Indian Journal of Experimental Biology Vol. 38, April 2000, pp. 373-378

Analogue-resistant mutants of Azotobacter chroococcum derepressed for nitrogenase activity and early ammonia excretion having

potential as inoculants for cereal crops

K Lakshminarayana*, Bela Shukla, S S Sindhu, Parveen Kumari , Neeru Narul a & R K Sheorana

Department of Microbiology, "Department of Plant Breedi ng, CCS Haryana Agricultural University, Hisar 125 004, India

Received 25 August 1999; revised I December 1999

Spontaneous mutants resistant to methionine sulfox imine (Msx), methyl alanine (Mal) and methyl ammoni um chloride (Mac) were derived from A.chroococc11111 strain A I 03. Msx and Mal-resistant mutants ex pressed 1.73 to I 0.98% of the full y derepressed nitrogenase activity when grown in Burk 's medium containing ammonium acetate. Mac-resistant mutants did not ex press nitrogenase acti vity in ammonium acetate supplemented medium. The mutants excreted ammonia even after 2 days of growth and some mutants excreted more ammoni a as compared to the parent. Selected mutants were inoculated on wheat (Tri ticum aestivum ) and barley (Hordeum vulgare) under fi eld conditions. Majority of the derepressed mutants increased grain yield of wheat and barley varyi ng from 1.2 to 33.3%. However, host-dependent effects on grain yield were observed with different mutants. Two mutants, Mal 27 and Mac 19 showed signi ficant increase in grain yields of both the crops. The resu lts suggest that metabolic analogue-resistant mutants of Azotobacter have potenti al fo r use as a biofertili zer fo r cereal crops.

Various species of Azotobacter fix dinitrogen in soil and have been reported to contribute towards nitrogen economy and fe rtility of soil s in the tropics and subtropics1.2. In these bacteria N2 fixation is lowered under high soil nitrogen level due to repression of bacterial nitrogenase by nitrogenous substances which include urea, ammonium (NH4 +) and nitrate (N03-) present in soil 3

. In Azotobacteriaceae, additi on of ammonium ions cause immediate inhibition of nitrogenase actiVIty which is recovered after exhaustion of ammonium from the medium4

. In A. vinelandii, NH4 + was reported to decrease the electrical component of the proton moti ve force and switch off the fl ow of electrons to nitrogenase5

.

Therefore, the use of soil iso lates showing high nitrogenase acti vity in chemically defined media may not serve useful purpose as biofertili zers for increasing crop producti vity since there are probably few places in nature which are free of nitrogenous compounds, espec iall y NO,- .

Streicher et a/.6 showed that glutamine or glutamate auxotrophs of Klebsiella were insensiti ve to nitrogenase repress ion by ammonium. Mutants of A. vinelandii were isolated which manifested partial

*Correspondent author : Fax : 0 1662-34952. E-mai I : hau @hry.ren.nic.i n

nitrogenase derepression7·8

. Some of the mutants also exhibited low level of ammoni a excretion7

. Cejudo et a /. 4 found that in A. chroococcum, the short-term inhibitory effect of ammonium on nitrogenase acti vi ty was prevented and reversed by inhibitors of the ammonium assimilation pathway, i.e . vi a glutamate synthetase-glutamine synthase. Compounds such as methionine sulfoximine (Msx), methyl alanine (Mal) and methyl ammonium chloride (Mac) were reported to select for mutations for derepressed nitrogenase activit/- 11

. Msx-resistant mutants were al so found to show early excretion of ammonia 111 Nostoc

12 muscorum . Azotobacter chroococcum has been long known to

act as a biofertilizer of agronomically important cereals and other non-legume crops2

·13

'14

• Reduced use of nitrogenous fertili zers and increased use of biofertilizers and organic manures are important elements of integrated plant-nutrient management. Therefore, deve lopment of nitrogenase-constituti ve (derepressed) mutants that fix dinitrogen in the presence of ammonium may provide a novel approach for development of biofertili zers for cereal crops. In this study, mutants, which could fix dinitrogen in presence of ammonium and had the ability to excrete ammonia, of A. chroococcum strain A I 03 were isolated by selection for resistance to the metabolic

374 INDIAN J EXP BIOL, APRIL 2000

analogues Msx, Mal and Mac. These mutants were tested as inoculants on wheat and barley under field conditions.

Materials and Methods

Isolation of metabolic analogue-resistant mutants -Azotobacter chroococcum strain A I 03 was used to obtain spontaneous mutants resistant to Msx, Mal and Mac. This strain showed high nitrogenase activity under cultural conditions and moderate ammonia excretion in Burk's medium 15

• The mtmmum inhibitory concentration (MIC) of each analogue on A. chroococcum was determined by observing growth in a 15 ml test tube containing 5 ml Burk's liquid broth supplemented with Msx (0-200 flg/ml ), Mal (0-1.5 mg/ml) or Mac (0-20 flg/ml). The MIC levels of Msx, Mal and Mac were found to be 40 flg/ml , 1.0 mg/ml and 10 fl g/ml, respectively .

An aliquot (0. 1 ml) of a 3 day-old suspension culture in Burk's liquid medium (about 107 cells/ml) was spread on agar plates of the same med ium supplemented with 50 flg/ml of Msx . Single cell isolates that developed on these plates were picked, suspended in water and vortexed for I min. The suspension was spread uniformly on plates containing 100 flg/ml of supplemented Msx. Similarly, spontaneously resistant mutants of Mal and Mac were isolated on medium plates supplemented with 1.2 mg/ml of Mal or 15 flg/ml of Mac, respectively. Clones showing resistance to the above concentrations of Msx, Mal and Mac were picked and maintained on Burk's medium slants.

Determination of nitrogenase activity-Nitro­genase actt vtty was determined by measuring acetylene (C2H2) reduction activity (ARA) of culture slants using Burk's medium as described by Sindhu et a/. 14

• Nitrogenase activity of each mutant culture was also determined on Burk's medium supplemented with 2 mM ammonium acetate. Three replicates of each culture were grown in 15 ml assay tubes ( 15x 125 mm), each tube containing 5 ml medium, at 28°±2°C for 2 days. The cotton plugs of tubes were then replaced with subaseals and I 0% C2H2 (v/v) was injected into each tube. The tubes were further incubated for a 24hr and ethylene (C2H.J) formed was determined in a Nucon gas chromatograph using Porapak N column (2M length) and dual fl ame ionisation detector (FlO) at I 05°C oven and I I ooc injector and detector temperatures.

Measurement of ammonia excretion - For detern ining ammonia excretion, I ml of 48hr old culture of Azotobacter was inoculated into 30 ml Burk's liquid medium in I 25 ml-capac ity flasks containing 2.5% sucrose and incubated at 28°±2°C under stationary conditions. Ammonia excretion was determined at 2 and 9 days of incubation 15

.16

. The suspension culture was centrifuged at I 0,000 rpm for I 0 min and ammonia (NH4 +) content in supernatant was determined colorimetrically using Chaykin's reagent after microdiffusion as described by Burris17

.

Inoculation of selected Azotobacter mutants on wheat and barley - The selected analogue-resistant mutan s of A. chroococcum along with parent strain A I 03 were tested on wheat (Triticum aestivum) var. WH29 1 and barley (Hordeum vulgare) var. BH75 under field conditions. Each bacterial strain (2 ml of 48 hr grown inoculum) was inoculated in 50 ml Burk's liquid broth and incubated on shaker at 28°±2°C for 3 days. A culture packet was made by mixing 20 ml growth suspens ion with 50 g charcoal taken as carrier1x, cured for 3 days and stored at 4 oc until use. This charcoal based Azotobacter culture was used to treat one ~g of seeds. Seeds were first treated with 2.5% jaggery (boi led and cooled) solut ion . The sticky seeds were then mixed thoroUJghly with the charcoal based culture for the attachment of Azotobacter cell s to the surface of seed~ The final bacterial count for each seed was 5.4x 106 viable cells. In case of control, the seeds were treated with only jaggery so lution .

Inoculated seeds of wheat and barley were sown at the University farm located at HAU, Hisar (p lot size each 5 x 0.92 M in four rows). The soil was sandy loam, having low available nitrogen ( 170-180 kg/ha), medium phosphorus ( 170-185 kg/ha) and rich amount of potassium (41 0-420 kg/ha). The observations for grain yield were recorded after 135 days of sowing.

Results Nitrogenase acttvtty and ammonia excretion in

derepressed mutants- Mutants of Azotobacter strain A I 03, resistant to the three metabolic analogues (Msx, Mal and Mac) were isolated and tested for ammonia excretion and nitrogen fixation in presence of ammonium. The parent strain A I 03 did not express ARA in Burk's medium supplemented with ammonium acetate and . showed ammonia excretion only after 9 days of incubation under stationary conditions. Forty six of total 50 purified Msx-

LAKSHMINARA YANA el a/.: ANALOGUE-RESISTANT MUTANTS OF AZOTOBACTER 375

resistant mutants showed nitrogenase activity in ammonium acetate supplemented medium and all of these mutants exhibited early ammonia excretion (Table I, data are given for only 5 selected mutants). The mutant Msx 21, which showed slightly more ARA than the parent strain (A I 03), and Msx I, which showed less ARA in Burk's medium, expressed more ARA in ammonium acetate supplemented medium compared to two other mutants Msx 27 and Msx 37. Maximum ammonia excretion (6.4 to 7.0 1-'g/ml) was observed in the mutants Msx I, Msx 21 and Msx 26 at 2 days of incubation . At 9 days of incubation, mutants Msx I, Msx 27 and Msx 37 showed more ammonia excretion ranging from 7.9 to I 0.21-'g/ml.

Among methyl alanine-resistant mutants, Mal 24 and Mal 27 mutants showed maximum derepressed ARA (0.8 to 1.0 1-1M C2H2 reduced/hr/mg protein). Mutants Mal I and Mal 27 showed more ammonia excretion compared to other mutants at 2 days of incubation whereas Mal 24 and Mal 30 showed 68 to 120% increase in ammonia excretion over the parent strain A I 03 at 9 days of incubation.

Derepressed nitrogenase activity in presence of ammonmm acetate was not observed in methyl ammomum chloride-resistant mutants. However,

mutant Mac 63 showed more ARA than the wild type parent strain (Table 1). Two mutants Mac 27 and Mac 63 excreted 5.6 and 5.2 1-'g/ml ammonia at 2 days of incubation and showed 128 to 154% increase in ammonia excretion over the parent strain at 9 days of incubation.

Effect of Azotobacter inoculation on the grain yield of wheat and barley - The selected mutants of A. chroococcum along with parent strain A I 03 were tested as inoculants under field conditions on wheat and barley. Parent strain A I 03 was found to increase the grain yield of wheat (16.3%) and barley (10.5 %) in comparison to uninoculated controls (Table 2) . The overall increase in grain yield varied from 1.2 to 30% in wheat and 4.8 to 33.3% in barley on inoculation with the analogue-resistant mutants in comparison to parent strain inoculated plants. The results also indicate host dependent response on grain yield due to inoculation of various analogue-resistant mutants. For example, two Msx-resistant mutants, Msx 26 and Msx 37 showed only 1.2 and 5.0% increase in grain yield on wheat but showed 33.3 and 24.8% increase in grain yield of barley respectively. Other mutants i.e. Msx I, Msx 27, Mal30 and Mac 27 were found to increase the grain yield of wheat (varying from 5.0 to

Table !-Nitrogenase activity and ammonia excretion of methionine sulfoximine, methyl alanine and methyl ammonium chloride resistant mutants

Strain/ Nitrogenase activity Ammonia excretion

Mutant Burk's Burk's medium 2 days 9days medium +ammonium

acetate

A I 03 (Parent) 17.3 5.0

Msx I 12.0 1.8 6.4 7.9

Msx 21 17.8 1.9 6.8 6.2

Msx 26 1.0 7.0 3.2

Msx 27 11.8 0.6 0.6 10.2

Msx 37 7.2 0.6 4.0 8.1

Mall 8.9 0.4 4.5 5.8

Mall6 10.4 0.3 2.7 7.9

Mal24 14.2 1.0 2.5 8.4

Mal27 12.0 0.8 4.0 3.2

Mal30 6.5 0.4 3.6 11.0

Mac 3 15.4 3.7 5.2

Mac 19 11.0 2.6 2.1

Mac 27 14.6 5.6 11.4

Mac 63 21.8 5.2 12.7

Mac 68 15.2 3.4 9.6

Nitrogenase activity Is expressed as J.IM C2H2 reduced/hR!mg cell protein. Ammonia excretion is expressed as J.lg of ammonia excreted/ml of the supernatant. Each given value is an average of 3 replications.

376 IN DIAN J EXP BIOL, APRIL 2000

15.0%) compared to the parent but did not increase the yield in barley. However, in some treatments e.g. Mal 24 decrease in grain yield was observed in both the crops . Two mutants Mal 27 and Mac 19 showed significant increase in grain yield in both the crops.

Discussion A limited number of soil isolates of A.

chroococcum are known to excrete varying amounts of ammonia when cultivated in nitrogen-free synthetic medium under stationary conditions 15

• The fixed dinitrogen (as ammonia) has been reported to be metabolized rapidly through ammonia assimi lating enzymes, releasing little or no free ammonium into the environment l.2 .

Nitrogenase synthesis is repressed by ammonium . A b . 4 19 Th f . rn zoto acter spectes · . ere ore, mtrogenous fertilizers can reduce bacterial effectiveness as

biofertilizers in the fi d d. In our study, three metabolic analogues viz., Msx , Mal and Mac were used to derive derepressed, ammonia excreting mutants. Msx- and Mal-resistant mutants could express low levels of nitrogenase acttvtty in ammonium acetate sup lemented medium (Table I). However, no derepression of nitrogenase activity was observed in Mac-resistant mutants . All the three classes of mutants exhibited early ammonia excretion (at 2 days) as compared to parent strain A I 03 which excreted ammonia on ly after 9 days of incubation (Table I). Similarly, constitutive mutants of asymbiotic N2-fixing bacteria resi stant to various metabolic analogues have previously been isolated9

'10

·12

. Methyl alanine was suggested to be gratuitious corepressor of nitrogenase and Mal­resistant mutants of A. vinelandii were shown to posse s constitutive nitrogenase activit/. Methyl

Table 2-Response of wheat and barl ey to inoculation with analogue-resi stant mutants of A. chroococcwn

Strain/mutant Grai n yield/plot (kg)

treatment Wheat % increase ( +) or Barl ey % increase(+) or decrease (-) over decrease (- ) over

parent strain parent strai n treatment treatment

Control 0.430 0.475

(uninoculated)

AI03 0.500 +16.3 0.525 +10.5

(parent strain)

Msx-resislant mutants

Msx I 0.575 +15.0 0.500 -4.8

Msx 21 0.472 - 5.6 0.525 0.0

Msx 26 0.506 +1.2 0.700 +33.3

Msx 27 0.550 +10.0 0.425 -1 9.0

Msx 37 0.525 +5 .0 0.655 +24.8

Mal-resistant mutwlls

Mall 0.450 - 10.0 0.500 - 4.8

Mall6 0.475 -5.0 0.550 +4.8

Mal24 0.450 - 10.0 0.475 -9.5

Mal 27 0.650 +30.0 0.650 +23 .8

Mal 30 0.575 +15 .0 0.475 -9.5

Mac-resistant mutants

Mac 3 0.450 -10.0 0.550 +4.8

Mac 19 0.650 +30.0 0.675 +28.6

Mac 27 0.550 +10.0 0.500 -4.8

Mac 63 0.475 - 5.0 0.575 +9.5

Mac 68 0.525 +5 .0 0.500 -4.8

SE = 0.067 0.080

CD= 0. 140 0. 169

Inoculation was carried out by bacterial seed coating (5.4x I Oh viable cell s/seed). Grain yield was determined at 135 days after sowing

LAKSHMINARA Y ANA eta/.: ANALOGUE-RESISTANT MUTANTS OF AZOTOBACTER 377

ammonium chloride is an analogue of ammonium and Mac-resistant mutants of A. vinelandii were shown to overproduce nitrogenase 10

• Methionine sulphoximine is an inhibitor of glutamate synthetase and Msx­resistant mutants have been found to cause early excretion of ammonia in Nostoc muscorum 12

.

The analogue-resistant mutants described in these studies were isolated as spontaneous mutants which expressed nitrogenase in ammonium contammg medium at level s up to 1.73 to I 0.98% of the full nitrogenase level observed in N2-free grown cultures of wild type. In contrast, cultures of the wild type grown in ammonium acetate supplemented medium did not express any nitrogenase activity. The highest level of constitutive nitrogenase activity reported for ammonium grown cultures of nonauxotrophic asymbiotic bacterium is 35% of the level found in N2

grown wild type20. Similarly, total nitrogen content of

the medium of a methyl ammonium- resistant mutant strain of A. vinelandii JK 219 was greater than that of the medium of the wild type by as much as 60% (ref. 10). It may be poss ible that the constitutive mutants of A. chroococcum isolated during these studies, might have altered ammonium transport systems such that ammonium efflux occurs at a greater than normal rate and therefore, the mutants would be capable of maintaining a higher level of nitrogenase than the wild type. Alternatively, because of the higher expression of nitrogenase activity , more nitrogen may be reduced than assimilated and consequently more ammonia is excreted into the medium.

Jensen21 reported that few soil s of New South Wales wheat belt contain sufficient diazotrophs such as Azotobacter spp. and Clostridium butyricum which make significant contribution to heterotrophic N2

fixation. The population of Azotobacter is generally low in North Indian soils due to poor organic matter content22

'23

. Sindhu and Lakshminarayana24 showed that nitrogen-fixing capacity of Azotobacter strains can be increased with the amendment of carbon sources in soil. Inoculat ion studies of wheat and barley with analogue-resistant mutants showed that mutants Msx 1, Msx 27, Mal 27, Mal 30, Mac 19 and Mac 27 gave better grain yield in wheat and Msx 26, Msx 37, Mal 27 and Mac 19 showed increased gra in yield in barley (Table 2). The highest grain yield of wheat was obtained by inoculation with the mutants Mal 27 and Mac 19 and of barley by inoculation with mutants Msx 26, Msx 37, Mal 27 and Mac 19.

Pandey and Kumar13 summarized the results of several field experiments and concluded that Azotobacter inoculation increased the yields of wheat, rice, maize, pearlmillet and sorghum by 0 to 72% over the uninoculated controls without any amendment and by 8 to 43% over control when farm yard manure and nitrogenous, phosphatic and potash fertilizers were added. Since these metabolic analogue-resistant mutants have acquired the ability to fix nitrogen in presence of combined nitrogen, they can serve as a useful biofertilizer for cereal crops under normal agronomic conditions.

Reference I Burris R H & Roberts GP, Biological nitrogen fixation. Amw

Rev Nutr, 13 (1993) 317. 2 Lakshminarayana K, Influence of Azotobacter on nitrogen

nutrition of plants and crop productivity. Proc Indian Nat/ Sci A cad, B59 ( 1993) 303.

3 Wu FJ , Moreno J & Vela GR, Growth of Azotobacter vinelandii on soi l nutrients. Appl Environ Microbial, 53 (1987) 489.

4 Cejudo FJ , De Ia Torre A & Paneque · A, Short term ammonium inhibition of nit rogen fixation in Azotobacter. Biochem Biophys Res Commun, 123 ( 1984) 240.

5 Laane C, Krone W, Konings W, Haaker H & Yeeger C, Short term effect of ammonium chloride on nitrogen fixation by Azotobacter vinelanclii and by bacteroids of Rhizobium leguminosarum. Eurl Biochem, 103 (1980) 39.

6 Streicher SL, Shanmugam KT, Ausubel F, Morandi C & Goldberg RB, Regul ation of nitrogen fix ation in Klebsiella pneumoniae: evidence for a role of glutamine synthetase as a regulator of nitrogenase synthesis . J Bacterial, 120 ( 1974) 815.

7 Gordon JK & Brill WJ , Mutants that produce nitrogenase in presence of ammonia. Proc Nat/ A cad Sci USA , 69 ( 1972) 350 1.

8 Terzaghi B, A method for isolation of Azotobacter mutants derepressed for nif. J Gen Microbial, 118 (1980) 275.

9 Sorger GJ , Regulation of nitrogen fixation in Azotobacter vinelandii OP and in an apparently partially constituti ve mutant. J Bacterial, 95 ( 1968) 1721.

I 0 Gordon JK & Jacobson MR, Isolati on and characterization of Azotobacter vinelandii mutant strains with potential as bacterial fertilizer. Can J Microbial, 29 ( 1983) 973.

II Revill a E, Cejudo FJ , Ll obell A & Paneque A, Short-term inhibition of nitrate uptake by Azotobacter chroococcum. Arch Microbial , 144 (1986) 187.

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13 Pandey A & Kumar S, Potential of azotobacters and Azospirillum as biofertilizer for upl and agriculture : A review. J Scient Indus Res, 48 ( 1989) 134.

14 Sindhu SS, Lakshminarayana K & Dalvir Singh, Expression of hydrogenase acti vity in Azotobacter chroococcum and its possible role in crop productivity. Indian J Expt Bioi, 32 (1994) 423.

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15 Naru la N, Laksh minarayana K & Tauro P, Ammonia excretion by Azotobacter chroococcum. Biotecllllol Bioeng, 23 ( 1981) 467.

16 Wallace CJ & Stokes 80, Assessment of nif derepressed microorganisms for commercial nitrogen llxati on. Biotechnol Bioeng Symp, 8 ( 1978) 153.

!7 Burris RH , Nitrogen fixation-Assay methoJs and techniques. in Methods in Enzymology, Volume 24, edited by SP Colowick and NO Kaplan (Academic Press, New York and London) 1972. 415.

18 Vincent JM, A manual for the practical study or root nodule ·bacteri a. International Biological Programme Hand book (Blackwell Scientilic Publisher, Oxford) 1970, 11 3.

19 Patridge COP, Walker CC, Yates MG & Postgate JR . The re lationship between hydrogenase and nitrogenase in Azotobacter chroococcum. Effect of nitrogen sources on hydr0'!t>nase activity. 1 Gen Microbial, 11 9 ( 19!i0) 313.

20 Gordon JK , Shah VK & Brill WJ , Feedback inhibition of nitrogenase. 1 Bacterial, 148 ( 198 1) 884.

2 1 Jensen HL, The nitrogen economy or wheat soils with special reference to non-symbioti c nitrogen fixation. 1 Aust Ins/ Agric Sci , 5 ( 1939) 154.

22 Sindhu SS & Lakshminarayana K, Di stribut ion of Azotobacter in the Haryana soi ls and effect or bacteriostasis on Azotobacter survival. Environ £col. 4 ( 1986) 536.

23 Gupta RD. Jha KK , Sethi AS & Saharan GS , Presence of Azotobacter in relati on to physico-chemical properties of some North Indian soil s. Proc Indian A cad Sci, 86 ( 1977) 389.

24 Sindhu SS & Lakshminarayana K, Effect of carbon sources amendments on acetylene reduction by Azotobacter strain in sandy loam soil. Annal Bioi, 8 ( 1992) 81.