Indian Journal of Experimental Biology Vol. 42, December 2004, pp. 1177-1185

Azide resistance in Rhizobium ciceri linked with superior symbiotic nitrogen fixation

V Vijay Bhaskar 1* & L R Kashyap2

1Centre for Cellular and Molecular Biology, Uppal Road, Hyderabad 500 007, India 2NRC on Plant Biotechnology, Indi an Agricultural Research Institute, New Delhi 110 012

Received 5 January 2004; revised 9 August 2004

Isolated azide resistant (AzR) nati ve R. ciceri strain 18-7 was resistant to sodium azide at 10 JLglml. To find if nil­reiteration is responsible for azide resistance and linked to superior symbiotic nitrogen fixation, transposon (Tn5) induced azide sensitive mutants were generated. Using 4 kb nif-reiterated Sinorhizobium meliloti DNA, a clone C4 that complemented azide sensitivity was iso lated by DNA hybridization from genomic library of chickpea Rhizobium strain Rcd30 l. EcoRI restriction mapping revealed the presence of 7 recognition sites with a total insert size of 19.17 kb. Restriction analysis of C4 clone and nif-reiterated DNA (pRK 290.7) with EcoRI and Xhol revealed similar banding pattern. Wild type strain 18-7, mutant M126 and complemented mutant M126(C4) were characterized for symbiotic properties (viz., acetylene reduction assay, total nitrogen content, nodule number and fresh and dry weight of the infected plants) and ex­planta nitrogenas:! activity. Our results suggested that azide resistance, nif~reiteration , and superior symbiotic effectiveness were interlinked with no correlation between ex-planta nitrogenase activity and azide resistance in R. ciceri.

Keywords : Azide resistance, Rhizobium ciceri, Symbiosis, Nitrogen fixati on

Symbiotic interaction between rhizobia and leguminous plants results in root nodules, where atmospheric dinitrogen is reduced to ammonia and that is utilized by host plant. Rhizobium legume symbiosis is a major source of biologically fixed nitrogen in diverse environments. On an average biological nitrogen fixation is estimated to contribute, globally, 140 million tonnes which is more than twice the amount produced through an alternative energy expensive industrial nitrogen fixation 1• So, improvement in the efficiency of legume - Rhizobium symbiotic systems is likely to result increase in production of high quality plant products.

Recombinant DNA technology improves the genetic resources of microbes. For example, specific DNA amplification (up to 2-3 copies) of nodDJ, common nodulation genes nodABC and operon essential for nitrogen fixation (nifN) from the nod regulon region of pSym-a plasmid in Sinorhizobium meliloti has increased alfalfa nodulation, nitrogen, and plant growth2

. Similarly, constitutive expression of nifA gene of Klebsiella pneumoniae on a multicopy plasmid has also enhanced nodulation competitiven~ss and nitrogen fixation by S. meliloti3

.

• Correspondent author E-mail : vvbr@ccmb.res.in Phone: 040-27192546

Accordingly, higher symbiotic expression of S. meliloti nifA driven by nifD promoter has resulted in addition of biomass (12%) in combination with dct genes4

. Recently, it has also been found that expression of fixNOQP operon (encoding cbb3 terminal oxidases) of Bradyrhizobium japonicum from a constitutive promoter in R. etli ntrC mutants has greatly enhanced the symbiotic performance of the strain5

.

Simple microbial technique to select for azide resistance has also been found to be a criterion to select superior nitrogen fixing strains of Rhizobium. Such azide resistant mutants of R. leguminosarum bv.viciae6

, mungbean-Rhizobium7, Cajanus­

Rhizobium8 and R. loti9 have been found to be superior over sensitive strains. However, except for these reports, not much is known regarding the molecular mechanisms underlying the azide resistance. A linkage has been reported between azide resistance and nodulation in R. leguminosarum 10

.

Expression of azide resistance has been reported to be dependent on fixABC in S. meliloti 11

• Recently, other findings have supported the involvement of fixNOQP genes (that encode symbiotic terminal oxidase cbb3) in R. etli5

•

Recognizing chickpea as the most important pulse crop of India covering about 30% of the total area and

1178 INDIAN J EXP BIOL, DECEMBER 2004

37% of the production among grain legumes and Rhizobium ciceri, a new introduction in grain legume symbiosis, we examined the role of azide resistance in relation to symbiotic nitrogen fixation.

Tryptone Yeast -extract (TY) medium 12. Triparental conjugal matings involving E. coli strains were done on TY medium. R. ciceri strains were also grown on minimal medium (RMM) 10 and nitrogen free medium (NF). NF medium used in the study was comprised­KH2P04, 200 mg; MgS04, 200 mg; K2HP04, 800 mg; CaCh (anhydrous), 90 mg; Fe-Mo (1 mg Fe and 0.1 mg Mo); and sucrose, 5 gin 11 of H20. Supplements were added to the autoclaved media at 50°C before plating. Medium was solidified by adding agar @ 15 g/1. For ex-planta nitrogenase assay, all strains were grown in CS7 medium 13

. Nitrogen-free Me knight's medium (MKS/4 was used for growing chickpea plants during nodulation assays.

Materials and Methods Bacterial strains-Twenty three native strains of

Rhizobium ciceri isolated from the chickpea plants grown in the fields of Indian Agricultural Research Institute, New Delhi were confirmed and used in the present study. List of strains used in the present study with their source has been listed in Table 1.

Culture mediwn--R. ciceri strains were grown either on Yeast Extract Mannitol (YMA) 10 or

Table !-Bacter ial strains and plasmids used in the study

Strain/plasmid

HB101

DH5a

WA803

HB 101(pLAFR1)

HB101 (pLAFR1-RC gene bank)

HB101 (pRK290.7)

HB101 (pRK2013)

W A803 (pGS9)

18-7

M126

M126(C4)

Azotobacter croococcum W5

Native R. ciceri strains: 15-5, 17-1, 17-2, 17-3, 17-4, 17-5, 17-

Relevant characteristics Source/Reference

recA, hsdR, hsdM, pro, leu, str', glaK2, Boyer and Dussoix 24

rpsL20, xyl5

supE44. L'l.LacU169( <jl80 lacz L'l.M 15) Sam brooker al. 16

hsdR17, recA1, endA1, gyrA96 thi-1, relA1

met, thi , supE, hsdR, hsdM Selvaraj and Iyer 17

inc p Tc' Friedman et al. 25

inc p Tc' Khanuja 13

HBI01 with additional nif DNA of Kashyap 11

Rhizobium meliloti that confers Az'

ColEl::pRK2 Tra+ Km' Ditta et al. 26

PGS9 with Tn5 transposon that confers Selvaraj and Iyer 17

Kan'!Neo' and Cam'

Azi'Nal' Spec' Thi s study

Azi ' Nal'Kan' I Neo' This study

Azi ' Nal' Kan' I Neo' Tc' Thi s study

Wild type Lab collection Shinde, S.T., Division of microbiology, IARJ, New Delhi .

7, 17-8, 17-9, 17-10, 17-11, 17- Wild type This study 12, 17-13, 17-14, 18-1 , 18-2, 18-3, 18-4, 18-5, 18-6, 18-8, 21-3, H-68, Rcd301

BHASKAR & KASHY AP: AZIDE RESISTANCE IN RHIZOBIUM CICERI 1179

Protocols and chemical compositions as described in laboratory manual of Sam brook et al. 15 were followed for molecular studies.

Isolation of azide resistant strains-A loopful of log phase cells from a fresh streak suspended in a drop of sterile water was spotted on NF agar medium supplemented with_ various concentrations (5, 10, 15, 20 J..Lg mr') of sodium azide. After 36 hr of incubation at 28°C, growth was monitored and resistant strain was purified on the same medium.

Nodulation and nitrogen fixation assays--Chickpea cultivar BG 256 seeds were surface sterilized with ethanol (70%) for 1 min followed by treatment for 3-5 min with acidified mercuric chloride [0.1% (w/v)] and rinse in sterile distilled water for 5-6 times. Seeds were soaked in bacterial suspension for 2 hr and sown in Leonard jars. Leonard jars containing germinated seedlings were placed in growth chamber with diurnal cycle of 12 hr light/dark cycle (light intensity of 65-67 J..Lmole of . photons/m2/sec). The day and night temperature was set at 23° and 15°C, respectively . Relative humidity was maintained at 60%. Plants were grown for 6-7 weeks by irrigating with MKS (1/4 strength), thrice a week.

After 6 weeks of growth, plants were harvested and collected data with respect to parameters - (i) nodule number per plant; (ii) acetylene reduction activity (ARA); (iii) shoot fresh and dry weight; (iv) root fresh and dry weight; and (v) total nitrogen content of shoot relating to symbiotic nitrogen fixation.

Acetylene reduction assays-ARA of the nodules was done using Nukon gas liquid chromatography. Using standard ethylene, acetylene reduced to ethylene was determined.

For ex-planta nitrogenase assay, bacterial cultures grown in CS7 slants were incubated for 16 hr at 30°C after injecting 1/lOth volume of acetylene by removing equal volume of air. Samples of 0.5 ml of gas per tube were analyzed by gas liquid chromatography for ethylene production.

Total nitrogen content of shoot-Total nitrogen content of the plant material was determined using a kjeltek nitrogen auto analyzer.

Leonard assemblies for plant assays to monitor symbiotic characteristics of various strains were laid out in completely randomized design. Each treatment was replicated for 5 times.

Results Twenty three native strains of R. ciceri isolated

from the root nodules of chickpea plants were screened for azide resistance at various concentrations. Only one strain designated as 18-7 was resistant t0 sodium azide at 10 ).!g/ml. Its generation time was about 2 hr (Fig. 1). This strain was fast grower as compare to some other R. ciceri strains, which had a generation time of 4-5 hr. Strain 18-7 was resistant to nalidixic acid, spectinomycin and chloramphenicol and partially to gentamycin. It was sensitive to kanamycin/neomycin and tetracycline.

Isolation of azide-sensitive mutants-Tn5 induced random mutagenesis of strain 18-7 was performed as described by Selvaraj and Iyer16 with slight modifications. Transconjugants of 18-7 and W A803 (pGS9) showed both NeoR and NaiR properties, otherwise the parental strain 18-7 was Neos and N al R. The neomycin resistance was due to the single copy integration of Tn5 into 18-7 genome, which was confirmed by a southern analysis wherein EcoRI digested genomic DNA of wild type and ·mutant strain was probed with transposon Tn5. As there is no EcoRI site on transposon Tn5, probe is expected to hybridize to one fragment for each of the insertion. Single hybridized band as seen in Fig. 2 suggested that the mutation had resulted due to single transposon insertion. The frequency of NeoR and NaiR transconjugants was 1 xl o-6 per recipient cell. Screening these transconjugants on NF medium supplemented with sodium azide led to the isolation of 13 mutants, at a mutational frequency of 1.3%. Out of these, one isolate designated M126 was found sensitive to 2 J..Lg mr' of sodium azide which was used for further studies.

§ 0 0 \0

~ Q 0

1.6

0.8

0.6

0.4

0.2

0

?I

0 10 15 20 25 30

Time (hr) Fig. 1-Growth-curve of R. ciceri strains 18-7(WT) and Ml26 (Az' mutant) grown in TY medium.

1180 INDIAN J EXP BIOL, DECEMBER 2004

Isolation of genomic clone that confers azide resistance--A genomic library of R. ciceri strain Red 301, resistant to sodium azide (20 11g mr'), was screened by colony hybridization using 4.5 kb Xhol fragment of pRK290.7 that catTied nif reiteration of S. meliloti, which confers azide resistance in S. meliloti 11

• After primary and secondary screenings, 4 putative clones designated C1, C2, C3 and C4 were isolated . Clone- C4 complemented azide sensitivity of M126 when mobilized by triparental mating using a helper plasmid pRK2013 (Fig. 3).

This clone was further examined for its characteristic restriction-banding pattern comparable to pRK290.7 by digesting with Xhol and EcoRI enzymes. A similar pattern as noticed for pRK290.7 was also observed for C4 clone suggesting that the clone C4 possibly represented nif DNA of R. ciceri (Fig. 4) due to the fact that probe DNA carried nif­reiterated DNA of S. meliloti 11

•

EcoRI restriction endonuclease map of C4 clone was prepared by partial digestion method (Fig. SA, B). On comparison, it was found that EcoRI map of C4 clone did not match with conventional nifHDK restriction pattern of either S. meliloti 17 or

>...

A Q)

X >... 0 ~

~

~

"0 U) c 1'- (\J

I co

K. pneumoniae 18• The sizes of their nifHDK EcoRI

fragments were 4 and 6 kb, respectively (Fig. 6). It is found to be 4.2 kb for B. japonicum20

. However, azide sensitive strain was affected for the nitrogenase activity in comparison to the wild type strain.

To examine the effect of identified gene in symbiotic nitrogen fixation, ARA and total nitrogen content of chickpea plants nodulated by the wild -type, mutant and complemented mutant strains were studied. Plants nodulated by strain 18-7 showed 59.49 11mole of ethylene produced per plant per hr. A comparable nitrogenase activity was shown by M126(C4); 51.18 )..!mole ethylene/plantlhr, while plants nodulated by M126 alone showed (30.48 11mole ethylene/plant/hr; Fig. 7). This observation suggested a link between nitrogenase enzyme activity and azide resistance.

In accordance with the above result, M126 containing C4 clone infected plants had similar amount of nitrogen [17.49 ± 0.96 mg of nitrogen/g dry wt; n=5] as that of plants infected with strain 18-7 [18.61 ± 0.96 mg of nitrogen/g dry wt; n=S)]. M126 nodulated plants had significantly lesser nitrogen [13.83 ± 0.96 mg of nitrogen/gm dry wt; n=5]. This

8

Fig. 2- Southern hybridisation to confirm mutagenesis by trasposon (Tn5) insertion. (a) Agarose gel lanes 1, A. Hind III marker (did not resolve properly); lanes 2&3, EcoRI digested genomic DNA of WT (18-7) and mutant (M126); and (b) Autoradiogram of a blot containing the above said DNA probed with 32p-Jabeled 3.3kb Hind III fragment of pGS9 corresponding to Tn5 transposon and with labeled A. Hind III DNA.

BHASKAR & KASHYAP: AZIDE RESISTANCE IN RHIZOBIUM CICERI 1181

meant that 18-7 and M126 (C4) infected plants had 34.56 and 26.46% higher nitrogen per g dry wt respectively than plants infected with M126 alone (Table 2).

Another symbiotic phenotype of the azide resistant mutants was found to be enhanced nodulation. Plants inoculated with strains 18-7 and M126 (C4) had 4 and 3.8 nodules on an average from 5 replications, respectively, while 2.8 on plants nodulated by strain M126 (Table 2). This observation, being statistically significant at 5% level, was in accordance with the finding that induction of azide resistance results in higher nodulation by R. loti9

.

It is hypothesized that azide, a respiratory inhibitor, being a substrate for nitrogenase gets reduced to a non-toxic product allowing bacteria to grow on azide supplemented medium. According to this, resistance to azide is possible when nitrogenase is active under free-living condition to reduce azide into ammonia thus, making it unavailable to inhibit energy generating electron transport pathway. To examine the link between azide resistance and ex-planta nitrogenase activity as reported in other rhizobia, ex­planta nitrogenase activity was studied for 18-7, M126, M126 (C4) and for Azotobacter croococcum strain W5 (positive control) grown on CS7 medium

Table 2-Total nitrogen content of chi ckpea plants nodulated with various strains

Rhizobium Resistance (R) Nodule Nodule Shoot Root N (mg g- 1

Strain Sensiti vity (S) to Azide number per dry wt/plant Fresh wt Dry wt Fresh wt Dry wt plant) plant (mg) (mg) (mg) (mg) (mg)

Control 0 1980 572 794 322 8.98 (0.7070)

18-7 R 4 5.98 2774 696 932 396 18.61 (2 .11 6)

Ml26 s 2.8 3.33 2378 654 844 384 13.83 (1.8 13)

M l26(C4) R 3.8 4.91 2532 678 938 404 17.49 (2.071)

CD at 5% 0.139 2.28 990 56 44 36 0.96

Values in parenthesis are transformed values

Fig. 3--Complementation of Az' mutant M126 with clone C4. R. ciceri strains 18-7(wt), M126 (Az' mutant) complemented strain Ml26 (C4) grown on (a) Nitrogen-free medium; and on (b) Nitrogen-free medium supplemented with 5 f,tgml' 1 sodium azide.

1182 INDIAN J EXP BIOL, DECEMBER 2004

A

8

23·0

9·4 6·5

4·3

2·">

23· 0 9·4 6·5 4·3

2 · 3 2·0

2 3 4 5

6 ·3 5·7 4·69 4·2

Fig. 4---Comparati ve restriction banding pattern of pRK290.7 and clones Cl, C3 and C4 cleaved---(a) with Xhof endonuclease: [J ane l , A Hind III ; 2, pRK290.7; 3, cloneC!; 4, cloneC3, and laneS, cloneC4]; (b) with EcoRJ endonuclease: [Lanes 1, A Hind III marker; 2, pLAFR!; 3, cloneC3; 4, clone C4 and 5, pRK290.7].

along with or without acetylene controls on slants containing only medium (negative controls). ARA estimates over 5 replications were 1.142, 1.124, 1.413 and 49.36 nmole of ethylene produced/hr/sample, respectively. Values for 18-7, M126 and M126(C4) being statistically insignificant at CD 5% level suggested the presence of a negligible level of acetylene reduction activity. So, in this case, ex­planta nitrogenase activity did not seem to play a role in conferring azide resistance/sensitivity. Therefore, our results were in accordance with the earlier views of a relationship between azide resistance and superior symbiotic nitrogen fixation, while did not support the correlation of azide resistance and ex­planta nitrogenase activity in R. ciceri.

Discussion It is hypothesized that respiratory inhibitor azide a

substrate for nitrogenase is reduced to a non-toxic product (NH3) allowing rhizobia to grow on an azide

supplemented medium6. Azide resis tance has been

found in many strains of rhizobia. A few of these were found superior N-fixers6

-9

. Resistance to lower concentrations of azide (5-10 11g mr') has been shown to be strong selection pressure for nitrogenase and linked to higher symbiotic nitrogen fixation 7. In the present study, twenty-three isolates of R. ciceri collected from the agricultural farm, Indian Agricultural Research Institute, New Delhi, were studied to know if azide resistance is related to symbiotic nitrogen fixation.

Mutants were generated by random transposon (Tn5) mutagenesis using pGS9, a suicide plasmid 16

•

Out of 13 azide sensitive mutants of wild type strain 18-7, an isolate (M126), showing higher sensitivity to sodium azide (2 11g mr 1

) was identified. A 4.5 kb nif­reiterated DNA (Xhol fragment of pRK 290.7) that confers azide resistance in R. meliloti11 was used as a probe in isolating its homologous clone from the R. ciceri Red 301 genomic library by colony hybridization. Four putative clones identified by primary and secondary screening, were mobilized into M 126 to see if they complemented for azide sensitivity. One clone C4 conferred azide resistance suggesting that it carried a gene that confers azide resistance. EcoRI restriction map of C4 clone was generated in order to find if it codes for nitrogenase (Fig. 5b). On comparison, it was found that EcoRI map of C4 clone did not match with the conventional nif HDK EcoRI restriction maps of S. meliloti and K. pneumoniae.

Whereas, in a study to find if the identified gene had nitrogenase activity conferring additional nitrogen fixation it was observed that the gene conferred 0.6 times additional nitrogen activity upon M126 resulting in higher total nitrogen content thus suggesting a link between azide resistance and nitrogen fixation.

Above results, gain support from the fact that azide, which is one of the substrates of nitrogenase enzyme encoded by nifHDK is detoxified by its action in diazotrophs . While the EcoRI map of C4 clone does not conform with already existing restriction maps of nif HDK of S. meliloti and K. pneumoniae, the clone showed similar Xhol (6 .3 and 5.7kb) and EcoRI (6.3 and 5kb) fragments as that of pRK290.7 (4.7, 4.2 kb Xhol and 4.0, 4.5 EcoRI fragments) that carried additional nif DNA used as a probe in DNA hybridization (Fig. 4). Thus, it is likely that azide resistance is linked to nif-reiteration. The existence of nif-reiterated DNA is a known phenomenon in

BHASKAR & KASHYAP: AZIDE RESISTANCE IN RHIZOBIUM CICERI 1183

diazotrophs . Functional multigene family of nitrogen reductase was known to exist in R. phaseoli. It was found to exist as two nif H copies in Rhizobium ORS 571. A symbiotic cluster on the pSym megaplasmid of S. meliloti was reported to carry a functional fix gene repeat and a nod locus (Reinelier et a/.

2 1). This is

reminescent of the existence of different copies of nif HDK in Azotobacter that encodes different types of nitrogenases for efficient nitrogen fixation under special conditions (Kennedy et a!. 22

) . In the light of these findings, if nif- reiterated DNA expresses in several diazotrophs under free-living conditions, azide resistance could be linked to nif- reiterated DNA.

On contrary to the above hypothesis , strains 18-7, M126 and M126 (C4) did not show ex-planta nitrogenase activity to a significant level, while strain

8

A

2 3·0 9 · 4 6 · 5 4· 3

E E

7

E E

K. pneumoniae nif region

Q I:JA L FMVSUN E K D H J I I I I I I I I I I I I I

R

niffragment B

H //,./ H

nif fragment A."E '_· _.L___,_K,__--.'1 __ Dr-...l-"-'H--I\ r I I R A1 H A2 B A3 R

R. meliloti Nif region Region of Nif homology .. X BH R H X H R B

Fig. 6-Restriction maps of nif regions from Klebsiella pnewnoniae and S. meliloti Endonuclease sites are indicated below each map by X, Xhoi ; R, EcoRI ; H, Hind III and B, Bam H I (Ruvkun and Ausubel, 1980)19

•

E

23 · 9· 6 · 4·

E E E

K b

7 90

-3·8 6 - 3 ·41

E

3 · 2 0

I · 9 4

rss:s ~~--------~------~--~,_----4---+-----~

0.58 5.34 4.58 0.76 0.99 2.2 1.21 3.51

• ..... 1.94

• • ~--. - - - - ____ ..,...

5.79

i·2 • • 3. ~

3.86

9.99 7.9

Fig. 5---(a) EcoRl restriction map of clone C4 digested at various time intervals. [Lanes l , )... Hind III marker; 2 to 8, clone C4 digested with EcoRI endonuclease for a period of 10, 20, 30, 40, 50, 60 min and for 3 hr. Sizes (kb) of partially restricted fragments are indicated];

(b) EcoRI restriction map of clone C4.

1184 INDIAN J EXP BIOL, DECEMBER 2004

Nitrogenase activity nitrogen fixation could be used for improvement of

60

~

~

.<: ~ <0 c: ·~ Q. ~ I N 30 u 0 " "' 0

20 E "-

10

Control 18-7{WT) M126 M126{C4)

Fig. ?-Nitrogenase activity (measured by ARA) shown by plants nodulated with strains 18-7(WT), M126 and M126 (C4).

Ml26 was affected in its symbiotic nitrogenase activity. This observation can be explained with another existing evidence that expression of fixNOQP is responsible for azide resistance and is linked to symbiotic nitrogen fixation in rhizobia. In the presence of azide-imposed inhibition on cytochrome­C oxidase, Cbb3 (terminal oxidase) encoded by fixNOQP operon completes electron transfer by a branch pathway22

. This pathway being active in AzR strains results in the generation of relatively more ATPs in azide resistant strains over sensitive ones. Since, N2 fixation requires around 20 ATP molecules to reduce one N2 molecule, azide resistant strains have selective advantage of more nitrogenase activity over sensitive mutants. This explanation supports our findings that azide resistant wild type as well as the mutant with C4 clone showed higher nitrogenase activity leading to more total nitrogen content in shoots.

An important hypothesis, for correlation between azide-resistance and higher symbiotic nitrogen fixatibn that emerges from this study is the presence of functional nif reiteration and/or the induction of derepressed expression of fixNOQP genes encoding symbiotic terminal oxidases23 that overcome the block imposed by azide on energy generating system. In the light of the current knowledge, it seems azide resistance is specified by many a loci and nifHDK and fixNOQP are two such loci which have direct bearing on superior symbiotic performance of the strain. Azide resistance being associated with superior

symbiotic effectiveness of various strains of Rhizobium.

Acknowledgement Financial Assistance in the form of JRF and SRF to

author from Council of Scientific and Industrial Research, New Delhi.

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BHASKAR & KASHY AP: AZIDE RESISTANCE IN RHIZOBIUM CICERI 1185

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