Urease biogenesis in Streptococcus thermophilus

Urease biogenesis in Streptococcus thermophilus

Research in Microbiology 156 (2005) 897–903 www.elsevier.com/locate/resmic Urease biogenesis in Streptococcus thermophilus Diego Mora a,∗ , Christoph...

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Research in Microbiology 156 (2005) 897–903 www.elsevier.com/locate/resmic

Urease biogenesis in Streptococcus thermophilus Diego Mora a,∗ , Christophe Monnet b , Carlo Parini a , Simone Guglielmetti a , Andrea Mariani a , Paola Pintus a , Francesco Molinari a , Daniele Daffonchio a , Pier Luigi Manachini a a Dipartimento di Scienze e Tecnologie Alimentari e Microbiologiche, Università degli Studi di Milano, via Celoria 2, 20133 Milan, Italy b Unité Mixte de Recherche Génie et Microbiologie des Procédés Alimentaires, Institut National de la Recherche Agronomique,

78850 Thiverval-Grignon, France Received 21 January 2005; accepted 26 April 2005 Available online 17 June 2005

Abstract Urease biogenesis was monitored in the lactic acid bacterium Streptococcus thermophilus during the growth cycle using in-gel detection and a phenol-hypochloride assay. Zymogram analysis, performed in a non-denaturing polyacrylamide gel, enabled visualization of a complex profile of bands whose number and intensity were dependent on the growth phase and culture conditions. The monitoring of urease biogenesis in batch fermentations revealed the onset of enzyme synthesis starting from the mid-exponential growth phase, with a maximum reached during the late exponential phase. Urease activity strongly increased at acidic pH but to a lesser extent when urea and nickel ions were added to the culture medium. When S. thermophilus cells were cultured with pH maintained at a neutral value, urease activity was detectable only in gel with extremely low signals. Evaluation of β-glucuronidase activity in strain DSM 20617T harboring a transcriptional fusion between a DNA fragment containing the putative urease promoter and the gusA reporter evidenced significant expression at neutral pH that strongly increased in an acidic environment. Further experiments carried out on pureI –gusA recombinant strain revealed that expression of ure genes was not affected by carbohydrates, nickel or urea availability. The presence of consistent expression of ure genes at neutral pH and the absence of induction of expression by carbohydrate availability demonstrated that the transcription of ure genes in S. thermophilus is regulated differently compared with that of the closely related S. salivarius. These differences are discussed taking into consideration the different habitats colonized by the two bacterial species.  2005 Elsevier SAS. All rights reserved. Keywords: Urease; In-gel detection; Expression; gusA-reporter gene; Streptococcus thermophilus

1. Introduction One of the main roles of Streptococcus thermophilus in milk fermentation is to provide rapid acidification by producing lactic acid from disaccharide lactose. Lactic acid plays an important role in milk coagulation and curd draining, imparting a fresh acidic flavor to fermented milk while helping to suppress the growth of pathogens and spoilage microorganisms. For these reasons, the rate of acidification is an important technological feature, since a delay in the time course of acidification may have dramatic effects upon * Corresponding author.

E-mail address: [email protected] (D. Mora). 0923-2508/$ – see front matter  2005 Elsevier SAS. All rights reserved. doi:10.1016/j.resmic.2005.04.005

the quality of the product or economic consequences for organization of the industrial process. The rate of acidification is a strain-dependent metabolic trait and is influenced in S. thermophilus by ureolytic activity [10,12]. Among lactic acid bacteria involved in dairy fermentation processes, urease activity is present and widely distributed only in S. thermophilus [11,20,21]. Urease (urea amidohydrolase, EC catalyzes the hydrolysis of urea to yield ammonia and carbamate, which spontaneously decompose to yield a second molecule of ammonia and carbonic acid. These reactions affect the rate of decrease in pH by neutralization of lactic acid production during the fermentation process with varying intensity depending on the urea concentration in milk [7,12,16]. Despite the importance of ure-


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ase in milk fermentation processes, this enzymatic activity has been poorly investigated in S. thermophilus and no information is available concerning the physiological role and the biogenesis of urease. Detailed molecular studies to determine the basis of regulated ure gene expression has been confined to only a few organisms [8]. In several microorganisms, urease gene expression is differentially regulated and influenced by the urea concentration in the medium and/or by the availability of nitrogen sources [8]. A novel form of urease gene regulation has been described in the oral bacterium Streptococcus salivarius [3–5]. In this organism, urea or nitrogen availability does not significantly affect urease expression, whereas growth at neutral pH results in nearly complete repression of transcription [3,4]. The characterization of urease gene clusters in S. thermophilus, whose closest phylogenetic neighbor is S. salivarius, has been recently described [12]. The sequence and transcription analysis of the urease cluster in S. thermophilus revealed the presence of eight open reading frames starting from ureI (putative membrane urea transporter gene) followed by ureABC (structural genes) and ureEFCD (accessory genes) with an operon organization [12]. The objective of the present study was the characterization of urease biogenesis in S. thermophilus by exploring the presence of differential expression of the ure operon in response to different culture conditions.

2. Materials and methods 2.1. Bacterial strains, media, growth conditions and reagents Wild-type Ure+ S. thermophilus DSM 20617T and the Ure− derivative A16(ureC3) were maintained in M17 broth [18], 2% (w/v) lactose, at 37 ◦ C. S. thermophilus strains harboring pNZ273 and pMI200 were maintained in M17 broth supplemented with 10 µg of chloramphenicol per ml. Escherichia coli VE7108 [12] strains harboring pNZ273 and pMI200 were routinely maintained at 37 ◦ C with aeration in Luria broth supplemented with 20 µg of chloramphenicol per ml. Urease biogenesis was evaluated in S. thermophilus strains grown in a PreludeTM fermenter (Pierre Guerin Technologies, France) with a working volume of 2 l in M17 medium at 37 ◦ C inoculated at 1% (v/v). The batch fermentation process was monitored evaluating pH and OD600 nm . When necessary, the batch fermentation process was set to operate at pH 6 or 7 by automatic addition of HCl (2 N) or NaOH (2 N). At different time points, cells from 100–200 ml of culture were harvested and total cellular proteins were extracted. 2.2. Extraction of cellular proteins Total bacterial proteins were extracted from cells harvested by centrifugation from 200 ml of M17 culture grown at 37 ◦ C for 12 h. Cells were washed twice in 50 mM sodium

phospate buffer (pH 7.5) and resuspended in 3 ml of the same buffer. Dithiothreitol (DTT) was added to the cell suspension at a final concentration of 20 mM. Cell disruption was then carried out in a French Press (SLM Instrument, Rochester, NY) and the resulting cellular extract was centrifuged at 20 000 g for 30 min. The supernatant was concentrated approximately fivefold using a centrifugal filter device, Microcon YM50 (50 000 Da nominal molecular weight limit) (Millipore Corporation, Bedford, MA) at 4 ◦ C. During concentration, the protein solution was washed with 50 mM potassium phosphate buffer (pH 7.5). Total protein was evaluated using the Bradford method [1]. 2.3. In-gel detection of urease activity The detection of urease activity in polyacrylamide gel was modified for bacteria, from the protocol developed for plant tissue by Witte and Medina-Escobar [19], as follows. Electrophoresis was carried out using 10 µg of total cell protein extract with a 7% polyacrylamide gel under nondenaturing conditions in a Mini-Protean III system apparatus (BioRad, Milano, Italy). Electrophoresis was performed at 100 V in a Tris–glycine buffer system containing Tris– HCl (25 mM) and glycine (250 mM) at pH 8.8. Thioglycolic acid was added to the cathodic running buffer at a final concentration of 100 µM. After electrophoresis, the gel was washed three times in cold acetate buffer (5 µM) and once in bidistilled water. The gel was then covered with 25 ml of a freshly prepared staining solution containing urea (0.10 M), p-nitroblue tetrazolium (Sigma–Aldrich, Milano, Italy) (0.08%), DTT (0.5 mM) and incubated at 37 ◦ C, from 1 to 12 h, until visualization of dark blue bands. After staining, the reaction was stopped by a 5 min incubation in HCl (20 mM). A gel image was then captured using a digital camera (CoolPix 990, Nikon, Nital S.p.A., Torino, Italy). 2.4. Evaluation of urease activity by the phenol hypochloride assay The assay was carried out by the quantitative determination of ammonium ions produced by urea. The assay was performed as described by Witte and Medina-Escobar [19], with some modifications, in a 200 µl volume containing: 10– 100 µg of cell protein extraction, 50 mM sodium phospate buffer (pH 7.5) and 50 mM urea. The tubes containing the reaction mixture were gently mixed and placed in a water bath at 37 ◦ C. At each time point, 20 µl of reaction sample were rapidly pipetted into 980 µl of distilled water, 200 µl of hypochloride reagent (NaOH, 370 mM; Na2 HPO4 , 80 mM; NaOCl, 13 mM, pH 12) and 100 µl of phenol nitroprusside reagent (Sigma–Aldrich). After addition of the phenol nitroprusside reagent, the tubes were inverted several times, incubated at 37 ◦ C for 30 min until the endpoint of color development. The amount of ammonium ions was calculated evaluating the OD at 636 nm and using standard solutions

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prepared with different concentrations of ammonium chloride. The urease reaction was monitored every 5–10 min for 30–40 min, sampling at least four times. One unit of urease activity was defined as the activity that releases one µmol of NH+ 4 per min. 2.5. Construction of pMI200 for analysis of the ure gene promoter region A 219-bp region located upstream from the ureI gene and containing the putative pureI promoter was amplified by PCR using primers 5 -TTATTACAGCTGTATCTGGGATTGAGCAAAGG-3 and 5 -TTATTACTGCAGCTAAGTAGGATGACACCTAACAT-3 with built-in PvuII and PstI sites (underlined), respectively. The 219-bp product was cloned into a PvuII-PstI-digested pNZ273 vector generating pMI200. The pNZ273 vector contains the promoterless gusA reporter gene coding for the β-glucuronidase enzyme [17]. The integrity of the amplified promoter region was confirmed by sequence analysis. The construct was initially made in E. coli VE7108 and subsequently used to transform S. thermophilus DSM 20617T as previously described [12]. The recombinant clones were selected on M17 agar containing chloramphenicol. Histochemical screening for β-glucuronidase activity by selecting for blue colonies with 5-bromo-4-chloro-3-indolyl-β-D-glucuronide (X-Gluc) (VWR International, Milano, Italy) was performed as described by Platteeuw and colleagues [17]. 2.6. β-glucuronidase assay Recombinant S. thermophilus strains were grown in M17 broth supplemented with 75 mM sodium phosphate buffer (pH 7) or in M17 adjusted to pH 6 by addition of HCl (2 N) until an OD600 nm value of 0.5. Cells were harvested by centrifugation, washed once with GusA buffer (10 mM β-mercaptoethanol, 1 mM EDTA, 0.1% Triton X-100 in 50 mM sodium phosphate buffer at pH 7) and then resuspended in 300 µl of the same buffer. Concentrated cell suspensions were subjected to mechanical disruption in the presence of 100 µl of glass beads (0.1 mm diameter) by homogenization for 3 min at 4 ◦ C. The amount of total protein of each lysate was measured using the Bradford method [1] with BSA as the standard. For the determination of β-glucuronidase activity, 50–100 µl of protein extract was added to 900–950 µl of GusA buffer containing 1.25 mM para-nitro-β-D-glucuronic acid (VWR International, Milano, Italy). Measurement of the β-glucuronidase activity was performed at 37 ◦ C, by monitoring the optical density at 420 nm with a microplatereader M680 (Bio-Rad Laboratories, Hercules, CA, USA) programmed for a reading set of 60 repetitions with intervals of 30 s. The β-glucuronidase activity was expressed in mOD420 nm per min per milligram of protein, as the mean of four independent determinations.


3. Results 3.1. In-gel detection of urease activity The detection of urease activity of S. thermophilus in native PAGE allowed the visualization of a complex enzymatic profile composed of several ureolytic signals, whose number was dependent on the culture conditions adopted. S. thermophilus DSM 20617T showed three activity signals designated a, b and c (Fig. 1). No additional signals were detected when the staining step was prolonged for more than 6 h. The activity band a was detectable only when cells were collected in the late exponential growth phase or when the culture broth was supplemented with Ni ions or urea at a final concentration of 10 µM and 0.5 g l−1 , respectively. In order to investigate the nature of the molecular species showing ureolytic activity in the zymogram, the total protein cell extract of the urease negative mutant A16, obtained from strain DSM 20617T by allelic exchange of the ureC gene with an in-frame deleted version ureC3 [12], was analyzed. The amount of total protein loaded on the native gel for wild-type and mutant samples was 10 µg, based on the Bradford method. As shown in Fig. 1B, no activity signals were visualized from the urease-negative mutant on native PAGE even after prolonging the staining step for 12 h at 37 ◦ C. These results demonstrated that UreC was necessary for the formation of the protein bands with ureolytic activity and that other enzymatic activities present in the total cell extract did not interfere with the NBT staining procedure. The presence of several activity signals in the zymogram analysis of S. thermophilus (Fig. 1) may be explained by the co-existence of several quaternary structures of the three main urease subunits α, β and γ (UreC, UreB and UreA)

Fig. 1. (A) In-gel detection of urease activity in the total cell protein extract of S. thermophilus DSM 20617T . Urease profiles obtained from cells grown for 18–24 h in M17 broth at 37 ◦ C, lane 1; cells harvested in the late exponential growth phase, after 6–8 h of incubation, lane 2. Ureolytic bands a, b, and c are shown. (B) In-gel detection of urease activity in the total cell protein extract of S. thermophilus strains grown for 18–24 h in M17 broth with NiCl2 at a final concentration of 10 µM. Lane 1, DSM 20617T wild type; lane 2, A16(∆ureC3).


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Fig. 2. Urease biogenesis by S. thermophilus DSM 20167T . (A) Bacterial growth as OD600 nm (white symbols) and culture pH (black symbols) in M17 broth, 37 ◦ C (1, 2), in presence of 0.5 g l−1 of urea (!, ") and with pH maintained at 6 (P, Q). In-gel detection of urease activity of cells grown in M17 (B), in M17 with urea 0.5 g l−1 (C) and with pH maintained at 6 (D). Zymogram was performed on total protein extract obtained from cells harvested at OD600 nm shown at the top of the figures. The same protein extracts were subjected to evaluation of urease activity by the phenol-hypochloride assay (E). Protein extracts from cells grown in standard M17 (2), in M17 with urea 0.5 g l−1 (") and with pH maintained at 6 (Q).

such as trimers, and trimers of trimers. Moreover, multiple activity signals may be interpreted as molecular complexes between urease and some accessory proteins, as observed in other bacterial species [8,9,14,15]. 3.2. Analysis of urease biogenesis in S. thermophilus Urease activity of S. thermophilus DSM20617T cells growing in M17 broth was detected starting from the late ex-

ponential growth phase (OD600 nm = 1.9), when the culture pH was lower than 6 (Fig. 2A, 2B and 2E). The zymogram analysis revealed the activity profile previously described, composed of signals a, b and c. The maximum level of activity detected was 36 ± 3 U g−1 (Fig. 2E). The addition of urea to the growth medium significantly increased urease activity, with signals b and c detectable in gel starting from the mid-exponential growth phase (OD600 nm = 1.5) (Fig. 2C) with culture pH near 6 (Fig. 2A) and an activity value of 70 ± 5 U g−1 (Fig. 2E). Moreover, urease activity reached a maximum of 232 ± 7 U g−1 (Fig. 2E) in the late exponential phase when urea was added to M17 broth. In the in-gel detection assay the highest values of urease activity were evidenced by the appearance of activity band a (Fig. 2C). To investigate the effect of culture pH on urease biogenesis, experiments were carried out at pH 6 and 7. A strong increase in urease biogenesis occurred from the early exponential phase (OD600 nm = 0.4) with an activity of 47 ± 3 U g−1 (Fig. 2A, 2D and 2E), when S. thermophilus was grown with a culture pH fixed at 6, a value quite close to the maximum of activity detected when S. thermophilus was grown without control of the culture pH. At pH 6 the maximum urease activity was 510 ± 15 U g−1 , which corresponded to an OD600 nm close to 0.9 (Fig. 2E). The high urease biogenesis in an acidic environment (pH 6) was also confirmed by the in-gel detection assay, in which activity signals a and b were visible in all samples tested, while the upper signal c was never visualized. Activity signals a and b showed an evident increase in intensity correlated with the levels of urease activity measured using the phenol hypochloride assay. While strong induction of urease biogenesis was measured at pH 6, no urease activity was detected by the phenol hypochloride assay when bacteria were grown at pH 7. At neutral culture pH, only extremely weak activity signals b and c were detected in gel even after prolonged staining for up to 12 h (data not shown). When cells were grown at neutral pH in the presence of 10 µM NiCl2 , urease activity was detected in the gel starting from the early exponential growth phase (OD600 nm = 0.5) with a value of 12.3 ± 0.5 U g−1 and reaching a maximum of 34.2 ± 0.7 U g−1 at OD600 nm = 1.7 (data not shown). The main activity signals detected in native gel were a and b with a very weak presence of band c. 3.3. pureI promoter activity Sequence comparison of the region located upstream the ureI gene of S. thermophilus DSM20617T (AJ544512) and S. salivarius 57.1 (U351248) revealed complete identity in the putative −10 and −35 sequences and in the direct repeat located immediately on the 5 side of the −35 region (Fig. 3). Interestingly, the S. thermophilus promoter region lacked the inverted repeat IR1 (Fig. 3) which was reported to be involved in the expression regulation of the low pHinducible ure operon of S. salivarius 57.1 [5]. A further stem loop was detected only in the S. thermophilus sequence at positions −101 and −115. The presence of IR2 and the ab-

Fig. 3. Sequence comparison of the promoter region located upstream the urel gene between S. salivarius 57.1 and S. thermophilus DSM20617T . The transcription initiation site of purel of S. salivarius is indicated with an arrow (+1). The putative −10 and −35 sequences are boxed. Inverted repeat sequences IR1 and IR2 and direct repeat sequences DR1 are underlined and indicated with arrows. The start codon of urel gene is underlined and typed in bold. Differences in the promoter region between S. salivarius and S. thermophilus are shown as bold typed nucleotides.

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sence of IR1 were also observed in the other four ure operon sequences of S. thermophilus available in the GenBank database (AF362908, AY374143, CP000023, CP000024). To evaluate the hypothesis of pH-inducible transcription of the urease operon, the entire promoter region shown in Fig. 3 plus an additional 27 bp on the 5 side were amplified and cloned in pNZ273 to obtain the new vector pMI200 carrying a transcriptional fusion with a gusA reporter gene. As expected, transformation of S. thermophilus DSM 20617T with pNZ273 did not allow both the isolation of blue colonies in M17-X-Gluc plates and the detection of β-glucuronidase activity using para-nitro-β-Dglucuronic acid as the substrate. The S. thermophilus strain DSM 20617T harboring pMI200 cultured in M17 broth buffered at neutral pH showed a reporter activity of 618 ± 42 mOD min−1 mg−1 of protein that increased to a value of 1015 ± 39 when grown in acidic M17 broth (pH 6). A culture pH lower than 6 was not used because the growth rate of S. thermophilus DSM 20617T did not reach a sufficient cell concentration suitable for enzymatic determination. To confirm the effect of the expression of pureI at neutral pH, and to minimize the effect of the lactic acid produced by S. thermophilus growing cells, strain DSM20617T harboring pMI200 was incubated in M17 buffered at pH 7 until the beginning of the exponential growth phase at OD600 nm = 0.2. Cells were subsequently collected by centrifugation, suspended in fresh M17 buffered at pH 7 and used to inoculate M17 buffered at pH 7 and M17 acidified to pH 6. The cells grown to OD600 nm = 0.5 were assayed for β-glucuronidase activity. Using this experimental protocol, the reporter activity proved to be only slightly different, 1013 ± 25 and 1201 ± 31 mOD min−1 mg−1 in neutral and acidic M17, respectively. To investigate whether the addition of Ni ions to the culture medium could enhance the transcription level by pureI , the level of expression was monitored in the recombinant pureI –gusA S. thermophilus strain. The addition of Ni ions at different final concentrations (1, 5, 10, 20 and 40 µM) in standard M17 did not result in a significant variation in the level of the reporter activity (data not shown). The growth of pureI –gusA recombinant strain in the presence of different amounts of glucose and lactose (20, 50, 100, 120 and 150 mM), in acidified and standard M17 broth, did not reveal significant variation in the β-glucuronidase activity. Culturing the pureI –gusA recombinant strain in M17, broth with urea at a final concentration of 0.5 g l−1 resulted in β-glucuronidase activity of 557 ± 32 mOD mg−1 min−1 , a value not significantly different from that obtained in standard M17 broth (595 ± 54 mOD min−1 mg−1 ).

4. Discussion In this study, an effective urease in-gel detection system was developed for the monitoring of urease biogenesis in S. thermophilus under different culture conditions. The


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in-gel detection assay led to the identification of different molecular species with urease activity in the total cell extract of S. thermophilus. The zymogram profiles obtained for strain DSM 20617T were composed of activity signals whose number was dependent on the growth phase and culture conditions. The nature of the various molecular forms showing ureolytic activity remains to be elucidated. When S. thermophilus was grown in M17 broth, urease activity was detected starting from the late exponential growth phase, when the culture pH fell below 6. The growth of S. thermophilus in an acidic environment (M17 broth, pH 6) resulted in approximately a tenfold increase in specific urease activity, underlining a consistent induction of expression of ure genes as already observed in S. salivarius [3–5]. However, in contrast to S. salivarius a reduction in active urease biogenesis, but not complete repression, was observed when growing S. thermophilus at neutral pH. The pH-regulated urease biogenesis of S. thermophilus is primarily governed at the transcriptional level, as demonstrated by analyzing the reporter activity of a pureI -gusA recombinant strain grown under different culture conditions. Nevertheless, the twofold enhancement of β-glucuronidase activity observed when growing cells in an acidic environment did not coincide with the much larger increase in urease activity evaluated in-gel and measured by the phenol hypochloride assay (Fig. 2), suggesting the presence of a translational or post-translational regulation system in urease biogenesis. To investigate whether the absence of repression of ure genes at neutral culture pH was a consequence of slight acidification occurring during the growth of S. thermophilus in batch condition, the culture pH reached at the end of the growth phase was measured. The M17 buffered culture maintained a stable pH 7 until the end of the growth phase (OD600 nm = 0.5), suggesting that ure genes of S. thermophilus were transcribed even if environmental acidification did not occur. Moreover, when β-glucuronidase activity was measured in standard M17 broth, the value obtained was not significantly different (595±54 mOD mg−1 min−1 ) from that evaluated in buffered M17 even if the final pH was 6.8. In this context, (i) the extremely low levels of urease detected in gel when cells were cultured at neutral pH, (ii) the significant increase in activity detected when nickel ions were added to the medium, and (iii) the absence of induction of expression by pureI with the addition of nickel, as verified using a pureI –gusA reporter system, suggests that the availability of nickel ions should be considered a key factor in the biogenesis of active urease when cells are cultured at neutral pH. Therefore, urease structural subunits (α, β, γ ) are also likely to be translated efficiently at pH 7. Expression of ure genes was not induced by urea but urea-dependent changes in urease activity were observed (Fig. 2C), suggesting that the activity level of the enzyme could be moderately regulated by the presence of the enzyme substrate as reported for other bacterial species [13].

Expression of ure genes in S. thermophilus DSM 20617T was not induced by an excess of carbohydrate, as otherwise reported for its closest neighbor S. salivarius [4]. This difference in the regulatory mechanism of the ure operon between S. salivarius and S. thermophilus could be related to the sequence differences observed in the urease promoter region of the two species, and in particular to the differences in sequence and position between the inverted repeats IR1 of S. salivarius and IR2 of S. thermophilus (Fig. 3). Moreover, the absence of regulation of expression of ure genes by carbohydrate availability in S. thermophilus may be analyzed from an ecological point of view. While S. salivarius is an oral bacteria able to produce acid from fermentable carbohydrates whose availability in the mouth is strictly linked to diet intake, S. thermophilus is an acidogenic lactic acid bacteria extremely adapted to life in a milk environment [2] where the carbohydrate availability, lactose, is practically unlimited (45–50 g l−1 ). As a consequence, unlike S. salivarius, S. thermophilus does not need to regulate the expression of urease, a stress response to low environmental pH, in function of environmental lactose concentration. In conclusion, the strong increase in urease biogenesis at pH 6 should be interpreted as a stress response of S. thermophilus metabolism when bacterial cells are grown in an acidic environment. The stressed condition of S. thermophilus growing at pH 6 was evidenced by a strong reduction in the growth rate (0.6OD600 nm per h in acidic medium versus 1.1OD600 nm per h in standard condition, Fig. 2A). Among known stress responses, urease activity is considered to be one of the mechanisms of resistance for counteracting low environmental pH developed by bacteria [6]. While S. thermophilus has acquired this mechanism of resistance to acidic environments, the majority of other lactic acid bacterial species have developed alternative strategies of resistance such as the arginine deiminase pathway, the H+ -ATPase proton pump and the glutamate decarboxylase system [6]. Another question which remains to be resolved is why urease is also produced at pH 7, albeit at low levels. An explanation could be linked to an alternative physiological mechanism used by S. thermophilus to supply ammonia and carbon dioxide for the production of amino acids and for the synthesis of important metabolic precursors such as carbamoylphosphate and oxaloacetate.

Acknowledgements This work was supported by a grant from the Ministry of the University and Technological and Scientific Research (First 2004). We thank Dr. Giorgio Giraffa (Istituto Sperimentale Lattiero Caseario di Lodi, Lodi, Italy) for his precious help in total cell protein extraction. We also thank Professor Giovanni Dehò (Dipartimento di Scienze Biomolecolari e Biotechnologie, Università degli Studi di Milano, Milano, Italy) for his helpful suggestions. We are grateful to

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Michela Besani and Stefania Arioli for their invaluable technical assistance.

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