Dual Role of the Oligopeptide Permease Opp3 during Growth of Staphylococcus aureus in Milk

Staphylococcus aureus RN6390 presents a diauxic growth in milk,due to amino acid limitation. Inactivation of the oligopeptidepermease Opp3 (dedicated to the nitrogen nutrition of the strain)not only affects the growth of the strain but also results inreduced expression levels of three major extracellular proteases.

INTRODUCTION

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INTRODUCTION

Staphylococcus aureus is a ubiquitous gram-positive pathogencommonly found in the environment. Some S. aureus strains areable to produce enterotoxins that cause food poisoning (6).In France, contaminated dairy products are the main source ofstaphylococcal food-borne disease outbreaks (3). Thus, the developmentof this bacterium in milk is a major concern for the safetyof dairy products. Our objective was to evaluate the growthof S. aureus in milk, with special attention focused on therole of the peptide transport systems.

Opp3 is involved in amino acid supply during growth of S. aureus RN6390 in milk.

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Opp3 is involved in...

S. aureus strain RN6390 (7) was grown at 37°C in reconstitutedskim milk (Nilac; Duiven, The Netherlands) with shaking (200rpm). The time courses of cell populations for seven independentreplicate cultures were evaluated by plating culture dilutionsafter disrupting bacterial clumps with an Ultra Turrax (IKA,Staufen, Germany).

Linear regression analysis performed for each growth experimentdiscarded the hypothesis of a single exponential growth phase,with the standard errors of the estimate in the same range asthe growth rates (from 0.70 to 1.19 h^–1). S. aureus presenteda diauxic growth in milk, with two distinct exponential growthphases separated by a short transition period (Fig. 1). Themean growth rates of the two exponential phases were 1.35 ±0.03 h^–1 and 0.60 ± 0.04 h^–1 (means for sevendeterminations ± confidence limits at P values of 0.95),with standard errors of the estimate lower than 0.23 and 0.17h^–1, respectively.

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FIG. 1. Growth of the S. aureus RN6390 wild-type strain in milk (•) or in milk supplemented with a mixture of 18 amino acids ( ${blacktriangleup}$ ).

When milk was supplemented with a mixture of 18 amino acids(10), growth of S. aureus RN6390 was clearly modified (Fig.1). Only one single exponential growth phase was observed (µ= 1.70 h^–1), whereas the final population was not affected(ca. 5 x 10⁹ CFU·ml^–1). These results suggest thatthe amino acid composition, rather than the concentration ofamino acids present in nonsupplemented milk, accounts for thegrowth limitation of the strain.

Five putative peptide transport systems are present in S. aureus:four ABC transporters, named Opp (oligopeptide permease), andone permease, DtpT. Among those, only Opp3 and DtpT exert anutritional function, ensuring the import of tri- to octapeptidesand di- and tripeptides, respectively (5). The role of thesetwo peptide transport systems in growth in milk was evaluatedby measuring the growth of opp3 and dtpT mutant strains. Alldeletion mutants were constructed by double crossover usingthe pMad vector as previously described (1, 5). While the dtpTmutant strain grew similarly to the wild-type strain, the growthof the opp3 mutant strain was altered, as the duration of thetransition period was fivefold longer (Fig. 2). This suggeststhat oligopeptides are used as a source of amino acids at thisstage of growth. The double opp3 dtpT mutant presented onlythe first exponential phase and a strongly reduced final population(about 10-fold), indicating that di- and tripeptides are a significantsource of amino acids when the strain is unable to import oligopeptides.These results were further supported by growths in milk supplementedwith a mixture of 18 amino acids in which all mutant strainsgrew as the wild-type strain (data not shown).

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FIG. 2. Growth of S. aureus RN6390 wild-type (•) and dtpT ( ${blacksquare}$ ), opp3 ( ${blacktriangleup}$ ), and opp3 dtpT ( ${diamondsuit}$ ) mutant strains during growth in milk.

The growth in milk of the opp1 opp2 opp4 dtpT mutant strainwas comparable to that for the wild type (data not shown), confirmingthe absence of significant nutritional function for Opp1, Opp2,and Opp4 systems, as reported previously (4).

Altogether, these results allow us to postulate the followingthree-stage scheme of nitrogen nutrition in S. aureus: (i) first,the bacteria utilize free amino acids from milk to reach a celldensity of about 3 x10⁸ to 5 x10⁸ CFU ml^–1; (ii) the poolof free amino acids in milk becomes too low, resulting in atransient growth arrest (this is supported by the fact thatnone of the amino acids required by S. aureus for an efficientgrowth, namely, Glu, Cys, Leu, Met, Gly, Val, Thr, Arg, andLys [3], could be detected anymore by high-performance liquidchromatography in their free form in the milk cultures at thisstage of growth [data not shown]); and (iii) a complementarysource of amino acids supplied as peptides allows bacteria torestart growth and to reach high cell densities (about 5 x 10⁹CFU·ml^–1).

Opp3 participates in extracellular protease regulation during growth in milk.

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Opp3 participates in...

Milk coagulation was observed during the transition period ofthe wild-type strain. As the culture was stirred, S. aureusdeveloped a respiratory metabolism that did not result in adecrease of pH. Consequently, milk clotting was most probablydue to casein proteolysis. This phenomenon was delayed in thecultures of opp3 and opp3 dtpT mutant strains (1.5 h and 6 h,respectively). As the cell densities of the strains were insimilar ranges at this growth stage (Fig. 2), this delay mightbe caused by reductions in the proteolytic activities of themutant strains. To evaluate this hypothesis, the extracellularproteolytic activities of the wild-type and opp3 mutant strainswere evaluated. Supernatants from transition period cultureswere settled onto cellulose disks placed onto agar (1.5%) platescontaining 3% casein. After incubation at 37°C, the haloof proteolysis (white precipitate of casein) observed with theopp3 mutant was smaller than that of the wild-type strain, indicatinga lower proteolytic activity (data not shown). Ten extracellularproteases (SspA and B; Aur; ScpA; and SplA, B, C, D, E, andF) have been identified in S. aureus. To test a putative deregulationin the transcription of one or several of the proteases encodedby genes in the opp3 mutant strain, quantitative real-time PCRon the sspA, aur, scpA, and splA genes was performed (sspB andthe splB to -F genes are cotranscribed with sspA and splA, respectively).RNAs and cDNA were prepared as previously described (5), andquantitative PCR was performed by using a SYBR green mastermix (Applied Biosystems) with 2 ng of cDNA (primers are listedin Table 1). gyrB was validated as a stably expressed gene underour conditions and used to normalize the quantity of each cDNAtested. Inactivation of opp3 did not affect the expression levelsof scpA and splA (data not shown). However, the levels of sspAand, to a lower extent, aur transcripts were reduced in theopp3 mutant (Fig. 3). As expected from the genetic organizationof ssp genes, the sspB transcript level was also decreased (datanot shown). We further observed a 1.5-h delay in milk coagulationin the culture of an sspA isogenic mutant strain (8), comparedto the level for the parental RN6390 strain, confirming theimplication of Ssp protease activity in the degradation of milkproteins (data not shown). Altogether, these results indicatethat the delay in coagulation with the opp3 mutant strain resultsfrom a reduction in proteolytic activity caused at least bya decrease in ssp transcription. It is worth noting that nodelay in coagulation could be observed between wild-type andopp3 mutant strains when milk was supplemented with a mixtureof amino acids. This suggests that Opp3 could be involved inenvironmental sensing by modulating intracellular amino acidpools acting on pleiotropic regulators, as already demonstratedfor other bacterial Opp systems (2, 4, 9). The amino acids andthe regulator involved in this mechanism remain to be elucidated.

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TABLE 1. Oligonucleotide primers used for quantitative PCR

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FIG. 3. Effect of opp3 deletion on expression of sspA (black bars) and aur (gray bars) by S. aureus RN6390 grown in milk. Expression levels of sspA and aur in the S. aureus wild-type and opp3 mutant strains were estimated by quantitative PCR. For each strain and each time point, gene expression was normalized using gyrB as the stably expressed reference gene. Results are presented as ratios of gene expression (wild-type [wt] strain/opp3 mutant). Vertical bars indicate the standard deviations.

In conclusion, peptides transported by Opp3 play a dual roleduring growth of S. aureus in milk, supplying bacteria withnitrogenous nutrients and influencing expression of genes encodingthree major extracellular proteases, most probably via an environmentalsensing mechanism. Thus, Opp3 might represent a new target forcontrolling S. aureus development in dairy products.

ACKNOWLEDGMENTS

We are grateful to Samira Makzhami and Claudia Bevilacqua (PICT,INRA Jouy en Josas, France) for their contribution to the quantitative-PCRanalyses.

A. Hiron received a fellowship from the Ministère del'Education Nationale, de l'Enseignement Supérieur etde la Recherche.

FOOTNOTES

* Corresponding author. Mailing address: UR888, Unité Bactéries Lactiques et Pathogènes Opportunistes, Institut National de la Recherche Agronomique, Domaine de Vilvert, 78352 Jouy en Josas cedex, France. Phone: 33 (0) 134 652 395. Fax: 33 (0) 134 652 065. E-mail: elise.durant{at}jouy.inra.fr

Published ahead of print on 13 March 2009.

These authors contributed equally to this work.

Present address: Biology of Gram-Positive Pathogens, CNRS URA2172, Department of Microbiology, Institut Pasteur, 25 Rue duDr Roux, 75724 Paris cedex 15, France.

REFERENCES

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