Effect of canavanine and other amino acid analogues on akinete formation in the cyanobacterium Anabaena cylindrica

Addition of the arginine analogue, canavanine, to cultures of nitrogen-fixing Anabaena cylindrica at the onset of akinete formation, resulted in the development of akinetes randomly distributed within the filament, in addition to those adjacent to heterocysts. The total frequency of akinetes increased up to five-fold. A feature of akinetes is their increased content of cyanophycin granules (an arginine-aspartic acid polymer) and addition of canavanine to cultures at an earlier stage resulted in entire filaments becoming agranular and containing agranular akinetes. The effects on akinete pattern appeared to be specific for canavanine since other amino acid analogues, although increasing the frequency of akinetes (approximately two-fold), had no effect on their position relative to heterocysts. In ammonia-grown, stationary phase cultures of A. cylindrica, akinetes were observed adjacent to proheterocysts and in positions more than 20 cells from any heterocyst. These observations indicate that nitrogen fixation and heterocysts are not essential for akinete formation in A. cylindrica, although the availability of a source of fixed nitrogen does appear to be a requirement.These results suggest that during exponential growth some aspect of the physiology of vegetative cells suppresses their development into akinetes and that the role of the heterocyst may not be one of direct stimulation of adjacent vegetative cells to form akinetes, but the removal or negation of the inhibition within them. A model for akinete formation and the involvement of canavanine is given.     

Pattern of akinete differentiation in the cyanobacteriumScytonema fritschii

The differentiation of akinetes inScytonema fritschii occurred adjacent to the newly developed heterocysts in late exponential phase. The filaments exhibited cell division leading to the formation of heterocysts, interspersed by the potential akinetes which could be identified by the accumulation of a large number of granules. Upon maturity, the akinetes acquired thick envelopes and were seen in elongated series interrupted by dead necridia which resulted from crumpling of the newly developed heterocysts. The formation of akinetes was accompanied by a change in color of cultures from blue-green to brown. Of the inorganic nitrogen sources tested, ammonium nitrate supported the formation of maximum percentage of akinetes. The incorporation of 7-azatryptophan and rifampicin in nitrate-free and nitrogen sources resulted in the production of heterocysts at a very high frequency in the late-exponential phase coinciding with akinete formation but the frequency of the latter was reduced. The activity of nitrogenase, nitrate reductase and glutamate-ammonia ligase was absent in mature akinetes. The absorption spectra of chlorophylla and phycobiliproteins revealed the presence of negligible amounts of the former white the latter were absent. The dry mass steadily increased during akinete differentiation with a concomitant decrease in C/N ratios.     

Structural aspects of akinete germination in the cyanobacterium Anabaena variabilis

Electronmicroscopical investigations of light activated akinetes in different phases before outgrowth of the germinating cell showed two alterations in the akinete envelope, obviously in connection with the germination process. After induction of germination the akinetes show formation of an expanding more or less electron dense layer between the outer cell wall layer (outer membrane, L_IV) and the condensed part of the akinete coat (the transformed sheath of the vegetative cell). Between this new formed layer and the mentioned part of the akinete coat thick laminar layers are deposited which contain alternately electron dense and electron transparent strata. The expanding layer is assumed to be a mucous layer which acts as swelling body causing, after bursting of the layered shell, the expulsion of the germinating cell in the manner characteristic for Anabaena variabilis.     

Germination of desiccated aged akinetes of alkaliphilic cyanobacteria

Morphological and biochemical changes associated with synchronous germination of mature, aged and desiccated akinetes of two alkaliphilic cyanobacteria, Cyanospira rippkae and Cyanospira capsulata, are described. Akinetes of both strains proved to be highly resistant to desiccation, being able to germinate, in the presence of either N₂ or nitrate as nitrogen source, with a germination frequency of more than 90% after seven years of storage in a dried state. The first cell division occurred after 8–10 h of incubation, thereafter the germlings of the two strains followed a different pattern of cell differentiation. Heterocysts were first noted, in a terminal position, at 16–18 h in three-celled germlings of C. capsulata and at 21–24 h in C. rippkae, when germlings were at least seven cells in length. Akinetes of both species possessed, on a per cell basis, almost identical amounts of all photosynthetic pigments but, under nitrogen fixing conditions, photosynthetic activity (oxygen evolution) was detected only after new proteins had been synthesized, before a functional heterocyst was developed and while total nitrogen remained constant. With energy provided by aerobic respiration, a wide range of intracellular amino acids characteristic of proteins was utilised to sustain the new protein synthesis. The end of this biosynthetic activity coincided with the timing of the first cell division. From this stage on, no changes in protein concentration occurred until mature heterocysts were developed. In the presence of nitrate, no significant changes in the major germination events were observed.     

Potential triggers of akinete differentiation in Nodularia spumigena (Cyanobacteriaceae) isolated from Australia

Nodularia spumigena, like many cyanobacteria, produces specialised reproductive structures, known as akinetes, which are believed to allow survival under unfavourable conditions. This study investigated the effects of salinity, nitrogen and phosphorus concentration at two irradiances on akinete differentiation in a N. spumigena isolate from the Gippsland Lakes, Victoria, Australia. A computer image analysis program was used to photograph filaments and assess production of akinetes over time in separate experiments for each environmental parameter. Heterocyst production and cell morphology were also examined. The results suggest that akinete production increases over time. Production of akinetes is further increased at low and high salinities and with the addition of nitrate. Higher irradiance increases akinete differentiation, although in combination with different phosphorus concentrations causes varied effects. The development and sedimentation of akinetes may provide an inoculum for reoccurring blooms. Heterocysts were only observed during experiments with varying salinity and nitrogen exposures. Light quantity appeared to play a large role in heterocyst production. The ability of N. spumigena to produce akinetes and heterocysts is likely to be part of the reason for its success and continual occurrence in estuarine environments low in nitrogen, such as the Gippsland Lakes, Victoria, Australia. Factors known to reduce heterocyst and akinete production will provide new insight to possible management controls for this species.     

AKINETE GERMINATION IN ANABAENA CIRCINALIS (CYANOPHTA)

Akinetes of a clonal culture of Anabaena circinalis Rabenhorst from Mt. Bold reservoir (eutrophic), South Australia, were isolated and the effects of light, phosphorus, and nitrogen availability on their germination were investigated. Light was required but there was no significant difference in percentage of germination after 72 h if akinetes were incubated in ASM-1 medium at irradiances of 15, 30, or 50 μmol.m^-2.s^-1. Maximum akinete germination occurred by 48 h. Nitrogen was not required, as 88% of akinetes germinated in the flasks without combined nitrogen added to the medium and without N₂ in the air. NH₄⁺-N at 28 mg N.L^-1 completely suppressed germination, whereas 28 mg NO₃ N.L^-1 had no effect relative to the controls without nitrogen. Phosphorus was required, and at 48 h percentage of germination in the flasks with 0.6 mg P.L^-1 added (78%) was significantly greater than in the flasks with 0.06 P.L^-1 (58%) and 0 mgP.L^-1 (24%) added. Germlings in the 0 mg P.L^-1 flasks were only 2–4 cells long and stunted in appearance, whereas germlings at all other P concentrations were 8–16 cells long. It is likely that the isolation process exposed some akinetes to intracellular phosphorus released from lysing vegetative cells, but this was insufficient to allow normal development in the 0 mg P.L^-1 flasks. The plot of percentage of germination vs. initial phosphorus concentration, in the medium showed a relationship analogous to Michaelis-Menten nutrient uptake kinetics, suggesting that a specific membrane-bound enzyme system(s) is involved, with phosphorus as the substrate. The half saturation value (K_S) for germination was 50 μg P.L^-1.     

AKINETE FORMATION IN PLANKTONIC ANABAENA SPP. (CYANOBACTEFUA) BY TREATMENT WITH LOW TEMPERATURE1

The effects of temperature, light intensity and nutrient depletion on akinete formation in seven strains of planktonic Anabaena spp.: A. mucosa TAC426; A. crassa TAC436; A. spiroides TAC443 and TAC444; A. flosaquae TAC446; and A. ucrainica TAC448 and TAC449 were examined. A Marked Pfft of temperature on akinete formation was observed at 40 μmol photons·m^?2·sec^?1 and nutrient-sufficient conditions. At 20° C, akinetes did not develop in A. mucosa TAC426, A. crassa TAC436, A. spiroides TAC443, A. flos-aquae TAC446, or A. ucrainica TAC449 but were formed at frequencies of a little over 11% (ratio of filaments with akinetes to total filaments) in A. spiroides TAC444 and A. ucrainica TAC448. None of the strains fmd akinetes or heterocysts at 30° C and 35° C. At lower temperature (10° C and 15° C), akinetes developed in all the strains at maximum frequencies of 13.4–77.4% during the late exponential phase or late exponential to stationary phases of growth. With only one exception, low light or nutrient deletion did not lead to the induction of akinete diferentiation at 20° C. Only akinete formation in A. flosaquae TAC446 was induced by nitrogen deletion with a frequency of 12.1%, similar to that induced by low temperature, but the initiation of akinete formation in the strain was delayed compared to treatment with low temperature. These results show that temperature was the most important environmental factor triggering akinete formation in these species. In A. crassa TAC436 and A. spiroides TAC443 and TAC444, akinetes developed during the late exponential growth phase even though heterocysts were formed at a 100% frequency (ratio of filaments with heterocysts to total filaments) throughout the entire growth phase. In A. mucosa TAC426, A. flos-aquae TAC446, and A. ucrainica TAC448 and TAC449, there was a positive correlation between heterocyst and akinete formation, suggesting that the presence of a heterocyst may play a role in akinete formation.     

Heterocyst and akinete induction with altered pattern in Anabaena cylindrica,caused by neo-peptone

Neo-peptone B119 (Difco) was found to have a significant effect on differentiation of heterocysts and akinetes in Anabaena cylindrica. On adding neopeptone (0.4 g/l) to exponential phase culture of A. cylindrica, the following effects were observed (i) increased heterocyst frequency with altered heterocyst spacing and presence of double and multiple heterocysts after 24 h in cultures grown on N-free medium, (ii) induction of regular pattern of heterocysts after 48 h, in culture grown on medium supplemented with NH₄Cl, (iii) induction of pro-akinetes after 48 h in both N-free and ammonium-grown cultures. The higher concentrations of neo-peptone were lytic to A. cylindrica, and, its lytic and inductive effects could be decreased by acid hydrolysis or supplementation of NH₄Cl. Gel-filtration of neo-peptone showed that the inductive as well as the lytic effect was associated with some active factor(s) with molecular weight between 10,000–20,000. The retention of the inductive effect on autoclavation but its loss on trypsin digestion suggested that active factor(s) may be heat stable polypeptide(s). The heterocyst induction by active factor(s) decreased and akinete induction increased with increasing culture age. The pro-akinetes induced during exponential phase divided before maturation, while those induced during late exponential phase, could achieve full maturity. Growth and nitrogenase activity was unaffected while there was an increase in mean cell length on treatment of A. cylindrica with active factor(s) from neo-peptone, indicating that the effect may be mediated through cell division process(es).Abbreviations used N Nitrogen - chl chlorophyll     

AKINETE DIFFERENTIATION IN ANABAENA CIRCINALIS (CYANOPHYTA)1

Three lines of evidence established conclusively that phosphorus limitation triggered akinetes to differentiate in Anabaena circinalis Rabenhorst. First, akinetes differentiated when phosphorus was limited, but not when nitrogen, inorganic carbon, iron, trace elements, or light were limited, or when dissolved oxygen concentration was increased. In the phosphorus limitation experiment, akinetes appeared first in the 0 mg P-L^?1 cultures, and the higher the initial concentration of phosphorus was, the longer it took for akinetes to differentiate. Second, akinete differentiation commenced when Q_p fell to the same critical concentration in all cultures. The critical Q_p for akinete differentiation in A. circinalis was 0.3-0.45 pg P·cell^?1, and there was no significant difference between cultures grown with 0.6, 0.2, 0.06, or 0 mg P · L^?1 (F= 5.48, of = 3, P > 0.05). Similarly, there were no significant differences between P cultures in internal cellular soluble reactive phosphorus (SRP) concentration (F= 0.63, df = 3, P > 0.05) or external SRP per cell in the medium (F= 5.16, df= 3, P > 0.05) when akinete differentiation commenced. Both were between 0.01 and 0.07 pg SRP-cell^?1. A thorough literature search indicates that this information has not been reported previously. The third line of evidence came from electron micrographs, which illustrated that polyphosphate was present in trichomes prior to akinete differentiation but was absent in trichomes with akinetes indicating that phosphorus reserves were depleted when akinetes differentiated. Lipid globules (carbon reserve) and cyanophycin granules (nitrogen reserve) increased in number in trichomes with akinetes, compared to trichomes without akinetes. Thus, the ratio of internal P:C:N was different in trichomes with akinetes compared to trichomes without akinetes and may be important in activating akinete-differentiating genes.     

Development of nitrogen-assimilating enzymes in sunflower cotyledons during germination as affected by the exogenous nitrogen source

Activities of nitrate reductase (NR; EC 1.6.6.1), nitrite reductase (NiR; EC 1.7.7.1), glutamine synthetase (GS; EC 6.3.1.2) and glutamate dehydrogenase (GDH; EC 1.4.1.3) were measured in cotyledons of sunflower (Helianthus annuus L. cv Peredovic) seedlings during germination and early growth under various external nitrogen sources. The presence of NO ₃ ^- in the medium promoted a gradual increase in the levels of NR and NiR activities during the first 7 d of germination. Neither NR nor NiR activities were increased in a nitrogen-free medium or in media with either NH ₄ ⁺ or urea as nitrogen sources. Moreover, the presence of NH ₄ ⁺ did not abolish the NO ₃ ^- -dependent appearance of NR and NiR activities. The increase of NR activity was impaired both by cycloheximide and chloramphenicol, which indicates that both cytoplasmic 80S and plastidic 70S ribosomes are involved in the synthesis of the NR molecule. By contrast, the appearance of NiR activity was only inhibited by cycloheximide, indicating that NiR seems to be exclusively synthesized on the cytoplasmic 80S ribosomes. Glutamine-synthetase activity was also strongly increased by external NO ₃ ^- but not by NH ₄ ⁺ or urea. The appearance of GS activity was more efficiently suppressed by cycloheximide than chloramphenicol. This indicates that GS is mostly synthesized in the cytoplasm. The cotyledons of the dry seed contain high levels of GDH activity which decline during germination independently of the presence or absence of a nitrogen source. Cycloheximide, but not chloramphenicol, greatly prevented the decrease of GDH activity.Abbreviations GDH glutamate dehydrogenase - GS glutamine synthetase - NiR nitrite reductase - NR nitrate reductase     

Nitrification and simultaneous denitrification byAzospirillum brasilense 12S

Azospirillum brasilense, an associative diazotrophs from sorghum roots grows autotrophically on NH ₄ ⁺ and CaCO₃. NH ₄ ⁺ a is also oxidized to NO ₂ ^- and then denitrified. Addition of malate to the autotrophic medium enhances both NH ₄ ⁺ oxidation as well as NONH ₂ ^- dissimilation. The incomplete nitrification linked denitrification results in a rapid loss of nitrogen from the growth medium. The bacterium also shows assimilatory NO ₃ ^- and NO ₂ ^- reductases and fixes nitrogen at 50 μg N/ml of NH ₄ ⁺ NO ₃ ^- or NO ₂ ^-     

Phosphorus deficiency,nitrogen assimilation and akinete differentiation in the cyanobacteriumAnabaena torulosa

Anabaena torulosa is unable to fix N₂ and to differentiate akinetes in a P-deficient nitrate-free medium. In a P-deficient medium with nitrate, the NO₃ ^? assimilation and period of akinete differentiation are of the same order of magnitude as in a P-containing nitrate medium. It is suggested that regulation of akinete differentiation in P-deficient organism proceeds through the regulation of the N-assimilating system. At the time of akinete differentiation, cellular P is excreted into the medium which leads to a decrease of cellular P.     

Long-term observations on Anabaena circinalis Rabenhorst (Cyanophyta)

Field observations on Anabaena circinalis Rabenhorst over six summer seasonal appearances in two dams have shown that the frequency of occurence of heterocysts became fairly constant soon after the appearance of the species and decreased just before the end of the growing season. By contrast, akinete frequency reached a maximum, very early in the season, then decreased rapidly. Drought led to a decrease in occurrence of both Anabaena and of akinetes in several dams; this was possibly associated with an increasing concentration of NOx in the water. At Carcoar dam this akinete reduction was shown first in end-of-season figures. Drought also led to an end-of-season decrease there in the occurrence of heterocysts. Variations in morphology were noted. The coiling of the trichome, shape of akinete and relative position of heterocyst were all variable, although these characters are often assumed to be of taxonomic importance.     

2-Methylhopanoids are maximally produced in akinetes of Nostoc punctiforme: geobiological implications

2-Methylhopanes, molecular fossils of 2-methylbacteriohopanepolyol (2-MeBHP) lipids, have been proposed as biomarkers for cyanobacteria, and by extension, oxygenic photosynthesis. However, the robustness of this interpretation is unclear, as 2-methylhopanoids occur in organisms besides cyanobacteria and their physiological functions are unknown. As a first step toward understanding the role of 2-MeBHP in cyanobacteria, we examined the expression and intercellular localization of hopanoids in the three cell types of Nostoc punctiforme : vegetative cells, akinetes, and heterocysts. Cultures in which N. punctiforme had differentiated into akinetes contained approximately 10-fold higher concentrations of 2-methylhopanoids than did cultures that contained only vegetative cells. In contrast, 2-methylhopanoids were only present at very low concentrations in heterocysts. Hopanoid production initially increased threefold in cells starved of nitrogen but returned to levels consistent with vegetative cells within 2 weeks. Vegetative and akinete cell types were separated into cytoplasmic, thylakoid, and outer membrane fractions; the increase in hopanoid expression observed in akinetes was due to a 34-fold enrichment of hopanoid content in their outer membrane relative to vegetative cells. Akinetes formed in response either to low light or phosphorus limitation, exhibited the same 2-methylhopanoid localization and concentration, demonstrating that 2-methylhopanoids are associated with the akinete cell type per se . Because akinetes are resting cells that are not photosynthetically active, 2-methylhopanoids cannot be functionally linked to oxygenic photosynthesis in N.   punctiforme .     

AKINETE PRODUCTION OF ANABAENA LEMMERMANNII AND A. CYLINDRICA (CYANOPHYCEAE) IN NATURAL POPULATIONS OF N‐ AND P‐LIMITED COASTAL MESOCOSMS1

A strong biomass increase of two Anabaena species was observed in natural plankton community enclosed into nine large mesocosms (51 m³) and manipulated with mineral nutrients and an organic carbon source during a 3‐week period in the coastal Baltic Sea. The water column and settled material from the bottom of the mesocosms were sampled at 2‐day intervals. Planktonic populations of Anabaena lemmermannii Richter and A. cylindrica Lemmermann and sedimentation rates of akinetes to the bottom were quantified. Comparing mesocosms with artificially induced nitrogen and phosphorus limitation, we found that during the third week of the experiment, the population size of A. lemmermannii was clearly higher in nitrogen‐limited units (by a factor of 2.4), whereas the production rate of akinetes was higher in the phosphorus‐limited units (by a factor of 2.5). Input of freshly produced A. lemmermannii akinetes to the benthos was on average 15 × 10⁶ and 6 × 10⁶ cells· m^?2·d^?1 in the P^? and N^? limited mesocosms, respectively. Our estimates of specific akinete production rate of A. lemmermannii in P^? and N^? limited mesocosms revealed an even larger divergence (a factor of 5.5), being on average 2.4 and 0.4 akinetes·10^?3 vegetative cells^?1·d^?1, respectively. The phosphorus addition effectively reduced akinete production of A. lemmermannii. Differences in the nutrient manipulation had no apparent effect on the biomass and akinete production of A. cylindrica. The akinete production pattern of A. cylindrica revealed a 1‐week delay compared with the vegetative population peak, whereas such a delay was not obvious in A. lemmermannii.     

The influence of light quality on akinete formation and germination in the toxic cyanobacterium Anabaena circinalis

Physiological control of akinete formation and subsequent germination is likely to be important in understanding and predicting how natural populations of cyanobacteria respond to their environment. While previous research has indicated nutrient limitation may be important in akinete formation new results presented here indicate that in the toxic and bloom-forming species Anabaena circinalis there was a profound effect of spectral quality. Under 40 μmol photons m^?2 s^?1 photosynthetically active irradiance (PAR) of predominately red irradiance akinete production was maximal at 2.1 × 10^?4 akinetes vegetative cell^?1 d^?1, some 3000 times greater than the 6.5 × 10^?8 akinetes vegetative cell^?1 d^?1 observed under equivalent PAR but predominately blue light. For cells grown under a range of predominantly red, white and green irradiance even short exposures to blue light reduced akinete formation rates by a factor of ten relative to controls, indicating that exposure to blue light inhibits akinete formation. Germination of akinetes was not influenced by the irradiance under which akinetes were formed: 88 ± 4.1% (mean ± 1 S.D.) of akinetes germinated with no evidence of an effect on germination success due to their production under predominately red, white or green irradiance (germination of akinetes produced under blue light was not tested). Spectral quality had a significant impact on both vegetative cell and germling growth rates. The results indicate a significant reduction in the cellular differentiation of A. circinalis vegetative cells into akinetes that is mediated by blue light. In an ecological context the production of akinetes will be greater in environments with less blue light; potentially including those with slower flow, more stratification, less vertical mixing and more turbidity. The resulting spatial pattern of akinete production is likely to influence the location of akinetes in sediments and the development of subsequent blooms from excysting germlings.     

Potassium deficiency triggers the development of dormant cells (akinetes) in Aphanizomenon ovalisporum (Nostocales,Cyanoprokaryota)1

Akinetes are spore‐like nonmotile cells that differentiate from vegetative cells of filamentous cyanobacteria from the order Nostocales. They play a key role in the survival and distribution of these species and contribute to their perennial blooms. Various environmental factors were reported to trigger the differentiation of akinetes including light intensity and quality, temperature, and nutrient deficiency. Here, we report that deprivation of potassium ion (K⁺) triggers akinete development in the cyanobacterium Aphanizomenon ovalisporum. Akinetes formation is initiated 3 d–7 d after an induction by K⁺ depletion, followed by 2–3 weeks of a maturation process. Akinete formation occurs within a restricted matrix of environmental conditions such as temperature, light intensity or photon flux. Phosphate is essential for akinete maturation and P‐limitation restricts the number of mature akinetes. DNA replication is essential for akinete maturation and akinete development is limited in the presence of Nalidixic acid. While our results unequivocally demonstrated the effect of K⁺ deficiency on akinete formation in laboratory cultures of A. ovalisporum, this trigger did not cause Cylindrospermopsis raciborskii to produce akinetes. Anabaena crassa however, produced akinetes upon potassium deficiency, but the highest akinete concentration was achieved at conditions that supported vegetative growth. It is speculated that an unknown internal signal is associated with the cellular response to K⁺ deficiency to induce the differentiation of a certain vegetative cell in a trichome into an akinete. A universal stress protein that functions as mediator in K⁺ deficiency signal transduction cascade, may communicate between the lack of K⁺ and akinete induction.     

Temperature control of nitrogen fixation in Klebsiella pneumoniae

At growth temperatures above 37°C, Klebsiella pneumoniae does not grow in a medium containing N₂ or NO ₃ ^- as nitrogen sources. However, both the growth in the presence of other nitrogen sources as well as the in vitro nitrogenase activity are not affected at this temperature. The inability to fix N₂ at high temperature is due to the failure of the cells to synthesize nitrogenase and other nitrogen fixation (nif) gene encoded proteins. When cells grown under nitrogen fixing conditions at 30°C were shifted to 39°C, there was a rapid decrease of the rate of de novo biosynthesis of nitrogenase (component 1), nitrogenase reductase (component 2), and the nifJ gene product. There was no degradation of nitrogenase at the elevated temperature since preformed enzyme remained stable over a period of at least 3 h at 39°C. Thus, temperature seems to represent a third control system, besides NH ₄ ⁺ and O₂, governing the expression of nif genes of K. pneumoniae.     

Proton translocation during denitrification in Rhodopseudomonas sphaeroides f. denitrificans

Proton translocation during the reduction of NO ₃ ^- , NO ₂ ^- , N₂O and O₂, with endogenous substrates, in washed cells of Rhodopseudomonas sphaeroides f. denitrificans was investigated by an oxidant pulse method. On adding NO ₂ ^- to washed cells, anaerobically in the dark, an alkalinization occurred in the reaction mixture followed by acidification. When NO ₃ ^- , N₂O or O₂ was added to cells in the dark or with these compounds and NO ₂ ^- in light an acidification only was observed. Proton translocation was inhibited by carbonyl cyanide-m-chlorophenyl hydrazone.Valinomycin treated cells produced acid in response to the addition of either NO ₃ ^- , NO ₂ ^- , N₂O or O₂. The proton extrusion stoichiometry ( ratios) in illuminated cells were as follows: NO ₃ ^- 0.5N₂, 4.82; NO ₂ ^- 0.5N₂, 5.43; N₂ON₂, 6.20; and O₂H₂O, 6.43. In the dark the comparable values were 3.99, 4.10, 4.17 and 3.95. Thus, illuminated cells produced higher values than those in the dark, indicating a close link between photosynthesis and denitrification in the generation of proton gradients across the bacterial cell membranes.When reduced benzyl viologen was the electron donor in the presence of 1 mM N-ethylmaleimide and 0.5 mM 2-n-heptyl-4-hydroxyquinoline-N-oxide in the dark, the addition of either NO ₃ ^- , NO ₂ ^- or N₂O to washed cells resulted in a rapid alkalinization of the reaction mixture. The stoichiometries for proton consumption, ratios without a permeant ion were NO ₃ ^- NO ₂ ^- ,-1.95; NO ₂ ^- 0.5 N₂O,-3.03 and N₂ON₂,-2.02. The data indicate that these reductions occur on the periplasmic side of the cytoplasmic membrane.Abbreviations BVH reduced benzyl viologen - CCCP carbonyl cyanide m-chlorophenyl hydrazone - DIECA N, N-diethyl-dithiocarbamate - HOQNO 2-n-heptyl-4-hydroxyquinoline-N-oxide - NEM N-ethylmaleimide