Maisuria Vimal B, Nerurkar Anuradha S
Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India; School of Life Sciences, Faculty of Health and Human Science, University of Hertfordshire, College Lane, Hatfield, Hertfordshire, United Kingdom.
Department of Microbiology and Biotechnology Centre, Faculty of Science, The Maharaja Sayajirao University of Baroda, Vadodara, Gujarat, India.
PLoS One. 2015 Sep 18;10(9):e0138034. doi: 10.1371/journal.pone.0138034. eCollection 2015.
Turf soil bacterial isolate Delftia sp. VM4 can degrade exogenous N-acyl homoserine lactone (AHL), hence it effectively attenuates the virulence of bacterial soft rot pathogen Pectobacterium carotovorum subsp. carotovorum strain BR1 (Pcc BR1) as a consequence of quorum sensing inhibition.
METHODOLOGY/PRINCIPAL FINDINGS: Isolated Delftia sp. VM4 can grow in minimal medium supplemented with AHL as a sole source of carbon and energy. It also possesses the ability to degrade various AHL molecules in a short time interval. Delftia sp. VM4 suppresses AHL accumulation and the production of virulence determinant enzymes by Pcc BR1 without interference of the growth during co-culture cultivation. The quorum quenching activity was lost after the treatment with trypsin and proteinase K. The protein with quorum quenching activity was purified by three step process. Matrix assisted laser desorption/ionization-time of flight (MALDI-TOF) and Mass spectrometry (MS/MS) analysis revealed that the AHL degrading enzyme (82 kDa) demonstrates homology with the NCBI database hypothetical protein (Daci_4366) of D. acidovorans SPH-1. The purified AHL acylase of Delftia sp. VM4 demonstrated optimum activity at 20-40°C and pH 6.2 as well as AHL acylase type mode of action. It possesses similarity with an α/β-hydrolase fold protein, which makes it unique among the known AHL acylases with domains of the N-terminal nucleophile (Ntn)-hydrolase superfamily. In addition, the kinetic and thermodynamic parameters for hydrolysis of the different AHL substrates by purified AHL-acylase were estimated. Here we present the studies that investigate the mode of action and kinetics of AHL-degradation by purified AHL acylase from Delftia sp. VM4.
We characterized an AHL-inactivating enzyme from Delftia sp. VM4, identified as AHL acylase showing distinctive similarity with α/β-hydrolase fold protein, described its biochemical and thermodynamic properties for the first time and revealed its potential application as an anti-virulence agent against bacterial soft rot pathogen Pectobacterium carotovorum subsp. carotovorum based on quorum quenching mechanism.
草坪土壤细菌分离株代尔夫特菌属VM4能够降解外源性N-酰基高丝氨酸内酯（AHL），因此由于群体感应抑制作用，它能有效减弱细菌性软腐病原菌胡萝卜软腐果胶杆菌胡萝卜亚种BR1菌株（Pcc BR1）的毒力。
方法/主要发现：分离得到的代尔夫特菌属VM4能够在以AHL作为唯一碳源和能源的基本培养基中生长。它还具有在短时间内降解各种AHL分子的能力。在共培养过程中，代尔夫特菌属VM4抑制了Pcc BR1的AHL积累和毒力决定酶的产生，且不干扰其生长。用胰蛋白酶和蛋白酶K处理后，群体猝灭活性丧失。通过三步法纯化了具有群体猝灭活性的蛋白质。基质辅助激光解吸/电离飞行时间（MALDI-TOF）和质谱（MS/MS）分析表明，AHL降解酶（82 kDa）与酸土代尔夫特菌SPH-1的NCBI数据库假设蛋白（Daci_4366）具有同源性。纯化后的代尔夫特菌属VM4的AHL酰基转移酶在20-40°C和pH 6.2时表现出最佳活性，以及AHL酰基转移酶类型的作用模式。它与α/β-水解酶折叠蛋白具有相似性，这使其在已知的具有N-末端亲核体（Ntn）-水解酶超家族结构域的AHL酰基转移酶中独一无二。此外，还估算了纯化后的AHL-酰基转移酶对不同AHL底物水解的动力学和热力学参数。在此，我们展示了对代尔夫特菌属VM4纯化的AHL酰基转移酶的AHL降解作用模式和动力学的研究。
我们对代尔夫特菌属VM4中的一种AHL失活酶进行了表征，鉴定为与α/β-水解酶折叠蛋白具有显著相似性的AHL酰基转移酶，首次描述了其生化和热力学性质，并揭示了其基于群体猝灭机制作为抗细菌性软腐病原菌胡萝卜软腐果胶杆菌胡萝卜亚种毒力剂的潜在应用。
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