Ecology and Survival of Microorganisms Research Group (ESMRG), Laboratorio de Ecología Molecular Microbiana (LEMM), Centro de Investigaciones en Ciencias Microbiológicas (CICM), Instituto de Ciencias (IC), Benemérita Universidad Autónoma de Puebla (BUAP), Puebla, Mexico.
Centro de Investigación en Biotecnología Aplicada, Instituto Politécnico Nacional, Tepetitla, Tlaxcala, Mexico.
PLoS One. 2019 Jul 19;14(7):e0219554. doi: 10.1371/journal.pone.0219554. eCollection 2019.
The potential of Pseudomonas putida KT2440 to act as a plant-growth promoter or as a bioremediator of toxic compounds can be affected by desiccation. In the present work, the bacterial survival ratio (BSR) in response to air desiccation was evaluated for P. putida KT2440 in the presence of different protectors. The BSR in the presence of nonreducing disaccharides, such as trehalose, was high after 15 days of desiccation stress (occurring at 30°C and 50% relative humidity), whereas in the absence of a protector the bacterial counts diminished to nondetectable numbers (ca 2.8 log CFU/mL). The LIVE/DEAD staining method showed that bacteria protected with trehalose maintained increased numbers of green cells after desiccation while cells without protection were all observed to be red. This indicated that nonprotected bacteria had compromised membrane integrity. However, when nonprotected bacteria subjected to 18 days of desiccation stress were rehydrated for a short time with maize root exudates or for 48 h with water (prolonged rehydration), the bacterial counts were as high as that observed for those not subjected to desiccation stress, suggesting that the cells entered the viable but nonculturable (VBNC) state under desiccation and that they returned to a culturable state after those means of rehydration. Interestingly an increase in the green color intensity of cells that returned to a culturable state was observed using LIVE/DEAD staining method, indicating an improvement in their membrane integrity. Cellular activity in the VBNC state was determined. A GFP-tagged P. putida strain expressing GFP constitutively was subjected to desiccation. After 12 days of desiccation, the GFP-tagged strain lost culturability, but it exhibited active GFP expression, which in turn made the cells green. Furthermore, the expression of 16S rRNA, rpoN (housekeeping), mutL, mutS (encoding proteins from the mismatch repair complex), and oprH (encoding an outer membrane protein) were examined by RT-PCR. All evaluated genes were expressed by both types of cells, culturable and nonculturable, indicating active molecular processes during the VBNC state.
铜绿假单胞菌 KT2440 具有促进植物生长和修复有毒化合物的潜力，但干燥会对其产生影响。在本研究中，评估了不同保护剂存在下铜绿假单胞菌 KT2440 在空气干燥胁迫下的细菌存活率（BSR）。在存在非还原二糖（如海藻糖）的情况下，BSR 在 15 天的干燥胁迫（在 30°C 和 50%相对湿度下发生）后仍然很高，而在没有保护剂的情况下，细菌数量减少到无法检测的水平（约 2.8 log CFU/mL）。LIVE/DEAD 染色法显示，用海藻糖保护的细菌在干燥后保持了更多的绿色细胞，而没有保护的细菌则全部呈现红色。这表明未受保护的细菌细胞膜完整性受损。然而，当未受保护的细菌在干燥胁迫下放置 18 天后，用玉米根分泌物进行短暂的水合（短暂水合）或用纯水进行 48 小时的水合（长时间水合）时，其细菌数量与未经历干燥胁迫的细菌数量相当，这表明在干燥胁迫下细胞进入了可存活但不可培养（VBNC）状态，并且在经过这些水合方式后，细胞恢复了可培养状态。有趣的是，使用 LIVE/DEAD 染色法观察到可培养状态下的细胞绿色强度增加，表明其细胞膜完整性得到改善。还测定了 VBNC 状态下的细胞活性。一株表达 GFP 的 GFP 标记铜绿假单胞菌被用于干燥胁迫实验。在干燥 12 天后，GFP 标记的菌株失去了可培养性，但表现出了活跃的 GFP 表达，从而使细胞变绿。此外，通过 RT-PCR 检测了 16S rRNA、rpoN（管家基因）、mutL、mutS（编码错配修复复合物的蛋白）和 oprH（编码外膜蛋白）的表达。两种类型的细胞（可培养和不可培养）均表达所有评估的基因，表明在 VBNC 状态下存在活跃的分子过程。
