Curr Microbiol (2010) 60:365–372 DOI 10.1007/s00284-009-9551-3
Identification of Actinomycetes Producing Phospholipase D with High Transphosphatidylation Activity Yozo Nakazawa • Rei Suzuki • Masataka Uchino • Yoshimasa Sagane • Takuji Kudo • Takeshi Nagai Hiroaki Sato • Katsumi Takano
•
Received: 25 October 2009 / Accepted: 10 November 2009 / Published online: 24 November 2009 Ó Springer Science+Business Media, LLC 2009
Abstract Previously we isolated six actinomycetes strains, 9-4, 10-1, 10-2, 10-3, 10-6, and 21-4, that produce phospholipase D (PLD) with high transphosphatidylation activity. In this study, we identified these strains, and the PLD activities were compared with those of reference strains. 16S rDNA sequences and DNA–DNA hybridization tests indicated taxonomic affiliations of strain 9-6 with Streptomyces senoensis, strains 10-1 and 10-6 with S. vinaceus, and strains 10-2 and 10-3 with S. racemochromogenes. Strain 21-4, though identified as a Streptomyces sp., could not be identified with any known species. Meanwhile, most of the culture supernatants of reference strains demonstrated no or very weak PLD activity, while those of our strains exhibited significantly higher activity.
Nucleotide sequences The DDBJ/EMBL/GenBank accession numbers for 16S rDNA sequences of strain 9-4, 10-1, 10-2, 10-3, 106, and 21-4 are AB222067, AB222068, AB222070, AB222071, AB222069, and AB222072, respectively. Y. Nakazawa (&) T. Nagai H. Sato Faculty of Bioindustry, Department of Food Science and Technology, Tokyo University of Agriculture, 196 Yasaka, Abashiri, Hokkaido 099-2493, Japan e-mail:
[email protected] R. Suzuki M. Uchino K. Takano Faculty of Applied Bioscience, Department of Applied Biology and Chemistry, Tokyo University of Agriculture, 1-1-1 Sakuragaoka, Setagaya-ku, Tokyo 156-8502, Japan Y. Sagane Sars International Centre for Marine Molecular Biology, Bergen High Technology Centre, Thormøhlensgt. 55, 5008 Bergen, Norway T. Kudo Japan Collection of Microorganisms, RIKEN BioResource Center, 2-1 Hirosawa, Wako, Saitama 351-0198, Japan
All of the strains in this study were identified as Streptomyces species. The PLD activity of our strains exceeded most of the reference Streptomyces strains. The findings in this study imply that the Streptomyces strains, although they are members of the same species, can produce different quantities of PLD enzyme.
Introduction Phospholipid (PL) is an amphipathic molecule having both a hydrophobic part in form of two long-chain fatty acids and a hydrophilic group in form of a phosphate ester, and thus is a natural surface-active agent that is widely utilized in foods and cosmetics as an emulsifier, stabilizer, and antioxidant [1]. Due to their ubiquitous distribution as a major component of the cell membrane in both plant and animal cells, the PLs are purified in large quantities from industrial oil production processes that utilize crude vegetable oil and animal fat to yield lecithin, one of the PLs with the most potential. The dominant component of native lecithin is phosphatidylcholine (PC). Lecithin contains other minor PL constituents such as phosphatidylglycerol (PG), phosphatidylserine (PS), phosphatidylethanolamine, and phosphatidylinositol. PG can provide effective emulsification stability to food production over a wide range of pHs and salt conditions compared with PC [2]. Additionally, PS, which can also be found in animal brains, has been proposed as a food supplement to improve and/or prevent senile dementia [3, 4]. Phospholipase D (PLD; EC.3.1.4.4) catalyzes the hydrolysis of the terminal phosphodiester bond in PL generating phosphatidic acid (PA) and free alcohol. When an alcohol is present in the reaction mixture, the PLD can also catalyze the transphosphatidylation reaction in which the
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PA moiety of the PL molecule is transferred to an alcohol molecule to form a phosphatidyl alcohol. Therefore, modification of the polar heads of natural PLs by PLD has been widely applied in the industrial preparation of less abundant PLs. PLD is a fairly ubiquitous enzyme that is widely distributed in bacteria, fungi, plant, and vertebrate species [5]. In particular, the PLD enzyme produced by some microorganisms, especially actinomycetes strains, dominantly displays high selectivity for transphosphatidylation [6–8]. Recently we isolated six bacterial strains using a screening strategy as follows: first, to obtain PLD-producing strains, microorganisms that grow in lecithinderived PC as the sole carbon source were screened; then, we examined the transphosphatidylation activities of culture supernatants using lecithin-derived PC as a PA moiety donor, in conjunction with various acceptors such as glycerol, L-serine, 2-aminoethanol hydrochloride [9]. Interestingly, all strains screened by this strategy appear to be actinomycetes strains based on the colony morphology on the selection plate, capability of forming spores, and the morphological behavior of the culture broth. In this study, we identified six strains based on their 16S rDNA sequences, phenotypic properties, and DNA–DNA hybridization tests with reference actinomycetes strains. Additionally, since all strains identified here were Streptomyces species, we also examined the distribution of PLD among the Streptomyces strains based on measuring extracellular PLD activity produced by the strains and detecting PLD genes by PCR analysis using a primer set targeting the highly conserved domain among the previously described Streptomyces pld genes.
extract agar (ISP 2), oatmeal agar (ISP 3), inorganic salts/ starch agar (ISP 4), and Pridham-Gottlieb carbon utilization agar medium (ISP 9). After incubation at 28°C for 14 days, morphological and cultural characteristics of strains were described according to the methods of the ISP [10]. The morphological observations of spores and mycelia were determined by scanning electron microscopy (S-2400; Hitachi). Color determination was done with color chips from A MYCOLOGICAL COLOUR CHART [11].
Materials and Methods Bacterial Strains, Medium, and Growth Conditions Actinomycetes strains 9-4, 10-1, 10-2, 10-3, 10-6, and 214, which were isolated previously [9], were used. S. senoensi NBRC 13843T, S. cirratus JCM 4738T (=NBRC 13398T), S. vinaceus NBRC 13425T, S. katrae JCM 4777T (=NBRC 13447T), S. racemochromogenes NBRC 12906T, S. polychromogenes JCM 4505T (=NBRC 13072T), S. tanashiensis NBRC 12919T, and S. nashvillensis NBRC 13064T were used as reference strains. These strains were grown in a GYP medium composed of 0.5% glucose, 0.5% yeast extract, 0.5% polypeptone, 0.2% K2HPO4, and 0.05% MgSO47H2O, at 28°C for 2 days with shaking at 200 rpm. Phenotypic Characterization The washed mycelia were inoculated onto International Streptomyces Project (ISP) media: yeast extract/malt
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Preparation of DNA Total DNA was extracted from 2-day cultured cells using the method of Saito and Miura [12] with the minor modifications of supplementing with achromopeptidase and lysozyme (Wako Pure Chemicals, Osaka, Japan) for lysing cells. Purity and concentration of the prepared DNA solution were measured with a NanoDrop spectrophotometer (ND-1000; Thermo Scientific). Solutions with an A260/A280 ratio above 1.9 were used for PCR and DNA–DNA hybridization. 16S rDNA Sequence Analysis The 16S rDNA was amplified using the PCR method with an Ex TaqÒ DNA polymerase (Takara) and primers 9F (positions 9–25, Escherichia coli numbering) and 1541R (1541–1525). Amplification was carried out using a DNA thermal cycler (GeneAmp PCR System 9700; Applied Biosystems) according to the following program: 95°C for 3 min followed by 30 cycles of denaturation (95°C, 30 s), primer annealing (55°C, 15 s), and primer extension (72°C, 1 min). At the end of the cycle, the reaction mixture was kept at 72°C for 5 min and then cooled to 4°C. The amplified 16S rDNA fragment was purified and directly subjected to cycle sequencing using an ABI PRISM BigDyeÒ Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems), according to the manufacturer’s protocol, with the following primers: 9F, 515F (515–531), 536R (536–519), 785F (785–805), 802R (802–785), 1099F (1099–1114), 1115R (1115–1100), and 1541R. The products were analyzed with an ABI PRISM 3100-Avant Genetic Analyzer (Applied Biosystems). The sequences were multiply aligned with selected sequences (Fig. 1) obtained from the DDBJ/EMBL/GenBank database using CLUSTAL X 1.81 software [13]. The alignment was manually verified and adjusted prior to construction of a phylogenetic tree. The tree was constructed using the neighborjoining method [14] from the nucleotide substitution rates (Knuc values) [15] calculated in CLUSTAL X. The topology of
Y. Nakazawa et al.: Identification of Actinomycetes Producing PLD Fig. 1 The phylogenetic relationships of the isolated strains and some Streptomyces strains based on 16S rDNA sequences. The branching pattern was generated by the neighbor-joining method. The bar indicates 0.01 nucleotide substitutions per site. Numbers indicate bootstrap values in percentages [70%. The sequence data were gathered from DDBJ/EMBL/GenBank databases. The numbers in parentheses are accession numbers for the DDBJ/EMBL/ GenBank nucleotide sequence databases
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86
T 96 "S. senoensis" NBRC 13843 (AB184525) Strain 9-4 (AB222067)
Ia
S. cirratus NBRC 13398T (AB184377)
I
S. vinaceus NBRC 13425T (AB184394)
Ib
Strain 10-1 (AB222068) Strain 10-6 (AB222069) S. nojiriensis NBRC 13794T (AB184485) 76 S. spororaveus NBRC 15456T (AB184682) S. xanthophaeus NBRC 12829T (AB184177) S. subrutilus NBRC 13388T ((AB184372)) S. avidinii NBRC 13429T (AB184395)
S. lavendulae subsp. lavendulae NBRC 12789T (AB184146) S. colombiensis NBRC 13454 (AB184415) 95
85 "S. citricolor" NBRC 13005 (AB184252) S. sporoverrucosus NBRC 15458T (AB184684) "S. verne" NBRC 3112T (AB184727) S. verne S. cinnamonensis NBRC 15873T (AB184707) 96 S. virginiae NBRC 12827T (AB184175) S. flavotricini NBRC 12770T (AB184132) "S. flaveus" NBRC 3359 (AB184749) S. globosus LMG 19896T (AJ781330)
84
S. roseochromogenus "S roseochromogenus" NBRC 3411 (AB184766) S. katrae NBRC 13447T (AB184409) 88
S. racemochromogenes NBRC 12906T (AB184235) S. polychromogenes NBRC 13072T (AB184292)
II
Strain 10-2 (AB222070) Strain 10-3 (AB222071) S i b i NBRC 12875T (AB184211) S. griseus subsp. griseus
83
S. roseoviridis NBRC 12911T (AB184239) S. showdoensis NBRC 13417T (AB184389) S. cinereoruber subsp. cinereoruber NBRC 12756T (AB184121) S. violaceorectus NBRC 13102T (AB184314) S. bikiniensis NBRC 14598T (AB184602)
92
S. roseolus NBRC 12816T (AB184168) 92
97
S. filamentosus NBRC 12767T (AB184130) S. nashvillensis NBRC 13064T (AB184286)
100
S. tanashiensis NBRC 12919T (AB184245) 100
III
Strain 21-4 (AB222072) S. avermitilis NBRC 14893T (AB184632)
S. albulus NBRC 13410T (AB184384) "S. acidomyceticus" NBRC 3125 (AB184731) 89
S. ambofaciens NBRC 12836T (AB184182)
0.01
S. coelicolor NBRC 12854T (AB184196)
the tree was evaluated using the bootstrap resampling method [16] with 1000 replicates.
the homologous DNA binding value. Standard deviation was \5%.
DNA–DNA Hybridization
PLD Assay for Culture Supernatant
DNA–DNA hybridization was performed using the method of Ezaki et al. [17] at 55°C in 29 SSC (19 SSC; 0.15 M NaCl plus 0.015 M sodium citrate, pH 7.0) containing 50% formamide. The experiment was performed at least three times. DNA–DNA relatedness was expressed as a mean of
To determine PLD productivity of Streptomyces strains, the PC hydrolyzing activity of the culture supernatants was measured according to the methods described by Imamura and Horiuti [18]. One unit (U) was defined as the amount of enzyme that liberated 1 lmol of choline per minute.
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Statistical analyses performed on data included analysis of variance (ANOVA) and differences among means determined by the Sheffe’s multiple range test with significance defined at P \ 0.001.
obtained by the neighbor-joining algorithm from Knuc values is shown in Fig. 1. The result revealed that the six isolates belong to three different clusters of sequences. We assigned the names cluster I, II, and III here to the three clusters. Isolates 9-4, 10-1, and 10-6 belonged to cluster I along with S. senoensis NBRC 13843T, S. cirratus NBRC 13398T, and S. vinaceus NBRC 13425T. Isolates 10-2 and 10-3 belonged to cluster II along with S. katrae NBRC 13447T, S. racemochromogenes NBRC 12906T, and S. polychromogenes NBRC 13072T, and isolate 21-4 belonged to cluster III along with S. nashvillensis NBRC 13064T and S. tanashiensis NBRC 12919T. Furthermore, cluster I could be divided into two sub-clusters: cluster Ia consisting of isolate 9-4 and S. senoensis NBRC 13843T, and cluster Ib including the remaining constituents. Of the three isolates in cluster I, the determined sequence for isolate 9-4 shared the highest identity (100%) with the 16S rDNA sequence of S. senoensis NBRC 13843T, and the remaining two isolates, 10-1 and 10-6, displayed the highest identity (100%) with S. cirratus NBRC 13398T and S. vinaceus NBRC 13425T. In cluster II, isolates 10-2 and 10-3 showed 100% similarity to the 16S rDNA sequences from S. racemochromogenes NBRC 12906T and S. polychromogenes NBRC 13072T. Isolate 21-4, belonging to cluster III, showed high similarity to the constituents of this cluster with 99.6–99.9% identity, but we could not find a strain that shared 100% similarity with isolate 21-4. Furthermore, cultural and phenotypic properties of our strains were compared with those of related reference Streptomyces strains revealed by 16S rDNA sequence analysis. The cultural and phenotypic properties of related strains were described in previous reports [25–28]. The aerial mass color of our strains, except for strain 21-4 on the tested ISP media, was pale purplish-gray to pale vinaceous, which could be defined as the ‘red color series’ proposed by Shiring and Gottlieb [10] and Nonomura [28]. No diffusible pigments were produced in any medium tested. By contrast, strain 21-4 exhibited an aerial mass color similar to the ‘gray color series’, and diffusible pigments that were grayish-yellow to yellowish-brown or olive brown on the tested ISP media. Scanning electron micrographs of strains assigned to cluster I (9-4, 10-1, and 10-6), cultured on ISP 4 medium at 28°C for 20 days, revealed aerial mycelia forming Retinaculiaperti, some of which formed primitive spirals or well-defined coils. The aerial mycelia of the strains assigned to cluster II (10-2 and 10-3) formed Rectiflexibiles spore chains dominantly terminated with loops or primitive spirals, some of which displayed well-defined coiled ends with several turns. The aerial mycelia of strain 21-4 (cluster III) formed Rectiflexibiles spores. The spore-surface ornamentations of all strains were smooth. Although the carbon utilization profiles of strains 10-1 and 10-6, and 10-2 and 10-3, were
PCR Amplification and Sequence Determination of the Streptomyces pld Gene The partial pld gene was amplified by PCR using PrimeSTARÒ HS DNA polymerase (Takara) and the primer set S300 Fw (50 -AAGGT(G/C)CG(G/C)ATCGT(G/C)GTCA G-30 ) and S300 Rv (50 -TA(G/C) CCGAAGTCCTG(G/C) AGCCA-30 ), which was designed based on the 314 bp highly conserved domain, tentatively designated as S300 here, revealed by a multiple alignment of the pld genes from S. acidmyceticus [19], S. antibioticus [20], Streptomyces sp. AA586 (PLDP) [21], and Streptomyces sp. PMF [22]. DNA was amplified under the following conditions: 30 cycles of denaturation at 98°C for 10 s, primer annealing at 55°C for 5 s, and primer extension at 72°C for 30 s, and a final step of cooling at 4°C. PCR products were run on a 2% (w/v) agarose gel to remove primers. Bands of amplified DNA (approx. 300 bp) were cut out with a scalpel and purified using a WizardÒ SV Gel and PCR Clean-Up System (Promega). Purified PCR products were subjected to cycle sequencing with the S300 Fw and S300 Rv primers, and directly sequenced with a DNA sequencer.
Results Identification of the Isolated Strains Previously, we isolated six actinomycetes strains by a unique screening method: the transphosphatidylation capacities of the PLD in the culture supernatants of the isolated strains were examined in a simple one-phase reaction mixture system [9]. The DNA fragments of nearly complete 16S rDNA, composed of 1515 nucleotides, were cloned and sequenced from the genomic DNA of our strains, and then compared with those of microorganisms deposited in DDBJ/EMBL/GenBank databases by a BLAST search [23]. Results showed that the sequences of our strains displayed high similarity to those of the Streptomyces species (data not shown). Thus, the nucleotide sequences for 16S rDNA from 37 Streptomyces strains were gathered from the database and aligned by the CLUSTAL X program; then the gaps and ambiguous nucleotides within the aligned sequences were removed. The resultant nucleotide sequences, each of which comprise 1434–1439 nucleotides corresponding to those from 45 to 1481 of the S. ambofaciens 16S rRNA gene [24], were used for construction of the phylogenetic tree. The phylogenetic tree
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similar to each other, the profiles of these strains did not correspond entirely with those of related reference strains. According to Stackebrandt and Goebel [29], two strains sharing a 16S rDNA sequence similarity of 97% and higher have a potential to be classified into the same taxon, and thus DNA pairing studies should be performed. Therefore, we attempted to match our strains to one of the candidates or a novel species in Streptomyces using the DNA–DNA hybridization (DDH) test. We used here the standard DDH value of 70% recommended by Wayne et al. [30] to delineate the species. Results are shown in Table 1, and are summarized as follows: (1)
(2)
(3)
Cluster Ia: the isolated strain 9-4 and S. senoensis NBRC 13843T shared 99% DDH value when strain 94 genomic DNA was used as the probe, and 80% DDH value when S. senoensis NBRC 13843T genomic DNA was used as the probe. Therefore, we matched isolate 9-4 to S. senoensis. Cluster Ib: since isolates 10-1 and 10-6 shared 100% DDH value, these strains were identified as the same species. Our isolated strains shared 73–77% DDH values against S. vinaceus NBRC 13425T, and therefore these strains were matched to S. vinaceus. Cluster II: isolates 10-2 and 10-3 were identified as the same species because they shared 100% DDH value. Since the isolated strains shared 91–97% DDH values against S. racemochromogenes NBRC 12906T, these strains were matched to S. racemochromogenes.
369
(4)
Cluster III: strain 21-4 shared only 37–44% DDH value against S. nashvillensis NBRC 13064T and 21– 53% DDH value against S. tanashiensis NBRC 12919T. Therefore, we could not match the strain to any known Streptomyces species.
This test indicated weak but significant relatedness (approximately 70% DDH value) between S. polychromogenes JCM 4505T and S. katrae JCM 4777T, and significant relatedness (70 and 100% DDH values) between S. nashvillensis NBRC 13064T and S. tanashiensis NBRC 12919T, implying a possibility that these strains are the same species. PLD Productivity of the Streptomyces Strains PLD activities in culture supernatants of the isolated strains were compared with those of reference strains. Isolate 9-4 was identified as a species of S. senoensis, but the PLD activity produced by the isolate (0.64 U ml-1) was 6.4-fold higher than reference strain S. senoensis NBRC 13843T. Isolates 10-1 and 10-6, identified as species S. vinaceus, displayed equivalent PLD activity (0.82 and 0.84 U ml-1) with the reference strain S. vinaceus NBRC 13425T. All the S. vinaceus strains (NBRC 13425T, 10-1 and 10-6) are closely related to the strain S. cirratus NBRC 13398T, but the PLD activity of the S. vinaceus strains was 3.9-fold higher than that of S. cirratus NBRC 13398T. Isolates 10-2 and 10-3 were identified as species S. racemochromogenes. However, isolates 10-2 and 10-3 (0.72 and 0.74 U ml-1)
Table 1 DNA–DNA relatedness values between the tested strains Cluster I S. senoensis NBRC 13843
T
NBRC 13843T
Strain 9-4
JCM 4738T
NBRC 13425T
Strain 10-1
100
99
36
35
37
Strain 9-4
80
100
37
36
39
S. cirratus JCM 4738T
55
38
100
35
46
S. vinaceus NBRC 13425T
53
27
43
100
77
Strain 10-1
44
31
37
73
100
Strain 10-6
49
30
37
74
100
Cluster II
JCM 4777
S. katrae JCM 4777T S. polychromogenes JCM 4505T
T
T
T
JCM 4505
NBRC 12906
100
74
63
62
69
100
60
58
S. racemochromogenes NBRC 12906T
50
55
100
97
Strain 10-2
51
64
91
100
Strain 10-3
53
63
91
100
Cluster III
NBRC 13064T
NBRC 12919T
Strain 21-4
T
100
100
37
S. tanashiensis NBRC 12919T
70
100
21
Strain 21-4
44
53
100
S. nashvillensis NBRC 13064
Strain 10-2
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Y. Nakazawa et al.: Identification of Actinomycetes Producing PLD
PLD activity (U/ml)
A 1.5
d c
1.0 0.5
e c
e d
f
b
a
f
b
a
0
Cluster Ia
Cluster Ib
Cluster II
Strain 21-4
NBRC 12919T
NBRC 13064T
Strain 10-3
Strain 10-2
JCM 4505T
NBRC 12906T
JCM 4777T
Strain 10-6
Strain 10-1
NBRC 13425T
JCM 4738T
Strain 9-4
NBRC 13843T
B
Cluster III
C Cluster Ia
Cluster Ib
Cluster II
Cluster III
Fig. 2 PLD productivity of the isolated and reference Streptomyces strains (a) and confirmation of the pld gene (b and c). a PC-hydrolytic activities of the culture supernatants of isolates and reference strains. Values indicated by same letters were not significantly different at P \ 0.001 according to Sheffe’s test. b The presence of the pld gene in genomic DNA from isolates and reference strains was verified by PCR using a primer set as described in ‘‘Materials and Methods’’
section. c Alignments of amino acid sequences deduced from the nucleotide sequences of partial pld genes of isolated and reference strains. The genomic DNA fragments of isolated strains were amplified by PCR using the primer pair S300 Fw/S300 Rv. The sequences were generated by elimination of the primer sequences. Alignments were performed against each cluster using the CLUSTAL X algorithm
produced PLD activities 3.8–3.9-fold higher than that of the reference strain S. racemochromogenes NBRC 12906T, respectively. Additionally, closely related strains S. polychromogenes JCM 4505T and S. katrae JCM 4777T produced 0.6- and 0.4-fold higher activity than the 10-2 and 10-3 strains, respectively. Isolate 21-4 produced
0.534 U ml-1 of PLD activity. This strain, which could not be matched to a known Streptomyces species, was related to reference strains S. nashvillensis NBRC 13064T and S. tanashiensis NBRC 12919T, but these strains produced only 0.075 U ml-1 (0.14-fold that of the 21-4 strain) and 0.025 U ml-1 (0.046-fold that of the 21-4 strain),
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371
respectively (Fig. 2a). As compared to other Streptomyces species well known as PLD producers, PLD activities of culture supernatants of S. antibioticus strain S-170 (3.2 U ml-1, 3-days cultivation at 28°C [31]; 3.4 U ml-1, 2-days cultivation at 26°C [32]) and Streptomyces sp. strain PMF (1.07 U ml-1, 2-days cultivation at 30°C [33]) are superior to those of our strains. However, our strains produced extracellular PLD activities as well or better than the PLD productive species Streptoverticillium cinnamoneum IFO 12852 (0.77 U ml-1, 3-days cultivation at 30°C [34]) and S. septatus strain TH-2 (0.18 U ml-1, 1-day cultivation at 34°C [35]; 0.28 U ml-1, 3-days cultivation at 37°C [36]). The pld gene in genomes from all the strains employed in this study was verified by PCR using a primer set targeting a 314 bp highly conserved region among the known Streptomyces pld genes followed by direct sequencing of amplified DNA fragments (Fig. 2b, c). Although all the strains employed for this study possess the pld gene in their genome, PLD activities produced by the strains showed significant variations. Within the determined nucleotide sequence consisting of 274 bp generated by elimination of the primer sequences, strains 10-1 and 10-6, and strains 102 and 10-3, shared identical nucleotide sequences. The fractional-deduced amino acid sequence of PLD from strain 9-4 exhibited the highest identities (98.9%) with the PLDs of S. senoensis NBRC 13843T, S. racemochromogenes NBRC 12906T, and strains 10-2 and 10-3. The amino acid sequences of the PLD from strains 10-1 and 10-6 exhibited the highest identity (97.8%) with that of S. vinaceus NBRC 13425T PLD. The PLDs of strains 10-2 and 10-3 displayed identical fractional-amino acid sequence as those of S. racemochromogenes NBRC 12906T and S. senoensis 13843T. The deduced sequence of PLD from strain 21-4 shared the highest identity (96.7%) with that of S. nashvillensis NBRC 13064T PLD.
strains 10-2 and 10-3 were matched to S. racemochromogenes. The three strains identified as S. vinaceus (S. vinaceus NBRC 13425T and strains 10-1 and 10-6) displayed similar intensities of PLD activity. However, the PLD activity of reference strains S. senoensis NBRC 13843T (0.16-fold that of strain 9-4) and S. racemochromogenes NBRC 12906T (0.26-fold that of strains 10-2 and 10-3) exhibited significantly lower PLD activities compared with our strains. Even though the determined sequences were limited to a 274 bp fragment coding a region that is the second of two HKD motifs of the PLD enzyme, the fractional amino acid sequences deduced from genomic DNA fragments displayed significantly higher identities of 97.8– 100% between the same species. The data imply that the Streptomyces strains can produce different quantities of PLD enzyme depending on the strain. Therefore, it is worthwhile to clone and sequence the pld genes, including the promoter region, of our strains to compare with known Streptomyces pld genes. Although essential residues for transphosphatidylation activity have remained obscure so far, there is a report demonstrating that the transphosphatidylation activity of S. cinnamoneum PLD was remarkably enhanced by substituting a serine residue into a GG motif that is close to the active center residues [40]. Thus, a difference in a few residues of the PLD enzyme may interfere with the transphosphatidylation capacity. The Streptomyces strains isolated and characterized here produced PLD enzyme with high transphosphatidylation activity, as previously shown. Further examination to characterize the enzymatic properties of the PLD enzymes produced by our strains would be of help to clarify the transphosphatidylation mechanism.
Discussion In the previous study, we isolated six strains that have a high capacity for production of PLD and have high transphosphatidylation activity [9]. All the strains were matched here to Streptomyces species based on the 16S rDNA sequences. PLD is widely distributed in animals, plants, and microorganisms. Of the PLDs from various organisms, the enzyme from the Streptomyces species is known to have high transphosphatidylation activity [37–39]. The PLD activities produced by our strains significantly exceeded those of the reference strains, except for S. vinaceus NBRC 13425T. Given the judging standard for the DDH test, our strain 9-4 was matched to S. senoensis, strains 10-1 and 10-6 were matched to S. vinaceus, and
Acknowledgments The authors are grateful to Prof. Toshio Nagashima, Tokyo University of Agriculture for his excellent contributions to this work.
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