Lysine decarboxylase activity of nonflat sour spore bearers resembling bacillus coagulans.
Key words: bacillus, aciduric, effervescence, spore-formers, lysine decarboxylase, cadaverine.
The activity of the lysine decarboxylase enzyme produced by bacteria results in breakdown of lysine and the formation of cadaverine with liberation of C[O.sub.2]. Gale and Epps (1944) described the specificity and reactions of the decarboxylase enzymes for six amino acids: lysine, ornithine, arginine, tyrosine, histidine, and glutamic acid. Yamakawa and Taira (1980) detected ornithine decarboxylase activity in B. subtilis cells during growth and sporulation. Ornithine decarboxylase showed two peaks of activity at pH levels of 5.5 and 8.5. This biphasic pattern of enzyme activity was paralleled by a biphasic pattern of polyamine level in the sporulating cells. Lysine decarboxylase is usually determined manometrically, as are some other decarboxylases (Gale 1974, Gale and Epps 1944, Najjar 1957, Neuberger and Sanger 1944, Soda and Moriguchi 1969). Another way to assay for amino acid decarboxylase is through the use of [sup.14]C-labelled substrates (Phan et al. 1982, Morris and Pardee 1965). These procedures involve trapping [sup.14]C[O.sub.2] in alkaline solution (Phan et al. 1982) or separating the substrates from the products by paper chromatography (Morris et al. 1965), electrophoresis (Phan et al. 1982), ion exchange resins (Tonelli et al. 1981), or spectrophotometric assay. Studies indicated that lysine decarboxylase might be subject to an end-product feedback inhibition mechanism in vivo (Pelosi et al. 1986, Stevens et al. 1978). Pelosi et al. (1986) reported that the influence of cadaverine on lysine decarboxylase was regulated by cadaverine at 10 mM concentration. This feedback inhibition obtained at 10 mM may not be relevant to the in vivo regulation of enzyme as in vivo cadaverine levels reported have been 0.1 mM. Kelland et al. (1986) reported that N-hydroxy and N-amino diamino-pimelates do not inhibit L-lysine decarboxylase from B. cadaveris.
Certain isolates of Bacillus elevate the pH of acid foods (Anderson 1984, Shamsudin 1984, Al-Dujaili and Anderson 1991). Based on physiological, biochemical, and genetic studies, these isolates share similar properties with B. coagulans. At the same time, they have other dissimilar properties, such as possessing lysine decarboxylase enzyme (Al-Dujaili and Anderson 1991) that may be linked to pH elevation. To our knowledge, there have been no published reports concerning the activity of lysine decarboxylase enzyme in these isolates. Therefore, the objectives of this study were to measure the activity and the optimum pH of lysine decarboxylase, as well as the pattern of lysine decarboxylase during different growth stages of Bacillus strains. In addition, the activity of this enzyme was compared among the isolates and with other B. coagulans strains.
MATERIALS AND METHODS
Criteria for Isolation
Desired colonies were those that grow on Bacillus Tomato Juice Agar (BTJA) and which produce zones of purple in the yellow, acidified medium. Single colonies with a purple margin and background were carefully picked for re-streaking on BTJA if they had a dry, flake-like appearance similar to the reference Fields, strain (FS) (Fields et al. 1977). Four isolates were used in this study. These isolates were 1-86 (Home-canned whole tomatoes), [Po.sub.1]-87 (potatoes), and [T.sub.1]-88, [T.sub.2]-88 (tomatoes).
B. coagulans 064-T-08 (FS) and B. coagulans NRS 54 were used as reference cultures. All cultures were aerobically grown on trypticase soy agar slants (TSA) (BBL Microbiology systems, Cockeysville, MD) or BTJA at 44[degrees]C. Stock cultures were maintained at 7[degrees]C in 16125 mm screw cap tubes on TSA slants.
Chemicals and Equipment
Except where noted, all chemicals were obtained from Sigma Chemical Co. Lysine (L-[U-[sup.14]C]) was purchased from Amersham Corporation, IL. Mcllvaine Buffer (citrate-phosphate buffer) was prepared according to the Mcllvaine method (Colowich and Kaplan 1955).
Enzyme Assay Equipment
A specifically designed 50 mi Erlenmeyer flask was used to conduct the lysine decarboxylase assay. A 9.5 cm glass stirring rod was fused to a 2 cm section of hollow glass tubing, which was closed at one end. A 3 cm x 1.0 cm square of #1 filter paper was inserted into the glass cup. This entire apparatus was tightly attached to the flask with a rubber stopper.
Cells were grown in a modified Schaeffer's medium (Schaeffer 1969) containing 10 g/liter nutrient broth (DIFCO), 0.5 g/liter MgS[O.sub.4]*7[H.sub.2]0, 2.0 g/liter KCl, [10.sup.-3] M Ca[(N[O.sub.3]).sub.2], 10-4 M Mn[Cl.sub.2], 10-6 M FeS[O.sub.4], and 0.1% glucose (Yamakawa and Taira 1980).
One colony from each strain was inoculated into 20 ml of modified Schaeffer's medium and incubated with shaking for 6 h at 37[degrees]C. A 10 ml aliquot of each culture was transferred to 200 ml of fresh modified Schaeffer's medium contained in a 2 liter Erlenmeyer flask. The culture was aerated vigorously at 37[degrees]C with a rotary shaker (New Brunswick Scientific). Cells were harvested after O h, 12 h, 16 h, 24 h, and 48 h.
Preparation of Lysine Decarboxylase Enzyme
Cells harvested at the desired periods of growth were centrifuged at 15,000 x g for 10 min, chilled, and washed with 100 mM Tris-HCl buffer solution (pH 7.4) containing 5 mM dithiothretol (DTT), 0.1 mM ethylenediamine-tetraacetate (EDTA). Suspensions were centrifuged at 15,000 g for 10 min, chilled and washed with 100 mM Tris HCl buffer solution (pH 7.4) containing 5mM dithiothretol (DTT), 0.1 mM ethylenediaminetetraacetate (EDTA) and 0.4 mM pyridoxal phosphate (PALP). The suspensions were centrifuged at 15,000 g for 10 min (Sorvall RC-5B refrigerated superspeed centrifuge, Dupont Co., Newton, CT) at 5[degrees]C and frozen overnight at -20[degrees]C. The frozen cells were homogenized in a mortar with pre-chilled quartz for 5 min and suspended in 10 mM Tris-HCl buffer solution (pH 7.4) containing 0.1 mM phenylmethyl-sulfonyl fluoride (PMSF), 5 mM DTT, 0.1 mM EDTA and 0.4 mM PALP. The extract was centrifuged at 15,000 g for 10 min, and the supernatant was dialyzed against 100 volumes of the Tris-HCl buffer solution (pH 7.4) containing 5 mM DTT, 0.1 mM EDTA and 0.4 mM PALP at 4[degrees]C for 16 h. Spectral Por 4 dialysis membrane tubing (Fisher Scientific) with molecular weight cutoff (MWCO) of 12,000-14,000 was used in the dialysis process. Dialyzed cell-free extracts were held at 4[degrees]C prior to decarboxylase tests and protein analysis.
The activity of lysine decarboxylase was determined using a modified version of the method used in the case of mammalian (Morey and Ho 1976), E. coli (Holtta et al. 1972), and B. subtilis cells (Yamakawa and Taira 1980). The [sup.14]C[O.sub.2] produced upon decarboxylation of [U-[.sup.14]C] lysine was measured. The reaction mixture consisted of McIlvaine buffer solution or 100 mM Tris HCl buffer solution (pH 7.5) with 5 mM DTT, 0.4 mM PALP, 10 mM L-lysine, 0.5 [micro]ci L-[sup.14]C-lysine (specific activity), and 0.5 ml enzyme preparation in a total volume of 0.2 ml. Duplicate cultures and other samples were used throughout this study.
The reaction was carried out at 37[degrees]C for 60 min in a glass Erlenmeyer flask. Filter paper containing 0.25 ml of ethanolamine and methylcellosolve mixture (2:1) was set in the glass cups and trapped the [sup.14]C[O.sub.2] released from the reaction mixture. The [sup.14]C[O.sub.2] production increased linearly with an increase of enzyme protein concentration for at least 60 min. Adding 0.25 ml of 50% trichloroacetic acid solution into the reaction mixture terminated the reaction. An additional incubation was performed at 37[degrees]C for 60 min to release all [sup.14]C[O.sub.2] liberated from L-[sup.14]C-lysine and to complete entrapment of [sup.14]C[O.sub.2] on the filter paper.
Filter papers were then removed to 20 ml vials, and 10 ml scintillant was added (multi-purpose pre-mixed liquid scintillation cocktail--Beckman). Radioactivity was counted with a liquid scintillation system LS 5801 (Beckman Instruments, Nuclear Systems Operation, Irvine, CA).
Protein was quantified using Bradford's (Bio-Rad) assay. Calorimetric determination was performed on a Perkin-Elmer Model 55B single-beam spectrophotometer (The Perkin-Elmer Co., Oak Brook, IL), with absorbency read at 595 nm. Bovine serum albumin (Sigma, #7656) was employed as the protein standard. Protein content of extracts was determined from a standard calibration curve utilizing StatGraphic[TM] (SAS Institute Inc.) software and a simple linear regression model.
Determination of pH Optima
The dialyzed supernatant from the selected strain was assayed at different pH levels. McIlvaine buffers, ranging in pH from 3.0 to 6.5, and Tris-HCl buffer solution (pH 7.5) were used under standard conditions.
Determination of Potential Inhibitor Effect on Lysine Decarboxylase Activity by Cadaverine
Two methods were used in this study. These methods were quantitative and qualitative. Among 24 isolates, isolate 1-86 was selected to represent all the isolates throughout this study, since there were no differences among all isolates in lysine decarboxylase activity.
I. Quantitative Method
Cadaverine was added at levels of 0, 10, 20, 30, and 40 mM to the standard assay described in the previous section. The decarboxlation of lysine was measured by trapping and counting the C[O.sub.2] evolved from L-[sup.14]-Clysine in the presence of different levels of cadaverine.
II. Qualitative Method
Cadaverine was added aseptically at levels of 0, 40, 80, and 120 mM to the sterilized BTJA medium. Strain 1-86 was aseptically inoculated in the medium. The culture was incubated aerobically at 37[degrees]C for 2-4 days. The effects of different levels of cadaverine on onset of strain 1-86 in BTJA were recorded after 8 h, 16 h, 24 h, and 48 h of incubation. Growth of isolate 1-86 and media color change (pH change) were recorded at each observation.
RESULTS AND DISCUSSION
The results of this study demonstrate the presence of lysine decarboxylase activity in all of the isolates and B. coagulans 064-T-08 (Tables 1 and 3). The occurrence, purification, and properties of inducible lysine decarboxylase have been shown in microorganisms (Sabo and Fisher 1974, Soda and Moriguchi, 1969). However, there has been no previous report of a constitutive lysine decarboxylase. The results of the present study strongly suggest that the synthesis of lysine decarboxylase is constitutive in the isolates. Further studies are desirable to clarify this possibility. In Escherichia coli, synthesis of the enzyme is not only inducible but also dependent on the concentration of glucose in the medium (Sabo and Fisher 1974). In contrast, the amount of enzyme synthesized in all isolate cells was independent of glucose concentration.
Lysine decarboxylase was synthesized in cells grown in modified Schaeffer's medium without lysine. Schaefer and colleagues (1969) observed that the sporulation of cells in growing cultures was affected by varying the nitrogen sources in the medium.
The results show that the optimum pH for lysine decarboxylase from the 24 h aged culture was 5.5 (Tables 1 and 3). However, the lysine decarboxylase activity in the acidic cultures (pH 5.5, 24 h) increased markedly up to the 48 h stage. At the same time, the results also indicate that the lysine decarboxylase specific activity was low at all pH levels for the control flat sour reference strains of B. coagulans (Table 2). This observation indicates that the flat sour strains do not possess the potential for lysine decarboxylase activity at all pH levels. Therefore, these strains have the ability to decrease the pH of acid foods. However, the isolates have the ability to grow and survive at the low pH levels of acid food; then they can increase the pH of the media by utilizing the amino acids and/or an organic acids.
However, it has been generally observed that regarding Bacillus, the pH of the medium drops to reflect the accumulation of organic acids from the vegetative cells and the pH rises from the induction of TCA cycle enzymes (Crabb et al. 1975, Jones and Spencer 1985) after the cells initiate sporulation. Lysine decarboxylase is found in our isolates at different growth stages. As the age of the culture increased, the activity of enzyme was increased, up to the 48 h culture. The activity of lysine decarboxylase was 1.0 nm/mg enzyme protein in 48 h culture age (Table 4). This enzyme is present at a relatively high specific activity level during growth, and the specific activity is maintained in the early sporulation stages (after 24 h in modified Schaeffer's medium). However, the fact that no great increase in specific activity occurs suggests that this enzyme does not have a catabolite-repressed function nor is it depressed by the environmental changes which occur when a cell goes from the log phase to the stationary phase of growth. This latter transition is similar to a step down from a rich to a poor medium (less in carbon and nitrogen sources).
Lysine decarboxylase is of interest to scientists concerned with sporulation of Bacillus strains because it displays significant activity in foods that are rich in lysine. These foods are beef and corn, which when canned in combination with tomato juice would hasten the increase in pH and create favorable conditions for outgrowth of C. botulinum if these foods are contaminated by Bacillus strains that elevate the pH of acidic food. This enzyme, when assayed with lysine (10 mM) as substrate, increased activity linearly with the age of the culture. The enzyme activity increased linearly with the pH of the assay media until pH 5.5. Enzyme activity was decreased as the pH increased to 7.5 (Table 4).
There are two possible mechanisms which may control the sequential appearance of lysine decarboxylase: (i) Repression of lysine decarboxylase requires a high concentration of an intracellular metabolite (cadaverine) and depression may set in immediately after the rapidly metabolizable carbon sources have been used up. (ii) Another mechanism of control occurs if some newly formed enzyme (i.e., diamine oxidase) produces and uses up an existing metabolite (cadaverine) and thereby induces the lysine decarboxylase in turn. Gerdes and Leistner (1979) demonstrated that the diamine oxidase was dehydrogenated cadaverine in B. cadoveris. Popkin and Maas (1980) indicated that the E. coli mutants blocked in putrescine synthesis are capable of slow residual growth without putrescine supplementation. When a polyamine auxotroph is grown under conditions of polyamine starvation, cadaverine is present in the perchloric acid-extractable pool. Presumably, cadaverine and its amino propyl-derivative are able to substitute partially for the growth-promoting properties of putrescine and spermidine. Thus, they concluded that cadaverine synthesis in polyamine starved polyamine auxotrophs must occur via release of feedback inhibition of lysine decarboxylase rather than by induction of this enzyme.
An extracellular cadaverine compound represses lysine decarboxylase. The repressor effect can also be seen by measuring the specific activity of lysine decarboxylase in the presence of cadaverine at different levels. The specific activity was decreased as the cadaverine levels increased from 10 to 40 mM. Occasionally, a biochemical reaction catalyzed by lysine decarboxylase is repressed by the cadaverine. This leads to inability to elevate the pH of the media in isolates of Bacillus strains that lack (depressed) lysine decarboxylase.
The effects of different levels of cadaverine as potential inhibitors of enzyme activity were observed in this study. These observations suggested that if lysine decarboxylase activity is regulated by feedback inhibition, the inhibition is not be exerted at a level of 10 mM cadaverine. However, at levels of 20, 30, and 40 mM cadaverine showed decreased enzyme activity. The results showed that at pH 5.59 the cadaverine is more effective as a potential inhibitor than at pH 5.0. The results also showed that of the isolates tested, cadaverine at 40 mM had the highest inhibitory effect on enzymes of isolate 1-86 at either pH 5.0 or 5.5 (Table 5).
The inhibition seen at 20-40 mM concentration may not be relevant to the in vivo regulation of lysine decarboxylase activity, because cadaverine concentration in vivo have been found to be very low (Table 5). Pelosi et al. found that 5 mM concentration of trans-2-hydroxy-4(3-hydroxy-4-methoxy phenyl) quinolizidine and Vertine inhibited activity to 28 and 16% of control, respectively, implying that lysine decarboxylase activity may be subject to an end-product feedback inhibition mechanism in vivo which allows alkaloid levels to reach 5 mM in the vicinity of their biosynthesis. It is also possible that such concentrations are never reached. Kelland et al. (1986) reported that neither N-hydroxy nor N-amino diaminopimelates inhibited lysine decarboxylase from B. cadavens.
The effect of cadaverine as a potential inhibitor was also observed on growth of isolates in BTJA. The effect of cadaverine was noted on the pH change of the BTJA. However, the results indicated that the 40 mM cadaverine had no inhibitory effect on growth of bacteria on BTJA. Hence the cadaverine had an inhibitory effect on lysine decarboxylase activity at levels of 80 and 120 mM. Therefore, there are correlations between the activity of lysine decarboxylase and the change in the pH of the medium from the acid (yellow color) to the alkaline range (pink color).
The activity of lysine decarboxylase in Bacillus strains was tested and compared with the flat sour strains of B. coagulans. This enzyme system is present in our Bacillus strains grown under a variety of conditions. At the same time, this study also indicated that the lysine decarboxylase activity was 0.05 nMole [sup.14]C[O.sub.2]/mg protein/min at all pH levels for the flat sour strains of B. coagulans. This observation strongly indicates that the flat sour strains do not possess lysine decarboxylase activity at any pH levels. Therefore, the flat sour strains have the ability to decrease the pH of acid foods. However, our isolates have the ability to grow and survive at the low pH levels of acid foods, and then can increase the pH of the acid foods by utilizing the amino acid.
Lysine decarboxylase is repressed by extracellular cadaverine. This resuited in depressed lysine decarboxylase activity of our isolates and a corresponding lack of ability to elevate pH. With use of BTJA, this lack of pH elevation was always repeated.
TABLE 1. Lysine decarboxylase activity of Isolate 1-86 (24 h culture) at various pH levels. Specific activity Initial pH (nMole [sup.14]C[O.sub.2]/mg protein/min) 3.08 0.097 4.0 0.577 4.5 0.630 5.5 1.050 6.5 0.754 7.5 0.633 TABLE 2. Lysine decarboxylase activity of Bacillus coagulans NRS-54 (24 h culture) at various pH levels. Specific activity Initial pH (nMole [sup.14]C[O.sub.2]/mg protein/min) 4.0 0.055 4.5 0.051 5.5 0.055 6.5 0.053 7.5 0.050 TABLE 3. Lysine decarboxylase activity of isolates (24 h culture) P[O.sub.1]-87, [T.sub.1]-88, [T.sub.2]-88, and B. coagulans 064-T-08 at various pH levels. Specific activity (nMole [sup.14]C[O.sub.2]/mg protein/min) Isolates Initial pH P[O.sub.1]-87 [T.sub.2]-88 [T.sub.2]-88 064-T-08 4.5 0.640 0.645 0.653 0.650 5.5 0.983 1.080 1.030 0.985 6.5 0.760 0.736 0.739 0.750 7.5 0.618 0.629 0.620 0.620 TABLE 4. Lysine decarboxylase specific activities at different ages of isolate 1-86, initial pH 5.5. Specific activity Hours of incubation (nMole [sup.14]C[O.sub.2]/mg protein/min) 0 0.031 12 0.798 16 0.987 24 1.050 48 1.142 TABLE 5. Effects of different levels of cadaverine on the activity of lysine decarboxylase from isolate 1-86 (24 h culture). Specific Activity (nMole [sup.14]C[O.sub.2]/mg protein/min) Cadaverine (mM) Initial pH 0 10 20 30 40 5.00 0.983 0.961 0.757 0.646 0.493 5.50 1.047 0.972 0.720 0.591 0.431
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Jameel S. Al-Dujaili Division of Sciences Louisiana State University at Eunice Eunice, LA 70535
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|Author:||Al-Dujaili, Jameel S.|
|Publication:||The Proceedings of the Louisiana Academy of Sciences|
|Date:||Jan 1, 2001|
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