Effect of High Temperature on Pseudomonas putida ... - Springer Link

Curr Microbiol (2008) 56:453–457 DOI 10.1007/s00284-008-9105-0

Effect of High Temperature on Pseudomonas putida NBRI0987 Biofilm Formation and Expression of Stress Sigma Factor RpoS S. Srivastava Æ A. Yadav Æ K. Seem Æ S. Mishra Æ V. Chaudhary Æ C. S. Nautiyal

Received: 13 September 2007 / Accepted: 26 November 2007 / Published online: 25 January 2008 Ó Springer Science+Business Media, LLC 2008

Abstract Pseudomonas is an efficient plant growth– promoting rhizobacteria; however, among the limiting factors for its commercialization, tolerance for high temperature is the most critical one. After screening 2,500 Pseudomnas sp. strains, a high temperature tolerant–strain Pseudomonas putida NBRI0987 was isolated from the drought-exposed rhizosphere of chickpea (Cicer arietinum L. cv. Radhey), which was grown under rain-fed conditions. P. putida NBRI0987 tolerated a temperature of 40°C for B 5 days. To the best of our knowledge, this is the first report of a Pseudomnas sp. demonstrating survival estimated by counting viable cells under such a high temperature. P. putida NBRI0987 colony-forming unit (CFU)/ml on day 10 in both the absence and presence of MgSO4.7H2O (MgSO4) in combination with glycerol at 40°C were 0.0 and 1.7 9 1011, respectively. MgSO4 plus glycerol also enhanced the ability of P. putida NBRI0987 to tolerate high temperatures by inducing its ability to form biofilm. However, production of alginate was not critical for biofilm formation. The present study demonstrates overexpression of stress sigma factor rS (RpoS) when P. putida NBRI0987 is grown under high-temperature stress at 40°C compared with 30°C. We present evidence, albeit indirect, that the adaptation of P. putida NBRI0987 to high temperatures is a complex multilevel

S. Srivastava A. Yadav K. Seem S. Mishra V. Chaudhary C. S. Nautiyal (&) Division of Plant Microbe Interactions, National Botanical Research Institute, Rana Pratap Marg, Lucknow 226001, India e-mail: [email protected]

regulatory process in which many different genes can be involved.

Introduction Pseudomonas is an efficient plant growth–promoting rhizobacteria, and certain isolates can enhance plant health [6– 8]. Pseudomonas can be found in many different environments, including soil, water, plant, animal, and human. To persist successfully in a changing environment a microorganism must sense such change and react appropriately. Among the limiting factors for its commercialization, tolerance for high temperature is the most critical, resulting in limited use because of its short shelf life [6, 12]. An understanding of the growth of Pseudomonas isolated from stressed conditions is likely only when the physiology of these organisms has been carefully studied under these suboptimal conditions. Moreover, Pseudomonas spp. with the genetic potential for increased tolerance to these adverse environmental stresses could enhance production of food in semiarid and arid regions of the world [6, 9]. In view of the sensitivity of Pseudomonas spp. to the high temperatures frequently encountered in the tropics and subtropics, an investigation was conducted to find a means by which to better protect or enhance the stress tolerance of Pseudomonas spp. A high temperature–tolerant P. putida NBRI0987 strain was isolated from the drought-exposed rhizosphere of chickpea, which was grown under rain-fed conditions. The goal of this study was to elucidate the phenotypic and genetic attributes of high temperature– tolerant P. putida NBRI0987 involved in enhanced biofilm formation and protecting Pseudomonas from high temperature.

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S. Srivastava et al.: High Temperature–Tolerant P. putida

Materials and Methods Bacterial Strain, Culture Media, and Growth Conditions Bacterial strains were isolated from the roots of fieldgrown chickpea (Cicer arietinum L. cv. Radhey) in a rain-fed area of Dholpur, Rajasthan, India, located at latitude 26°420 north, longitude 77°540 east. High temperature–tolerant rhizosphere–competent P. putida NBRI0987 was isolated from field-grown chickpea (Cicer arietinum L. cv. Radhey) rhizosphere as previously described [6] and was identified using the Biolog system (Biolog, Hayward, CA). Unless otherwise stated, P. putida NBRI0987 was grown and maintained on nutrient broth (NB) or nutrient agar (NA) (HI-MEDIA Laboratories, Bombay, India).

High-temperature Stress and Quantification of Biofilm Formation and Alginate Production The stress tolerance of Pseudomonas strains toward temperature was tested by growing them on NB broth in 150-ml Erlenmeyer flasks containing 50 ml NB with an initial inoculation of approximately 1 9 107 CFU/ml as previously described [8]. The flasks were incubated on a New Brunswick Scientific (Edison, NJ) Innova model 4230 refrigerated incubator shaker at 180 rpm. Populations at each time point in the Figs. 1 through 3 represent the means of three independent experiments. An SD of ± 0.25 log CFU/ml was found for the viable cell counts. The method for determining the extent of bacterial adherence to the microtiter well surfaces has been described elsewhere [10]. Briefly, the bacterial supernatants were discarded after incubation, and loosely adherent bacteria were removed by three washes with phosphate-buffered saline (pH 7.2). The microtiter plates were then inverted and allowed to dry before each well was filled with 25 ll 0.1% (w/v) crystal violet (CV) solution and incubated at room temperature for 30 minutes. Unbound CV was removed by three washes with water, and the plates were inverted to dry. Cell-bound CV was released from bacterial cells by the addition of 200 ll 95% ethanol and, after incubation at room temperature for 30 minutes on a rotary shaker, the concentration of CV in each solution was determined by the optical density reading at 590 nm (Tecan Infinite 200 Microplate Reader, Ma¨nnedorf, Switzerland). Similarly, wells containing only NB but no bacteria were used as negative controls. Levels of alginate were determined as previously described [17]. Bacterial population, bacterial adherence, and alginate measurements represent the means of three independent experiments.

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Fig. 1 Effect of MgSO4.7H2O (MgSO4) (25 mM) plus glycerol (5% v/v) supplementation on the growth of P. putida NBRI0987. Control, MgSO4, glycerol, and glycerol plus MgSO4 at (A) 30°C and (B) 40°C. Bacterial survival was determined at the indicated times in triplicate, and results are the means of three independent experiments. SD ± 0.25 log CFU/ml was found for the viable cell counts. Variation (SD) was within symbol dimensions

Isolation of Total RNA and Reverse Transcriptase Reaction–Polymerase Chain Reaction P. putida NBRI0987 cells were grown in NB at 30°C and 40°C for 20 hours. One milliliter of the culture was taken to prepare RNA. The cells were immediately frozen at -80°C. Total RNA was isolated from the frozen cells using RNAEasy Mini Kit (Qiagen, Hilden, Germany) as described by the manufacturer. Residual DNA was digested using DNase (Qiagen) treatment. Thus, total RNA obtained was checked on 1.5% formamide denaturing gel and quantified using a spectrophotometer (Shimadzu, Kyoto, Japan). Using equal amount of RNA (5 lg) from each sample, cDNA synthesis was performed using the RevertAid H Minus First-Strand cDNA Synthesis Kit (Fermentas UAB, Vilnius, Lithuania) as described by the manufacturer. Primer sequences used for 16S [2] and rpoS [5] were as previously described. Primer sequences used for AlgT, AlgD, and GreA were designed using gene sequences from gene accession numbers GI:24982893 [5’-


gttgcaagcctgaacgatg-3’], GI:24982742 [5’-actgtctggagcct ttgcat-3’], and GI:24986475 [5’-cgacatggaatacccacagg-3’], respectively. All experiments were independently repeated three times. Oligonucleotides used in this study were synthesized by Bangalore Genei (Banglore, India). Polymerase chain reaction amplification of the respective genes were performed using equalized cDNA concentration (approximately 25 ng) from each RNA sample in a 20-ll reaction mixture containing 2.0 ll Taq buffer (10 mM Tris HCl, pH 9.0, 50 mM KCl, 1.5 mM MgCl2, and 0.01% gelatin), 0.5 mM each of forward and reverse primers, deoxyribonucleoside triphosphate (0.1 mM each), and 0.3 U Taq polymerase from Bangalore Genei. After 3 minutes of initial denaturation at 94°C, the reaction involved 30 cycles of 94°C for 1 minute, 1 minute at annealing temperature 63°C for rpoS and 60°C for rest of the genes, extension at 72°C for 1 minute, and final extension for 10 minutes at 72°C in a Flexigene thermocycler (Techne, Cambridge, UK). Amplified products were loaded on 1.2% agarose gel, and the molecular mass of the amplified DNA was estimated by comparing with the 500-bp DNA ladder (Fermentas UAB, Vilnius, Lithuania). Scanning the gels was performed on the Gel-Documentation System (Uvitec, Cambridge, UK). All the experiments were independently repeated three times.

Results and Discussion After screening a total of [ 2,500 Pseudomnas sp. strains, a high temperature–tolerant strain in P. putida NBRI0987 was isolated that tolerated a temperature of 40°C for B 5 days (Fig. 1). To the best of our knowledge, this is the first report of a Pseudomnas sp. demonstrating survival estimated by counting viable cells under such a high temperature. We studied the effects of various carbon, nitrogen, and metals alone and in various combinations on the survival of P. putida NBRI0987 at 40°C (data not provided). Survival of the strain was monitored at 30°C (Fig. 1A) and 40°C (Fig. 1B), in the presence of MgSO4.7H2O (MgSO4; 25 mM) plus glycerol (5% v/v) for B 10 days. P. putida NBRI0987 survived in NB containing MgSO4 plus glycerol for B 10 days (Fig. 1). Enhanced cell survival at 30°C (Fig. 1A) and 40°C (Fig. 1B), was observed in the presence of MgSO4 plus glycerol for B 10 days compared with MgSO4 and glycerol used alone. P. putida NBRI0987 CFU/ml on day 10 in the presence of 0, MgSO4, glycerol, and MgSO4 plus glycerol at 30°C were 1.7 9 109, 1.3 9 1011, 1.3 9 1011 and 1.7 9 1013/ml, respectively (Fig. 1A). At 40°C, P. putida NBRI0987 CFU/ml on day 10 in the presence of 0, MgSO4, glycerol, and MgSO4 plus glycerol were 0, 1.2 9 109, 8.5 9 108 and 1.7 9 1011, respectively (Fig. 1 B). In general, P. putida NBRI0987 efficiently tolerated

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high temperature (40°C) in the presence of MgSO4 plus glycerol: CFU/ml of P. putida NBRI0987 were greater in the presence of MgSO4 plus glycerol compared with its absence (Fig. 1). Bacterial biofilms have a significant impact in medical, industrial, and environmental settings. Numerous environmental parameters influence whether biofilms are successfully established in these settings, as was found for P. fluorescens [10] and Sinorhizobium meliloti [11]. Because we noted enhanced survival of P. putida NBRI0987 grown in the presence of NB supplemented with MgSO4 plus glycerol, and observation suggested that biofilm formation may be involved because it represents a survival strategy, we tested this possibility. The effect of MgSO4 plus glycerol was studied on the biofilm-formation ability of P. putida NBRI0987 in the wells of the microtiter plates at 30°C and 40°C. Fig. 2 shows that maximal biofilm formation was observed 48 hours after incubation in the microtiter plate wells in the presence of MgSO4 plus glycerol for up to 48 hours compared with their use separately. The results indicate that the combination of MgSO4 plus glycerol enhances the ability of P. putida NBRI0987 to tolerate high temperature by inducing it to a sessile mode of life, i.e., a biofilm (Fig. 2). The present study demonstrates that rpoS expression is induced when P. putida NBRI0987 is grown under temperature stress at 40°C compared with 30°C (Fig. 3). The regulation and function of stress sigma factor rS (also known as r38), encoded by rpoS, has been studied in a variety of Gram-negative bacteria, especially in Escherichia coli and Pseudomonas spp. [3–5, 10]. Certain functions of RpoS are similar in Pseudomonas spp. and in

Fig. 2 Effect of MgSO4.7H2O (MgSO4) (25 mM) plus glycerol (5% v/v) supplementation on biofilm formation of NBRI0987. Control, MgSO4, glycerol, and glycerol plus MgSO4 (column A) at 24 hours at 30°C, (column B) at 48 hours at 30°C, (column C) at 24 hours at 40°C, and (column D) at 48 hours at 40°C. Values represent the means of three independent experiments, and vertical bars indicate SE

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Fig. 3 Agarose gel analysis of expression of Sigma 70 (rpoS), alginate structural gene (algD), alternative sigma factor (algT), and transcription elongation factor (greA) at 30°C and 40°C of Pseudomonas putida NBRI0987

enteric bacteria. In particular, survival during osmotic, heat, or oxidative stress is decreased in the rpoS mutants of P. aeruginosa, P. putida, and P. fluorescens [12]. rpoS has been reported as being an important factor for adaptation to stress conditions because it produce alginate, which is important for survival under stress conditions [5]. Therefore, the affect of high temperature on alginate biosynthesis pathway was also studied. In this study, we examined the role of alginate production at 30°C and 40 °C and in the presence and absence of MgSO4 plus glycerol. Alginate produced was 0.396 ± 0.035 lg/mg wet biomass at 30 °C and 0.347 ± 0.067 lg/mg wet biomass at 40 °C after 20 hours of incubation. Although supplementation of MgSO4 plus glycerol served as a stress reliever by supporting biofilm formation and better survival, it decreased alginate formation: The amount of alginate produced was 0.204 ± 0.07 at 30°C and 0.218 ± 0.03 at 40°C after 20 hours of incubation. Quantitative estimation of alginate demonstrated that supplementation of MgSO4 plus glycerol relieves the stress imposed by high temperature and supports growth by forming more biofilm but not by the synthesis of alginate. Our data is in accordance with earlier findings that stressed environmental response results in more alg D expression, but because Pseudomonas are nonmucoid bacteria, they produce more or less equal alginate under either condition, indicating that the production of alginate is not critical for biofilm formation [14, 17].

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Alternative sigma factor (algT) was overexpressed, followed by algD the structural gene of the alginate biosynthetic pathway, at 40°C compared with 30°C (Fig. 3). The function of algT in the regulation of alginate synthesis has been well documented [1, 15]. Results are in agreement with previous reports stressing that tolerance ability is imparted by the overexpression of algT, which in turn takes over the charge of rpoS and thereby governs the synthesis of alginate biosynthetic gene algD [15]. This results in more biofilm formation, thus protecting cells from stress produced by high temperatures. Overexpression of rpoS and algT at 40°C was further supported by the overexpression of the greA family of transcription elongation factor with regard to temperature tolerance in our study (Fig. 3). greA is known to support the growth of E. coli at high temperatures, and it has also been reported to be induced by heat shock, salt shock, and oxidative stress in Bacillus subtilis [13, 16]. Therefore, data are indicative of the role of the transcription elongation factor greA in conferring stress tolerance in P. putida NBRI0987 toward high temperatures in accordance with rpoS. Our results suggest that the adaptation of P. putida NBRI0987 to high temperatures is a complex multilevel regulatory process in which many different genes can be involved. Our group has initiated molecular studies to isolate temperature-tolerant defective mutants and to identify the genes from which they derive, which will lead to a better understanding of the mechanism of temperature tolerance in bacteria. Acknowledgments Thanks are due to the director of the National Botanical Research Institute, Lucknow, for necessary support of this study. The study was supported by Task Force Grant No. SMM-002 from the Council of Scientific and Industrial Research, New Delhi, India, awarded to C. S. N.

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