Effect of phenol on protein and amino acid content of Xanthomonas oryzae pv. oryzae

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1 nd ian Journal of Experimental Biology Vol. 39, Otober 2, pp Effet of phenol on protein and amino aid ontent of Xanthomonas oryzae pv. oryzae Mohan & A Mahadevan* Centre for Advaned Study in Botany, University of Madras, Chennai 625, ndi a Reeived 16 Otober 2; revised 26 June 21 Leaf bli ght di sease of rie (O ryza sariva) is aused by the baterium Xanrh o111onas oryzae pv. oryzae. Phenol ( to 4 mm) indued hanges in protein profiles of X. o. pv. orvzae and a stress protein with a moleular mass of 69, appeared. HPLC analysis indi ated ourrene of amino aids suh as asparagine, alan ine, methionine and ystine in phenol treated e ll s. Proton MR analysis also revealed vari ati on on the presene of amino aids in the ells treated with phenol. Bateria, when exposed to different environmental stresses display variations in several maromoleular strutures, physiologial funtions and phenotypi haraters. Stresses inlude hemials, temperature, osmoti shok and total or partial starvation 1 Two important ways in whih ellular adaptation in bateria manifests itself during unfavourable growth onditions are by-(i) indution of stress proteins; and or (ii) aumulation of intraellular ompatible solutes like potassium, glutamate, proline, glyinebetaine et. 2. Groat et a/ 3 have reported indution of 3 proteins in Esherihia oli ells subjeted to arbohydrate starvation. Addition of gluose to the arbon starved ulture led to initiati on of a reovery phase, repression of starvation-indued proteins as we ll as indution of a new lass of transiently expressed proteins. Salinity indued hanges in the protein profiles in Rhizobium sp., exhibited alterations in the expression of as many as 19 proteins whih either showed an enhaned rate of synthesis or a deline in the levels as ompared to ontrols 4. Several responses in bateria to osmoti hanges have been reported 5. One of th em is hange in the level of amino aids. Passive efflu x of amino aids in response to a hypo-osmoti shift has been observed for proline, glutamate, lysine and other amino aids in di verse bateria. Effet of aromati substanes suh as phenols and their polymers impliated in the resistane of plants against miroorgani sms has not been studied at th e moleular levelr'. 7 espeially on b:1terial pl ant pathogens. n this paper, results have been presented *Correspondent author- Cenlre for Theoretial Bi ology, 4 (old), 26 ( ew), First Main Road, ndira agar, Adyar, Chennai 62, ndi a. E-m ail : on the effet of phenol on protein and amino aid ontent of Xanthomonas oryzae pv. oryzae, the ausal agent of baterial blight of paddy. Materials and Methods Xanthomonas oryzae pv. oryzae was obtained from th e Culture Coll etion Centre of the nstitute. Experiments were anied out by growing the ells in a syntheti medium (K 2 HP4-1.2 g; KH 2 P4-.8 g; MgS 4 7H g; glutami aid g; surose -2. g; distilled water-! 1; ph-7) 8 and inubated at 3 C. After 24 hr of growth, the ell s were olleted in sterile entrifuge tubes by enttifugation at 1, g in a Bekman High Speed Centrifuge (Model J2-21) fo r min. The ells were washed twie and suspended in sterile distilled water. OD of the ell suspension was adjusted to.5 at 54 nm in a Bekman spetrophotometer (Model DU-4) and used as inoulum. Effet of phenol on protein ontent was determined by amending the syntheti medium (without surose) with suitable volume of phenol stok soluti on to give a final onentrati on of 1, 2 and 4 mm. Protein esti111ation-control and phenol treated ells were harvested after every 12 hr and entrifuged at 1, g for min, twie with phosphate buffer (ph 7.). The pellet was suspended in 5 ml of buffer and disintegrated using an ultrasoni disintegrator (Braun-Labsoni 2) for 2 min. t was entrifuged agai n and the pell et was disarded. To ml of the supernatant, 1 ml of 1% TCA solu tion was added and left for 3 min. The preipitate vvas separated by entrifugation at 1, g for 1 min and di ssolved in 1 ml water. To 1 J..l.l fration, ml of Coomassie 9 blue reagent was added. The olour was read at

2 156 DA J EXP l3 OL, OCTOBER nm in a spetrophotometer (Bekman-DU4). A standard graph was drawn wi th bovine serum albumin. Polyarylamide gel eletrophoresis-eletrophoresis of protein samples was anied out aording to Mahadevan and Sridhar 1. Solution A-polyarylamide (3 g) and bis-arylamide (.8 g) were dissolved in 5 ml of di still ed water, made up to 1 ml, filtered and stored in a brown bottle at 4 C. Solution B-tris (36.6 g) was di ssolved in 48 ml of HC, ph was adjusted to 8.8, filtered and stored in a brown bottle at 4 C. Solution C-ammonium persulphate (14 g) was dissolved in 1 ml of water. Fresh solution was prepared and used. Eletrophoresis buffer (1 X): tri s (6 g) and glyine (28.8 g) were dissolved in 8 ml of water, ph was adjusted to 8.6, made up to 1 l and stored at 4 C. At the time of eletrophoresis, 1 X buffer was prepared from this stok and used as tank buffer. Solutions A (3 ml), B (6 ml) and C (1 ml) and distilled water (3 ml) were plaed in a side arm flask and mixed thoroughly. Air bubbles were removed by onneting the side arm to a vauum pump. After 1 min, TEMED was added and the gel was ast immediately. A.75 mm separating gel was ast and a film of water was layered on separation gel to prevent air ontat. A 5% staking gel was ast on the separation gel. Samples were mi xed with sample buffer (tris-hcl buffer ph ml ; SDS -.1 ml; surose -1 g; meraptoethanol -.5 ml ; bromophenol blue -.2 ml; water-2 ml) in the ratio of 1 :3 and kept in a water bath for 3-5 min at 9 C. They were loaded on to the wells of polyarylamide gel and eletrophoresed at 25 ma urrent for 4 hr at 4 C. The gel was stained with.2% Coomassie brilliant blue-r25 (Sigma) dye solution for 3 hr and was destained in methanol : aeti aid : water (4:1:5 v/v) till a white bakground appeared. The gel was transferred to a Hoefer Sanning Densitometer (Model-GS3) and sanned. Amino aid analysis- Protein samples vvere hydrolyzed and derivat ized for th e detetion of ami no aids in Shimadzu HPLC with SPD loa detetor and Simpak C-1 8 olumn. The mobil e phase onsisted of MeOH: H 2 : TEA buffer (55 : :.25 v/v), ph 7.7; the flow rate was ml/min and amino aids were deteted at 254 nm. Aid hydrolysis, derivati zati on and detetion of the samples were performed as desri bed in the operation manual of the above HPLC. Aid hydrolysis-protein sample (5 mg eah) was kept in glass vials with narrow nek. One ml of 6 HC was added to eah tube and nek of the vial was sealed under vauum. The samples were hydrolyzed at ll C for 22 hr in an oven. After ooling, the ontents were transferred to a new vial, the residue was washed with fresh 6 HCl and pooled. HC was evaporated in a vauum desiator. Derivatisation-The derivati sation reagent onsisted of 7:1:1:1 solution (by vol ume) of ethanol: triethanolamine: water: ph en y isoth ioyanate (PTC). The mixture was vortexed for a few seonds. To eah dried sample, 2 j..tl of deri vatisati on reagent was added, mixed thoroughly, allowed to stand for 2 min at 25 C and again dried thoroughly in a vauum desiator to remove traes of PTC. Final ontent was di ssolved in 1 ml of the sample diluent and passed through.45!jm filter for HPLC analysis. Sample diluent was prepared by dissolving 71 mg of disodium hydrogen phosphate (a 2 HP 4 ) in 1 1 of water and ph was adjusted to 7.4 with 1% phosphori aid. The resulting solution was mixed with aetonitrile to get 5% by volume of aetonitrile. Proton MR analysis-to study the metaboli hanges ourring in the phenol treated ells, proton MR analysis was done using lyophilized perhlori aid extrad 1 of ontrol and phenol treated ells at 24 hr. The lyophilized samples were dissolved in deuterium oxide (Sigma, USA) in a MR tube prior to analysis in Bruker 3 MR Spetrometer. Suitable ontrols were kept for eah treatment. All the experiments were repeated at least thrie and reproduibl e results are presented. Results Extraellular protein from the ulture filtrate was below the detetion level. n th e ells exposed to lower onentrations of phenol, intraellul ar protein level was low (Fig. 1 ). n th e ontrol ells, protein level inreased up to 24 hr but delined at 36th hr and again inreased till 6th hr. Similar trend was reorded in th e phenol treated ell s, however protei n level was low at 4 mm, ompared with and 2 mm ph enol treatment. SDS-PAGE of proteins from baterial ells Sanning of gels re vealed th e presene of 2 maj or proteins in ontrol ells i.e., peak numbers 7 and 1 at 12th hr, peak numbers 8 and at 24th hr and peak numbers 9 and 12 at 36th hr. The total number of peaks also inreased during 24 and 36th hr (Fig.2). Protein pattern of ontrol el ls did not reveal any

3 MOHA & MAHADEV A: EFFECT OF PHEOL O PROTE AD AMO ACD 157 remarkable hange exept the formation of peak numbers 7, 11 and 12 during 24th hr, whih is the ative period of growth. When the ells were treated with mm phenol, the quantity of proteins delined as indiated by the size of peaks. n 2 mm phenol treated ells, quantity of protein further delined, several small peaks appeared and in 4 mm phenol treatment, protein level was least (Fig. 2). The prominent peaks of ontrol ells i.e., number 1 at 12th and 24th hr, number 12 at 36th hr delined in the phenol treated ells at all onentrations during 12th hr but slightly inreased in size during 24 and 36 hr. Further, peak number 1 of 12th and 24th hr treated ells and peak number 12 of ells olleted at 36th hr appeared broken into several small peaks in the phenol treated ells. A speifi feature noted only in the phenol treated 1/) v u 3 l 25 T"" 2 >< r--. Ol 15 ~. ~ 1 C1l Control --1mM -k-2mm nubation Time (hr) Fig. -Effet of phenol on protein ontent of Xanthomonas oryzae pv. oryzae ells was the presene of a small peak i.e., peak number 3 (1 mm/12th hr), peak number 4 ( mm/24th hr), peak number 3 (1 rnm/36th hr); peak number 5 (2 rnm112th hr), peak number 4 (2 rnm/24th hr), peak number 5 (2 rnm/36th hr); peak number 3 (4 mm/12th hr), peak number 4 (4 rnm/24th hr), and peak number 3 (4 mm/36th hr). This peak did not appear in the ontrol ells but indued by phenol. This stress protein had a moleular weight of a 69,, as measured by omputer software (GS365W- Hoefer Sientifi, USA.) Amino aid pattern-control ells ontained fewer amino aids than phenol treated ells. Amino aids like histidine, leuine, proline, serine, and valine were present in both ontrol and treated ells whereas alanine, asparagine, ystine, glyine and methionine were deteted only in the phenol treated ells (Table 1, Fig. 3). Lysine was deteted only in the ontrol ells and not in the treated ells. Asparagine was the only amino aid found in high onentration, (42.9 ~g/mg protein), than any others. ext to asparagine, serine and proline were present in abundane and they existed in the ells treated with phenol. Hi stidine, lysine, phenylalanine and tyrosine were present in low onentration. Other amino aids were present 111 traes and their identity ould not be established. 1 H MR analysis-three peaks were obtained in the ontrol at 24th hr and identified as proline, tyrosine and valine. o peak was present in 4 mm treated ells at 24th hr. Six peaks appeared in the ells suspended in 2 mm phenol at 24th hr and they were glyine, proline, glutamine, glutami aid, leuine and valine (Fig. 4). Table 1-Effet of phenol on amino aid ontent of X.o. pv. oryzae Phenol (mm) Amino aid Control 2 4 (j..lg/mg protein) Asparagine Proline Serine Glyine Valine Leuine Histidine Lysine.3.3 Phenylalanine Alanine Tyrosine Methionine Cystine

4 158 DA J EXP BOL, OCTOBER 21 Disussion Clearly phenol altered the protein ontent of X. o. pv. oryzae and it delined with inreasing onentration of phenol. As early as 1945, Fogg and Lodge 12 proposed that the protein preipitating ability of phenol depends upon its onentration. Further, the rate of sy nthesis of various enzymes would be redued by the phenol, leading to deline in metabolism. The bateriidal ation of phenol, aordi ng to Cooper 13, is by preipitating proteins. Lipid profile of the ells of X. o. pv. oryzae was markedly affeted by phenol treatment 14. COTROL lmm SDS-PAGE eletrophoresis of protein samples of ontrol and phenol treated ells revealed a marked differene between them. Further, the peaks of ontrol ells, whih were of large protein moleules beame broken to several smaller peaks (or small peptide moleules) in the phenol treated ells. Certainly phenol interferes with the protein metabolism of X. o. pv. oryzae. A stress protein with a moleular weight of a 69, daltons might be result of the phenol treatment. Although aumulation of several new proteins has been reported as a result of stress 2mM 4mM E 7 24hr 1 o () :::::> ~5 <f) : Q) hr Length of the gel ( mm) Fig. 2- Densitometer sanning of SDS gels showing protein pattern of X. o. pv. oryzae

5 MOHA & MAHADEV A: EFFECT OF PHEOL O PROTE AD AMO ACD ~1 "' COTROL,..., Gl, lmm,..,),;., 2mM Si grti rti 4mM 12hr 1 a;.,; ": 2 1\i 5- - f----' w~. ;:::; Q..::!.,"' "'.-i$ 'Of ~, "?~~ G>.,..:.; DS ~ ui J-.-.-, '" onid o o o q,._1 1 on o. "' ~a) 15 s,,.;«! "' '1"": r<i"' r<l ": 24hr E - > = ' 8 ii,..:,..: 'Of, -!.., w..,,.., "' "'.n,._..: D f-./ v '" ": on., '",Do Gl,..:. - Gl 15 l'lid "':"'! on,.;,.;,...""!o ":~,j ~..., ": 36hr J w Gl CZ) "': 'Of ~ ~... ::: 8,...ui~ ai,._q Gl,._. ai minutes 15 T Fig. 3-Amino aid pattern of X. o. pv. oryzae

6 16 DA J EXP BOL, OCTOBER 21 COTROL (24 h) u 2... <) -ro > PPM 2mM (24 h) (,) "' > PPM mm (24 h) :..'). :. ~~1~~~~~~~~~~''...,,,.~ '~~.. ~~~~ PPM Fig. 4-1 H MR spetrum of the PCA extrat of X. o. pv. oryzae

7 MOHA & MAHADEVA: EFFECT OF PHEOL O PROTE AD AMO ACD 161 onditions 3.4, our study showed a single protein. Purifiation and haraterization of this stress protein might give more information on the mode of ation of phenol on this baterium. When there is an alteration in protein profile, the involvement of amino aids annot be overlooked. Proline inreased in the ells exposed to phenol and its aumulation is onsidered as a response to stress. Proline might aumulate due to stimulated synthesis from glutamate and/or due to inhibition of proline oxidation 15 n general, gram-negative bateria ahieve high intraellular onentrations of proline during osmoti stress only by enhaned transport 2. Measures 16 has reported the aumulation of glutami aid and proline in mi xed non-halophili bateria subj eted to stress. More number as well as inreased amount of amino aids was deteted from the protein samples of phenol treated ells. The reason for the aumulati on of alanine, asparagine, glyine, methionine and ystine in phenol treated ells is not lear. 31 P, 13 C and 1 H MR spetrosopy studies are presently used to study baterial metabolism 17 The suessful appliation of 1 H MR to th e study of maromoleules suh as proteins depends on the ability to assign resonane to speifi amino aid residues or to speifi protons of protein bound ligands 18. Our results on PCA extrat of ontrol and phenol treated ells by 1 H MR revealed the presene of 3 amino aids in the ontrol, 6 in 2 mm and none in 4 mm phenol treated ell s. However, this result did not orroborate with the analytial data obtained from HPLC analysis. Certainly the mode of ation of phenol involves substantial hanges in the protein metabolism of X. o. pv. oryzae, ulminating in the death of ells. Aknowledgement We thank University Grants Commission, ew Delhi for providing funds to proure Densitometer with omputer progamme and Dr Chandrakumar, Central Leather Researh nstitute, Chennai for providing MR failities. Referenes Graham P H, Stress tolerane in Rhizobium and Bradyrhizobium, and nodulation under adverse soi l onditions. Can J Mirobial, 38 (1992) Csonka L, Physiologial and geneti responses of bateria to osmoti stress. Mirobial Rev, 53 (1989) Groat R G, Shultz J E, Zyhlinsky E, Bookman A & Matin A, Starvation protein in E. oli : Kinetis of synthesis and role in starvation survival. J Baterial, 168 (1986) Saxena D, Amin M & Khanna S, Modulation of protein profiles in Rhizobium sp. under salt stress. Can J Mirobial, 42 (1995) Wong L S, Johnson M S, Sangberg L B & Taylor B L, Amino aid effl ux in response to hemotati and osmoti signals in Baillus subtilis. J Bat, 177 ( 1995) Mahadevan A, Biohemial aspets of plant disease resistane. Vol.2. Post-infetional defense mehanisms. (Today & Tomorrows, ew Delhi) 199 1, p Rangaswamy G & Mahadevan A, Diseases of Crop Plants in ndia. (Prentie-Hall ndia) 1999, 7. 8 Rao S R K & ayudu M V, n vitro nutrition of Xanthomonas ampestris pv. oryzae (Uyeda and shi ya) Dowson, induing baterial leaf bli ght. ndian J Exp Bioi. 16 (1978) Bradford M M, A rapid and sensiti ve method of quantifiation of mirogram quantities of proteins utilising the priniple of dye binding. Anal Biohem, 72 (1976) Mahadevan A & Sridhar R, Methods in physiologial plant pathology. (S ivakami Publiations, Chennai, ndia) 1996, De Jo ng S, Mulder H, de Vries E C E & Robillard G T, MR spetrosopy anal ysis of phosphorous metabolites and the effet of adriamyin on these metabo li te levels in an adriamyin sensitive and resi stant human small ell lung arinoma ell line. B r J Caner, 63 ( 199 1) Fogg A H & Lodge R M, The mode of antibaterial ation of phenols in relation to drug-fastness. Trans Faraday So, 41 (1945) Cooper E A, On the relations of phenol and metaresol to proteins: a ontribution to our knowledge of the mehanism of di sinfetion. Biohem. J, 6 (191 2) Mohan & Mahadevan A, Effet of phenol on lipid and fatty aid ontent of Xanthomonas Ol) zae pv. oryzae. ndian J Exp bioi, (in press). 15 akamura H, Speifi proline aumulati on in an area mutant of E. oli K12 grown in salt-hypertoni medium. J Cen Mirobial, 113 (1979) Measures J C, Role of amino aids in osmoregul ation of nonha lophili bateria. ature (London), 257 (1975) Aguayo J B, Gamsik M P & Dik J D, Hi gh reso lu tion deuterium MR studies of baterial metaboli sm. J Bioi Ch em, 263 ( 1988) Aull J L, Crespi H L, Williams D E & Evanohko W T, Purifiation of deuterated th ymidylate synthase and proton nulear magneti resonane studies. Biohim. Biophys Ara. 79 (1982) 31.