Indian Journal of Experimental Biology Vol. 39, May 2001, pp. 464-468
Siderophore production by fluorescent pseudomonads colonizing roots of certain crop plants R D Yeole, B P Dave & H C Dube* Department of Life Sciences, Bhavnagar University, Bhavnagar 364 002, India
Received 8 Seprember 2000: revised 23 .January 2001 Twelve fluorescen t Pseudomonas isolates colonizing roots of four crop plants, chill i, cotton, groundnut and soybean , were examined for extracellular siderophore production in different media under iron deficient conditio ns. While all the orga nisms produced siderophores, they varied in the quantity of siderophores produced and in their preference to the medium. The siderophores were invariably hydroxamates (pyoverdine) of trihydroxamate type which formed bide ntate ligands with Fe Ill ions.
Siderophores (sid=iron, phores=bearers) are low molecular weight (< 1000 D) iron chelating compounds produced by microorganisms under iron stress conditions 1.2. No system analogous to siderophores has been found for any other metal ion thus, making iron unique in requiring such specific ligands. A large number of bacteria and fungi are known to produce siderophores under iron limiting conditions in the soil. The root coloniz~ng bacteria, called rhizobacteria, produce siderophores that determine their role as deleterious rhizobacteri a (ORB) or as plant growth promoting rhizobacteria (PGPR). PGPR produce siderophores having greater binding potentials which deprive the ORB of their iron nutrition 3 . Based on this mechanism, the siderophore-producing organisms (mostly fluorescent Pseudomonas) have been used in biological control of plant pathogens as well as for 4 obtaining higher yield from crop plants . In the present study, we have examined siderophore production by 12 rhizobacterial isolates (fluorescent Pseudomonas) obtained from chilli, cotton, groundnut, soybean, characterized their chemical nature and iron binding properties.
Materials and Methods Organisms-Twelve fluorescent
Pseudomonas
isolates used in this study were obtained from the rhizoplanes of 4 crops and grown on King's B mediums. The method of their isolation and identification have been described earlier6 . The organisms were designated as CHRBl, CHRB2, CHRB3 (from chilli); *Correspondent author: Email:
[email protected]
CORB 1, CORB2, CORB3, CORB4 (from cotton); GNRB 1, GNRB2, GNRB3 (from groundnut); and SBRBI, SBRB2 (from soybean). Siderophore production-Four media used were Sand's succinate medium 7 , Mayer & Abdallah medium8, Philson & Llinas medium 9 and Scher & Baker 10 medium • The glasswares were washed with HCI (6 M) to remove traces of iron, while the media were rendered iron free by treating them with 8 hydroxyquinoline dissolved in chloroform as suggested by 11 Messenger and Ratledge • Aliquots (50 mL) of the media were dispensed in conical fla ks (100 mL) and autoclaved at !Sib psi. These were inoculated with 106 cells/mL of fluorescent pseudo monads obtained from 18 hr old cultures grown on King's B mediums at 28°-30°C. Forty-eight hr old cul:ures were centrifuged at 10,000 rpm, and the cell-free supernatants were examined for extracelluLar siderophore production by standard assays, as given in following paragraphs. FeC/3 test 12-To 0.5 mL of cell free culture supernatant was added to 0.5 mL of 2% of aqueous FeCI 3 solution. Appearance of orange or red-brown colour indicated the presence of siderophore. Chrome azurol sulphonate {CAS) assa/ 3--To 0.5 mL of CAS assay solution wa added 0.5 mL of cell free culture supernatant. The change in colour of the blue dye to orange indicated the presence of siderophores. CAS assay solution was prepared as-A 6 mL of hexadecyl trimethylammoniumbromide (10 mM; HDTMA) solution was placed in a volumetric flask (100 mL) and diluted with water. A mixture of 1.5 mL iron (III) solution (1 mM FeCb.6H 20 in 10 mM
YEOLE eta/.: SIDEROPHORE PROUDCTION BY FLUORESCENT PSEUDOMONADS COLONIZING ROOTS
HCI) and 7.5 mL of 2 rnM aqueous CAS dye solution was slowly added under stirring. Anhydrous piperazine (4.307 g) was dissolved in water and 6.25 mL of HCI (12 M) was added to get a buffer solution (pH 5.6). It was added to above volumetric flask made to 100 mL with distilled water to make CAS assay solution. CAS agar plate test 13-Pseudomonas isolates were streaked on CAS agar plates for 48 hr at 30°C. Formation of orange halos around the colonies indicated the presence of siderophores. CAS agar plates were prepared as-CAS (60.5 mg) was dissolved in distilled water (50 mL) and mixed with 10 mL of iron (III) solution (1 mM FeCI 3.6H 20 in 10 rnM HCI) with stirring. This solution was slowly added to 72.9 mg of HDTMA dissolved in 40 mL of water. The resultant dark blue liquid was autoclaved at 1S lb psi. Also, a mixture of 750 mL, H 20; 100 mL, (10xMM9 salts); 15 g, agar; 30.2 g, and pipes, (12.0 g of SO% wlw NaOH solution to raise the pH of pipes to 6.8) were also autoclaved. After cooling to 50°C, 30 mL of casamino acid (10%), the carbon source and supplements like vitamjns and antibiotics (0.2%) after making a sterile solution were added. The dye solution was finally added as sterile solution, along the glass wall, with enough agitation to achieve mixing without generation of foam. Each plate contained 30 mL of blue agar. Spectrophotometric assa/-Cell-free culture supernatants were examined for their absorption maximum in Shimadzu UV-Vis 160 A spectrophotometer. A peak at or near 405 nm indicated the presence of siderophore. Chemical nature of siderophores
Hydroxamate, catecholate, and carboxylate nature of the siderophores was examined by following tests. Hydroxamate nature Neilands' spectrophotometric assay 2-To l mL of cell-free supernatant, was added 1-5 mL of freshly prepared 2% aqueous FeCI 3 solution, and absorbance between 400-600 nm was noted. A peak between 420450 nm, indicated the hydroxamate nature of the siderophores. Tetrazolium salt test 14- To a pinch of tetrazolium salt, was added 1-2 drops of 2 N NaOH and 0.1 mL of the test culture supernatant. Instant appearance of a red to deep-red colour indicated the presence of hydroxamate siderophores.
465
Catecholate nature Neilands' spectrophotometric assa/-Formation of wine-coloured complex by addition of 1-5 mL of freshly prepared 2% aqueous FeCI 3 to 1mL of the test sample, that absorbed maximally at 495 nm, indicated catecholate nature of siderophores. Arnow's test 15-To 1 mL of cell-free culture supernatant, was added 1 mL of 0.5 N HCI, 1 mL of nitrite molybdate reagent and 1 mL of 1 N NaOH, followed by distilled water to make the volume to S mL. Absorbance was read at 500 nm using 2, 3 dihydrox ybenzoic acid as standard. Carboxylate nature Vogel's chemical test 16-To 3 drops of 2 N NaOH, was added 1 drop of phenolphthalein and then water was added until light pink colour developed. Disapearance of pink colour on addition of test sample, indicated carboxylate nature of siderophores. Spectrophotometric assay 17-To 1 mL of cell free culture supernatant was added 1 mL of 250 11M CuS0 4 and 2 mL of acetate buffer (pH 4). Copper complex was observed for absorption maximum between 190-280 nm. There is no specific wavelength for absorption of copper complex and, thus, the entire wavelength (190-280 nm) was scanned to observe the peak of absorption of siderophore. Measurement of siderophore concentration 18Standard curves were prepared for absorbance (630 nm) of desferroxamine mesylate (DFOM; standard for hydroxamate siderophores) divided by the absorbance (630 nm) of the reference solution as a function of siderophore concentration (20-100 ~.that yielded a linear relationship. Reference solution contained all the ingredients except the siderophore. Cell-free culture filtrate (0.5 mL) wa~ added to 0.5 mL of CAS assay solution and NArer was measured at 630 nm. Siderophore concentration was calculated from the standard curve. Determination of mono-, di-, and trihydroxamate nature of siderophores
Spectrophotometric method12-pH dependent absorption maxima of ferrate hydroxamate siderophores have been used to distinguish ferric complexes of mono-, di- and trihydroxamates. Ferric complexes were examined spectrophotometrically for a shift in Arnax (nm) at different pH. Little (3-8 nm) or no shift when pH of the growth medium varied from 4 to 7 indicated trihydroxamate nature, while a wide shift (up to 80 nm) indicated dihydroxamate nature. Mono
INDIAN J EXP BIOL, MAY 200 I
466
hydroxamate siderophores showed a shift (500-520 nm) when pH dropped to 4. Electrophoretic method 12-Paper electrophoresis was performed with a flat-bed device, with 4% formic acid at pH 2. Cell free supernatants of 48 hr old cultures, grown at 30°C, were spotted on Whatman No. 3 paper. The electrophoresis was run at approximately 30 Ycm· 1 for 1-2 hr. The paper was dried to remove traces of formic acid and sprayed on both sides with 2% aqueous FeCJ 3 solution. Ferric monohydroxamate and dihydroxamate complexes formed deep-purple and pink-purple colours, respectively, while ferric trihydroxamates gave red colour reaction. 19 Binding properties of hydroxamate siderophores -Binding property i.e., the number of bonds the ligand formed with metal ions was detected by stable/unstable nature of colour of ferrate siderophores at different pH. It is stable (red) over a wide pH range for hexadentates. Tetra-, and bidentate siderophores
are unstable and show a change in colour from red to pink-purple or deep-purple colour respectively.
Results and Discussion Results of siderophore productio by twelve fluorescent Pseudomonas isolates on 4 media (Table 1) suggested that succinate medium supported siderophore production by all the test isolates as evidenced by positive FeCI 3 test, CAS assay, CAS agar plate test and spectrophotometric assay. Only 3 organisms (CORB 1, GNRB2 and SBRB2), produced siderophores on all the media, while other 3 (CHRB 1, GNRB I and GNRB3) produced siderophores on 3 media (except Scher and Baker medium). These six organisms (CHRB 1, CORB 1, GNRB 1, GNRB2, GNRB3 and SBRB2) were examined for detailed studies related to siderophore production on succinate medium which emerged as the most preferred medium and supported siderophore production by all the isolates.
Table 1-Siderophore production by 12 Pseudomonas isolates in four media after 48 hr of growth Pseudomonas isolates
CHRBI
Media
FeC1 3 test
CAS assay
CAS agar plate test
I
+ + +
+ + +
+
408 406 404
CORB4
+ +
+
+
+
406
GNRBI
2 3 4 CHRB2
I
Spectra- Pseudomonas Photo- isolates metric assay Amax (nm)
4 I
+
+
+
407
GNRB2
4
CORB3
+
405
SBRBI
+ + + +
I 2 3 4
+
+
+
+
+
+
I 2 3 4
+
+
+
405
3 4
CORB2
GNRB3
+
406 406 407 405
+ +
+ + + +
I
2
CAS assay
CAS agar plate test
SpectraPhotometric assay Amax (nm)
I 2
+
+
+
403
I 2
+ +
+
3 4
+
+
+ + +
404 404 403
I
+ + + +
+ +
402 405 406
+
+ + + + + + +
402 405 406
2 3 4
2 3 COREl
FeC1 3 test
3 4
2 3
CHRB3
Media
+
+
+
+
I 2
+
3 4
+
+ + +
I
+
+
+
404
+ + +
+ +
+ +
+
+
+
+
+
407 404 405 407
+
2 3 4 SBRB2
I
2 3 4
Media taken were-(1}-Succinate medium; (2}-Mayer & Abdallah; (3}-Philson& Llinas; and (4}-Scher & Bake_r_ _ _ _ _.
467
YEOLE eta/.: SIDEROPHORE PROUDCTION BY FLUORESCENT PSEUDOMONADS COLONIZING ROOTS
Results of the tests for the chemical nature of siderophores (Table 2) suggested that the siderophores produced by all the six Pseudomonas isolates were of hydroxamate type, as indicated by positive Neilands' spectrophotometric assay and tetrazolium salt test. Pyoverdine nature of hydroxamate nature was evident by their absorption maximum between 407-412 nm (Table 1). These results corroborate the findings of Messenger and Ratledge'' who have reported hydroxamate nature for pyoverdine siderophores. Mayer and Abdallah 8 have reported production of mixed type of siderophores (hydroxamate and catecholate) by fluorescent Pseudomonas isolates. Among the 6 isolates, CHRB I was the highest siderophore producer (21.7xl0 4 g/mL), and SBRB2 the least (16.8xl0-4 g/mL). According to the amount of siderophores pro-
duced, the organisms could be arranged in a sequence as-CHRBl > CORBl > GNRBI > GNRB2 = GNRB3 > SBRB2. Difference in the quantity in siderophore produced is a logical happening, and several reports have indicated such variations 20 , which changes with time, space and environment in which the organism operates. Results of detection of mono-, di- and trihydroxamate nature of siderophores produced by three potent isolates CHRBl, COREl and GNRBI and (Table 3), indicated that the siderophores were trihydroxamates as they showed a shift in Amax (3-7 nm) at different pH. Dihydroxamate with a broad shift in Amax (6-80 nm) and monohydroxamate (500-520 nm at pH 4) could not be detected. Trihydroxamate nature is
Table 2-Detection of chemical nature (hydroxamate, catechol ate, carboxylate) of siderophores of 6 fluorescent Pseudomonas isolates Pseudomonas isolates
Hydroxamate Neilands' spectroTetrazolium photometric salt test assay
Carboxylate SpectraVogel's test photometric assay
+ + + + + +
420 425 430 440 428 445
CHRBl CORSI GNRBI GNRB2 GNRB3 SBRB2
Catechol ate Neilands' spectroArnow's photometric test assay
Siderophore concentration xJ0-4 g/mL 80.7 21.7 19.2 18.2 18.2 16.8
Table 3-Determination of mono-, di-, and trihydroxamate nature of siderophores by spectrophotometric and electrophoretic methods and their binding properties Pseudomonas isolates
"-max
pH CHRBI
CORBI
GNRBI
(nm) of ferrate
sidero~hores
"-max
(nm) shift
Amax
(nm)
4 5 6 7 8 9
420 420 421 426 427 420
7
4
421 421 425 425 426 426
5
5 6 7 8 9 4 5 6 7 8 9
420 421 422 425 427 427
7
Colour of ferrate siderophores in electrophoresis
Inference
Colour of ferrate hydroxamates
Red
Trihydroxamate
Red Red Red Deep blue Deep blue Deep blue
Red
Reddish brown
Trihydroxamate
Trihydroxamate
Red Red Red Deep blue Deep blue Deep blue Red Red Red Deep blue Deep blue Dee blue
Binding properities
Bidentate
Bidentate
Bidentate
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INDIAN 1 EXP BIOL, MAY 2001
known to be more common, which by virtue of presence of 2 atoms of 0 2 in each hydroxamic acid allows formation of hexadentate ligand, as each Oz forms a bidentate ligand 21 • This is a common property among the hydroxamates. Results of electrophoretic mobilities of hydroxamate siderophores with 4% formic acid at pH 2, also suggested the trihydroxamate nature as evidenced by formation of reddish-brown colour, on spraying with 2% aqueous FeCh solution. In tests for ligand properties i.e., stable/unstable nature of coloured ferrate siderophores at different pH (Table 3), the siderophores of 3 Pseudomonas isolates CHRB 1, CORB I and GNRB 1 formed red to deep blue colour at pH range 4-9, indicating their bidentate nature. If the number of hydroxamates and ligands produced was correlated, it was found all trihydroxamates formed hexadentate ligands, but the present siderophores, which were trihydroxamates showed bidentate ligands. This was an important observation which was difficult to interpret, as the available literature does not provide any explanation.
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