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).

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

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.

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.

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 e_m 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).

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-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

Data analysis

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).

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).

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).

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).

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