A cyanobacterial mutant resistant against a bleaching herbicide

J. Basic Microbiol. 40 (2000) 4, 279–288

(1Photobiological Nitrogen Fixation Research Laboratory, Department of Genetics and Plant Breed-ing, Institute of Agricultural Sciences, Banaras Hindu University, Varanasi-221005 India and 2Institutfür Botanik und Pharmazeutische Biologie, Friedrich-Alexander-Universität, Staudtstr. 5, D-91058Erlangen, Germany)

A cyanobacterial mutant resistantagainst a bleaching herbicide

A. VAISHAMPAYAN1, R. P. SINHA1, 2, A. K. GUPTA1 and D.-P. HÄDER2

(Received 25 January 2000/Accepted 17 April 2000)

The nitrogen fixing cyanobacterium Nostoc muscorum has been found to be sensitive to the herbicideSAN 6706 [4-chloro-5 (dimethylamino)-2-(a,a,a,-trifluoro-m-tolyl)-3-(2H) pyridazinon] at 30–45 µMwithin 15 min. The toxicity was more severe in combined nitrogen-free (Ncomb-free) medium than in acombined nitrogen medium; this enhancement was reversible by supplementation of the medium with3 mM glucose or 5 µM ATP, serving as carbon and/or energy source in this organism. A mutant of thiscyanobacterium resistant to 3 mM SAN 6706 has been isolated and characterized to perform nitroge-nase activity in exogenous ATP supplemented Ncomb-free medium. However, it exhibited a moderategrowth in combined nitrogen media in the absence of external ATP. The resistance factor is higherthan 100. Simultaneously, this strain possesses a cross-resistance to methylamine, a well-knowninhibitor of photophosphorylation, irrespective of the exogenous ATP supply. The behavior of themutant suggests a defective phosphorylation in its photosynthetic system.

Application of herbicides in agriculture is a wide spread practice to suppress weed growthand increase crop yields (CHATTOPADHYAY 1987). However, massive use of these chemi-cals poses a potential danger to agriculturally important N2-fixing cyanobacteria (GREAVE1982, ZARGAR and DAR 1990, VAISHAMPAYAN et al. 1998). Most of the earlier studies onthis aspect have been confined to screening for the lethal and semi-lethal concentrations ofvarious agro-chemicals in cyanobacteria with little emphasis on the possible mode(s) ofaction in these organisms (VENKATARAMAN and RAJYALAKSHMI 1972, MEHTA andHAWXBY 1979, ROGER and KULASOORIYA 1980).

The present work attempts to improve the situation, reporting results on the biologicaleffects of the herbicide SAN 6706 [4-chloro-5 (dimethylamino)-2-(a,a,a,-trifluoro-m-tolyl)-3-(2H) pyridazinon] on the N2-fixing cyanobacterium Nostoc muscorum by isolation andphysiological characterization of a unique mutant resistant to 3 mM SAN 6706 showingan absolute dependence on exogenous ATP for N2 fixation and growth in Ncomb-free me-dium.

Materials and methods

The organism: The filamentous, heterocystous and N2-fixing cyanobacterium Nostoc muscorum,isolated from a paddy field in Darbhanga, India (axenic strain No. Nm 01 of the L. N. M. UniversityCulture Collection Centre, Darbhanga, India), was cultured in modified Chu 10 medium (GERLOFFet al. 1950) supplemented with 5 mM KNO3. The organism neither forms heterocysts nor fixes nitro-gen in the presence of external nitrogen, such as NO3

–, NO2– or NH4

+, but on transfer from a combined Nmedium to a fresh Ncomb-free medium it starts to form heterocysts (with a frequency of 5–6 per hun-dred vegetative cells) and fixing nitrogen within 20–24 h. Like other wild type isolates of Nostoc

280 A. VAISHAMPAYAN et al.

muscorum, this Nostoc strain is non-mucilagenous and forms a homogeneous suspension in liquidculture and discrete colonies on solid agar medium. This property makes this organism suitable forphysiological and genetic studies, and facilitates its use as a test material in the described investiga-tion.

Test of physiological effects: For assessing the physiological effects of SAN 6706 on Nostoc musco-rum, increasing concentrations of the herbicide were prepared in double distilled water and applied toan exponential-phase culture of N. muscorum (strain Nm 01) for 15 min. The controls and the treatedexperimental samples were washed and inoculated in sterile Ncomb-free medium as well as mediumenriched with 5 mM NO3

–, supplemented or not with 3 mM glucose and/or 5 µM ATP. ExogenousNADPH

2 was not used here as it had no protective effects on SAN 6706-induced toxicity in this

organism.

Determination of growth and heterocyst frequency: All experimental samples were inoculated in agrowth chamber at a temperature of 28 � 2 °C and a fluorescent light irradiance (400–700 nm) ofabout 10 W/m2 under aseptic conditions, after VAISHAMPAYAN (1981). Growth was measured byoptical density determinations at 663 nm after 8 days of inoculation (which is the peak day of expo-nential growth phase) as well as on every second day (starting from day zero). The maximum hetero-cyst frequency, generally obtained between 4–6 days after inoculation was determined microscopi-cally as the number of heterocysts per hundred vegetative cells, after VAISHAMPAYAN (1982a). Theuptake of exogenous ATP was measured in terms of the concentration left unutilized in the mediumafter 8 days of growth. It was determined biochemically using the luciferase method of LEHNINGER(1982).

Determination of survival: Survival studies were determined after treating Nm 01 strain populationsof equal known cell densities for 15 min with different concentrations of the herbicide, ranging be-tween 0–6 µM. The liquid suspensions of the controls and treated samples were fragmented to indi-vidual cells by continuous one-hour gentle shaking with sterilized glass beads, after VAISHAMPAYAN(1983), which were inoculated in PETRI dishes in five replicates containing 1% agar medium (Ncomb-free or 5 mM NO3

–) � 3 mM glucose and/or 5 µM ATP. The inoculates were transferred to a growthchamber and showed colonial growth of the viable cells after two weeks of incubation. The number ofcolonies was determined and compared with the untreated populations for the assessment of the sur-vival under the various treatments. The data were plotted as per cent survival on a log graph and fittedwith smooth curves from which the LD50 values for the herbicide were determined for the differentgrowth conditions (Ncomb-free or NO3

– medium � glucose and/or ATP).

Selection of mutant: The LD50 of the herbicide was found to be close to 30 µM. A mutant of Nm 01,resistant to 3 mM SAN 6706 was selected following the selective plating technique of VAISHAMPAYANand SINGH (1981a, b). For this purpose an exponentially growing culture of Nm 01 was harvested inmass, separated into single cells by gentle shaking with sterilized glass beads and spread on Ncomb-freeor 5 mM NO3

– solid (1% agar) medium supplemented with 3 mM SAN 6706. These samples, alongwith the untreated controls (also Ncomb-free or NO3

– agar media) were incubated in the growth chamberfor two weeks. The treated samples, inoculated on herbicide-supplemented PETRI dishes, exhibitedvery fast killing, and no colonial growth was obtained till 15 days after inoculation. Spontaneouslyoccurring SAN 6706-resistant putative mutant colonies were located exclusively on NO3

– medium(there was no mutant colonial growth on Ncomb-free medium). The number of putative mutant colonieson the NO3

–-supplemented set of herbicide-containing PETRI dishes was compared with that of thecontrol to determine the spontaneous mutation frequency for 3 mM SAN 6706 resistance. Many suchputative mutant colonies were separately raised to axenic clonal cultures and subjected to stabilitytests for 10 successive generations in herbicide-free NO3

– medium (the mutant failed to grow in Ncomb-free medium). All those which maintained the SAN 6706 resistance up to the 11th transfer to 3 mMSAN 6706-containing NO3

– medium were regarded as stable. The best performing one was designatedas Nm 62 mutant strain and deposited at the L. N. M. University Culture Collection Centre, Darb-hanga, India.

Cross resistance examination: Since SAN 6706 induced effects similar to those observed earlier formethylamine (MA) in Nostoc muscorum (ORTEGA et al. 1977, SINGH et al. 1979, VAISHAMPAYAN and

Herbicide-resistant cyanobacterial mutant 281

SINGH 1981a, VAISHAMPAYAN 1982b), the cross resistance to the toxic concentration of this chemicalwas examined in wild type (Nm 01) and the SAN 6706-resistant mutant (Nm 62). Furthermore, themutant was characterized for its growth with or without 3 mM SAN 6706 in Ncomb-free and NO3

– media,supplemented or not with 3 mM glucose and/or 5 µM ATP/1 mM MA. The heterocyst frequency andN2-fixing (acetylene reducing) activity of wild type Nostoc muscorum and its Nm 62 mutant weredetermined, after VAISHAMPAYAN (1983).

Determination of LD50 and resistance factor of mutant: The LD50 of the mutant Nm 62 was deter-mined by inoculating equal cell concentrations on Ncomb-free and 5 mM NO3

– agar media supplementedwith 0–6 mM SAN 6706 � 5 µM ATP or 1 mM MA, and plotting the survival logarithmically withfitting curves. The resistance factor of the Nm 62 mutant for SAN 6706-resistance was determined bydividing the LD50 value for SAN 6706 by that obtained for the wild type strain Nm 01.

Statistical analysis and chemicals: Mean values and standard error were determined from five repli-cates for each treatment (WALPOLE and MYERS 1978). ATP was obtained from SIGMA Chemical Co.,St. Louis, Missouri, USA. All other analytical chemicals and medium constituents were of C.P. grade,B.D.H., India. The herbicide was supplied by courtesy of LAXAMI Chemical Co., Phatuha, India.

Results and discussion

The herbicide inhibited growth in the wild type Nostoc muscorum (Nm 01) in both Ncomb-free and NO3

– media depending on its concentrations used (Figs. 1 and 2, respectively).Apparently, growth inhibition was higher in Ncomb-free medium than in NO3

– supplementedmedium. However, the wild type strain could grow in neither Ncomb-free nor NO3

– supple-mented medium in the presence of 60 µM SAN 6706. The organisms exhibited a decrease inper cent survival in dependence of herbicide concentration (Fig. 3). Herbicide concentra-tions of 28.5 µM and 33.3 µM were found to permit 50% survival of the wild type organism(LD50) in Ncomb-free and NO3

– media, respectively.

Fig. 1Growth of wild type Nostoc muscorum (Nm 01), untreated (solid line) or treated for 15 min with30 µM (broken line), 45 µM (broken line with one dot) or 60 �M (broken line with two dots) SAN 6706,in Ncomb-free medium, without (�) or with 3 mM glucose (�) or 5 µM ATP (�); the range of SE was� 0.006 to 0.020; the effect of the simultaneous application of glucose and ATP was significantly notmuch different than that of glucose alone, thus data for the additive effect of glucose plus ATP are notshown


Fig. 2Growth of wild type Nostoc muscorum (Nm 01), untreated (solid line) or treated for 15 min with30 µM (broken line), 45 µM (broken line with one dot) or 60 �M (broken line with two dots) SAN 6706,in 5 mM NO3

– medium, without (�) or supplemented with 3 mM glucose (�) or 5 µM ATP (�); the rangeof SE was � 0.012 to 0.018; the effect of the joint application of glucose plus ATP was significantlynot much different than that of glucose alone, thus data for the additive effect of glucose plus ATP arenot shown

Fig. 3Percent survival of wild type Nostoc muscorum (Nm 01), treated for 15 min with graded concentra-tions of the herbicide SAN 6706, on Ncomb-free (solid line) or 5 mM NO3

– containing (broken line) agarmedium, supplemented (�) or not with 3 mM glucose (�) or 5 µM ATP (�); the range of SE was � 4 to10%; the effect of the joint application of glucose plus ATP was significantly not much different fromthat of glucose alone, thus data for the additive effect of glucose plus ATP are not shown;the vertical arrows pointing to the X-axis indicate the LD50 of the herbicide in Ncomb-free and NO3

–

media


In parallel to suppressing growth, the herbicide was found to inhibit the heterocyst-forming activity of the wild type organism in Ncomb-free medium. The heterocyst frequencyof Nm 01 gradually declined with increasing concentrations of the chemical, and no hetero-cyst was detected after treatment with 60 µM SAN 6706 (Table 1).

However, the inhibitory effects of the herbicide at concentrations of up to 45 µM ongrowth in Ncomb-free (Fig. 1) or NO3

– (Fig. 2) medium and its heterocyst-forming activity inNcomb-free medium (Table 1) were significantly reversed on supplementation with exoge-nous glucose or ATP. In addition, a recovery of nearly 12–24% survival was found insamples treated with even the highest concentration of the herbicide used (60 µM) afterexogenous supplementation with glucose and/or ATP (Fig. 3). It is important to point outthat Nostoc muscorum is a photoheterotrophic organism (SINGH and VAISHAMPAYAN1978); it has been shown to utilize exogenous glucose as a carbon and energy source(VAISHAMPAYAN 1982b). However the present results indicate that the SAN 6706 treatedwild type culture is primarily energy-limited and that it can also utilize exogenous ATP as aready energy source. The uptake of externally applied ATP has been found to be equal inboth Ncomb-free and NO3

– media (Table 1). Glucose is preferred when jointly applied withATP (Table 1). Recovery of growth and heterocyst frequency in the herbicide-treatedsamples with glucose plus ATP was not much different from that of glucose alone (Figs. 1and 2; Table 1).

Moreover, experimental samples treated with higher concentrations of the herbicideshowed lower consumption of ATP, coupled with lower recovery of growth and heterocyst

Table 1Data* on maximum heterocyst frequency (generally obtained between 4–6 days of inoculation asnumber of heterocysts per hundred vegetative cells)**) of the wild type Nostoc muscorum (Nm 01)untreated or treated for 15 min with graded concentrations of the herbicide San 6706, in Ncomb-free or5 mM NO3

– medium, with or without 3 mM glucose and/or 5 µM ATP***

Heterocyst frequencyHerbicidetreatment(µM for 15 min)

Exogenoussupplement(s)

Ncomb-free NO3–

0 NILGlucoseATP***Glucose + ATP***

5.8 � 0.16.2 � 0.15.9 � 0.2 (1.9 � 0.1)***6.3 � 0.2 (4.1 � 0.2)***

0.00.00.0 (2.0 � 0.1)***0.0 (4.1 � 0.1)***


5.5 � 0.16.1 � 0.05.8 � 0.1 (2.5 � 0.1)***6.0 � 0.1 (4.0 � 0.1)***

0.00.00.0 (2.6 � 0.2)***0.0 (4.1 � 0.1)***


3.3 � 0.15.1 � 0.14.3 � 0.1 (3.2 � 0.1)***5.2 � 0.1 (4.0 � 0.2)***

0.00.00.0 (3.5 � 0.1)***0.0 (4.1 � 0.2)***


0.0 (Dead)0.0 (Dead)0.0 (Dead) (4.6 � 0.3)***0.0 (Dead) (4.8 � 0.1)***

0.0 (Dead)0.0 (Dead)0.0 (Dead) (4.7 � 0.1)***0.0 (Dead) (4.8 � 0.0)***

* The values are means of five independent readings with standard error** Heterocyst frequency on day zero was zero; heterocysts were never formed in NO3

– medium*** The values in parentheses show the quantity of ATP (µM) left unutilized in the medium after

8 days of incubation


frequency (Table 1), suggesting that higher concentrations of SAN 6706 adversely affectsthe transport of exogenous ATP into the cells. However, it is also possible that parts of thepopulation might have already died.

The observed effects of SAN 6706 appear to be similar to those of MA which is knownto act as an inhibitor of photophosphorylation in Nostoc muscorum (ORTEGA et al. 1977)and other cyanobacteria such as Plectonema boryanum (PADAN and SCHULDINER 1978).This inhibitory effect of MA has been found to be reversed by exogenous supplementationwith ATP, serving as a carbon and/or energy source in Nostoc muscorum (VAISHAMPAYAN1982b). Since the same exogenous substrates effectively reduced the inhibitory effects ofSAN 6706 in this cyanobacterium, and since SAN 6706 contains the dimethylamino groupin its chemical constitution with a biochemical action known to be similar to methylamine(DODGE 1975), it is likely that SAN 6706 operates as an inhibitor of photophosphorylation.Further, since the inhibitory effect of the herbicide was observed to be more pronouncedunder N2-fixing conditions, and since energy for N2 fixation is supplied by cyclic photo-phosphorylation (FOGG et al. 1973), the herbicide appears to primarily affect cyclic photo-phosphorylation in this organism. Detailed biochemical studies are, however, needed tosubstantiate this proposition.

The spontaneous mutation frequency of the wild type Nostoc muscorum (Nm 01) to3 mM SAN 6706 resistance was found to be 2.06 � 0.49 � 10–9, and the mutant appearedexclusively in NO3

– medium (Table 2). No mutant colony emerged in Ncomb-free medium.This mutant designated as Nm 62 strain of Nostoc muscorum, was highly stable in NO3

–

medium. This stability was found to be similar in Ncomb-free medium but only if supple-mented exogenously with ATP or MA (Table 2). The LD50 of this mutant was found to be4.25–4.65 mM SAN 6706 in Ncomb-free medium supplemented with 5 µM ATP/1 mM MA(the mutant did not survive at all without ATP or MA in Ncomb-free medium) or NO3

– me-dium �5 µM ATP/1 mM MA (Fig. 4). The resistance factor was thus found to be in the

Fig. 4Percent survival of 3 mM San 6706-resistant Nostoc muscorum (Nm 62), on Ncomb-free (solid line) or5 mM NO3

– containing (broken line) agar medium with no exogenous supplement (�), or with 5 µMATP (�) or 1 mM methylamine (�), supplemented with zero to 6 mM concentrations of the herbicideSAN 6706; the range of SE was �3 to 14%; at such high concentrations no additive effect of glucosecould be detected, and thus data are not shown for the survival of the mutant with glucose; the verticalarrows pointing to the X-axis indicate the LD50 of the herbicide in the mutant in Ncomb-free +ATP/methylamine and NO3

– � ATP/methylamine media


Table 2Data* on (i) spontaneous mutation frequency to 3 mM SAN 6706-resistance, (ii) per cent stabilityof San 6706-resistant mutant (strain No. Nm 62) and (iii) SAN 6706-resistance factor (LD50 ofSAN 6706 in Nm 62/LD50 of SAN 6706 in Nm 01) in Ncomb-free or 5 mM NO3

– medium � 5 µM ATP or1 mM methylamine (MA)

Medium Supplement Spontaneous mutationfrequency

Per cent stability** Resistance factor

Ncomb-free NILATPMA

ND***NC****NC****

Nil18.71/25 (74.84%)17.04/25 (68.16%)

Nil149.12 � 14.17161.40 � 23.08

NO3– NIL

ATPMA

2.06 � 0.49 � 10–9

NC****NC****

14.22/17 (83.65%)23.44/25 (89.76%)20.61/22 (93.68%)

150.15 � 18.38133.63 � 11.55139.64 � 20.72

* The values are the means of five independent readings with standard errors** Mean number of stable mutant colonies tested for stability per replicate (values in parentheses

are those for the per cent stable colonies for 3 mM SAN 6706-resistance).*** Not detected.**** Not conducted.

range between 150–160 in Ncomb-free plus ATP/MA medium and 130–150 in NO3– medium

� ATP/MA, which makes it exciting enough for detailed future genetic analysis and bio-chemical characterization of the mutant.

While the wild type strain Nm 01 shows growth, heterocyst differentiation and nitroge-nase activity strictly in herbicide-free medium, the Nm 62 mutant has shown these activitiesalmost equally well in medium supplemented or not with 3 mM SAN 6706 (Figs. 5 and 6;Table 3). However, it failed to grow in Ncomb-free medium (Fig. 5) and grew slower than thewild type strain in herbicide-free NO3

– medium (Fig. 2). Remarkably, exogenous supple-mentation with ATP supported considerable growth of the mutant in Ncomb-free medium(Fig. 5) and little change in growth upon NO3

– supplementation (Fig. 6). Further, in sharpcontrast to the wild type strain, the Nm 62 mutant could not fix nitrogen unless the Ncomb-free medium was supplemented with ATP (Fig. 3), suggesting that the mutant had possiblya defective cyclic photophosphorylation which is the prime source of energy for N2 fixationand growth of cyanobacteria in Ncomb-free medium (FOGG et al. 1973).

Another important feature of the Nm 62 mutant is its observed simultaneous cross-resistance to 1 mM MA in both Ncomb-free and NO3

– media, in sharp contrast to the wild typestrain which could not survive at this MA concentration in the medium (Table 3). It hasbeen reported earlier that MA is metabolized as a nitrogen source in 5 mM MA-resistantmutants (Nm 09, Nm 18 and Nm 19) of N. muscorum (SINGH et al. 1979, VAISHAMPAYANand SINGH 1981a, VAISHAMPAYAN 1982b). In the present class of 3 mM SAN 6706-resistant Nm 62 mutant too, we have evidence for the metabolic utilization of MA as anitrogen source. This is in view of the fact that MA could support a significant growth ofthe Nm 62 mutant in Ncomb-free medium without supplementation with any exogenous ATP(Fig. 5). Here, MA definitely serves as a metabolizable nitrogen source like NO3

– rather thanan energy source. This is substantiated by the finding that application of MA completelysuppressed heterocyst differentiation and nitrogenase activity, as typically observed withNO3

– (Table 3), exactly similar to the observations reported for the various sources of com-bined nitrogen (VAISHAMPAYAN 1983).

The results, therefore, strongly suggest that there is a link between SAN 6706 and MA,the former of which is a very likely an inhibitor of photophosphorylation in the N2-fixingcyanobacterium Nostoc muscorum, and that the 3 mM SAN 6706-resistant Nm 62 mutantmay have a defective cyclic photophosphorylation conferring resistance to the SAN 6706


Fig. 5Growth of 3 mM SAN 6706-resistant Nostoc muscorum mutant (Nm 62) in Ncomb-free medium with noexogenous supplement (�), or with 5 µM ATP (�) or 1 mM methylamine (�), not supplemented (solidline) or supplemented with 3 mM (broken line), 4.5 mM (broken line with one dot) or 6 mM (brokenline with two dots) SAN 6706; the range of SE was �0.011 to 0.074; at such high concentrations noeffect of glucose could be detected, and data for growth of the mutant with glucose are not shown.In the wild type (Nm 01), methylamine (1 mM used here) proved acutely toxic, while ATP had amarginal additive effect (data not shown)

Fig. 6Growth of 3 mM SAN 6706-resistant Nostoc muscorum mutant (Nm 62) in 5 mM NO3

– medium withno exogenous supplement (�), or with 5 µM ATP (�) or 1 mM methylamine (�), without (solid line) orsupplemented with 3 mM (broken line), 4.5 mM (broken line with one dot) or 6 mM (broken line withtwo dots) SAN 6706; the range of SE was �0.010 to 0.034; at such high herbicide concentrations noeffect of glucose could be detected, and thus data are not shown for growth of the mutant with glu-cose. In the wild type (Nm 01), methylamine (1 mM used here) proved acutely toxic, while ATP had amarginal additive effect (data not shown)


Table 3Data* on maximum heterocyst frequency (HF) (obtained between 4–6 days of inoculation as numberof heterocysts per hundred vegetative cells)** and nitrogenase activity/acetylene reducing activity(ARA) (n moles ethylene per mg protein per hour, assessed on the day of maximum heterocyst fre-quency) of wild type Nostoc muscorum (Nm 01) and its 3 mM SANn 6706-resistant mutant (Nm 62)in herbicide-free or 3 mM SAN 6706 containing Ncomb-free or 5 mM NO3

– medium, supplemented or notwith 5 µM ATP or 1 mM methylamine (MA)

Supplement StrainParameter N source �3 mM San6706

Parent (Nm 01) Mutant (Nm 62)

HF Ncomb-free

NO3–

(–)(–)(–)(+)(+)(+)(–) or (+)

NilATPMANilATPMANil, ATP or MA

5.8 � 0.15.9 � 0.20.00.00.00.00.0

7.2 � 0.45.8 � 0.20.07.3 � 0.35.7 � 0.20.00.0

ARA Ncomb-free

NO3–

(–)(–)(–)(+)(+)(+)(–) or (+)

NilATPMANilATPMANil, ATP or MA

41.450 � 2.37743.720 � 2.8140.00.00.00.00.0

0.040.013 � 5.1070.00.042.126 � 6.0110.00.0

* The values are the means of five independent readings with standard error** Heterocyst frequency on the day zero was zero in view of the use of NO3

– grown non-heterocystous mother culture of the respective strains. Heterocysts were never found in NO3

–

medium in either of the two strains, supplemented or not with MA or ATP

sensitive site in this organism. Further experiments on ATP yield, catabolic end productsand the capacity of Nm 62 mutant to form glutamine from MA and glutamate (by a direct invitro glutamine synthetase assay) are in progress in attempt to confirm the existence andnature of an MA-metabolizing enzyme in the Nm 62 mutant.

Acknowledgements

A.V. gratefully acknowledges the receipt of financial assistance from the ICAR, New Delhi (GrantNo. 7-15-SW & DF) for this work as a part of All-India Coordinated Research Project on BiologicalNitrogen Fixation. This work was financially supported in part by the CSIR, New Delhi, India (GrantNo. 9/13(795)/96-EMR-I(RK)213108) to R. P. S. We thank M. SCHUSTER for excellent technicalassistance.

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Mailing address: Prof. Dr. D. P. HÄDER, Institut für Botanik und Pharmazeutische Biologie, Friedrich-Alexander-Universität, Staudtstr. 5, D-91058 Erlangen, GermanyTel.: +49 9131 852 8216; Fax: +49 9131 852 8215e-mail: [email protected]

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A cyanobacterial mutant resistant against a bleaching herbicide