United Graduate School of Agricultural Science, Tokyo University of Agriculture and Technology , 3-5-8 Saiwai-cho, Fuchu, Tokyo 183-8509 , Japan.
Braz J Microbiol. 2011 Apr;42(2):576-91. doi: 10.1590/S1517-838220110002000023. Epub 2011 Jun 1.
Cells of Ralstonia solanacearum were exposed to Cu in distilled water, and the resulting Cu-stressed non-culturable cells were inoculated to natural (non-pasteurized) and pasteurized soils in order to examine their culturability and recovery. Exposing the cells to 20 μM CuSO4 produced transitory non-culturable cells, which exhibited a remarkable recovery in culturability after incubation in the solution for 36 h, reaching a density near the initial level by 108 h. To determine whether such non-culturable cells actually "resuscitated" or multiplied after adapting to Cu toxicity, growth curves were constructed in order to contrast the rates of increase in culturable cell numbers between Cu-stressed or non-stressed inocula. Additionally, fresh non-stressed cells were exposed to CuSO4 in the presence of nalidixic acid by adding the antibiotic at different times after the onset of Cu stress to verify any cell multiplication during the population increase. The results revealed that the non-culturable cells surviving Cu toxicity adapted very quickly to Cu and began multiplying within 12 h, because only the Cu-stressed cells that were increasing in the exponential growth phase, but not those in the stationary phase, were killed by the antibiotic. Such cells exhibited an apparent tolerance to this metal when inoculated to a freshly prepared solution of CuSO4, and also detoxified the ion in the solution in which they grew. The presence of nutrients greatly counteracted the effect of Cu in water microcosms, since culturable cells were detected and increased in number even when exposed to 40 μM CuSO4. In contrast, when inoculated to non-pasteurized soil, Cu-stressed cells showed no such recoveries. However, when the soil was pasteurized before inoculation or added with nutrients, culturable cells were recovered and increased in number. This indicates that increased nutrient availability in soil allows Cu-stressed cells to quickly overcome the stress and increase in culturable populations.
将罗尔斯通氏菌细胞暴露于蒸馏水中的铜，将由此产生的铜胁迫非可培养细胞接种到天然（未巴氏消毒）和巴氏消毒土壤中，以检查它们的可培养性和恢复能力。将细胞暴露于 20 μM CuSO4 会产生短暂的非可培养细胞，这些细胞在溶液中孵育 36 小时后表现出显著的可培养性恢复，到 108 小时时接近初始水平。为了确定这些非可培养细胞在适应铜毒性后是否真的“复苏”或增殖，构建了生长曲线，以对比可培养细胞数量增加率，比较铜胁迫或非胁迫接种物之间的差异。此外，新鲜的非胁迫细胞在添加萘啶酸后暴露于 CuSO4 中，通过在铜胁迫开始后不同时间添加抗生素来验证种群增加过程中的任何细胞增殖。结果表明，在铜毒性中存活下来的非可培养细胞对铜适应得非常快，并在 12 小时内开始增殖，因为只有在指数生长阶段增加的铜胁迫细胞，而不是处于静止期的细胞，才会被抗生素杀死。这些细胞在接种到新鲜的 CuSO4 溶液中时表现出对这种金属的明显耐受性，并且还可以在其生长的溶液中解毒该离子。当在水微宇宙中存在养分时，养分极大地抵消了铜的作用，因为即使暴露于 40 μM CuSO4 也可以检测到可培养细胞并增加其数量。相比之下，当将其接种到未巴氏消毒的土壤中时，铜胁迫细胞没有这种恢复能力。然而，当在接种前对土壤进行巴氏消毒或添加养分时，可以回收可培养细胞并增加其数量。这表明土壤中增加的养分可用性可以使铜胁迫细胞迅速克服压力并增加可培养种群。
