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Desiccation-induced viable but nonculturable state in Pseudomonas putida KT2440, a survival strategy.

  • Pazos-Rojas, Laura Abisaí1, 2, 3
  • Muñoz-Arenas, Ligia Catalina4
  • Rodríguez-Andrade, Osvaldo1
  • López-Cruz, Lesther Emanuel1
  • López-Ortega, Orestes5
  • Lopes-Olivares, Fábio6
  • Luna-Suarez, Silvia2
  • Baez, Antonino1
  • Morales-García, Yolanda Elizabeth1, 3
  • Quintero-Hernández, Verónica1, 7
  • Villalobos-López, Miguel Angel2
  • De la Torre, Jesús8
  • Muñoz-Rojas, Jesús1
  • 1 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. , (Mexico)
  • 2 Centro de Investigación en Biotecnología Aplicada, Instituto Politécnico Nacional, Tepetitla, Tlaxcala, Mexico. , (Mexico)
  • 3 Licenciatura en Biotecnología, Facultad de Ciencias Biológicas, BUAP, Puebla, Mexico. , (Mexico)
  • 4 Facultad de Ingeniería Ambiental, Universidad Popular Autónoma de Puebla, Puebla, Mexico. , (Mexico)
  • 5 Inserm U932, Institute Curie, Paris, France. , (France)
  • 6 Núcleo de Desenvolvimento de Insumos Biológicos para a Agricultura (NUDIBA), Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), Rio de Janeiro, Brazil. , (Brazil)
  • 7 CONACYT, ESMRG, LEMM, CICM, IC, BUAP, Puebla, México.
  • 8 Department of Environmental Protection, CSIC-Estación Experimental del Zaidín, Granada, Spain. , (Spain)
Published Article
Public Library of Science
Publication Date
Jan 01, 2019
DOI: 10.1371/journal.pone.0219554
PMID: 31323038


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.

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