/5.6


MS MS MS MS

MS MS MS/MS MS MS MS MS

MS MS MS/MS MS

MS

MS MS MS

MS MS MS MS MS MS

MS/MS MS

MS MS MS MS MS/MS MS/MS MS

identif. typeb seq. cov. (%)

no. of peptides

locus tag

65 66 63 83

Miscellaneous 11 5 17 5 10 4 15 6

LGG_01300 LGG_01265 LGG_01061 LGG_01837

Csd MreB MvaK MvaS

WelG Wze Glf RmlB RmlB RmlC RmlC

Exopolysaccharide Biosynthesis 78 17 7 LGG_02045 51 15 4 LGG_02052 461 37 5 LGG_02050 111 19 7 LGG_01997 111 19 7 LGG_02038 125 30 7 LGG_01998 92 22 6 LGG_02039

12 11 7 4

p40

AgaS DacA GlmU

GroEL DnaK GroEL GroEL GroEL GroEL

Def

AgaS DacA MurD p40

32 28 24 11

LGG_00031

LGG_00333 LGG_00254 LGG_02562

Cell Wall Biosynthesis 32 12 28 11 24 10 4

LGG_02239 LGG_01604 LGG_02239 LGG_02239 LGG_02239 LGG_02239

Chaperones 45 28 26 16 59 30 38 23 23 11 18 8

11

LGG_01861 LGG_02081

Regulation 36 4 20 5

OppD PepF PepX PepD PepX PepX YuxL

name

LGG_00333 LGG_00254 LGG_01282 LGG_00031

149 163 969 63

63

149 163 123

277 166 266 249 88 101

425 64

Proteolytic Enzyme System 71 9 4 LGG_01941 79 11 6 LGG_00984 81 9 6 LGG_01695 69 12 4 LGG_01158 455 15 5 LGG_01695 77 2 2 LGG_01695 323 37 24 LGG_01864

Mowse score

Cysteine desulfurase/Selenocysteine lyase Rod shape-determining protein mreB Phosphomevalonate kinase Hydroxymethylglutaryl-CoA synthase

Putative galactofuranosyltransferase Tyrosine-protein kinase UDP-galactopyranose mutase dTDP-glucose 4,6-dehydratase dTDP-glucose 4,6-dehydratase dTDP-4-dehydrorhamnose 3,5-epimerase dTDP-4-dehydrorhamnose 3,5-epimerase

Galactosamine-6-phosphate deaminase serine-type carboxypeptidase Glucosamine-1-phosphate acetyltransferase/ UDP-N-acetylglucosamine pyrophosphorylase Putative secreted antigen GbpB/SagA, putative peptidoglycan hydrolase Galactosamine-6-phosphate deaminase D-alanyl-D-alanine serine-type carboxypeptidase UDP-N-acetylmuramoylalanine - D-glutamate ligase Putative secreted antigen GbpB/SagA, putative peptidoglycan hydrolase D-alanyl-D-alanine

60 kDa chaperonin GROEL Chaperone protein dnaK 60 kDa chaperonin GROEL 60 kDa chaperonin GROEL 60 kDa chaperonin GROEL 60 kDa chaperonin GROEL

Transcriptional regulator, GntR family Transcriptional regulator

Oligopeptide transport ATP-binding protein oppD Oligoendopeptidase F Xaa-Pro dipeptidyl-peptidase Dipeptidase A Xaa-Pro dipeptidyl-peptidase Xaa-Pro dipeptidyl-peptidase Acylamino-acid-releasing enzyme

function

protein

research articles Koskenniemi et al.

log log log stat stat stat stat stat stat stat stat 147 148 149 150 151 152 153 154 155 156 157

1.1 1.9 8.9 4.1 1.3 2.9 1.8 7.9 8.0 2.6 1.9 growth phasea spot no.

Table 2. Continued

Two proteins were identified from these spots. It remains unclear which of these proteins was more c

MS, MALDI-MS; MS/MS, LC-MS/MS. b a Log, logarithmic growth phase; stat, stationary growth phase. abundant.

GTP-binding protein TypA/BipA Zn-dependent hydrolase Zn-dependent hydrolase Cell division protein ftsZ Glutamine synthetase D-2-hydroxyisocaproate dehydrogenase Phosphomevalonate kinase Hydroxymethylglutaryl-CoA synthase Topoisomerase IV subunit A Multicopper oxidase family protein Hydrolase (HAD superfamily)

function name

TypA YkqC YqgA FtsZ GlnA LdhD MvaK MvaS ParC SufI YqeK LGG_01327 LGG_01311 LGG_01340 LGG_01286 LGG_01696 LGG_00158 LGG_01061 LGG_01837 LGG_01420 LGG_02415 LGG_01735 12 14 5 7 8 5 4 10 8 6 5 21 20 9 18 37 11 10 25 16 12 20 141 172 62 79 468 55 63 148 323 58 89 MS MS MS MS MS/MS MS MS MS MS/MS MS MS 68.0/4.9 61.6/6.7 62.3/6.8 44.6/4.5 50.4/5.7 37.1/5.6 39.3/6.6 42.9/5.6 90.9/6.5 57.1/6.0 22.7/6.4 2.26 1.67 1.52 1.52 1.57 1.62 2.10 1.66 1.70 1.72 1.68 10-5 10-4 10-6 10-4 10-2 10-3 10-6 10-4 10-3 10-5 10-5

locus tag no. of peptides seq. cov. (%) Mowse score identif. typeb theoretical Mw (kDa)/pI fold change t test

× × × × × × × × × × ×

protein


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Several proteins involved in exopolysaccharide biosynthesis were more abundant in MRS conditions. Exopolysaccharides of LAB can improve the texture and viscosity of fermented food products (e.g., Ruas-Madiedo et al.58), increase the tolerance of the bacterium to some antimicrobial factors,59 and may have some positive health effects (e.g., immunostimulation) on human.60 The production of exopolysaccharides might thus be a desired feature for probiotic LAB. According to present study, exopolysaccharide biosynthesis proteins Glf, RmlB, RmlC, WelG, and Wze were more abundant when LGG was grown in MRS than in the whey medium. Differences in exopolysaccharide production of LAB have earlier been found to be caused by differences in carbon/nitrogen ratio61 and sugar composition of the growth medium62 as well as rate of growth.63 MRS and whey media differ from each other both in their sugar composition, and nitrogen content and based on this study, MRS is more optimal than the whey medium for production of some exopolysaccharide biosynthesis proteins of LGG, and possibly also for the production of exopolysaccharides. Previously, LGG has been demonstrated to produce substantially more exopolysaccharides in a milkbased AOAC medium than in MRS medium.64 However, the AOAC medium contains several nitrogen and carbon sources besides milk, and thus, it is not fully comparable with the whey medium used in this study. The observed higher exopolysaccharide biosynthesis protein production may also partly be explained by the higher growth rate in MRS, since exopolysaccharide production has been shown to be growth rate dependent.63,65 Several proteins involved in cell wall and peptidoglycan biosynthesis were more abundant during growth in MRS than in whey medium. This might cause differences in cell surface structure of LGG and thereby also in the probiotic properties of LGG. In addition, p40 protein was 1.5-fold as abundant in MRS as in whey medium conditions. Excreted p40 protein has been demonstrated to promote intestinal epithelial homeostasis,66 which is a desired feature for a probiotic organism. Previous work indicates that p40 protein is synthesized as a preprotein from which a 28 amino acid signal peptide is processed prior secretion to culture medium.66 Most probably the p40 protein observed here represents the intact preprotein, since the migration of the p40 protein spot on our 2D-gels indicates molecular weight of approximately 42 kDa, which is very close to the theoretical molecular weight of the preprotein (42.6 kDa). On the other hand, the location of the p40 spots in gel is not consistent with the theoretical pI value (7.7), since calculation with DeCyder program indicates slightly more acidic pI (∼6.9) for the corresponding spot. Possible explanations for this discrepancy include the effect of charged posttranslational modification. The Components of the Proteolytic System Demonstrate Varying Response to MRS and Whey Media. LAB typically have multiple amino acid auxotrophies and, therefore, are dependent on the transport of amino acids and/or transport and hydrolysis of exogenous peptides for growth. Growth to high cell-densities in milk relies on casein-derived peptides and amino acids provided by the proteolytic system that have been intensively studied, especially in L. lactis (reviewed recently in Savijoki et al.67). In the present study, several components of the proteolytic system were identified as exhibiting growth media-dependent production. Two aminopeptidases, PepN and PepC, as well as an oligopeptidase PepF1, were more abundant in cells grown in whey, whereas Journal of Proteome Research • Vol. 8, No. 11, 2009 5003

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Figure 3. (A) The effect of growth media on the relative abundance of purine biosynthesis proteins. For each detected purine biosynthetic protein, a Cy3 or Cy5 scan image of the protein spot is represented both at logarithmic (log) and stationary (stat) growth phase in whey and MRS media. The fold change of each spot is indicated. The following abbreviations are used: PRPP, phosphoribosyl pyrophosphate; PRA, phosphoribosyl amine; GAR, phosphoribosyl glycinamide; FGAR, phosphoribosyl N-formylglycinamide; FGAM, phosphoribosyl N-formylglycinamidine; AIR, phosphoribosyl aminoimidazole; CAIR, phosphoribosyl carboxyaminoimidazole; SAICAR, phosphoribosyl succinocarboxamide aminoimidazole; AICAR, phosphoribosyl aminoimidazole carboxamide; FAICAR, phosphoribosyl formamidoimidazole carboxamide; IMP, inosine monophosphate; XMP, xanthosine monophosphate; GMP, guanosine monophosphate; sAMP, adenylosuccinate; AMP, adenosine monophosphate; PurF, amidophosphoribosyltransferase; PurD, phosphoribosylamine-glycine ligase; PurL, phosphoribosylformylglycinamidine synthase; PurM, phosphoribosylformylglycinamidine cyclo-ligase; PurK, phosphoribosylaminoimidazole carboxylase; PurC, phosphoribosylamidoimidazolesuccinocarboxamide synthase; PurB, adenylosuccinate lyase; PurH, phosphoribosylaminoimidazolecarboxamide formyltransferase/IMP cyclohydrolase; GuaB, IMP dehydrogenase; GuaA, GMP synthase; GuaC, GMP reductase. (B) Genetic organization of the purine biosynthesis genes in LGG. Locations of putative PurBoxes are indicated with asterisks (*). The genes encoding the differentially produced proteins represented in panel A are marked in bold and with a gray arrow. Stem and loop structures stand for putative transcription termination sites. The LGG gene numbers are indicated. (C) Location of purine biosynthesis operons and genes in the LGG genome.

a dipeptidase PepD, a dipeptidyl aminopeptidase PepX, and an oligopeptidase PepF2, as well as the oligopeptide transport protein OppD were more abundant in LGG cells during growth in MRS (Tables 1 and 2). The peptide and amino acid content of growth medium is known to affect the expression of the components of the proteolytic system of LAB.68,69 A proteomics approach revealed that PepO1, PepN, PepC, PepF, as well as the peptide transport proteins Opp and OptS were more abundant in L. lactis during growth in a medium lacking free amino acids and peptides.32 Intriguingly, a protein identified as glutamine synthetase, GlnA was produced more during growth in MRS. Previous reports have indicated that the glnA gene is cotranscribed with pepX in L. rhamnosus 1/6,70 and since the same gene organization of pepX and glnA is conserved in LGG (data not shown), and PepX and GlnA exhibit similar protein expression profiles, it is tempting to speculate that these proteins may be part of the same regulon in LGG. 5004


The transcriptional regulator CodY negatively regulates the expression of several components of the proteolytic system of L. lactis, and the intracellular pool of branched-chain amino acids modulates the strength of repression.71-73 The differential production of the proteolytic enzymes of LGG in the present study most likely reflect the differences in free amino acid and peptide pools of MRS and whey medium. Recently, Smeianov et al.33 used microarrays to analyze gene expression in dairy starter strain L. helveticus CNRZ32 during growth in milk versus MRS. These results revealed induction of the opp genes, pepN, and pepX in milk, whereas the expression of pepD was induced under MRS conditions. Furthermore, the expression profiles for pepC and pepF appeared to be independent of media type in L. helveticus CNRZ32.33 Thus, the regulation of expression of Opp, PepX, PepC, and PepF in LGG may be different from that in L. helveticus CNRZ32, and could reflect the adaptation of LGG to a different niche compared to the dairy strain CNRZ32.


Conclusions In the present study, we compared the adaptation of probiotic LGG to a hydrolyzed whey-based medium and a rich MRS laboratory medium using the quantitative 2-D DIGE technique. The advantage of 2-D DIGE technology is the possibility of multiplexing due to differential protein labeling with fluorescent CyDyes. Furthermore, the ability to compare parallel gels due to an internal standard included in every gel enables detection of statistically highly significant differences in protein abundance. Here, remarkable differences were observed in the growth medium-dependent proteomes of LGG, as 100 separate proteins were detected to be differentially produced when the proteomes of LGG cells grown in whey and MRS media were compared. A couple of differentially produced proteins, like p40, enolase, glyceraldehyde-3 phosphate dehydrogenase and exopolysaccharide biosynthesis enzymes, have been linked to probiotic features, but their exact role in probiotic characteristics of LGG is not fully known. The major proteins more abundant in cells growing in whey medium were purine biosynthesis proteins, since milk-based whey medium does not supply LGG with purines, while MRS is a source of purines for them. Second, galactose metabolizing enzymes were detected in higher amounts in cells grown in whey medium, which is probably a consequence of differences in sugar compositions of the two different growth media. According to our data, LGG may have the capability to ferment the galactose moiety of lactose using two different pathways, the Leloir and tagatose-6-phosphate pathway. Growth medium may also affect the balance between mixed-acid and homolactic acid fermentation of LGG, since the proteins required for mixedacid fermentation, including alcohol dehydrogenase/ acetaldehyde dehydrogenase, pyruvate dehydrogenase, and acetolactate synthase, were all more abundant in cells growing in MRS. These alterations in protein abundance may be related to possible differences in redox balance between the LGG cultures under two different growth media conditions. This study clearly demonstrates that the proteome of LGG grown in hydrolyzed whey-based medium differs significantly from that grown in rich MRS laboratory medium. Since milk protein containing media are commonly used in commercial LAB production, this type of media could be recommended for use in physiological studies of probiotics to elucidate a more direct relationship with conditions the probiotics are exposed to prior to consumption. Abbreviations: LAB, Lactic acid bacteria; LGG, L. rhamnosus GG.

Acknowledgment. Elina Ahola-Iivarinen, Marko Hukka, and Hanna Jefremoff are acknowledged for their technical assistance. This work was supported financially by the Academy of Finland (grant 210740). Supporting Information Available: Supplementary Figure S1, motifs used to screen for binding elements; Supplementary Figures S2-S6 show the genomic regions and sequence alignments and highlight the locations of putative PurRbinding elements; Supplementary Table S1, setup of DIGE experiment; Supplementary Table S2, PurBox matches; Supplementary Table S3, the final list of PurBox matches. This material is available free of charge via the Internet at http://pubs.acs.org.

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Proteome Analysis of Lactobacillus rhamnosus GG Using 2-D DIGE

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