(ACC) based on ninhydrin reaction for rapid ... - Wiley Online Library

Letters in Applied Microbiology ISSN 0266-8254

ORIGINAL ARTICLE

A colorimetric assay of 1-aminocyclopropane-1-carboxylate (ACC) based on ninhydrin reaction for rapid screening of bacteria containing ACC deaminase Z. Li1, S. Chang1, L. Lin2,3, Y. Li2,3 and Q. An1 1 Key Laboratory of Molecular Biology of Crop Pathogens and Insects, Ministry of Agriculture, Institute of Biotechnology, Zhejiang University, Hangzhou, China 2 Agricultural College, Guangxi University, Nanning, China 3 Guangxi Crop Genetic Improvement and Biotechnology Laboratory, Guangxi Academy of Agricultural Sciences, Nanning, China

Keywords 1-aminocyclopropane-1-carboxylate, ACC deaminase, chimney-top PCR plate, ninhydrin reaction, plant growth-promoting bacteria. Correspondence Qianli An, Institute of Biotechnology, Zhejiang University, Hangzhou 310058, China. E-mail: [email protected]

2011 ⁄ 0449: received 16 March 2011, revised 27 April 2011 and accepted 16 May 2011 doi:10.1111/j.1472-765X.2011.03088.x

Abstract Aims: 1-Aminocyclopropane-1-carboxylate (ACC) deaminase activity is an efficient marker for bacteria to promote plant growth by lowering ethylene levels in plants. We aim to develop a method for rapidly screening bacteria containing ACC deaminase, based on a colorimetric ninhydrin assay of ACC. Methods and Results: A reliable colorimetric ninhydrin assay was developed to quantify ACC using heat-resistant polypropylene chimney-top 96-well PCR plates, having the wells evenly heated in boiling water, preventing accidental contamination from boiling water and limiting evaporation. With this method to measure bacterial consumption of ACC, 44 ACC-utilizing bacterial isolates were rapidly screened out from 311 bacterial isolates that were able to grow on minimal media containing ACC as the sole nitrogen source. The 44 ACCutilizing bacterial isolates showed ACC deaminase activities and belonged to the genus Burkholderia, Pseudomonas or Herbaspirillum. Conclusions: Determination of bacterial ACC consumption by the PCR-plate ninhydrin–ACC assay is a rapid and efficient method for screening bacteria containing ACC deaminase from a large number of bacterial isolates. Significance and Impact of the Study: The PCR-plate ninhydrin–ACC assay extends the utility of the ninhydrin reaction and enables a rapid screening of bacteria containing ACC deaminase from large numbers of bacterial isolates.

Introduction 1-Aminocyclopropane-1-carboxylate (ACC) deaminase catalyses the cleavage of ACC, the immediate precursor of ethylene in plants, to a-ketobutyrate and ammonia (Honma and Shimomura 1978). Bacteria containing ACC deaminase attach to plant cells, act as a sink for plant ACC to uptake and cleave the ACC secreted by plant cells and thus reduce plant ACC concentration, ethylene evolution and the extent of ethylene inhibition of plant growth, particularly, under a variety of abiotic and biotic stresses (Glick et al. 1998). Moreover, bacteria containing ACC deaminase can down-regulate the plant genes involved in ethylene-induced stress responses and 178

defence signalling pathways and up-regulate the plant genes involved in growth and protein production (Hontzeas et al. 2004). Glick et al. (1995) have developed a rapid procedure to isolate pseudomonads that are able to grow on a minimal medium containing ACC as the sole nitrogen source and significantly promote plant growth. Thereafter, a growing number of bacterial isolates in a wide range containing ACC deaminase have been isolated by laboratories worldwide and have shown statistically significant plant growth-promoting activity (Blaha et al. 2006; Glick et al. 2007; Onofre-Lemus et al. 2009). ACC deaminase activity has long been a key marker for identifying the plant growth-promoting bacteria (Glick et al. 2007).

ª 2011 The Authors Letters in Applied Microbiology 53, 178–185 ª 2011 The Society for Applied Microbiology

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ACC deaminase is present in the cytoplasm of bacteria at a low level until it is induced by ACC, and the induction of enzyme activity is a relatively slow process (Jacobson et al. 1994). ACC deaminases do not bind ACC with a high affinity; the Km values range from 1Æ5 to 17Æ4 mmol l)1 (Hontzeas et al. 2006). ACC deaminase activity is usually analysed according to a colorimetric 2,4-dinitrophenylhydrazine assay of the product a-ketobutyrate (Honma and Shimomura 1978; Penrose and Glick 2003). Recently, Fu et al. (2009) measured the ACC deaminase activity of a hydrogen-oxidizing bacterium using a colorimetric ninhydrin assay to determine consumption of the substrate ACC after 24-h growth in a minimal medium with an initial ACC concentration of approx. 5 mmol l)1 (0Æ5 g l)1). Because the 24-h consumption period includes an uncertain enzyme induction period, and the ACC concentration decreases during the 24-h consumption and even the initial ACC concentration may be below the Km of the ACC deaminase, this assay is not appropriate for determining ACC deaminase activity. However, the ninhydrin assay of ACC, an a-amino acid with a cyclopropane ring, enables the determination of bacterial utilization of ACC. In this study, we improved the colorimetric ninhydrin– ACC assay and adapted it by heating the ACC and ninhydrin regent in the wells of a polypropylene chimney-top 96-well PCR plate on boiling water and used the novel PCR-plate ninhydrin–ACC assay to rapidly screen bacteria containing ACC deaminase from a large number of bacterial isolates. Materials and methods Bacteria and media Forty-three nitrogen-fixing bacterial isolates were isolated from rhizosphere soils and surface-sterilized roots of a main sugarcane cultivar ROC22 cultivated in Guangxi, China. Sixty-eight bacterial isolates were isolated by a modified NFb medium [adding 5 g of sucrose and 5 g of glucose into 1 l of NFb medium (Do¨bereiner et al. 1976)] from surface-sterilized stems of seven maize cultivars cultivated in Heilongjiang, China. Two hundred bacterial isolates were isolated from vigorously washed roots of a rice cultivar C2 grown in a field in Zhejiang University. Among the 200 bacterial isolates obtained from rice roots, 100 isolates were isolated by plating on the nitrogen-deficient DF medium (Dworkin and Foster 1958), the other 100 isolates were isolated by plating on the DF-ACC medium. The DF medium was prepared as described by Penrose and Glick (2003). A 0Æ5 mol l)1 solution of ACC (Yurlic Chemical S&T Co. Ltd, Shanghai, China) was filter-sterilized through a 0Æ2-lm membrane, aliquoted

PCR-plate ninhydrin–ACC assay

and frozen at )20C. Just prior to use, the frozen ACC solution was thawed and added into autoclaved DF minimal medium to obtain the DF-ACC medium with a final ACC concentration of 3Æ0 mmol l)1. Solid DF and DFACC media were supplemented with 1Æ5% (w ⁄ v) ultrapure agar (Sangon Biotech Co. Ltd, Shanghai, China). Luria–Bertani (LB) medium (Sambrook and Russell 2001) was used as the rich medium for bacterial culture and storage. All 311 bacterial isolates formed visible colonies after streaking on the DF-ACC agar and incubation for 48 h at 28C. Ninhydrin reagent Five hundred milligrams of ninhydrin and 15 mg of ascorbic acid were dissolved in 60 ml of ethylene glycol, stored at )20C and mixed with 60 ml of 1 mol l)1 citrate buffer (pH 6Æ0) prior to use. Standard colorimetric ninhydrin assay of ACC with test tubes and cuvettes The DF-ACC medium (with an ACC concentration of 3Æ0 mmol l)1) was diluted with the DF medium to respective ACC working concentrations of 0Æ005, 0Æ01, 0Æ015, 0Æ02, 0Æ03, 0Æ04, 0Æ05, 0Æ10, 0Æ15, 0Æ20, 0Æ25, 0Æ30, 0Æ40 and 0Æ50 mmol l)1. After the addition of 1 ml of ACC working solution and 2 ml of ninhydrin reagent, glass test tubes were capped and shaken and placed in a boiling water bath. After 15 min, the tubes were moved into a water bath at room temperature for 2 min and then shaken for 30 s (Lamothe and McCormick 1972). After standing at room temperature for 10 min, the solution was transferred into a cuvette and absorbance was measured at 570 nm with an SP-721E spectrophotometer (Spectrum Shanghai, Shanghai, China). The DF medium was used as a blank. Each working solution was run in triplicate. In addition, 1 ml of a tenfold diluted supernatant of a bacterial culture was used to determine ACC in bacterial cultures with the standard ninhydrin assay. Ninhydrin assay of ACC with polypropylene chimneytop 96-well PCR plates and microplates (96-well PCR-plate ninhydrin–ACC assay) Sixty microlitres of ACC working solutions ranging from 0Æ005 to 0Æ50 mmol l)1 and 120 ll of ninhydrin reagent were dispensed into wells of a polypropylene nonskirted chimney-top 96-well PCR plate (Bio Basic Inc., Markham, Canada) and mixed by pipetting. The PCR plate floated with an ethylene–vinyl acetate plastic sheath was heated on a boiling water bath for 30 min. The DF


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medium was used as a blank. Each working solution was run in triplicate. After heating, solutions in wells were transferred into a Costar polystyrene flat bottom 96-well microplate (Corning Inc., Corning, NY, USA). The absorbance at 570 nm in microplate wells was measured with a SpectraMax spectrophotometer (Molecular Devices, Sunnyvale, CA, USA).

Bacterial single colonies

28°C, rotation, overnight growth to late-log phase

Rich medium (5 ml) 2 ml

Screening of ACC-utilizing bacterial isolates

Harvested by centrifugation

A single colony maintained on LB agar was picked into 5 ml of liquid LB medium and incubated overnight at 28C on a shaker at 200 rev min)1. Two millilitres of each culture was harvested in a 2Æ0-ml microcentrifuge tube by centrifugation at 8000 g for 5 min. The cell pellet was washed twice with 1 ml of liquid DF medium and then suspended in 2 ml of DF-ACC medium in a 12-ml culture tube and incubated at 28C on the shaker at 200 rev min)1 for 24 h. A 2-ml sample of DF-ACC medium without inoculation was incubated in parallel. One millilitre of each culture was centrifuged in a 1Æ5-ml microcentrifuge tube at 8000 g for 5 min. One hundred microlitres of each supernatant was diluted to 1 ml with liquid DF medium in a 1Æ5-ml microcentrifuge tube. Sixty microlitres of each tenfold diluted supernatant was used for the 96-well PCR-plate ninhydrin–ACC assay. The DF medium was used as a blank. Each diluted supernatant was run in triplicate. The resulted Ruhemann’s Purple depth from each diluted supernatant and the diluted DF-ACC medium without inoculation was compared by eyes and recorded. After transfer of 100 ll of the remaining reaction solution, the absorbance of microplate wells was measured at 570 nm with the SpectraMax spectrophotometer. The procedure is shown in Fig. 1. A bacterial isolate corresponding to a visibly reduced colour depth or a statistically lower absorbance of supernatant compared with that of the DF-ACC medium without inoculation was regarded as an ACC-utilizing bacterial isolate.

Washed twice with 1 ml of DF medium DF-ACC medium (2 ml)

Suspended with 2 ml of DF-ACC medium

28°C, rotation, 24 h 100 µl

1 ml

Centrifugation 100 µl 10-fold dilution with DF medium 60 µl

60 µl

Mixed with 120 µl of ninhydrin reagent floating on boiling water, 30 min Comparison of color depth

100 µl

Visually determination of ACC-utilizing bacteria

Comparison of absorbance at 570 nm Determination of ACC-utilizing bacteria Figure 1 Procedure diagram for screening of ACC-utilizing bacteria via the 96-well PCR-plate ninhydrin–ACC assay. ACC, 1-aminocyclopropane-1-carboxylate.

Measurement of ACC deaminase activity The ACC deaminase activity of six ACC-utilizing bacterial isolates isolated from sugarcane rhizosphere and maize stems was measured after induction in the DF-ACC medium for 8, 16 and 24 h by a 2,4-dinitrophenylhydrazine assay of a-ketobutyrate, as described by Penrose and Glick (2003). The ACC deaminase activity of 38 ACC-utilizing bacterial isolates obtained from rice roots was measured after 8-h induction. In addition, bacterial isolates of NN145S and SZ6-5 that were unable to utilize ACC and were isolated from sugarcane rhizosphere and maize stem were used as negative controls. 180

Amplification and sequencing of 16S rRNA genes from ACC-utilizing bacterial isolates PCR amplification of near full-length 16S rRNA gene sequences was performed with the universal 27F ⁄ 1492R primers (Lane 1991). Approximately 1500 bp of PCR amplicons were respectively cloned into the pMD18-T vector (TaKaRa Biotechnology Co., Ltd, Dalian, China) and screened as described by the manufacturer. The clones containing inserts of the correct size were chosen for sequencing by ABI 377 automated DNA sequencers


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Table 1 ACC-utilizing bacteria tested in this study


Bacterial isolate CZ152S DN248-14 SZ6-1 SZ6-2 SZ6-3 SZ6-4 Os8 Os9 Os13 Os14 Os17 Os18 Os19 Os20 Os21 Os23 Os24 Os25 Os26 Os27 Os28 Os30 Os31 Os32 Os33 Os34 Os35 Os37 Os38 Os40 Os41 Os43 Os44 Os45 Os46 Os47 Os48 Os49 Os50 Os51 Os52 Os53 Os54 Os36

Source Sugarcane rhizosphere Maize stem Maize stem Maize stem Maize stem Maize stem Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root Rice root

Genus affiliation

16S rRNA gene sequence accession no.

Burkholderia

HQ204278

Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Burkholderia Pseudomonas Pseudomonas Pseudomonas Pseudomonas Herbaspirillum Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas Herbaspirillum Herbaspirillum Pseudomonas Burkholderia Herbaspirillum Pseudomonas Herbaspirillum Herbaspirillum Burkholderia Burkholderia Herbaspirillum Herbaspirillum Herbaspirillum Burkholderia Burkholderia Burkholderia Herbaspirillum Burkholderia Pseudomonas Pseudomonas Pseudomonas Pseudomonas Pseudomonas

HQ624994 HQ624992 HQ624993 HQ624995 HQ624996 HQ728548 HQ728549 HQ728550 HQ728551 HQ728552 HQ728553 HQ728554 HQ728555 HQ728556 HQ728557 HQ728558 HQ728559 HQ728560 HQ728561 HQ728562 HQ728563 HQ728564 HQ728565 HQ728566 HQ728567 HQ728568 HQ728569 HQ728570 HQ728571 HQ728572 HQ728573 HQ728574 HQ728575 HQ728576 HQ728577 HQ728578 HQ728579 HQ728580 HQ728581 HQ728582 HQ728583 HQ728584 HQ840775

ACC deaminase activity (nmol a-ketobutyrate per mg protein per h)* 7556 ± 273 9037 8462 7564 11 139 7703 16 575 7362 6063 1906 2912 1003 577 1312 710 694 722 435 572 848 675 853 752 490 12 122 969 621 528 1534 977 559 718 2080 2628 4851 3704 2433 1434 1969 3738 11 827 571 309 309

± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ± ±

394 287 442 250 190 280 441 245 372 214 49 132 298 58 40 243 67 26 225 26 89 174 285 541 111 146 4 177 234 50 38 39 73 313 308 178 209 359 434 72 4 163 71

ACC, 1-aminocyclopropane-1-carboxylate. *ACC deaminase activity was measured after 8-h induction in the DF-ACC medium.

(Invitrogen, Shanghai, China). The obtained sequences were compared with sequences in GenBank using the Blast program (Altschul et al. 1997). The 16S rRNA gene sequences were deposited in GenBank under the accession numbers HQ204278, from HQ624992–HQ624996, HQ728548–HQ728584 and HQ840775 (Table 1).

Data analysis Data plotting and t-tests were carried out using Microsoft Excel 2007, to compare the ACC concentrations of a given sample determined by the standard ninhydrin assay and the PCR-plate assay, the absorbance values between the


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(a)

0·05

0·10 0·15 0·20 0·25 0·30 ACC concentration (mmol l–1)

0·35

Figure 2 Standard curves of ACC concentrations ranging from 0Æ05 to 0Æ3 mmol l)1 determined by both standard colorimetric ninhydrin assay (s, y = 2Æ854x, R2 = 0Æ999) and 96-well PCR-plate ninhydrin assay (d, y = 1Æ796x, R2 = 0Æ999). Each data point represents the mean from triplicate determinations, and the error bar represents the standard deviation. ACC, 1-aminocyclopropane-1-carboxylate.

reacted tenfold diluted noninoculated DF-ACC medium after incubation and the diluted supernatants of bacterial cultures or diluted DF-ACC medium stored at )20C.

(b)

ACC concentration (mmol l–1)

1·0 0·9 0·8 0·7 0·6 0·5 0·4 0·3 0·2 0·1 0·0 0·00

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ACC deaminase activity (nmol α-ketobutyrate mg protein–1 h–1)

Optical density at 570 nm


3·0 2·5 2·0 1·5 1·0 0·5 0·0 –0·5

CZ152S DN248-14 SZ6-1

SZ6-2

SZ6-3

SZ6-4

Isolates

14 000 12 000 10 000 8000 6000 4000 2000 0

CZ152S DN248-14 SZ6-1

SZ6-2

SZ6-3

SZ6-4

Isolates

Results ACC determination by colorimetric ninhydrin assays Ethylene glycol used as the solvent stabilized the ninhydrin reagent and colour development. Adding ascorbic acid in the ninhydrin reagent prevented the hydrindantin from oxidation. The absorbance values of ACC solutions ranging from 0Æ015 to 0Æ3 mmol l)1 at 570 nm after reaction were highly correlated with the ACC concentrations (R2 = 0Æ999) and resulted in linear calibration curves by both standard ninhydrin assay and 96-well PCR-plate ninhydrin assay (Fig. 2). The time of period for maximum development of Ruhemann’s Purple in plate wells was twice as long as in test tubes. The absorbance value of each ACC working concentration at 570 nm in the PCR-plate assay was lower than that of the standard assay (Fig. 2). The ACC concentrations in a given bacterial culture after a period of incubation in the DF-ACC medium determined by both assays were statistically identical at the 0Æ05 confidence level (data not shown). The results of the PCR-plate assay on tenfold diluted supernatants of six ACC-utilizing bacterial isolates are shown in Fig. 3a. Screening ACC-utilizing bacteria by the PCR-plate ninhydrin–ACC assay The absorbance value at 570 nm of the tenfold diluted noninoculated DF-ACC medium after 24-h incubation at 28C was slightly lower than that of the diluted DF-ACC 182

Figure 3 (a) ACC concentrations remaining in the DF-ACC medium containing 3Æ0 mmol l)1 ACC after incubation of each ACC-utilizing bacterial isolate for 8 ( ), 16 ( ) and 24 h (h) measured by the PCRplate ninhydrin assay. (b) ACC deaminase activity of each ACC-utilizing bacterial isolate measured by the 2,4-dinitrophenylhydrazine assay after induction in the DF-ACC medium for 8 ( ), 16 ( ) and 24 h (h). Each data point represents the mean from triplicate determinations, and the error bar represents the standard deviation. ACC, 1aminocyclopropane-1-carboxylate.

medium stored at )20C. However, the difference was not statistically significant at the 0Æ05 confidence level (data not shown). The absorbance values of the tenfold diluted supernatants of 44 bacterial isolates from the 311 tested bacterial isolates were significantly lower than that of the diluted noninoculated DF-ACC medium. All the 44 ACC-utilizing bacterial isolates, represented by the six bacterial isolates obtained from sugarcane rhizosphere and maize stems, consumed all or nearly all the 3 mmol l)1 of ACC in the DF-ACC medium after the 24-h incubation (Fig. 3a). Consequently, the colour depth of the diluted supernatants from the 44 bacterial cultures was visibly weaker compared with that of the diluted noninoculated DF-ACC medium. Among the 100 bacterial isolates obtained from rice roots using the DF medium, four isolates of Os8, Os9, Os13 and Os14 (4%) utilized ACC; among the other 100 bacterial isolates obtained from rice roots using the DF-ACC medium, 34 isolates (34%) utilized ACC.


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ACC deaminase activities of ACC-utilizing bacteria The colorimetric 2,4-dinitrophenylhydrazine assay detected ACC deaminase activity from all the 44 ACC-utilizing bacterial isolates (Table 1) but not from the isolates of NN145S and SZ6-5 that were unable to utilize ACC (data not shown). Moreover, represented by the six ACCutilizing bacterial isolates obtained from sugarcane rhizosphere and maize stems, the ACC deaminase activity of each isolate varied with the induction period and was generally lower after 24 h of induction than after 8 h (Fig. 3b). Therefore, the ACC deaminase activities of the 38 ACC-utilizing bacterial isolates obtained from rice roots were measured after 8 h of induction. Genus affiliation of bacteria containing ACC deaminase by analysis of 16S rRNA gene sequences Analysis of the 16S rRNA gene sequences indicated that the 44 bacterial isolates containing ACC deaminase were closely related to the bacterial strains belonging to the genus Burkholderia, Pseudomonas or Herbaspirillum (Table 1). The highest sequence similarities to the strains of the respective genus were all more than 99Æ5%. Therefore, the CZ152S isolate obtained from sugarcane rhizosphere soils, the five isolates obtained from maize stems and the 12 isolates obtained from rice roots were affiliated to the genus Burkholderia; 16 isolates obtained from rice roots were affiliated to the genus Pseudomonas, and the other ten isolates obtained from rice roots were affiliated to the genus Herbaspirillum. Discussion Ninhydrin reactions have been widely used to analyse amino acids, peptides and proteins as well as numerous other ninhydrin-positive compounds and have undergone and continue to undergo major developments (Friedman 2004). Fu et al. (2009) prepared an aqueous ninhydrin solution without a reducing reagent to quantify ACC. Here, we improved the assay using ethylene glycol as the solvent to stabilize the ninhydrin reagent and colour development (Starcher 2001) and used ascorbic acid as the reducing regent to prevent the oxidation of the hydrindantin (Yokoyama and Hiramatsu 2003). ACC is unstable in solution and degrades by approx. 5% per day at 4C (Penrose et al. 2001). However, we have not determined a significant reduction in ACC concentration from the tenfold diluted medium after incubation of the medium at 28C for 24 h. Perhaps, the ninhydrin–ACC assay is not sensitive as the Waters AccQÆTag Amino Acid Analysis Method (Waters Corp., Milford, MA) as used by Penrose et al. (2001) to detect the


degradation, and ⁄ or the products of ACC degradation are ninhydrin-positive. Several microplate ninhydrin assays have been developed (Starcher 2001; Iyamu et al. 2008). Starcher (2001) heated a polystyrene Nunc MaxiSorp microplate (Nalge Nunc Int., Rochester, NY) using a boiling water bath. Iyamu et al. (2008) overlaid reaction solutions in wells with 50 ll of optically inert oil and heated a polystyrene Costar microplate at 100C in a temperature-controlled oven. However, these polystyrene microplates were deformed when we heated them on boiling water or at 100C in an oven for 15 min. We obtained consistent and reproducible results by heating polypropylene nonskirted chimney-top 96-well PCR plates on boiling water. Polypropylene PCR plates are heat resistant. The nonskirted chimney-top PCR plate can be easily floated on boiling water by supporting the surrounding edges of the plate with an ethylene–vinyl acetate plastic sheath; the bottom halves of all wells can be evenly heated by boiling water; the elevated well top prevents accidental contamination from boiling water and limits evaporation to some extent. The PCR-plate ninhydrin assay requires a solution transfer from the PCR plate to a microplate to quantify ACC, increasing the cost. However, the solution transfer and the absorbance measurement using a microplate spectrophotometer are likely dispensable to determine ACC-utilizing bacteria because all ACC-utilizing bacterial isolates used up, or almost used up, the initial 3 mmol l)1 ACC after the 24-h incubation, leading to a visible reduction in final colour depth compared with the noninoculated DF-ACC medium. Skipping solution transfer and microplate reading, the PCR-plate ninhydrin–ACC assay becomes a simple and low-cost processing to rapidly screen ACC-utilizing bacterial isolates. Using triplicate wells for each sample, a 96-well plate accommodates a blank, a tenfold diluted noninoculated DF-ACC medium after 24-h incubation and up to 30 supernatant samples of bacterial cultures. Importantly, all the ACC-utilizing bacterial isolates showed ACC deaminase activities, suggested a high efficiency of the screening processing. Moreover, most of the ACC-utilizing bacterial isolates belong to genus Burkholderia and Pseudomonas in which ACC deaminase is widespread (Blaha et al. 2006; Onofre-Lemus et al. 2009), and the others belong to genus Herbaspirillum in which ACC deaminase activity is increasingly being reported (Rothballer et al. 2008; Rashedul et al. 2009; Sun et al. 2010). The respective screening rate of 4% and 34% on the bacterial isolates obtained from rice roots with DF medium and DF-ACC medium shows that ACC enriches bacteria containing ACC deaminase. However, growth on minimal media containing ACC as the sole nitrogen


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source is not a sufficient determination for a bacterial isolate to possess ACC deaminase; identification of a bacterial isolate containing ACC deaminase requires demonstration of ACC deaminase activity. Here, bacterial utilization of ACC determined by the ninhydrin–ACC assay presents a direct correlation with bacterial possessing of ACC deaminase activity. ACC deaminase activity is an efficient marker for plant-associated bacteria to promote plant growth by lowering the levels of ethylene inhibition in plants. Numerous potential plant growth-promoting bacteria have been isolated worldwide based on markers other than ACC deaminase activity, such as sugarcane-associated nitrogenfixing bacteria tested in this study. The PCR-plate ninhydrin–ACC assay extends the utility of ninhydrin reaction and facilitates the screening of bacteria containing ACC deaminase from large numbers of old bacterial collections and new bacterial isolates. Acknowledgements This work was supported by Science Foundation of Chinese University (grant no. 2009QNA6030), Zhejiang Provincial Natural Science Foundation of China (grant no. Y307143), and National Science & Technology Specific Project (grant no. 2009ZX08009-134B). References Altschul, S.F., Madden, T.L., Scha¨ffer, A.A., Zhang, J., Zhang, Z., Miller, W. and Lipman, D.J. (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25, 3389–3402. Blaha, D., Prigent-Combaret, C., Mirza, M.S. and Moe¨nneLoccoz, Y. (2006) Phylogeny of the 1-aminocyclopropane-1-carboxylic acid deaminase encoding gene acdS in phytobeneficial and pathogenic Proteobacteria and relation with strain biogeography. FEMS Microbiol Ecol 56, 455–470. Do¨bereiner, J., Marriel, I.E. and Nery, M. (1976) Ecological distribution of Spirillum lipoferum Beijerinck. Can J Microbiol 22, 1464–1473. Dworkin, M. and Foster, J. (1958) Experiments with some microorganisms which utilize ethane and hydrogen. J Bacteriol 75, 592–603. Friedman, M. (2004) Applications of the ninhydrin reaction for analysis of amino acids, peptides, and proteins to agricultural and biomedical sciences. J Agric Food Chem 52, 385–406. Fu, B., Wang, W., Tang, M. and Chen, X. (2009) Isolation and identification of hydrogen-oxidizing bacteria producing 1-aminocyclopropane-1-carboxylate deaminase and the determination of enzymatic activity. Acta Microbiol Sin 49, 395–399.

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