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The development of the heterocyst by filamentous nitrogen-fixing cyanobacteria provides an attractive model system for studying cellular differentiation. Heterocyst synthesis is repressed by the presence of exogenous combined nitrogen. In this report, it is shown that the tryptophan analog, D,L-7-azatryptophan (Aza-T), is capable of relieving the repressive effect of exogenous NH4NO3 on heterocyst and nitrogenase synthesis. In nitrogen-fixing cultures, the presence of 20 micron Aza-T increases the heterocyst frequency twofold. The glutamate analog, L-methionine-D,L-sulfoximine (MSX), has also been shown to cause a derepression in the synthesis of heterocysts and nitrogenase. However, unlike MSX, Aza-T does not appear to exert its effects by inhibiting the activity of glutamine synthetase. Therefore, glutamine synthetase may not be the sole key to the derepression of heterocyst and nitrogenase development in the cyanobacteria. It is hoped that a study of Aza-T action may lead to the elucidation of a novel control mechanism.

Metabolically active heterocysts were isolated from a mutant of Anabaena sp. strain CA with fragile vegetative cells. Heterocysts isolated from cultures grown in 1% CO2 in air reduced C2H2 at 57 and 10 nmol of C2H2 per mg (dry weight) per min under H2 and Ar, respectively. However, if whole filaments were sparged with 1% CO2 in 99% Ar for 12 h before heterocyst isolation, these heterocysts showed C2H2 reduction rates of 83 nmol of C2H4 per mg (dry weight) per min under either H2 or Ar, or 40% the activity of whole filaments grown in 1% CO2 in air. Heterocysts isolated from cultures sparged with 100% Ar or 1% CO2 in 99% N2 had the same C2H2 reduction pattern as heterocysts from cultures grown in 1% CO2 in air, i.e., low activity under Ar and high activity under H2. Labeling of whole filaments incubated with NaH14CO3 for 12 h under 1% CO2 in air or 1% CO2 in 99% Ar resulted in a twofold higher accumulation of 14C-labeled compounds in vegetative cells and heterocysts of Ar-incubated cells. Our results suggest that during incubation under 1% CO2 in 99% Ar, presumably a nitrogen starvation condition, continuing photosynthetic fixation of CO2 leads to accumulation of material(s) in the heterocysts that supports a high, persistent endogenous rate of C2H2 reduction. This material appears to be, in part, glycogen.

A mutant has been isolated from Anabaena sp. strain CA by treatment with N-methyl-N'-nitro-N-nitrosoguanidine, which has the unusual phenotypic characteristic of growth only under N2-fixing conditions. Growth of the mutant was completely inhibited by NO3- or NH4+ at concentrations routinely used for growth of the wild type, and sensitivity to NH4+ was especially pronounced. The inhibitory effect of NH4+ could not be overcome by glutamine, glutamate, or casein hydrolysate. Ammonia had no immediate inhibitory effect on protein synthesis, CO2 fixation, or O2 evolution, and the gradual inhibition of C2H2 reduction activity by NH4+ resembled a repression phenomenon. The glutamine synthetase activity of N2-fixing cultures appeared normal, yet the mutant was incapable of utilizing exogenous NH4+ for growth. Preliminary evidence suggests a possible alteration of glutamine synthetase, which could result in sensitivity to exogenous NH4+ by progressive inactivation of the enzyme or repression of its synthesis.

Changes in protein composition were noted when heterocysts of Anabaena sp. strain CA were isolated from filaments grown in 1% CO2-99% N2 and subsequently exposed to oxygen. Immunospecific Western blot analysis showed that the Fe protein of nitrogenase is altered. In cells grown under microaerobic conditions, the Fe protein was found in a form with an apparent molecular weight of 30,000. Exposure to oxygen caused a shift in the migration of this polypeptide to a position corresponding to an apparent molecular weight of 31,500. This modification was reversible upon removal of oxygen from the culture. Chloramphenicol did not inhibit the alteration in either direction. Suppression by ammonium nitrate of the recovery of nitrogenase activity from the effects of oxygen did not prevent the alteration of the protein. Other inhibitors of nitrogenase activity, (metronidazole, carbonyl cyanide m-chlorophenylhydrazone, and phenazine methosulfate) were tested for their effect on Fe protein modification. Alteration of the Fe protein may relate to the protection of nitrogenase from the deleterious effects of oxygen.

An alternative route for the primary assimilation of ammonia proceeds via glutamine synthetase-carbamyl phosphate synthetase and its inherent glutaminase activity in Anabaena sp. strain 1F, a marine filamentous, heterocystous cyanobacterium. Evidence for the presence of this possible alternative route to glutamate was provided by the use of amino acid analogs as specific enzyme inhibitors, enzymological studies, and radioistopic labeling experiments. The amino acid pool patterns of continuous cultures of Anabaena sp. strain 1F were markedly influenced by the nitrogen source. A relatively high concentration of glutamate was maintained in the amino acid pools of all cultures irrespective of the nitrogen source, reflecting the central role of glutamate in nitrogen metabolism. The addition of 1.0 microM azaserine increased the intracellular pools of glutamate and glutamine. All attempts to detect any enzymatic activity for glutamate synthase by measuring the formation of L-[14C]glutamate from 2-keto-[1-14C]glutarate and glutamine failed. The addition of 10 microM DL-7-azatryptophan caused a transient accumulation of intracellular citrulline and alanine which was not affected by the presence of chloramphenicol. The in vitro activity of carbamyl phosphate synthetase and glutaminase increased severalfold in the presence of azatryptophan. Results from radioisotopic labeling experiments with [14C]bicarbonate and L-[1-14C]ornithine also indicated that citrulline was formed via carbamyl phosphate synthetase and ornithine transcarbamylase. In addition to its effects on nitrogen metabolism, azatryptophan also affected carbon metabolism by inhibiting photosynthetic carbon assimilation and photosynthetic oxygen evolution.

The addition of DL-7-azatryptophan (AZAT), a tryptophan analog, to continuous cultures of Anabaena sp. strain CA grown with 10 mM nitrate as the nitrogen source resulted in the differentiation of heterocysts. Analysis of the intracellular amino acid pools of Anabaena sp. strain CA after the addition of AZAT showed a marked decline in the intracellular glutamate pool and a slight increase in the levels of glutamine. The in vitro activity of glutamate synthase, the second enzyme involved in primary ammonia assimilation in Anabaena spp., was partially inhibited by the presence of AZAT at concentrations which are effective in triggering heterocyst formation (15% inhibition at 10 microM AZAT and up to 85% inhibition at 1.0 mM AZAT). Azaserine, a glutamine analog and potent glutamate synthase inhibitor, had no effect on the triggering of heterocyst development from undifferentiated batch and continuous cultures of Anabaena sp. strain CA. However, the presence of 1.0 microM azaserine significantly decreased the intracellular glutamate pool and increased the glutamine pool. The addition of AZAT also caused a decrease in the C-phycocyanin content of Anabaena sp. strain CA as a result of its proteolytic degradation. AZAT also had an inhibitory effect on the nitrogenase activity of Anabaena sp. strain CA. All these results suggest that AZAT causes a general nitrogen starvation of Anabaena sp. strain CA filaments, triggering heterocyst synthesis.

Metabolically active heterocysts isolated from wild-type Anabaena sp. strain CA showed high rates of light-dependent acetylene reduction and hydrogen evolution. These rates were similar to those previously reported in heterocysts isolated from the mutant Anabaena sp. strain CA-V possessing fragile vegetative cell walls. Hydrogen production was observed with isolated heterocysts. The ratio of C2H4 to H2 produced ranged from 0.9 to 1.2, and H2 production exhibited unique biphasic kinetics consisting of a 1 to 2-min burst of hydrogen evolution followed by a lower, steady-state rate of hydrogen production. This burst was found to be dependent upon the length of the dark period immediately preceding illumination and may be related to dark-to-light ATP transients. The presence of 100 nM NiCl2 in the growth medium exerted an effect on both acetylene reduction and hydrogen evolution in the isolated heterocysts from strain CA. H2-stimulated acetylene reduction was increased from 2.0 to 3.2 mumol of C2H4 per mg (dry weight) per h, and net hydrogen production was abolished. A phenotypic Hup- mutant (N9AR) of Anabaena sp. strain CA was isolated which did not respond to nickel. In isolated heterocysts from N9AR, ethylene production rates were the same under both 10% C2H2-90% Ar and 10% C2H2-90% H2 with or without added nickel, and net hydrogen evolution was not affected by the presence of 100 nM Ni2+. Isolated heterocysts from strain CA were shown to have a persistent oxygen uptake of 0.7 mumol of O2 per mg (dry weight) per h, 35% of the rate of whole filaments, at air saturating O2 levels, indicating that O2 impermeability is not a requirement for active heterocysts.

Aerobic photoproduction of H2 was demonstrated in Anabaena spp. strains CA and 1F when cells were growing under nitrogen-fixing conditions. The rates of production, measured either by the hydrogen electrode or in a flow system by gas chromatography, were 10 to 15% of the rate of photosynthetic O2 evolution or 50 to 80% of the rates of acetylene reduction. Strains CA and 1F differed in several respects. In strain CA, H2 production was immediately partially sensitive to 3-(3,4-dichlorophenyl)-1,1-dimethylurea, whereas strain 1F was not immediately affected. Strain CA also showed a consistently higher rate of H2 production than did strain 1F. H2 production in strain CA was also markedly influenced by the light intensity used for growth, although the growth rates indicated that the light intensities used were essentially saturating.

Anabaena sp. CA fails to synthesize heterocysts and nitrogenase when grown with KNO3 as the nitrogen source. By contrast, both heterocysts and proheterocysts are synthesized in NH4Cl-containing media to a level nearly commensurate with cells grown in the absence of combined nitrogen. The growth rate of the organism in NH4Cl-containing media was similar to that obtained with KNO3 as the nitrogen source and was independent of the presence of N2 in the atmosphere. Thus, our results indicate that the organism assimilated nitrate and ammonium nitrogen equally well to meet the nitrogen requirements for growth. Moreover, in contrast to previous studies with other cyanobacteria, the repressor singal for heterocyst differentiation in Anabaena sp. CA is not derived from the metabolism of ammonia but appears to be involved with nitrate metabolism. Nitrogenase activity was partially expressed in NH4Cl-grown cultures. Increasing the level of nitrogenase activity to a value representative of a N2-grown culture required both the inhibition of ammonia assimilation and de novo protein synthesis. An increase in the number of mature heterocysts was not required. The fact that high levels of exogenous ammonia only partially repress the synthesis of proteins required for the maximum expression of nitrogenase activity in Anabaena sp. CA has important implications.

Mutants of Anabaena strain CA impaired in nitrogenase activity and growth on N2 were isolated and characterized. One mutant was selected for resistance to L-methionine-DL-sulfoximine, and others were selected for resistance to DL-7-azatryptophan or for requirements for combined nitrogen. The mutants varied in sensitivity of growth and nitrogenase activity to atmospheric 02. Several of the mutants whose growth on N2 was impaired under aerobic conditions could grow and reduce acetylene at rates comparable to the wild type when grown microaerobically under N2-CO2 (99:1). The acetylene reduction activity of some of the strains grown under N2-CO2 was immediately and completely lost upon exposure to atmospheric O2, but in at least one strain this loss was reversed when the O2 concentration was lowered, even after 10 h of exposure to air. The characteristics of the O2-sensitive mutants suggest that there may be several sites sensitive to O2 and that the protective mechanism involves several different phenomena.

Photoautotrophically grown cells of the blue-green alga (cyanobacterium) Nostoc sp. strain Mac assimilated and oxidized both glucose and fructose in the dark at different rates. The rate of fructose metabolism in these cells could be stimulated by casein hydrolysate, the effect being most pronounced at low sugar concentrations. This stimulation was not seen in cells grown heterotrophically in the dark, suggesting that it is a transitory phenomenon which disappears during the autotrophy-heterotrophy growth transition. The stimulation of fructose assimilation by casein hydrolysate was abolished by chloramphenicol or streptomycin, suggesting there are rate-limiting steps in protein biosynthesis in the dark that ultimately lead to inhibition of fructose uptake. Glucose metabolism did not show these phenomena, indicating there are differences in the metabolism of the two sugars.

The peptidoglycan from two types of filamentous cell division mutants of Agmenellum quadruplicatumstrain BG1 has been compared to that of the parent organism. Small variations in the total peptidoglycan composition on a dry-weight basis were found in the mutants. The reduced level of peptidoglycan in the serpentine mutant is consistent with a general decrease in the ratio of surface area to volume as compared to the parent organism. The increased peptidoglycan content in the septate mutant confirms previous ultrastructural observations of the greatly enlarged peptidoglycan septum between adjacent cells. A comparison of peptidoglycan composition and cross-linking in the two types of filamentous mutants of A. quadruplicatumand in drug-induced phenocopies suggests that structural alterations of the peptidoglycan are not involved in the apparent impairments of cellular division. Furthermore, data concerning the relative susceptibilities of the parent and mutants to antibiotics indicate that neither mutant exhibits a gross alteration of permeability.

A tryptophan-requiring auxotroph of Agmenellum quadruplicatumstrain BG1, a species of blue-green bacteria, was isolated by means of a nitrosoguanidine-penicillin procedure. Its growth characteristics were determined, and the enzymological block was identified in the A activity of tryptophan synthetase. Starvation of the auxotroph for tryptophan resulted in the derepression of the synthesis of all five enzymes. The first four enzymes derepressed 2- to 3-fold, and tryptophan synthetase B derepressed 20-fold. In the parental prototroph, BG1, anthranilate synthetase was active in crude extracts with ammonia as the amino donor reactant, but not with glutamine.

A unicellular blue-green alga, Agmenellum quadruplicatum, and a filamentous blue-green alga, Lyngbya lagerheimíi, were grown heterotrophically in dim light with glucose as major source of carbon and possibly energy. The dim-light conditions did not support autotrophic growth. The two blue-green algae appeared to have the same metabolic block, namely an incomplete tricarboxylic acid cycle, as has been found in other obligately phototrophic blue-green algae. Under dim-light conditions, glucose made a greater contribution to cell constituents (amino acids) of A. quadruplicatumand L. lagerheimiithan under high-light conditions.

Filamentous mutants were induced in a coccoid blue-green alga, Agmenellum quadruplicatumstrain BG1, after treatment with N-methyl-N′-nitro-N-nitrosoguanidine (NTG). The mutants fall into two general classes: filaments with cross walls and filaments without cross walls. All mutants of these general types derived from BG1 are stable and have growth rates the same as or very similar to the wild type under a variety of conditions. Detailed examination of one mutant, 53SB2, revealed no difference in deoxyribonucleic acid content nor in base ratios. Mutant 53SB2 did not revert to the normal cell size and shape when grown under different physical conditions nor upon the addition of potential reversing agents to the basal medium. It is our general experience that filamentous mutants such as those described here in BG1 are commonly induced in other coccoid blue-green algae after NTG treatment.

The unicellular blue-green bacterium Agmenellum quadruplicatumstrain BG-1 was found to be capable of rapid photoheterotrophic growth but unable to grow in the dark on a variety of reduced organic substrates. The generation time on glycerol was 12 h, and on CO2, 3 h. Glycerol carbon was converted into cellular carbon with a very high efficiency. This high efficiency of carbon conversion, the action spectrum for growth on glycerol, cell pigmentation, gas exchange measurements, and immediate ability of photoheterotrophically grown cells to evolve O2(upon the addition of CO2) suggest the involvement of both photosystems I and II of photosynthesis during photoheterotrophic growth.

Nostocsp. (strain Mac) was shown to be capable of using glucose, fructose, or sucrose as a sole source of carbon and energy in the dark. In the light in the absence of exogenously supplied CO2, this strain exhibited a more versatile metabolism. In addition to the three sugars above, glycerol and acetate served as sole sources of carbon. This photoheterotrophic growth in the absence of exogenously supplied CO2appears to involve O2-evolving photosynthesis. The action spectrum for photoheterotrophic growth on acetate closely resembles the action spectrum for photosynthesis. The physiology of photoheterotrophic growth was further investigated through determinations of stable carbon isotope ratios and measurements of gas exchanges. These investigations suggest that respired CO2from substrate oxidation is assimilated by the photosynthetic machinery.