Purification and Characterization of a Keratinase from a

APPLIED AND ENVIRONMENTAL MICROBIOLOGY, OCt. 1992, p. 3271-3275 0099-2240/92/103271-05$02.00/0 Copyright 1992, American Society for Microbiology Vol. 58, No. 10 Purification and Characterization of a Keratinase from a Feather-Degrading Bacillus licheniformis Strain XIANG LIN,t CHUNG-GINN LEE,4 ELLEN S. CASALE, AND JASON C. H. SHIH* Department of Poultry Science and University Biotechnology Program, North Carolina State University, Raleigh, North Carolina 27695-7608 Received 27 April 1992/Accepted 21 July 1992 A keratinase was isolated from the culture medium of feather-degrading Bacilus licheniformis PWD-1 by use of an assay of the hydrolysis of azokeratin. Membrane ultrafiltration and carboxymethyl cellulose ion-exchange and Sephadex G-75 gel chromatographies were used to purify the enzyme. The specific activity of the purified keratinase relative to that in the original medium was approximately 70-fold. Sodium dodecyl sulfatepolyacrylamide gel electrophoresis analysis and Sephadex G-75 chromatography indicated that the purified keratinase is monomeric and has a molecular mass of 33 kda. The optimum ph and the pi were determined to be 7.5 and 7.25, respectively. Under standard assay conditions, the apparent temperature optimum was 50 C. The enzyme is stable when stored at -20 C. The purified keratinase hydrolyzes a broad range of substrates and displays higher proteolytic activity than most proteases. In practical applications, keratinase is a useful enzyme for promoting the hydrolysis of feather keratin and improving the digestibility of feather meal. The major component of feathers is keratin. Because of a high degree of cross-linking by cystine disulfide bonds, hydrogen bonding, and hydrophobic interactions, keratin is insoluble and not degradable by proteolytic enzymes, such as trypsin, pepsin, and papain (7, 8, 14). Despite the unusual stability of keratin, feathers do not accumulate in nature. Our laboratory has reported the isolation and characterization of a feather-degrading bacterium, Bacillus licheniformis PWD-1 (23, 24). The bacterium grows on feathers as the primary organic substrate for supplying carbon, sulfur, and energy. Biodegradation of feathers by this bacterium represents a method for improving the utilization of feathers as, for example, a feed protein. Recently, feeding experiments with chickens demonstrated a significantly better growth response when the bacterial fermentation product feather lysate (18) was added to the diet than when untreated feathers or commercial feather meal was added (18, 22). This study reports the purification and characterization of the keratinase secreted by feather-degrading B. lichenifornis PWD-1. MATERIALS AND METHODS Organism and growth conditions. The bacterium used in this study was a patented strain of B. licheniformis PWD-1 isolated in our laboratory (19, 23). All culture conditions and the feather culture medium were as previously described. The medium contained, per liter, the following: 0.5 g of NH4Cl, 0.5 g of NaCl, 0.3 g of K2HPO4, 0.4 g of KH2PO4, 0.1 g of MgCl2 6H20, 0.1 g of yeast extract, and 10 g of hammer-milled chicken feathers. The ph was adjusted to 7.5. Feathers were washed, dried, and hammer milled prior to being added to the medium. The medium was sterilized by autoclaving. The bacterium was cultured in a test tube (20 by 1.5 cm) * Corresponding author. t Permanent address: Shenyang Agricultural University, Shenyang, Liaoning, People's Republic of China. t Present address: Department of Animal Production, Council of Agriculture, Taipei, Taiwan, Republic of China. 3271 containing 10 ml of culture medium. After 4 days of incubation, 10 ml of the culture medium was transferred to a 3-liter Fernbach flask containing 1.0 liter of medium. After 4 days of incubation, the medium was collected for keratinase purification. All incubations were done at 50 C with shaking at 120 rpm in a controlled-environment shaker (New Brunswick Scientific Co., New Brunswick, N.J.). Enzyme purification. The culture medium was prefiltered through glass wool to remove residual undegraded feathers. The medium was then filtered through a 0.45-,um-pore-size membrane with a Pellicon cassette system (Millipore Corp., Bedford, Mass.) to remove bacterial cells and other particles. The filtrate was concentrated by membrane ultrafiltration (molecular weight cutoff, >10,000) with the same (Pellicon) system. The crude concentrated keratinase solution was applied to a column of carboxymethyl cellulose (CMcellulose) (2.5 by 60 cm) at 4 C. The column was equilibrated with buffer A (25 mm potassium phosphate buffer [ph 5.8]). Approximately 50 mg of protein was loaded on the column. Elution was begun with 500 ml of buffer A. The eluted proteins were monitored by measuring the A280 of the fractions. When the 280-nm reading was a continuous baseline, elution with buffer B (25 mm potassium phosphate buffer [ph 6.2], 20 mm NaCl) was begun. Approximately 300 ml was used for this elution. Finally, buffer C (25 mm potassium phosphate buffer [ph 6.8], 20 mm NaCl) was used. The elution flow rate was 0.4 ml/min, and 5-ml fractions were collected. Fractions were screened with the milk-agarose plate assay. Fractions exhibiting proteolytic activity were assayed for keratinase activity with the azokeratin hydrolysis test. Keratinase active fractions were pooled and concentrated with an Amicon stirred cell filtration system with Diaflo ultrafilters (molecular weight cutoff, >10,000) (Amicon Div., W. R. Grace and Co., Beverly, Mass.) at 4 C. Purification of keratinase was continued with a Sephadex G-75 column (1.5 by 90 cm) at 4 C. Equilibration and elution were carried out at 4 C with 50 mm potassium phosphate buffer (ph 7.0) at a flow rate of 0.3 ml/min. Fraction volumes of 3 ml were collected. Protein elution was followed by monitoring of the A280 of each fraction. Fractions were screened with the milk-agarose plate assay, and

3272 LIN ET AL. then the active fractions were assayed for keratinase activity with the azokeratin hydrolysis test. Keratinase active fractions were pooled and concentrated with the Amicon stirred cell unit. Enzyme hydrolysis of azokeratin. Azokeratin was prepared by reacting ball-milled feather powder (24) with sulfanilic acid and NaNO2 by use of a method similar to that described by Tomarelli et al. for azoalbumin (21). For a standard assay, 5 mg of azokeratin was added to a 1.5-ml centrifuge tube along with 0.8 ml of 50 mm potassium phosphate buffer (ph 7.5). This mixture was agitated until the azokeratin was completely suspended. A 0.2-ml aliquot of an appropriately diluted enzyme solution was mixed with the azokeratin, and the mixture was incubated for 15 min in a 50 C water bath. The reaction was terminated by the addition of 0.2 ml of 10% trichloroacetic acid, and the mixture was filtered. The A450 of the filtrate was measured with a UV-160 spectrophotometer (Shimadzu, Columbia, Md.). A control was prepared by adding trichloroacetic acid to a reaction mixture before adding the enzyme solution. Without knowing the molar extinction coefficient of azopeptides, we defined 1 U of keratinase activity as an increase in the A450 of 0.01 after 15 min in the test reaction compared with the control reaction. Milk-agarose plate assay. A method that was an alternative to the azokeratin hydrolysis assay was used to screen large numbers of fractions collected from CM-cellulose and Sephadex G-75 columns. One gram of agarose was dissolved in 98 ml of 50 mm potassium phosphate buffer (ph 7.5). After the agarose solution was cooled to 60 to 70 C, 2 ml of evaporated whole milk was added with gentle agitation. The mixture was poured rapidly onto a clean glass plate (20 by 20 cm) and spread evenly. After the agarose had solidified, 81 (9 by 9) small wells (5 mm in diameter) were punched in the agarose. A 25-,l aliquot of a sample was applied to each well. The plate was incubated in a moist atmosphere either overnight at 37 C or for 2 h at 50 C. Clear zones around wells were indicative of proteolytic activity. A similar method was previously published (17). Keratinase activity was further identified by azokeratin hydrolysis as described above. Free amino group assay. For the native feather keratin and other protein substrates, including casein, elastin, collagen, and bovine serum albumin (BSA) (all from Sigma Chemical Co., St. Louis, Mo.), the proteolytic activities of keratinase were determined on the basis of the production of free amino groups. A modified ninhydrin method was used for detecting free amino groups (16). Five milligrams of a protein substrate was placed in a 1.5-ml tube with 0.8 ml of buffer and 0.2 ml of enzyme solution containing 8,ug of purified keratinase. After incubation at 50 C for 30 min, the reaction was stopped by the addition of trichloroacetic acid. The reaction mixture was filtered with a 5.0-,um-pore-size syringe filter (Micron Separations, Inc., Westboro, Mass.) or centrifuged to remove insoluble protein, and a 0.5-ml aliquot was removed for a reaction with ninhydrin reagent to determine the production of free amino groups. The standard curve was determined with leucine at 0.1 to 0.5,umol. Both the free amino group assay and the azokeratin hydrolysis assay were used to compare the keratinolytic activities of a variety of proteases (Sigma). The enzymes tested were papain (EC 3.4.22.2), bovine trypsin (EC 3.4.4.4), porcine elastase (EC 3.4.21.36), Clostridium histolyticum collagenase (EC 3.4.24.3), Tntirachium proteinase K (EC 3.4.21.14), and keratinase. Protein determination. Protein concentrations were determined by the Bio-Rad (Richmond, Calif.) protein assay method as described by Bradford (3). The microassay pro- APPL. ENVIRON. MICROBIOL. cedure was performed, and BSA was used as the standard protein. Enzyme properties. The native molecular weight of keratinase was estimated by Sephadex G-75 column chromatography; calibration was done with standard proteins from a kit obtained from Pharmacia Fine Chemicals (Piscataway, N.J.). To examine the purity and determine the subunit molecular weight, we performed discontinuous sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS- PAGE) as described by Laemmlli (9). The protein bands were stained with a solution of Coomassie blue R-250. Isoelectric focusing, as described in Bio-Rad Instruction Manual 161-0310, was used to determine the isoelectric point of keratinase. The experiment was conducted with an LKB horizontal electrophoresis unit (2117 Multiphor) and LKB Ampholines. The protein bands were stained with a Bio-Rad silver stain kit by use of the method developed by Mehta and Patrick (11) and Confavreux et al. (4). The ph and apparent temperature optimum for keratinase activity were assayed by the azokeratin hydrolysis assay. To determine whether a prosthetic group is an integral part of keratinase, we scanned the enzyme solution with a Shimadzu UV-160 UV-visible recording spectrophotometer. A BSA solution of the same protein concentration was used for comparison. Determination of the production of sulfhydryl groups. Reduction of disulfides to sulfhydryls in keratin may be part of the mechanism of enzymatic keratinolysis. To test this hypothesis, we determined the production of sulfhydryl groups by use of the Ellman reaction (5) during the enzymatic reaction, while hydrolysis was monitored as the increase in the production of free amino groups as described above. One hundred milligrams of feather keratin was incubated at 50 C with 50,ug of keratinase in 15 ml of 50 mm phosphate buffer (ph 7.5). Every 10 min, 1.6 ml of reaction mixture was removed for analysis, 1.0 ml for sulfhydryls and 0.6 ml for free amino groups. RESULTS Enzymatic hydrolysis of azokeratin. Insoluble azokeratin as the chromogenic substrate was incubated with the keratinase solution to produce a soluble colored product. The supernatant demonstrated an increase in the A450 in a linear fashion in the first 30 min because of the release of azopeptide derivatives into the solution. This assay, in conjunction with prescreening by the milk-agarose plate assay, was used for the purification of keratinase. Purification of keratinase. A summary of the purification of keratinase from the culture medium of B. licheniformis PWD-1 is presented in Table 1. Membrane ultrafiltration and CM-cellulose ion-exchange and Sephadex G-75 gel chromatographics yielded a purified keratinase fraction having an overall purification factor of 70-fold. The final product had a specific activity of about 6,000 U/mg. The ultrafiltration step removed approximately 60% of the total protein while maintaining 70% of the enzyme activity. CM-cellulose column chromatography purified the enzyme 43-fold and removed 98% of the original total protein. Elution from the Sephadex G-75 column yielded a homogeneous protein, as shown by a single protein band in SDS-PAGE (Fig. 1A). Enzyme properties. The purified PWD-1 keratinase is a monomeric protein with a molecular mass of 33 kda, as estimated by both Sephadex G-75 chromatography (data not shown) and SDS-PAGE (Fig. 1). The ph optimum and apparent temperature optimum of keratinase activity, as determined by the azokeratin hydrolysis assay, are 7.5 and

VOL. 58, 1992 TABLE 1. Purification of keratinase from the medium of B. licheniforinis PWD-1 Total Total Sp act Purifi- Step protein ua (U/mg of % of U cation (mg) protein) (fold) Medium 142 12,200 86 100 1.0 Membrane concen- 54.5 8,450 150 69.3 1.8 tration CM-cellulose chro- 3.3 12,080 3,720 99 43 matography Sephadex G-75 1.5 8,910 5,990 73.1 70 chromatography a One unit is defined as an increase in the A450 of 0.01 after reaction with azokeratin for 15 min. 50 C, respectively. Isoelectric focusing determined that the isoelectric point is 7.25. The keratinase is fairly stable upon storage at low temperatures. A solution sample (30,ug/ml) was found to lose only 7% of its activity at -20 C and 20% of its activity at 4 C after 19 days of storage. However, at room temperatures (20 to 25 C), the half-life of keratinase activity is 4 to 5 days. The loss of enzyme activity is largely due to enzymatic autolysis, which was detected by SDS- PAGE (data not shown). The UV-visible absorption spectrum of the keratinase was found to be identical to that of BSA. No absorption peak besides the typical absorption peaks at 280 and <220 nm for a protein molecule was detected. Enzyme specificity. A free amino group assay was used to compare the substrate specificities of a variety of proteases. The keratinase was capable of hydrolyzing all the proteins tested, including BSA, casein, collagen, elastin, and feather keratin. As demonstrated in Table 2, both the native feather keratin and synthetic azokeratin were specific substrates for keratinase. They were degraded less by other proteases. Sulfhydryl analysis. During the enzymatic hydrolysis of feather keratin, sulfhydryl groups in the reaction mixture were monitored (Fig. 2). No increase in the production of free sulfhydryl groups was detected during the reaction. On the other hand, the production of free amino groups increased as a result of peptide bond cleavage in a linear fashion over a 1-h period. DISCUSSION The combination of the milk-agarose plate assay and the azokeratin hydrolysis assay was demonstrated to be effective for the purification of the keratinase from the culture medium of B. licheniformis PWD-1. The milk-agarose plate assay is a simple method for screening large numbers of fractions for proteolytic activity. For the identification of keratinase activity, the azokeratin hydrolysis assay is simple and specific. The two methods correlated well in that the active fractions showing proteolytic activity on the milkagarose plate also showed keratinase activity. Therefore, only one major protease, the keratinase, was present in the bacterial medium. To further confirm the keratinolytic activity, we also used a third method, measuring the increase in the production of free amino groups upon hydrolysis of the native feather keratin. Both the native feather keratin and synthetic azokeratin have been shown to be more specifically hydrolyzed by keratinase than by other proteases (Table 2). The purification of keratinase was effective and efficient. 1 0 5 0 4 Iv n 4 B. LICHENIFORMIS KERATINASE 3273 M.W(KDa) 1 97.4 66.2 2 3 4 5 4-3 1 -m -_ 21.5 1 4.4 m phosphorylase b BSA ovalbumin A keratinase w. M carbonic anhydrase lysozyme trypsin inhibitor T 0.0 0.2 0.4 0.6 0.8 1.0 Relative mobility B FIG. 1. SDS-PAGE of purified keratinase. (A) SDS-polyacrylamide gel. Lanes: 1 and 3, purified keratinase; 2, molecular mass standards; 4, CM-cellulose-treated preparation; 5, crude enzyme preparation. (B) Molecular mass standard curve. Standards: phosphorylase b, 97.4 kda; BSA, 67 kda; ovalbumin, 43 kda; carbonic anhydrase, 31 kda; soybean trypsin inhibitor, 21.5 kda; lysozyme, 14.4 kda. About 73% of the total activity was retained, while only 1% of the original total protein remained. However, concentration of the medium by membrane ultrafiltration resulted in a decrease in the total enzyme units. The total enzyme units returned to the original level after ion-exchange chromatography (Table 1). This phenomenon was repeatedly observed. The concentration of the proteins may be such that the

3274 LIN ET AL. APPL. ENVIRON. MICROBIOL. Substrate Temp ( C) TABLE 2. Relative keratinolytic activities of proteases in two different assays Keratinolytic activity of: Papain Trypsin Collagenase Elastase Proteinase K Keratinasea Feather keratin 37 0 13 0 17 36 41 50 0 16 0 27 51 100 Azokeratin 37 8 23 2 35 39 64 50 7 27 1 47 52 100 a For the feather keratin substrate, the specific activity of purified keratinase is 118 pmol of leucine equivalents released per h per mg of enzyme protein; for the azokeratin substrate, the specific activity is 4,700 U, and 1 U is defined as an increase in the A450 of 0.01 after reaction for 15 min under standard assay conditions. keratinase is inhibited by an unknown but coconcentrated factor. The keratinase molecule is monomeric. Molecular weights were found to be identical when they were determined by two different methods, SDS-PAGE and Sephadex G-75 gel chromatography. The enzyme is relatively thermostable, with an apparent optimum temperature of 50'C. Cold storage at -20 C is stable. At room temperatures, autolysis results in a 50% loss of activity in 4 to 5 days. On the basis of its pl of 7.25, determined by isoelectric focusing, keratinase is a nearly neutral protein. This conclusion is verified by the fact that keratinase binds to CMcellulose at an acidic ph (5.8) and is not released until ph 6.8 is reached. The optimum ph (7.5) for keratinase is close to its pi, indicating that keratinase is most active when it carries the least net. The keratinase not only can hydrolyze keratin but also has the ability to hydrolyze a wide variety of soluble and insoluble protein substrates. The soluble substrates BSA and casein are readily degradable, while the insoluble substrates collagen, elastin, and keratin are less so. The keratinase is 0.4-0.2 E 0.3-[-NH 2] 0~~~~~~~~~ E =10. 2 0.2 0.1 Lo 0 U)0.1 Time(min)~~~~~~~~~~~m- [-HJ 0.0 ~ * I 0.0 0 2 0 4 0 6 0 8 0 Time (min) FIG. 2. Analysis of free amino and sulfhydryl groups during enzymatic keratinolysis. ID CD more hydrolytic for elastin and collagen than some elastases and collagenases (data not shown). Elastin and collagen are similar to keratin in that they are insoluble, highly structured, and recalcitrant to many proteases. However, examination of the individual structures of these proteins reveals quite different assemblies. Collagen, like keratin, is a fibrous protein and consists of three a chains assembled in a triple helix conformation, resulting in a rod-like shape. Elastin is not a fibrous protein but rather is a polymer of globular proteins assembled in a fibrous arrangement. Both elastin and collagen are stabilized by covalent intermolecular crosslinkages involving lysine and lysine derivatives (1, 2, 13, 15, 20). These properties provide some degree of resistance to peptide hydrolysis. The unique structure of keratin makes it very resistant to proteolytic digestion. The resistance is due not only to the supercoiled helical structure of polypeptide chains but also to the strength of intermolecular disulfide bonds and other molecular interactions (6, 12). One of the possible mechanisms in breaking down keratin is the reduction of disulfide bonds. For instance, pretreatment of keratin with a reducing agent, such as thioglycolate, disrupts the disulfide bonds and increases digestibility by trypsin (7). However, such a mechanism is not likely in the case of the keratinase. First, it degrades keratin without a reducing agent or coenzyme, and second, enzymatic keratinolysis is not coupled with an increase in the production of detectable sulfhydryls (Fig. 2). The molecular mechanism of the enzymatic action remains to be determined. In summary, a simple colorimetric assay for keratinase was developed. With this method, a keratinase was purified from newly discovered B. licheniformis PWD-1. The keratinase was characterized in terms of its biochemical properties and found to have a broad substrate specificity. The reduction of disulfide bonds to sulfhydryls was not detected in the hydrolysis process. The use of keratinase as a feed additive to improve the digestibility of feather meal in chickens has been demonstrated in our laboratory (10). The purification and characterization studies of the keratinase have provided the basis to develop further the production and uses of this enzyme. ACKNOWLEDGMENT We appreciate the grant support from Southeastern Poultry and Egg Association. REFERENCES 1. Anwar, R. A., and G. Oda. 1966. Biosynthesis of desmosine and isodesmosine. J. Biol. Chem, 241:4638. 2. Bailey, A. J., S. P. Robins, and G, Balian. 1974. Biological significance of the intermolecular cross-links of collagen. Na-

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