INTRODUCTION

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Lactococcus lactis subsp. lactis is used widely in the cheese industry as a starter culture for cheese production. Starter cultures face carbohydrate starvation conditions with less than 0.1% lactose in the cheese curd after pressing (34). Starvation conditions decrease the ability to synthesize ATP, generate proton motive force (PMF), and accumulate nutrients necessary to maintain viability over time (13).

In optimum growth, L. lactis is a homofermentative lactic acid bacterium that ferments lactose to lactic acid and ATP. Lactococci lack a cytochrome system and are unable to produce ATP by oxidative phosphorylation (29). They rely on glycolysis and substrate-level phosphorylation to generate compounds that serve as energy donors for solute transport and growth (31). In lactococci, lactose is transported into the cell by a phosphoenolpyruvate (PEP)-mediated phosphotransferase system (PTS), sugar transport ATPases, ion-linked sugar transport mechanisms, and sugar exchange mechanisms for a net yield of four ATP molecules per lactose molecule via glycolysis (19, 28, 33).

The absence of lactose causes immediate energy starvation because lactococci do not contain carbohydrate storage polymers (18, 22). During starvation, the intracellular levels of the glycolytic intermediates PEP, 3-phosphoglycerate (3-PG), and 2-phosphoglycerate increase and constitute the PEP potential, which provides a link between sugar transport and energy-yielding reactions of glycolysis (28, 29). During starvation, PEP is metabolized slowly to pyruvate and ATP due to the regulation of pyruvate kinase (28). This maintenance of large PEP pools may provide necessary maintenance energy (ATP) for the organism during starvation as well as permit the rapid accumulation of PTS sugars when they become available again (28, 29).

Upon energy starvation of L. lactis, the PMF dissipates, the pH gradient collapses, and ATP levels decrease below 0.1 mM (13, 18). Nevertheless, many organisms have the ability to use alternate carbon sources for energy. In response to carbohydrate starvation, many bacteria become viable but nonculturable (VBNC) rather than die and lyse, as suggested for lactococci (6). This state has been observed for bacteria such as Vibrio, Pseudomonas, Micrococcus, Enterococcus, Brevibacterium, Escherichia coli, and others (16). During the VBNC state, cells continue to transport and metabolize nutrients but do not form colonies on solid agar. Shigella dysenteriae becomes VBNC during starvation, and the cells continue to transport and incorporate methionine into cellular proteins (23).

Transport of amino acids in lactococci occurs via three different mechanisms. The majority of amino acids are transported by the PMF-driven transport that links amino acid uptake to the PMF. The driving force for H⁺ translocation and the PMF is usually supplied by the free energy of ATP hydrolysis (12). L. lactis catalyzes the translocation of leucine, isoleucine, valine, alanine, glycine, serine, threonine, methionine, histidine, proline, cysteine, lysine, phenylalanine, tyrosine, tryptophan, and arginine together in symport with one proton via separate mechanisms (12, 19). During starvation of lactococci, the proton-linked amino acid transporter and the arginine or ornithine antiporter still function and are only moderately affected during incubation (13).

The driving force for arginine uptake into cells is supplied by the arginine and ornithine concentration gradient (20). This transport mechanism is beneficial because no additional metabolic energy is required for the transport of arginine across the membrane (12). The excess arginine may be metabolized by the arginine deiminase (ADI) pathway to produce energy (ATP) in L. lactis (4). The ADI pathway is widely distributed among bacteria and serves either as the sole or as an additional source of energy, carbon, and nitrogen in lactic acid bacteria, bacilli, Pseudomonas, Aeromonas, clostridia, Mycoplasma, and halobacteria (4).

In this study, the influence of lactose and arginine on the amino acid metabolism and culturability of L. lactis was examined. This study was initiated to determine if L. lactis could become VBNC after the exhaustion of lactose when present with arginine and other amino acids. Amino acids may be utilized for protein synthesis during starvation as well as provide an additional energy source. Our results indicate that lactococci remain viable in the absence of lactose or arginine. The cells were able to use other amino acids to survive, produce ATP, and maintain cellular integrity without being culturable on agar.

	MATERIALS AND METHODS

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Bacterial strains and media. L. lactis subsp. lactis ML3 was obtained from the Utah State University culture collection. L. lactis subsp. lactis 11454 was obtained from the American Type Culture Collection (Rockville, Md.). Strains were propagated in Elliker broth (Difco Laboratories, Detroit, Mich.) for 16 h at 30°C, harvested by centrifugation (11,750 × g for 5 min at 25°C), washed twice with sterile saline, resuspended in 2 ml of sterile saline, and inoculated (1%) into sterile chemically defined basal medium (CDM) (7, 11). CDM was supplemented with lactose and arginine to make four media: 0.2% lactose and 2% arginine, 0.2% lactose and 0% arginine, 0% lactose and 2% arginine, and 0% lactose and 0% arginine. The media were then adjusted to pH 7.0, buffered with 190 mM sterile 3-[N-morpholino]-propanesulfonic acid (MOPS; Sigma Chemical Co., St. Louis, Mo.), and filter sterilized through a 0.2-µm-pore-size bottle-top filter (Corning Costar, Corning, N.Y.). All cultures were grown in 200 ml of media at 30°C.

Lactose determination. The lactose concentration was determined by high-pressure liquid chromatography. The sample was prepared by filtration through a sterile 0.2-µm-pore-size syringe filter (Nalge Company, Rochester, N.Y.) to remove cells. The filtered sample (10 µl) was injected with a model 507 chromatograph (Beckman Instruments, Inc., Fullerton, Calif.) autosampler into a Benson carbohydrate BC100 Ca²⁺ column (0.78 by 30 cm) fitted with a guard cartridge in a cartridge holder (Alltech, Deerfield, Ill.). The column and guard cartridge were heated to 86°C in a Bio-Rad Laboratories (Hercules, Calif.) column heater. Lactose was eluted with water at a flow rate of 0.5 ml/min during a 14-min run with a model 125 pump (Beckman). Detection was done with an LC 30 refractive-index detector (The Perkin-Elmer Corp., Norwalk, Conn.). Peak areas were determined with a model 427 integrator (Beckman) set at an attenuation of 16. A linear standard curve (R² = 0.99) was observed with 0.125, 0.25, 0.5, 1.0, and 2.5 mg of lactose per ml (data not shown).

Culturable cell estimation. Samples were taken at various times from the culture suspensions and diluted in sterile saline dilution blanks. From these dilutions, 100 µl was spread on Elliker agar plates with a Spiral System CU (Cincinnati, Ohio). The plates were incubated anaerobically for 48 h at 30°C. The colony count was determined with duplicate plates in accordance with the manufacturer's instructions.

Viable cell estimation. Samples were collected from the cell suspensions and stained in accordance with the manufacturer's instructions by use of the LIVE/DEAD BacLight bacterial viability kit (Molecular Probes, Inc., Eugene, Oreg.). The kit estimated bacterial viability with two nucleic acid stains that differed in their spectral characteristics and ability to penetrate healthy bacterial cell membranes. The fluorescence emission of each cell suspension was measured with an RF-1501 spectrofluorophotometer (Shimadzu, Pleasanton, Calif.) at an excitation of 480 nm and an emission of 520 nm for the live dye and an excitation of 550 nm and an emission of 580 nm for the dead dye. Results are expressed in relative fluorescence units.

ATP quantitation. The ATP concentration in cell extracts was quantified by measuring luminescence with an ATP assay kit (Calbiochem-Novabiochem Corporation, San Diego, Calif.) as described by the manufacturer. The assay is based on the luciferase-catalyzed oxidation of D-luciferin in the presence of an ATP-magnesium salt and oxygen to produce light. Luminescence was measured with an LS6500 scintillation counter (Beckman).

Amino acid determination. Samples were prepared by filtering cell suspensions through a 0.2-µm-pore-size syringe filter (Nalge) and were centrifuged (5,000 × g for 3 h at 4°C) through a 1K Centricon (Pall Filtron, Ann Arbor, Mich.). An aliquot was derivatized with 3-(4-carboxybenzoyl)quinoline-2-carboxaldehyde in accordance with the instructions enclosed in the ATTO-TAG CBQ derivatization kit (Molecular Probes). Norleucine was added as an internal standard to each reaction mixture prior to the addition of the derivatizing agent.

	RESULTS AND DISCUSSION

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Lactose utilization. The lactose concentration during the growth of L. lactis subsp. lactis ML3 decreased to nondetectable levels irrespective of arginine content within 3 days (Fig. 1A) and did not change the pH of the media due to the use of MOPS buffer. The culture containing lactose and arginine grew to higher numbers (10⁹ CFU/ml) than did the culture without arginine (10⁸ CFU/ml) after lactose depletion (Fig. 2A). The presence of arginine appeared to play a beneficial role in cell growth and viability when present with lactose.

View larger version (16K): [in this window] [in a new window]   FIG. 1.   Lactose concentrations in spent media for L. lactis ML3 (A) and L. lactis 11454 (B). Cells were grown in 0.2% lactose and 2% arginine () and 0.2% lactose and no arginine ().

View larger version (19K): [in this window] [in a new window]   FIG. 2.   Culturable cell counts for L. lactis ML3 (A) and L. lactis 11454 (B) grown in various media. Cells were grown in 0.2% lactose and 2% arginine (), 0.2% lactose and no arginine (), no lactose and 2% arginine (), and no lactose or arginine ().

Viability and culturability. The culturability of L. lactis was estimated by determining the cell population from the plate counts, while viability was determined with the use of fluorescence as a result of healthy cell membranes and the amount of ATP present. L. lactis ML3 grown in CDM/L⁺/A⁺ reached a cell density of 10⁹ CFU/ml, which decreased to 10⁵ CFU/ml after 14 days (Fig. 2A). Conversely, in CDM/L/A (CDM lacking lactose [L] and arginine [A]), strain ML3 was culturable for 2 days and then became nonculturable on solid agar. The addition of either lactose or arginine increased cell density and culturability time for ML3.

View larger version (25K): [in this window] [in a new window]   FIG. 3.   Estimation of viability with fluorescence for L. lactis ML3 (A) and L. lactis 11454 (B) grown in various media. Cells were grown in 0.2% lactose and 2% arginine (), 0.2% lactose and no arginine (), no lactose and 2% arginine (), and no lactose or arginine (). The inset in panel A depicts data from days 0 to 3 for ML3. RFU, relative fluorescence units.

ATP concentration. As a third measure of viability, the ATP concentration was determined during growth. The ATP concentration during the growth of L. lactis ML3 followed the same pattern as plate counts and viable fluorescent population estimations (compare Fig. 2A, 3A, and 4A). However, in CDM/L/A, the ATP concentration decreased initially and then increased to the original level. The presence of ATP with the plate counts and fluorescence viability estimations showed viable cells present at a low concentration, but no cells could be cultured on solid agar. These observations suggested that L. lactis ML3 cells were viable but nonculturable, since ATP was still present in the cells.

View larger version (21K): [in this window] [in a new window]   FIG. 4.   ATP concentrations for L. lactis ML3 (A) and L. lactis 11454 (B). Cells were grown in 0.2% lactose and 2% arginine (), 0.2% lactose and no arginine (), no lactose and 2% arginine (), and no lactose or arginine ().

Amino acid profile. Extracellular amino acid concentrations were estimated by capillary electrophoresis and laser-induced fluorometry. Concentrations of histidine, methionine, glutamine, asparagine, threonine, tyrosine, alanine, leucine, phenylalanine, proline, and isoleucine were unchanged during 14 days of incubation with all media and strains tested (data not shown). In L. lactis ML3 cultures, glycine-valine and glutamate concentrations decreased by as much as 67% in CDM/L⁺/A⁺ until the lactose was depleted, after which they decreased only slightly (Fig. 5A). The decrease in extracellular glutamate and glycine or valine concentrations is in agreement with the observation of an increase in the intracellular concentrations of glutamate and glycine at the onset of starvation (22). Rapid but variable use of glutamate by lactococci was also found with L. lactis 133 and is associated with osmotic stress and ATP content for transport (12, 19, 21, 31). The rate of glutamate uptake by L. lactis increases more than 30-fold when the intracellular pH is raised from 6.0 to 7.4 due to osmotic stress (21). These observations have also been made for E. coli and Salmonella typhimurium in connection with the intracellular accumulation of potassium and glutamate, which enhances survival by regulating osmotic pressure and intracellular pH (1, 8). Hence, observations with ML3 suggest that glutamate utilization may signal stress during the incubation period (Fig. 5 and 6).

View larger version (18K): [in this window] [in a new window]   FIG. 5.   Extracellular amino acid profiles for L. lactis ML3 with 0.2% lactose and 2% arginine (A) and without lactose and 2% arginine (B). Amino acids shown are serine (), glycine or valine (), and glutamate (). RFU, relative fluorescence units.

View larger version (17K): [in this window] [in a new window]   FIG. 6.   Extracellular amino acid profiles for L. lactis 11454 with 0.2% lactose and 2% arginine (A) and without lactose and 2% arginine (B). Amino acids shown are serine (), glycine or valine (), and glutamate (). RFU, relative fluorescence units.