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Applied and Environmental Microbiology, February 2006, p. 1402-1409, Vol. 72, No. 2
0099-2240/06/$08.00+0     doi:10.1128/AEM.72.2.1402-1409.2006
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Glutamate Dehydrogenase Activity Can Be Transmitted Naturally to Lactococcus lactis Strains To Stimulate Amino Acid Conversion to Aroma Compounds

Catherine Tanous,¹ Emilie Chambellon,¹ Dominique Le Bars,¹ Gilbert Delespaul,² and Mireille Yvon^1*

Institut National de la Recherche Agronomique, Unité de Biochimie et Structure des Protéines, 78352 Jouy en Josas, France,¹ R & D Fromageries Bel, Vendôme, France²

Received 15 September 2005/ Accepted 12 December 2005

Top Abstract Introduction Materials and Methods Results Discussion References

 
Amino acid conversion to aroma compounds by Lactococcus lactis is limited by the low production of -ketoglutarate that is necessary for the first step of conversion. Recently, glutamate dehydrogenase (GDH) activity that catalyzes the reversible glutamate deamination to -ketoglutarate was detected in L. lactis strains isolated from a vegetal source, and the gene responsible for the activity in L. lactis NCDO1867 was identified and characterized. The gene is located on a 70-kb plasmid also encoding cadmium resistance. In this study, gdh gene inactivation and overexpression confirmed the direct impact of GDH activity of L. lactis on amino acid catabolism in a reaction medium at pH 5.5, the pH of cheese. By using cadmium resistance as a selectable marker, the plasmid carrying gdh was naturally transmitted to another L. lactis strain by a mating procedure. The transfer conferred to the host strain GDH activity and the ability to catabolize amino acids in the presence of glutamate in the reaction medium. However, the plasmid appeared unstable in a strain also containing the protease lactose plasmid pLP712, indicating an incompatibility between these two plasmids.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Over the past few years, several groups have attempted to intensify or diversify the production of aroma compounds by stimulating the amino acid catabolism by lactic acid bacteria (LAB), which is a major route in the flavor formation in cheese and in other fermented food products (37). For example, the use as an adjunct of a Lactococcus lactis strain that overproduces cystathionine ß-lyase, an enzyme involved in the conversion of methionine into volatile sulfur compounds, induced an additional sulfur/thermophilic flavor to Gouda cheese (J. E. T. van Hylckama Vlieg, R. van Kranenburg, and P. Bruinenberg, Abstr. IDF Symp. Cheese, Prague, 2004). Also the overall intensification of amino acid catabolism in cheese or in fermented sausages, by addition of -ketoglutarate (-KG) in the products before maturation, highly stimulated aroma production (1, 15, 35). Indeed, in LAB, -KG production is a limiting factor for amino acid transamination, which initiates the amino acid conversion to aroma compounds (10, 38). This transamination reaction generates -ketoacids, which are then degraded through one or two additional steps into different aroma compounds such as aldehydes, alcohols, carboxylic acids, and sulfur compounds (37). Most of these compounds are produced by enzymatic degradation, but a few of them result from chemical degradation, in particular oxidation (Fig. 1) (20, 21). As an alternative to adding exogenous -KG to the cheese, the use of an LAB strain with glutamate dehydrogenase (GDH) activity, which catalyzes the reversible oxidative deamination of glutamate to -ketoglutarate, increased the amino acid conversion to aroma compounds. In fact, the importance of GDH activity in amino acid conversion to aroma compounds was revealed in a cheese model by using as a starter an L. lactis strain overexpressing the gene encoding the catabolic NAD-dependent GDH of Peptostreptococcus asaccharolyticus (25). Considering that this strain could not be used in food industry since it was a genetically modified organism, GDH activity has been searched for in natural LAB strains and found in several strains, including lactococci, mesophilic lactobacilli, and Streptococcus thermophilus (14, 18, 32). Interestingly, these GDH-positive strains were capable of degrading the amino acid precursor of aroma compounds in a medium containing glutamate, while strains without GDH activity did not. However, the relationship between GDH activity of LAB and their ability to convert amino acids to aroma compounds needs to be confirmed with isogenic strains with different levels of GDH activity and without GDH activity.

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FIG. 1. Phenylalanine catabolism pathways in Lactococcus lactis. -KG, -ketoglutarate; Glu, glutamate; AT, aminotransferases; HADH, hydroxy acid dehydrogenase; KADH, ketoacid dehydrogenase; KADC, ketoacid decarboxylase; Alc.DH, alcohol dehydrogenase; Ald.DH, aldehyde dehydrogenase; CoA, coenzyme A; Ox, chemical oxidation; –2C, loss of 2 carbons.

Recently, the gene responsible for GDH activity of an L. lactis strain isolated from peas was identified and characterized. This gene encodes a GDH that belongs to the family I of hexameric GDHs (2) and functions mainly in glutamate biosynthesis. The gdh gene is located on a large plasmid of about 70 kb and is part of a remnant transposon also containing functional genes (cadA and cadC) encoding cadmium resistance (30). This plasmid localization appears to be promising for transferring GDH activity into other L. lactis strains used in the dairy industry. Indeed, large plasmids of L. lactis are often self-transmissible by mating procedures, and such plasmids carrying industrially significant traits have been exploited in the construction of industrially useful transconjugants (11). Moreover, the presence of cadmium resistance genes on the plasmid provides a potential selectable marker for its transfer.

In this study, inactivation of the gdh gene in L. lactis NCDO1867 and construction of a strain with inducible expression of gdh at different levels confirmed the direct impact of L. lactis GDH activity on amino acid catabolism in vitro at pH 5.5, the pH of cheese. Moreover, we demonstrate that GDH activity and consequently the ability to catabolize amino acids could be transmitted to other L. lactis strains by natural transfer of the plasmid carrying the gdh gene.

Top Abstract Introduction Materials and Methods Results Discussion References

 
Bacterial strains, plasmids, and culture conditions.
The bacterial strains, constructs, and plasmids used in this study are listed in Table 1.

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TABLE 1. Strains and plasmids used in this study

Lactoccocus strains were grown at 30°C in M17 medium (Difco) (33) supplemented with 0.5% (wt/vol) glucose (M17-G) or lactose (M17-L), except for strain TIL487, which was grown at 37°C. For the overexpression experiments, an L. lactis strain (TIL488) harboring a plasmid with the gdh gene under the control of the nisin-inducible promoter (PnisA) was grown in M17-L to an optical density at 600 nm (OD₆₀₀) of 0.5 and induced with a final nisin concentration of 0 to 5 ng ml^–1 for 4 h. For growth experiments in milk, L. lactis strains NCDO1867 and TIL506 were cultivated at 30°C in 75 mM ß-glycerophosphate-buffered milk (reconstituted with NILAC milk powder [NIZO, Ede, The Netherlands] at 10% [wt/vol] in distilled sterilized water) supplemented or not supplemented with yeast extract (YE; Difco), respectively.

When necessary, erythromycin (Ery) and tetracycline (Tet) were added to the media at a final concentration of 5 µg ml^–1. Plate media were prepared by adding agar (Difco) to liquid media at a final concentration of 1.5% (wt/vol).

Preparation of cells for amino acid degradation and GDH activity determination.
For all experiments, the same cell preparations were used for both amino acid degradation tests and GDH activity determinations. Cells in the late exponential growth phase were harvested by centrifugation (4,100 x g for 15 min at 4°C), washed twice with 50 mM sodium ß-glycerophosphate buffer (pH 7.5), and suspended in the same buffer to an OD₄₈₀ of 200. Aliquots of cell suspensions were stored at –20°C until used. They were directly used for amino acid degradation tests, while they were pelleted by centrifugation and suspended in the same volume of 50 mM triethanolamine buffer (TEA; pH 7) for GDH activity determinations.

DNA techniques.
DNA restriction and modification enzymes were purchased from GIBCO-BRL (Cergy Pontoise, France), Eurogentec (Seraing, Belgium), or Boehringer (Mannheim, Germany) and used as recommended by the suppliers. The oligonucleotides used in this study were synthesized by Eurogentec and are listed in Table 2.

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TABLE 2. Oligonucleotides used in this study

L. lactis electrocompetent cells were prepared and transformed as described previously by Holo and Nes (16). Plasmid DNA was prepared according to the method of O'Sullivan and Klaenhammer (24). Southern blot hybridization was performed as described by the supplier of the ECL enhanced chemiluminescence kit (Amersham, Little Chalfont, Buckinghamshire, United Kingdom). The gdh probe was prepared by using the ECL kit from a PCR-amplified fragment of gdh which was obtained with primers 13F and 22R.

PCR amplifications were carried out on a Perkin-Elmer (Courtaboeuf, France) DNA thermal cycler 2400, using Taq polymerase (Qbiogen, Illkirch, France). Samples for sequencing were prepared with the PRISM Ready Reaction dye deoxy terminator cycle sequencing kit (Applied Biosystems, Courtaboeuf, France), and the sequences were determined with an automatic sequencer (370A DNA sequencer; Applied Biosystems).

Inactivation and overexpression of the gdh gene.
The construction of the gdh mutant (TIL487) was described previously (30). Briefly the gdh gene was disrupted in NCDO1867 by a single crossing over with the thermosensitive pGhost9 vector containing a 1-kb internal fragment of the 1,344-bp gdh gene.

The gdh gene was overexpressed by using the nisin-controlled expression (NICE) system (9), in which expression is induced by adding nisin (Sigma Chemicals, St. Louis, MO) to the culture medium. To do this, the gdh gene was cloned under the control of the nisin-inducible promoter (PnisA) in pMSP3545 containing nisR and nisK genes, which activate PnisA by nisin-mediated signal transduction (5). More precisely, a 1.41-kb DNA fragment containing the complete gdh gene was amplified from L. lactis NCDO1867 by using primers in which cloning sites were introduced at the start codon and downstream of the gene by PCR-mediated primer mutagenesis. An NcoI restriction site was introduced at the ATG start codon of the gene by using the primer Nis1bF, containing three substitutions generating the new NcoI site. Similarly, an SphI restriction site was introduced 30 bp downstream from the TAA stop codon of the gene by using the primer Nis2R, containing two substitutions generating the new SphI site (Table 2). The PCR product was cloned as an NcoI-SphI fragment in pMSP3545 digested with NcoI-SphI. The ligation mixture was used to transform electrocompetent cells of TIL46 and create TIL488. Transformed cells were selected on M17-L agar plates containing Ery at 5 µg ml^–1. A control strain (TIL339) was constructed by introducing pMSP3545 without the gdh gene into TIL46. Constructs were checked by PCR with specific primers of pMSP3545 (pMSP1F/pMSP2R) that amplify fragments of 2,042 bp or 686 bp with or without the gdh insert, respectively. Sequencing of the 2-kb fragment confirmed the integration site of the gdh gene in pMSP3545 at an ATG codon and the correct sequence of the integrated gdh gene.

Transfer of pGdh442 and pLP712 in L. lactis MG1363.
pGdh442 was transferred in the plasmid-free strain L. lactis MG1363, generating TIL504. The transfer was performed in two steps. First, the plasmid DNA preparation of the mutant strain (TIL487) was used to transfer by electroporation pGdh442 plasmid, in which pGhost9 (Ery^r) was inserted, into L. lactis strain MG1363. Then pGhost9 was excised from one transformant containing only the plasmid of interest according to the protocol developed by Biswas et al. (3) to restore the initial plasmid pGdh442. Colonies in which pGhost9 was excised were Ery sensitive, and the gdh integrity was verified by PCR (oligonucleotides 37F and 38R) and sequencing of amplified fragments.

To obtain the L. lactis strain (TIL506) harboring both pGdh442 and a protease/lactose (pLP712) plasmid, L. lactis TIL504 electrocompetent cells were electroporated with the pLP712 isolated from L. lactis TIL672 (7). Transformants were selected by plating on fast strain differencing agar (FSDA), which selects clones capable of utilizing casein and lactose (17), containing 0.1 mM CdCl₂. The positive clones were checked for their plasmid content by plasmid DNA preparation and PCR amplifications with appropriate oligonucleotides derived from sequences of gdh (13F/22R) and prtP (prt1F/prt2R).

Conjugative transfer of pGdh442.
Transfer of pGdh442 by conjugation was tested by using L. lactis TIL504 containing pGdh442 and L. lactis TIL193 (MG1363 containing pGhost8 Tet^r) as donor and recipient strains, respectively. TIL504 and TIL193 were cultivated separately overnight with the appropriate resistance selection: i.e., cadmium (Cd; 0.1 mM) and Tet (5 µg ml^–1), respectively. Cells were mixed in a donor/recipient ratio of 1:1, washed twice with a 0.85% (wt/vol) NaCl solution, and suspended in 100 µl of M17-G. The mixture was plated on M17-G and incubated at 30°C for 7 or 14 h. Cells on plates were suspended in 5 ml of 10% M17-G solution, and serial dilutions were then plated onto media that selected for the donor (M17-G containing Cd) and transconjugant (M17-G containing Cd and Tet). The transfer frequency was calculated as the number of transconjugants per recipient cell. The plasmid content of one selected transconjugant (TIL507) was verified by a plasmid DNA preparation which was used as template for Southern blot hibridization with the gdh probe and by PCR amplifications with oligonucleotides from sequences of gdh (37F/38R) and pGhost8 (P91/P96).

Growth inhibition assays.
The cadmium and zinc sensitivity of L. lactis strains was evaluated in M17-G medium containing 0, 0.05, 0.1, 0.2, 0.3, 0.4, and 0.5 mM cadmium chloride (CdCl₂; Sigma) and 0, 5, 10, 20, 30, and 40 mM zinc chloride (ZnCl₂; Sigma). Different media were inoculated with exponentially growing cultures and then incubated at 30°C. The growth was assessed by measuring the absorbance at 480 nm after incubation for 16 h. Results are expressed as the MIC (i.e., the minimal concentration of CdCl₂ or ZnCl₂ which inhibited by 90% the growth of strains).

Plasmid stability tests.
The stability of pGdh442 in the different strains was assessed by two methods. The first consisted of the determination of the percentage of the total population resistant to cadmium chloride after growth for 10, 30, and 60 generations in M17 (or in milk for TIL506). The strains were inoculated in M17-G or in buffered milk and grown for 12 h. Then they were diluted (1%) in fresh medium and incubated for another 12 h. Under these conditions, each phase of growth corresponded to approximately 10 generations. At the initial inoculation step (i.e., generation 0) and after 10, 30, and 60 generations of growth, the cultures were serially plated on both M17-G medium and M17-G medium containing 0.1 mM or 0.05 mM cadmium chloride (CdCl₂).

The second method consisted of determination of the presence or absence of pGdh442 (and pLP712 for TIL506), after growth for 6 generations in M17, by PCR on 50 isolated colonies with oligonucleotides specific for gdh (13F and 22R) and prtP (prt1F and prt2R). For each strain, the M17 culture was inoculated from a colony isolated on plate containing the selective medium to maintain the plasmids (M17 plus CdCl₂ for TIL504 and NCDO1867 and FSDA plus CdCl₂ for TIL506).

Determination of GDH activity.
GDH activity was determined in cell extracts (CEs) prepared as previously described (38). Briefly, cells from 100 ml of culture at late log phase were harvested by centrifugation (4,100 x g for 15 min at 4°C) and washed twice with 50 mM TEA. The pellet was resuspended in 5 ml of TEA containing 1.6 mg ml^–1 of lysozyme and 0.2 mg ml^–1 of saccharose and incubated at 30°C for 2 h. The spheroplast pellet was harvested by centrifugation (4,100 x g for 15 min at 4°C) and resuspended in hypotonic buffer TEA to provoke spheroplast lysis. Cell debris was eliminated by centrifugation at 20,000 x g for30 min. The supernatant was filtered through a 0.45-µm-pore-size filter (Millipore Corporation, Bedford, Mass.) and was used as the CE. The protein concentrations of CEs were determined by the Bradford method (4) by using bovine serum albumin as the standard. NAD- and NADP-dependent GDH activities were determined in fresh cell extracts by using a test based on the colorimetric glutamic acid assay of Boehringer, as previously described (18). Quantification was made with calibration curves established with pure NADH and NADPH. One unit of GDH activity corresponds to the production of 1 nmol of NAD(P)H per min of reaction. The results are the mean of three determinations (± the standard deviation).

Amino acid catabolism in vitro.
Phenylalanine (Phe), glutamate (Glu), pyridoxal phosphate, and standard amino acid metabolites were obtained from Sigma.

Amino acid catabolism by different strains was studied as previously described (26) by incubating resting cells in a reaction medium containing L-[2,6-3H]phenylalanine (60 Ci mmol^–1) as a tracer (Isotopchim, Peyruis, France). The reaction mixture contained 100 mM phosphate buffer, pH 5.5, 2 mM unlabeled Phe, 0.05 µM radiolabeled Phe, 0.05 mM pyridoxal phosphate, and 10 mM glutamate (Glu). Fifty microliters of a cell suspension corresponding to an OD₄₈₀ of 200 was added to 450 µl of reaction mixture and incubated for 40 h at 37°C. Aliquots (100 µl) of reaction mixture were taken after 0, 20, and 40 h of incubation, and cells were removed by centrifugation (5,000 x g, 5 min). Metabolites formed were then analyzed by reverse-phase high-performance liquid chromatography with both UV detection (214 nm) and radioactivity detection as previously described (38). The data reported are the means of three determinations (±the standard deviation).

Top Abstract Introduction Materials and Methods Results Discussion References

 
Impact of gdh inactivation and overexpression on GDH activity.
As previously described (30), gdh inactivation in NCDO1867 led to a total loss of GDH activity (Fig. 2A), indicating that only this gene was responsible for NAD- and NADP-dependent GDH activities of the strain.

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FIG. 2. Effect of gdh inactivation and overexpression on NAD- and NADP-dependent GDH activities (A) and in vitro Phe degradation in the presence of Glu at pH 5.5 (B). WT, wild-type strain (NCDO1867), with the growth medium indicated in parentheses; GDH–, gdh mutant strain (TIL487); GDH++, gdh-overexpressing strain (TIL488) induced with different levels of nisin indicated in parentheses. Error bars indicate standard deviations of triplicate determinations.

The gdh gene from NCDO1867 was also overexpressed with the NICE system in L. lactis TIL46, which does not exhibit GDH activity. Overexpression was induced with increasing concentrations of nisin. The maximum level of GDH activity was reached in cells induced with 2.5 ng ml^–1 of nisin (Fig. 2A), while the noninduced cells as well as the nisin-induced cells of TIL339 (containing pMSP3545 without gdh gene) did not exhibit any activity (data not shown). The optimal overexpression led to a GDH activity about 20-fold higher than the activity of the wild-type strain, NCDO1867. Both NAD- and NADP-dependent GDH activities were recovered in the overexpressing strain, confirming that the gdh gene encodes a dually specific GDH which is more active with NADP.

Besides the impact of the gdh-controlled overexpression on GDH activity, we observed that NCDO1867 grown in milk complemented with yeast extract exhibited a 2.5-fold-higher GDH activity than the strain grown in M17-G (Fig. 2A).

Impact of gdh inactivation and overexpression on Phe catabolism at pH 5.5.
The impact of gdh inactivation and overexpression on Phe catabolism was studied by incubating resting cells in a reaction medium at pH 5.5 containing radiolabeled Phe as tracer and glutamate. The cells were the same as those used for GDH activity determination. We chose to present in Fig. 2B the results obtained after incubation for 40 h since the Phe degradation still increased between 20 and 40 h. First, the wild-type strain, NCDO1867, degraded about 12% of Phe at pH 5.5, while less than 2% degradation was observed with the gdh mutant strain (TIL487), indicating that the lack of GDH activity in NCDO1867 results in the loss of ability to degrade amino acids (Fig. 2B). NCDO1867 produced from Phe noticeable quantities of aroma compounds such as phenylacetaldehyde, phenylethanol, and phenylacetate. These productions indicate that NCDO1867 possesses an -ketoacid decarboxylase (-KADC) activity that transforms the -ketoacid resulting from Phe transamination (phenylpyruvate) to phenylacetaldehyde, which is further reduced to phenylethanol by an alcohol dehydrogenase (Fig. 1) (8, 29). The production of phenylacetate is due to an -ketoacid dehydrogenase activity that is common in L. lactis (28). Second, the overexpressing strain, TIL488, in which gdh expression was induced with increasing concentrations of nisin, was capable of degrading Phe up to 29% (Fig. 2B), while the noninduced strain as well as the control strain, TIL339, did not catabolize Phe (data not shown). The maximum level of degradation (around 30%) was reached by cells induced with 2.5 ng ml^–1 of nisin. The overexpressing strain mainly produced phenylacetate, phenyllactate, and benzaldehyde from Phe and did not produce phenylacetaldehyde and phenylethanol, contrary to NCDO1867.

As previously observed for GDH activity, L. lactis NCDO1867 grown in milk-YE degraded Phe 2.5-fold more than NCDO1867 grown in M17-G (Fig. 2B). In addition to the metabolites produced by the cells grown in M17-G, cells grown in milk produced phenyllactate, which is the reduction product of phenylpyruvate.

Natural transfer of pGdh442 using cadmium resistance as a selectable marker.
Previously we demonstrated that gdh was located on a large plasmid of about 70 kb (pGdh442) and was part of a remnant transposon also containing functional genes encoding Cd resistance (30) that could be used as tool to detect the presence of the plasmid. We tried to transfer pGdh442 into another L. lactis strain naturally by a mating procedure. We used L. lactis strain TIL504, an MG1363 derivative strain containing only pGdh442, as the donor strain, and L. lactis TIL193, an MG1363 derivative strain containing pGhost8 with Tet^r, as the recipient strain. Conjugant clones were selected on M17-G supplemented with Tet (5 µg ml^–1) and CdCl₂ (0.1 mM). Conjugants were found at a frequency of 1 x 10^–7 per recipient cell. These conjugants were verified by PCR amplifications with specific gdh and pGhost8 oligonucleotides and by Southern hybridization analysis of plasmid DNA with the gdh gene as a probe. These analyses revealed that both gdh and pGhost8 genes were present in the conjugants and that the conjugants contained the whole plasmid. The conjugant was named TIL507. The transfer of the whole plasmid pGdh442 by mating suggests that the plasmid contains conjugative elements or was mobilized by the chromosomal sex factor of MG1363 (13).

Stability of pGdh442 in the different strains.
We assessed pGdh442 stability in the wild-type strain, NCDO1867, and in two L. lactis derivative strains of MG1363, one containing both plasmids pGdh442 and pLP712 (which is a protease/lactose plasmid), TIL506, and the other containing only pGdh442, TIL504. First, the stability of pGdh442 was determined during growth in M17-G or milk by counting the percentage of the population exhibiting cadmium resistance. Only 10% ± 5% of TIL506 colonies were cadmium resistant after M17-G growth for 10 generations, while 100% ± 15% of TIL504 or NCDO1867 colonies were resistant after growth for 60 generations. After growth of TIL506 on milk for 10 generations, no colony was resistant. Second, we looked for the presence of pGdh442 by PCR on 50 colonies of the different strains isolated from M17-G cultures corresponding to about 6 generations. The presence of pLP712 was also determined in colonies of TIL506. In the M17-G culture of TIL506, only 16% of the colonies contained both plasmids pGdh442 and pLP712, 16% contained only pGdh442, and 68% contained only pLP712. The stability of pGdh442 in NCDO1867 and TIL504 was confirmed since 100% of the colonies contained pGdh442 after growth in M17 for 6 generations.

Impact of pGdh442 in L. lactis strains containing or not containing pLP712 on Cd/Zn resistance, GDH activity, and amino acid catabolism.
In pGdh442, gdh is part of a remnant transposon also containing functional genes (cadA and cadC) encoding cadmium resistance (30). Generally, cadA/cadC genes also code for zinc resistance (23). Therefore, cadmium and zinc resistance, GDH activity, and the capacity to degrade the Phe of strains in which pGdh442 was transferred were compared to the same properties of the pGdh442 donor strain, NCDO1867, and the plasmid recipient strains, MG1363, TIL193, and TIL672.

The CdCl₂ and ZnCl₂ MICs of the three strains TIL504, TIL506, and TIL507 in which pGdh442 was transferred were higher than those of the control strains MG1363, TIL193, and TIL672 without pGdh442 (Table 3), confirming the role of pGdh442 in cadmium and zinc resistance. The CdCl₂ and ZnCl₂ MICs of the three strains in which pGdh442 was transferred were a little lower than those of NCDO1867, suggesting the presence of other resistance genes in NCDO1867.

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TABLE 3. Cadmium and zinc resistance of L. lactis strains harboring pGdh442 and control strains without pGdh442

The GDH activity of the different strains was determined after cultures in the presence or the absence of CdCl₂ (Fig. 3A). After growth in medium containing 0.1 mM CdCl₂, all strains containing pGdh442 exhibited approximately the same GDH activity as NCDO1867, while no GDH activity was detected in the control strains. However, the level of GDH activity in cells grown in the absence of CdCl₂ was about 1.5-fold lower than that for NCDO1867, twice as low as that for TIL504 and TIL507, and close to zero for TIL506. Also, no activity was detected in TIL506 grown in milk (data not shown). The absence of activity in TIL506 grown in M17 (or in milk) is in agreement with the rapid loss of pGdh442 after growth in M17. On the contrary, the higher GDH activity of TIL504, TIL507, and NCDO1867, in which pGdh442 is stable, in cells grown in the presence of cadmium may be due to a stimulation of GDH activity by CdCl₂, as was previously reported for GDH of tomato plants under cadmium stress (6).

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FIG. 3. Effect of the transfer of pGdh442 in derivative strains of L. lactis MG1363 on NAD- and NADP-dependent GDH activities (A) and in vitro Phe degradation in the presence of Glu at pH 5.5 (B). NCDO1867, wild-type strain; TIL504, MG1363 containing pGdh442; TIL507, transconjugant obtained from TIL193 with pGdh442; TIL506, MG1363 containing pGdh442 and pLP712; MG1363, TIL193, and TIL672, control strains without pGdh442. The presence or absence of cadmium in the growth medium is indicated by Cd+ and Cd–, respectively. Error bars indicate standard deviations of triplicate determinations.

Considering that the presence of CdCl₂ in growth medium seemed to be necessary to maintain pGdh442 in certain strains, we investigated Phe catabolism by the three strains TIL504, TIL506, and TIL507 after growth in the presence or the absence of CdCl₂. Almost no degradation was observed with all strains grown in the presence of CdCl₂ (data not shown), indicating that CdCl₂ probably inhibited enzymes involved in amino acid catabolism, as previously observed in Staphylococcus aureus (34).

After growth in the absence of CdCl₂, TIL504 and TIL507 were capable of degrading Phe to a lower extent than NCDO1867 but more than the original strains MG1363 and TIL193 (Fig. 3B), confirming that the transfer of pGdh442 conferred to L. lactis strains the capacity to degrade Phe. TIL506 only slightly degraded Phe because of the loss of pGdh442.

Top Abstract Introduction Materials and Methods Results Discussion References

 
GDH appears to be a key enzyme in the production of aroma compounds from amino acids by LAB since it is the one enzyme capable of producing -ketoglutarate for amino acid transamination (31). Previously, a few L. lactis strains exhibiting GDH activity had been found, and the gdh gene encoding this activity was identified and localized on a plasmid (30, 32). The present study shows that, although the gdh gene of L. lactis encodes a GDH mainly active in glutamate biosynthesis, it is responsible for the capacity of L. lactis strains to catabolize amino acids to aroma compounds. In the wild-type strain, NCDO1867, the expression of the gdh gene seems to be regulated by nutritional effectors since cells grown in milk-YE exhibited 2.5-fold-higher GDH activity than cells grown in M17-G. Similar to the gene overexpression with the NICE system, this natural induction enhanced Phe catabolism. Although an increase in the level of GDH activity leads to an increase in the level of Phe catabolism, the two parameters are not proportional. In fact, the two situations for Phe catabolism and GDH activity measurements are totally different since Phe catabolism is determined with whole cells while GDH activity is determined in cell extracts with substrate and cofactor in excess. But interestingly, even a small increase in GDH activity highly stimulated Phe catabolism.

Whereas the level of amino acid degradation by L. lactis is related to the level of GDH activity of the strains, the nature of the metabolites produced depends on the enzyme content of each GDH-positive strain. Indeed, L. lactis strains NCDO1867 and TIL488 produced different metabolites because TIL488 does not possess the -KADC activity that converts -ketoacids to aldehydes in NCDO1867 (Fig. 1). But the two strains produced phenyllactate and phenylacetate since both strains possess activities that are responsible for reduction and oxidative decarboxylation of phenylpyruvate. These results clearly show the interest in transferring GDH activity into other L. lactis strains exhibiting interesting catabolic activities.

The location of gdh on a large plasmid suggested that this gene might be naturally transmitted to other strains. Our mating experiments show that, actually, pGdh442 can be naturally transmitted to other L. lactis strain by using cadmium resistance as a selectable marker. The genes encoding cadmium resistance also confer to the strain zinc resistance that could also be used as a selectable marker. Contrary to plasmids carrying antibiotic resistance, plasmids encoding heavy metal resistance are safe for utilization in the food industry. pGdh442 that had been transferred by electroporation (TIL504) or by conjugation (TIL507) in the L. lactis plasmid-free strain MG1363 or in its derivative TIL193 strain containing pGhost8 appeared to be stable after several subcultures of the strains without selection pressure. However, in the presence of the protease/lactose plasmid pLP712 isolated from L. lactis NCDO763, pGdh442 was unstable. Due to the incompatibility between these two plasmids, only the plasmid necessary for growth is maintained. The incompatibility may be due to the same origin of replication in both plasmids. Therefore, to study the impact of pGdh442 in the strain growing in milk, it will be necessary to transfer pGdh442 in an L. lactis strain that contains the genes coding for lactose and casein utilization in chromosomal DNA, such as in the strains described by Nissen-Meyer et al. (22) and Leenhouts et al. (19), or carried by a plasmid with a replication origin different from that of pGdh442.

The results obtained in this work provide new prospects to intensify and diversify the production of aroma compounds in cheese. First, wild-type GDH-positive strains can be used, and identification of effectors that naturally increase gdh expression should permit intensification of their ability to catabolize amino acids. Considerable amino acid catabolism intensification can also be obtained by using food-grade vectors for gdh overexpression in different strains. Finally, pGdh442 transfer to other L. lactis strains that exhibit varied profiles of amino acid catabolism appears to be an interesting way to diversify aroma production, even if the recipient strain is not capable of growing in milk alone. Indeed, pGdh442 also contains the oppABCDF and pepO genes (unpublished data), which allow utilization of peptides resulting from casein degradation. Therefore, the strains containing pGdh442 should be capable of growing in milk when associated with protease-positive strains and could be used as adjunct cultures. The interest in L. lactis strains as adjunct cultures is linked to their varied profiles of amino acid catabolism. They possess a high level of aminotransferase activities with different specificities toward aromatic amino acids (ArAA) and branched-chain amino acids (BcAA) resulting in a large diversity of metabolites produced. ArAA are mainly precursors of floral aromas, while BcAA mainly produce malty or strong cheese aromas (27, 36, 38). Moreover, some L. lactis strains have -KADC activity that transforms -ketoacids to various aroma compounds such as aldehydes and alcohols that are rarely produced by Lactobacillus, another group of species used as an adjunct (29). However, all of these proposed ways to modify naturally the amino acid catabolism should be further tested in cheese trials.

 
This work was funded by a Eureka research grant (E!2536). We are also grateful to Danisco and the Fromageries Bel for their financial support.

We would like to thank Philippe Horvath, Anne-Marie Sépulchre, and Véronique Monnet for their critical reading of the manuscript; Christine Mezange for technical assistance; and G. M. Dunny, who kindly provided the expression vector inducible with nisin, pMSP3545.

 
* Corresponding author. Mailing address: INRA, Unité de Biochimie et Structure des Protéines, 78352 Jouy en Josas, France. Phone: 33 1 34 65 21 59. Fax: 33 1 34 65 21 63. E-mail: mireille.yvon{at}jouy.inra.fr.

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Applied and Environmental Microbiology, February 2006, p. 1402-1409, Vol. 72, No. 2
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This article has been cited by other articles:

• Tanous, C., Chambellon, E., Yvon, M. (2007). Sequence analysis of the mobilizable lactococcal plasmid pGdh442 encoding glutamate dehydrogenase activity. Microbiology 153: 1664-1675 [Abstract] [Full Text]  

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