Combined Nitrogen Removal Research Articles

Excision of the 59-kb fdxN DNA element is required for transcription of the nifD gene in Anabaena PCC 7120 Heterocysts

ABSTRACTIn the heterocyst-forming cyanobacterium Anabaena (also Nostoc) sp. strain PCC 7120, the nitrogen-fixation (nif) genes are expressed specifically in heterocysts during the late stages of development. We used gfp-reporter fusions to examine the regulation of the nifH and nifD genes. Plasmid-borne reporter fusions containing up to 500-bp of the nifH upstream region, which extended into the nifU gene, did not show developmentally regulated GFP fluorescence after removal of a nitrogen source from the growth medium, which indicated that sequences essential for transcriptional regulation are outside of the tested regions. Therefore, a gfp-reporter fusion was engineered into the chromosome at the 5′ end of nifD to produce strain AMC1774. This reporter construct at the native locus showed strong developmentally regulated GFP expression in differentiating heterocysts by 18 h after removal of combined nitrogen. We then screened for UV-induced mutants of AMC1774 that differentiated heterocysts but failed to show GFP-reporter fluorescence. Several dark mutants were obtained and then complemented with an expression library. One mutant was complemented with a clone that contained the xisF and alr1460 genes. The xisF gene is required for excision of a 59,428-bp DNA element present within the fdxN gene in the nifB-fdxN-nifS-nifU gene cluster, which is upstream of the nifHDK gene cluster. Analysis of the original UV-induced mutant showed that it was defective for excision of the fdxN DNA element. Together, our data shows that transcription of the nifHDK genes requires a distant promoter upstream of the fdxN DNA element. We used a gfp transcriptional reporter to demonstrate developmentally regulated promoter activity from the intergenic region upstream of nifB. We also show that the promoter for a second nifH gene, nifH2 (alr0874), lies proximal to nifH2 in its upstream intergenic region.

Integration of methane removal in aerobic anammox-based granular sludge reactors

ABSTRACTCombined partial nitritation-anaerobic ammonium oxidation (anammox) processes have been widely applied for nitrogen removal from anaerobic digestion reject water. However, such streams also contain dissolved methane that can escape to the atmosphere, hence contributing to global warming. This study investigates the possibility of integrating methane removal in aerobic anammox-based granular sludge reactors, through modelling and simulation. Methane removal could be established through aerobic methane-oxidizing bacteria (MOB), denitrifying anaerobic methane-oxidizing bacteria (damoB, NO2− + CH4 → N2 + CO2), and/or archaea (damoA, NO3− + CH4 → NO2− + CO2). The simulation results demonstrated that the combined removal of nitrogen and methane was feasible at low dissolved oxygen conditions. Aerobic MOB were the main responsible microorganisms for removing methane. A sensitivity analysis of key kinetic parameters showed a shift in the methanotrophic populations depending on the most favourable parameters for each microbial group, while keeping high nitrogen and methane removal efficiencies. Possible methane stripping during aeration could be limited by increasing the depth within the reactor column at which aeration was supplied. Overall, the integration of methane removal in aerobic anammox-based granular sludge reactors seems to be a promising process option to reduce the carbon footprint from wastewater treatment.

Quantitative impact of influent characteristics on nitrogen removal via anammox and denitrification in a landfill bioreactor case

In this paper, Quantitative Polymerase Chain Reaction (qPCR), Illumina MiSeq sequencing and 15N stable isotopic tracing methods were applied to study the effects of influent organic loading, BOD5/TN and NO2-N/NH4-N ratios on the combined nitrogen removal through denitrification and anammox. The results show that, the total nitrogen removal was above 90% at the optimum reaction condition: organic loading 0.04kg/m3·day, BOD5/TN 0.2, and NO2-N/NH4-N 1.0. Approximately 9.43% of the nitrogen removal occurred through anammox process when BOD5/TN was 0.1, and increased to 21.46% when BOD5/TN was 0.2. The anammox accounted for 10% when NO2-N/NH4-N was 0.5, and that increased to 20.72% when NO2-N/NH4-N was 1.0–1.5. hzsA was directly proportional to the influent BOD5/TN. A positive correlation between BOD5/TN and anammox microorganisms existed. Similarly, there was a positive correlation between NO2-N/NH4-N and denitrification microorganisms. Therefore, it is feasible to regulate the influent to improve nitrogen removal efficiency.

The trpE gene negatively regulates differentiation of heterocysts at the level of induction in Anabaena sp. strain PCC 7120.

Levels of 2-oxoglutarate (2-OG) reflect nitrogen status in many bacteria. In heterocystous cyanobacteria, a spike in the 2-OG level occurs shortly after the removal of combined nitrogen from cultures and is an integral part of the induction of heterocyst differentiation. In this work, deletion of one of the two annotated trpE genes in Anabaena sp. strain PCC 7120 resulted in a spike in the 2-OG level and subsequent differentiation of a wild-type pattern of heterocysts when filaments of the mutant were transferred from growth on ammonia to growth on nitrate. In contrast, 2-OG levels were unaffected in the wild type, which did not differentiate under the same conditions. An inverted-repeat sequence located upstream of trpE bound a central regulator of differentiation, HetR, in vitro and was necessary for HetR-dependent transcription of a reporter fusion and complementation of the mutant phenotype in vivo. Functional complementation of the mutant phenotype with the addition of tryptophan suggested that levels of tryptophan, rather than the demonstrated anthranilate synthase activity of TrpE, mediated the developmental response of the wild type to nitrate. A model is presented for the observed increase in 2-OG in the trpE mutant.

Fluorescence spectroscopy study of heterocyst differentiation in Anabaena PCC 7120 filaments

Filamentous Anabaena PCC 7120 differentiates nitrogen-fixing specialized cells called heterocysts at regular intervals following removal of combined nitrogen from the medium. Phycobiliproteins are degraded during differentiation. Heterocyst differentiation was followed at the single cell level by using confocal fluorescence microscopy. The presence of an enhanced fluorescence emission peak from allophycocyanin (APC) indicates that the degradation of the phycobilisomes during nitrogen deprivation possibly initiates at the linker between APC and photosystem II in a bottom-to-top disassembly model. Furthermore, the fluorescence emission peak around 650 nm provides an advantageous marker to identify early candidates for differentiation.

Diazocyte development in the marine diazotrophic cyanobacterium Trichodesmium

The establishment of non-diazotrophic cultures of the filamentous marine cyanobacterium Trichodesmium erythraeum IMS101 enabled the first detailed investigation of the process leading to the development of its unique nitrogen-fixing cell type, the diazocyte. Trichome heterogeneity was apparent already within 3-8 h, while the differentiation of mature diazocytes, containing the nitrogenase enzyme, required 27 h after the removal of combined nitrogen. The distribution of 'pro-diazocytes' within the trichomes correlates with the localization of mature diazocytes, which suggests that pattern regulation is an early event during diazocyte development. The development was initially identified as changes in the subcellular ultrastructure, most notably the degradation of glycogen granules and gas vacuoles. These changes were preceded by the induced expression of the global nitrogen regulator ntcA at an early stage of combined nitrogen deprivation, followed by elevated expression of genes related to nitrogen metabolism and their corresponding proteins. The strongest induction (10-fold) was related to the transcription of the respiratory gene coxB2, apparent already at an early stage, which suggests an important role for respiration and the subsequent energy generation in the subcellular changes found, and in the creation of the reducing environment required for nitrogen fixation in diazocytes.

Directional RNA deep sequencing sheds new light on the transcriptional response of Anabaena sp. strain PCC 7120 to combined-nitrogen deprivation

BackgroundCyanobacteria are potential sources of renewable chemicals and biofuels and serve as model organisms for bacterial photosynthesis, nitrogen fixation, and responses to environmental changes. Anabaena (Nostoc) sp. strain PCC 7120 (hereafter Anabaena) is a multicellular filamentous cyanobacterium that can "fix" atmospheric nitrogen into ammonia when grown in the absence of a source of combined nitrogen. Because the nitrogenase enzyme is oxygen sensitive, Anabaena forms specialized cells called heterocysts that create a microoxic environment for nitrogen fixation. We have employed directional RNA-seq to map the Anabaena transcriptome during vegetative cell growth and in response to combined-nitrogen deprivation, which induces filaments to undergo heterocyst development. Our data provide an unprecedented view of transcriptional changes in Anabaena filaments during the induction of heterocyst development and transition to diazotrophic growth.ResultsUsing the Illumina short read platform and a directional RNA-seq protocol, we obtained deep sequencing data for RNA extracted from filaments at 0, 6, 12, and 21 hours after the removal of combined nitrogen. The RNA-seq data provided information on transcript abundance and boundaries for the entire transcriptome. From these data, we detected novel antisense transcripts within the UTRs (untranslated regions) and coding regions of key genes involved in heterocyst development, suggesting that antisense RNAs may be important regulators of the nitrogen response. In addition, many 5' UTRs were longer than anticipated, sometimes extending into upstream open reading frames (ORFs), and operons often showed complex structure and regulation. Finally, many genes that had not been previously identified as being involved in heterocyst development showed regulation, providing new candidates for future studies in this model organism.ConclusionsDirectional RNA-seq data were obtained that provide comprehensive mapping of transcript boundaries and abundance for all transcribed RNAs in Anabaena filaments during the response to nitrogen deprivation. We have identified genes and noncoding RNAs that are transcriptionally regulated during heterocyst development. These data provide detailed information on the Anabaena transcriptome as filaments undergo heterocyst development and begin nitrogen fixation.

Ligninolytic and antioxidative enzymes of a marine cyanobacterium Oscillatoria willei BDU 130511 during Poly R-478 decolourization

Removal of combined nitrogen and addition of Poly R-478 to the growth medium enhanced oxidative stress, and altered the activities of ligninolytic enzymes of Oscillatoria willei BDU 130511. The activities of ligninolytic and antioxidative enzymes (LiP-like, LAC, PPO, SOD, POD, CAT, and APX) were increased upon nitrogen limitation and dye supplementation. The metabolic enzymes tested (GR, GPX, EST, and MDH) showed differential expressions under varied growth conditions. Up on nitrogen limitation, O. willei BDU 130511 showed enhanced ligninolytic activity as shown by α-keto-γ-methylthiolbutyric acid (KTBA) oxidation and increased H 2O 2 production. The organism decolourized 52% of Poly R-478 due to partial degradation and adsorption of dye particles from dye-added medium after 7 days of growth. This manuscript discusses the responses of ligninolytic and antioxidative enzymes of O. willei BDU 130511 during Poly R-478 decolourization/degradation, and the organism’s potential in bioremediation.

Tu-P10:416 Circadian pattern of homocystein, interleukin-6 and C-reactive protein in patients with hypertension and in patients with obstructive sleep APNEA

Removal of combined nitrogen and addition of Poly R-478 to the growth medium enhanced oxidative stress, and altered the activities of ligninolytic enzymes of Oscillatoria willei BDU 130511. The activities of ligninolytic and antioxidative enzymes (LiP-like, LAC, PPO, SOD, POD, CAT, and APX) were increased upon nitrogen limitation and dye supplementation. The metabolic enzymes tested (GR, GPX, EST, and MDH) showed differential expressions under varied growth conditions. Up on nitrogen limitation, O. willei BDU 130511 showed enhanced ligninolytic activity as shown by α-keto-γ-methylthiolbutyric acid (KTBA) oxidation and increased H2O2 production. The organism decolourized 52% of Poly R-478 due to partial degradation and adsorption of dye particles from dye-added medium after 7 days of growth. This manuscript discusses the responses of ligninolytic and antioxidative enzymes of O. willei BDU 130511 during Poly R-478 decolourization/degradation, and the organism’s potential in bioremediation.

CcbP, a calcium-binding protein from Anabaena sp. PCC 7120, provides evidence that calcium ions regulate heterocyst differentiation.

Although it is known that calcium is a very important messenger involved in many eukaryotic cellular processes, much less is known about calcium's role in bacteria. CcbP, a Ca(2+)-binding protein, was isolated from the heterocystous cyanobacterium Anabaena sp. PCC 7120, and the ccbP gene was cloned and inactivated. In the absence of combined nitrogen, inactivation of ccbP resulted in multiple contiguous heterocysts, whereas overexpression of ccbP suppressed heterocyst formation. Calmodulin, which is not present in Anabaena species, could also suppress heterocyst formation in both Anabaena sp. PCC 7120 and Anabaena variabilis. HetR induction upon nitrogen step-down was slow in the strain overexpressing ccbP. The Ca(2+) reporter protein obelin was used to show that mature heterocysts had a high intracellular free Ca(2+)concentration {[Ca(2+)](i)}, and immunoblotting showed that CcbP was absent from heterocysts. A regular pattern of cells with higher [Ca(2+)](i) was established during heterocyst differentiation before the appearance of proheterocysts. A rapid increase of [Ca(2+)](i) could be detected 4 h after the removal of combined nitrogen, and this increase was suppressed by excessive CcbP. These results suggest that Ca(2+) ions play very important roles in hetR induction and heterocyst differentiation.

Identification of the active site of HetR protease and its requirement for heterocyst differentiation in the cyanobacterium Anabaena sp. strain PCC 7120.

HetR is a serine-type protease required for heterocyst differentiation in heterocystous cyanobacteria under conditions of nitrogen deprivation. We have identified the active Ser residue of HetR from Anabaena sp. strain PCC 7120 by site-specific mutagenesis. By changing the S152 residue to an Ala residue, the mutant protein cannot be labeled by Dansyl fluoride, a specific serine-type protein inhibitor. The mutant protein showed no autodegradation in vitro. The mutant hetR gene was introduced into Anabaena strain 884a, a hetR mutant. The resultant strain, Anabaena strain S152A, could not form heterocysts under conditions of nitrogen deprivation even though the up-regulation of the mutant hetR gene was induced upon removal of combined nitrogen. The Anabaena strain 216, which carries a mutant hetR gene encoding S179N HetR and could not form heterocysts, also produced HetR protein upon induction. Sequence comparison shows that Ser152 is conserved in all cyanobacterial HetR. Immunoblotting was used to study HetR induction in both the wild-type and mutant strains. The amount of mutant HetR in strain S152A and in strain 216 increased continuously for 24 h after nitrogen step-down, while the amount of HetR in wild-type cells reached a maximum level within 6 h after nitrogen step-down. Our results show the Ser152 is the active site of HetR. The protease activity is required for heterocyst differentiation and might be needed for repression of HetR overproduction under conditions of nitrogen deprivation.

The presence and expression of hetR in the non-heterocystous cyanobacterium Symploca PCC 8002

The filamentous cyanobacteria Symploca PCC 8002 ( Symploca) and Trichodesmium spp. fix nitrogen aerobically in the light in a light/dark cycle, without forming specialized thick-walled cells (heterocysts). Even though they do not form heterocysts, we amplified and sequenced a segment of a key regulatory gene in heterocyst differentiation, the hetR gene, from Symploca, Trichodesmium erythraeum and Leptolyngbya PCC 73110 (which fixes nitrogen anaerobically) using degenerate oligonucleotides. The transcriptional level of hetR in Symploca PCC 8002 was examined in relation to nifH expression during nitrogen step-down. The expression pattern of hetR suggests that it was not induced during removal of combined nitrogen, as is the case with the heterocystous cyanobacteria. This is the first report of sequences corresponding to a portion of hetR from within the group of non-heterocystous cyanobacteria.

The presence and expression of hetR in the non-heterocystous cyanobacterium Symploca PCC 8002.

The filamentous cyanobacteria Symploca PCC 8002 (Symploca) and Trichodesmium spp. fix nitrogen aerobically in the light in a light/dark cycle, without forming specialized thick-walled cells (heterocysts). Even though they do not form heterocysts, we amplified and sequenced a segment of a key regulatory gene in heterocyst differentiation, the hetR gene, from Symploca, Trichodesmium erythraeum and Leptolyngbya PCC 73110 (which fixes nitrogen anaerobically) using degenerate oligonucleotides. The transcriptional level of hetR in Symploca PCC 8002 was examined in relation to nifH expression during nitrogen step-down. The expression pattern of hetR suggests that it was not induced during removal of combined nitrogen, as is the case with the heterocystous cyanobacteria. This is the first report of sequences corresponding to a portion of hetR from within the group of non-heterocystous cyanobacteria.

Tidal effect on dynamics of pore water nitrate in intertidal sediment of a eutrophic Estuary

To evaluate the effect of the tidal cycle on the pore water nitrate dynamics in intertidal sediment, concentrations of inorganic nitrogen in water and sediment were monitored during tidal cycles in the mud flat of Tama Estuary, Japan. During submergence, nitrate concentration was highest in the overlying water and decreased monotonically with increasing depth in the sediment, suggesting that the primary source of nitrate in the sediment was nitrate transported from the overlying water. Pore water nitrate decreased remarkably during the initial 3–4 hours after the onset of exposure. Thereafter, it was constant or slightly increased until tidal flooding.In situ accumulation of nitrate at the end of exposure, however, did not exceed the nitrate concentrations in the overlying water. The inhibition of nitrate reduction and the stimulation of nitrification would explain the change of nitrate concentration, both consistent with the input of oxygen into the sediment following a 10 mm drop of the water table. In Tama Estuary sediments, the effect of the tidal cycle on the removal of combined nitrogen is rather negative, because high nitrate concentrations in the overlying water canceled the positive effect of nitrate accumulation by nitrification during exposure, while tidal oxygen intrusion have an inhibitory effection sedimentary denitrification.

Isolation and characterization of the gene encoding the principal sigma factor of the vegetative cell RNA polymerase from the cyanobacterium Anabaena sp. strain PCC 7120

The filamentous cyanobacterium Anabaena sp. strain PCC 7120 responds to combined nitrogen deprivation by forming specialized nitrogen-fixing cells at regular intervals along the filament. Genetic and biochemical studies have indicated that regulation of gene expression during differentiation occurs at the transcriptional level. As part of a characterization of RNA polymerase during differentiation, the gene encoding the 52-kDa principal sigma factor of the Anabaena sp. strain PCC 7120 vegetative-cell RNA polymerase was isolated by using an oligonucleotide probe based on the sequence of the N-terminal seven amino acids of the purified protein. sigA codes for a 390-amino-acid polypeptide that has a predicted molecular weight of 45,641. The amino acid sequence of the polypeptide encoded by sigA contains four regions corresponding to conserved domains of the principal RNA polymerase sigma factors of Escherichia coli (sigma 70) and Bacillus subtilis (sigma 43). Thus, although the subunit composition of cyanobacterial RNA polymerase core differs from that of other eubacteria (G. J. Schneider and R. Haselkorn, J. Bacteriol. 170:4136-4140, 1988), the principal sigma factor of at least one cyanobacterium is typically eubacterial. In contrast to sigma 70 and sigma 43 operon organization, sigA is monocistronic and encodes two transcripts of 1.7 and 2.2 kb. The abundance of the 1.7-kb transcript remains constant under both nitrogen-replete and nitrogen-limiting conditions, whereas the 2.2-kb transcript is induced following the removal of combined nitrogen. Continued or enhanced transcription of sigA under nitrogen starvation conditions is consistent with the observation that the principal RNA polymerase in differentiating cells contains SigA.

High Nitrite Levels off Northern Peru: A Signal of Instability in the Marine Denitrification Rate

During February and March 1985, nitrite levels along the northern (approximately 7 degrees to 10 degrees S) Peruvian coast were unusually high. These accumulations occurred in oxygen-deficient waters, suggesting intensified denitrification. In a shallow offshore nitrite maximum, concentrations were as high as 23 micromoles per liter (a record high). Causes for the unusual conditions may include a cold anomaly that followed the 1982-83 El Niño. The removal of combined nitrogen (approximately 3 to 10 trillion grams of nitrogen per year) within zones of new or enhanced denitrification observed between 7 degrees to 16 degrees S suggests a significant increase in oceanic denitrification.

Biological and Cultural Characteristics of the Effective Frankia Strain HFPCcl3 (Actinomycetales) from Casuarina cunninghamiana (Casuarinaceae)

From homogenates prepared from surface-sterilized nodules of seedlings of Casuarina cunninghamiana grown aeroponically, a strain of Frankia designated HFPCc13 was isolated and has been grown in pure filamentous culture in a defined synthetic nutrient medium. Vesicle and sporangium formation can be induced by removal of combined nitrogen from the medium.Frankia strain HFPCc13 nodulates young seedlings of C. cunninghamiana and C. equisetifolia within three weeks of inoculation with an optimum root medium pH of 6–7 for nodulation and optimum temperature of 30–35 °C. The presence of combined nitrogen in the root medium inhibits nodulation with NH4+ more inhibitory than NO3−. Frankia HFPCc13 does not nodulate Allocasuarina species within the same family nor several other possible actinorhizal plants tested. Thus this strain is quite precise in its host specificity. The rate of acetylene reduction was greater in C. cunninghamiana than the closely related species C. equisetifolia. In neither of these host species were vesicles observed to occur within the infected root nodules which had been demonstrated to be actively fixing dinitrogen. Root nodules were shown to be active in acetylene reduction over a range of O2 concentration in the gaseous environment with an optimum at about 20 per cent O2, the ambient PO2 of the air. The mechanism(s) for oxygen protection of nitrogenase within the filamentous form of Frankia within these nodules remains to be explained.

Initiation and ontogeny of vesicles in cultured Frankia sp. strain HFPArI3.

Removal of combined nitrogen from the medium of Frankia sp. strain HFPArI3 induced the formation of specialized structures, called vesicles, which are the proposed site of nitrogen fixation. After 5 to 6 h of culture on N-free medium, newly formed vesicles, termed provesicles, arose from the tips of some hyphae. These structures were spherical, phase dark, ca. 1.5 to 2.0 micron in diameter, and were not associated with acetylene reduction (nitrogenase) activity. Provesicles reached their greatest frequency after ca. 24 h of N-free culture. Provesicles increased in size to become mature vesicles which first appeared after 18 to 20 h of N-free culture. They were ca. 2.5 micron in diameter, phase bright, and reached their greatest frequency after 5 to 6 days, at which time nitrogenase activity peaked. Some vesicles eventually became damaged structurally and took on the appearance of ghosts. Transmission electron micrographs revealed an increase in size from provesicle to mature vesicle. Also evident with the micrographs were the presence of a septum between the young provesicle and parental hypha, the presence of glycogen in some young vesicles, the development of internal septations as vesicles matured, and the degradation of cytoplasm and internal septae in ghost vesicles. The extent to which the formation of vesicles is reversible by the addition of NH4+ was investigated. Commitment times of 3.2 and 6.5 h were obtained for provesicles and vesicles, respectively. A concentration-dependent inhibition of nitrogenase by NH4+ was demonstrated. The structure of preexisting vesicles was also affected by addition of NH4+ to the culture medium.

Control of phycobiliprotein proteolysis and heterocyst differentiation in Anabaena.

Phycobiliprotein degradation can be initiated in cultures of the cyanobacterium Anabaena by removal of combined nitrogen from the medium. Certain strains of Anabaena differentiate cells specialized for aerobic nitrogen fixation (heterocysts) under such conditions. We describe here a procedure for the preparation of extracts from heterocysts or vegetative cells that contain an activity capable of degrading only the phycobiliproteins in a mixture of soluble Anabaena proteins in vitro. This activity increased under nitrogen starvation conditions or in ammonia-replete cultures treated with the glutamine synthetase inhibitor methionine sulfoximine. The increase in activity induced by nitrogen starvation was prevented by chloramphenicol or by carbon starvation. Under all these conditions, phycobiliprotein degradative activity assayed in vitro was correlated with the loss of phycobiliprotein absorbance in vivo. Finally, starvation of a met auxotroph of Anabaena for methionine (in the presence of ammonia) did not induce phycobiliprotein degradation in vivo or the increase in proteinase activity. Together with direct measurements of ppGpp, these results indicate that proteolysis in Anabaena is not controlled by compounds associated with the stringent response in Escherichia coli. Since the increase in proteinase activity appears to be regulated by the same variables that control heterocyst differentiation, the activity should provide a useful biochemical marker for the early events of differentiation.

Dynamic aspects of phycobilisome structure

In exponentially growing cells of Synechococcus sp. 6301, over 95% of the phycobiliproteins are located in phycobilisomes, and the remainder is present in the form of low molecular weight aggregates. In addition to the subunits of the phycobiliproteins (C-phycocyanin, allophycocyanin, allophycocyanin B), the phycobilisomes of this unicellular cyanobacterium contain five non-pigmented polypeptides. During the initial phase of starvation (24 h after removal of combined nitrogen from the growth medium), the phycobiliproteins in the low molecular weight fraction largely disappeared. Phycocyanin was lost more rapidly from this fraction than allophycocyanin. Simultaneous changes in the phycobilisome were (1) a decrease in sedimentation coefficient, (2) a decrease in phycocyanin: allophycocyanin ratio, (3) a shift in the fluorescence emission maximum from 673 to 676 nm, and (4) a selective complete loss of a 30,000 dalton non-pigmented polypeptide. Upon extensive nitrogen starvation (72 h), the intracellular level of phycocyanin decreased by over 30-fold. These results indicate that in the early stage of nitrogen starvation, the free phycobiliproteins of the cell are degraded, as well as a significant proportion of the phycocyanin from the periphery of the phycobilisome. However, the structures partially depleted of phycocyanin still function efficiently in energy transfer. On extended starvation, total degradation of residual phycobilisomes takes place, possibly in conjunction with the detachment of these structures from the thylakoids. None of the effects of the absence of combined nitrogen were seen when cells were starved in the presence of chloramphenicol, or in a methionine auxotroph starved for methionine.