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

Abstract

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.

Leveraging Pseudomonas Stress Response Mechanisms for Industrial Applications

“…Viable but non-culturable (VBNC) cells have low metabolic activity and intact membranes, but they are unable to replicate until they enter a more hospitable environment ( Ayrapetyan et al, 2018 ). Pseudomonas strains can enter this state as a response to unfavorable environmental conditions such as nutrient limitation, temperature extremes, and desiccation ( Lowder et al, 2000 ; Arana et al, 2010 ; Pazos-Rojas et al, 2019 ). Resuscitation methods that allow VBNC cells to repair and transition into a culturable state can vary greatly depending on the type of microorganism and environmental conditions ( Bédard et al, 2014 ).…”

“…Resuscitation methods that allow VBNC cells to repair and transition into a culturable state can vary greatly depending on the type of microorganism and environmental conditions ( Bédard et al, 2014 ). The plant growth-promoting rhizobacterium P. putida KT2440 was found to return to a cultivable state after interaction with maize root exudates or a 48 h rehydration in water ( Pazos-Rojas et al, 2019 ). The persistence of VBNC Pseudomonas in the natural environment was demonstrated with the biocontrol strain P. protegens CHA0 which survived in an uncovered field plot as a combination of dormant and VBNC cells for 72 days ( Troxler et al, 2012 ).…”

A Bacterial Consortium Interacts With Different Varieties of Maize, Promotes the Plant Growth, and Reduces the Application of Chemical Fertilizer Under Field Conditions

“…The root of each plant was placed in 50 mL conical tubes with 10 mL of sterile water for 1.5 h. To determine the bacterial population extracted from the roots, the MSDP technique was used with each selection media for each bacterium; the incubation conditions of the plates were 24 to 48 h at 30 • C (Rodríguez-Andrade et al, 2015; Molina-Romero et al, 2017). The vermiculite strongly adhered to the root was removed and its dry weight determined to calculate the parameter Colony-Forming Units / gram of Vermiculite (CFU/gV) (Morales-García et al, 2011;Rodríguez-Andrade et al, 2015;Molina-Romero et al, 2017;Pazos-Rojas et al, 2019).…”

Narrative of a versatile and adept species Pseudomonas putida

“…Despite the high metabolic and adaptive versatility, the survival of P. putida decreases drastically after the loss of water [102,103]. The recent study by Pazos-Rojas and colleagues demonstrated that during desiccation, P. putida suffers damage to its membranes and enters the VBNC state, but the cells return to the culturable state after prolonged rehydration when the membranes are repaired [104]. Thus, the entering P. putida in the VBNC state could allow survival of this organism in its natural environment under conditions of desiccation.…”