The redox-sensitive R-loop of the carbon control protein SbtB contributes to the regulation of the cyanobacterial CCM.

SbtB is a PII-like protein that regulates the carbon-concentrating mechanism (CCM) in cyanobacteria. SbtB proteins can bind many adenyl nucleotides and possess a characteristic C-terminal redox sensitive loop (R-loop) that forms a disulfide bridge in response to the diurnal state of the cell. SbtBs also possess an ATPase/ADPase activity that is modulated by the redox-state of the R-loop. To investigate the R-loop in the cyanobacterium Synechocystis sp. PCC 6803, site-specific mutants, unable to form the hairpin and permanently in the reduced state, and a R-loop truncation mutant, were characterized under different inorganic carbon (Ci) and light regimes. Growth under diurnal rhythm showed a role of the R-loop as sensor for acclimation to changing light conditions. The redox-state of the R-loop was found to impact the binding of the adenyl-nucleotides to SbtB, its membrane association and thereby the CCM regulation, while these phenotypes disappeared after truncation of the R-loop. Collectively, our data imply that the redox-sensitive R-loop provides an additional regulatory layer to SbtB, linking the CO2-related signaling activity of SbtB with the redox state of cells, mainly reporting the actual light conditions. This regulation not only coordinates CCM activity in the diurnal rhythm but also affects the primary carbon metabolism.

From an Hsp90 - binding protein to a peptide drug.

The Lpl proteins represent a class of lipoproteins that was first described in the opportunistic bacterial pathogen Staphylococcus aureus, where they contribute to pathogenicity by enhancing F-actin levels of host epithelial cells and thereby increasing S. aureus internalization. The model Lpl protein, Lpl1 was shown to interact with the human heat shock proteins Hsp90α and Hsp90ß, suggesting that this interaction may trigger all observed activities. Here we synthesized Lpl1-derived peptides of different lengths and identified two overlapping peptides, namely, L13 and L15, which interacted with Hsp90α. Unlike Lpl1, the two peptides not only decreased F-actin levels and S. aureus internalization in epithelial cells but they also decreased phagocytosis by human CD14+ monocytes. The well-known Hsp90 inhibitor, geldanamycin, showed a similar effect. The peptides not only interacted directly with Hsp90α, but also with the mother protein Lpl1. While L15 and L13 significantly decreased lethality of S. aureus bacteremia in an insect model, geldanamycin did not. In a mouse bacteremia model L15 was found to significantly decreased weight loss and lethality. Although the molecular bases of the L15 effect is still elusive, in vitro data indicate that simultaneous treatment of host immune cells with L15 or L13 and S. aureus significantly increase IL-6 production. L15 and L13 represent not antibiotics but they cause a significant reduction in virulence of multidrug-resistant S. aureus strains in in vivo models. In this capacity, they can be an important drug alone or additive with other agents.

Roles of second messengers in the regulation of cyanobacterial physiology: the carbon-concentrating mechanism and beyond.

Second messengers are a fundamental category of small molecules and ions that are involved in the regulation of many processes in all domains of life. Here we focus on cyanobacteria, prokaryotes playing important roles as primary producers in the geochemical cycles due to their capability of oxygenic photosynthesis and carbon and nitrogen fixation. Of particular interest is the inorganic carbon-concentrating mechanism (CCM), which allows cyanobacteria to concentrate CO2 near RubisCO. This mechanism needs to acclimate toward fluctuating conditions, such as inorganic carbon availability, intracellular energy levels, diurnal light cycle, light intensity, nitrogen availability, and redox state of the cell. During acclimation to such changing conditions, second messengers play a crucial role, particularly important is their interaction with the carbon control protein SbtB, a member of the PII regulator protein superfamily. SbtB is capable of binding several second messengers, uniquely adenyl nucleotides, to interact with different partners in a variety of responses. The main identified interaction partner is the bicarbonate transporter SbtA, which is regulated via SbtB depending on the energy state of the cell, the light conditions, and different CO2 availability, including cAMP signaling. The interaction with the glycogen branching enzyme, GlgB, showed a role for SbtB in the c-di-AMP-dependent regulation of glycogen synthesis during the diurnal life cycle of cyanobacteria. SbtB has also been shown to impact gene expression and metabolism during acclimation to changing CO2 conditions. This review summarizes the current knowledge about the complex second messenger regulatory network in cyanobacteria, with emphasis on carbon metabolism.

Pleiotropic effects of PipX, PipY, or RelQ overexpression on growth, cell size, photosynthesis, and polyphosphate accumulation in the cyanobacterium Synechococcus elongatus PCC7942.

The cyanobacterial protein PipY belongs to the Pyridoxal-phosphate (PLP)-binding proteins (PLPBP/COG0325) family of pyridoxal-phosphate-binding proteins, which are represented in all three domains of life. These proteins share a high degree of sequence conservation, appear to have purely regulatory functions, and are involved in the homeostasis of vitamin B6 vitamers and amino/keto acids. Intriguingly, the genomic context of the pipY gene in cyanobacteria connects PipY with PipX, a protein involved in signaling the intracellular energy status and carbon-to-nitrogen balance. PipX regulates its cellular targets via protein-protein interactions. These targets include the PII signaling protein, the ribosome assembly GTPase EngA, and the transcriptional regulators NtcA and PlmA. PipX is thus involved in the transmission of multiple signals that are relevant for metabolic homeostasis and stress responses in cyanobacteria, but the exact function of PipY is still elusive. Preliminary data indicated that PipY might also be involved in signaling pathways related to the stringent stress response, a pathway that can be induced in the unicellular cyanobacterium Synechococcus elongatus PCC7942 by overexpression of the (p)ppGpp synthase, RelQ. To get insights into the cellular functions of PipY, we performed a comparative study of PipX, PipY, or RelQ overexpression in S. elongatus PCC7942. Overexpression of PipY or RelQ caused similar phenotypic responses, such as growth arrest, loss of photosynthetic activity and viability, increased cell size, and accumulation of large polyphosphate granules. In contrast, PipX overexpression decreased cell length, indicating that PipX and PipY play antagonistic roles on cell elongation or cell division. Since ppGpp levels were not induced by overexpression of PipY or PipX, it is apparent that the production of polyphosphate in cyanobacteria does not require induction of the stringent response.

Carbon signaling protein SbtB possesses atypical redox-regulated apyrase activity to facilitate regulation of bicarbonate transporter SbtA.

The PII superfamily consists of widespread signal transduction proteins found in all domains of life. In addition to canonical PII proteins involved in C/N sensing, structurally similar PII-like proteins evolved to fulfill diverse, yet poorly understood cellular functions. In cyanobacteria, the bicarbonate transporter SbtA is co-transcribed with the conserved PII-like protein, SbtB, to augment intracellular inorganic carbon levels for efficient CO2 fixation. We identified SbtB as a sensor of various adenine nucleotides including the second messenger nucleotides cyclic AMP (cAMP) and c-di-AMP. Moreover, many SbtB proteins possess a C-terminal extension with a disulfide bridge of potential redox-regulatory function, which we call R-loop. Here, we reveal an unusual ATP/ADP apyrase (diphosphohydrolase) activity of SbtB that is controlled by the R-loop. We followed the sequence of hydrolysis reactions from ATP over ADP to AMP in crystallographic snapshots and unravel the structural mechanism by which changes of the R-loop redox state modulate apyrase activity. We further gathered evidence that this redox state is controlled by thioredoxin, suggesting that it is generally linked to cellular metabolism, which is supported by physiological alterations in site-specific mutants of the SbtB protein. Finally, we present a refined model of how SbtB regulates SbtA activity, in which both the apyrase activity and its redox regulation play a central role. This highlights SbtB as a central switch point in cyanobacterial cell physiology, integrating not only signals from the energy state (adenyl-nucleotide binding) and the carbon supply via cAMP binding but also from the day/night status reported by the C-terminal redox switch.

Millet-inspired systems metabolic engineering of NUE in crops.

The use of nitrogen (N) fertilizers in agriculture has a great ability to increase crop productivity. However, their excessive use has detrimental effects on the environment. Therefore, it is necessary to develop crop varieties with improved nitrogen use efficiency (NUE) that require less N but have substantial yields. Orphan crops such as millets are cultivated in limited regions and are well adapted to lower input conditions. Therefore, they serve as a rich source of beneficial traits that can be transferred into major crops to improve their NUE. This review highlights the tremendous potential of systems biology to unravel the enzymes and pathways involved in the N metabolism of millets, which can open new possibilities to generate transgenic crops with improved NUE.

Native metabolomics identifies the rivulariapeptolide family of protease inhibitors.

The identity and biological activity of most metabolites still remain unknown. A bottleneck in the exploration of metabolite structures and pharmaceutical activities is the compound purification needed for bioactivity assignments and downstream structure elucidation. To enable bioactivity-focused compound identification from complex mixtures, we develop a scalable native metabolomics approach that integrates non-targeted liquid chromatography tandem mass spectrometry and detection of protein binding via native mass spectrometry. A native metabolomics screen for protease inhibitors from an environmental cyanobacteria community reveals 30 chymotrypsin-binding cyclodepsipeptides. Guided by the native metabolomics results, we select and purify five of these compounds for full structure elucidation via tandem mass spectrometry, chemical derivatization, and nuclear magnetic resonance spectroscopy as well as evaluation of their biological activities. These results identify rivulariapeptolides as a family of serine protease inhibitors with nanomolar potency, highlighting native metabolomics as a promising approach for drug discovery, chemical ecology, and chemical biology studies.

The impact of the cyanobacterial carbon-regulator protein SbtB and of the second messengers cAMP and c-di-AMP on CO2 -dependent gene expression.

The amount of inorganic carbon (Ci ) fluctuates in aquatic environments. Cyanobacteria evolved a Ci -concentrating mechanism (CCM) that is regulated at different levels. The regulator SbtB binds to the second messengers cAMP or c-di-AMP and is involved in acclimation to low Ci (LC) in Synechocystis sp. PCC 6803. Here, we investigated the role of SbtB and of associated second messengers at different Ci conditions. The transcriptome of wild-type (WT) Synechocystis and the ΔsbtB mutant were compared with Δcya1, a mutant defective in cAMP production, and ΔdacA, a mutant defective in generating c-di-AMP. A defined subset of LC-regulated genes in the WT was already changed in ΔsbtB under high Ci (HC) conditions. This response of ΔsbtB correlated with a diminished induction of many CCM-associated genes after LC shift in this mutant. The Δcya1 mutant showed less deviation from WT, whereas ΔdacA induced CCM-associated genes under HC. Metabolome analysis also revealed differences between the strains, whereby ΔsbtB showed slower accumulation of 2-phosphoglycolate and ΔdacA differences among amino acids compared to WT. Collectively, these results indicate that SbtB regulates a subset of LC acclimation genes while c-di-AMP and especially cAMP appear to have a lesser impact on gene expression under different Ci availabilities.

New views on PII signaling: from nitrogen sensing to global metabolic control.

PII proteins are multitasking information-processing proteins occurring in bacteria, archaea, and plastids, decoding the metabolic state of the cells and providing this information to various regulatory targets. Research in recent years identified a wide range of novel PII targets mainly through ligand fishing assays, indicating that PII proteins evolved into major regulatory hubs of cellular metabolism. PII proteins orchestrate not only key steps of nitrogen and carbon metabolism but rather control a wide range of transporters and can also regulate the production of signaling molecules (c-di-GMP) and cofactors (NAD+). A recently identified class of PII-interacting proteins, which by themselves have no enzymatic activity, modulate cellular processes through protein interactions, further extending the regulatory range of PII proteins.

Diurnal metabolic control in cyanobacteria requires perception of second messenger signaling molecule c-di-AMP by the carbon control protein SbtB.

Because of their photosynthesis-dependent lifestyle, cyanobacteria evolved sophisticated regulatory mechanisms to adapt to oscillating day-night metabolic changes. How they coordinate the metabolic switch between autotrophic and glycogen-catabolic metabolism in light and darkness is poorly understood. Recently, c-di-AMP has been implicated in diurnal regulation, but its mode of action remains elusive. To unravel the signaling functions of c-di-AMP in cyanobacteria, we isolated c-di-AMP receptor proteins. Thereby, the carbon-sensor protein SbtB was identified as a major c-di-AMP receptor, which we confirmed biochemically and by x-ray crystallography. In search for the c-di-AMP signaling function of SbtB, we found that both SbtB and c-di-AMP cyclase­deficient mutants showed reduced diurnal growth and that c-di-AMP­bound SbtB interacts specifically with the glycogen-branching enzyme GlgB. Accordingly, both mutants displayed impaired glycogen synthesis during the day and impaired nighttime survival. Thus, the pivotal role of c-di-AMP in day-night acclimation can be attributed to SbtB-mediated regulation of glycogen metabolism.

Efficacy of Bottle Gourd Seeds' Extracts in Chemical Hazard Reduction Secreted as Toxigenic Fungi Metabolites.

Bottle gourd seeds are surrounded by innumerable bioactive components of phytochemicals. This work aimed to evaluate the effectiveness of bottle gourd extracts as antimicrobial and an-ti-mycotoxigenic against toxigenic fungi and mycotoxins. Polar and nonpolar extracts were made from the seeds. The polar eco-friendly extract was prepared by an ultrasonication-assisted technique utilizing aqueous isopropanol (80%), whereas the non-polar extract was obtained using petroleum ether (40-60). The antioxidant efficacy, total phenolic content, and flavonoid content of the extracts were all measured. The fatty acid profile was measured using GC equipment, and the influence on toxigenic fungus and mycotoxin release was also investigated. The antioxidant efficacy of the polar extract is reflected. The total phenolic values of the oil and polar extract were 15.5 and 267 mg of GAE/g, respectively. The total flavonoid content of the oil was 2.95 mg catechol/g, whereas the isopropyl extract of seeds contained 14.86 mg catechol/g. The polar extract inhibited the DPPH more effectively than oil. When compared to other seed oils, the fatty acid composition differed. The pathogens were distinguished by the MIC and MFC for the polar extract. Three sterols were found in the oil, with a high concentration of B-sitosterols. The oil's valuable -carotene content and tocopherol content were recorded. When compared to traditional antibiotics, the polar extract has shown promising antimicrobial activity against infections and toxigenic fungi. Bottle gourd extracts, as a non-traditional bioactive source, are viewed as a potentially promising alternative that might contribute to increased food safety, shelf-life, and security.

The Effect of Wall Material Type on the Encapsulation Efficiency and Oxidative Stability of Fish Oils.

Fish oil is the primary source of long-chain omega-3 fatty acids, which are important nutrients that assist in the prevention and treatment of heart disease and have many health benefits. It also contains vitamins that are lipid-soluble, such as vitamins A and D. This work aimed to determine how the wall material composition influenced the encapsulation efficiency and oxidative stability of omega fish oils in spray-dried microcapsules. In this study, mackerel, sardine waste oil, and sand smelt fish oil were encapsulated in three different wall materials (whey protein, gum Arabic (AG), and maltodextrin) by conventional spray-drying. The effect of the different wall materials on the encapsulation efficiency (EE), flowability, and oxidative stability of encapsulated oils during storage at 4 °C was investigated. All three encapsulating agents provided a highly protective effect against the oxidative deterioration of the encapsulated oils. Whey protein was found to be the most effective encapsulated agent comparing to gum Arabic and maltodextrin. The results indicated that whey protein recorded the highest encapsulation efficiency compared to the gum Arabic and maltodextrin in all encapsulated samples with EE of 71.71%, 68.61%, and 64.71% for sand smelt, mackerel, and sardine oil, respectively. Unencapsulated fish oil samples (control) recorded peroxide values (PV) of 33.19, 40.64, and 47.76 meq/kg oil for sand smelt, mackerel, and sardine oils after 35 days of storage, while all the encapsulated samples showed PV less than 10 in the same storage period. It could be concluded that all the encapsulating agents provided a protective effect to the encapsulated fish oil and elongated the shelf life of it comparing to the untreated oil sample (control). The results suggest that encapsulation of fish oil is beneficial for its oxidative stability and its uses in the production of functional foods.

Kinetic Analysis of a Protein-protein Complex to Determine its Dissociation Constant (KD) and the Effective Concentration (EC50) of an Interplaying Effector Molecule Using Bio-layer Interferometry.

Biolayer interferometry (BLI) is an emerging analytical tool that allows the study of protein complexes in real time to determine protein complex kinetic parameters. This article describes a protocol to determine the KD of a protein complex using a 6×His tagged fusion protein as bait immobilized on the NTA sensor chip of the FortéBio® Octet K2 System (Sartorius). We also describe how to determine the half maximal effective concentration (EC50, also known as IC50 for inhibiting effectors) of a metabolite. The complete protocol allows the determination of protein complex KD and small molecular effector EC50 within 8 h, measured in triplicates. Graphic abstract: Principle of the Biolayer interferometry measurement. (Middle, top) Exemplary result of the BLI measurement using Octet® (Raw Data). Wavelength shift (Δλ) against time. (A) Baseline 1. Baseline measurement. When the sensor is equilibrated in the kinetics buffer. The light is reflected with no difference. (B) Load. The his-tagged proteins (ligand) are loaded onto the sensor surface. The light is reflected with a shift of the wavelength. (C) Baseline 2. The loaded sensor is equilibrated in the kinetics buffer. No further wavelength shift appears. (D) Association. The loaded sensor is dipped into the analyte solution. The analyte binds to the immobilized ligand along with an increased wavelength shift. (E) Dissociation. Afterward, the sensor is dipped again into the kinetics buffer without the analyte. Some analyte molecules dissociate. The wavelength shift decreases. (Subfigures A-E) The left side shows the position of the sensor during the measurement seen in the representative BLI measurement, marked with the figure label. The right side shows the light path in the sensor. Black waves represent the light emitted to the sensor surface. The red waves show the light reflected from the sensor surface back to the detector.

Ultra-fast, high throughput and inexpensive detection of SARS-CoV-2 seroconversion using Ni2+ magnetic beads.

To monitor the levels of protecting antibodies raised in the population in response to infection and/or to immunization with SARS-CoV-2, we need a technique that allows high throughput and low-cost quantitative analysis of human IgG antibodies reactive against viral antigens. Here we describe an ultra-fast, high throughput and inexpensive assay to detect SARS-CoV-2 seroconversion in humans. The assay is based on Ni2+ magnetic particles coated with His tagged SARS-CoV-2 antigens. A simple and inexpensive 96 well plate magnetic extraction/homogenization process is described which allows the simultaneous analysis of 96 samples and delivers results in 7 min with high accuracy.

The exo-ß-N-acetylmuramidase NamZ from Bacillus subtilis is the founding member of a family of exo-lytic peptidoglycan hexosaminidases.

Endo-ß-N-acetylmuramidases, commonly known as lysozymes, are well-characterized antimicrobial enzymes that catalyze an endo-lytic cleavage of peptidoglycan; i.e., they hydrolyze the ß-1,4-glycosidic bonds connecting N-acetylmuramic acid (MurNAc) and N-acetylglucosamine (GlcNAc). In contrast, little is known about exo-ß-N-acetylmuramidases, which catalyze an exo-lytic cleavage of ß-1,4-MurNAc entities from the non-reducing ends of peptidoglycan chains. Such an enzyme was identified earlier in the bacterium Bacillus subtilis, but the corresponding gene has remained unknown so far. We now report that ybbC of B. subtilis, renamed namZ, encodes the reported exo-ß-N-acetylmuramidase. A ΔnamZ mutant accumulated specific cell wall fragments and showed growth defects under starvation conditions, indicating a role of NamZ in cell wall turnover and recycling. Recombinant NamZ protein specifically hydrolyzed the artificial substrate para-nitrophenyl ß-MurNAc and the peptidoglycan-derived disaccharide MurNAc-ß-1,4-GlcNAc. Together with the exo-ß-N-acetylglucosaminidase NagZ and the exo-muramoyl-l-alanine amidase AmiE, NamZ degraded intact peptidoglycan by sequential hydrolysis from the non-reducing ends. A structure model of NamZ, built on the basis of two crystal structures of putative orthologs from Bacteroides fragilis, revealed a two-domain structure including a Rossmann-fold-like domain that constitutes a unique glycosidase fold. Thus, NamZ, a member of the DUF1343 protein family of unknown function, is now classified as the founding member of a new family of glycosidases (CAZy GH171; www.cazy.org/GH171.html). NamZ-like peptidoglycan hexosaminidases are mainly present in the phylum Bacteroidetes and less frequently found in individual genomes within Firmicutes (Bacilli, Clostridia), Actinobacteria, and γ-proteobacteria.

Cellular and Molecular Strategies in Cyanobacterial Survival-"In Memory of Prof. Dr. Wolfgang Lockau".

Aerobic life on Earth evolved about 3 [...].

Magnetic Bead-Based Immunoassay Allows Rapid, Inexpensive, and Quantitative Detection of Human SARS-CoV-2 Antibodies.

Immunological methods to detect SARS-CoV-2 seroconversion in humans are important to track COVID-19 cases and the humoral response to SARS-CoV-2 infections and immunization to future vaccines. The aim of this work was to develop a simple chromogenic magnetic bead-based immunoassay which allows rapid, inexpensive, and quantitative detection of human antibodies against SARS-CoV-2 in serum, plasma, or blood. Recombinant 6xHis-tagged SARS-CoV-2 Nucleocapsid protein was mobilized on the surface of Ni2+ magnetic beads and challenged with serum or blood samples obtained from controls or COVID-19 cases. The beads were washed, incubated with anti-human IgG-HPR conjugate, and immersed into a solution containing a chromogenic HPR substrate. Bead transfer and homogenization between solutions was aided by a simple low-cost device. The method was validated by two independent laboratories, and the performance to detect SARS-CoV-2 seroconversion in humans was in the same range as obtained using the gold standard immunoassays ELISA and Luminex, though requiring only a fraction of consumables, instrumentation, time to deliver results, and volume of sample. Furthermore, the results obtained with the method described can be visually interpreted without compromising accuracy as demonstrated by validation at a point-of-care unit. The magnetic bead immunoassay throughput can be customized on demand and is readily adapted to be used with any other 6xHis tagged protein or peptide as antigen to track other diseases.

Functional and structural characterization of PII-like protein CutA does not support involvement in heavy metal tolerance and hints at a small-molecule carrying/signaling role.

The PII-like protein CutA is annotated as being involved in Cu2+ tolerance, based on analysis of Escherichia coli mutants. However, the precise cellular function of CutA remains unclear. Our bioinformatic analysis reveals that CutA proteins are universally distributed across all domains of life. Based on sequence-based clustering, we chose representative cyanobacterial CutA proteins for physiological, biochemical, and structural characterization and examined their involvement in heavy metal tolerance, by generating CutA mutants in filamentous Nostoc sp. and in unicellular Synechococcus elongatus. However, we were unable to find any involvement of cyanobacterial CutA in metal tolerance under various conditions. This prompted us to re-examine experimentally the role of CutA in protecting E. coli from Cu2+ . Since we found no effect on copper tolerance, we conclude that CutA plays a different role that is not involved in metal protection. We resolved high-resolution CutA structures from Nostoc and S. elongatus. Similarly to their counterpart from E. coli and to canonical PII proteins, cyanobacterial CutA proteins are trimeric in solution and in crystal structure; however, no binding affinity for small signaling molecules or for Cu2+ could be detected. The clefts between the CutA subunits, corresponding to the binding pockets of PII proteins, are formed by conserved aromatic and charged residues, suggesting a conserved binding/signaling function for CutA. In fact, we find binding of organic Bis-Tris/MES molecules in CutA crystal structures, revealing a strong tendency of these pockets to accommodate cargo. This highlights the need to search for the potential physiological ligands and for their signaling functions upon binding to CutA. DATABASES: Structural data are available in Protein Data Bank (PDB) under the accession numbers 6GDU, 6GDV, 6GDW, 6GDX, 6T76, and 6T7E.

Heavy Metal Stress Alters the Response of the Unicellular Cyanobacterium Synechococcus elongatus PCC 7942 to Nitrogen Starvation.

Non-diazotrophic cyanobacteria are unable to fix atmospheric nitrogen and rely on combined nitrogen for growth and development. In the absence of combined nitrogen sources, most non-diazotrophic cyanobacteria, e.g., Synechocystis sp. PCC 6803 or Synechococcus elongatus PCC 7942, enter a dormant stage called chlorosis. The chlorosis process involves switching off photosynthetic activities and downregulating protein biosynthesis. Addition of a combined nitrogen source induces the regeneration of chlorotic cells in a process called resuscitation. As heavy metals are ubiquitous in the cyanobacterial biosphere, their influence on the vegetative growth of cyanobacterial cells has been extensively studied. However, the effect of heavy metal stress on chlorotic cyanobacterial cells remains elusive. To simulate the natural conditions, we investigated the effects of long-term exposure of S. elongatus PCC 7942 cells to both heavy metal stress and nitrogen starvation. We were able to show that elevated heavy metal concentrations, especially for Ni2+, Cd2+, Cu2+ and Zn2+, are highly toxic to nitrogen starved cells. In particular, cells exposed to elevated concentrations of Cd2+ or Ni2+ were not able to properly enter chlorosis as they failed to degrade phycobiliproteins and chlorophyll a and remained greenish. In resuscitation assays, these cells were unable to recover from the simultaneous nitrogen starvation and Cd2+ or Ni2+ stress. The elevated toxicity of Cd2+ or Ni2+ presumably occurs due to their interference with the onset of chlorosis in nitrogen-starved cells, eventually leading to cell death.