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Strains of Bacillus licheniformis and Bacillus subtilis may synthesize a water-soluble, viscous slime material containing D- and L-glutamic acid residues. This polyglutamic acid (PGA) is polymerized via amide linkages between the -amino and -carboxylic groups of the amino acid residues. PGA is the principal component of "Itohiki-natto," a traditional Japanese food prepared from steamed soybean by the biological action of PGA-producing strains of B. subtilis, which are generally referred to as "Bacillus natto" or B. subtilis (natto) (17). PGA has other biotechnological applications in cosmetics, medicines, and foods. However, the synthesis of even small amounts of PGA can be a problem in the fermentation industry, most notably in the production of extracellular enzymes from bacilli, where PGA accumulation causes increased viscosity of the fermentation broth, reduced enzyme yield, uncontrollable foaming, and complications in product recovery. The unpredictable nature of PGA synthesis is particularly troublesome.

Cultural conditions affecting PGA biosynthesis have been studied using various poorly identified strains of B. licheniformis and B. subtilis in complex, ill-defined media, and these studies have resulted in conflicting conclusions. In general, strains have been classified into two categories: (i) those that require exogenous L-glutamate for PGA synthesis, for example, B. subtilis strains IFO 3335 (6) and F-2-01 and B. licheniformis 9945 (16) (although Birrer et al. reported that strain 9945 does not require exogenous glutamate for PGA synthesis [2]), and (ii) strains that do not require exogenous glutamate (often described as de novo synthesis), such as B. licheniformis A35 (3) and B. subtilis TAM-4 (10).

The mechanisms of PGA biosynthesis in B. licheniformis and B. subtilis have been elusive until recently. A membranous synthetase complex from B. licheniformis which catalyzes the activation, racemization, and polymerization of L-glutamate into exclusively poly-D-glutamate has been partially characterized (5), but B. subtilis (natto) has been studied more extensively in this context. A gene originally thought to code for -glutamyltranspeptidase (GGT) (8) but later referred to as a PGA "stimulating factor" (7) has been cloned and sequenced, although its role in PGA synthesis is unknown. More recently, an unrelated gene coding for an extracellular GGT has been cloned and sequenced from B. subtilis (natto) (12) which is essentially identical to a ggt gene previously cloned from B. subtilis (18). However, the recent cloning and characterization of three genes, pgsA, -B, and -C, that are homologs of the Bacillus anthracis cap genes (1) casts doubt on the relevance of the ggt genes and GGT in PGA synthesis.

In this study we show that PGA synthesis is repressed by exogenous L-glutamate in strains of B. licheniformis but not in B. subtilis strains. Moreover, a role for extracellular GGT in PGA production has been shown to be unlikely through the poor correlation between enzyme activity and PGA yield and the finding that B. subtilis S317 synthesizes large amounts of PGA in the absence of detectable GGT activity.

Analytical procedures. B. licheniformis and B. subtilis strains (Table 1) were grown in basal salts comprising (per liter) glucose (20 g), K₂HPO₄ (14 g), KH₂PO₄ (6 g), MgSO₄ · 7H₂O (0.2 g), and 1 ml of trace element solution (FeSO₄ · 7H₂O, CaCl₂ · 2H₂O, MnSO₄ · 4H₂O, ZnCl₂; 1 mM each) at 37°C. Nitrogen sources were added as described in Tables 1 and 2. Medium (20 ml) in 100-ml flasks was inoculated with 100 µl of a frozen spore suspension in 20% glycerol and was shaken at 250 rpm. All data are average results from three separate flask cultures with less than 12% variation. PGA was determined in culture supernatants after clarifying cultures by centrifugation at room temperature. The supernatant was dialyzed against water for 24 h at 4°C and hydrolyzed with an equal volume of 6 M HCl at 100°C overnight. The hydrolysate was neutralized with 6 M NaOH, and the quantity of glutamic acid in the solution was determined using ninhydrin with glutamic acid as a standard. Glutamate was confirmed as the sole product in the hydrolysate by thin-layer chromatography on Silica Gel-60 plates (Merck) using n-butanol-acetic acid-water (12:3:5). The plates were dried and sprayed with acetone containing 0.2% ninhydrin and 2% collidine to visualize the amino acids (data not shown). The amount of PGA is given as glutamic acid equivalent to the difference between the hydrolyzed and unhydrolyzed samples. The degree of polymerization of PGA was analyzed by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The polymer was purified from culture supernatants using ethanol precipitation and deoxyribonuclease I treatment (10) before SDS-PAGE in a 10% gel. The protein standards (ovalbumin, 45 kDa; serum albumin, 66.2 kDa; phosphorylase b, 97 kDa) were stained with Coomassie brilliant blue and, after destaining in 7% acetic acid-10% methanol, the gel was stained for PGA with 0.5% methylene blue in 3% acetic acid and destained in water.

Effect of glutamate on PGA biosynthesis in B. licheniformis and B. subtilis. The effect of exogenous L-glutamate on PGA accumulation and GGT activity in various strains of B. licheniformis and B. subtilis is shown in Table 1. The typical culture density (optical density at 650 mm [OD₆₅₀]) after incubation for 24 h was between 2.4 and 3.5 with (NH₄)₂SO₄ as the nitrogen source, between 1.0 and 3.5 with glutamate as the nitrogen source, and between 2.0 and 4.0 with casein hydrolysate. PGA accumulation was determined at the beginning of stationary phase, when the yield was likely to be maximal (Fig. 1). This is a considerably shorter incubation period than that used by others, which varied between 48 and 96 h (2, 3, 5), but this probably reflects the simplicity and low concentrations of medium components used here. Although depolymerization of PGA ensues as the bacterium enters stationary phase, this is a slow process (2, 4, 6), and electrophoresis of precipitated PGA showed that there was no substantial degradation until incubation beyond 30 h (Fig. 2).

View larger version (22K): [in this window] [in a new window]   FIG. 1.   Growth of B. licheniformis S173 and PGA biosynthesis in medium containing casein hydrolysate as the nitrogen source. Symbols: , OD660; , PGA accumulation. At the times indicated by the arrows, the cultures were equally divided into two flasks and sodium glutamate was added to 4 g/liter to one of the flasks. Incubation of both flasks continued for 26 h, and the final yield of PGA after incubation in the presence of sodium glutamate is given in milligrams per milliliter above the arrows.

View larger version (68K): [in this window] [in a new window]   FIG. 2.   Molecular size of extracellular PGA from B. licheniformis S 173 as a function of culture age (numbers above lanes represent hours since inoculation). PGA was precipitated from culture supernatant and examined by SDS-PAGE (see text for details).

Factors affecting PGA synthesis in B. licheniformis S173. Given the industrial importance of B. licheniformis in the extracellular enzyme industry (14), we examined the synthesis of PGA in B. licheniformis S173 in more detail. PGA synthesis was estimated after growth in basal salts medium containing (NH₄)₂SO₄ (4 g/liter) as the nitrogen source with various carbon sources. Glucose, maltose, sucrose, and xylose supported similar levels of PGA synthesis (0.6 to 0.9 mg/ml). Citric acid stimulated maximal synthesis (1.0 mg/ml), and with galactose as the carbon source, PGA was undetectable. Citrate has been noted previously for supporting high levels of PGA synthesis in B. licheniformis 9945a (11) and B. subtilis IFO 3335 (6), which suggests that the requirement for the tricarboxylic acid cycle for metabolism of citrate enhances the production of -ketoglutarate as the direct precursor for glutamate and PGA (4).