Biomacromolecules 2003, 4, 424-428
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First-Order Kinetics Analysis of Monomer Composition Dependent Polyhydroxyalkanoic Acid Degradation in Pseudomonas spp. Mun Hwan Choi,† Jong Kook Rho,† Ho-Joo Lee,† Jae Jun Song,§ Sung Chul Yoon,*†,‡ and Sang Yeol Lee†,‡ Biomaterials Science Laboratory, Division of Life Science at the College of Natural Sciences, Gyeongsang National University, Chinju 660-701, Korea, Division of Applied Life Sciences (BK21) at the Graduate School, Gyeongsang National University, Chinju 660-701, Korea, and Environmental Bioresources Laboratory, Korea Research Institute of Bioscience and Biotechnology (KRIBB), Yusong, Taejon 305-600, Korea Received November 2, 2002; Revised Manuscript Received January 18, 2003
The intracellular degradation of polyhydroxyalkanoic acid (PHA) in pseudomonads was investigated by first-order kinetics analysis using the initial rate method. One type of PHA was accumulated in five Pseudomonas spp., P. oleoVorans, P. aeruginosa, P. fluorescens, P. citronellolis, and P. putida, by growing them on octanoic acid. The monomer compositions of the five PHA were not significantly different from one another: 85-90 mol % 3-hydroxyoctanoic acid (3HO), 7-12 mol % 3-hydorxycaproic acid (3HC), and 3-6 mol % 3-hydroxydecanoic acid (3HD). The first-order degradation rate constants (k1) for the octanoate-derived PHA (designated P(3HO)) in the five species were in a similar range between 0.060 and 0.088 h-1. This may indicate the similar specificities of the five intracellular depolymerases. In addition, the similar k1 among the different species may correlate with the high degree of amino acid sequence identities (over 85%) among the intracellular PHA depolymerase phaZ genes. Six other chemically different types of PHA were accumulated in P. putida from n-nonanoic acid, n-decanoic acid, 5-phenyvaleric acid, or 11phenoxyundecanoic acid as a single or a mixed carbon source. The calculated k1 values were characteristic to each PHA, reflecting their chemical structures. In comparison with P(3HO), an increase in the levels of the two minor monomers 3HC and 3HD as in P(21 mol % 3HC-co-56 mol % 3HO-co-23 mol % 3HD) significantly slowed the rate of intracellular degradation. From the comparison of k1 values, it is suggested that the P. putida intracellular depolymerase is most active against P(3HO). Introduction Bacteria degrade previously accumulated polyhydroxyalkanoate (PHA) mostly in the absence of an exogenous carbon source1-3 and in the presence of nitrogen4-6 and utilize the degradation products for growth and survival if the PHA in cells can be degraded by intracellular depolymerase(s).6,7 However, despite its physiological and biotechnological significance, the degradation mechanism of the amorphous8 PHA granules in cells is still poorly understood,3-5 probably because of the difficulty in the isolation and purification of the enzymes without loss of activity, whereas the degradation mechanism of several denatured PHAs including poly(3hydroxybutyrate) (P(3HB)) by extracellular PHA depolymerases has been extensively studied.9 An intracellular P(3HB) depolymerase gene (phaZ) has been cloned from Ralstonia eutropha H16 and characterized.5 The crude extract * To whom correspondence may be addressed. Phone: +82-55-7515942. Fax: +82-55-753-0765. E-mail:
[email protected]. † Division of Applied Life Sciences (BK21) at the Graduate School, Gyeongsang National University. ‡ Division of Life Science at the College of Natural Sciences, Gyeongsang National University. § Environmental Bioresources Laboratory, Korea Research Institute of Bioscience and Biotechnology.
of Escherichia coli containing the P(3HB) depolymerase gene digested artificial amorphous P(3HB) granules and released mainly oligomeric D-(-)-3-hydroxybutyrate. However, native P(3HB) granules isolated from chromosomal knockout mutant of the intracellular P(3HB) depolymerase gene of R. eutropha H16 showed a reduced but not completely eliminated activity of 3HB release.4 From this, they suggested the possible presence of intracellular P(3HB) depolymerase isoenzymes. It has been suggested that, without isolation and purification of the intracellular PHA depolymerase and native PHA substrates, the relative specificity of the enzyme6,10,11 as well as the microstructural heterogeneity6,10-12 of the PHA prepared from two or more precursors could be determined by measuring in situ the first-order degradation rate constants (k1) of the PHA in cells. The in vivo kinetic analysis really enables us to circumvent the inherent difficulty in the study of the intracellular enzymes located on the surfaces of PHA granules in cells. The validity of the suggestion is based on the fact that the depolymerization by the intracellular depolymerase is the rate-determining step in the intracellular degradation reaction, which was proved by NMR local sequence analysis revealing the local sequence specific PHA
10.1021/bm0257199 CCC: $25.00 © 2003 American Chemical Society Published on Web 02/19/2003
Polyhydroxyalkanoic Acid Degradation
degradation in Hydrogenophaga pseudoflaVa cells.6 The fact that the reaction is first order with respect to the concentration of the monomer units has been suggested to probably imply a constant level of depolymerase that is active on the surface of granules during degradation.6 For the three types of copolymers, P(3HB-co-3-hydroxyvalerate (3HV)) and P(3HBco-4-hydroxybutyrate (4HB))6 and P(3HV-co-4HB),11 the monomer specificity of the depolymerase was found to decrease in the order 3HB > 3HV > 4HB. The same specificity order was demonstrated by the two methods of NMR local sequence and first-order kinetic analysis. This strong correlation between the two methods gives us a rationale for the suggestion described above. That is, the firstorder rate constant, k1, can be used as a measure of the relative specificity of the PHA depolymerase within a bacterial species.6,10,11 To find whether the suggestion is applicable to different species in a genus, our study was extended to the pseudomonads because the Pseudomonas spp. belonging to rRNA homology group I can produce medium-chain-length (MCL) PHAs which are composed of at least two or more types of the 3-hydroxyalkyl or aromatic monomers with various lengths of side chains.1-3,13 Thus the degradation kinetics analysis of such structurally diverse MCL PHAs was expected to reveal more details on the specificity nature developed by the first-order kinetic analysis, specifically in terms of the chemical structure of a PHA. Furthermore, in a genetic study of P. putida U, disruption of the gene encoding phaZ prevented the mobilization of the polymer accumulated intracellularly.14 This may indicate that the pseudomonads have no isozymes of their intracellular PHA depolymerases. In this study, seven structurally different PHAs were accumulated in Pseudomonas putida BM01 utilizing carboxylic acid compounds such as octanoic acid, nonanoic acid, decanoic acid, 5-phenylvaleric acid, and 11-phenoxyundecanoic acid as a single or a mixed carbon source and their intracellular degradation kinetics was investigated to understand the specificity nature of the intracellular PHA depolymerase. A comparison of the degradation rate constant for a PHA with overall similar monomer ratio among different species in a genus was also expected to disclose a structural relationship among the depolymerases. Actually, the complete nucleotide sequences and derived amino acid sequences of the phaZ genes in the three pseudomonads P. oleoVorans,15 P. aeruginosa,16,17 and P. putida14 have been reported. The three phaZ genes have a high degree of structural homology (in amino acid identities over 85%). Thus, to find the proposed implication of the enzyme specificity in the degradation rate constants, a similar type of MCL PHA mainly composed of 3-hydroxyoctanoic acid (3HO) units (designated P(3HO)) was separately accumulated in the five pseudomonads, P. citronellolis (ATCC 13674), P. oleoVorans (ATCC 29347), P. aeruginosa (ATCC 27853), P. fluorescens BM07, and P. putida BM01, and their degradation rates were determined and analyzed in terms of first-order reaction kinetics.
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Materials and Methods Microorganisms and Reagents. Two bacterial strains, Pseudomonas putida BM0118 and Pseudomonas fluorescens BM07,19 isolated in our lab and three ATCC cultures, Pseudomonas citronellolis (ATCC 13674), Pseudomonas oleoVorans (ATCC 29347), and Pseudomonas aeruginosa (ATCC 27583), were used for the MCL PHA degradation study. Several carbon sources such as n-alkyl fatty acids, 5-phenylvaleric acid, 11-phenoxyundecanoic acid, etc., were purchased from Sigma Chemical Co. and used without further purification. PHA Accumulation in Cells and Its Quantitative Assay. PHA was accumulated in cells by growing a preculture in a modified M1 medium20 containing a carbon source or a mixture of two carbon sources and 8 mM (or higher) ammonium sulfate. All growth experiments for PHA accumulation were performed under aerobic conditions in a temperature-controlled shaker (Korea Instrument Co., Seoul, Korea) at 30 °C and 190 rpm. Cells were harvested by centrifugation in a Beckman J2-HS (rotor JA-10, 7000 rpm for 10 min). The isolated cells for PHA analysis were washed with methanol and dried under vacuum for 2 days. The monomer composition of the polyesters in the cells was determined by analyzing the methyl esters recovered from a sulfuric acid/methanol treatment of the cells using a HewlettPackard HP5890A gas chromatograph equipped with a HP-1 capillary column and a flame ionization detector.18,20 A typical GC run condition was as follows: initial temperature 80 °C, 2 min; heating rate 10 °C/min; final temperature 230 °C, 2 min; carrier (He) flow rate, 3 mL/min; injector temperature, 230 °C; detector temperature, 280 °C. For quantitative GC analyses, several PHA were isolated, purified, and characterized using quantitative 13C NMR20 and 1H NMR for the use as the standards. Poly(3-hydroxy-5phenylvalerate) (P(3HPV)) homopolymer was efficiently accumulated in P. putida by cultivating it on a mineral salts medium containing 5-phenylvalerate mixed with butyrate which supported only cell growth but not induced PHA accumulation.21 The aromatic polyester bearing a phenoxy group in the side chain was accumulated in the cells from 11-phenoxyundecanoic acid.22 Other polymers were prepared according to the procedures in the literature.18-22 Intracellular PHA Degradation and Kinetics Analysis. The harvested cells containing various types of polyesters were transferred to a carbon-source-free mineral medium (a medium containing the same mineral as a PHA synthesis mineral medium) containing ammonium sulfate (1.0 g/L). The cells were incubated under an aerobic shaking condition at 30 °C and 190 rpm. Five milliliters of a culture was removed with appropriate time intervals in order to analyze the medium and the cells. The amount of NH4+ remaining in the medium was measured by the Nessler reagent method.20 The monomer-unit composition of the PHA remaining in cells was determined by gas chromatography as described above. The initial rate method6,10,11 was used in the calculation of first-order degradation rate constants (k1) throughout this study. The data points used in all the calculations were taken for the ones over the initial cultivation period just before reaching a steady state, which gave a
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Table 1. Degradation of P(3HO) in Pseudomonas spp. Which Were Grown on 40 mM Octanoic Acid as the Sole Carbon Source at 30 °C for 30-48 h
organism
sampling period
dry cell wt (g/L)
PHA wt (g/L)
Pseudomonas putida BM01 Pseudomonas citronellolis ATCC 13674 Pseudomonas oleovorans ATCC 29347 Pseudomonas aeruginosa ATCC 27583 Pseudomonas fluorescens BM07
BDb AD BD AD BD AD BD AD BD AD
3.75 2.27 2.13 1.63 2.38 1.85 1.83 1.51 2.85 2.19
2.18 0.17 0.61 0.03 0.65 0.05 0.39 0.02 0.79 0.10
initial PHA (wt %) 58.1 28.6 27.3 21.3 27.7
monomer composition (mol %) 3HC
3HO
3HD
7 6 12 11 7 8 10 9 8 6
88 89 85 86 90 90 86 88 86 88
5 5 3 3 3 2 4 3 6 6
k1 (h-1)
corr coeff (r2)
0.083c
0.973
9d (8.4)
0.088
0.978
9 (7.9)
0.080
0.967
11 (8.7)
0.087
0.967
10 (8.0)
0.060
0.984
11 (11.6)
half-life t1/2 (h)
remaining nitrogena (g/L) 1.02 0.00 1.01 0.11 1.01 0.08 1.00 0.18 1.02 0.25
a Determined as ammonium sulfate. b BD, before degradation; AD, after degradation. The upper values were measured before degradation experiments and the lower values were measured after degradation experiments. c The initial rate method was used in the calculation. See text for details. d Determined as a midpoint between the time zero and the time when the steady-state degradation was reached. The t1/2 values in parentheses were calculated using t1/2 ) 0.693/k1.
high fitting correlation coefficient (r2) over minimum 0.93 for most cases. Results Degradation of P(3HO) in Pseudomonas spp. The polyester termed P(3HO) was accumulated in cells by growing a bacterium on 40 mM octanoic acid in a mineral salts medium.20 All the P(3HO) polymers synthesized by the five pseudomonads contained 3-hydroxyoctanoic acid unit (3HO) as the major monomer (85-90 mol %) and both 3-hydroxyhexanoic acid (3HC) (7-12 mol %) and 3-hydroxydecanoic acid (3HD) (3-6 mol %) as the minor monomers (Table 1). The degradation experiment was carried out in the presence of 1.0 g/L ammonium sulfate. A further increase in the level of ammonia in media had little effect on the degradation rate even when increased up to 2.0 g/L. The remaining PHA level was analyzed and fitted into a firstorder kinetics plot. The calculated slope gives the first-order degradation rate constant. The data points used for a linear fitting analysis were taken only from those in the initial active degradation period (the initial rate method). This sounds reasonable that little limitation to the initial degradation may play long before a steady state is reached.6 All five sets of data fit nicely into a first-order kinetics equation. This confirms our previous suggestion that intracellular PHA degradation follows a first-order kinetics.6 Each fitted calculation showed a very high correlation coefficient (r2) higher than 0.97 (Figure 1B). The degradation rate was very similar irrespective of the type of species and the content of PHA in cells. As shown in Table 1, the molar monomer compositions of PHA in the cells before the degradation experiments were similar. Furthermore, the compositions of the PHA remaining after degradation did not change, which means all the polymers were real random copolymers. Thus, the similar degradation rate for the five species suggests that the specificities of the intracellular PHA depolymerases may be quite similar. Degradation of Various Types of MCL-PHA in Pseudomonads putida BM01. Six other different types of aliphatic or aromatic PHA were accumulated in P. putida
Figure 1. First-order degradation of P(3HO) in MCL-PHA-producing microorganisms. Data points for linear fitting analysis were taken only for those in the initial active degradation period (the initial rate method).
BM01, and their intracellular degradation experiments were carried out (Table 2). Overall, the intracellular PHA degradation is a function of the monomer composition of PHA. Among the PHAs studied, the P(3HO) (P(7 mol % 3HC-
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Table 2. Degradation of Various Types of PHA in Pseudomonas putida BM01 that Was Grown on the Corresponding Carbon Sources at 30 °C carbon source used for PHA synthesis
wt (g/L)
monomer composition (mol %)
k1
dry cell PHA 3HC 3HH 3HO 3HN 3HD 3HPV 3HPOV 3HPOH
octanoic acid (40 mM)
3.75b 2.27
2.18 0.17
nonanoic acid (40 mM)
2.07 1.65
0.89 0.11
decanoic acid (40 mM)
3.53 2.34
1.61 0.15
21 28
56 49
23 23
5-phenylvaleric acid/octanoic acid (20 mM/20 mM)
3.12 2.03
1.62 0.35
d
60 26
d
5-phenylvaleric acid/butyric acid (20 mM/20 mM)
1.79 1.33
0.66 0.16
11-phenoxyundecanoic acid/ octanoic acid (10 mM/15 mM)
3.17 2.63
1.06 0.42
11-phenoxyundecanoic acid (10 mM)
1.89 1.73
0.52 0.34
7 6
88 89 49 46
d
5 5
d
r2
Na (g/L)
0.083 (3HO)
0.973 1.02 0.00
0.039 (3HH)c 0.034 (3HN)
0.969 1.01 0.965 0.00
0.043 (3HC) 0.048 (3HO) 0.050 (3HD)
0.923 1.03 0.938 0.00 0.937
40 74
0.087e (3HO) 0.020f (3HO) 0.015 (3HPV)
0.995 1.00 0.965 0.00 0.898
100 100
0.019 (3HPV)
0.982 1.02 0.00
51 54
53 44
(h-1)
24 32
23 24
0.031 (3HO) 0.985 1.02 0.021 (3HPOV) 0.975 0.00 0.030 (3HPOH) 0.955
63 67
37 33
0.013 (3HPOV) 0.980 1.03 0.014 (3HPOH) 0.980 0.28
a Remaining nitrogen, determined as ammonium sulfate. b The upper values are for before degradation and the lower values for after degradation. The incubation time for the degradation experiment was 60 h. c k1 was separately determined against each comonomer unit in the PHA. d Less than 5 mol %. e The determined value for the first period. f The determined value for the second period. (See text and ref 10 for details.)
co-88 mol % 3HO-co-5 mol % 3HD)) synthesized from octanoic acid degraded at the fastest rate (0.083 h-1 for 3HO unit). In comparison with the P(3HO) from octanoic acid, an increase in the levels of the two comonomers 3HC and 3HD as in the PHA (P(21 mol % 3HC-co-56 mol % 3HOco-23 mol % 3HD)) from decanoic acid significantly slowed the rate of degradation (0.048 h-1 for 3HO unit). This may suggest that an optimum length of the side chain for degradation is five carbons linked in series. As for the PHA from nonanoic acid, the PHA having odd numbers of sidechain carbon degraded at a much slower rate (average 0.036 h-1 for both 3-hydroxyheptanoate (3HH) and 3-hydroxynonanoate (3HN) unit). 3HH is one methylene unit shorter than 3HO, and 3HN is one methylene unit longer than 3HO. Thus, this may suggest that the intracellular depolymerase has the highest specificity against 3HO unit. In addition, for the three aliphatic PHAs, the monomer composition was relatively constant before and after degradation, indicative of randomness in their copolymerization. The PHA with aromatic groups in their side chains degraded at a much slower rate than the aliphatic PHA described. The degradation rate also depended on the ratio of aliphatic/aromatic monomers. Poly(3-hydroxy-5-phenylvalerate) (P(3HPV) homopolymer degraded at the rate of 0.019 h-1, four times or more slower than P(3HO). The PHA with phenoxy groups poly(3-hydroxy-5-phenoxyvalerate-co-3-hydroxy-7-phenoxyheptanoate), P(3HPOV-co3HPOH), most slowly degraded among the PHAs studied, six times slower than P(3HO). The phenoxy PHA was a random copolymer considering the similar degradation rate constants determined for the two comonomer units (0.013 and 0.014 h-1). The PHA with both aromatic and aliphatic groups showed different kinetic behavior in their degradation. The PHA composed of phenoxy and aliphatic comonomers degraded faster than the aliphatic-monomer-free 3HPOV/
3HPOH copolymer, and the degradation rate constant determined for each monomer was similar, indicative of a random copolymer nature. However, the PHA composed of 3HPV and 3HO exhibited totally different two degradation rate constants 0.087 for 3HO and 0.015 h-1 for 3HPV.10 These two constants are quite close to the values determined for P(3HO) and P(3HPV), respectively. On the basis of the degradation rate constants alone, it could be concluded that the 3HO/3HPV polymer was not a random copolymer but a blend of two polymers, P(3HO) and P(3HPV). Furthermore, the initial monomer composition changed significantly from 60 to 26 mol % for 3HO and from 40 to 74 mol % for 3HPV after degradation. A fractionation experiment proved that the PHA synthesized in P. putida BM01 from mixtures of octanoic acid and 5-phenylvalerate was a blend of 3HOrich and 3HPV-rich chains, not a real copolymer.10 Such structural heterogeneity was found to be caused by the significantly different assimilation rates of the two precursors. All these clearly show that the intracellular depolymerase is highly specific against P(3HO) but poorly active against the aromatic PHA. Discussion Each k1 Value Is Characteristic and Unique to a Specific Type of PHA. There is experimental evidence supporting the view that the first-order degradation rate constant is a characteristic and unique value and that it does directly reflect the specific nature of the intracellular PHA depolymerase in a bacterium. First, the pseudomonads belonging to rRNA homology group Ι have a similar k1 in the degradation of P(3HO) in cells. In addition, the aromatic homopolymer P(3HPV) accumulated in both P. putida and P. citronellolis also degraded at the same rate.10 This may suggest a similar level of the depolymerase coverage on the
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surface of PHA granules in each bacterium as well as a similar specificity nature of the pseudomonads depolymerases (phaZ genes). Second, P(3HPV) and P(3HO) degraded in separately accumulated cells as fast as in the blend-type PHA (a mixture of P(3HPV) and P(3HO)) cell as shown for P. putida.10 This additionally means that the coverage of the intracellular depolymerase is independent of the chemical type of MCL PHA and thus the number density of the intracellular depolymerase on a PHA granule may be relatively constant irrespective of the culture parameters. Third, as in the case of P. putida, the substrate-type dependence of the intracellular degradation clearly reveals the intracellular degradability systematically reflecting the chemical structural characteristics of the substrates (Table 2). Fourth, the degradation rate of a copolymeric PHA is generally controlled by the comonomer unit that has a slower degradation rate. Finally, as reported earlier for the case of SCL PHA degradation in H. pseudoflaVa,6 the degradation rate constants determined kinetically are known to correlate well with the local sequence specificity determined by analyzing NMR signal changes before and after PHA degradation. This correlation strongly suggests that the intracellular depolymerization reaction by the intracellular depolymerase is the rate-determining step in the PHA degradation.6 All these suggest that the first-order rate constant can be used as a determinant for the PHA substrate specificity of the intracellular depolymerase concerned. The uniqueness of each k1 was further supported by our Blast search result for phaZ gene in P. aeruginosa excluding any probable existence of phaZ isozymes at the level of protein sequence homology. The Similar k1 Values for P(3HO) among the Pseudomonads May Reflect the High Structural Homology of phaZ Genes Reported. The structural factors affecting the activity of the pseudomonads intracellular depolymerase may include the side-chain length, uniformity of the side-chain length, type of neighboring monomer unit and its recognizable number of units, and chemical nature of the side groups (Table 2). On the basis of the relative magnitude of the degradation rate constant, P(3HO) may be the best substrate for the depolymerases. That is, the depolymerase is P(3HO) depolymerase. Solely from the similar degradation rate constant for P(3HO) in all pseudomonads studied, it can be suggested that the five pseudomonads intracellular depolymerases might have a high structural homology. Actually, the complete nucleotide sequences and derived amino acid sequences of the depolymerase phaZ gene in the three pseudomonads P. oleoVorans,15 P. aeruginosa,16,17 and P. putida14 have been reported. The three phaZ genes have a very high degree of structural homology (identity), over 85%. In conclusion, the first-order degradation rate constant determined by the initial rate method represents, in a
Choi et al.
quantitative sense, the relative specificity of the intracellular PHA depolymerase involved in the initial degradation. However, this suggestion is based on the assumption that the level of the intracellular depolymerase on the surface of PHA granules is relatively constant independent of the type of strain and the type of PHA. Acknowledgment. This work was supported in part by the Korea Science and Engineering Foundation Grant R012000-000-00070-0. M.H.C. thanks for the postdoctoral fellowship through BK21 program to Korea Research Foundation (KRF). H.-J.L. and J.K.R. acknowledge the graduate scholarship through BK21 program to KRF. References and Notes (1) Anderson, A. J.; Dawes, E. A. Microbiol. ReV. 1990, 54, 450-472. (2) Doi, Y. Microbial polyesters; VCH Publishers: New York, 1990. (3) Madison, L. L.; Huisman, G. W. Microbiol. Mol. Biol. ReV. 1999, 63, 21-53. (4) Handrick, R.; Reinhardt, S.; Jendrossek, D. J. Bacteriol. 2000, 182, 5916-5918. (5) Saegusa, H.; Shiraki, M.; Kanai, C.; Saito, T. J. Bacteriol. 2001, 183, 94-100. (6) Yoon, S. C.; Choi, M. H. J. Biol. Chem. 1999, 274, 37800-37808. (7) Choi, M. H.; Yoon, S. C.; Lenz, R. W. Appl. EnViron. Microbiol. 1999, 65, 1570-1577. (8) Song, J. J.; Yoon, S. C.; Yu, S. M.; Lenz, R. W. Int. J. Biol. Macromol. 1998, 23, 165-173. (9) Jendrossek, D.; Schirmer, A.; Schlegel, H. G. Appl. Microbiol. Biotechnol. 1996, 46, 451-463. (10) Chung, D. M.; Choi, M. H.; Song, J. J.; Yoon, S. C.; Kang I. K.; Huh, N. E. Int. J. Biol. Macromol. 2001, 29, 243-250. (11) Choi, M. H.; Lee, H.-J.; Rho, J. K.; Yoon, S. C.; Nam, J. D.; Lim, D.; Lenz, R. W. Biomacromolecules, 2003, 4, 38-45. (12) Choi, M. H.; Song, J. J.; Yoon, S. C. Can. J. Microbiol. 1995, 41 (Suppl. 1), 60-67. (13) Steinbu¨chel, A.; Valentin, H. E. FEMS Microbiol. Lett. 1995, 128, 219-228. (14) Garcı´a, B.; Olivera, E. R.; Min˜ambres, B.; Ferna´ndez-Valverde, M.; Can˜edo, L. M.; Prieto, M. A.; Garcı´a, J. L.; Martı´nez, M.; Luengo, J. M. J. Biol. Chem. 1999, 274, 29228-29241. (15) Huisman, G. W.; Wonink, E.; Meima, R.; Kazemier, B.; Terpstra, P.; Witholt, B. J. Biol. Chem. 1991, 266, 2191-2198. (16) Stover, C. K.; Pham, X. Q.; Erwin, A. L.; Mizoguchi, S. D.; Warrener, P.; Hickey, M. J.; Brinkman, F. S. L.; Hufnagle, W. O.; Kowalik, D. J.; Lagrou, M.; Garber, R. L.; Goltry, L.; Tolentino, E.; WestbrookWadman, S.; Yuan, Y.; Brody, L. L.; Coulter, S. N.; Folger, K. R.; Kas, A.; Larbig, K.; Lim, R. M.; Smith, K. A.; Spencer, D. H.; Wong, G. K. S.; Wu, Z.; Paulsen, I. T.; Reizer, J.; Saier, M. H.; Hancock, R. E. W.; Lory, S.; Olson, M. V. Nature 2000, 406, 959-964. (17) Timm, A.; Steinbu¨chel, A. Eur. J. Biochem. 1992, 209, 15-30. (18) Song, J. J.; Yoon, S. C. J. Microbiol. Biotechnol. 1994, 4, 126133. (19) Lee, H.-J.; Choi, M. H.; Kim, T.-U.; Yoon; S. C. Appl. EnViron. Microbiol. 2001, 67, 4963-4974. (20) Choi, M. H.; Yoon, S. C. Appl. EnViron. Microbiol. 1994, 60, 32453254. (21) Song, J. J.; Choi, M. H.; Yoon, S. C.; Huh, N. E. J. Microbiol. Biotechnol. 2001, 11, 435-442. (22) Song, J. J.; Yoon, S. C. Appl. EnViron. Microbiol. 1996, 62, 536-544.
BM0257199
