Shenzhen Key Laboratory of Marine Bioresource and Eco-environmental Science, College of Life Science, Shenzhen University, Shenzhen, 518060, China.
Microb Ecol. 2012 Jan;63(1):64-73. doi: 10.1007/s00248-011-9928-2. Epub 2011 Aug 18.
The viable but nonculturable (VBNC) state has been found to be a growth strategy used by many aquatic pathogens; however, few studies have focused on VBNC state on other aquatic bacterial groups. The purpose of this study was to explore the VBNC state of cyanobacteria-lysing bacteria and the conditions that regulate their VBNC state transformation. Three cyanobacteria-lysing heterotrophic bacterial strains (F1, F2 and F3) were isolated with liquid infection method from a lake that has experienced a cyanobacterial bloom. According to their morphological, physiological and biochemical characteristics and results of 16SrDNA sequence analysis, F1, F2 and F3 were identified as strains of Staphylococcus sp., Stappia sp. and Microbacterium sp., respectively. After being co-cultured with the axenic cyanobacterium, Microcystis aeruginosa 905, for 7 days, strains F1, F2 and F3 exhibited an inhibition effect on cyanobacterial growth, which was expressed as a reduction in chlorophyll concentration of 96.0%, 94.9% and 84.8%, respectively. Both autoclaved and filtered bacterial cultures still showed lytic effects on cyanobacterial cells while centrifuged pellets were less efficient than other fractions. This indicated that lytic factors were extracelluar and heat-resistant. The environmental conditions that could induce the VBNC state of strain F1 were also studied. Under low temperature (4°C), distilled deionized water (DDW) induced almost 100% of F1 cells to the VBNC state after 6 days while different salinities (1%, 3% and 5% of NaCl solution) and lake water required 18 days. A solution of the cyanobacterial toxin microcystin-LR (MC-LR) crude extract also induced F1 to the VBNC state, and the effect was stronger than DDW. Even the lowest MC-LR concentration (10 μg L(-1)) could induce 69.7% of F1 cells into VBNC state after 24 h. On the other hand, addition of Microcystis aeruginosa cells caused resuscitation of VBNC state F1 cells within 1 day, expressed as an increase of viable cell number and a decrease of VBNC ratio. Both VBNC state and culturable state F1 cells showed lytic effects on cyanobacteria, with their VBNC ratio varying during co-culturing with cyanobacteria. The findings indicated that VBNC state transformation of cyanobacteria-lysing bacteria could be regulated by cyanobacterial cells or their toxin, and the transformation may play an important role in cyanobacterial termination.
可培养但不可培养（VBNC）状态已被发现是许多水生病原体使用的一种生长策略；然而，很少有研究关注其他水生细菌群体的 VBNC 状态。本研究旨在探索蓝藻裂解细菌的 VBNC 状态以及调节其 VBNC 状态转化的条件。使用液体感染法从经历过蓝藻水华的湖泊中分离出三种蓝藻裂解异养细菌菌株（F1、F2 和 F3）。根据它们的形态、生理和生化特征以及 16SrDNA 序列分析结果，F1、F2 和 F3 分别被鉴定为葡萄球菌属、Stappia 属和微杆菌属的菌株。与无菌蓝藻铜绿微囊藻 905 共培养 7 天后，菌株 F1、F2 和 F3 对蓝藻生长表现出抑制作用，表现为叶绿素浓度分别降低 96.0%、94.9%和 84.8%。巴氏杀菌和过滤的细菌培养物仍对蓝藻细胞表现出裂解作用，而离心沉淀则不如其他部分有效。这表明裂解因子是细胞外的且耐热的。还研究了诱导菌株 F1 进入 VBNC 状态的环境条件。在低温（4°C）下，蒸馏去离子水（DDW）在 6 天后几乎将 100%的 F1 细胞诱导至 VBNC 状态，而不同的盐度（1%、3%和 5%的氯化钠溶液）和湖水需要 18 天。蓝藻毒素微囊藻毒素-LR（MC-LR）粗提物的溶液也诱导 F1 进入 VBNC 状态，其效果强于 DDW。即使是最低的 MC-LR 浓度（10μg/L）也能在 24 小时后诱导 69.7%的 F1 细胞进入 VBNC 状态。另一方面，添加铜绿微囊藻细胞可在 1 天内使 VBNC 状态的 F1 细胞复苏，表现为活菌数增加和 VBNC 比例降低。VBNC 状态和可培养状态的 F1 细胞均对蓝藻表现出裂解作用，其 VBNC 比例在与蓝藻共培养过程中发生变化。研究结果表明，蓝藻裂解菌的 VBNC 状态转化可以被蓝藻细胞或其毒素调节，这种转化可能在蓝藻终止中发挥重要作用。
