Fofia Microbiol. 44 (2), 196-200 (1999)
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Growth and Siderophore Production in Bradyrhizobium (Lupin) Strains under Iron Limitation ~I.H. ABD-ALLA* Department of Botany, Faculty of Science, Assiut University,Assiut 71M6, Egypt Received September 8~ 1998 Revised version October 23, 1998 ABffI~CT. Six Bradyrhizobium (lupin) strains were evaluated for their ability to produce siderophores using four chemical assays. Two strains gave positive reactions with chrome azurol S assay (CAS) and produced hydroxamate-type siderophores. The other four strains gave negative results for siderophore production using the four assays. Generation time, growth yield and hydroxamate production of one strain (WPBS 3201 D) were affected by the iron concentration of the culture medium and the previous culture history of the cells. Resuspension of washed ceils grown previously in media supplemented with 0 and 20 IJmol/L Fe into differing iron regimes (0, 0_5, 1, 2, 4, 8, 10, 15 and 20 lamol/L Fe) suggest that the extent of hydroxamate production depended on the growth history of the cells. Cells pregrown in 20 lamol/L Fe produced a high amount of hydroxamares compared with cells pregrown in iron-free medium when resuspended in medium containing up to 4 Izmol/L Fe. Cells pregrown in 20 lamol/L Fe were more sensitive to iron repression than those pregrown in 0.5 lamoi/L Fe. Mannitol was the best carbon source for siderophore production. Siderophore synthesiswas inhibited by 4-chloromercuribenzenesuifonieacid, 2,4-dinitrophenol, sodium azide and MgCI2 suggesting that an energized membrane and a mercapto group are essential and required for hydroxamate synthesis in strain WPB5 3201 D. Iron deficiency in nodulated legumes is very common on alkaline soils and affects such comm o n agricultural crops as chick pea (Rai et al. I982), French bean (Hemantaranjan and Garg i986) and peanut ( O ' H a r a et aL 1988). Although nodule initiation may occur normally in peanut ( O ' H a r a et aL ~988) it is drastically curtailed in lupins under iron deficiency (Tang et al. I99o). There is, however, iittle evidence that iron deficiency in soil actually decreases the number of root-nodule bacteria, imp136ng either that these organisms have lower demands for iron during normal growth and survival than the plant, or that they have other mechanisms for acquiring iron under deficiency conditions. Iron is essential for the growth of living organisms and is a common element in the rhizosphere. However, in oxlc environments, iron is readily oxidized to Fe 3+ and forms insoluble ferric hydroxides which decrease its biological availability. Faced with iron deficiency, many Gram-negative bacteria have developed high-affinity iron-uptake mechanisms. These organisms produce and excrete high-affinity ferric chelators termed siderophores and possess m e m b r a n e transport systems to take up the siderophore-iron complex (Neilands I982). It has been suggested that the excretion of siderophores by rhizosphere bacteria may stimulate plant growth by improving the Fe nutrition of the plant (Crowley et aL I987). Iron is required to support the process of nitrogen fixation: the key enzymes, dinitrogenase and dinitrogen reductase, contain about 38 molecules of iron per enzyme complex. The need for reductase activity during N2 fixation increases demand for the iron-rich components of the electron transport chain. Leghemoglobins, produced to control 02 tension, are also iron-containing compounds. These factors combine to increase an organism's demand for iron under nitrogen-fixing conditions. Siderophore-mediated iron uptake has been reported for several root-nodule bacteria (Guerinot I99I). Phenolate siderophores have been reported in iron-limited, but not iron-replete, cultures of Rhizobium leguminosamrn, R. trifolii and cowpea Rhizobhtm (Modi et aL I985; Rioux et aL i986). R. leguminosantm may also produce a hydroxamate siderophore under iron-limiting conditions (Carson et aL x992). R. meliloti produces a rhizobactin which lacks both catechol and hydroxarnate functional groups (Smith and Neilands I984). Bradyrhizobium ]aponicurn utilize citric a d d as a siderophore (Guerinot et aL I99o). So far, there is no report on siderophores from Bradyrhizobium ([upin) strains. In this investigation six strains of Bradyrhizobiurn (lupin) strains were examined for their ability to grow and produce siderophores under iron-limiting conditions.
*Current mailingaddress: Institute of Plant Nutrition, Justus-Liebig University, SiManlaye 6, 35390 Gieflen, Germany. fax + 0641 99 391 69 e-mail
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1999
SIDEROPHORE PRODUCTION IN Bradyrhizobium STRAINS 197
M A T E R I A L S AND METHODS
Organisms. Bradyrhizobium (lupin) strains USDA 3040, 3041, 3042 were obtained from Plant Physiology and Genetic Research Unit, U.S. Department of Agricultural Research Service (331 Edward Madigan Laboratory, Urbana, IL, USA). Strain CB 2272 was obtained from Cunningham Laboratory, St. Lucia (Queensland, Australia) and strains WPBS 3201 D and 3211 D were obtained from WPBS
Rhizobium collection, AFRO Institute of Grassland and Environmental Research, Dfyed (Pals Gogerddan, Aberystwyth, Great Britain). Glassware preparation. All glassware used was cleaned in 20 % HC1 to remove iron and rinsed in deionized water. Media and culture. The minimal salts medium (MSM) of Brown and Dilworth (1975) contained (4 retool/L) NH4CI as nitrogen source and phosphate at 0.3 mmol/L. The carbon source was 20 mmol/L mannitol. Iron was added as 0 or 20 gmol/L FeC13. The pH was adjusted to 6.9 before autoclaving. Flasks containing 25 mL medium supplemented with the appropriate iron concentration and incubated in an orbital shaker at 1.8 Hz and 28 ~ Cultures were harvested after 5 d and centrifuged at 67 Hz for 10 rain. The pellets were resuspended in iron-free MSM and centrifuged as before. The washed cells were then resuspended in MSM of the required iron concentration (0, 0.5, 1, 2, 4, 8, 10, 15 and 20 gmol/L Fe). Cell concentration was measured at 600 nm using Spectronic 2000 (Bausch & Lomb). Protein was assayed according to Lowry. Siderophore assay. Catechol-type phenolates were assayed on ethyl acetate extracts of the culture supernatant of Bradyrhizobium (lupin) strains using a modification of the ferric chloride-tripotasslum hexacyanoferrate reagent of Hathway (Reeves et al. I983). Ethyl acetate extracts were prepared by extracting 20 mL of supernatant twice with an equal volume of solvent at pH 2. Hathway's reagent was prepared by adding 1 mL of 100 mmol/L ferric chloride in 0.1 mol/L HCI to 100 mL of distilled water, and to this was then added 1 mL 100 mmol/L tripotassium hexacyanoferrate (Reeves et aL I983). For the assay, one volume of the reagent was added to one volume of sample and absorbance was determined at 560 nm for salicylates with sodium salicylate as standard and at 700 nm for dihydroxy phenols with 2,3-dihydroxybenzoic acid as a standard. Hydroxamate-type siderophores were determined by the method of Gibson and Magrath (I969). To 0.5 mL of culture supernatant was added 0.5 mL 6 mol/L H2SO4 and the mixture was autoclaved in a glass tube. One mL of 1 % sulfanilic acid (W/V) in .30 % acetic acid (V/V) and 0.5 mL of .3 % iodine was destroyed by addition of I mL of 2 % (W/V) Na3AsO4 solution. A I mL solution of i-naphthylamine (0.3 % in 30 % acetic acid) was then added and the total volume made up to 10 mL with distilled water. After 30 min, absorbance at 526 nm was measured. Hydroxylamine hydrochloride was used as standard and 1.0 ~tmol/L of the compound gave an absorbance of 0.1. Chrome azurol S (CAS) agar medium was prepared by the method of Alexander and Zuberer ('~99x). CAS agar was prepared from four solutions which were sterilized separately before mixing, q-he F e - C A S indicator solution (solution 1) was prepared by mixing 10 mL of 1 mmol/L FeCIy6H20 (in 10 mmol/L HC1) with 50 mL of an aqueous solution of CAS (1.21 g/L). The resulting dark purple mixture was added slowly, with constant stirring, to 40 mL of an aqueous solution of hexadecyltrimethylammonium bromide (1.821 g/L). This yielded a dark blue solution which was autoclaved, then cooled to 50 ~ All of the reagents in the indicator solution were freshly prepared for each batch CAS agaro The buffer solution (solution2) was prepared by dissoMng 30.24g of piperazineN,N-bis(2-ethanesulfonic acid) (PIPES) in 750 mL of salt solution containing 0.3 g K2HPO4, 0.5 g NaCl and Io0 g NH4C1. The pH was adjusted to 6.8 with 50 % (W/V) KOH and water was added to bring the volume to 800 mL. The solution was autoclaved after adding 15 g of agar, then cooled to 50 ~ Solution 3 contained the following (in 70 mL water): 2 g glucose, 2 g mannitol, 493 mg MgSO4"7H20, 11 nag CaC12, 1.17 rng MnSO4"2H20, 1.4 mg H3BO3, 40gg CuSO4"5H20, 1.2 mg ZnSO4"TH20, 1.0 mg Na2MoO4-2H20~ Solution 3 was autoclaved, cooled to 50 ~ then added to the buffer solution along with 30 mL filter-sterilized 10 % (W/V) casamino acids (solution 4). The indicator solution was added last, with sufficient stirring to mix the ingredients without forming bubbles. A yellow halo surrounding a bacterial colony indicated a positive CAS reaction. Free citric acid was determined enzymically using the citrate-malate dehydrogenase assay and following the disappearance of NADH at 340 nm (Moellering and Grubcr 1966). The effects of different carbor~ sources (mannitol, glucose, succinate, citrate and fumarate) on siderophore production
198 M.H.ABD-ALLA
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were investigated. Also the effects of a number of potential inhibitors on siderophore production were also monitored, 4-chloromercuribenzenesulfonic acid (PCMBS), 2,4-dinitrophenol (2,4-D), carbony! cyanide 3-chlorophenylhydrazone (CCCP), sodium azide and HgC12.
RESULTS AND DISCUSSION
Production of siderophores. Among the six strains of Bradyrhizobium (lupin) the WPBS 3201 D and WPBS 3211 D gave a strong reaction in the CAS assay; the first of these reacted strongly, the second moderately in the hydroxamate assay. No reaction Was observed in the phenolate and citrate assay. The other four strains gave negative results for siderophore production using all four assays. Effect of iron on growth and siderophoreproduction. Generation time, growth yield and hydroxamate production of strain WBPS 3201 D were affected by the iron concentration of the culture medium and the previous culture history of the ceils. "l-hose that had been grown in iron-free medium and then in media containing different concentrations of iron, showed marked differences in mean generation time. The mean generation time of cells grown in the iron-free medium was 12 h compared with 3 2 h for cells grown in medium supplemented with 20 ~tmol/L. In contrast, cells that had been grown previously in a medium containing 20 vtmol/L Fe and then resuspended in media containing Fe concentrations over the range of 0 - 2 0 Ixmol/L showed only small differences in the mean generation time (Table I). However, there were marked differences in growth yield; they were 5-fold higher in a n~edium containing 20 ~tmol/L than in the iron-free medium. Generally, growth yield increased with added iron for cells pregrown in iron-free medium or a medium containing 20 lamol/L Fe, but were higher for cells grown previously in 20 ~tmol/L Fe. Cells pregrown in excess iron are able to maintain a rapid growth rate when resuspended in a medium without iron. Cells pregrown in 20 ~tmol/L Fe produced a high amount of hydroxamates compared with cells pregrown in iron-free medium. Strain WPBS 3201 D synthesizes a hydroxamate when resuspended in a medium containing iron up to 4 or 2 lamol/L Fe for cells pregrown in 0 or 20 ~tmol/L Fe, respectively. Increased iron concentration above 4 p m o l / L resulted in a complete inhibition of hydroxamate synthesis (Table I). When cells of WPBS 3201 D pregrowm in a medium containing 0.5 p m o l / L Fe were resuspended in a Fe-free medium, hydroxamate production was detected within 1 h. Addition of 10 p m o l / L Fe (final concentration) to cells actively producing hydroxamate caused no change in the rate of hydroxamate synthesis and no difference between the control culture and those treated with 10 ~tmol/L Fe at 5 h (Fig. 1). However, in cells previously grown in a medium supplemented with 20 9 m o l / L Fe and resuspended in an iron-free medium, hydroxamate synthesis was repressed for 4 h. This lag may reflect utilization of small repressive amounts of iron. When 10 I.tmol/L Fe was added at 5 h to these cells, the rate of hydroxamate synthesis was repressed after 2 h (Fig. 1). Table I. Effect of the previous culture history of Bradyrhizobiumstrain WPBS 3201 Dt on generation time (GT), yield of dry matter (DM) and hydroxamateproduction (HP) when the cells grown in MSM with different iron concentration Cells pregrown on MSM of 0 pmol/L Fe
Added Fe ~tmol/L GT, h 0 0.5 1 2 4 8 10 15 20
12 t0 9.2 8.2 6.4 5.3 4.2 3.5 3.2
DM, g/L HP, pmol/L* 0.04 0.13 0.24 0.50 0.59 0.61 0.69 0.72 0.74
8.5 8.4 8.1 6.3 1.2 0 0 0 0
20 lamol/LFe HP/DM
GT, h
212 65 34 12.6 2.0 0 0 0 0
4.6 4.5 4.2 4.4 3.8 3.5 3.2 3.2 3.1
*Grown in iron-free MSM or MSM containing20 lamol/Liron.
DM, g/L HP, pmol/L* tIP/DM
*Per mg protein.
0.18 0.25 0.46 0.62 0.69 0.73 0.79 0.82 0.89
15.5 10.8 8.8 6.9 0 0 0 0 0
86 43 19.1 11.1 0 0 0 0 0
S I D E R O P H O R E P R O D U C T I O N IN Bradyrhizobhtm STRAINS
1999
5
I
199
I
nrnol /mg 2
1
Fig. 1. Hydroxamate production (nmol/mg protein of
Bradyrhizobium (lupin) strains WPBS 3201 D grown in 0.5 lamol/L Fe (1) and 20 lamol/L Fe (2) and resuspended in iron-free medium. Iron (10 lamol/L Fe final concentration) was added at 5 h and hydroxamate was measured in cells pregrown in 20 ~tmol/L Fe (3). /,
8
12
h
It is apparent that it is not just the external iron concentration that regulates the derepression of siderophore synthesis in this strain. In iron-deficient cells hydroxamate production appears to be less sensitive to iron repression than when cells were pregrown in excess-iron. An explanation for these observations may be that the cell has a mechanism that attempts to maintain minimum intracellular pools of iron. The regulation of siderophore production may involve 'immediately available-iron' and 'storage-iron' components. Siderophore production would be repressed when both components are present in the cell in excess. In cells grown in 20 Ixmol/L Fe which have a high total cell iron, when transferred to zero iron, the 'immediately available-iron' would be depleted and siderophore biosynthes is derepressed. The addition of 10/amol/L Fe to such ceils may result in repression of siderophore synthesis after 2 h. This could be attributed to the presence of 'immediately-available iron' and 'storageiron' in repressive amounts. However, cells grown in 0.5 ~tmol/L Fe and resuspended in zero iron contain low levels of both iron components. Addition of 10 ~tmol/L Fe to those cells would maintain siderophore production without any depression (Fig. 1). Carrillo-Castaned and Peralta 0988) reported that 19 out of 52 strains of R. phaseoli showed siderophore-like activity when bioassayed against Xanthomonas campestris and Pseudomonas syringae. However, further testing to verify the production of a siderophore by chemical assay was not reported. Gueriont et al. 0990) reported that one out of 20 strains of B. japonicum produced one type of siderophore using five different assays and two different growth conditions. Carson et aL (I992) reported that R. leguminosamm excretes a hydroxamate siderophore when grown in a low-iron medium. Effect of carbon source on siderophore production. Hydroxamate synthesis was affected by the carbon Table II. Effect of carbon source on siderophore source in the growth medium. Mannitol was the best production in Bradyrtu'zobium strain WPBS 3201 D source for siderophore production. However, cells grown on fumarate, succinate or malate and citrate Carbon source tlydroxamate produced significantly less hydroxamate (Table II). The production t amount and the type of siderophore produced by an organism depends on the availability of organic and inMannitol 15.9 organic nutrients (Neilands I982). Glucose 11.3 Effect of metabolic inhibitors on siderophore Fumarate 5.3 production. Siderophore production was inhibited by Succinate 4.2 Malate 4.1 PCMBS, 2,4-D, CCCP, sodium azide and HgCI2 Citrate 1.2 (Table III). The result of this study indicates that an energized membrane and a mercapto group are essentlamol/L per mg protein. tial and required for hydroxamate synthesis.
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M.H. ABD-ALLA
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Table III. Effect of some metabolic inhibitor agents on sideroNkrogenase is a key enzyme in biophore production logical nitrogen fixation. Nitrogenase is made up of two proteins, both rich in iron and essential for activity. Nonheme-iron Concentration Hydroxamate Compound in growth medium production electron-transfer proteins, such as ferredoxmmol/L % of control in and flavodoxin, are essential in nitrogen fixation (Eady and Postgate I974). TherePCMBS 0.1 24 fore, the possible role of siderophores in 0.2 16 delivering physiologically active iron in bio2,4-D 2 38 logical nitrogen fixation can be further ex4 25 plored. Moreover, the Bradyrhizobium and CCCP 0.1 23 Rhizobium siderophore may be involved in 0.2 12 HgCI2 1 26 the biosynthesis of leghemoglobin and iron2 16 containing enzymes, such as hydrogenase, Sodium azide 2 59 peroxidase and catalase. High levels of cata4 30 lase have been reported in the effective nodules of some legumes (Francis and Alexander I972). The ecological advantage of the synthesis of siderophore may enable Bradyrhizobhtrn and Rhizobium to compete with other species for iron uptake.
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