A Genetic Insight Into Peptide and Amino-Acid ...

CURRENT MICROBIOLOGY Vol. 52 (2006), pp. 464–468 DOI: 10.1007/s00284-005-0353-y

Current Microbiology An International Journal ª Springer Science+Business Media, Inc. 2006

A Genetic Insight Into Peptide and Amino-Acid Utilization by Propionibacterium freudenreichii LMG 16415 Franca Rossi, Veronica Gatto, Marta Marzotto, Sandra Torriani Dipartimento Scientifico e Tecnologico, Universit( degli Studi di Verona, Strada Le Grazie 15, 37134, Verona, Italy Received: 18 October 2005 / Accepted: 10 February 2006

Abstract. In this note the genetic characterization of the peptide degrading system of Propionibacterium freudenreichii was addressed. Genomic fragments of P. freudenreichii subsp. freudenreichii LMG 16415 were cloned in Escherichia coli XL1 Blue, and those leading to an increase in peptidase-like activity using chromogenic substrates aminoacyl-b-naphtylamides (aminoacyl-bNA) were isolated and sequenced. This strategy allowed the identification of partial gene regions of P. freudenreichii LMG 16415 with significant similarity to proteins directly or indirectly involved in peptide and amino acid metabolism, i.e., an oligopeptide transporter, a D-amino acid oxidase, a muropeptidase, and an ABC transporter involved in osmoregulation similar to glycine betaine transporters.

Bacterial strains belonging to the species Propionibacterium freudenreichii are used as starters in the manufacture of Swiss-type cheeses. Their maximal growth follows the development of thermophilic starter lactic acid bacteria. Their main technologic role is forming the characteristic holes of Emmental cheese by release of carbon dioxide from the fermentation of lactate. They are able to hydrolyze N-terminal proline from peptides [5, 6, 14]. One of the enzymes involved in proline release from peptides, a proline iminopeptidase, Pip, from P. freudenreichii subsp. shermanii ATCC 9617, is the only peptidase of dairy propionibacteria characterized at the sequence level to date [6]. The contribution of other enzymatic components to free proline formation by P. freudenreichii has been also demonstrated [11, 14]. However, some investigations indicated that P. freudenreichii strains exert only slight or not significant effects on the increase of free proline in cheeses [15, 16]. The role of propionibacteria in other aspects of proteolysis and cheese flavor compounds formation from amino acids has not been investigated extensively, but some studies have reported that P. freudenreichii strains were found to influence the qualitative and

Correspondence to: S. Torriani; email: [email protected]

quantitative peptide profile of Cheddar cheese [2], were active on casein oligopeptides [3], and were able to hydrolyze Leu-bNA, Lys-bNA and Phe-Pro-bNA [10]. In a recent investigation on the aroma compounds formed in Raclette and Swiss-type cheese with addition of P. freudenreichii strain mixtures, branched chain volatile substances derived from isoleucine and leucine catabolism were detected [15, 17]. Given the importance of such bacterial species in cheese-making technology, there is a need for further characterization of the genetic asset and regulation of its peptide-degrading system. From a practical point of view, this could allow the development of simple and rapid polymerase chain reaction (PCR), or reverse transcriptase-PCR (RT-PCR), based tests for the definition of activities expressed by single strains in cheese, thus helping the selection of those that might have an impact in the dairy technology. To identify genetic determinants involved in peptide and amino-acid utilization by P. freudenreichii, a cloning and chromogenic screening strategy was applied. Escherichia coli XL1-Blue was transformed with a plasmid vector containing gene inserts from P. freudenreichii LMG 16415; the gene fragments conferring an increased capacity to hydrolyze aminoacyl-bNA substrates to the host strain were isolated for sequencing.

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Materials and Methods Bacterial strains and growth conditions. P. freudenreichii subsp. freudenreichii LMG 16415 (afterward named P. freudenreichii LMG 16415) was used as a source of DNA fragments to be cloned in E. coli XL1-Blue (Stratagene, La Jolla, CA). It was cultured in sodium lactate (SL) broth (1% sodium lactate, 1% tryptone, 1% yeast extract, 0.25% K2HPO4, and 0.12% KH2PO4, pH 7.0) at 30
C for 48 to 96 hours in anaerobiosis obtained with an Anaerocult A system (VWR International, Milan, Italy). E. coli XL1-Blue was used for the isolation of P. freudenreichii gene fragments cloned in pGEM-5Zf(+) vector (Promega, Madison, WI). Such strain was grown in Luria Bertani (LB) medium at 37
C in aerobiosis. Transformed cells were plated on LB medium supplemented with 100 lg/mL ampicillin, 1 mM isopropyl-bthiogalactopyranoside, and 80 mg/mL 5-bromo-4-chloro-3-indolyl-bD-galactopyranoside. E. coli clones harboring pGEM-5Zf(+) with genomic DNA inserts were recognized by the blue–white colony screening. Isolation of P. freudenreichii genome fragments. Genomic DNA extraction, restriction, and ligation reactions and E. coli electrotransformation were carried out according to standard methods [12]. Plasmid DNA was purified using commercial mini preparation kits (QIAGEN, Germany). Gene fragments from P. freudenreichii DNA were isolated by restriction of approximately 1 lg genomic DNA with PstI, fractionation of restriction products with sizes >1 kb from agarose gels using the Ultra Clean DNA Purification Kit (MoBio, Solana Beach, CA), and ligation with approximately 10 ng pGEM-5Zf(+) vector digested with the same enzyme. Screening for substrate hydrolyzing activity. All of the white colonies obtained from a single transformation event, i.e., 600 clones, presumptively hosting recombinant plasmids, were replicated singularly on LB medium with sterile toothpicks. A plate-screening procedure was applied for the identification of E. coli transformants expressing hydrolysis activity against a pool of chromogenic substrates (L-prolineb-naphthylamide, L-leucine-b-naphthylamide, L-phenylalanyl-Lproline-b-naphthylamide, L-arginine-b-naphthylamide, L-lysine-bnaphthylamide, and N-acetyl-DL-phenylalanine-b-naphthyl ester; all from Bachem, Bubendorf, Switzerland), each at concentration 0.2 mg/mL in 20 mM sodium phosphate at pH 7.0. Ten mL of the substrates mixture were added to the plate after 24 hours of growth, then plates were flooded with 5 mL Fast Garnet GBC salt (Sigma), 1 mg/mL water solution, and incubated at room temperature for 10 minutes. Colonies developing a purple color and halo were isolated for plasmid extraction, PCR amplification, and sequencing of the DNA insert. PCR program. For amplification of DNA inserts in pGEM-5Zf(+) vector, cycling conditions comprised 40 cycles of denaturation at 94
C for 1 minute, annealing at 45
C for 1 minute, and extension at 72
C for 2 minutes. The PCR reaction mixture contained 0.5 lmol/L each primer T7 (5¢-GTAATACGACTCACTATAGGGC-3¢) and SP6 (5¢ATTTAGGTGACACTATAGAATAC-3¢), 100 lmol/L dNTPs, 1.5 mmol/L MgCl2, 0.05 U/lL Taq DNA polymerase (Polymed, Florence, Italy), 10% (v/v) dimethyl sulfoxide (Sigma), and approximately 10 ng plasmid DNA, extracted by the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI), in 1 · PCR reaction buffer. PCR products were separated on 1.5% agarose gel in TAE buffer, and, when required, they were excised and purified by the Wizard SV Gel and PCR Clean-Up System (Promega, Madison, WI). Sequencing and sequence analysis. PCR products and primers were vacuum dried and provided to the external sequencing service (Centro

Ricerche Biotecnologiche, BMR, University of Padova, Padova, Italy). Sequencing was carried out on PCR products obtained from recombinant plasmids with primers T7 and SP6. The same primers were used for sequencing both strands of the plasmid inserts by the cycle sequencing method with the Big Dye terminator v3.1 chemistry on an ABI 3700 sequencer. Sequence annotation was carried out by database homology searches with the BLASTX alignment tool at NCBI. The presence of open reading frames (ORFs) and conserved domains was determined using the ORF Finder analysis tool.

Results and Discussion P. freudenreichii subsp. freudenreichii LMG 16415 was selected as a source of DNA fragments among other P. freudenreichii strains for its higher hydrolysis activities demonstrated toward a variety of aminoacyl-bNA substrates [10]. Application of the screening Fast Garnet GBC salt/aminoacyl-bNA substrates on a small gene library from such strain was done using E. coli XL1Blue host strain with an unaltered peptidolytic system. The same E. coli strain, devoid of any hydrolyzing activity against the tested substrates, has been already used for isolation of peptidase-encoding genes with the chromogenic test applied in this study [18]. From transformation of E. coli XL1-Blue with ligation reactions of pGEM-5Zf(+) vector plus genomic DNA fragments of such strain, 600 white colonies were replicated and tested for the development of a purple color in the presence of Fast Garnet GBC salt after incubation with aminoacyl-bNA substrates. Based on such screening, 24 clones were selected for plasmid extraction, amplification of the insert by PCR with primers T7/SP6, and sequencing. None of the analyzed clones contained gene regions coding for proteins belonging to known peptidase families. Instead, sequences coding for partial conserved domains of proteins that also indirectly might have influenced peptide-degrading activities of the host E. coli cells were identified (Table 1). The peptide utilization system of bacteria comprises proteases, peptidases, cell membrane–associated transporters for the uptake of peptides, and amino acids and enzymes of the amino-acid catabolism. All of them may enhance the capacity of bacterial cells to use peptide or amino-acid substrates; this can explain why the chromogenic plate assay resulted in the isolation of gene fragments not encoding peptidases. The plate test presented a background of ‘‘false positive’’ results with plasmid inserts that had no apparent direct relation with peptidolytic activity; among these were fragments encoding proteins of diverse functions, such as transposases, oxidoreductases, phosphatases, enzymes involved in carbohydrate metabolism, DNA replication, protein synthesis, and other activities that could not be

1380 bp

829 bp

366 bp

180 AM110694

312 AM110696

503 AM110697

COG1173, DppC, ABC-type dipeptide/oligopeptide/nickel transport systems, permease components (Amino-acid transport and metabolism/inorganic ion transport and metabolism), E = 5e)05, positions 1–43 COG0665, DadA, glycine/D-amino-acid oxidases (deaminating) (amino acid transport and metabolism), E = 1e)06, positions 3–312 None

COG1125, OpuBA, ABC-type proline/glycine betaine transport systems, ATPase components (amino-acid transport and metabolism), E = 1e)18, positions 1–100 COG1174, OpuBB, ABC-type proline/glycine betaine transport systems, permease component (amino-acid transport and metabolism), E = 5e)05, positions 1–192

Positions 161–392 of an extracellular protein gamma-D-glutamate-meso-diaminopimelate muropeptidase (putative) (Lactobacillus plantarum WCFS1), length = 496, E = 2e)07 Positions 4–113 of a predicted metal-dependent hydrolase of the COG3618, predicted metal-dependent hydrolase of the TIM-barrel fold (Pseudomonas aeruginosa TIM-barrel fold, E = 1e)12, positions 4–115 UCBPP-PA14), length = 319, E = 3e)28

Positions 23–392 of the glycine/D-amino acid oxidases (deaminating) (Brevibacterium linens BL2), length = 416, E = 3e)32

Positions 201–292 of the glycine betaine transport system ProV-like ATP-binding protein (Leifsonia xyli subsp. xyli strain CTCB07) length = 321, E = 3e)22 Positions 33–212 of the ProW-like ABC-type glycine betaine transport system ATP binding and permease components (Leifsonia xyli subsp. xyli strain CTCB07), length = 229, E = 5e)23 Positions 120–161 of a putative dipeptide transport system permease ABC transporter protein (Sinorhizobium meliloti 1021), length = 310, E = 3e)04

Conserved domains and corresponding amino-acid positions in the putative translations

b,c

Length of the ORF portions, regions of homology in the most similar proteins, and positions comprised in conserved domains are indicated. Designations of two different ORFs present in the DNA insert of clone 72. E-expectation value.

a

129 bp

576 bpc

357 bpb

161 AM110695

72 AM110698

Clones and accession numbers Size of the ORF regions Alignment positions on proteins with highest similarity

Table 1. Genes involved in peptide and amino-acid utilization by P. freudenreichii LMG 16415 identified in this study

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correlated with the targets of this research. However, on the whole, the chromogenic plate assay proved useful for opening an insight into the peptide and amino-acid utilization system studied; the ratio of purple colonies (n = 24) versus the total number of clones analyzed (n = 600) was rather low; therefore, the isolation of genetic determinants of interest did not require extensive sequencing. Not restricting the search to specific genes allowed the identification of various genetic traits that can be involved in peptide and amino-acid utilization. Two clones exhibiting the strongest reactions turned out to be false positives since they contained a plasmid pGEM-5Zf(+) without any insert. It could depend on the possible loss of the exogenous sequences by the host strain because of a recombination event. A third clone, which showed a fluctuating strong reaction in different subculturing steps, contained an insert encoding for proteins with maximum similarity with ProV and ProW proteins of a glycine betaine ATP-binding cassette transport system of Leifsonia xyli subsp. xyli, strain CTCB07 (clone 72, Table 1). It could be of interest to determine experimentally the substrate specificity of such ABC transporter. Although it is predicted to transfer the compatible solute glycine betaine for osmoregulation, the strong peptide-hydrolyzing capacity conferred in some instances to the host strain could be explained by the uptake of Pro-bNA or other aminoacyl-bNAs present; hence it might be involved also in the uptake of some amino acids as reported for the hystidine transport system of Sinorhizobium meliloti [1]. The identification of a small region encoding the permease component of a dipeptide transport system (clone 161, Table 1) from P. freudenreichii LMG 16415 can be the start for sequencing the operon comprising such gene and elucidate its specificity, regulation, and regulatory role for the expression of other components of the proteolytic system as has been done in Lactococcus lactis [13]. The gene encoding a putative D-amino acid oxidase (clone 180, Table 1) has no homology in P. acnes KPA171202, the most related to P. freudenreichii among bacteria with publicly available whole genome sequence; according to literature data, it could have a role either in amino-acid catabolism only or also in specific biosynthetic patterns [9]. The putative gamma-D-glutamate-meso-diaminopimelate muropeptidase (clone 312, Table 1) could enhance peptide hydrolysis capacity in cheese by the release of intracellular peptidases because of cell autolysis. In fact, the role of such enzyme type in autolysis has been demonstrated in Bacillus spp. strains [8]. A complete characterization of the genetic deter-

minant coding for this protein can elucidate its contribution to autolysis in addition to other described autolysins of P. freudenreichii [7]. Indeed, this type of physiologic trait has relevance for the selection of bacteria used in the dairy technology. Finally, the putative hydrolase with the conserved domain of predicted metal-dependent hydrolase of the TIM-barrel fold (clone 503, Table 1) might be a peptidase according to the findings on the isoaspartyl dipeptidase from E. coli [4]. With exception of the putative D-amino acid oxidase in the DNA insert of clone 180, all of the ORFs identified are incomplete. Successive sequencing of the flanking regions by strategies such as inverse PCR will permit cloning or the development of gene inactivation systems for P. freudenreichii that can elucidate substrate specificity, role in peptide-utilization capacity, and cell physiology of the gene products identified in this study. The gene isolation method described could be applied to fragments obtained with different restriction enzymes to increase the number and type of available genome loci involved in amino-acid and peptide utilization by P. freudenreichii. Literature Cited 1. Boncompagni E, Dupont L, Mignot T, ØsterPs M, Lambert A, Poggi M-C, et al. (2000) Characterization of a Sinorhizobium meliloti ATP-binding cassette histidine transporter also involved in betaine and proline uptake. J Bacteriol 182:3717– 3725 2. Fernandez-Espla MD, Fox PF (1998) Effect of adding P. freudenreichii subsp. shermanii NCDO 853 or Lactobacillus casei ssp. casei IFPL 731 on proteolysis and flavor development of cheddar cheese. J Agric Food Chem 46:1228–1234 3. Gagnaire V, MollQ D, Sørhaug T, LQonil J (1999) Peptidases of dairy propionibacteria. Lait 79:43–57 4. Jozic D, Kaiser JT, Huber R, Bode W, Maskos K (2003) X-ray structure of isoaspartyl dipeptidase from E. coli: A dinuclear zinc peptidase evolved from amidohydrolases. J Mol Biol 332:243–56 5. Langsrud T, Reinbold GW, Hammond EG (1977) Proline production by Propionibacterium shermanii P59. J Dairy Sci 60:16– 23 6. Leenhouts K, Bolhuis A, Boot J, Deutz I, Toonen M, Venema G, et al. (1998) Cloning, expression and chromosomal stabilization of the Propionibacterium shermanii proline iminopeptidase gene (pip) for food-grade application in Lactococcus lactis. Appl Environ Microbiol 64:4736–4742 7. Lemee R, Lortal S, Cesselin B, van Heijenoort J (1994) Involvement of an N-acetylglucosaminidase in autolysis of Propionibacterium freudenreichii CNRZ 725. Appl Environ Microbiol 60:4351–4358 8. Margot P, Pagni M, Karamata D (1999) Bacillus subtilis 168 gene lytF encodes a gamma-D-glutamate-meso-diaminopimelate muropeptidase expressed by the alternative vegetative sigma factor, sigmaD. Microbiology 145:57–65 9. Mçrtl M, Diederichs K, Welte W, Molla G, Motteran L, Andriolo G, et al. (2004) Structure-function correlation in

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CURRENT MICROBIOLOGY Vol. 52 (2006) 15. Thierry A, Maillard MB, HervQ C, Richoux R, Lortal S (2004) Varied volatile compounds are produced by Propionibacterium freudenreichii in Emmental cheese. Food Chem 87:439–446 16. Thierry A, Maillard MB, Bonnarme P, Roussel E (2005) The addition of Propionibacterium freudenreichii to Raclette cheese induces biochemical changes and enhances flavor development. J Agric Food Chem 53:4157–4165 17. Thierry A, Maillard MB, Richoux R, Kerjean JR, Lortal S (2005) Propionibacterium freudenreichii strains quantitatively affect production of volatile compounds in Swiss cheese. Lait 85:57–74 18. Varmanen P, Savijoki K, Uvall S, Palva A, Tynkkynen S (2000) X-prolyl dipeptidyl aminopeptidase gene (pepX) is part of the glnRA operon in Lactobacillus rhamnosus. J Bacteriol 182:146– 154