Production of polyhydroxybutyrate by the cyanobacterium cf. Anabaena sp.

Polyhydroxybutyrate (PHB) production by the cyanobacterium cf. Anabaena sp. was here studied by varying the medium composition and the carbon source used to induce mixotrophic growth conditions. The highest PHB productivity (0.06 gPHB gbiomass-1 d-1) was observed when cultivating cf. Anabaena sp. in phosphorus-free medium and in the presence of sodium acetate (5.0 g L-1 concentration), after an incubation period of 7 days. A content of 40% of PHB on biomass, a dry weight of 0.1 g L-1, and a photosynthetic efficiency equal to the control were obtained. The cyanobacterium was then grown on a larger scale (10 L) to evaluate the characteristics of the produced PHB in relation to the main composition of the biomass (the content of proteins, polysaccharides, and lipids): after an incubation period of 7 days, a content of 6% of lipids (52% of which as unsaturated fatty acids with 18 carbon atoms), 12% of polysaccharides, 28% of proteins, and 46% of PHB was reached. The extracted PHB had a molecular weight of 3 MDa and a PDI of 1.7. These promising results demonstrated that cf. Anabaena sp. can be included among the Cyanobacteria species able to produce polyhydroxyalkanoates (PHAs) either in photoautotrophic or mixotrophic conditions, especially when it is grown under phosphorus-free conditions.

A TonB-Like Protein, SjdR, Is Involved in the Structural Definition of the Intercellular Septa in the Heterocyst-Forming Cyanobacterium Anabaena.

Cyanobacteria are photosynthetic organisms with a Gram-negative envelope structure. Certain filamentous species such as Anabaena sp. strain PCC 7120 can fix dinitrogen upon depletion of combined nitrogen. Because the nitrogen-fixing enzyme, nitrogenase, is oxygen sensitive, photosynthesis and nitrogen fixation are spatially separated in Anabaena. Nitrogen fixation takes place in specialized cells called heterocysts, which differentiate from vegetative cells. During heterocyst differentiation, a microoxic environment is created by dismantling photosystem II and restructuring the cell wall. Moreover, solute exchange between the different cell types is regulated to limit oxygen influx into the heterocyst. The septal zone containing nanopores for solute exchange is constricted between heterocysts and vegetative cells, and cyanophycin plugs are located at the heterocyst poles. We identified a protein previously annotated as TonB1 that is largely conserved among cyanobacteria. A mutant of the encoding gene formed heterocysts but was impaired in diazotrophic growth. Mutant heterocysts appeared elongated and exhibited abnormal morphological features, including a reduced cyanophycin plug, an enhanced septum size, and a restricted nanopore zone in the septum. In spite of this, the intercellular transfer velocity of the fluorescent marker calcein was increased in the mutant compared to the wild type. Thus, the protein is required for proper formation of septal structures, expanding our emerging understanding of Anabaena peptidoglycan plasticity and intercellular solute exchange, and is therefore renamed SjdR (septal junction disk regulator). Notably, calcium supplementation compensated for the impaired diazotrophic growth and alterations in septal peptidoglycan in the sjdR mutant, emphasizing the importance of calcium for cell wall structure. IMPORTANCE Multicellularity in bacteria confers an improved adaptive capacity to environmental conditions and stresses. This includes an enhanced capability of resource utilization through a distribution of biochemical processes between constituent cells. This specialization results in a mutual dependency of different cell types, as is the case for nitrogen-fixing heterocysts and photosynthetically active vegetative cells in Anabaena. In this cyanobacterium, intercellular solute exchange is facilitated through nanopores in the peptidoglycan between adjacent cells. To ensure functionality of the specialized cells, septal size as well as the position, size, and frequency of nanopores in the septum need to be tightly established. The novel septal junction disk regulator SjdR characterized here is conserved in the cyanobacterial phylum. It influences septal size and septal nanopore distribution. Consequently, its absence severely affects the intercellular communication and the strains' growth capacity under nitrogen depletion. Thus, SjdR is involved in septal structure remodeling in cyanobacteria.

Phytohormone up-regulates the biochemical constituent, exopolysaccharide and nitrogen metabolism in paddy-field cyanobacteria exposed to chromium stress.

BACKGROUND: Cyanobacteria are well known for their inherent ability to serve as atmospheric nitrogen fixers and as bio-fertilizers; however, increased contaminants in aquatic ecosystem significantly decline the growth and function of these microbes in paddy fields. Plant growth regulators play beneficial role in combating the negative effects induced by heavy metals in photoautotroph. Current study evaluates the potential role of indole acetic acid (IAA; 290 nm) and kinetin (KN; 10 nm) on growth, nitrogen metabolism and biochemical constituents of two paddy field cyanobacteria Nostoc muscorum ATCC 27893 and Anabaena sp. PCC 7120 exposed to two concentrations of chromium (CrVI; 100 µM and 150 µM). RESULTS: Both the tested doses of CrVI declined the growth, ratio of chlorophyll a to carotenoids (Chl a/Car), contents of phycobiliproteins; phycocyanin (PC), allophycocyanin (APC), and phycoerythrin (PE), protein and carbohydrate associated with decrease in the inorganic nitrogen (nitrate; NO3- and nitrite; NO2-) uptake rate that results in the decrease in nitrate and ammonia assimilating enzymes; nitrate reductase (NR), nitrite reductase (NiR), glutamine synthetase (GS), glutamate synthase (GOGAT) except glutamate dehydrogenase (GDH). However, exogenous supplementation of IAA and KN exhibited alleviating effects on growth, nitrogen metabolism and exopolysaccharide (EPS) (first protective barrier against metal toxicity) contents in both the cyanobacteria, which probably occurred as a result of a substantial decrease in the Cr uptake that lowers the damaging effects. CONCLUSION: Overall result of the present study signifies affirmative role of the phytohormone in minimizing the toxic effects induced by chromium by stimulating the growth of cyanobacteria thereby enhancing its ability as bio-fertilizer that improved fertility and productivity of soil even in metal contaminated condition.

Screening of microalgae for integral biogas slurry nutrient removal and biogas upgrading by different microalgae cultivation technology.

The microalgae-based technology has been developed to reduce biogas slurry nutrients and upgrade biogas simultaneously. In this work, five microalgal strains named Chlorella vulgaris, Scenedesmus obliquus, Selenastrum capricornutum, Nitzschia palea, and Anabaena spiroides under mono- and co-cultivation were used for biogas upgrading. Optimum biogas slurry nutrient reduction could be achieved by co-cultivating microalgae (Chlorella vulgaris, Scenedesmus obliquus, and Nitzschia palea) with fungi using the pelletization technology. In addition, the effects of different ratio of mixed LED light wavelengths applying mixed light-emitting diode during algae strains and fungi co-cultivation on CO2 and biogas slurry nutrient removal efficiency were also investigated. The results showed that the COD (chemical oxygen demand), TN (total nitrogen), and TP (total phosphorus) removal efficiency were 85.82 ± 5.37%, 83.31 ± 4.72%, and 84.26 ± 5.58%, respectively at red: blue = 5:5 under the co-cultivation of S. obliquus and fungi. In terms of biogas upgrading, CH4 contents were higher than 90% (v/v) for all strains, except the co-cultivation with S. obliquus and fungi at red: blue = 3:7. The results indicated that co-cultivation of microalgae with fungi under mixed light wavelengths treatments was most successful in nutrient removal from wastewater and biogas upgrading.

Transcriptomic and Proteomic Profiling of Anabaena sp. Strain 90 under Inorganic Phosphorus Stress.

Inorganic phosphorus (Pi) is one of the main growth-limiting factors of diazotrophic cyanobacteria. Due to human activity, the availability of Pi has increased in water bodies, resulting in eutrophication and the formation of massive cyanobacterial blooms. In this study, we examined the molecular responses of the cyanobacterium Anabaena sp. strain 90 to phosphorus deprivation, aiming at the identification of candidate genes to monitor the Pi status in cyanobacteria. Furthermore, this study increased the basic understanding of how phosphorus affects diazotrophic and bloom-forming cyanobacteria as a major growth-limiting factor. Based on RNA sequencing data, we identified 246 differentially expressed genes after phosphorus starvation and 823 differentially expressed genes after prolonged Pi limitation, most of them related to central metabolism and cellular growth. The transcripts of the genes related to phosphorus transport and assimilation (pho regulon) were most upregulated during phosphorus depletion. One of the most increased transcripts encodes a giant protein of 1,869 amino acid residues, which contains, among others, a phytase-like domain. Our findings predict its crucial role in phosphorus starvation, but future studies are still needed. Using two-dimensional difference in gel electrophoresis (2D-DIGE) and liquid chromatography-tandem mass spectrometry (LC-MS/MS), we found 43 proteins that were differentially expressed after prolonged phosphorus stress. However, correlation analysis unraveled an association only to some extent between the transcriptomic and proteomic abundances. Based on the present results, we suggest that the method used for monitoring the Pi status in cyanobacterial bloom should contain wider combinations of pho regulon genes (e.g., PstABCS transport systems) in addition to the commonly used alkaline phosphatase gene alone.

[Effect of light and temperature on growth kinetics of Anabaena flosaquae under phosphorus limitation].

Phosphorus, light and temperature are the key environmental factors leading to algae growth. But the effects of interaction between light and temperature on the growth of Anabaena flosaquae under phosphorus limitation were not well documented in literature. Anabaena flosaquae was selected for the study and lab-scale experiment and simulation were carried out. The results showed that the optimal temperature of Anabaena flosaquae was 20 degrees C under phosphorus limitation when the light intensity was constant, and the optimal light intensity (illuminance) of Anabaena flosaquae was 3 000 lx under phosphorus limitation when the temperature was constant. Based on model fitting and parameter calibration, the optimal temperature and light intensity of Anabaena flosaquae were 21.03 degress C ± 1.55 degrees C and 2 675.12 lx ± 262.93 lx, respectively. These data were close to the actual water environmental condition at the end of spring. Results of this study will provide important foundation for prediction of Anabaena blooms.

Inactivation of agmatinase expressed in vegetative cells alters arginine catabolism and prevents diazotrophic growth in the heterocyst-forming cyanobacterium Anabaena.

Arginine decarboxylase produces agmatine, and arginase and agmatinase are ureohydrolases that catalyze the production of ornithine and putrescine from arginine and agmatine, respectively, releasing urea. In the genome of the filamentous, heterocyst-forming cyanobacterium Anabaena sp. strain PCC 7120, ORF alr2310 putatively encodes an ureohydrolase. Cells of Anabaena supplemented with [(14) C]arginine took up and catabolized this amino acid generating a set of labeled amino acids that included ornithine, proline, and glutamate. In an alr2310 deletion mutant, an agmatine spot appeared and labeled glutamate increased with respect to the wild type, suggesting that Alr2310 is an agmatinase rather than an arginase. As determined in cell-free extracts, agmatinase activity could be detected in the wild type but not in the mutant. Thus, alr2310 is the Anabaena speB gene encoding agmatinase. The ∆alr2310 mutant accumulated large amounts of cyanophycin granule polypeptide, lacked nitrogenase activity, and did not grow diazotrophically. Growth tests in solid media showed that agmatine is inhibitory for Anabaena, especially under diazotrophic conditions, suggesting that growth of the mutant is inhibited by non-metabolized agmatine. Measurements of incorporation of radioactivity from [(14) C]leucine into macromolecules showed, however, a limited inhibition of protein synthesis in the ∆alr2310 mutant. Analysis of an Anabaena strain producing an Alr2310-GFP (green fluorescent protein) fusion showed expression in vegetative cells but much less in heterocysts, implying compartmentalization of the arginine decarboxylation pathway in the diazotrophic filaments of this heterocyst-forming cyanobacterium.

Influence of phosphorus and pH on the fungicidal potential of Anabaena strains.

The genus Anabaena is known to be a rich source of bioactive metabolites, but the biocontrol potential of this genus, mediated through hydrolytic enzymes is less investigated. In our investigation, five Anabaena strains - A. laxa RPAN8, A. iyengarii RPAN9, A. variabilis RPAN59 and A. oscillarioides RPAN69 (with A. variabilis RPAN16 serving as negative control) were evaluated in time course studies involving incubation under three levels of phosphorus and pH conditions. Total chlorophyll, proteins, chitosanase, endoglucanase and CMCase activity were measured and inhibition assayed against phytopathogenic fungi. The four weeks old RPAN69 culture showed significantly higher chlorophyll which was 41% higher than control. This was also linked with an enhancement of 18.26% and 9.18% in chitosanase and CMCase activity respectively over control in the treatment involving half dose of phosphorus. Chlorophyll and CMCase activity showed a high degree of correlation with highest values at pH 9.5. A pH of 5.5 was the most suitable condition for the maximum activity of chitosanase for all the strains except RPAN16. The strains RPAN8 and RPAN9 showed the highest activity of endoglucanase at pH 5.5 while the other strains exhibited maximum activity at pH 7.5. This study provides insight into the role of P and pH in modulating fungicidal activity in different Anabaena strains, which can be valuable for enhancing their efficiency as a biocontrol agent.

Facultative diazotrophy increases Cylindrospermopsis raciborskii competitiveness under fluctuating nitrogen availability.

Relative fitness of three bloom-forming and potentially toxic cyanobacteria from the subtropical St. John's River, Florida was investigated under a range of nutrient conditions, during a bloom dominated by Cylindrospermopsis raciborskii. Nitrogen (N) was the primary nutrient limiting phytoplankton primary productivity and biomass. Phytoplankton biomass was also enhanced by phosphorus (P) added either alone or jointly with N, suggesting different components of the phytoplankton experienced distinct nutrient limitations. Based on quantitative PCR, the diazotrophic cyanobacteria Anabaena sp. and C. raciborskii were responsible for the primary production response to P additions, while the nondiazotrophic Microcystis aeruginosa appeared to benefit from N released from the diazotrophs. Cylindrospermopsis raciborskii maintained high net growth rates under diazotrophic and nondiazotrophic conditions, while Anabaena sp. growth was significantly reduced under DIN enrichment. C. raciborskii appears to be a generalist with regard to N source, a lifestyle traditionally not considered a viable ecological strategy among diazotrophs. Using facultative diazotrophy, C. raciborskii gains a growth advantage under fluctuating DIN conditions, such as systems that are under the influence of anthropogenic N loading events. The described niche differentiation may be a key factor explaining the recent global expansion of C. raciborskii.

Nitrogen status dependent oxidative stress tolerance conferred by overexpression of MnSOD and FeSOD proteins in Anabaena sp. strain PCC7120.

The heterocystous nitrogen-fixing cyanobacterium, Anabaena sp. strain PCC7120 displayed two superoxide dismutase (SOD) activities, namely FeSOD and MnSOD. Prolonged exposure of Anabaena PCC7120 cells to methyl viologen mediated oxidative stress resulted in loss of both SOD activities and induced cell lysis. The two SOD proteins were individually overexpressed constitutively in Anabaena PCC7120, by genetic manipulation. Under nitrogen-fixing conditions, overexpression of MnSOD (sodA) enhanced oxidative stress tolerance, while FeSOD (sodB) overexpression was detrimental. Under nitrogen supplemented conditions, overexpression of either SOD protein, especially FeSOD, conferred significant tolerance against oxidative stress. The results demonstrate a nitrogen status-dependent protective role of individual superoxide dismutases in Anabaena PCC7120 during oxidative stress.

[Influence of nutrient sources on Anabaena spiroides growth and odorous compounds production characteristics].

The occurrence of taste and odors, produced by secondary metabolites of cyanobacteria, has been one of the major water quality problems in drinking water. However, the odorous compounds produced by cyanobacteria usually differ significantly with different species. One cyanobacterium isolated from Yanghe reservoir was identified as Anabaena sp., which can produce high level of geosmin consistently during laboratory culture. By culture expanding experiments, the algal growth and geosmin production characteristics of the Anabaena sp. were studied on different conditions of nitrogen and phosphorus sources. The results indicated that geosmin mainly remained in the intracellular algal cells regardless of the nutrient sources, and the extracellular content was only in th range of 0.2% - 9.6%. Compared with ammonia nitrogen conditions, the growth of Anabaena sp. in nitrate nitrogen conditions was much higher, with a 1.4-fold variation in geosmin production. While ammonia nitrogen concentration was 0.5 mg/L, the algal biomass and geosmin production achieved the highest level of 3.8 x 10(4) cells, mL(-1) and 1.1 x 10(4) ngL(-1), respectively. When the nitrate nitrogen concentration was 2.0 mg/L, the algal biomass and geosmin production achieved the highest level of 6.6 x 10(4) cells x mL(-1) and 1.3 x 10(4) ng x L(-1), respectively. Compared with nitrogen sources, the growth of Anabaena sp. could be promoted significantly until phosphorus level attained 0.12 mg/L, indicating that phosphorus is the main limiting nutrient source for Anabaena sp.. For Yanghe reservoir, the nutrient level has already been enough for the growth of Anabaena sp. Therefore, the nutrient source content, especially phosphorus, should be reduced effectively to control the cyanobacterium bloom and taste and odor problems.

Stimulation of pigment accumulation in Anabaena azollae strains: effect of light intensity and sugars.

The influence of high light intensity on the growth and pigment accumulating ability of Anabaena azollae was investigated. A. azollae responded positively to high light intensity (6 klx) and was further evaluated at higher intensities (10 and 15 klx), in the presence of glucose, sucrose and jaggery +/- DCMU. Significant enhancement in phycobiliproteins and carotenoids was observed in the sugar supplemented cultures at high light intensities. SDS-PAGE profiles of whole cell proteins revealed the presence of unique bands in such treatments. Sucrose supplementation induced a 30-90 % increase in carotenoids, phycocyanin and phycoerythrin content at 10 klx. Molecular analysis of the stimulatory and interactive role of sugars on pigment enhancement at high light intensity may aid in better exploitation of cyanobacteria as a source of pigments.

Algal extracellular products suppress Anabaena flos-aquae heterocyst spacing.

Intra- and interspecific chemical signals allow bacteria to respond to environmental conditions by regulating gene transcription. In cyanobacteria, gene products and the presence of fixed nitrogen regulate heterocyst frequency. In this paper, we describe a chemical made by a green alga, Chlamydomonas reinhardtii, that suppresses heterocyst formation in the co-occurring cyanobacterium, Anabaena flos-aquae. Cyanobacterial heterocyst frequencies were reduced in the presence of water-soluble, proteinase- and heat-resistant molecules greater than 15 kDa in molecular size. Green algal cells in all phases of growth made the suppressor. Ammonium and nitrate concentrations in the medium did not correlate with this change in phenotype. In addition, growth rate was not enhanced by the extracellular products. Therefore, C. reinhardtii extracellular products acted as a heterocyst inhibitor, not as a fixed nitrogen source. Chemical interactions between green algae and cyanobacteria influence heterocyst formation, an important consideration in understanding the outcome of competition between these organisms and the dynamics of phytoplankton communities.

Toxicity of two carbamate insecticides to the cyanobacterium Anabaena PCC 7120 and computations of partial lethal concentrations by the probit method.

Toxicity studies of two commercial carbamate insecticides, carbaryl and carbofuran with the nitrogen-fixing filamentous cyanobacterium Anabaena PCC 7120, are described. Under nitrogen-fixing conditions and with calcium nitrate supplementation, 100 and 120 ppm carbaryl were the respective lethal concentrations (LC100), while 20 to 80 ppm (nitrogen-fixing conditions) and 20 to 100 ppm (with nitrate supplementation) were the partial lethal doses (

Differential effects of thiobencarb toxicity on growth and photosynthesis of Anabaena variabilis with changes in phosphate level.

A thiobencarb dose of 3 mg L(-1) reduced the protein content of Anabaena variabilis, whereas it elevated the carbohydrate content. Measurements of Anabaena growth, photosynthetic activity, and respiration rate revealed that the high dose of phosphate (0.53 mM) exerted no additional toxic effect to thiobencarb toxicity. Recovery of Anabaena cells from the inhibitory effect of thiobencarb occurred immediately after its reculture in herbicide free-medium. Maximum uptake of thiobencarb was associated with high biomass yield.

Pleiotropic effects of potassium deficiency in a heterocystous, nitrogen-fixing cyanobacterium, Anabaena torulosa.

Omission of potassium from the growth medium caused multiple metabolic impairments and resulted in cessation of growth of the filamentous, heterocystous, nitrogen-fixing cyanobacterium Anabaena torulosa, during both diazotrophic and nitrogen-supplemented growth. Prominent defects observed during potassium deprivation were: (i) the loss of photosynthetic pigments, (ii) impairment of photosynthetic functions, (iii) reduced synthesis of dinitrogenase reductase (Fe-protein), (iv) inhibition of nitrogenase activity, and (v) specific qualitative modifications of protein synthesis leading to the repression of twelve polypeptides and synthesis and accumulation of nine novel polypeptides. The observed metabolic defects were reversible, and growth arrested under prolonged potassium deficiency was fully restored upon re-addition of potassium. Such pleiotropic effects of potassium deficiency demonstrate that apart from its well-known requirement for pH and turgor homeostasis, K+ plays other vital specific roles in cyanobacterial growth and metabolism.

Variation of microcystins, cyanobacterial hepatotoxins, in Anabaena spp. as a function of growth stimuli.

Cyanobacterial hepatotoxins, microcystins, are specific inhibitors of serine/threonine protein phosphatases and potent tumor promoters. They have caused several poisonings of animals and also pose a health hazard for humans through the use of water for drinking and recreation. Different strains of the same cyanobacterial species may variously be nontoxic, be neurotoxic, or produce several microcystin variants. It is poorly understood how the amount of toxins varies in a single strain. This laboratory study shows the importance of external growth stimuli in regulating the levels and relative proportions of different microcystin variants in two strains of filamentous, nitrogen-fixing Anabaena spp. The concentration of the toxins in the cells increased with phosphorus. High temperatures (25 to 30 degrees C), together with the highest levels of light studied (test range, 2 to 100 mumol m-2 s-1), decreased their amount. Different structural variants of microcystins responded differently to growth stimuli. Variants of microcystin (MCYST)-LR correlated with temperatures below 25 degrees C, and those of MCYST-RR correlated with higher temperatures. Nitrogen added into the growth medium and increasing temperatures increased the proportion of microcystin variants demethylated in amino acid 3. All variants remained mostly intracellular. Time was the most important factor causing the release of the toxins into the growth medium. Time, nitrogen added into the growth medium, and light fluxes above 25 mumol m-2 s-1 significantly increased the concentrations of the dissolved toxins. According to the results, it thus seems that the reduction of phosphorus loads in bodies of water might play a role in preventing the health hazards that toxic cyanobacterial water blooms pose, not only by decreasing the cyanobacteria but also by decreasing their toxin content.

Growth of the cyanobacterium Anabaena on molecular nitrogen: NifJ is required when iron is limited.

The nifJ gene of Klebsiella pneumoniae encodes an oxidoreductase required for the transfer of electrons from pyruvate to flavodoxin, which reduces nitrogenase. The nifJ gene of Anabaena 7120, isolated from a cosmid bank, was found to contain an open reading frame encoding a 1197-aa protein. The deduced amino acid sequence shows 50% identity to the Klebsiella homolog. The nifJ gene in Anabaena 7120 was inactivated by chromosomal interruption. The resulting mutant was unable to grow on medium depleted of both iron and combined nitrogen but grew normally, fixing nitrogen, when iron was present. NifJ transcripts of 2.7 and 4.3 kb are induced by iron depletion irrespective of nitrogen status. One particular stretch of the Anabaena 7120 nifJ gene encodes 12 aa with no complementary matches in the Klebsiella protein. This insert contains five tandem repeats of the heptamer CCCCAGT. These heptamers, as well as heptamers and octamers of other related sequences, have been located in a number of cyanobacterial genomes but are usually not found within the coding region of a gene. The site of the Anabaena 7120 heptamers in the nifJ genes of other filamentous cyanobacteria contains a surprising diversity of repeated sequences, both octamers and heptamers. The corresponding protein inserts range in length from 1 to 21 aa, relative to Klebsiella NifJ.

Directed mutagenesis of an iron-sulfur protein of the photosystem I complex in the filamentous cyanobacterium Anabaena variabilis ATCC 29413.

In oxygenic photosynthetic organisms the PSI-C polypeptide, encoded by the psaC gene, provides the ligands for two [4Fe-4S] centers, FA and FB, the terminal electron acceptors in the photosystem I (PSI) complex. An insertion mutation introduced in the psaC locus of the filamentous cyanobacterium Anabaena variabilis ATCC 29413 resulted in the creation of a mutant strain, T398-1, that lacks the PSI-C polypeptide. In medium supplemented with 5 mM fructose, the mutant cells grew well in the dark. However, when grown in the same medium under light, the doubling rate of T398-1 cells was significantly decreased. In intact cells of T398-1, bicarbonate-dependent whole-chain electron transport (PSII and PSI) could not be detected, although partial electron transport reactions involving either one of the two photosystems could be measured at significant rates. The low-temperature EPR signals attributed to the [4Fe-4S] centers FA and FB were absent in the mutant cells. Chemical titration measurements indicated that the ratios of chlorophyll to the primary donor P700 were virtually identical in membranes from the wild-type and mutant cells. Moreover, room-temperature optical spectroscopic analysis of the thylakoid membranes isolated from T398-1 showed flash-induced P700 oxidation followed by dark rereduction, indicating primary photochemistry in PSI. Thus stable assembly of the reaction center of PSI can occur in the absence of the Fe-S cluster cofactors FA and FB. These studies demonstrate that Anabaena 29413 offers a useful genetic system for targeted mutagenesis of the PSI complex.