ISOLATION attracted growing attention because of several problems associated

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ISOLATIONOF ACC DEAMINASE PRODUCING Pseudomonassp. FROM TEXTILE EFFLUENT 1.INTRODUCTION                                                               Microbialpopulations in heavy metal polluted environments contains microorganisms whichhave been adapted to toxic concentrations of heavy metals and become resistantto metal. Such microorganisms have developed diverse mechanisms for survival inthe occurrence of heavy metals, and acquired genetic properties that counteractthe effects of high metal ion concentrations. The use of heavy metal resistantmicroorganisms for the decontamination of heavy metals from contaminated waterand soil has attracted growing attention because of several problems associatedwith pollutant removal using conventional methods and this pollution cause thesoil bacteria to tolerate or adapt the condition and help the plants in goodmanner.    Many industrial practicesrelease toxic heavy metal ions in the environment, such practices include directapplication of industrial effluents that may contain high concentrations ofheavy metals to pond, river, agricultural land, or irrigating agricultural landwith untreated wastewater.

Accumulated heavy metals in the environmentconstitute potential health hazards to humans, harm to living resources andecology. Heavy metals including cadmium, lead, zinc, mercury, copper, cobaltand nickel, which act as soluble compounds or exchangeable elements represent arisk of toxicity depending on the rate of transfer from polluted areas to soilsolution, plants, ground water, soil microflora and to the food chains. thetextile effluents released from the industries can directly introduced intorivers and lands which leads to toxic of the soil as well as eutrophicationhappens in the water bodies due to excess nutrients availability.

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    Itwas reported that a small number of soil Pseudomonas possess the enzyme1- aminocyclopropane 1 carboxylase (ACC) deaminase (Klce el al.,1991)this enzyme cleaves ACC, the immediate biochemical precursor of ethylene inplants, to ammonia and ? – ketobutyrate (Honma and Shimomura 1978). The use ofthe enzyme by Klec el al., (1991) to alter gene expression in transgenicplants carrying a functional ACC deaminase gene by lowering ethylene levelsprompted us to question whether plant growth promoting rhizobacteria (PGPR)such as Pseudomonas putida (Lifshitz et al., 1987) might alsoposses ACC deaminase activity and if they did, whether this enzyme was somehowinvolved in the promotion of plant growth by these PGPR. Some of these strainswere able to produce the enzyme 1-aminocyclopropane-1-carboxylic acid (ACC)deaminase and the plant growth regulatory hormone indole-3-acetic acid (IAA).

Some strains were also able to chelate ferric iron and solubilize potassium,phosphorus and zinc, and produce ammonia.    The important drawback in agricultural isthe ripening of the fruits as early age by the ethylene gas which is naturallyproduce by plants when is needed. Ethylene is a plant aging hormone. It isnaturally occurring and produce with ripening .

It is responsible for changingtexture, taste, colour, softening and other process in ripening. Recent studiessuggest two different strategies used by Rhizobia to reduce the amount ofethylene synthesized by their legume symbionts. One strategy utilizes thecompound through rhizobitoxine which acts to inhibit the enzyme ACC synthase andhence ethylene biosynthesis. In addition, the enzyme1-aminocyclopropane-1-carboxylate (ACC) deaminase which catalyzes the cleavageof ACC to ?-ketobutyrate and ammonia decreases ethylene levels in host rootsand thereby enhances nodulation (Ma et al. 2003a; Ma et al. 2004).1.

1 Plant growth promoting bacteria    Plantgrowth promoting bacteria have a positive influence n plant growth anddevelopment. Many of the bacteria found in soil are bound to the surface ofsoil particles and are found in soil aggregates and interact specifically withthe roots of plants. The interaction between bacteria and the roots of plantsmay be beneficial, harmful or neutral for the plant and effect of a particularbacterium may vary as a consequence of soil conditions. For example, aparticular organism that facilitates plant growth by fixing nitrogen, which isusually present in the soil in limited amounts, is unlikely to provide benefitto a plant in a setting where exogenous fixed nitrogen is added to soil.    PGPR can promote plant growth and development by indirect or directmeans (Glick et al.

1999; Nelson 2004). “Direct mechanisms can be demonstratedin the absence of plant pathogens or other rhizosphere microorganisms, whileindirect promotion of plant growth involves these bacteria reducing hedeleterious effects of plant pathogens” (Nelson 2004). There are several waysin which plant growth-promoting bacteria can directly enhance plant growth and development(Glick 1995). For example, they chelate irons by producing siderophore,solubulizes minerals, fix atmospheric nitrogen, produce hormones, and enzymeswhich can enhance or inhibit the plant growth and development.    PGPR (Plant growth promoting rhizobacteria)can affect plant growth in two different ways, indirectly or directly. Theindirect promotion of plantgrowth occurs when PGPR lessen or prevent thedeleterious effect of one or more phytopathogenic organisms.

The direct promotionof plant growth by PGPR for the most part entails either providing the plantwith compound that is synthesized by the bacterium or facilitating the uptakeof certain nutrients from the environment. 2.REVIEWOF LITERATURE    2.1 Isolation of Pseudomonas fluorescence    Microorganismsare generally found in nature as mixed populations.

To study the specific roleplayed by a specific microorganism in the environment, it should be isolated aspure culture. And the pure culture should be maintained.    Theisolation and screening of PGPR by the production of phytohormones like IAA(Auxin).

In addition, most PGPR has ACC Deaminase enzyme that cleaves ACC whichis a intermediate precursor of ethylene synthesis, to ?-ketobutyrate andammonia thereby lowers the ethylene level. This enzymes only produce when thebacteria under stress such as salinity, heavy metal.     ACCdeaminase was  first discovered in bacteria.

Some plant growth-promoting bacteria are capable of processing the plant-borne ACCby converting it into ammonia and ?-ketobutyrate using the enzyme ACC deaminase(HonmaandShimomura,1978). ACC deaminase was retrieved. Pseudomonas sp.

strainACP (Honma and Shimomura,1978), Pseudomonas chloroaphis  6G5 (Klee etal., 1991), Pseudomonas putida GR12-2 (Jacobsonetal.,1994) and Pseudomonasputida UW4 (Hontzeasetal.,2004). ACC deaminase containing bacteria canreduce stress susceptibility of plants during flooding (Barnawal et al., 2012;Li et al.

, 2013), drought (Mayak et al., 2004a), salinity (Mayak et al., 2004b;Nadeem et al., 2007, 2010), flower senescence (Nayani et al., 1998; Ali et al.,2012), metal pollution (Glick, 2010), organic pollution (Gurska et al.

, 2009)and pathogens (Glick, 2014 and references therein). In addition, it has beenreported that the presence of ACC deaminase can increase the symbioticperformance of Rhizobial strains (Ma et al., 2003).     Pseudomonas fluroscencs strains such as ps.fuorescens Pf2, Ps. fluroscens TDK1 & RMD1 were isolated from soilsample  and the efficacy of the bacteriato promote plant growth under stress condition (Kumar D S 2006). Enterobacter cloacae,  Pseudomonas putida and Pseudomonasfluorescens were isolated from the soil near the rhizosphere where have themore ACC deaminase producing bacteria with ACC as a nitrogen source in themedia.(Donna m 2002).

     Pseudomonas isolation agar, is a selectivemedia of pseudomonas auroginosa. aswell as pseudomonas fluorescence wasisolated by pigmentation and biochemical characters. ACCD producing pseudomonas fluorescence was isolateddirectly by adding ACC into the medium, where the ACCD producing bacteria growusing ACC as nitrogen. IN most of the articles Kings B medium and Tryptic soybroth were used as selective media for pseudomonasfluorescence. instead of the selective media we can use the Pseudomonasisolation agar  where pseudomonas fluorescence grow as a whitecolourless pigment whille pseudomonasauroginosa produce greenish yellow pigmentation on media. 2.

2Ethylene Biosynthesis   Ethylenewas discovered as a plant regulator in e work done in dark green peaseedlings  showed reduced growth ofhypocotyl (Neljubov 1901). Biosynthetic pathway of ethylene was came upon whenS-adenosyl-L- methionine (SAM) was an intermediate between methionine andethylene( Adams & Yang 1977). The major discovery that made the methioninecycle in plants unique from all other organisms, was the characterization of1-aminocyclopropane-1-carboxylic acid (ACC) as the intermediate between SAM andethylene (Adams and Yang, 1979). The identification of ACC as the precursor forethylene by feeding experiments on apple tissue, using radio-labeled methionine.

     As mentioned above, ACC is produced fromSAM, this reaction is catalyzed by the enzyme ACC-synthase (ACS; Boller et al.,1979). The ethylene synthesisis carrued out by two major enzymes are ACCsynthase and ACC oxidase. ACS is a gene which is encoded by plant involved inethylene biosynthesis, encoes ACC synthase converts the SAM  into ACC ( VanDER straeten et al., 1992). The second enzyme is ACC oxidase (ACCO)  acts in the presence of oxygen,  converts ACC into ethylene. and it wasisolated by addition of ascorbic acid (vitamin C) to the media ( Ververidis& John 1991).

             figure1- Biosynthetic pathway of ethylene via intermediate precursors SAM and ACC.2.3 ACC Deaminase    ACC deaminase is anenzyme which breaks down ACC. Specifically ACCDcleaves the cyclopropane ring of ACC and removes anamino group to produce ?-Ketobutyrate and ammonia (Penrose and Glick, 2001).

TheEnzyme has been only found in microorganisms. Yeast and plant growth promotingbacteria have been studied so far.     ACCdeaminase production may be helpful in the nodulation process and therebyincrease the nitrogen supply for legume plants due to a more effectivenodulation. This may be especially important when plants are growing understressful conditions so that ethylene may attain levels that are highlyinhibitory to nodulation(Nascimenti et al., 2016).     It canreduce ethylene generation in plants; in this way they can reduce the extent ofgrowth inhibition caused by high-level ethylene, particularly under abioticstresses (Glick, Penrose and Li 1998).

ACCD-mediated plant growth promotion hasstimulated tremendous interest in research into the isolation and applicationof ACCD-producing bacteria (Glick et al. 2007, 2014). 2.3 ACC deaminase enzyme activity assay     Theassessment of bacterial ACCD activity requires growth conditions with theinduction of ACC deaminase. Activity of ACCD was assayed by measuring?-ketobutyrate and ammonia, the hydrolysis product of ACC (khan A L 2016).Ina series of known ?-ketobutyrate concentrations, 2 mL of the2,4-dinitrophenyl-hydrazine reagent (0.

2% 2, 4-dinitrophenyl-hydrazine in 2 moLL-1 HCl) was added, the contents were vortexed and incubated at 30°C for 30min, during which the ?-ketobutyrate was derivitized as aphenylhydrazine. Thecolor of phenyl hydrazine was developed by the addition of 2 mL, 2 moL L-1 ofNaOH, the absorbance of the mixture was measured after mixing by usingspectrophotometer at 540 nm.( khan A L 2016).

     The level of ACC deaminase activity thatis observed when strain P. putida GR12-2 is grown on minimal medium plusammonium sulfate represents a basal level of activity of ?5% of the total activity that ismeasured in extracts grown on minimal medium containing ACC (as a nitrogensource) instead of ammonium sulfate. A few other amino acids including L-alanine,DL-alanine and DL-valine can also induce ACC deaminase enzyme activity, albeitto a limited extent, while  ?-aminoisobutyricacid can induce activity to nearly the same level that is found with ACC.Optimum pH and temperature for activity is 8.0 and 30?C respectively (Shimomuraand Honma, 1978). ACC deaminase requires pyridoxyl phosphate as a cofactorwhich represents the aminotransferase family of enzymes. It is also reactivewith other D-amino acids but is competitively inhibited by L-serine (Penroseand Glick).

2.4 Objectives    However,most researchers has focused on the regulation of biosynthesis of ethylene. Theexistence of ACC deaminase in soil bacteria has been discovered, it was assumedthat may play an important role to reduce growth inhibition caused byhigh-level ethylene, particularly under abiotic stresses. ACCD producing pseudomonas sp. were identified as efficient strainin pseudomonads thus the objectives are 1. To isolate ACC Deaminase  producing Pseudomonassp from the textile effluent.

 2. To characterize Psuedomonas fluorescensisolates from their morphological and biochemical properties. 3.

To assess the activity of the enzyme bycalorimetric assay. 4. To sequence the 16S rRNA of pseudomonas toassign the species. 3.

MATERIALSAND METHODS MATERIALS AND METHODS  3.1. Sample    Textileeffluent was collected from the textile industry, kerala; stored at 4?.  3.2 Isolation of pseudomonas fluorescens     Forisolation, 1 mL of effluent was diluted in the range of 10-1 to 10-6.  10-2  and 10-3  spread on sterilized pseudomonas isolationagar (Peptic digest of animal tissue 20.0g, Magnesium chloride 1.

4g,Potassium sulphate 10.0g, Irgasan 0.025g, Agar 13.6g per litre).

Incubated at room temperatures for 24-48 hrs. The plates were examined dailyupto 3 days for visible colonies. Pure culture were obtained by the streakplate methed.3.3 Characterization ofisolate.

3.3.1. Morphologicalcharacterization    Morphological characteristicsof the colony of  isolate were examinedonPseudomonas  isolation agar medium. Culturalcharacterization of  isolates observed bydifferent characteristics of colonies such as shape,  size, surface, margin, colour, pigmentationetc., were recorded. 3.3.

2 Gram’s staining    A drop of steriledistilled water was placed in the center of glass slide. A loopfulof inoculum fromyoung culture was taken, mixed with water, and placed in the centerof the slide.The suspension was spread out on slide using the tip of inoculation needleto make a thinsmear. The smear was dried in air and fixed through heating bypassing theslide 3 to 4 times over the flame. The smear was then flooded with Crystalviolet solutionfor 1 min and washed gently with flow of tap water. Then the slide wasflooded withIodine solution. After incubation at room temperature for 1 min, Iodinesolution wasdrained out followed by washing with 95% decolorizer. After that, it waswashed withwater within 15 to 30 sec and blot carefully.

The smear was incubated withSafranin  solution for 1 min. The slide was washedgently in flow of tap water and driedin air. Theslide was examined under microscope at 100X power with oil immersion anddata wasrecorded.3.3.4Biochemical characterization    Motility,IMViC, catalase, oxidase, nitrate reduction, gelatin hydrolysis were performedto characterize the isolates and recorded.

3.3.5 ACC deaminase activityassay     ACCdeaminase activity was determined for isolated strain according to the protocol described by Penrose and Glick(2003) with a standard curve of ?-ketobutyrate between 0.1 and 1 µM. he number of µM of ?- ketobutyrateproduced were determined by comparing the absorbance at 540nm of the sample to the standard curve.

10µM?-ketobutyrate  used to generate standardcurve. Each in a series of known concentration of ?-keobutyrate  with 3 ml of 2,4- dinitroohenylhydrazinereagent (0.2% of 2,4-dinitrophenylhydrazine in 2M HCl) is added, votexed andincubated at 30?C for 30 mins, during this time ?-ketobutyrate is derived asphenylhydrazone. The colour is developed by the addition of 2ml of 2M NaOH, aftergently mixed the absorbance at 540nm is measured.3.3.6 Induction of ACCDand Bacterial extract     The enzyme activity is measured inbacterial extracts in the following procedure. The bacteria are cultured insutable rich medium and transferred to the minimal medium with ACC as the solenitrogen source.

Bacterial culture are grown to mid to late log phase in 15 mlof rich medium 10 µl of culture strain. Incubated at suitable temperature. Thecontents are centrifuged at 8000g for 10 min at 4?C. The pelleted cells arewashed with DF ( Dworkin & Foster 1958) salts minimal medium per litre ( 4gKH2PO4, 6g Na2HPO4, 0.2g MgSO4.

7HO,2g glucose, 2g gluconic acid,2g citric acid, 1mg FeSO4, 10g H3BO3,11.1g MnSO4. H2O,  124.6g      ZnSO4.H2O,78.22g CuSO4. 5H2O, 10g MoO3, pH 7.

2& (NH4)2SO4 as a nitrogen source). Centrifugedat 8000g for 10 min at 4?C. The cells are suspended in 7.5 ml of DF saltsminimal medium with 45 µl of 0.5 M ACC ( to obtain final concentration of 3 mM). the tube was incubated inshaking incubator for 24hr.

the cells are collected by centrifugation at 800gfor 10 min at 4?C. The cells are washed by 0.1 M Tris-HCl,pH 7.6 for enzymeassay and suspended in 0.03 M MgSO4 for Gonotobiotic root elongationassay. 3.3.

7 Gonotobiotic rootelongation assay     This method is used as a method of assessingbacterial strain to produce ACC deaminase by inhibiting ethylene which in turnproducing long roots. ACC deaminase producing sol bacteria av been isolated wasassayed by root elongation assay and shown to promote can alo seedlings ( Glicket al., 1995, Belimov et al., 2001). The bacterial cell pellet was suspended in0.

5 ml sterile 0.03 M MgSO4 and placed on ice. A 0.

5 ml sample wasremoed and diluted 8-10 times in 0.03 M MgSO4; the absorbance ismeasured at 600nm. This measured is used to adjust the absorbance to 0.15 with0.03 M MgSO4.      Canola seeds are disinfected before use. Theseeds are soaked in 70 % ethanol for 1 min followed by 1% sodium hypochloritefor 10 mins and washed with sterile distilled water 5-6 times. Each petri dishis filled with 12 ml sterile distilled water and 0.

03 M  MgSO4. 6 seeds areplaced with bacterial strain, and without bacteria as control. The seed arecultured at 25?C  with a cycle of 12hrdark & 12hr light.

The primary root length are measured after 5thday f growth and recorded. Typically, the length of the roots of canola seedsare 40 – 60%. Greater than the untreated seedlings.3.4 Strainidentification3.4.1 DNA isolation                                                                                  

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