Ryu, Du-Hwan
(Department of Biomedicinal Science and Biotechnology, Paichai University)
,
Lee, Sang-Won
(Department of Biomedicinal Science and Biotechnology, Paichai University)
,
Mikolaityte, Viktorija
(Department of Biomedicinal Science and Biotechnology, Paichai University)
,
Kim, Yea-Won
(Department of Biomedicinal Science and Biotechnology, Paichai University)
,
Jeong, Haeyoung
(Infectious Disease Research Center, Korea Research Institute of Bioscience and Biotechnology (KRIBB))
,
Lee, Sang Jun
(Department of Systems Biotechnology, Chung-Ang University)
,
Lee, Chung-Hak
(School of Chemical and Biological Engineering, Seoul National University)
,
Lee, Jung-Kee
(Department of Biomedicinal Science and Biotechnology, Paichai University)
N-acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) plays a major role in development of biofilms, which contribute to rise in infections and biofouling in water-related industries. Interference in QS, called quorum quenching (QQ), has recieved a lot of attention in recent years. Rhodococcu...
N-acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) plays a major role in development of biofilms, which contribute to rise in infections and biofouling in water-related industries. Interference in QS, called quorum quenching (QQ), has recieved a lot of attention in recent years. Rhodococcus spp. are known to have prominent quorum quenching activity and in previous reports it was suggested that this genus possesses multiple QQ enzymes, but only one gene, qsdA, which encodes an AHL-lactonase belonging to phosphotriesterase family, has been identified. Therefore, we conducted a whole genome sequencing and analysis of Rhodococcus sp. BH4 isolated from a wastewater treatment plant. The sequencing revealed another gene encoding a QQ enzyme (named jydB) that exhibited a high AHL degrading activity. This QQ enzyme had a 46% amino acid sequence similarity with the AHL-lactonase (AidH) of Ochrobactrum sp. T63. HPLC analysis and AHL restoration experiments by acidification revealed that the jydB gene encodes an AHL-lactonase which shares the known characteristics of the α/β hydrolase family. Purified recombinant JydB demonstrated a high hydrolytic activity against various AHLs. Kinetic analysis of JydB revealed a high catalytic efficiency (kcat/KM) against C4-HSL and 3-oxo-C6 HSL, ranging from 1.88 x 106 to 1.45 x 106 M-1 s-1, with distinctly low KM values (0.16-0.24 mM). This study affirms that the AHL degrading activity and biofilm inhibition ability of Rhodococcus sp. BH4 may be due to the presence of multiple quorum quenching enzymes, including two types of AHL-lactonases, in addition to AHL-acylase and oxidoreductase, for which the genes have yet to be described.
N-acyl-homoserine lactone (AHL)-mediated quorum sensing (QS) plays a major role in development of biofilms, which contribute to rise in infections and biofouling in water-related industries. Interference in QS, called quorum quenching (QQ), has recieved a lot of attention in recent years. Rhodococcus spp. are known to have prominent quorum quenching activity and in previous reports it was suggested that this genus possesses multiple QQ enzymes, but only one gene, qsdA, which encodes an AHL-lactonase belonging to phosphotriesterase family, has been identified. Therefore, we conducted a whole genome sequencing and analysis of Rhodococcus sp. BH4 isolated from a wastewater treatment plant. The sequencing revealed another gene encoding a QQ enzyme (named jydB) that exhibited a high AHL degrading activity. This QQ enzyme had a 46% amino acid sequence similarity with the AHL-lactonase (AidH) of Ochrobactrum sp. T63. HPLC analysis and AHL restoration experiments by acidification revealed that the jydB gene encodes an AHL-lactonase which shares the known characteristics of the α/β hydrolase family. Purified recombinant JydB demonstrated a high hydrolytic activity against various AHLs. Kinetic analysis of JydB revealed a high catalytic efficiency (kcat/KM) against C4-HSL and 3-oxo-C6 HSL, ranging from 1.88 x 106 to 1.45 x 106 M-1 s-1, with distinctly low KM values (0.16-0.24 mM). This study affirms that the AHL degrading activity and biofilm inhibition ability of Rhodococcus sp. BH4 may be due to the presence of multiple quorum quenching enzymes, including two types of AHL-lactonases, in addition to AHL-acylase and oxidoreductase, for which the genes have yet to be described.
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제안 방법
Kinetic analysis was carried out to characterize the purified JydB using phenol red, a pH indicator, by monitoring the release of H+ during the enzymatic degradation of C4-HSL, C6-HSL, and oxo-C6-HSL. Lineweaver-Burk plot was used to determine the kinetic constants (KM and kcat).
대상 데이터
The genome sequencing of Rhodococcus sp. BH4 was carried out at the National Instrumentation Center for Environmental Management in Seoul National University (Republic of Korea) using the PacBio RSII platform with P6-C4 chemistry (Pacific Biosciences, USA). RS_HGAP_Assembly.
Samples were chromatographed on an HPLC system with a UV/visible light (VIS) detector set at 205 nm by use of a ZORBAX Eclipse XDB-C18 column (4.6 × 250 mm) (Agilent Technologies).
The N-acyl-homoserine lactones used in this study were; N-butyryl-l-homoserine lactone (C4-HSL), N-hexanoyl-l-homoserine lactone (C6-HSL), N-octanoyl-l-homoserine lactone (C8-HSL), N-decanoyl-l-homoserine lactone (C10-HSL), N-dodecanoyl-l-homoserine lactone (C12-HSL), N-(3-oxohexanoy-(3-homoserine lactone (3-oxo-C6-HSL), N-(3-oxooctanoyl)-l-homoserine lactone (3-oxo-C8-HSL) and N-(3-oxododecanoyl)-l-homoserine lactone (3-oxo-C12-HSL), which were purchased from BNPharm Co, Ltd (Daejeon, South Korea). In order to determine the activity of JydB, 6 μg of purified enzyme was mixed with AHLs (final concentrations depended on the biosensor: 20 μM for CV026, 5 μM and 1 μM for NT1 and 1 μM for A136) in Tris-HCl buffer (10 mM pH 7.
데이터처리
The one-tailed Student’s t-test was used to assess significant difference between groups.
이론/모형
The purity and size of the protein were confirmed by SDS-PAGE. Concentration was estimated using the Bradford assay.
JydB is indicated with an asterisk. The dendrogram was constructed using the neighbor-joining method with MEGA X software (http://www.megasoftware.net/). The scale bar represents 0.
성능/효과
Five candidate genes for the QQ enzymes were amplified by PCR, besides the qsdA gene (A0W34_26390) which possesses a 99% amino acid sequence identity with QsdA from the Rhodococcus erythropolis strain W2 [20]. These five genes (A0W34_26705, A0W34_24065, A0W34_00835, A0W34_31420, A0W34_03910) shared a 35%-45% amino acid sequence identity with sequences of known AHL-lactonases, AHL-acylases, and oxidoreductases (Table S2). These genes were cloned into pMD20 T vector and expressed in E.
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