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Hydroxy, n-saturated, branched, dioic, and unsaturated fatty acids in six freshwater wild cyanobacteria (Chroococcus minutus, Lyngbya ceylanica, Merismopedia glauca, Nodularia sphaerocarpa, Nostoc linckia, and Synechococcus aeruginosus) collected from different lakes and springs of Israel have been identified by gas chromatography-mass spectrometry (GC-MS).
The PsaC subunit of Photosystem I (PS I) is a 9.3-kDa protein that binds two important cofactors in photosynthetic electron transfer: the [4Fe-4S] clusters FA and FB. The g-tensor orientation of FA- and FB- is believed to be correlated to the preferential localization of the mixed-valence and equal-valence (ferrous) iron pairs in each [4Fe-4S]+ cluster. The preferential position of the mixed-valence and equal-valence pairs, in turn. can be inferred from the study of the temperature dependence of contact-shifted resonances by 1H NMR spectroscopy. For this, a sequence-specific assignment of these signals is required. The 1H NMR spectrum of reduced, unbound PsaC from Synechococcus sp. PCC 7002 at 280.4 K in 99% D2O solution shows 18 hyperfine-shifted resonances. The non-solvent-exchangeable, hyperfine-shifted resonances of reduced PsaC are clearly identified as belonging to the cysteines coordinating the clusters FA- and FB- by their downfield chemical shifts, by their temperature dependencies, and by their short T1 relaxation times. The usual fast method of assigning the 1H NMR spectra of reduced [4Fe-4S] proteins through magnetization transfer from the oxidized to the reduced state was not feasible in the case of reduced PsaC. Therefore, a de novo self-consistent sequence-specific assignment of the hyperfine-shifted resonances was obtained based on dipolar connectivities from 1D NOE difference spectra and on longitudinal relaxation times using the X-ray structure of Clostridium acidi urici 2[4Fe-4S] cluster ferredoxin at 0.94 A resolution as a model. The results clearly show the same sequence-specific distribution of Curie and anti-Curie cysteines for unbound, reduced PsaC as established for other [4Fe-4S]-containing proteins; therefore, the mixed-valence and equal-valence (ferrous) Fe-Fe pairs in FA- and FB- have the same preferential positions relative to the protein. The analysis reveals that the magnetic properties of the two [4Fe-4S] clusters are essentially indistinguishable in unbound PsaC, in contrast to the PsaC that is bound as a component of the PS I complex.
Chlorophyll- b-possessing cyanobacteria of the genus Prochlorococcus share the presence of high amounts of alpha- and beta-carotenoids with green algae and higher plants. The branch point in carotenoid biosynthesis is the cyclization of lycopene, for which in higher plants two distinct enzymes are required, epsilon- and beta-lycopene cyclase. All cyanobacteria studied so far possess a single beta-cyclase. Here, two different Prochlorococcus sp. MED4 genes were functionally identified by heterologous gene complementation in Escherichia coli to encode lycopene cyclases. Whereas one is both functionally and in sequence highly similar to the beta-cyclase of Synechococcus sp. strain PCC 7942 and other cyanobacteria, the other showed several intriguing features. It acts as a bifunctional enzyme catalyzing the formation of epsilon- as well as of beta-ionone end groups. Expression of this cyclase in E. coli resulted in the simultaneous accumulation of alpha- beta-, delta-, and epsilon-carotene. Such an activity is in contrast to all lycopene epsilon-cyclases known so far, including those of the higher plants. Thus, for the first time among prokaryotes, two individual enzymes were identified in one organism that are responsible for the formation of cyclic carotenoids with either beta- or epsilon-end groups. These two genes are suggested to be designated as crtL-b and crtL-e. The results indicate that both enzymes might have originated from duplication of a single gene. Consequently, we suggest that multiple gene duplications followed by functional diversification resulted several times, and in independent lineages, in the appearance of enzymes for the biosynthesis of cyclic carotenoids.
Five Merismopedia-like cyanobacterial strains were collected from microbial mats at Norderney Island, subcultured in the laboratory, and finally grown as unicyanobacterial cultures. As a sixth strain, Merismopedia glauca from the rising dbl quote, left (low)Sammlung von Algenkulturen" at Gottingen (SAG) was used for comparisons. According to morphological and physiological characteristics initially observed in the field and during initial subculturing, the five strains were assigned to the species Merismopedia glauca, Merismopedia punctata, or Merismopedia elegans. However, after prolonged maintenance under laboratory conditions, the formation of platelet-like colonies stopped, whereas cell sizes, production of extracellular polymeric substances, and division patterns were stably maintained. These physiological and morphological parameters allowed us to divide the six strains into two clusters. This division was further supported by the profiling of total cell protein and phycobilisomes using SDS-PAGE. The nearly complete 16S rDNA sequence of three of the six isolates was determined. The comparative sequencing analysis revealed an almost 100% identity of these three Merismopedia-like strains. The evolutionary distance dendrogram constructed placed this Merismopedia cluster into a common line of descent with Synechocystis sp. strain PCC6906. Based on the analysis of common stretches of 1,050 nucleotides, the overall similarity between the sequence types of rising dbl quote, left (low)Merismopedia" and rising dbl quote, left (low)Synechocystis" is 96-97%. The values of the different methods for taxonomic classification of unicyanobacterial strains, the relationship of the cyanobacterial genera Merismopedia, Synechococcus, Synechocystis, and Eucapsis sp., and the functional role of different Merismopedia morphologies within microbial mats are discussed. It is suggested that all analyzed Merismopedia strains be combined into one species, namely Merismopedia punctata Meyen (1839).
A Tn5-1063-derived mutant of Nostoc punctiforme strain ATCC 29133 was unable to fix N2 in air although it reduced acetylene in the absence of O2. Mutant strain UCD 307 formed cells morphologically similar to heterocysts, but it failed to synthesize the characteristic heterocyst glycolipids. Sequence analysis around the site of insertion revealed an ORF of 3,159 base pairs, termed hglE. hglE putatively encodes a 115.4-kDa protein containing two domains with conserved amino acid sequences identified with acyl transferase and the chain length factor variation of beta-ketoacyl synthase active sites. These active sites are characteristic of polyketide and fatty acid synthases. The N. punctiforme strain 29133 hglE gene is transcribed only under nitrogen-limiting growth conditions. The hglE gene, or similar sequences, was found in all other heterocyst-forming cyanobacteria surveyed and was absent in unicellular Synechococcus sp. strain PCC 7942. Based on these results, we propose that the synthesis of heterocyst glycolipids follows a pathway characteristic of polyketide synthesis and involves similar large, multienzyme complexes.
Previous studies largely carried out with environmental samples or axenic and non-axenic cultures suggested that cyanobacteria may be a rich source of hitherto unexplored bioactive compounds. This has been confirmed in the present study by a screening of 146 axenic strains from the Pasteur Culture Collection (PCC) of cyanobacteria. Use of degenerate PCR primers, designed on the basis of conserved sequence motifs in the aminoacyl-adenylation domain of peptide synthetases, revealed the presence of the corresponding genes in the majority (75.3%) of the strains examined. Among unicellular cyanobacteria, only Chamaesiphon sp. strain PCC 6605, two strains of Gloeocapsa and most Microcystis isolates (22 out of 24) contained these genes; no amplicons were detected for any members of the genera Cyanothece, Gloeobacter and Gloeothece and the genetically diverse representatives of Synechococcus and Synechocystis. By contrast, eight out of ten pleurocapsalean members, 16 out of 25 oscillatorian strains, and all but two of the 63 filamentous heterocystous cyanobacteria tested gave positive amplification results. This information will be highly valuable for further exploring the corresponding cyanobacterial peptides and for elucidating the bioactivity of such non-ribosomally synthesized molecules.
Bumetanide-sensitive Na-K-Cl cotransporters and thiazide-sensitive Na-Cl cotransporters comprise a family of integral membrane transport proteins, the Na-K-Cl cotransporter (NKCC) family. Each of the members of this family is over 1,000 amino acids in length. We have multiply aligned the ten currently sequenced members of this family from human, rabbit, rodent, shark, flounder, moth, worm and yeast sources. Phylogenetic analyses suggest the presence of at least six isoforms of these full length proteins in eukaryotes. Average hydropathy and average similarity plots have been derived revealing that each of these proteins possesses a central, well conserved, hydrophobic domain of almost invariant length, possibly consisting of twelve transmembrane alpha-helical spanners, an N-terminal, poorly conserved, hydrophilic domain of variable length, and a C-terminal, moderately conserved, hydrophilic domain of moderately constant length. A functionally uncharacterized homologue of this family occurs in the cyanobacterium Synechococcus sp. Limited sequence similarity of these proteins with members of a family of basic amino acid transporters suggest that the NKCC family may be distantly related to the previously characterized, ubiquitous, amino acid-polyamine-choline (APC) family of facilitators. These observations suggest that the NKCC family is an old family that has its roots in the prokaryotic kingdom.
Hydrogenases are important enzymes in the energy metabolism of microorganisms. Therefore, they are widespread in prokaryotes. We analyzed the occurrence of hydrogenases in cyanobacteria and deduced a FeFe-hydrogenase in three different heliobacterial strains. This allowed the first phylogenetic analysis of the hydrogenases of all five major groups of photosynthetic bacteria (heliobacteria, green nonsulfur bacteria, green sulfur bacteria, photosynthetic proteobacteria, and cyanobacteria). In the case of both hydrogenases found in cyanobacteria (uptake and bidirectional), the green nonsulfur bacterium Chloroflexus aurantiacus was found to be the closest ancestor. Apart from a close relation between the archaebacterial and the green sulfur bacterial sulfhydrogenase, we could not find any evidence for horizontal gene transfer. Therefore, it would be most parsimonious if a Chloroflexus-like bacterium was the ancestor of Chloroflexus aurantiacus and cyanobacteria. After having transmitted both hydrogenase genes vertically to the different cyanobacterial species, either no, one, or both enzymes were lost, thus producing the current distribution. Our data and the available data from the literature on the occurrence of cyanobacterial hydrogenases show that the cyanobacterial uptake hydrogenase is strictly linked to the occurrence of the nitrogenase. Nevertheless, we did identify a nitrogen-fixing Synechococcus strain without an uptake hydrogenase. Since we could not find genes of a FeFe-hydrogenase in any of the tested cyanobacteria, although strains performing anoxygenic photosynthesis were also included in the analysis, a cyanobacterial origin of the contemporary FeFe-hydrogenase of algal plastids seems unlikely.
The partitioning of the widely used gasoline additive methyl tert-butyl ether (MTBE) between liquid growth media and gaseous phase was measured daily under laboratory conditions to determine how closely dissolved MTBE concentrations matched nominal concentrations. Total (gaseous and dissolved) MTBE averaged across 6 days for 29.6, 503.2, and 1005.7 mg L-1 MTBE treatments were 89.9, 90.3, and 73.0% of nominal, respectively, and mean dissolved MTBE in these same treatments were 74.6, 73.8, and 69.6% of total MTBE, respectively. This suggests that dissolved MTBE concentrations can vary substantially from nominal. The effect of MTBE on the growth of selected algae was also evaluated under laboratory conditions. Three unicellular algae, Selenastrum capricornutum (Chlorophyta), Navicula pelliculosa (Bacillariophyta), and Synechococcus leopoliensis (= Anacystic nidulans, Cyanophyta = Cyanobacteria), representative of three taxonomic groups, were used as test organisms. Toxicity tests were acute and increase in cell number was used as an indicator of growth. Algal species were exposed by injection of MTBE into sealed vessels containing defined liquid growth media. The growth of N. pelliculosa and S. leopoliensis was negatively affected at nominal 2400 mg L-1 MTBE, whereas the growth of S. capricornutum was negatively affected at nominal 4800 mg L-1 MTBE and positively affected at nominal 600 mg L-1 MTBE. The differential sensitivity of the growth of these representative species suggests that MTBE may alter algal community composition in the natural environment.
Bacterivory by heterotrophic nanoflagellates and ciliates has been widely studied in aquatic environments, but data on the grazing of amoebae, are still scarce. From the water samples of Dianchi Lake (Kunming, Yunnan Province, China), we isolated an amoeba, designated as Naegleria sp. strain W2, which had potent grazing effects on some kind of cyanobacteria. The food selection mechanism and the digestion process of the amoeba were investigated in batch experiments. Predation experiments showed that filamentous cyanobacteria (e.g., Anabaena, Cylindrospermum, Gloeotrichia, and Phormidium) were readily consumed, with clearance rates ranging from 0.332 to 0.513 nL amoeba(-1) h(-1). The tight threads (Oscilltoria) and aggregates (Aphanizomenon) could not be ingested; however, their sonicated fragments were observed inside food vacuoles, suggesting that their morphologies prevent them from being ingested. Live video microscopy noted that unicellular Chroococcaceae (e.g., Synechococcus, Aphanocapsa, and Microcystis) were excreted after ingestion, indicating that food selection takes place inside food vacuoles. To determine whether the tastes or the toxins prevented them from being digested, heat-killed cells were retested for predation. Digestion rates and ingestion rates of the amoebae for filamentous cyanobacteria were estimated from food vacuole content volume. Through a "cold-chase" method, we found that the food vacuole contents declined exponentially in diluted amoebae cells, and digestion rates were relatively constant, averaging about 1.5% food vacuole content min(-1) at 28 degrees Celsius. Ingestion strongly depended on the satiation status of the amoebae, starved amoebae fed at higher rates compared with satiated amoebae. Our results suggest that the food selection and food processing mechanisms of the amoeba are similar to those of interception feeding flagellates; however, filamentous cyanobacteria cannot obtain a refuge under the grazing pressure of phagotrophic amoebae, which may widen our knowledge on the grazing of protists.
The influence of prey characteristics such as motility and size as well as of predator characteristics such as satiation and preculturing diet on the feeding process of interception feeding heterotrophic nanoflagellates was investigated. Three species of gram-negative bacteria, one species of gram-positive bacteria, two species of cyanobacteria (Synechococcus) and inert latex particles were fed as prey particles for three species of heterotrophic nanoflagellates (Spumella, Ochromonas, Cafeteria). Ingestion rates depended on the satiation of the flagellates and especially on the filling status of the food vacuoles. In addition, the ingestion rates depended on the characteristics of the food particle and were modified by pre-culturing the flagellates on either Pseudomonas putida or Bacillus subtilis. Digestion was found to be particle-specific. Cyanobacteria were excreted a few minutes after ingestion whereas heterotrophic bacteria were stored and digested in the food vacuoles. The spectrum of ingested particles is not identical to that of digested particles and thus neither the diet of the flagellates nor their impact on bacterial communities can be calculated simply from food vacuole content. "Selective digestion" could be shown to be an important selection mechanism concerning natural food particles. The digestion strategies of Cafeteria on the one hand and Spumella and Ochromonas on the other hand may be an important factor to explain protozoan species composition and succession in the field. In addition to bacterial abundance and grazing pressure by metazooplankton, the bacterial speciescomposition as well as biochemical variations within bacterial species may influence protozoan species composition and abundance.
Cyanobacteria are oxygenic photosynthetic bacteria that have been used increasingly to study diverse biological processes, including photosynthesis and its regulation; cell differentiation and N2 fixation; metabolism of nitrogen, carbon, and hydrogen; resistance to environmental stresses; and molecular evolution. Many vectors and other genetic tools have been developed for unicellular and filamentous strains of cyanobacteria. Transformation, electroporation, and conjugation are used for gene transfer. Diverse methods of mutagenesis allow the isolation of many sought-for kinds of mutants, including site-directed mutants of specific genes. Reporter genes permit measurement of the level of transcription of particular genes, and assays of transcription within individual colonies or within individual cells in a filament. Complete genomic sequences have been obtained for the unicellular cyanobacterium, Synechocystis sp. strain PCC 6803 and the filamentous, heterocyst-forming cyanobacterium, Anabaena sp. strain PCC 7120. Genomic sequence projects are under way for Nostoc punctiforme strain PCC 73102 (ATCC 29133) and strains of the unicellular genera, Synechococcus, Prochlorococcus, and Gloeobacter. Genomic sequence data provide the opportunity for global monitoring of changes in genetic expression at transcriptional and translational levels in response to variations in environmental conditions. The availability of genomic sequences accelerates the identification, study, modification and comparison of cyanobacterial genes, and facilitates analysis of evolutionary relationships, including the relationship of chloroplasts to ancient cyanobacteria. The many available genetic tools enhance the opportunities for possible biotechnological applications of cyanobacteria.
Abstracts. The heat shock protein HtpG is homologous to members of the Hsp90 protein family of eukaryotes and is essential for basal and acquired thermotolerances in cyanobacteria. In this study we have examined the role of HtpG in the cyanobacterium, Synechococcus sp. PCC 7942, in the acclimation to low temperatures. The inactivation of the htpG gene resulted in severe inhibition of cell growth and of the photosynthetic activity when the htpG mutant was shifted to 16 degrees C from 30 degrees C. Wild-type cells were able to resume growth without a lag period when shifted to 30 degrees C after 5 days at 16 degrees C, while the mutant displayed a detectable lag. The HtpG protein was induced in the wild-type cells at 16 degrees C. Electrophoresis in the absence of sodium dodecyl sulfate (SDS) showed that a novel, high-molecular-weight complex containing GroEL and DnaK accumulated at 16 degrees C, but the accumulation was strongly inhibited in the htpG mutant. Our results demonstrate that the HtpG protein contributes significantly to the ability of cyanobacteria to acclimate to low temperatures.
The heat shock protein HtpG, which is a homolog of HSP90, is essential for basal and acquired thermotolerances in cyanobacteria. Recently we demonstrated that HtpG was involved in the acclimation to low temperatures in cyanobacteria. In this study, we elucidated a role of HtpG in the cyanobacterium Synechococcus sp. PCC 7942, in the acclimation to oxidative stress that was caused by high irradiance and/or methyl viologen. The inactivation of the htpG gene resulted in a decrease in the survival rate and an increase in the photoinhibition of photosystem II when cells in a liquid medium were incubated under high light conditions. The htpG mutant was highly sensitive to methyl viologen when it was grown on an agar plate. High irradiance and/or methyl viologen greatly increased the expression of the htpG gene as well as the groEL gene in the wild-type strain. Taken together, our results suggest that HtpG may play a role by itself or with other molecular chaperones in the acclimation to oxidative stress.
In this study, fatty-acid content and patterns were analyzed in order to distinguish Nostoc flagelliforme, an edible terrestrial cyanobacterium, from other Nostoc species and representatives typical of its close neighbors (genera Anabaena, Microcystis, and Synechococcus). According to the Kenyon-Murata classification system, all the Nostoc species were assigned to Group II due to the presence of C18:2n3 and C18:3n3, and the absence of C18:3n6. Hierarchical cluster analysis was also employed to separate N. flagelliforme and other Nostoc species or strains. A dendrogram calculation of all fatty-acid components manifested phenetic characteristics, showing that the degree of relatedness of two strains of N. flagelliforme aggregated them within a small subgroup. Another dendrogram, calculated from seven comprehensive parameters (including ratios of different fatty-acid categories, degree of fatty-acid unsaturation, etc.), also clearly delimited the minute difference in fatty-acid profiles between the tested organisms. Our results suggest that profiling fatty acids could be a useful approach in the taxonomic or phylogenetic study of the genus Nostoc and might serve as a valuable supplement to the current morphology-based classification system.
Cyanobacteria respond to nutrient-limiting conditions by degrading their phycobilisomes (PBS), the light-harvesting complexes for photosynthesis. In Synechococcus sp. PCC 7942, the expression of nblA, an essential gene in this process, is controlled by the response regulator NblR and the sensor NblS. Here we study the effect of inactivation of dspA (an nblS homologue) and an nblR-like gene on phycobilisome degradation in Synechocystis sp. PCC 6803, under nitrogen starvation. In each mutant, the expression of nblA was found to be unaffected and sequential PBS degradation occurred after nitrogen deprivation (although it was slightly delayed). Our results demonstrate that dspA and nblR-like do not exert a major control of PBS degradation in Synechocystis sp. PCC 6803.
Phosphoenolpyruvate carboxylase (EC 4.1.1.31) from Synechococcus vulcanus (SvPEPC) is a unique enzyme, being almost insensitive to feedback inhibition at neutral pH. In order to assess its usefulness in metabolic engineering of plants, SvPEPC was expressed in Arabidopsis thaliana (L.) Heynh. under the control of the cauliflower mosaic virus 35S promoter. About one-third of the transformants of the T1 generation showed severe visible phenotypes such as leaf bleaching and were infertile when grown on soil. However, no such phenotype was observed with Arabidopsis transformed with Zea mays L. PEPC (ZmPEPC) for C4 photosynthesis, which is normally sensitive to a feedback inhibitor, L-malate. For the SvPEPC transformants of the T2 generation, which had been derived from fertile T1 transformants, three kinds of phenotype were observed when plants were grown on an agar medium containing sucrose: Type-I plants showed poor growth and a block in true leaf development; Type-II plants produced a few true leaves, which were partially bleached; Type-III plants were apparently normal. In Type-I plants, total PEPC activity was increased about 2-fold over the control plant but there was no such increase in Type-III plants. The phenotypes of Type-I plants were rescued when the sucrose-containing agar medium was supplemented with aromatic amino acids. Measurement of the free amino acid content in whole seedlings of Type-I transformants revealed that the levels of the aromatic amino acids Phe and Tyr were lowered significantly as compared with the control plants. In contrast, the levels of several amino acids of the aspartic and glutamic families, such as Asn, Gln and Arg, were markedly enhanced (4- to 8-fold per plant fresh weight). However, when the medium was supplemented with aromatic amino acids, the levels of Asn, Gln, and Arg decreased to levels slightly higher than those of control plants, accompanied by growth recovery. Taken together, it can be envisaged that SvPEPC is capable of efficiently exerting its activity in the plant cell environment so as to cause imbalance between aromatic and non-aromatic amino acid syntheses. The growth inhibition of Type-I plants was presumed to be primarily due to a decreased availability of phosphoenolpyruvate, one of the precursors for the shikimate pathway for the synthesis of aromatic amino acids and phenylpropanoids. The possible usefulness of SvPEPC as one of the key components for installing the C4-like pathway is proposed.
Protein phosphorylation catalysed by protein kinases is an important mechanism for signal transduction in both prokaryotes and eukaryotes. A novel gene, pknD, encoding a protein similar to eukaryotic-type protein kinases, was cloned from Anabaena sp. PCC7120. The N-terminal region of PknD is 60% identical to that of PknA, another putative Ser/Thr kinase from the same strain. Both PknA and PknD have C-terminal regions that are rich in Pro and Thr residues. Expression of pknD was undetectable by RNA/DNA hybridisation and was thus examined by RT-PCR. The pknD transcript was detected in filaments cultured in the presence of either nitrate or ammonium as a source of combined nitrogen, and also in filaments transferred from nitrate-sufficient to N2-fixing conditions. pknD mutants were created, and their growth characteristics under different nitrogen regimes and their capacity for heterocyst development were investigated. The growth rates of the mutants were similar to those of the wild-type strain in the presence of either nitrate or ammonium, but were only 20% that of the wild type under N2-fixing conditions. The rate of nitrogenase activity is normal in pknD mutant under aerobic conditions. Under nitrogen-fixing conditions, the inactivation of pknD led to enhanced modification of the PII protein compared to the weak phosphorylation of PII observed in the wild-type strain. This high level of PII phosphorylation in the pknD mutant is reminiscent of the situation in nitrogen-starved Synechococcus PCC7942 cells. PknD might be involved in regulating nitrogen metabolism or nitrogen trafficking from heterocysts to vegetative cells.