Ultrastructure of the membrane systems in the unicellular cyanobacterium Synechocystis sp. strain PCC 6803

Summary. Among prokaryotes, cyanobacteria are unique in having highly differentiated internal membrane systems. Like other Gram-negative bacteria, cyanobacteria such as Synechocystis sp. strain PCC 6803 have a cell envelope consisting of a plasma membrane, peptidoglycan layer, and outer membrane. In addition, these organisms have an internal system of thylakoid membranes where the electron transfer reactions of photosynthesis and respiration occur. A long-standing controversy concerning the cellular ultrastructures of these organisms has been whether the thylakoid membranes exist inside the cell as separate compartments, or if they have physical continuity with the plasma membrane. Advances in cellular preservation protocols as well as in image acquisition and manipulation techniques have facilitated a new examination of this topic. We have used a combination of electron microscopy techniques, including freeze-etched as well as freeze-substituted preparations, in conjunction with computer-aided image processing to generate highly detailed images of the membrane systems in Synechocystis cells. We show that the thylakoid membranes are in fact physically discontinuous from the plasma membrane in this cyanobacterium. Thylakoid membranes in Synechocystis sp. strain PCC 6803 thus represent bona fide intracellular organelles, the first example of such compartments in prokaryotic cells. Supplementary material to this paper is available in electronic form at Correspondence and reprints: Department of Biology, CB1137, Washington University, St. Louis, MO 63130, U.S.A.     

The deletion of Ffh in Escherichia coli induces adverse physiological and morphological changes

The Escherichia coli Ffh protein is homologous to the SRP54 subunit of the eukaryotic signal recognition particle (SRP) that is involved in targeting and translocation of membrane proteins. The functions of Ffh in E. coli were investigated using the mutant with the Ffh deficiency. The mutant showed lower growth rate at 30°C and rapidly lost viability at the non-permissive temperature of 42°C. In addition, the amount of the total membrane proteins decreased sharply in the mutant. The mutant cells cultured at either 30 or 42°C appeared to have an elongated shape as compared to the wild type cells. Transmission electron microscopy revealed that the membrane layer of the mutant cells was thinner than that of the wild type cells. The article is published in the original.     

Reconstitution of the spinach oxygen-evolving complex with recombinant Arabidopsis manganese-stabilizing protein

The psbO gene of cyanobacteria, green algae and higher plants encodes the precursor of the 33 kDa manganese-stabilizing protein (MSP), a water-soluble subunit of photosystem II (PSII). Using a pET-T7 cloning/expression system, we have expressed in Escherichia coli a full-length cDNA clone of psbO from Arabidopsis thaliana. Upon induction, high levels of the precursor protein accumulated in cells grown with vigorous aeration. In cells grown under weak aeration, the mature protein accumulated upon induction. In cells grown with moderate aeration, the ratio of precursor to mature MSP decreased as the optical density at induction increased. Both forms of the protein accumulated as inclusion bodies from which the mature protein could be released under mildly denaturing conditions that did not release the precursor. Renatured Arabidopsis MSP was 87% as effective as isolated spinach MSP in restoring O₂ evolution activity to MSP-depleted PSII membranes from spinach; however, the heterologous protein binds to spinach PSIIs with about half the affinity of the native protein. We also report a correction to the previously published DNA sequence of Arabidopsis psbO (Ko et al., Plant Mol Biol 14 (1990) 217–227).     

Identification of Photosystem I components from a glaucocystophyte,Cyanophora paradoxa: The PsaD protein has an N-terminal stretch homologous to higher plants

Thylakoid membranes and Photosystem I (PS I) complexes were isolated from a glaucocystophyte, Cyanophora paradoxa, which is thought to have the most primitive ‘plastids’, and the proteins related to PS I were examined. The intrinsic light-harvesting chlorophyll protein complexes of PS I (LHC I) were not detected by an immunological method. The PS I complexes consisted of at least eight low-molecular-mass proteins in addition to PS I reaction center proteins. The N-terminal sequence of the PsaD protein has higher homology to that of Chlamydomonas reinhardtii and land plants, than to that of other algae or cyanobacteria. On the other hand, the PsaL sequence has the highest homology to those of cyanobacteria. Taking into account the other sequences of PS I components whose genes are encoded in the cyanelle genome, and the fact that LHC I is not detected, it is concluded that PS I of C. paradoxa has chimeric characteristics of both ‘green’ lineages and cyanobacteria. This revised version was published online in June 2006 with corrections to the Cover Date.     

Cloning and sequence analysis of a signal peptidase I from the thermophilic cyanobacterium Phormidium laminosum

Type I signal peptidases are a widespread family of enzymes which remove the presequences from proteins translocated across cell membranes, including thylakoid and cytoplasmic membranes of cyanobacteria and thylakoid membranes of chloroplasts. We have cloned and sequenced a signal peptidase gene from the thermophilic cyanobacterium Phormidium laminosum which is believed to encode an enzyme common to both membrane systems. The deduced amino acid sequence is 203 residues long and although the overall similarity among signal peptidases is rather low there are a number of identifiable conserved regions present. The P. laminosum enzyme is predicted to have a single transmembrane domain, in contrast to other Gram-negative bacterial sequences, but similar to other type I signal peptidases.     

Isolation and characterization of outer and inner envelope membranes of cyanelles from a glaucocystophyte, Cyanophora paradoxa

Envelope membranes were isolated by sucrose density gradient floatation centrifugation from the homogenate of cyanelles prepared from Cyanophora paradoxa. Two yellow bands were separated after 40 h of centrifugation. The buoyant density of one of the two fractions (fraction Y2) coincided with that of inner envelope membranes of spinach or plasma membranes of cyanobacteria. The other yellow fraction (fraction Y1) migrated to top of sucrose-gradient even at 0% sucrose. Pigment analysis revealed that the heavy yellow fraction was rich in zeaxanthin while the light fraction was rich in β-carotene, and the both fractions contained practically no chlorophylls. Another yellow fraction (fraction Y3) was isolated from the phycobiliprotein fraction, which was the position where the sample was placed for gradient centrifugation. Its buoyant density and absorption spectra were similar to outer membranes of cyanobacteria. We have assigned fractions Y2 and Y3 as inner and outer envelope membrane fractions of cyanelles, respectively. Protein compositions were rather different between the two envelope membranes indicating little cross-contamination among the fractions. H. Koike and Y. Ikeda contributed equally.     

Effects of Cyanobacteria on Survival and Reproduction of the Littoral Crustacean Gammarus zaddachi (Amphipoda)

Toxic cyanobacteria can have harmful or fatal impacts on aquatic organisms. In the archipelagos of the northern Baltic Sea, the open sea blooms often drift into littoral areas, where they decompose and release toxins and other chemical compounds in the water. However, the effects of cyanobacteria on the littoral organisms have not previously been investigated. We studied the effects of three cyanobacteria species (toxic Nodularia spumigena, non-toxic N. sphaerocarpa and non-toxic Aphanizomenon flos-aquae) and purified dissolved nodularin (produced by N. spumigena) on a common littoral amphipod Gammarus zaddachi. Nodularin was transferred to eggs, juveniles and adults of G. zaddachi, but no significant negative effects of dissolved nodularin were detected on adults, eggs or juveniles. However, survival of adults decreased by the exposure to toxic N. spumigena cells. The egg hatching rate and juvenile survival were not affected when exposed to the three cyanobacteria species. In contrast, a weak decrease in the egg production and an increased abortion of embryos from the brood pouch of females was observed, the later indicating a failure in parental care. Further, a decrease in grazing rate on the filamentous green alga Enteromorpha intestinalis was observed. The results suggest that toxic cyanobacteria blooms are not extremely fatal, but may have, in high concentrations, negative effects on the adult survival, fecundity, and feeding behaviour of gammarids inhabiting the littoral zone.     

Electron microscopic structural analysis of Photosystem I,Photosystem II,and the cytochromeb6/f complex from green plants and cyanobacteria

Electron microscopy (EM) in combination with image analysis is a powerful technique to study protein structure at low- and high resolution. Since electron micrographs of biological objects are very noisy, substantial improvement of image quality can be obtained by averaging individual projections. Crystallographic and noncrystallographic averaging methods are available and have been applied to study projections of the large protein complexes embedded in photosynthetic membranes from cyanobacteria and higher plants. Results of EM on monomeric and trimeric Photosystem I complexes, on monomeric and dimeric Photosystem II complexes, and on the monomeric cytochromeb6/f complex are discussed.     

A comparative structural and functional analysis of cyanobacterial plastocyanin and cytochrome c 6 as alternative electron donors to Photosystem I

Plastocyanin and cytochrome c ₆ are two soluble metalloproteins that act as alternative electron carriers between the membrane-embedded complexes cytochromes b ₆ f and Photosystem I. Despite plastocyanin and cytochrome c ₆ differing in the nature of their redox center (one is a copper protein, the other is a heme protein) and folding pattern (one is a β-barrel, the other consists of α-helices), they are exchangeable in green algae and cyanobacteria. In fact, the two proteins share a number of structural similarities that allow them to interact with the same membrane complexes in a similar way. The kinetic and thermodynamic analysis of Photosystem I reduction by plastocyanin and cytochrome c ₆ reveals that the same factors govern the reaction mechanism within the same organism, but differ from one another. In cyanobacteria, in particular, the electrostatic and hydrophobic interactions between Photosystem I and its electron donors have been analyzed using the wild-type protein species and site-directed mutants. A number of residues similarly conserved in the two proteins have been shown to be critical for the electron transfer reaction. Cytochrome c ₆ does contain two functional areas that are equivalent to those previously described in plastocyanin: one is a hydrophobic patch for electron transfer (site 1), and the other is an electrically charged area for complex formation (site 2). Each cyanobacterial protein contains just one arginyl residue, similarly located between sites 1 and 2, that is essential for the redox interaction with Photosystem I. This revised version was published online in June 2006 with corrections to the Cover Date.     

The Evolutionary History of Nitrogen Fixation, as Assessed by NifD

The evolutionary history of nitrogen fixation has been vigorously debated for almost two decades. Previous phylogenetic analyses of nitrogen fixation genes (nif) have shown support for either evolution by vertical descent or lateral transfer, depending on the specific nif gene examined and the method of analyses used. The debate centers on the placement and monophyly of the cyanobacteria, proteobacteria, and Gram-positive bacteria (actinobacteria and firmicutes). Some analyses place the cyanobacteria and actinobacteria within the proteobacteria, which suggests that the nif genes have been laterally transferred since this topology is incongruent with ribosomal phylogenies, the standard marker for comparison. Other nif analyses resolve and support the monophyly of the cyanobacteria, proteobacteria, and actinobacteria, supporting vertical descent. We have revisited these conflicting scenarios by analyzing nifD from an increased number of cyanobacteria, proteobacteria, and Gram-positive bacteria. Parsimony analyses of amino acid sequences and maximum likelihood analysis of nucleic acid sequences support the monophyly of the cyanobacteria and actinobacteria but not the proteobacteria, lending support for vertical descent. However, distance analysis of nucleic acid sequences placed the actinobacteria within the proteobacteria, supporting lateral transfer. We discuss evidence for both vertical descent and lateral transfer of nitrogen fixation.     

A comparison of the major lipid classes and fatty acid composition of marine unicellular cyanobacteria with freshwater species

The fatty acid composition of two motile (strains WH 8113 and WH 8103) and one nonmotile (strain WH 7803) marine cyanobacteria has been determined and compared with two freshwater unicellular Synechocystis species (strain PCC 6308 and PCC 6803). The fatty acid composition of lipid extracts of isolated membranes from Synechocystis PCC 6803 was found to be identical to that of whole cells. All the marine strains contained myristic acid (14:0) as the major fatty acid, with only traces of polyunsaturated fatty acids. This composition is similar to Synechocystis PCC 6308. The major lipid classes of the nonmotile marine strain were identified as digalactosyl diacylglycerol, monogalactosyl diacylglycerol, phosphatidylglycerol, and sulfoquinovosyl diacylglycerol, identical to those found in other cyanobacteria.Abbreviations DGDG Digalactosyl diacylglycerol - MGDG Monogalactosyldiacylglycerol - PG Phosphatidylglycerol - SGDG sulfoquinovosyl diacylglycerol - gc gas chromatography - ms mass spectrometry     

Glutamine synthetase specific activity and protein concentration in symbiotic Anabaena associated with Azolla caroliniana

Glutamine synthetase (GS) is the primary NH₄ ⁺ assimilating enzyme of cyanobacteria. The specific activities and cellular protein concentration of GS in symbiotic cyanobacteria associated with the water fern Azolla caroliniana were determined and compared to free-living cultures of Nostoc sp. strain 7801, a strain originally isolated from symbiotic association with the bryophyte Anthoceros punctatus. Both the in vitro specific activity and concentration of GS in symbiotic cyanobacteria separated from A. caroliniana were approximately 3-fold lower than the free-living Nostoc sp. strain 7801 culture. These results imply depressed synthesis of GS by the symbiont associated with A. caroliniana.     

Partial sequence of ribulose-1,5-bisphosphate carboxylase/oxygenase and the phylogeny of Prochloron and Prochlorococcus (Prochlorales)

The prochlorophytes, oxygenic photosynthetic prokaryotes having no phycobiliprotein but possessing chlorophylls a and b, have been proposed to have a common ancestry with green chloroplasts, yet this is still controversal. We report here that partial sequence comparisons of the large subunit of ribulose-1,5-bisphosphate carboxylase/oxygenase, including sequence data from two prochlorophytes, Prochlorococcus and Prochloron, indicate that Prochlorococcus is more closely related to a photosynthetic bacterium, Chromatium vinosum (-purple bacteria), than to cyanobacteria, while Prochloron is closely related to the prochlorophyte Prochlorothrix and to cyanobacteria. The molecular phylogenetic tree indicates that a common ancestor of Prochlorococcus and -purple bacteria branched off from the land plant lineage earlier than Prochloron, Prochlorothrix, and cyanobacteria.Correspondence to: A. Shimada     

A Homolog of Bacillus subtilis Trigger Factor in Listeria monocytogenes Is Involved in Stress Tolerance and Bacterial Virulence

Molecular chaperones play an essential role in the folding of nascent chain polypeptides, as well as in the refolding and degradation of misfolded or aggregated proteins. They also assist in protein translocation and participate in stress functions. We identified a gene, designated tig, encoding a protein homologous to trigger factor (TF), a cytosolic ribosome-associated chaperone, in the genome of Listeria monocytogenes. We constructed a chromosomal Δtig deletion and evaluated the impact of the mutation on bacterial growth in broth under various stress conditions and on pathogenesis. The Δtig deletion did not affect cell viability but impaired survival in the presence of heat and ethanol stresses. We also identified the ffh gene, encoding a protein homologous to the SRP54 eukaryotic component of the signal recognition particle. However, a Δffh deletion was not tolerated, suggesting that Ffh is essential, as it is in Bacillus subtilis and Escherichia coli. Thus, although dispensable for growth, TF is involved in the stress response of L. monocytogenes. The Δtig mutant showed no or very modest intracellular survival defects in eukaryotic cells. However, in vivo it showed a reduced capacity to persist in the spleens and livers of infected mice, revealing that TF has a role in the pathogenicity of L. monocytogenes.     

Production,by filamentous,nitrogen-fixing cyanobacteria,of a bacteriocin and of other antibiotics that kill related strains

Colonies of sixty-five filamentous cyanobacteria were screened for the production of temperate phages and/or antibiotics on solid medium. None of them was observed to release phages. However, seven N₂-fixing strains were found to produce antibiotics very active against other cyanobacteria. The antibiotic produced by Nostoc sp. 78-11 A-E represents a bacteriocin of low molecular weight. Nostoc sp. ATCC 29132 appears to secrete, together with an antibiotic, a protein that inhibits its action.     

DoMicrocystis aeruginosa toxins accumulate in the food web: a laboratory study

The accumulation ofMicrocystis aeruginosa hepatotoxins (microcystin-LR) in the phantom midge larvaChaoborus was studied in a 16 d laboratory experiment. In the cyanobacteria treatment,Chaoborus larvae were fed withDaphnia pulex juveniles which had been feeding for two days on a mixture ofScenedesmus obtusiusculus and toxicMicrocystis aeruginosa. In the control treatment theChaoborus larvae were offeredD. pulex raised onScenedesmus only. An HPLC analysis failed to detect any cyanobacteria toxin in theChaoborus larvae, indicating that the toxin was metabolized or excreted byD. pulex andChaoborus. There was a statistically significant increase in mortality of larvae in the cyanobacteria treatment, but no difference between treatments in pupation success ofChaoborus was observed.     

Exogenous Hexoses Cause Quantitative Changes of Pigment and Glycerolipid Composition in Filamentous Cyanobacteria

Cyanobacteria Spirulina platensis and Nostoc linckia were grown in the presence of 5 mM and 50 mM glucose or 5 mM mannose, non-metabolisable glucose analogue that effectively triggers the repression of photosynthesis. Glucose evoked active cyanobacterial growth but chlorophyll (Chl) content decreased to some extent and porphyrins were excreted. The content of monogalactosyldiacylglycerol decreased in glucose-grown cyanobacteria and that of phosphatidylglycerol increased substantially. Mannose inhibited cyanobacteria growth as well as Chl synthesis, however, phosphatidylglycerol contents were higher than in respective control samples. In cyanobacterial cells glucose may not only inhibit photosynthetic processes, but also cause structural transformations of membranes which may be necessary for the activity of respiratory electron transport chain components under heterotrophic conditions.     

Daphnia vertical distribution and the presence of toxic cyanobacteria

Daphnia can alter its vertical position in the water column in response to chemical cues from predators. In this study we tested the hypothesis that a Daphnia pulex clone with little evolutionary exposure to cyanobacteria would move away from patches of cyanobacteria (Anabaena affinis and A. flos-aquae) which contain potent endotoxins. Daphnia was censused at 2 h intervals for 6 h in laboratory columns in which there was a steep vertical gradient of cyanobacteria. Data were analyzed by repeated-measures ANOVA. Control (no Anabaena) and experimental columns showed no significant differences in Daphnia distributions. Daphnia in experimental columns frequently moved into areas with dense concentrations of Anabaena and stayed there for long periods of time. Our results show that this D. pulex clone does not exhibit a rapid (within 6 h) avoidance response to toxic Anabaena.     

Cyanobacteria dynamics in a restored Finnish lake: a long term simulation study

Several explanations have been presented for the formation of lacustrine cyanobacteria blooms. These include elevated water temperature, nutrient enrichment, low N/P ratios, low light energy requirements, high pH and/or low carbon dioxide concentration, selective zooplankton grazing, excretion of compounds which suppress the growth of competing algae, and migration. The impact of the first four were studied in Lake Tuusulanjärvi, a eutrophic Finnish lake, using deterministic simulation. The study, which covered 15 years, included a sequence of cases on the recovery of the lake after the implementation of two restoration measures: aeration of the hypolimnion, and termination of municipal waste discharge, both of which were done during the study period. The focus was on Microcystis and Oscillatoria, and on the N-fixing cyanobacteria, Anabaena and Aphanizomenon. Eukaryotic phytoplankton were also included in the study. Monitoring data were used. The roles of the hypotheses were discussed, from the point of view of the utility of the information in water resources management.