Linker polypeptides of the phycobilisome from the cyanobacterium Mastigocladus laminosus: amino-acid sequences and relationships

Three linker polypeptides of the phycobilisome from the cyanobacterium Mastigocladus laminosus were isolated: A 8.9-kDa polypeptide, L8.9R(C), which is probably associated with C-phycocyanin, a 34.5-kDa polypeptide, L34.5,PCR, which forms a complex with C-phycocyanin, and a 34.5-kDa polypeptide, L34.5,PECR, which is linked to phycoerythrocyanin. The complete amino-acid sequence (80 residues) of the L8.9R(C) polypeptide was determined as well as the N-terminal 44 residues of both L34.5R polypeptides and the 114 C-terminal residues of L34.5,PECR. L8.9R(C) is homologous to L8.9C (Füglistaller et al. (1984) Hoppe-Seyler's Z. Physiol. Chem. 365, 1085-1096) and to the C-terminal sequence of L34.5,PECR. The N-terminal sequences of L34.5,PECR and L34.5,PCR exhibit 34% homology. The 44 N-terminal residues of L34.5,PECR are related to the beta-subunit of phycoerythrocyanin (23% homology), while the C-terminal sequence of L34.5,PECR is more related to alpha PEC (21% homology within 60 residues). This suggests that the 30-kDa-linker polypeptide family originates from a fusion of the alpha- and beta-subunit genes and the corresponding intercistronic DNA sequence, as might have arisen through mutation in the stop-codon of the beta-subunit gene. Hence, all polypeptides of the phycobilisome (including perhaps the anchor polypeptide) may be derived from an early ancestor phycobiliprotein subunit, which itself is also related to myoglobin (Schirmer et al. (1985) J. Mol. Biol. 184, 251-277).     

Isolation of hydrogenase from the thermophilic cyanobacterium Mastigocladus laminosus

Summary A soluble, cytoplasmic hydrogenase was detected and partially purified from Mastigocladus laminosus. It was found to be unstable but could be stabilized to some extent by Mg⁺⁺; 50% of the activity remained after one week in air at 4 °C.     

Isolation and biliprotein characterization of phycobilisomes from the thermophilic cyanobacterium Mastigocladus laminosus Cohn

A method for the effective isolation of functionally intact phycobilisomes from the thermophilic cyanobacterium M. laminosus is presented, using an unconventional high buffer molarity for stabilizing the aggregates and introducing a DNAse treatment of the disrupted cells to obtain sharp banding of the phycobilisomes in the linear sucrose density gradients.The structural integrity of the isolated phycobilisomes is demonstrated by a fluorescence emission maximum at 673 nm of aggregated allophycocyanin and by electron microscopy.Besides C-phycocyanin and allophycocyanin, phycoerythrocyanin is a constituent pigment of the phycobilisomes. These pigments indicated in the absorption spectrum of phycobilisomes with a maximum at 610 nm and two shoulders at 650 and 580 nm, respectively, were characterized by spectral data and isoelectric points.     

Isolation and characterization of phycoerythrocyanin and chromatic adaptation of the thermophilic cyanobacterium Mastigocladus laminosus

The thermophilic cyanobacterium Mastigocladus laminosus exhibits chromatic adaptation: In green light the production of the phycobiliprotein phycoerythrocyanin is enhanced drastically.Phycoerythrocyanin was characterised with respect to its molecular weight, isoelectric point, absorption spectra and size of its aggregates. The two subunits of the protein were separated and characterised according to these criteria. Their chromophore contents, amino acid compositions and N-terminal amino acid sequences were also determined. The sequences were compared with those of allophycocyanin and C-phycocyanin from Mastigocladus laminosus.Abbreviations PEC phycoerythrocyanin - -PEC -subunit of PEC - -PEC -subunit of PEC - PE phycoerythrin - PC phycocyanin - APC allophycocyanin - SDS PAGE sodium dodecyl sulfate polyacrylamide gel electrophoresis - chl a chlorophyll a     

Heterocyst differentiation in the cyanobacterium Mastigocladus laminosus.

The morphological and ultrastructural aspects of heterocyst differentiation in the branching, filamentous cyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The earliest differentiation stages involved cytoplasmic changes, including (i) rapid degradation of carboxysomes, (ii) degradation of polysaccharide granules, and (iii) accumulation of electron-dense ribosomal or protein material (or both). Intermediate differentiation stages involved synthesis of a homogeneous extra wall layer, development of necks leading to adjacent cells, and elaboration of a complex system of intracytoplasmic membranes. Late differentiation stages included further development of necks and continued elaboration of membranes. Mature heterocysts possessed a uniformly electron-dense cytoplasm that contained large numbers of closely packed membranes, some of which were arranged in lamellar stacks. Mature heterocysts lacked all of the inclusion bodies present in undifferentiated vegetative cells, but contained a number of unusual spherical inclusions of variable electron density. Cells in both narrow and wide filaments were capable of differentiating. No regular heterocyst spacing pattern was observed in the narrow filaments; the number of vegetative cells between consecutive heterocysts of any given filament varied by a factor of 10. Heterocysts developed at a variety of locations in the wide, branching filaments, although the majority of them were situated adjacent to branch points. M. laminosus displayed a marked tendency to produce sets of adjacent heterocysts or proheterocysts (or both) that were not separated from each other by vegetative cells. Groups of four or more adjacent heterocysts or proheterocysts occurred frequently in wide filaments, and in some of these filaments virtually all of the cells appeared to be capable of differentiating into heterocysts.     

Isolation and localization of N4-methylasparagine in phycobiliproteins from the cyanobacterium Mastigocladus laminosus

The occurrence of post-translationally methylated asparagine residues in beta AP from Anabaena variabilis, Synechococcus PCC 6301 and Porphyridium cruentum has recently been reported (Klotz, A.V., Leary, J.A. & Glazer, A.N. (1986) J. Biol. Chem. 261, 15891-15894). We reinvestigated the amino-acid compositions of all phycobiliproteins from Mastigocladus laminosus. During total hydrolysis of beta AP, beta 16.2 and beta PC one mol methylamine per mol protein was released. These proteins were chemically and enzymatically fragmented and the sequences of the fragments containing the modified asparagine residue were determined by automated Edman degradation. Residues beta AP72, beta 16.2 72 and beta PC 72 were identified as N4-methylasparagine. This derivative of asparagine was also found at a homologous position in beta PE of Calothrix. In the x-ray structure model of C-phycocyanin (PC) the residue beta PC 72 points towards the chromophore beta 84, presumably having an effect on the spectroscopic characteristics of this light harvesting pigment protein complex.     

Phylogeography of the thermophilic cyanobacterium Mastigocladus laminosus

We have taken a phylogeographic approach to investigate the demographic and evolutionary processes that have shaped the geographic patterns of genetic diversity for a sample of isolates of the cosmopolitan thermophilic cyanobacterial Mastigocladus laminosus morphotype collected from throughout most of its range. Although M. laminosus is found in thermal areas throughout the world, our observation that populations are typically genetically differentiated on local geographic scales suggests the existence of dispersal barriers, a conclusion corroborated by evidence for genetic isolation by distance. Genealogies inferred using nitrogen metabolism gene sequence data suggest that a significant amount of the extant global diversity of M. laminosus can be traced back to a common ancestor associated with the western North American hot spot currently located below Yellowstone National Park. Estimated intragenic recombination rates are comparable to those of pathogenic bacteria known for their capacity to exchange DNA, indicating that genetic exchange has played an important role in generating novel variation during M. laminosus diversification. Selection has constrained protein changes at loci involved in the assimilation of both dinitrogen and nitrate, suggesting the historic use of both nitrogen sources in this heterocystous cyanobacterium. Lineage-specific differences in thermal performance were also observed.     

Ultrastructure of the cyanobacterium,Mastigocladus laminosus

The morphology and ultrastructure of the thermophilic cyanobacteriumMastigocladus laminosus were examined by scanning and transmission electron microscopy. Mature cultures consisted of relatively old, wide filaments that branched frequently to form younger, thinner filaments. The cells of the younger filaments had a consistently cylindrical morphology, while those of older filaments were rounded and pleomorphic. The internal ultrastructure of the cells depended somewhat on their age. As young cells became larger and wider, their thylakoids underwent slight rearrangement and spread out toward the center of the cytoplasm. Polyphosphate bodies, carboxysomes (polyhedral bodies), and lipid-body-like structures increased in number as the cells aged, but ribosomes and cyanophycin granules were depleted. Cell division involved septum formation followed by ingrowth of the outer membrane and sheath. Cells in older filaments were separated from each other by a complete layer of sheath material. Septum formation in older cells was also seen to occur parallel to the long axis of the filament, thereby confirming that true branching took place.     

Crystallization of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus and preliminary characterization of two crystal forms

The light-harvesting protein phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus Cohn has been crystallized in two different crystal forms by vapour diffusion. In 5% (w/v) polyethylene glycol at pH 8.5, hexagonal crystals of space group P63 with cell constants a = b = 158 A, c = 40.6 A were obtained, which turned out to be almost isomorphous with the hexagonal crystals of C-phycocyanin from the same organism. Consequently, the conformation of both phycobiliproteins must be very similar. From 1.5 M-ammonium sulfate (pH 8.5), orthorhombic crystals of space group P2221 with cell constants a = 60.5 A, b = 105 A, c = 188 A could be grown. Density measurements of these crystals indicate that the unit cell contains 18 (alpha beta)-units. A detailed packing scheme is proposed that is consistent with the observed pseudo-hexagonal X-ray intensity pattern and with the known size and shape of (alpha beta)3-trimers of C-phycocyanin. Accordingly, disc-like (alpha beta)3-trimers are associated face-to-face and stacked one upon another in rods with a period of 60.5 A, corresponding to the cell dimension a.     

Significance of braided trichomes in the cyanobacterium Mastigocladus laminosus.

Loops and braids in filaments of the cyanobacterium Mastigocladus laminosus were observed. Braided filaments were generally in the form of a right-handed helix (87%) but were occasionally observed as left-handed helices (13%). It was demonstrated by time-lapse photomicrography that braids continued to grow as braids and that loops coiled into braids as growth proceeded. Measurements of the distance between grooves in 74 braids yielded an average distance of 13 +/- 3 micron, a result which suggests that braid formation is not random. We propose that the braids arise as a consequence of the helical growth of cells that make up the filaments of M. laminosus.     

Structural basis for the thermostability of ferredoxin from the cyanobacterium Mastigocladus laminosus

Plant-type ferredoxins (Fds) carry a single [2Fe-2S] cluster and serve as electron acceptors of photosystem I (PSI). The ferredoxin from the thermophilic cyanobacterium Mastigocladus laminosus displays optimal activity at 65 degrees C. In order to reveal the molecular factors that confer thermostability, the crystal structure of M.laminosus Fd (mFd) was determined to 1.25 A resolution and subsequently analyzed in comparison with four similar plant-type mesophilic ferredoxins. The topologies of the plant-type ferredoxins are similar, yet two structural determinants were identified that may account for differences in thermostability, a salt bridge network in the C-terminal region, and the flexible L1,2 loop that increases hydrophobic accessible surface area. These conclusions were verified by three mutations, i.e. substitution of L1,2 into a rigid beta-turn ((Delta)L1,2) and two point mutations (E90S and E96S) that disrupt the salt bridge network at the C-terminal region. All three mutants have shown reduced electron transfer (ET) capabilities and [2Fe-2S] stability at high temperatures in comparison to the wild-type mFd. The results have also provided new insights into the involvement of the L1,2 loop in the Fd interactions with its electron donor, the PSI complex.     

Development of motility in cultures of the cyanobacterium Mastigocladus laminosus

Abstract The time course for the development of motility in cultures of the cyanobacterium Mastigocladus laminosus was established quantitatively using a slicer tool as described here. The slicer tool produces samples of trichomes from centrifuged pellets that, under identical conditions, shed comparable numbers of hormogonia. The number of hormogonia formed in liquid culture rises steeply between 24 and 31 h of incubation, returning to essentially zero in the next 24 h. The initial lag may be devoted to the cell divisions needed to form the cells of the hormogonium. The drop in motility could be due to one or more heat-stable substance(s) accumulated in the medium, since used media inhibited motility and the effect resisted autoclaving. The fact that the inoculum needed to be ground in order for motility to occur suggests that the structure of the clump inhibits the shedding of hormogonia. Some ecological implications are proposed, assuming that the clump structure interferes with light and mass transfer.     

Refined three-dimensional structure of phycoerythrocyanin from the cyanobacterium Mastigocladus laminosus at 2.7 A

The structure of the phycobiliprotein phycoerythrocyanin from the thermophilic cyanobacterium Mastigocladus laminosus has been determined at 2.7 A resolution by X-ray diffraction methods on the basis of the molecular model of C-phycocyanin from the same organism. Hexagonal phycoerythrocyanin crystals of space group P6(3) with cell constants a = b = 156.86 A, c = 40.39 A, alpha = beta = 90 degrees, gamma = 120 degrees are almost isomorphous to C-phycocyanin crystals. The crystal structure has been refined by energy-restrained crystallographic refinement and model building. The conventional crystallographic R-factor of the final model was 19.2% with data to 2.7 A resolution. In phycoerythrocyanin, the three (alpha beta)-subunits are arranged around a 3-fold symmetry axis, as in C-phycocyanin. The two structures are very similar. After superposition, the 162 C alpha atoms of the alpha-subunit have a mean difference of 0.71 A and the 171 C alpha atoms of the beta-subunit differ by 0.51 A. The stereochemistry of the chiral atoms in the phycobiliviolin chromophore A84 is C(31)-R, C(4)-S. The configuration of the chromophore is C(10)-Z, C(15)-Z and the conformation C(5)-anti, C(9)-syn and C(14)-anti like the phycocyanobilin chromophores in phycoerythrocyanin and C-phycocyanin.     

Morphology and ultrastructure of the cyanobacterium Mastigocladus laminosus growing under nitrogen-fixing conditions

The morphological and ultrastructural characteristics of the cyanobacterium Mastigocladus laminosus growing under N₂-fixing conditions were examined with light and electron microscopy. Vegetative cells in narrow filaments contained randomly arranged segments of thylakoid membrane, centrally located carboxysomes (polyhedral bodies), peripherally located lipid bodies, and large numbers of polysaccharide granules in addition to nuclear material and ribosomes. The ultrastructural characteristics of cells in wide filaments were similar, except for increased numbers of carboxysomes and lipid bodies. Heterocytes and proheterocysts developed at a variety of locations in narrow filaments, wide filaments, and the lateral branches off of wide filaments. Akinetes were not observed in any of the filaments. The morphological characteristics of heterocysts and proheterocysts were variable and depended on those of the vegative cells from which the heterocysts and proheterocysts developed. Mature M. laminosus heterocysts were somewhat similar to those formed in other cyanobacterial genera, but they possessed a number of distinct and unique ultrastructural characteristics, including (i) the absence of a fibrous and, possibly, a laminated wall layer, (ii) the presence many closely packed membranes throughout most of the cytoplasm, and (iii) the presence of unidentified, spherical inclusion bodies of variable electron density.     

The complete amino acid sequence of both subunits of C-phycocyanin from the cyanobacterium Mastigocladus laminosus.

The amino acid sequences of both subunits of the C-phycocyanin from the thermophilic cyanobacterium Mastigocladus laminosus have been determined. The alpha-chain consists of 162 amino acid residues and has a molecular weight of 18000, whereas the beta-chain consists of 172 residues and has a molecular weight of 19400. For the first three quarters of their length the polypeptide chains are 31% homologous, whereas there is no significant homology in the final quarter up to the C-terminus. This could mean that the introduction of an additional chromophore binding site in the last quarter of the beta-chain during evolution was achieved via a large number of point mutations or by exchange of the whole C-terminal part in an ancestral gene.     

Effect of nitrogen starvation on the morphology and ultrastructure of the cyanobacterium Mastigocladus laminosus.

The effects of nitrogen starvation on the morphology and ultrastructure of the branching, filamentous cyanobacterium Mastigocladus laminosus were examined with light and electron microscopy. The internal ultrastructural characteristics of vegetative cells changed markedly during nitrogen starvation. Carboxysomes were degraded, while polyphosphate bodies and lipid bodies accumulated. The ultrastructure of mature heterocysts was also affected by nitrogen starvation; their intracytoplasmic membranes vesiculated to form vacuolelike structures and, eventually, large empty regions in the cytoplasm. Nitrogen starvation stimulated extensive heterocyst differentiation in M. laminosus, producing heterocyst frequencies of 17.5% in narrow filaments and 28.3% in wide filaments within 44 h after transfer to N-free conditions. Cells in wide filaments differentiated so extensively that only 16.8% of them failed to initiate the differentiation process within 44 h.     

Allophycocyanin complexes of the phycobilisome from Mastigocladus laminosus. Influence of the linker polypeptide L8.9C on the spectral properties of the phycobiliprotein subunits

The following phycobiliproteins and complexes of the allophycocyanin core were isolated from phycobilisomes of the thermophilic cyanobacterium Mastigocladus laminosus: alpha AP, beta AP, (alpha AP beta AP), (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C, (alpha APB alpha AP2 beta AP3)L8.9C. The six proteins and complexes were characterised spectroscopically with respect to absorption, oscillator strength, extinction coefficient, fluorescence emission, relative quantum yield, fluorescence emission polarisation and fluorescence excitation polarisation. The interpretation of the spectral data was based on the three-dimensional structure model of (alpha PC beta PC)3 (Schirmer et al. (1985) J. Mol. Biol. 184, 257-277), which is related to the allophycocyanin trimer. The absorption and CD spectra of the complexes (alpha AP beta AP)3, (alpha AP beta AP)3L8.9C and (alpha APB alpha AP2 beta AP3)L8.9C could be deconvoluted into the spectra of the phycobiliprotein subunits. The assumptions made for the deconvolution could be checked by the synthesis of the spectra of (alpha APB beta AP)3. The synthesised spectra are in good agreement with the corresponding measured spectra published by other authors. Considering the deconvoluted spectra the following influences on the chromophores could be ascribed to L8.9C: L8.9C neither influences the alpha AP nor the alpha APB chromophores. L8.9C shifts the absorption maximum of the beta AP chromophore to longer wavelength than the absorption maximum of the alpha AP chromophore in trimeric complexes. L8.9C increases the oszillator strength of the beta AP chromophores to about the value of the alpha AP chromophores in trimeric complexes. L8.9C turns the beta AP chromophores from sensitizing into weak fluorescing chromophores. By means of the hydropathy plot and the predicted secondary structure, a postulated three-fold symmetry in the tertiary structure of L8.9C could be confirmed.     

Photosynthetic activities of a membrane preparation of the thermophilic cyanobacterium Mastigocladus laminosus

Photosynthetically active membranes have been prepared from the thermophilic cyanobacterium Mastigogladus laminosus by treatment with lysozyme. The membranes were active in electron transport through photosystem I and II as well as in photophosphorylation and proton uptake. Cells were grown at 40°, 45° and 55°C respectively. The temperature optimum of oxygen evolution of whole cells was about 10°C higher than the growth temperature. In isolated membranes the temperature optimum for cyclic photophosphorylation was identical to the growth temperature of the cells whereas the optimum for photosystem II electron transport never exceeded 40°C. Photophosphorylation was inhibited by N, N-dicyclohexyl carbodiimide (DCCD), carbonyl-cyanide-m-chlorophenylhydrazone and NH₄Cl, whereas proton uptake was enhanced by DCCD. Electron transport was slightly inhibited by these treatments. The membranes could be stored for several weeks at-20°C in 50% glycerol without any loss in the activities.Abbreviations DPIP 2,6-dichlorophenolindophenol - CCCP Carbonyl-cyanide-m-chlorophenylhydrazone - DCCD N,N-dicyclohexyl carbodiimide - PMS N-methylphenazonium methosulfate - DCMU 3-(3-4-dichlorophenyl)-1,1-dimethylurea - TMP 20 mM Tris-HCl buffer pH 7.8, 10 mM MgCl₂, 5 mM phosphate buffer pH 7.8