Glutamine Synthesis Is Essential for Growth of Streptococcus thermophilus in Milk and Is Linked to Urea Catabolism

Growth of a glutamine synthetase-deficient mutant of Streptococcusthermophilus was compared to that of the parent strain in milkthat was not supplemented or was supplemented with ammoniumchloride, glutamine, or the urease inhibitor flurofamide. Itwas concluded that one of the functions of urease is to supplyammonia for the synthesis of glutamine.

INTRODUCTION

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Introduction

In contrast to the other lactic acid bacteria present in startercultures, Streptococcus thermophilus possesses a urease, whichconverts urea into ammonia and carbon dioxide (5, 10). Evenif this enzyme is not required for the growth of S. thermophilusin milk (8), we observed recently that its inhibition by flurofamidedecreased the growth of most of the strains tested (9). Onehypothesis to explain this result is that ammonia and carbondioxide produced from urea could be used in several biosynthesisroutes, such as amino acid production. In the present study,we investigated the importance of glutamine synthesis for thegrowth of S. thermophilus in milk and its relation with thecatabolism of urea.

Inactivation of the gene encoding a glutamine synthetase.

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Inactivation of the gene...

The nucleotide sequence of the putative glutamine synthetasegene, named glnA, was obtained from the S. thermophilus LMD-9genome sequence, produced by the U.S. Department of Energy JointGenome Institute (http://www.jgi.doe.gov/). The glnA gene wasPCR amplified from DNA of S. thermophilus CNRZ385 (Table 1)using the oligonucleotides olgln01 (5' AGCAATACAATGTTCGG 3')and olgln02 (5' GTAAAGATACCACCTCTGG 3'). The resulting 2.6-kbPCR fragment was then cloned into the TOPO XL vector (Invitrogen,Groningen, The Netherlands) and transferred into Escherichiacoli TOP10. The resulting plasmid was named pGS21. A 1.1-kbdeletion within the glnA gene was performed by digestion ofpGS21 with BglII (Eurogentec, Seraing, Belgium), which has twocutting sites in glnA. After ligation (fast-link DNA ligationkit; Epicentre Technologies, Madison, Wis.), the resulting plasmid,named pGS39, was digested with HindIII and PstI to release a1.6-kb fragment containing the deleted glnA gene. This fragmentwas inserted in the plasmid pG⁺host9::ISS1 (6), previously digestedwith the same restriction enzymes (this also eliminated theISS1 element from the vector). The resulting plasmid, namedpGS45, was obtained in E. coli TIL206 and then introduced intoS. thermophilus CNRZ385 using a previously described protocol(4). The procedure for gene replacement described by Biswasand coworkers (2) was then applied to the glnA gene, exceptthat all culture media were supplemented with 0.25 g liter^–1glutamine. The resulting glutamine synthetase-negative mutantwas named GS200. Presence of the deletion and absence of thevector in GS200 was checked by PCR.

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TABLE 1. Strains and plasmids

Glutamine synthetase activity.

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Glutamine synthetase activity.

After growth of the parent and mutant strains at 37°C, cellswere harvested by centrifugation, washed with 0.9% NaCl, andresuspended in imidazole-HCl buffer (100 mM, pH 7.15). Glutaminesynthetase activity was then measured using the "forward reactionassay" described by Bender and coworkers (1). One unit of glutaminesynthetase activity was defined as the amount of enzyme producing1 µmol of glutamyl hydroxamate per min at 37°C. Theparent strain displayed a similar glutamine synthetase activity,whether the medium was supplemented with glutamine or not (Table2). No activity could be detected when measurements were donein the presence of 15 mM of the glutamine synthetase inhibitormethionine sulfoximine. As expected, no glutamine synthetaseactivity could be detected in strain GS200.

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TABLE 2. Glutamine synthetase activity of S. thermophilus CNRZ385 and GS200

Growth of the parent strain in milk.

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Growth of the parent...

After growth in M17 broth at 37°C, cells were harvestedby centrifugation, washed twice with 0.9% NaCl, and inoculated,at a concentration equivalent to 0.007 absorbance unit (575nm), in reconstituted skim milk (100 g liter^–1; EPI ingrédients,Ancenis, France) that had been heated for 10 min at 80°C.Sterile 8-ml tubes were filled with 7.8 ml of inoculated milkand hermetically sealed. They were then incubated at 37°Cand sampled regularly during 12 h. Growth in milk was measuredafter clarification of the medium (3). Some cultures were alsodone in the presence of 10 mM ammonium chloride, 0.25 g liter^–1L-glutamine, or 10 µM of flurofamide. Flurofamide is apotent inhibitor of urease, and growth of S. thermophilus strainsin the presence of this compound is equivalent to that of mutantstrains deficient in urease (7). Figure 1A shows that growthof strain CNRZ385 in milk is lower when urease is inhibited.Indeed, final biomass concentration is approximately 23% lowerin the presence of flurofamide. The same result was obtainedwhen the cultures were repeated (mean value of 2.43 ±0.11 in the absence of flurofamide and 1.77 ± 0.12 inthe presence of flurofamide). In the presence of flurofamide,i.e., when urease was inhibited, glutamine and ammonia stimulatedthe growth of the strain. Indeed, after 12 h of growth, themean biomass concentration was 1.77 ± 0.12, 2.03 ±0.14, and 2.30 ± 0.16 for the cultures in the presenceof flurofamide, of flurofamide plus glutamine, and of flurofamideplus ammonium chloride, respectively. The stimulating effectof ammonium chloride and glutamine was not observed in the absenceof the urease inhibitor. It may thus be proposed that the stimulatingeffect of the metabolism of urea could be due, at least partly,to the production of ammonia, which may be used in biosyntheticreactions, such as glutamine synthesis.

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FIG. 1. Effect of ammonia and glutamine on the growth of S. thermophilus CNRZ385 (A) or the glutamine synthetase-deficient mutant GS200 (B) in milk and in milk supplemented with flurofamide (representative graphs selected from three repeated experiments). Cultures in milk ( ${circ}$ ), milk plus ammonium chloride ( ${square}$ ), milk plus glutamine ( ${triangleup}$ ), milk plus flurofamide (•), milk plus flurofamide plus ammonium chloride ( ${blacksquare}$ ), and milk plus flurofamide plus glutamine ( ${blacktriangleup}$ ).

Concentrations of ammonia and urea were assayed using an enzymatickit (r-biopharm, Saint Didier Au Mont d'Or, France). As expected,no consumption of urea occurred in the cultures supplementedwith flurofamide (Table 3). The amount of nitrogen present inurea and ammonia (N_urea+ammonia) was calculated using the followingrelation: N_urea+ammonia = 2 x [urea] + 1 x [ammonia]. It isnoteworthy that when CNRZ385 was cultivated in nonsupplementedmilk, there was a significant decrease of N_urea+ammonia, comparedto the amount present initially in milk (6.78 mM versus 8.41mM). During its growth in milk, S. thermophilus was thus ableto assimilate nitrogen present in milk ammonia and urea. Nodecrease of N_urea+ammonia could be highlighted (Student test)in cultures supplemented with ammonium chloride, flurofamide,or glutamine. This does not necessarily mean that ammonia wasnot assimilated in these culture media. Indeed, this resultmay be explained by the fact that the relative amount of ammoniaused by S. thermophilus was too low compared to the level ofprecision of the assays of ammonia and urea.

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TABLE 3. Ammonia and urea concentration in milk and in cultures of S. thermophilus CNRZ385 after 12 h of growth^a

Growth of the glutamine synthetase-deficient mutant in milk.

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Growth of the glutamine...

Strain GS200 had only a very limited growth in milk (Fig. 1B),indicating that glutamine synthesis is essential for the growthof S. thermophilus. The addition of glutamine, but not of ammoniumchloride, stimulated the mutant. It is noteworthy that the growthof GS200 in the presence of glutamine was slightly lower whenmilk was also supplemented with flurofamide (P < 0.05).

In milk, glutamine is present in peptides and caseins and isalso present as a free amino acid. In the present study, weshowed that these sources of glutamine do not fulfill the requirementsof S. thermophilus and that some glutamine has to be producedvia the glutamine synthetase. Furthermore, as glutamine additionstimulated the wild-type strain only when the catabolism ofurea was inhibited by the addition of flurofamide, it is likelythat ammonia production from urea is essential for the glutaminesynthetase to have a sufficient in vivo activity. These resultssupport the idea that one of the physiological functions ofurease in S. thermophilus is to supply ammonia for the synthesisof glutamine. To our knowledge, this is the first study showingthe importance of ammonia assimilation for the growth of lacticacid bacteria in milk.

Nucleotide sequence accession number.

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Nucleotide sequence accession...

The nucleotide sequence of the S. thermophilus CNRZ385 glnAgene has been deposited in GenBank under the accession numberAY764257.

ACKNOWLEDGMENTS

We thank Michèle Nardi for helpful suggestions, AmélieDorvillez for technical assistance, and Christine Young forcritically reading the manuscript.

FOOTNOTES

* Corresponding author. Mailing address: Unité Mixte de Recherche Génie et Microbiologie des Procédés Alimentaires, Institut National de la Recherche Agronomique, 78850 Thiverval-Grignon, France. Phone: 33 1 30 81 54 91. Fax: 33 1 30 81 55 97. E-mail: monnet{at}grignon.inra.fr.

REFERENCES

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