K. C. Carson, S. Holliday, A. R. Glenn, and M. J. Dilworth. Nitrogen Fixation Research Group, School of Environmental and Life Sciences, Murdoch University, ...
Arch Microbiol (1992) 157:264-271
Archives of
Micrnbiology
© Springer-Verlag 1992
Siderophore and organic acid production in root nodule bacteria K. C. Carson, S. Holliday, A. R. Glenn, and M. J. Dilworth Nitrogen Fixation Research Group, School of Environmental and Life Sciences, Murdoch University, Murdoch, Western Australia 6150 Received May 31, 1991/Accepted October 30, 1991
Abstract. Nineteen strains of root nodule bacteria were grown under various iron regimes (0.1, 1.0 and 20 gM added iron) and tested for catechol and hydroxamate siderophore production and the excretion of malate and citrate. The growth response of the strains to iron differed markedly. For 12 strains (Bradyrhizobium strains NC92B and 32H 1, B. japonicum USDA 110 and CB 1809, B. lupini WU8, cowpea Rhizobium NGR234, Rhizobium meliloti strains U45 and CC169, Rhizobium leguminosarum bv viciae WU235 and Rhizobium leguminosarum bv trifolii strains TA1, T1 and WU95) the mean generation time showed no variation with the 200-fold increase in iron concentration. In contrast, in Bradyrhizobium strains NC921, CB756 and TALl000, B.japonicum strain 61A76 and R. leguminosarum bv viciae MNF300 there was a 2 - 5 fold decrease in growth rate at low iron. R. meliloti strains WSM419 and WSM540 showed decreased growth at high iron. All strains of root nodule bacteria tested gave a positive CAS (chrome azurol S) assay for siderophore production. No catechol-type siderophores were found in any strain, and only R. leguminosarum bv trifolii T1 and bv viciae WU235 produced hydroxamate under low iron (0.1 and 1.0 gM added iron). Malate was excreted by all strains grown under all iron regimes. Citrate was excreted by B. japonicum USDA110 and B. lupini WU8 in all iron concentrations, while Bradyrhizobium TALl000, R. leguminosarum bv viciae MNF300 and B.japonicum 61A76 only produced citrate under low iron (0.1 and/or 1.0 gM added iron) during the stationary phase of growth. Key words: Siderophore - Organic acids - Hydroxamate - Rhizobium
Two thirds of the nitrogen input into the world's soils is accomplished by biological processes (Postgate 1982), Offprint requests to: K. C. Carson Abbreviations." CAS, chrome azurol S; HDTMA, hexadecyltrime-
thylammonium bromide
among which the symbiotic association between root nodule bacteria and their respective host legumes is especially important. This relationship is iron dependent, since iron is required both for the manufacture of key proteins such as the nitrogenase complex and leghaemoglobin and for nodule formation. In Lupinus angustifolius L. cv Yandee inoculated with Bradyrhizobium lupini and grown in solution culture, it was found that the numbers of nodule initials were depressed when the iron concentration was low, but once initiation had occurred nodules developed normally (Tang et al. 1990a). In contrast, iron deficiency in peanuts results in arrested nodule development with fewer bacteroids in nodules, decreased amounts of leghaemoglobin and decreased nitrogenase activity (O'Hara et al. 1988a). An adequate supply of iron is therefore essential for the establishment and function of the nodule symbiosis. Effective nodulation may also be dependent upon the ability of the infecting rhizobia to obtain iron from their environment. Once infection has occurred all the nutritional requirements of the root nodule bacterium, including iron, are supplied by the host plant. While iron is not mobile in lupins, (foliar iron application has no effect on nodulation (Tang et al 1990b)), peanuts translocate foliar iron applied to iron deficient plants allowing nodule development to proceed once initiation has occurred (O'Hara et al. 1988a). How iron is supplied to the infecting bacterium is not understood, but it has been suggested that differences in nodule development under iron deficient conditions may be due to varying abilities of different strains of root nodule bacteria to acquire iron for nodule initiation and development (O'Hara et al. 1988b). Siderophores are known to have a high affinity for ferric iron (Neilands 1981) and siderophore production has been reported in various species of root nodule bacteria (Guerinot 1991). Such strains differ in their abilities to produce siderophores, and it is thought that this may be related to the effectiveness of strains in the establishment of symbiosis with their host plants (Nambiar and Sivaramakrishnan 1987). Rhizobium meliloti DM4 produces rhizobactin (Smith et al 1985) and R. meliloti 1021 another type of rhizobac-
265 tin, r h i z o b a c t i n
1021
(Schwyn and Neilands
1987).
Rhizobium leguminosarum bv. viciae (hereafter referred to as R. leguminosarum) I A R I 102 (Patel et al. 1988), c o w p e a Rhizobium R A - 1 ( M o d i et al. 1985) a n d Bradyrhizobium strains N C 9 2 a n d 5a/70 ( N a m b i a r a n d S i v a r a m a k r i s h n a n 1987) a n d a n o n - n o d u l a t i n g strain R. leguminosarurn b v trifolii (hereafter referred to as R. trifolii) A R 6 (Skor u p s k a et al. 1989) are all r e p o r t e d to p r o d u c e c a t e c h o l type siderophores. No hydroxamate-type siderophores have been r e p o r t e d f r o m a n y r o o t n o d u l e b a c t e r i a . O r g a n i c acids such as c i t r a t e a n d a n t h r a n i l a t e have also been r e p o r t e d to act as s i d e r o p h o r e s in Bradyrhizobium japonicum strain 61A152 (Bosch et al. 1988) a n d R. leguminosarum ( R i o u x et al. 1986 a, b), respectively. S i d e r o p h o r e p r o d u c t i o n , identified o n l y b y the c h r o m e a z u r o l m e t h o d (CAS), h a s been r e p o r t e d in o t h e r strains o f R. meliloti, R. leguminosarum a n d R. trifolii ( S c h w y n a n d N e i l a n d s 1987; R e i g h a n d O ' C o n n e l l 1988; A m e s - G o t t fred et al. 1989). In this w o r k 19 strains o f r o o t n o d u l e b a c t e r i a were screened for s i d e r o p h o r e p r o d u c t i o n a n d e x c r e t i o n o f o r g a n i c acids in a n a t t e m p t to b e t t e r u n d e r s t a n d h o w free-living r h i z o b i a o b t a i n i r o n f r o m their e n v i r o n m e n t .
Bacterial strains Bacterial strains used are listed in Table 1.
Media and growth conditions All strains were grown in the minimal salts medium (MSM) of Brown and Dilworth (1975) with 0.01% Difco yeast extract (Fe assay 7 gg g 1), NH4C1 (10 raM) as nitrogen source and phosphate at 0.3 raM. Carbon was supplied as 10 mM arabinose plus 10 mM succinate; the medium was maintained at pH 6.5 with 20mM HEPES. Iron was added as FeC13 in HC1 to the required level; this did not affect the pH. To remove contaminating iron from the buffer, carbon sources and other salts in the MSM, stock solutions of HEPES, succinate and arabinose (all at 1 M), and salts solution (MgSO 4 . 7 H20 2.5 g, NaC1 2.0 g, CaC12 • 2 H20 0.2 g and K2HPO4 0.5 g in 500 ml) were passed through a CPG/8-hydroxyquinoline column (Smith and Neilands 1984). The hydroxyquinoline was obtained from Pierce Chemical Co. (Ill., USA).
Cell density Cell density was measured at 600 nm using a Beckman DU-64 spectrophotometer.
Protein assay Protein was assayed using the Coomassie Brillant Blue microprotein method of Bradford (1976) using serum albumin as a standard.
Methods
Glassware preparation Siderophore assays All glassware used was cleaned in 16% HC1 to remove residuaI iron and rinsed in deionised water from a reverse osmosis desalinator from Osmotron (Australia) Ltd. This quality deionised water was used in all growth media and in the preparation of all reagents. Table 1. Strains of root nodule bacteria used in this study
Catechol-type siderophores were measured using a modification of the method of Arnow (1937). A four-fold increase in sensitivity was achieved by using 0.25 ml 2 M HCI, 0.5 mI nitrite-molybdate
Bacteria
Source
Bradyrhizobium sp. NC92B Bradyrhizobium sp. NC92I Bradyrhizobium sp. TALl000 Bradyrhizobium sp. 32H1 Bradyrhizobium sp. CB756 Bradyrhizobium japonicum USDA110 Bradyrhizobium japonicum CB 1809 Bradyrhizobium japonicum 61 A76 Bradyrhizobium lupini WU8
BNF Resource Centre, Bangkok. Thailand ICRISAT BNF Resource Centre, Bangkok. Thailand Nitragin Co., Milwaukee, Wisconsin, USA CSIRO, Brisbane, Queensland. Australia BNF Resource Centre, Bangkok. Thailand BNF Resource Centre, Bangkok, Thailand Nitragin Co., Milwaukee, Wisconsin, USA School of Agriculture, University of W.A., Perth, Western Australia John Innes Research Institute, Norwich, UK School of Agriculture, University of W.A., Perth, Western Australia CSIRO, Canberra, A.C.T., Australia
Rhizobium leguminosarum MNF300 Rhizobium leguminosarum WU235 Rhizobium leguminosarum biovar trifolii T1 Rhizobium Ieguminosarum biovar trifolii TA1 Rhizobium leguminosarum biovar trifolii WU95 Rhizobium meliloti WSM419 Rhizobium meliloti WSM540 Rhizobium meliloti CC169 Rhizobium meliloti U45 Rhizobium (cowpea) sp. NGR234
Department of Agriculture, Perth, Western Australia School of Agriculture, University of W.A., Perth, Western Australia Department of Agriculture, Perth, Western Australia Department of Agriculture, Perth, Western Australia Department of Agriculture, Perth, Western Australia Department of Agriculture, Perth, Western Australia CSIRO, Canberra, A.C.T., Australia
266 reagent (20 g sodium nitrite and 20 g sodium molybdate per 100 ml) with 4.0 ml of supernatant. Hydroxamate-type siderophores were determined by the Csaky method (Gillam et al. 1981) and also using ferric perchlorate in dilute acid (Atkin et al. 1970). Half a ml of 10 mM ferric perchlorate in 0.2 M perchloric acid was added to each 1.0 ml of supernatant, and the absorbance at 450 nm measured after 10 rain. Desferal was used as a standard to quantify the assay with a 1.0-mM solution giving an absorbance of 1.0 at 450 nm.
C A S top-layer technique Root nodule bacteria were grown on MSM agar containing 1.0 g - 1 ~ yeast extract, then overlaid with 10.0 ml of 0.7% agar in 20 mM MES pH 6.5 to which 2.0 ml of CAS reagent (Schwyn and Neilands 1987) was added. Within 24 h a yellow halo surrounded the bacterial colony if the strain was CAS positive.
U S D A 1 1 0 , B. lupini W U 8 , c o w p e a Rhizobium N G R 2 3 4 , R. meliloti U 4 5 a n d CC169, R. leguminosarum W U 2 3 5 a n d R. trifolii T A 1, T 1 a n d W U 9 5 ) s h o w e d little v a r i a t i o n in m e a n g e n e r a t i o n time w i t h a 200-fold increase in a d d e d i r o n c o n c e n t r a t i o n in the m e d i u m (0.1 to 20 laM) (Fig. 1, T a b l e 2 ) . H o w e v e r , others (Bradyrhizobium N C 9 2 I , CB756 a n d T A L l 0 0 0 , B.japonicum 61A76 a n d R. leguminosarum M N F 3 0 0 ) s h o w e d a m a r k e d decrease in m e a n g e n e r a t i o n time, s o m e t i m e s as g r e a t as 2 - 5 fold, as the a d d e d i r o n c o n c e n t r a t i o n i n cr eased f r o m 0.1 to 20 g M ) (Fig. 1, T a b l e 2). In c o n t r a s t to all o t h e r strains tested, t w o a c i d - t o l e r a n t strains o f R. meliloti, W S M 4 1 9 a n d W S M 5 4 0 , s h o w e d
Table 2. Mean generation times (h) in 0.1, 1.0 and 20 gM added iron
Organic acid assays Citrate was determined using the method of Mollering and Gruber (1966) as modified by Dagley (1974) except that 0.1 M Tris-HC1 buffer pH 8.2 was used instead of triethanolamine. This assay detects both the free acid and its salts, such as ferric citrate. The minimum detectable concentration is 16 gM. The presence of MES buffer in the growth medium resulted in apparent concentrations of citrate and malate which were elevated by as much as ten fold. To avoid this problem HEPES buffer, which did not interfere with the assays, was used in all the growth media. Malate was assayed using a technique derived from Bernt and Bergmeyer (1974) for glutamate estimation. In a final volume of 1.5 ml there was 0.75 mmol glycine, 0.6 mmol hydrazine monohydrate, 4 ~tmol NAD + and 3 units of malate dehydrogenase. The samples were incubated at 30 °C for 90 rain before reading at 340 nm. Malate concentrations were determined from a standard curve (20-100 nmol) and minus enzyme controls were used as blanks.
Results and discussion
Growth of root nodule bacteria in differing concentrations of iron T w e l v e strains o f r o o t n o d u l e b a c t e r i a (Bradyrhizobium strains N C 9 2 B a n d 32H1, B. japonicum CB1809 a n d
Bacteria
0.1gMFe
1.0~tMFe 2 0 g M F e
R. leguminosarum WU235 R. trifolii WU95 B.japonicum CB1809 R. trifolii T1 Bradyrhizobium NC92B B. lupini WU8 B. japonicum USDA 110 R meliloti CC169 Bradyrhizobium 32H1
4 4 4.5 4.5 5 6.5 4 10 23 12 10 9
4 3.8 4.5 4 4.5 5.6 3 9 23 11 9 8.5
4 3.6 4 4 4 5.5 3 9 21 10 8 7
Bradyrhizobium TALl000 Bradyrhizobium NC92I B.japonicum 61A76 R. leguminosarurn MNF300 Bradyrhizobium CB756
10 28 32 12 48
8 15 26 5 36
6.6 13 10 3 9
R. meliloti WSM419 R. meliloti WSM540
15 13
15 17
46 22
R. meliloti U45 R. sp. NGR234 R. trifolii TAI
b
a
D- - ' ' - - - I n
1.0
1.0
//
0.5
oo ~o .
