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  • The Internet Journal of Genomics and Proteomics
  • Volume 1
  • Number 1

Original Article

Growth Promotion Of Mung Beans By Bacterial Pyrroloquinoline

S Shahab, N Ahmed

Citation

S Shahab, N Ahmed. Growth Promotion Of Mung Beans By Bacterial Pyrroloquinoline. The Internet Journal of Genomics and Proteomics. 2003 Volume 1 Number 1.

Abstract

Pyrroloquinoline (PQQ) is an important cofactor of bacterial dehydrogenases, linking the oxidation of many different compounds to the respiratory chain. There have been very few studies of the functional role of PQQ in plant growth. CMG 860 is a native bacterial isolate having multidimensional biofertilizing abilities. CMG 860 has been found to have pqq operon in two fragments A_D and E whose sequences are homologous to those of earlier reports. To scrutinize the role of pqq as a main plant growth promoting factors in CMG860, mutants were produced which had mutation in pqq gene, this was confirmed by HPLC analysis. These mutants when compared with wild type showed a 22-25 % decrease in plant growth activity. However when pqq was introduced in the mutants they regained their ability of plant growth promotion perhaps in the similar way as it promotes growth of mammals.

 

Introduction

Pyrroloquinoline quinone [4, 5-dihydro-4, 5-dioxo-1H-pyrrolo-[2, 3-f] quinoline-2, 7, 9-tricarboxylic acid is an aromatic, tricyclic ortho quinone that serves as the redox cofactor for several bacterial dehydrogenases. PQQ was the first of the class of quinone cofactors that have been discovered in the last 18 years and make up the prosthetic group of quinoproteins[3,6,7.] PQQ is water soluble, heat stable, and has the ability to carry out redox cycles [15,17]. It has been reported that PQQ acts as a reactive oxygen species (ROS) scavenger by directly neutralizing reactive species in Escherichia coli [10]

Although plants and animals do not produce PQQ themselves, PQQ has invoked considerable interest because of its presence in human milk and its remarkable antioxidant properties. PQQ was the vitamin identified after 50 years in 2003 [3, ,2]. Although biological functions of PQQ are not fully understood [17] PQQ has attracted considerable interest because of its presence in a wide variety of foods and its remarkable antioxidant properties [8,9,14].

There have been few studies of the functional roles of PQQ in plants. It is known that PQQ stimulates pollen germination in vitro in the plant species Lilium, Tulipa, and Camellia [16,14]but the mechanisms are unclear. PQQ is at least 100 times more efficient than ascorbic acid, isoflavonoids, and poly phenolic compounds in assays assessing redox cycling potentials [20, 15] In addition to scavenging superoxide, PQQ could also scavenge other toxic free radicals, as do vitamin E, b-carotene and carotenoids, vitaminC, flavonoids, conjugated linoleic acid, and phenolic compounds[11] PQQ is found in plant and animal tissues in the nanogram to gram range even though plants and animals do not produce PQQ themselves[10,11]

Genes involved in PQQ synthesis have been cloned from Acinetobacter calcoaceticus[5], K. pneumoniae,[1] Pseudomonas fluorescens CHA0 [ 5] Methylobacterium organophilum DSM 760 [1]and M. extorquens AM1 (Morris et al., 1994). In A. calcoaceticus, five pqq genes were identified and sequenced, designated IV, V, I, II and III[5,20]. In K. pneumoniae, genes analogous to those were identified and designated pqqABCDE, and in addition, a sixth gene was found immediately downstream of pqqE, designated pqqF [12,21] In Methylobacterium strains, a five gene cluster (designated pqqDGCBA) was identified by complementation analysis [1, 13], and sequence data from M. extorquens AM1 showed that the first three of these genes (pqqDGC) were analogous to pqqABC of K. pneumoniae [22,13] In P. fluorescens, genes analogous to pqqFAB of K. pneumoniae have also been sequenced [18,24].

Figure 1
Alignment of pqq gene cluster. Equivalent genes have the same pattern (Source: Felder et al., 2000)

Materials and Methods

Reaction mixture , Cycling condition Amplification and Sequencing

PCR amplification of pqq gene of CMG860 was carried out with thermocycler (Eppendorf) by using Epicentre PCR core kit catalog no. amp 4670. Polymerease chain reaction was carried out in total volume of 50 µl in 0.5 ml microfuge tubes. Colony PCR was performed by initially denaturing the DNA at 96°C for 10 minutes followed by 30 cycles at 95°C for 30 seconds. Annealing temperature was set at 52°C -62°C (gradient was applied). The pqqA-D gene of CMG860 (EU037096) was amplified by using primer pair of Af2S (forward) and DRS (backward) primers. and pqqE by EF1 and ERS. Sequencing was done commercially by using ABI prism 377 automated DNA Sequencer. Sequences Data obtained were analyzed by blastaligothirm (www.ncbi.nlm.nih.gov/blast/cgi).

Mutation in pqq Gene

A stock solution of acridine orange (5mg/25ml) as a mutagenic agent was prepared. An aliquot of 10 µl overnight grown culture was inoculated in test tubes containing variable concentrations of acridine orange (10µl, 50µl, 100 µl, 200 µl, and 400 µl). These test tubes were incubated at 37°C for 24 hours at 100 rpm. A100µl culture from 10-5, 10-6 and 10-7 dilutions were spread over nutrient agar plates and incubated for 24 hrs at 37°C to get isolated colonies. About 100 colonies were randomly selected and tooth picked on to a control (nutrient agar plate) and test plate (tris minimal agar plate having 5mM zinc phosphate). The colonies which lost phosphate solubilization activity were initially selected and designated as CMG860 mutant colonies CMG860 M2 and CMG860 M4.

Auxin Production Activity

Auxin production activity of the wild type CMG860 and mutants CMG860M2 and CMG860M4 were carried out by using salkovski reagent method [12]

Isolation of pqq Mutants

Mutant strains M2 and M4 were studied for the growth promotion of the mung beans seedlings. Briefly, the surface sterilized seeds were incubated with late log phase cultures of wild type CMG860 and mutant strains CMG860M2 and CMG860M4 by incubating them with 10ml of respective bacterial suspension (10-7cfu per seed) in a sterile 15ml test tube. Non inoculated control seeds were incubated with 15 ml of nutrient broth. The mutant strains CMG860M2 and CMG860M4 were then finally selected on the basis of their demolished plant growth promotion activities.

Revival of Plant Growth Promotion activities of Mutants in Pots

Mutant strains CMG860M2 and CMG860M4 were analyzed by the method of Choi et al., 2008 [1]with slight modification for the revival of their plant growth promotion activities via addition of synthetic pqq. Following treatments were made.

Set A contained: O/N grown culture of CMG860 wild type

Set B contained: O/N grown culture of CMG860M2/CMG860M4

Set C contained: synthetic PQQ +O/N grown culture of CMG860M2 /CMG860M4

Two days old mung beans seedlings were immersed in 10 ml O/N culture of wild type CMG860 (Set A) , mutants CMG860M2 / CMG860M4 (Set B) and in 10 ml of 100nM of synthetic PQQ (Set C) for 1 hr, and then transferred into the plastic pots contained 200gm autoclave sand, and the surplus synthetic PQQ solution that remained after treatment was poured into the pot of Set C. Pots were placed in a green house and set to a 16-h light/8-h-dark cycle with a relative humidity of 60%. Shoot length and root length of the plants were recorded after 10 days.

Analysis of PQQ

To analyze PQQ production of wild type and mutant strains a method of Choi et al., 2008 was applied. Wild type CMG860 and mutant strains CMG860M2 and CMG860M4 were grown for 48 hrs at 20°C in tris minimal medium containing 1% glucose. One volume of cell culture was diluted with nine volumes of methanol and the precipitated materials were removed by centrifugation. After evaporation of the methanol, a Sep-Pak C18 cartridge (Waters) was washed with 10 ml of methanol and subsequently with 10 ml of water. The sample was acidified with HCl to pH 2.0 and loaded onto the cartridge. After washing with 10 ml of 2mM HCl, PQQ was eluted with 70% methanol. To identify the peak of PQQ, 200 ml of the sample were mixed with 100 ml of 0.2 M Na2B4O7 buffer and adjusted to pH 8.0 with HCl and 90 ml of 0.5% (v/v) acetone. RP-HPLC was performed using a Shimadzu LC-6A HPLC system as described previously (Van derMeer et al., 1990) with a fluorescence detector. Fluorescence was monitored at e x 5=360 and em 5=480 nm. A C18 column (150 mm 3x4.6 mm i.d., 5-mm particle size; Phenomenex) was used for analytical separation. Fractions corresponding to the acetone adduct (5-acetonyl-PQQ) were analyzed using ESI-MS (JEOL).

Extraction of protein from mung bean plants

A 5 day old leaves of mung beans were plucked and immediately placed in vials containing liquid nitrogen. These samples were further dried for 24h in freeze dry system using freezone 6liter benchtop model 77520. Extraction of the plant protein was done by using sigma kit model .The extraction was done according the vendors recommendation. Briefly, the freeze dry samples of mung bean approximately 50 mg were ground in liquid nitrogen to a fine powder. Tissue samples were transferred to 2ml eppendorf held at –20 °C. A 1.5 ml Methanol Solution was added in sample. The suspension was briefly vortex for 15-30 seconds and then placed at –20 °C. The mixture was incubated for 5 minutes at –20 °C. and then centrifuged at 16,000 x g for 5 minutes at 4 °C. Add 1.5 ml of acetone pre-chilled to –20°C in pellet. the mixture was again incubated for 5 minutes at –20 °C. The mixture was Centrifuge at 16,000 x g for 5 minutes at 4 °C. The freezer vials were weighed and the predetermined tare mass of the vial subtracted to determine the plant tissue mass. A 4 μl of Reagent Type 2 Working Solution was added for each mg of plant tissue. The mixture was centrifuged at 16,000 x g for 30 minutes to pellet plant tissue debris. The supernatant (total protein sample) was removed by pipette and place in a clean, labeled tube. The total protein sample is then proceed for SDS-PAGE analysis.

SDS-PAGE Electrophoresis

SDS-polyacrylamide gels (12 % w/v) were prepared according to Laemmli (1 1). Samples were diluted in Laemmli sample buffer ( 11) and boiled for 3 min before loading. gels were run for 90 min and then run at room temperature at 100 V . Gels were stained with Coomassie blue staining method. An electrophoresis cell (model:DYCZ_24E) was used. Prestained mol wt markers were purchased from Sigma

Results

Sequence Homology of Amplified PCR Product (pqqA-D)and pqq E of CMG860

Data analysis

The PCR product was purified using quick PCR purification kit. The ABI373 DNA sequencing system was used for sequence pqqA –D operon. A total of 2212 bases were determined which showed 95% homology with pqqBC gene of Pseudomonas aeruginosa LESB58 and 97% homology to pqqAB gene of Pseudomonas aeruginosaPAO1 and 77% of pqqA_Dgene of Pseudomonas fluorescens strain B16. The purified PCR product of pqqE was also sequenced. A total of 1026 bases were determined which showed 98% homology with pqqE gene of Pseudomonas aeruginosa PAO1 and 98% Pseudomonas aeruginosa LESB58 and 98% of Pseudomonas aeruginosa UCBPP- PA14. Nucleotide analysis was performed by BLASTn and all overlapping sequences were removed. Gene sequences of pqqA-D and pqqE were submitted to genbank, their accession no are given in Table 1.

Isolation of pqq Mutants

Following random mutagenesis of Pseudomonas aeruginosa CMG860 with acridine orange, mutant strains CMG860M2 and CMG860M4 were isolated primarily on the basis of their failure in halos formation (Figure1).

Auxin Production Activity

The auxin production activity of wild type and mutants was performed by using salkovski assay reagent method. Both mutant strains (CMG860M2 and CMG860M4) and Wild type CMG860 showed auxin production activity (Figure 2).

Growth Promotion of Mung Been Seedling

Effect of Wild type and mutant strains were checked on the growth of mung beans seedlings. Heights of the plants were measured every 3 days up to 10 days after the treatments. Mutant strains CMG860M2 and CMG860M4 were failed to promote growth of mung beans seedlings while revival of plant growth promotion activities was observed (Figure3 and Figure4) when synthetic pqq was added in O/N culture of mutant strains (CMG860M2 and CMG860M4). The height of mung beans plants treated with wild type CMG860 was increased by approximately 25% at 10 days after treatment (Figure5 and Figure6).

Identification and Quantification of PQQ via RP-HPLC in CMG860 wild type and mutants

Production of PQQ from wild type CMG860 and mutant stains CMG860M2 and CMG860M4 were confirmed via RP- HPLC. The retention time of standard PQQ is 1.8. Quantification of PQQ in wild type CMG 860 was also performed via HPLC by the integration of peak heights. CMG60 wild type produced 2.44 mg/ml of PQQ (Figure7 and 8). No PQQ production was detected in mutant strains CMG860M2 and CMG860M4 (Figure 7 and 8).

SDS-PAGE Electrophoresis

The total extracted protein was analyzed via SDS-PAGE Electrophoresis. A band of 37 kb was isolated which was absent from control sample. The presences of 37kb band clearly indicate the difference at protein level.

Figure 2
Table 1

Figure 3
Table 2

+*= A BAND WAS VISUALISED

Figure 4
Figure 1: Following Random Mutagenesis of Pseudomonas aeruginosa CMG860 with Acridine Orange, Mutant Strains CMG860M2 and CMG860M4 Were Isolated Primarily on the Basis of Their Failure in Halos Formation

KEYS

A=Growth of M2on nutrient agar plate
B= Growth of M4on nutrient agar plate
C= Growth of M2on Tris agar plate
D= Growth of M4on Tris agar plate

Figure 5
Figure 2: Auxin Production Activity of wild type CMG860 and mutants

KEYS

A= CONTROL
B= CMG860
C= CMG860M2
D= CMG860M4

Figure 6
Figure 3: Mutant Strains M2 and M4 were Studied for the Growth Promotion of the Mung Beans Seedlings.

Keys:A = Wild Type 860B = Mutant M4
C = Mutant Supplemented with Synthetic pqq

Figure 7
Figure 4: Mutant Strains M2 and M4 Were Studied for the Growth Promotion of the Mung Beans Seedlings.

Keys A = Wild Type 860B = Mutant M4 C = Mutant Supplemented with Synthetic pqq

Figure 8
FIGURE 5: SDS_PAGE Analysis

KEYS :a= ladder,b= control, c,d= cmg860

Discussion

Gene encoding PQQ cofactor was detected in CMG860 by PCR (Figure1). The purified PCR product of CMG860pqqA-D (EU72017) was sequenced. A Total of 2212 bases were determined which showed 95% homology with pqqBC gene of Pseudomonas aeruginosa LESB58 and 97% homology to pqqAB gene of Pseudomonas aeruginosaPAO1 and 77% of pqqA_D gene of Pseudomonas fluorescens strain B16. The purified PCR product of CMG860pqqE (EU72016) was also sequenced. A total of 1026 bases were determined which showed 98% homology with pqqE gene of Pseudomonas aeruginosa PAO1 and 98% Pseudomonas aeruginosa LESB58 and 98% of Pseudomonas aeruginosa UCBPP- PA14.

There have been few studies of the functional roles of PQQ in plants. It is known that PQQ stimulates pollen germination in vitro in the plant species Lilium, Tulipa, and Camellia [16,17]but the mechanisms are unclear. To investigate the role of PQQ as a main plant growth promoting factor in CMG860, mutagenesis experiments were performed. Results of mutagenesis clearly indicated that wild type CMG860 showed better plant growth as compared to mutants (Figure3-4). Wild type CMG860 markedly increased the shoot length of mung beans seedlings up to 114 % and 87 % respectively over mutants M2 and M4 while wild typeCMG860 increased the root length of mung beans seedlings up to 200% over mutants. Cultural analysis of the mutant and wild type CMG860 via HPLC clearly indicated the production of PQQ as main agent responsible for plant growth promotion activities (Figure 3 and 4). The revival of plant growth in presence of synthetic PQQ clearly indicated that PQQ is a plant growth promoter. Therefore it was assumed that the biochemical basis of plant growth promotion mediated by PQQ is more or less similar to that of its growth promotion in mammals .[1] It was likely to propose here that many PGPR produce PQQ, which would illuminate previously unknown plant growth promotion mechanisms. As in mammals, PQQ has great potential to be used as a growth promoting factor in plants. Plant growth promotion by PGPR has received attention for academic and practical reasons because beneficial interactions between PGPR and plants offer tremendous potential for field applications. In order to study the mechanism of plant growth promotion at protein level, The total protein extraction from young leaves of mung bean plants was carried out as the RNA analysis was difficult because of highly degrading potential of RNA hence protein extraction from the mung beans leaves were performed. Total protein was extracted from treated and untreated control samples via Plant protein extraction kit. A band of approximately 37kb was found in test samples which was absent in control samples. These results were in good harmony with those obtained by Adam (1999) and Abdel monem et al (2000). These result led us to identify a new plant growth promotion factor, PQQ, from Pseudomonas aeruginosa CMG860 which could be used as potential biofertilizer. A band of 37kb clearly unrevealed the mechanism of plant growth promoting activity at protein level .it was presumed that this band might be help ful in understanding the plant growth promoting abilities at protein level.

References

1. Abdel –Monem, M.A.S.,H.E, Khalifa, M. Beider,I. A. El-Ghandour and Y.G. Galal, 2001. Using biofertilizers for maize production: response and economic return under different irrigation treatments. J. Sustainable Agric.,19:41-48.
2. Adam, M.S.,1999 . The productive effect of the cyanobacterium Nostoc muscorum on the growth of some crop plants. Acta Microbial.Poloncia, 48:163-171.
3. Biville, F., E. Turlin and F. Gasser, 1989. Cloning and genetic analysis of six pyrroloquinoline quinone biosynthesis genes in Methylobacterium organophilum DSM760. J. Gen. Microbiol. 135:2917–2929.
4. Choi et al, 2008 Choi, O., K. Jinwoo, K. Jung-Gun, J. Yeonhwa, J. S. Moon, C. S. Park and I. Hwang, 2008. Pyrroloquinoline Quinone Is a Plant Growth Promotion Factor Produced by Pseudomonas fluorescens B161. Plant Physiol. 146: 657–668.
5. Duine, J. A., 1991. Quinoproteins: enzymes containing the quinonoid cofactor pyrroloquinoline quinone, topaquinone or tryptopha tryptophan quinone. Eur J Biochem. 200:271-284.
6. Duine J.A., 1999. The PQQ story. Journal of Bioscience and Bioengineering. 88:231-236.
7. Felder, M., A. Gupta, V. Verma, A. kumar, G. N. Qazi, J. Cullum, 2000.The pyrrolo quinoline quinone synthesis genes of gluconobacter oxydans. FEMS Microbiol. Lett.193:231-236
8. Goosen, N., H. P. A. Horsman, R. G. M. Huinen and P. VandePutte, 1989. Acinetobacter calcoaceticus genes involved in biosynthesis of the coenzyme pyrroloquinoline-quinone: nucleotide sequence and expression in Escherichia coli K-12. J. Bacteriol. 171:447-455.
9. Kasahara, T. and T. Kato, 2003. Nutritional biochemistry: A new redox-cofactor vitamin for mammals. Nature 422: 832-836
10. Klinman &Mu, 1994 Klinman, J. P. and D. Mu, 1994. Quinoenzymes in biology. Annu. Rev. Biochem. 63:299-344.
11. Klinman, J. P., 1996. New quinocofactors in eukaryotes. J. Biol. Chem. 271:27189-27192.
12. Kumazawa T, Sato K, Seno H, Ishii A, Suzuki O 1995. Levels of pyrroloquinoline quinone in various foods. Biochem J. 307: 331–333.
13. Kumazawa T, Seno H, Urakami T, Matsumoto T, Suzuki O .1992. Trace levels of pyrroloquinoline quinone in human and rat samples detected by gas chromatography /mass spectrometry. Biochim Biophys Acta 1156:62–66.
14. Meulenberg, J. J. M., E. Sellink, N. H. Riegman and P. W. Postma, 1992.
Nucleotide sequence and structure of the Klebsiella pneumoniae pqq

operon.Mol. Gen. Genet. 232:284–294.
15. Morris, C. J., F. Biville, E. Turlin, E. Lee, K. Ellermann, W. H. Fan, R. Ramamoorthi, , A. L. Springer and M. E. Lidstrom,1994. Isolation, phenotypic characterization and complementation analysis of mutants in ethylobacterium extorquensAM1unable to synthesize pyrroloquinoline quinone and sequence of pqqD, pqqG, and pqqC. J. Bacteriol. 176:1746-1755.
16. Misra HS, Khairnar NP, Barik A, Priyadarsini K, Mohan H, Apte SK
(2004) Pyrroloquinoline-quinone: a reactive oxygen species scavenger
in bacteria. FEBS Lett 578: 26–30.
17. McIntire WS (1998) Newly discovered redox cofactors: possible nutritional, medical, and pharmacological relevance to higher animals. AnnuRev Nutr 18: 145–177.
18. Shahab and Ahmed 2008 Shahab. S. and N. Ahmed, 2008. Effect of various parameters on the efficieny of zinc phosphate solubilization. AF. J. Biotechnol. 17: 1543-1547.
19. Sadaf Shahab, Nuzhat Ahmed and Nasreen S. Khan 2009. Indole acetic acid production and enhanced plant growth promotion by indigenous PSBs . Accepted in Af. J. of Agricultural Research December.2009
20. Schnider, U., C. Keel, , C. Voisard, , G. Defago, and D. Haas, 1995. Tn5-directed cloning of pqq genes from Pseudomonas fluorescens CHA0: mutational inactivation of the genes results in overproduction of the antibiotic pyoluteorin. Appl. Environ. Microbiol. 61: 3856-3864.
21. Smidt CR, Steinberg FM, Rucker RB (1991) Physiological importance of pyrroloquinoline quinone. Proc Soc Exp Biol Med 197: 19–26
22. Stites TE, Mitchell AE, Rucker RB (2000) Physiological importance of quinoenzymes and the O-quinone family of cofactors. J Nutr 130: 719–727.
23. Xiong LB, Sekity J, Shimose N .1988. Stimulation of Lillium pollen germination by pyrroloquinoline quinine. Agric Biol Chem 52: 1065–1066
24. Xiong LB, Sekity J, Shimose N .1990. Occurrence of pyrroloquinoline quinone (PQQ) pistils and pollen grains of higher plants. Agric Biol Chem 54: 249–250
25. Paz, M.A., P.M. Gallop, B.M. Torrelio and R. Flueckiger, 1988. The amplified detection of free and bound methoxatin (PQQ) with nitroblue tetrazolium redoc reactions. Biochem. Biophys. Res. Commun. 154:1330–1337.

Author Information

Sadaf Shahab
Centre for Molecular Genetics, University of Karachi

Nuzhat Ahmed
Centre for Molecular Genetics, University of Karachi

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